CN117545768A

CN117545768A - Method for screening and expressing disulfide bond-bound binding polypeptides

Info

Publication number: CN117545768A
Application number: CN202280042228.XA
Authority: CN
Inventors: D·麦克格力戈尔; 黄芮祺; G·瓦内尔简金斯; V·斯密德
Original assignee: Institute Of Applied Biomedical Sciences
Current assignee: Institute Of Applied Biomedical Sciences
Priority date: 2021-05-12
Filing date: 2022-05-11
Publication date: 2024-02-09

Abstract

The present disclosure relates to methods of generating and screening display libraries of disulfide-bound binding polypeptides, e.g., to identify binding peptides specific for a target molecule. In some embodiments, the binding peptides comprise ultralong CDR3. These binding peptides may be derived from bovine antibodies comprising ultralong CDR3, or they may be synthetic or semi-synthetic. Also provided herein are display libraries comprising disulfide-bonded binding polypeptides. The disclosure also relates to methods of producing or expressing soluble disulfide-bound binding polypeptides, e.g., using suitable host cells. Also provided herein are compositions comprising soluble disulfide-bonded binding polypeptides.

Description

Method for screening and expressing disulfide bond-bound binding polypeptides

Statement of government interest

The present invention was made with government support under R01 GM105826 and R01 HD088400 awarded by the national institutes of health (National Institutes ofHealth). The government has certain rights in this invention.

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/187,931, filed on day 5 and 12 of 2021, and U.S. provisional application No. 63/288,992, filed on day 13 of 12 and 12 of 2021, the respective contents of which are hereby incorporated by reference in their entirety.

Sequence listing provided as text file

The present application is filed with a sequence listing in electronic format. The sequence listing is provided as a file created at 5.10.2022 under the name 165772000440seqlist. Txt, having a size of 128 kilobytes. The information in the sequence listing in electronic format is incorporated by reference in its entirety.

Technical Field

The present disclosure relates to methods of generating and screening display libraries of disulfide-bound binding polypeptides, e.g., to identify binding peptides specific for a target molecule. In some embodiments, the binding peptide comprises an ultralong CDR3. The binding peptides may be derived from bovine antibodies comprising ultralong CDR3, or they may be synthetic or semi-synthetic. Also provided herein are display libraries comprising disulfide-bonded binding polypeptides. The disclosure also relates to methods of producing or expressing soluble disulfide-bound binding polypeptides, e.g., using suitable host cells. Also provided herein are compositions comprising soluble disulfide-bonded binding polypeptides.

Background

Antibodies are natural proteins formed by the vertebrate immune system in response to foreign substances (antigens), primarily for the defense against infection. Antibodies contain Complementarity Determining Regions (CDRs) that mediate binding to a target antigen. Some bovine antibodies have abnormally long variable heavy chain (VH) CDR3 sequences compared to other vertebrates. These long CDR3 (up to 70 amino acids) can form unique domains that protrude from the antibody surface, thereby allowing for a unique antibody platform. There is a need for improved methods for screening and producing antibodies or portions thereof containing long CDR3, as well as for screening and producing other disulfide-bonded polypeptides.

Disclosure of Invention

In some embodiments, provided herein is a method of making a bovine ultralong CDR3 antibody display library, comprising: (a) Amplifying sequences encoding various variable heavy chain (VH) regions of the IgHV1-7 family from a library of bovine antibody VH chain complementary DNA (cDNA) templates; (b) Constructing a plurality of replicable expression vectors for the plurality of VH regions, wherein each replicable expression vector comprises a first nucleic acid sequence encoding a single chain variable fragment (scFv) comprising an amplified VH region joined to a lambda VL region selected from the group consisting of variable light chain (VL) regions of BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8 and F18, or a humanized variant thereof; (c) Transforming a suitable host cell with the plurality of replicable expression vectors under conditions suitable for producing amplified display particles; and (d) collecting the amplified display particles, wherein the amplified display particles comprise display particles displaying a fusion protein comprising an scFv.

In some of any of the embodiments, the VL region is a BLV1H12 VL region.

In some embodiments, provided herein is a method of making a bovine ultralong CDR3 antibody display library, comprising: (a) Amplifying sequences encoding various variable heavy chain (VH) regions of the IgHV1-7 family from a library of bovine antibody VH chain complementary DNA (cDNA) templates; (b) Constructing a plurality of replicable expression vectors for the plurality of VH regions, wherein each replicable expression vector comprises a first nucleic acid sequence encoding a single chain variable fragment (scFv) comprising an amplified VH region that is ligated to a BLV1H12 lambda variable light chain (VL) region or a humanized variant thereof; (c) Transforming a suitable host cell with the plurality of replicable expression vectors under conditions suitable for producing amplified display particles; and (d) collecting the amplified display particles, wherein the amplified display particles comprise display particles displaying a fusion protein comprising an scFv.

In some of any of the embodiments, the cDNA template library is prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle. In some of any of the embodiments, the method further comprises preparing a cDNA template library from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from the immunized cattle. In some of any of the embodiments, the method further comprises immunizing the cow with a target antigen.

In some of any of the embodiments, the amplified display particles comprise bacterial display, yeast display, mammalian display, phage display, mRNA display, ribosome display, or DNA display particles. In some of any of the embodiments, the amplified display particles are phage display particles. In some of any of the embodiments, the amplified display particle is a phagemid particle. In some of any of the embodiments, each replicable expression vector further comprises a second nucleic acid sequence encoding at least a portion of a phage coat protein, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding a phage coat protein in an amount sufficient to produce a phagemid particle, whereby the fusion protein comprises at least a portion of a phage coat protein.

In some embodiments, provided herein is a method of making a bovine ultralong CDR3 antibody phage display library, comprising: (a) immunizing a bovine with a target antigen; (b) Preparing a library of antibody Variable Heavy (VH) strand complementary DNA (cDNA) templates from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle; (c) Amplifying sequences encoding a plurality of VH regions of the IgHV1-7 family from the cDNA template library; (d) Constructing a plurality of replicable expression vectors for the plurality of VH regions, wherein each replicable expression vector comprises: (1) A first nucleic acid sequence encoding a single-chain variable fragment (scFv) comprising an amplified VH region joined to a BLV1H12 lambda variable light chain (VL) region or a humanized variant thereof, and (2) a second nucleic acid sequence encoding at least a portion of a phage coat protein; (e) Transforming a suitable host cell with the plurality of replicable expression vectors; (f) Infecting the transformed host cell with a helper phage having a gene encoding a phage coat protein in an amount sufficient to produce amplified phagemid particles; and (g) collecting the amplified phagemid particles, wherein the amplified phagemid particles comprise phagemid particles displaying a fusion protein comprising the at least part of the phage coat protein and the scFv.

In some of any of the embodiments, the BLV1H12 lambda VL region is set forth in SEQ ID NO. 2. In some of any of the embodiments, the BLV1H12 VL region is a humanized variant of the VL region of BLV1H 12. In some of any of the embodiments, the humanized variant comprises one or more of the amino acid substitutions I29V and N32G and/or DNN to GDT in the CDR2 region based on the amino acid substitutions of Kabat numbering S2A, T5N, P S, A12G, A S and amino acid substitutions in the P14L, CDR1 region. In some of any of the embodiments, the humanized variant comprises the sequence set forth in SEQ ID NO. 107.

In some of any of the embodiments, the amplified VH region is indirectly joined to the BLV1H12 lambda VL region via a peptide linker. In some of any of the embodiments, the peptide linker is (Gly ₄ Ser) ₃ (SEQ ID NO:94)。

In some of any of the embodiments, the various VH regions of the IgHV1-7 family are amplified from a cDNA template library using a forward primer comprising the sequence set forth in SEQ ID NO. 84 and a reverse primer comprising the sequence set forth in SEQ ID NO. 85.

In some of any of the embodiments, prior to construction, the method further comprises size separating sequences encoding the plurality of amplified VH regions to enrich for VH regions with ultralong CDRs 3. In some of any of the embodiments, the size separation is performed by gel electrophoresis. In some of any of the embodiments, the gel electrophoresis is performed using 1.2%, 1.5% or 2% agarose gel, optionally using 2% agarose gel. In some of any of the embodiments, size separation comprises separating a sequence of length, about or greater than 550 base pairs from sequences encoding the plurality of amplified VH regions, wherein the sequence of length, about or greater than 550 base pairs comprises sequences encoding VH regions with ultralong CDR 3.

In some of any of the embodiments, the gel electrophoresis is performed using a 2% agarose gel.

In some embodiments of any of the embodiments, at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region. In some of any of the embodiments, at least or at least about 30% of the amplified particles display an scFv comprising a VH region comprising an ultralong CDR3 region. In some of any of the embodiments, at least or at least about 40% of the amplified particles display an scFv comprising a VH region comprising an ultralong CDR3 region. In some of any of the embodiments, at least or at least about 50% of the amplified particles display an scFv comprising a VH region comprising an ultralong CDR3 region.

In some of any of the embodiments, the ultralong CDR3 is a 25-70 amino acid peptide sequence comprising a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

In some of any of the embodiments, the ultralong CDR3 is 40 to 60 amino acids in length. In some of any of the embodiments, the ultralong CDR3 is at least 42 amino acids in length. In some of any of the embodiments, the ultralong CDR3 is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

In some of any of the embodiments, the ultralong CDR3 comprises at least 4 cysteine residues. In some of any of the embodiments, the ultralong CDR3 comprises 4 cysteine residues. In some of any of the embodiments, the ultralong CDR3 comprises 6, 8, 10, or 12 cysteine residues.

In some of any of the embodiments, the ultralong CDR3 has at least 2 disulfide bonds. In some of any of the embodiments, the ultralong CDR3 has 2 disulfide bonds. In some of any of the embodiments, the ultralong CDR3 has 3, 4, or 5 disulfide bonds. In some of any of the embodiments, the method further comprises identifying a CDR 3-knob (knob) sequence in the scFv sequence.

In some embodiments, provided herein is a method of preparing an ultralong CDR 3-knob display library, the method comprising: (a) Amplifying sequences encoding a plurality of CDR 3-only knob antibodies from a bovine antibody Variable Heavy (VH) chain complementary DNA (cDNA) template library using forward and reverse primers specific for the upstream stem (walk) domain and downstream stem domain of the bovine ultralong CDR3 region; (b) Constructing a plurality of replicable expression vectors for the plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises a first nucleic acid sequence encoding an amplified CDR3 knob; (c) Transforming a suitable host cell with the plurality of replicable expression vectors under conditions suitable for producing amplified display particles; and (d) collecting the amplified display particles, wherein the amplified display particles comprise display particles displaying a fusion protein comprising an amplified CDR3 knob.

In some embodiments, provided herein is a method of preparing an ultralong CDR 3-knob phage display library, comprising: (a) immunizing a bovine with a target antigen; (b) Preparing a library of antibody Variable Heavy (VH) strand complementary DNA (cDNA) templates from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle; (c) Amplifying sequences encoding a plurality of CDR 3-only knob antibodies from a cDNA template library using forward and reverse primers specific for the upstream and downstream stem domains of the bovine ultralong CDR3 region; (d) Constructing a plurality of replicable expression vectors for the plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises (1) a first nucleic acid sequence encoding an amplified CDR3 knob and (2) a second nucleic acid sequence encoding at least a portion of a phage coat protein; (e) Transforming a suitable host cell with the plurality of replicable expression vectors; (f) Infecting the transformed host cell with a helper phage having a gene encoding a phage coat protein in an amount sufficient to produce amplified phagemid particles; and (g) collecting the amplified phagemid particles, wherein the amplified phagemid particles comprise phagemid particles displaying a fusion protein comprising at least a portion of a phage coat protein and an amplified CDR3 knob.

In some of any of the embodiments, the primer comprises or consists of any one of the sequences set forth in SEQ ID NOS: 7-11 and 121-130.

In some of any of the embodiments, the primer comprises or consists of any one of the sequences set forth in SEQ ID NOS.7-11. In some of any of the embodiments, the primer comprises or consists of any one of the sequences set forth in SEQ ID NOS.8-11. In some of any of the embodiments, the primer comprises or consists of any one of the sequences set forth in SEQ ID NOS.121-130. In some of any of the embodiments, the primer comprises or consists of any one of the sequences set forth in SEQ ID NOS: 123, 127 and 128.

In some of any of the embodiments, the primers comprise two or more of the primers set forth in SEQ ID NOS: 7-11 and 121-130. In some of any of the embodiments, the primers comprise two or more of the primers set forth in SEQ ID NOS 8-11 and 123, 127 and 128. In some of any of the embodiments, the primers comprise three or more of the primers set forth in SEQ ID NOS 8-11 and 123, 127 and 128. In some of any of the embodiments, the primer comprises four or more of the primers set forth in SEQ ID NOS 8-11 and 123, 127 and 128.

In some of any of the embodiments, the primer comprises a primer consisting of the sequence set forth in SEQ ID NO. 8, a primer consisting of the sequence set forth in SEQ ID NO. 9, a primer consisting of the sequence set forth in SEQ ID NO. 10, and a primer consisting of the sequence set forth in SEQ ID NO. 11.

In some of any of the embodiments, the primer comprises a primer consisting of the sequence set forth in SEQ ID NO. 123, a primer consisting of the sequence set forth in SEQ ID NO. 127, and a primer consisting of the sequence set forth in SEQ ID NO. 128.

In some of any of the embodiments, the primer comprises a primer consisting of the sequence set forth in SEQ ID NO. 8, a primer consisting of the sequence set forth in SEQ ID NO. 9, a primer consisting of the sequence set forth in SEQ ID NO. 10, a primer consisting of the sequence set forth in SEQ ID NO. 11, a primer consisting of the sequence set forth in SEQ ID NO. 123, a primer consisting of the sequence set forth in SEQ ID NO. 127, and a primer consisting of the sequence set forth in SEQ ID NO. 128.

In some of any of the embodiments, the method further comprises identifying a CDR 3-knob from a bovine antibody variable weight (VH) chain template sequence. In some of any of the embodiments, the CDR 3-knob is identified from the antibody sequence by an algorithm comprising: identifying a conserved cysteine in frame 3 and a conserved tryptophan in frame 4; and determining the sequence of the CDR-3 knob, wherein: the length of the amino acid sequence of the CDR-3 knob is K; the sequence starts at position X+1 and ends at X+K; and k=l-2X; wherein L is the number of amino acids in the amino acid sequence starting from the conserved cysteine in frame 3 and ending in the conserved tryptophan in frame 4, and X is the number of amino acids from the first cysteine in frame 3 to the first conserved cysteine encoded by the DH region in CDR H3.

In some of any of the embodiments, the antibody sequence is a bovine antibody. In some of any of the embodiments, the identified CDR 3-knob extends one, two, three, four, or five amino acids at the N and/or C terminus as compared to the identified sequence.

In some of any of the embodiments, each of the plurality of CDR 3-only knob antibodies comprises a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds. In some of any of the embodiments, the peptide sequence is 40 to 60 amino acids in length. In some of any of the embodiments, the peptide sequence is at least 42 amino acids in length. In some of any of the embodiments, the peptide sequence is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

In some of any of the embodiments, the peptide sequence comprises at least 4 cysteine residues. In some of any of the embodiments, the peptide sequence comprises 4 cysteine residues. In some of any of the embodiments, the peptide sequence comprises 6, 8, 10, or 12 cysteine residues.

In some of any of the embodiments, the peptide sequence has at least 2 disulfide bonds. In some of any of the embodiments, the peptide sequence has 2 disulfide bonds. In some of any of the embodiments, the peptide sequence has 3, 4, or 5 disulfide bonds.

In some of any of the embodiments, the target antigen is a non-toxic bacterium, virus, viral protein, immunomodulatory protein (e.g., a checkpoint molecule), cancer antigen, human IgG, or recombinant protein thereof. In some of any of the embodiments, the immunomodulatory protein is a checkpoint molecule.

In some of any of the embodiments, cDNA template libraries are synthesized using IgM (SEQ ID NO: 4), igA (SEQ ID NO: 5) and IgG specific (SEQ ID NO:3 and 6) primer libraries. In some of any of the embodiments, cDNA template libraries are synthesized using IgM, igA and IgG specific libraries comprising primers comprising or consisting of the sequences set forth in SEQ ID NO. 4, primers comprising or consisting of the sequences set forth in SEQ ID NO. 5, primers comprising or consisting of the sequences set forth in SEQ ID NO. 3 and primers comprising or consisting of the sequences set forth in SEQ ID NO. 6.

In some embodiments, provided herein is a method of preparing an ultralong CDR 3-knob display library, the method comprising: (a) Constructing a plurality of replicable expression vectors for a plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises a first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds; (b) Transforming a suitable host cell with the plurality of replicable expression vectors under conditions suitable for producing amplified display particles; and (c) collecting the amplified display particles, wherein the amplified display particles comprise display particles displaying a fusion protein comprising a CDR3 knob.

In some embodiments, provided herein is a method of preparing an ultralong CDR 3-knob phage display library, comprising: (a) Constructing a plurality of replicable expression vectors for a plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises: (1) A first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds, and (2) a second nucleic acid sequence encoding at least a portion of a bacteriophage coat protein; (b) Transforming a suitable host cell with a plurality of replicable expression vectors; (c) Infecting the transformed host cell with a helper phage having a gene encoding a phage coat protein sufficient to produce amplified phagemid particles; and (d) collecting the amplified phagemid particles, wherein the amplified phagemid particles comprise phagemid particles displaying a fusion protein comprising at least a portion of a phage coat protein and a CDR3 knob.

In some of any of the embodiments, at least one of the plurality of CDR 3-knob antibodies is identified from an antibody sequence by an algorithm comprising: identifying a conserved cysteine in frame 3 and a conserved tryptophan in frame 4; and determining the sequence of the CDR-3 knob, wherein: the length of the amino acid sequence of the CDR-3 knob is K; the sequence starts at position X+1 and ends at X+K; and k=l-2X; wherein L is the number of amino acids in the amino acid sequence starting from the conserved cysteine in frame 3 and ending in the conserved tryptophan in frame 4, and X is the number of amino acids from the first cysteine in frame 3 to the first conserved cysteine encoded by the DH region in CDR H3. In some of any of the embodiments, the antibody sequence is a bovine antibody. In some of any of the embodiments, the at least one CDR 3-knob antibody has a sequence that extends one, two, three, four, or five amino acids at the N-and/or C-terminus as compared to the identified sequence.

In some of any of the embodiments, the peptide sequence comprises an upstream stem domain and a downstream stem domain, wherein the cysteine motif is between the upstream stem domain and the downstream stem domain.

In some of any of the embodiments, the peptide sequence is amplified from DNA from a bovine immunized with the target antigen. In some of any of the embodiments, the peptide sequence is amplified from a variable heavy chain cDNA library from an immunized cow using primers specific to either side of the stem domain of the bovine ultralong CDR3 region.

In some of any of the embodiments, the peptide sequence does not comprise the upstream stem domain of the N-terminus of the cysteine motif. In some of any of the embodiments, the peptide sequence does not comprise the downstream stem domain of the C-terminal end of the cysteine motif.

In some of any of the embodiments, the upstream stem domain comprises the sequence CX ₂ TVX ₅ Q, wherein X ₂ And X ₅ Is any amino acid. In some of any of the embodiments, X ₂ Ser, thr, gly, asn, ala or Pro, and X ₅ Is His, gln, arg, lys, gly, thr, tyr, phe, trp, met, ile, val or Leu. In some of any of the embodiments, X ₂ Is Ser, ala or Thr, and X ₅ Is His or Tyr.

In some of any of the embodiments, the peptide sequence is a synthetic CDR 3-knob. In some of any of the embodiments, the peptide sequence is a cyclic peptide or a modified cyclic peptide. In some of any of the embodiments, the peptide sequence is a semisynthetic CDR 3-knob derived from a bovine CDR 3-knob.

In some of any of the embodiments, the peptide sequence is 40 to 60 amino acids in length. In some of any of the embodiments, the peptide sequence is at least 42 amino acids in length. In some of any of the embodiments, the peptide sequence is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

In some of any of the embodiments, the plurality of CDR3 knobs are mutated at one or more selected positions within a nucleic acid sequence encoding a peptide sequence, wherein the plurality of replicable expression vectors are a family of mutated vectors.

In some of any of the embodiments, the expression vector further comprises a secretion signal sequence. In some of any of the embodiments, the secretion signal sequence is a pelB signal sequence.

In some embodiments of any embodiment, a suitable host cell is an E.coli cell. In some of any of the embodiments, a suitable host cell is a TG 1-competent cell.

In some of any of the embodiments, the phagemid particles are derived from M13 phage. In some of any of the embodiments, the coat protein is the M13 phage gene III coat protein (pIII). In some of any of the embodiments, the helper phage is selected from the group consisting of M13K07, M13R408, M13-VCS, and Phi X174. In some of any of the embodiments, the helper phage is M13K07.

In some of any of the embodiments, the display particle displays one copy of the fusion protein on average on the surface of the particle.

In some embodiments, provided herein are libraries of display particles produced by any one of the provided methods.

In some embodiments, provided herein is a replicable expression vector comprising a gene fusion encoding a fusion protein comprising a first nucleic acid sequence encoding a single-chain variable fragment comprising a bovine variable heavy chain (VH) region comprising an ultralong CDR3 joined to a lambda VL region of a variable light chain (VL) region selected from BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8 and F18, or a humanized variant thereof.

In some embodiments, provided herein is a replicable expression vector comprising a gene fusion encoding a fusion protein comprising a first nucleic acid sequence encoding a single-chain variable fragment comprising a bovine variable heavy chain (VH) region comprising an ultralong CDR3 joined to a BLV1H12 λ variable light chain (VL) region or a humanized variant thereof.

In some of any of the embodiments, the replicable expression vector further comprises a second nucleic acid sequence encoding at least a portion of a bacteriophage coat protein.

In some embodiments, provided herein are display particles encoded by any one of the provided replicable expression vectors.

In some embodiments, provided herein is a library of display particles comprising any one of a plurality of provided display particles.

In some embodiments of any of the embodiments, at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the display particles in the library comprise an scFv comprising a VH region comprising an ultralong CDR3 region. In some of any of the embodiments, at least or at least about 30% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region. In some of any of the embodiments, at least or at least about 40% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region. In some of any of the embodiments, at least or at least about 50% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region.

In some embodiments, provided herein is a replicable expression vector comprising a gene fusion encoding a fusion protein comprising a first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming a disulfide bond.

In some of any of the embodiments, the display particle is a phage display particle. In some of any of the embodiments, the display particle is a phagemid particle.

In some embodiments, provided herein is a method for selecting an antibody binding protein, the method comprising: (1) Contacting any one of the provided libraries of display particles with a target molecule under conditions that allow binding of the display particles to the target molecule; and (2) separating the bound display particles from unbound display particles, thereby selecting display particles comprising antibody binding proteins that bind the target molecule.

In some of any of the embodiments, the target molecule is a non-toxic bacterium, virus, viral protein, immunomodulatory protein (e.g., a checkpoint molecule), cancer antigen, human IgG, or recombinant protein thereof. In some of any of the embodiments, the target molecule is a coronavirus, a coronavirus pseudovirus, a recombinant coronavirus spike protein, or a Receptor Binding Domain (RBD) of a coronavirus spike protein. In some of any of the embodiments, the coronavirus is selected from the group consisting of 229E, NL, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV2. In some of any of the embodiments, the coronavirus is SARS-CoV2 selected from the group consisting of a Wuhan-Hu-1 isolate, a B.1.351south African variant, or a B.1.1.7UK variant.

In some of any of the embodiments, the method further comprises (i) infecting a suitable host cell with a replicable expression vector encoding the selected display particles bound in (2); (ii) collecting amplified display particles; and (iii) repeating steps (1) and (2) using the amplified display particles as a library of display particles. In some of any of the embodiments, the display particle is a phagemid particle, and the method further comprises infecting the transformed host cell with an amount of a helper phage having a gene encoding a phage coat protein sufficient to produce an amplified phagemid particle.

In some of any of the embodiments, these steps are repeated one or more times. In some embodiments of any of the embodiments, the steps are repeated with the same target molecule or different target molecules. In some of any of the embodiments, the steps are repeated with a different target molecule, and the different target molecule is associated with the target molecule. In some embodiments of any of the embodiments, the different target molecules are the same type of pathogen as the target molecule, pathogens in the same pathogen group, or variants of the target molecule.

In some of any of the embodiments, the method further comprises sequencing the fusion gene in the selected display particle to identify the antibody binding protein.

In some of any of the embodiments, the method further comprises producing full length IgG or Fab from the selected antibody binding proteins.

In some of any of the embodiments, the antibody binding protein is an scFv, and the method comprises constructing a heavy chain or portion thereof comprising ligating a VH region of the scFv with a constant region or portion thereof. In some of any of the embodiments, the method comprises constructing the humanized VH region by replacing the knob region of the ultralong CDR3 region of the humanized bovine VH region with the ultralong CDR3 region of the selected antibody binding protein. In some of any of the embodiments, the ultralong CDR3 region of the selected antibody binding protein is replaced between the upstream and downstream stem chains of a humanized bovine VH region. In some of any of the embodiments, the VH region comprises the formula V1-X-V2, wherein the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO. 111; the X region comprises the ultralong CDR3 of the selected antibody binding protein; and the V2 region comprises the sequence shown in SEQ ID NO. 112. In some of any of the embodiments, the method further comprises constructing a heavy chain or portion thereof comprising ligating the humanized VH region to the constant region or portion thereof. In some of any of the embodiments, the heavy chain or portion thereof is a human IgG1 heavy chain or portion thereof.

In some of any of the embodiments, the method further comprises coexpression of the heavy chain or portion thereof with the light chain. In some of any of the embodiments, the light chain is a bovine light chain of BLVH12, BLV5D3, BLV8C11, BF1H1, BLV5B8, or F18, or a humanized variant thereof. In some of any of the embodiments, the light chain is the BLV1H12 light chain (SEQ ID NO: 113) or a humanized variant thereof. In some of any of the embodiments, the light chain is a humanized light chain as set forth in SEQ ID NO. 114. In some of any of the embodiments, the light chain is the BLV5B8 light chain (SEQ ID NO: 115) or a humanized variant thereof. In some of any of the embodiments, the light chain is a human light chain. In some of any of the embodiments, the light chain is selected from the group consisting of VL1-47, VL1-40, VL1-51 and VL 2-18. In some of any of the embodiments, the light chain is shown in any of SEQ ID NOS 116-120.

In some of any of the embodiments, the light chain is a BLV1H12 light chain comprising the sequence set forth in SEQ ID NO. 113 or a humanized variant thereof. In some of any of the embodiments, the light chain is a BLV5B8 light chain comprising the sequence set forth in SEQ ID NO. 115 or a humanized variant thereof.

In some embodiments, provided herein is a method for producing a soluble ultralong CDR3 knob, the method comprising: (a) Transforming E.coli with an expression vector encoding a fusion protein comprising an ultralong CDR3 knob and a bacterial chaperone joined by a cleavable linker, wherein the ultralong CDR3 knob is a 25-70 amino acid peptide sequence having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds; (b) Culturing the bacterium under conditions allowing expression of the fusion protein; (c) Isolating the fusion protein from the supernatant of the bacterial cell lysate; and (d) cleaving the cleavable linker of the fusion protein, thereby producing a soluble ultralong CDR3 knob comprising 1-6 disulfide bonds that is free of bacterial chaperones.

In some of any of the embodiments, the ultralong CDR3 knob is an antibody binding protein selected by any of the provided methods.

In some of any of the embodiments, the ultralong CDR3 knob is an antibody binding protein identified by any of the provided methods.

In some of any of the embodiments, the fusion protein has increased solubility relative to an ultralong CDR3 knob alone. In some of any of the embodiments, the bacterial chaperonin is thioredoxin a (TrxA).

In some of any of the embodiments, the cleavable linker is an enterokinase cleavable tag having the amino acid sequence DDDDK (SEQ ID NO: 106). In some of any of the embodiments, cleaving the cleavable linker comprises adding enterokinase to the supernatant.

In some of any of the embodiments, the soluble ultralong CDR3 knob comprises an additional linker to allow cyclization of the soluble ultralong CDR3 knob via chemical or enzymatic methods. In some of any of the embodiments, the additional linker allows for sortase-mediated cyclization. In some of any of the embodiments, the method further comprises cyclizing the soluble ultralong CDR3 knob.

In some of any of the embodiments, the method further comprises (e) removing enterokinase and/or bacterial chaperones from the solution comprising the soluble ultralong CDR3 knob.

In some of any of the embodiments, the method further comprises enriching the soluble ultralong CDR3 knob from a solution comprising the soluble ultralong CDR3 knob. In some of any of the embodiments, enriching comprises size exclusion chromatography.

In some of any of the embodiments, the method further comprises generating a multispecific binding molecule comprising a soluble ultralong CDR3 knob.

In some of any of the embodiments, the ultralong CDR3 knob size is 3-8kDa. In some of any of the embodiments, the ultralong CDR3 knob size is 4-5kDa.

In some embodiments, provided herein is a fusion protein comprising an ultralong CDR3 knob and a bacterial chaperone joined by a cleavable linker, wherein the ultralong CDR3 knob is a 25-70 amino acid peptide sequence having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

In some of any of the embodiments, the bacterial chaperonin is thioredoxin a (TrxA).

In some of any of the embodiments, the cleavable linker is an enterokinase cleavable tag having the amino acid sequence DDDDK (SEQ ID NO: 106).

In some of any of the embodiments, the ultralong CDR3 knob comprises 1-6 disulfide bonds.

In some embodiments, provided herein is a composition comprising any one of the provided fusion proteins.

Provided herein is a method of identifying CDR3 knob sequences from antibody sequences, the method comprising identifying a conserved cysteine in frame 3 and a conserved tryptophan in frame 4; and determining the sequence of the CDR-3 knob, wherein: the length of the amino acid sequence of the CDR-3 knob is K; the sequence starts at position X+1 and ends at X+K; and k=l-2X; wherein L is the number of amino acids in the amino acid sequence starting from the conserved cysteine in frame 3 and ending in the conserved tryptophan in frame 4, and X is the number of amino acids from the first cysteine in frame 3 to the first conserved cysteine encoded by the DH region in CDR H3. In some of any of the embodiments, the antibody sequence is a bovine antibody. In some of any of the embodiments, the CDR 3-knob antibody has a sequence that extends one, two, three, four, or five amino acids at the N-and/or C-terminus as compared to the identified sequence.

In some embodiments, provided herein is a purified soluble ultralong CDR3 knob produced by any one of the provided methods, wherein the soluble ultralong CDR3 is 25-75 amino acids in length and comprises 1-6 disulfide bonds.

In some embodiments, the ultralong CDR3 knob has an amino acid sequence length K; and the sequence starts at position x+1 and ends at x+k; and k=l-2X; and wherein L is the number of amino acids in the amino acid sequence of the antibody starting at the conserved cysteine in frame 3 and ending at the conserved tryptophan in frame 4, and X is the number of amino acids from the first cysteine in frame 3 to the first conserved cysteine encoded by the DH region in CDR H3. In some embodiments, the antibody sequence is a bovine antibody. In some embodiments, the knob sequence has a sequence further extended by one, two, three, four, or five amino acids at the N and/or C terminus.

Provided herein are peptide knobs having a sequence of length K, wherein: the knob has an amino acid sequence length K; the sequence starts at position X+1 and ends at X+K; and k=l-2X; and wherein L is the number of amino acids in the amino acid sequence of the antibody starting at the conserved cysteine in frame 3 and ending at the conserved tryptophan in frame 4, and X is the number of amino acids from the first cysteine in frame 3 to the first conserved cysteine encoded by the DH region in CDR H3. In some embodiments, the antibody sequence is a bovine antibody. In some embodiments, the knob sequence has a sequence further extended by one, two, three, four, or five amino acids at the N and/or C terminus.

In some embodiments, provided herein are compositions comprising any of the provided purified soluble ultralong CDR 3.

In some of any of the embodiments, the composition further comprises a pharmaceutically acceptable carrier.

In some of any of the embodiments, the composition is formulated for parenteral administration. In some of any of the embodiments, the composition is formulated for intravenous, intramuscular, topical, otic, conjunctival, nasal, inhalation, or subcutaneous administration. In some of any of the embodiments, the composition is formulated for administration by inhalation.

Drawings

FIG. 1 depicts a schematic diagram of an exemplary ultralong CDR3 bovine antibody, including a "knob" peptide of between 4kDa and 6kDa in size.

FIG. 2A depicts the binding of immune calf serum to the RBD domain of SARS CoV-2S protein via ELISA. The neutralizing activity of serum IgG against SARS-CoV-2 pseudovirus is shown in FIG. 2B.

FIG. 3A depicts pIII phage fusion constructs (i.e., scFv and "knob" displays) in each display library.

FIG. 3B shows a schematic representation of the pTAU1 phage vector multiple cloning site for the direct cloning of bovine CDR3 knob DNA fragments into NcoI-NotI fragments. A schematic representation of the pTAU1-BLV1H12 (-VH) phage scFv vector multiple cloning sites for cloning the bovine VH DNA fragment in-frame with BLV1H 12V-lambda DNA into the NcoI-XhoI fragment is shown in FIG. 3C. FIG. 3D depicts the separation on agarose gel between an ultralong VH fragment and a shorter VH fragment without ultralong CDR3 regions.

Exemplary ultralong antibodies, R2C1 (SKD, SEQ ID NO: 68), R2C3 (SKM, SEQ ID NO: 69), R4C1 (SEQ ID NO: 70), R5C1 (SEQ ID NO: 71), SR3A3 (SEQ ID NO: 72), RR2F12 (SEQ ID NO: 73), and RR2G3 (SEQ ID NO: 74) are aligned in FIG. 4. The germline sequence (SEQ ID NO: 75) is also shown.

FIG. 5A depicts binding of an exemplary chimeric bovine-human IgG1 antibody to spike protein, and binding to RBD is also shown in FIG. 5B. Figure 5C shows ELISA binding of IgG antibodies to recombinant stabilized spike proteins derived from several SARS CoV strains. Figure 5D shows ELISA binding curves of selected IgG antibodies against omigram Rong Bianti RBD (left) or recombinant stabilized spike trimer (right).

FIG. 5E reflects exemplary ELISA data for R4C1 and R2D9 versus SARS-CoV-2 versus SARS-CoV-1. FIG. 5F shows ELISA binding activity for three different exemplary antibody knob candidates against WT (Wuhan) SARS CoV-2 spike protein. Fig. 5G depicts a modified western blot using SDS and biotinylated RBD detection.

FIG. 6A shows a schematic representation of the pET32b vector cloning site for trxA-CDR 3-knob fusion and CDR 3-knob expression. A schematic representation of the purification process from bacterial lysates is shown in fig. 6B. FIG. 6C depicts CDR 3-knob SDS-PAGE showing efficient purification of soluble CDR 3-knobs from E.coli lysates. FIG. 6D depicts an exemplary SDS-PAGE gel of several purified ultralong CDR H3 knob peptides.

FIG. 7A shows the results of a Wuhan-Hu-1 spike protein capture ELISA using serial dilutions of IMAC purified trxA-fusion. Binding of the TrxA-R2G3 fusion protein is also shown in figure 7B.

FIG. 8A depicts a background-subtracted ELISA of soluble biotinylated RBD bound to an exemplary purified R2-G3 CDR 3-knob. Soluble R2G3 knob binding relative to reference anti-spike antibody (CR 3022) is shown in fig. 8B.

The amino acid sequence of an exemplary truncated R2G3 mutant is shown in fig. 8C. Exemplary truncated R2G3 mutants include R2G3 TRUNC1 (SEQ ID NO: 87), R2G3 TRUNC2 (SEQ ID NO: 88), R2G3 TRUNC3 (SEQ ID NO: 89), R2G3 TRUNC3A (SEQ ID NO: 90), R2G3 TRUNC3B (SEQ ID NO: 91), R2G3 TRUNC4 (SEQ ID NO: 92), and R2G3 TRUNC5 (SEQ ID NO: 93). Also shown are parent R2G3 variants (SEQ ID NO: 86) from which exemplary truncation mutants are derived.

FIG. 8D depicts SDS-PAGE of R2G3 truncations after bacterial expression and purification. ELISA binding results for biotinylated RBDs truncated by coated CDR 3-knobs are shown in FIG. 8E.

FIG. 9A depicts size exclusion chromatography of a purified R4C1 knob. The gel electrophoresis gel of the two fractions (A4 and A7) is shown in fig. 9B.

Fig. 9C depicts size exclusion chromatography of the purified R2G3 knob. The gel electrophoresis gel of fraction (A6) is shown in fig. 9D.

The results of the pseudoviral luciferase assays for four exemplary ultralong CDR3 antibodies (F12, G3, SKD and SKM) for viruses expressing wild-type (FIG. 10A), variant "UK" (FIG. 10B), variant "484K" (FIG. 10C) and variant "SA" (FIG. 10D) SARS CoV-2 spike protein are shown in FIG. 10.

Fig. 11A shows IC50 values of different IgG antibodies against pseudoviruses from various coronavirus strains. FIG. 11B shows a comparison of R2G3 IgG, fab and knob in neutralizing wild type SARS-CoV-2 pseudovirus.

FIG. 12 is a depiction of multi-specific knob peptide compositions and formats. A variety of paratope knob peptides can be attached to an immunoglobulin, including as homodimers or heterodimers, to provide a multispecific binding polypeptide. A variety of paratope knob peptides may also be directly connected in series, such as via a linker. The plurality of knob peptides may also be combined into a mixture or mixture to provide a combined polyclonal composition.

FIG. 13A depicts the crystal structure of BLV1H12 Fab (PDB 4k3 d), with an enlarged view of the stem and knob regions with the framework 3 cysteine, knob position 1 cysteine and framework 4 tryptophan side chains shown in FIG. 13B.

The sequence alignment of the stem and knob regions of the 12 exemplary antibodies is shown in fig. 14, flanked by the upstream and downstream stem regions shown with black raised white letters.

FIG. 15 is a schematic representation of a stem and knob domain (L) containing three residues of CDR H3 plus the N-terminus.

Binding to biotinylated RBD by coated CDR 3-knob truncation as assessed by ELISA is shown in fig. 16A. An exemplary SDS-PAGE of the R2G3 truncations after bacterial expression and purification is shown in FIG. 16B.

FIG. 17A shows ELISA binding to biotinylated RBD by coated CDR 3-knob N-terminal truncations, and exemplary SDS-PAGE of R2G 3N-terminal truncations after bacterial expression and purification is shown in FIG. 17B.

FIG. 18A shows sequence alignment of primers specific for the upstream and downstream stem domains of the bovine ultralong CDR3 region. FIG. 18B shows PCR products obtained by amplification using primers.

Detailed Description

In some embodiments, provided herein are display libraries and methods of making display libraries (including bovine or synthetic ultralong CDR3 display libraries or cyclic peptide display libraries), as well as methods of screening the libraries to obtain binding molecules specific for a target molecule. In some embodiments, the display library is derived from sequences that are selectively amplified from cDNA of an immunized cow, e.g., to enrich or select for sequences encoding ultralong CDR 3. In some embodiments, methods of producing soluble peptides, in some cases soluble ultralong CDR3 knobs, are also provided herein. The soluble ultralong CDR3 knob produced may be bovine or synthetic. The soluble peptides produced according to the provided methods also include cyclic peptides.

In some aspects, the provided methods allow screening and generation of disulfide-bonded knob peptides, including those derived from bovine antibodies comprising ultralong CDR3, which can be independently expressed and generated as independent binding units according to the provided methods. In some aspects, the provided methods provide a simple immune-based discovery platform. The platform provides greater structural diversity of peptides than in vitro display based platforms, where each knob peptide screened and generated potentially has its own new disulfide-bonded structure. The platform also allows for rapid hit discovery for target molecules.

As described herein, bovine antibodies have a unique structure comprising an ultralong CDR3 sequence, which forms a structure in which subdomains with unusual architecture are formed by a "stem" consisting of two 12-residues, antiparallel β -strands (uplink and downlink) and a longer, e.g., 39-residue, disulfide-rich "knob" located at the top of the stem away from the paratope of the canonical antibody. The knob region of the ultralong CDR3 confers antigen binding. Unlike antibodies from other species, such as human and mouse, the CDR regions L1, L2, L3, H1 and H2 of bovine or bovine-derived antibodies exhibit less sequence diversity because most of their sequence diversity is in CDR H3 (Stanfield et al 2016Sci.Immunol,1 (1): doi:10.1126/sciimmunol.aaf 7962). Thus, for bovine or bovine-derived antibodies, antigen binding is primarily or solely through CDR H3, while other CDRs do not contribute to antigen binding.

The available methods of analyzing and utilizing unique ultralong CDR H3 structures are not entirely satisfactory. In many cases, the method requires excision and purification of the isolated knob domain (Macpherson et al 2020PLOS Biology,18 (9): e 30000821). Such methods are not easily amenable to good manufacturing practices for the production of therapeutic molecules and are also inefficient in terms of the amount of knob protein that can be produced. In addition, the use of an enzymatic excision knob may also compromise the integrity of the isolated protein.

Notably, it was discovered herein that disulfide-bonded knob peptides derived from the ultralong CDR-H3 of bovine antibodies can be expressed and produced independently as independent binding units according to the provided methods, and retain picomolar binding affinity and neutralizing activity towards target molecules (e.g., SARS-CoV 2). The knob peptide is only about 4-5kDa, e.g., about 4.4kDa, and represents the smallest individual antigen binding domain. It exhibits high affinity and epitope coverage similar to larger antibodies. Its small size approaches that of a small molecule and thus exploits the utility of antigen binding domains as new and novel therapeutic agents. For example, its small size allows for better tissue penetration and also allows alveolar delivery. Furthermore, the knob peptides provided are stable due to their rigid disulfide-bonded small domains. This stable structure avoids aggregates seen in nanobodies and other immunoglobulin domain-based fragments. As also demonstrated herein, it was found that it can be produced in high yield in e.coli according to the provided methods, making knob peptides highly developable as therapeutic molecules. Peptides produced according to the provided methods can be targeted to a known virus or virus type as mabs or as knobs. In some aspects, in the case of a pandemic outbreak, the mAb and knob may be ready for rapid discovery and production, and in the case of a new disease strain, may be rapidly diverted. In some aspects, mAb and knob production according to the provided methods can quickly transition to GMP standards. In some aspects, the knob may be used in a "hybrid" of treatment protocols.

Also provided herein are compositions containing any one of the knob peptides screened and produced according to the provided methods. In some embodiments, the composition may be monoclonal, providing a single knob peptide to provide a single paratope for binding to a desired antigen (such as SARS-CoV 2). In other embodiments, the provided compositions are polyclonal and contain a mixture or mixture of different knob peptides directed against different epitopes of an antigen or different antigens (fig. 12).

In addition, multi-specific binding formats utilizing knob peptides of small and unique sizes are provided herein (fig. 12). For example, different knob paratopes may be engineered into the backbone of a human or humanized ultralong CDR-H3 full length antibody, wherein dimerization of Fc provides bivalent or multivalent forms. In some cases, a "knob-in-hole" (pestle) Fc engineering strategy can be used to generate heterodimeric bispecific or multispecific forms containing two, three, four, or more different knob peptides, each providing a different paratope for binding to a desired antigen, such as a spike protein of SARS-CoV 2.

Also provided herein are methods of treatment and uses of the provided binding polypeptides (including antibodies or antigen binding fragments or knob polypeptides) and compositions thereof.

I. Definition of the definition

Unless defined otherwise, all technical, symbolic, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ease of reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is commonly understood in the art.

As used herein, the articles "a" and "an" refer to one or more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, where a range of values is provided, it is to be understood that each intervening value, to the extent that it is between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of the limits included are also included in the claimed subject matter. This applies regardless of the width of the range.

As used herein, the term "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, when referring to a measurable value (such as an amount, duration, etc.), the term "about" is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1% and still more preferably ±0.1% relative to the specified value, as such variations are suitable for carrying out the disclosed methods.

An "ultralong CDR3" or "ultralong CDR3 sequence" as used interchangeably herein comprises a CDR3 or CDR3 sequence that is not derived from a human antibody sequence. The ultralong CDR3 may be 35 amino acids or more in length, for example 40 amino acids or more in length, 45 amino acids or more in length, 50 amino acids or more in length, 55 amino acids or more in length, or 60 amino acids or more in length. In some embodiments, the ultralong CDR3 is 25-70 amino acids in length, such as 40-70 amino acids in length. Typically, the ultralong CDR3 is a heavy chain CDR3 (CDR-H3 or CDRH 3). Ultralong CDR3H3 exhibits the characteristics of CDRH3 of ruminant (e.g., bovine) sequences. The structure of an ultralong CDR3 includes a "stem" consisting of an uplink and a downlink (e.g., about 12 amino acids in length per chain), and a disulfide-rich "knob" located at the top of the stem. The unique "stem and knob" structure of ultralong CDR3 results in two antiparallel β chains (up and down stem chains) of the knob supporting disulfide bonding protruding out of the antibody surface to form a tiny antigen binding domain. In some embodiments, the ultralong CDR3 antibody comprises an upstream stem region, a knob region, and a downstream stem region in that order.

As used herein, "CDR 3-knob" or "knob" interchangeably refers to a portion of an ultralong CDR3 that is a peptide sequence of 40-70 amino acids in length, wherein the CDR 3-knob has at least 4 non-canonical Cys residues, such as 6, 8, 10, or up to 12 non-canonical cysteine residues, and forms 2-6 disulfide bonds. Typically, the knob contains an initial cysteine residue with the amino acid motif cysteine-proline (CP). In some cases, the CDR 3-knob may be located between an upstream stem (stem a) or a downstream stem (stem B) in an antibody or antigen binding fragment containing the ultralong CDR3, wherein the CDR 3-knob protrudes beyond the antibody interface to form an antigen binding site with the antigen. In other cases, the CDR 3-knob may be independently generated as a "knob" peptide as described herein.

As used herein, the terms "knob peptide", "CDR 3-knob peptide" or "knob-only peptide" are used interchangeably to refer to independently generated linear disulfide-bonded peptides that are 40-70 amino acids in length and contain 2-6 disulfide bonds formed by at least 4 non-canonical Cys residues (such as 6, 8, 10, or up to 12 non-canonical cysteine residues). Knob peptides may be derived from ultralong CDR3 or may be synthetically produced. Typically, the first cysteine of the peptide sequence contains the initial cysteine residue with the amino acid motif cysteine-proline (CP). Knob peptides are linear molecules that cannot undergo cyclization to form cyclic molecules.

"substantially similar" or "substantially identical" refers to a sufficiently high degree of similarity between two values (typically one associated with an antibody disclosed herein and the other associated with a reference/comparison antibody) that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance in the context of the biological property measured by the values (e.g., kd values). The difference between the two values is preferably less than about 50%, preferably less than about 40%, preferably less than about 30%, preferably less than about 20%, preferably less than about 10% as a function of the value of the reference/comparison antibody.

"binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to an inherent binding affinity that reflects a 1:1 interaction between binding pair members (e.g., antibodies and antigens). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant. Low affinity antibodies typically bind antigen slowly and tend to dissociate easily, while high affinity antibodies typically bind antigen faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which may be used for the purposes of this disclosure.

"percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MegAlign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared.

"polypeptide", "peptide", "protein" and "protein fragment" are used interchangeably to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

"amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

"conservatively modified variants" applies to both amino acid and nucleic acid sequences. "amino acid variant" refers to an amino acid sequence. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acids do not encode an amino acid sequence, essentially identical or related (e.g., naturally contiguous) sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, both codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to another of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence encoding a polypeptide herein also describes silent variations of that nucleic acid. The skilled artisan will recognize that in some cases, each codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) may be modified to produce a functionally identical molecule. Thus, silent variations of a nucleic acid which encodes a polypeptide are implicit in the described sequence with respect to the expression product, not with respect to the actual probe sequence. With respect to amino acid sequences, the skilled artisan will recognize that a single substitution, deletion, or addition of a nucleic acid, peptide, polypeptide, or protein sequence that alters, adds, or deletes a single amino acid or a small percentage of amino acids in the coding sequence is a "conservatively modified variant" including where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions providing functionally similar amino acids are well known in the art. Such conservatively modified variants are complements of, and do not exclude, polymorphic variants, interspecies homologs, and alleles disclosed herein. Typical conservative substitutions include: 1) Alanine (a), glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) Serine (S), threonine (T); 8) Cysteine (C), methionine (M) (see, e.g., cright on, proteins (1984)).

A "humanized" or "human engineered" form of a non-human (e.g., bovine) antibody is a chimeric antibody that contains amino acids representing the sequence of a human immunoglobulin, including, for example, where the smallest sequence is derived from a non-human immunoglobulin. For example, a humanized or human engineered antibody may be a non-human (e.g., bovine) antibody in which some residues are substituted with residues from similar sites in a human antibody (see, e.g., U.S. Pat. No. 5,766,886). The humanized antibody optionally may further comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones et al, nature 321:522-525 (1986); riechmann et al Nature332:323-329 (1988); and Presta, curr.Op.struct.biol.2:593-596 (1992). See also the following review articles and references cited therein: vaswani and Hamilton, ann. Allergy, asthma & immunol.1:105-115 (1998); harris, biochem. Soc. Transactions 23:1035-1038 (1995); hurle and Gross, curr.op.Biotech.5:428-433 (1994).

By "variable domain" of an antibody is meant a specific Ig domain of an antibody heavy or light chain, which contains amino acid sequences that vary among different antibodies. Each light chain and each heavy chain has one variable region domain (VL and VH). The variable domains provide antigen specificity and are therefore responsible for antigen recognition. Each variable region contains CDRs and Framework Regions (FR) as part of an antigen binding site domain.

"constant region domain" refers to a domain in an antibody heavy or light chain that contains amino acid sequences that are relatively more conserved in antibodies than variable region domains. Each light chain has a single light chain constant region (CL) domain and each heavy chain contains one or more heavy chain constant region (CH) domains, including CH1, CH2, CH3, and in some cases CH4. Full length IgA, igD and IgG isotypes contain CH1, CH2, CH3 and hinge regions, while IgE and IgM contain CH1, CH2, CH3 and CH4. The CH1 and CL domains extend the Fab arm of the antibody molecule, thus facilitating interaction with the antigen and rotation of the antibody arm. The antibody constant regions may serve effector functions such as, but not limited to, clearing antigens, pathogens, and toxins to which the antibodies specifically bind, for example, through interactions with various cells, biomolecules, and tissues.

The terms "complementarity determining region" and "CDR," synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous amino acid sequences that confer antigen specificity and/or binding affinity within an antibody variable region. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Typically, there are four FRs (FR-H1, FR-H2, FR-H3 and FR-H4) in each full-length heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3 and FR-L4) in each full-length light chain variable region.

The exact amino acid sequence boundaries for a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by: kabat et al (1991), "Sequences of Proteins of Immunological Interest," 5 th edition Public Health Service, national Institutes ofHealth, bethesda, MD ("Kabat" numbering scheme); al-Lazikani et Al, (1997) JMB 273,927-948 ("Chothia" numbering scheme); macCallum et al, J.mol. Biol.262:732-745 (1996), "anti-body-antigen interactions: contact analysis and binding site topography," J.mol. Biol.262,732-745 "(" Contact "numbering scheme); lefranc MP et al, "IMGT unique numbering for immunoglobulin and T cell receptor variabledomains and Ig superfamily V-like domains," Dev Comp Immunol, month 1 2003; 27 (1) 55-77 ("IMGT" numbering scheme); honyger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variabledomains: an automatic modeling and analysis tool," J Mol Biol, 6/8/2001; 309 (3) 657-70, ("Aho" numbering scheme); and Martin et al, "Modeling antibody hypervariable loops: a combined algorithm," PNAS,1989,86 (23): 9268-9272, ("AbM" numbering scheme).

The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. Numbering of both Kabat and Chothia protocols is based on the most common antibody region sequence length, with insertions achieved by insert letters, e.g. "30a", and deletions occurring in some antibodies. These two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is based on a compromise between Kabat and Chothia definitions used by Oxford Molecular AbM antibody modeling software.

Table 1 below lists exemplary location boundaries for CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 identified by the Kabat, chothia, abM and Contact schemes, respectively. For CDR-H1, the residue numbers are listed using the Kabat and Chothia numbering schemes. FR is located between the CDRs, e.g., FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, etc. It should be noted that because the Kabat numbering scheme shown will be inserted at H35A and H35B, the ends of the Chothia CDR-H1 loop vary between H32 and H34 when numbered using the Kabat numbering convention shown, depending on the length of the loop.

TABLE 1 boundary of CDRs according to various numbering schemes。

1-Kabat et Al (1991), "Sequences of Proteins of Immunological Interest," 5 th edition Public Health Service, national Institutes of Health, bethesda, MD2-Al-Lazikani et Al, (1997) JMB 273,927-948

Thus, unless otherwise indicated, a given antibody or region thereof is such as"CDR" or "complementarity determining region" of its variable region or a separately specified CDR (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass a complementarity determining region as defined (or specified) by any one of the preceding schemes. For example, when it is stated that a particular CDR (e.g., CDR-H3) contains a given V _H Or V _L Where the amino acid sequences of corresponding CDRs in a region amino acid sequence, it is to be understood that such CDRs have the sequences of the corresponding CDRs (e.g., CDR-H3) within the variable region, as defined by any one of the preceding schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes, but it should be understood that the provided antibodies may include CDRs as described according to any of the other foregoing numbering schemes or other numbering schemes known to the skilled artisan

Likewise, unless otherwise indicated, the FR or individually specified FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4) of a given antibody or region thereof, such as the variable region thereof, is to be understood as encompassing a (or a specific) framework region as defined by any of the known schemes. In some cases, schemes for identifying a particular CDR, FR, or CDR are specified, such as the CDR defined by Kabat, chothia, abM or Contact methods. In other cases, specific amino acid sequences of CDRs or FR are given.

An antibody containing ultralong CDR3 is an antibody containing a Variable Heavy (VH) chain with ultralong CDR 3. Antibodies may also include pairing of VH chains with Variable Light (VL) chains. In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region. Thus, the term antibody includes full length antibodies and portions thereof, including antibody fragments, wherein such antibodies contain heavy chains or portions thereof and/or light chains or portions thereof. An antibody may contain two heavy chains (which may be represented as H and H ') and two light chains (which may be represented as L and L'), wherein each L chain is linked to an H chain by a covalent disulfide bond, and the two H chains are linked to each other by a disulfide bond. The terms "full length antibody" or "intact antibody" are used interchangeably to refer to an antibody in substantially intact form as opposed to an antibody fragment. Full length antibodies are antibodies that typically have two full length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH 4) and two full length light chains (VL-CL) and hinge regions.

The term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F (ab') ₂ Fragments, fab' fragments, fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) capable of specific binding _H ) Regions and single chain variable fragments (scFv).

An "antibody fragment" comprises a portion of an intact antibody, an antigen-binding region and/or a variable region of an intact antibody. Antibody fragments include, but are not limited to, fab fragments, fab 'fragments, F (ab') ₂ Fragments, fv fragments, disulfide-linked Fv (dsFv), fd fragments, fd' fragments; single chain antibody molecules, including single chain Fv (scFv) or single chain Fab (scFab); antigen binding fragments of any of the above and multispecific antibodies derived from antibody fragments.

"Fab fragments" are antibody fragments produced by digestion of full-length immunoglobulin with papain, or fragments of identical structure produced synthetically (e.g., by recombinant means). Fab fragments contain the light chain (containing V _L And C _L ) And another chain comprising the variable domain of the heavy chain (V _H ) And a constant region domain of the heavy chain (C _H 1)。

"scFv fragment" refers to a polypeptide comprising a variable light chain (V _L ) And a variable heavy chain (V _H ) Is a fragment of an antibody of (a). The length of the linker is such that the two variable domains bridge without substantial interference. Exemplary linkers are (Gly-Ser) _n Residues, some of which are dispersed throughout to increase solubility.

The term "corresponding to" with respect to the position of a protein, e.g., reciting a nucleotide or amino acid position "corresponding to" a nucleotide or amino acid position in a disclosed sequence, such as shown in the sequence listing, refers to a nucleotide or amino acid position identified upon alignment of the disclosed sequence based on structural sequence or using standard alignment algorithms (such as the GAP algorithm). For example, corresponding residues of similar sequences (e.g., fragments or species variants) can be determined by structural alignment methods with reference sequences. By aligning the sequences, the person skilled in the art can identify the corresponding residues, for example using conserved and identical amino acid residues as guidance.

As used herein, the term "effective amount" or "therapeutically effective amount" means an amount of a pharmaceutical composition sufficient to significantly and positively alter the symptoms and/or conditions to be treated (e.g., provide a positive clinical response). The effective amount of the active ingredient used in the pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient employed, the particular pharmaceutically acceptable excipients and/or carriers employed, and the knowledge and expertise of the attending physician.

As used herein, the term "pharmaceutically acceptable" refers to a material that does not abrogate the biological activity or properties of the compound and that is relatively non-toxic, such as a carrier or diluent, i.e., the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, a composition refers to any mixture of two or more products, substances, or compounds (including cells). It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound of the present invention with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. There are a variety of techniques in the art for administering compounds including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary and topical administration, and administration via inhalation.

As used herein, "disease or disorder" refers to a pathological condition in an organism caused by a cause or disorder including, but not limited to, infection, acquired disorder, genetic disorder, and characterized by identifiable symptoms.

As used herein, the term "treatment", "treatment" or "treatment" refers to ameliorating a disease or disorder, e.g., slowing or preventing or reducing the progression of the disease or disorder, e.g., the root cause of the disorder or at least one clinical symptom thereof.

As used herein, the term "subject" refers to animals, including mammals, e.g., humans. The terms subject and patient are used interchangeably.

As used herein, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or substituted.

Display library and selection method

In some aspects, provided herein are methods of making an ultralong CDR3 antibody display library. In some aspects, provided herein are also methods of making an ultralong CDR 3-knob display library. In some embodiments, the display library is a phage display library. In some embodiments, the ultralong CDR3 antibody or knob is derived from a bovine antibody, e.g., an antibody produced based on a bovine immunized with the target antigen. In some embodiments, the ultralong CDR3 antibody or knob is synthetic. In some embodiments, the ultralong CDR3 antibody or knob comprises a cyclic peptide or modified cyclic peptide, e.g., containing an exogenous peptide sequence.

A. Library generation method

Techniques for manipulating nucleic acids, such as creating mutations in sequences, subcloning, labeling, probing, sequencing, hybridization, etc., are described in detail in scientific publications and patent literature. See, e.g., sambrook J, russell DW (2001) Molecular Cloning: a Laboratory Manual, 3 rd edition Cold Spring Harbor Laboratory Press, new York; current Protocols in Molecular Biology, ausubel, john Wiley & Sons, inc., new York (1997); laboratory Techniques in Biochemistry and Molecular Biology: hybridization With Nucleic Acid Probes, section I, theory and Nucleic Acid Preparation, tijssen, eds., elsevier, N.Y. (1993).

Any known method for generating a library containing variant polynucleotides and/or polypeptides may be used with the provided methods and vectors to generate a display library, e.g., a phage display library, and select binding proteins from the library. The library may be used in a screening assay to select binding proteins from the library for any antigen, including, for example, any virus, bacteria, other pathogenic, immunomodulatory proteins (e.g., checkpoint molecules), or cancer antigens. To facilitate screening, antibody libraries are typically screened using display technology such that there is a physical link between the individual molecules (phenotypes) of the library and the genetic information (genotypes) encoding them. Such methods include, but are not limited to, cell display, including bacterial display, yeast display, mammalian display, phage display (Smith, G.P. (1985) Science 228:1315-1317), mRNA display, ribosome display, and DNA display.

In some embodiments, the library provided is a phage display library. In some embodiments, the display library is a phage display library. In some embodiments, phage display libraries are generated using phagemids encoding at least a portion of phage coat proteins, in addition to encoding polypeptides for display. In some embodiments, the phagemid particles are derived from M13 phage. In some embodiments, the coat protein is the M13 phage gene III coat protein (pIII).

In some embodiments, phage display libraries are generated by fusing a candidate binding polypeptide as described herein, such as an ultralong CDR3 scFv antibody fragment or an ultralong CDR3 knob peptide, with the gene III small coat protein (Ff: F1, M13, or fd) of an F-specific filamentous phage of escherichia coli. Alternatively, other bacterial species may be used to generate phage display libraries, including Pseudomonas fluorescens. In some embodiments, gene III is a small coat protein (also known as pIII) of M13 phage. The gene III small coat protein (present in about 5 copies at one end of the virion) is involved in correct phage assembly and infection by attachment to the pili of e.coli. Phage display methods are known.

In some embodiments, a nucleic acid encoding a candidate binding polypeptide as described herein (such as an ultralong CDR3 scFv antibody fragment or an ultralong CDR3 knob peptide) is inserted into or constructed as part of a replicable expression vector, wherein the nucleic acid is fused to a nucleic acid encoding at least a portion of a phage coat protein (e.g., pIII). In some embodiments, a nucleic acid encoding a candidate binding polypeptide as described herein (such as an ultralong CDR3 scFv antibody fragment or an ultralong CDR3 knob peptide) is fused to pIII.

In some embodiments, the replicable expression vector is a plasmid vector that typically contains a variety of components including a promoter, a signal sequence, a phenotypically selectable gene, an origin of replication site, and other necessary components known to those of ordinary skill in the art. The most commonly used promoters in prokaryotic vectors include the lac Z promoter system, alkaline phosphatase pho A promoter, phage lambda PL promoter (temperature sensitive promoter), tac promoter (hybrid trp-lac promoter regulated by lac repressor), tryptophan promoter, phage T7 promoter or other suitable microbial promoters. Examples of promoter systems include lacz, λpl, TAC, T7 polymerase, tryptophan and alkaline phosphatase promoters, and combinations thereof. Suitable prokaryotic signal sequences may be obtained from genes encoding, for example, the lamB or OmpF (Wong et al, gene,68:193 1983), malE, phoA, E.coli thermostable enterotoxin II (STII) signal sequences or the Pel B secretion signal sequences. In some embodiments, the expression vector will further contain a secretion signal sequence operably fused to the nucleic acid encoding the polypeptide. In some embodiments, the secretion sequence is a Pel B secretion signal sequence. In some embodiments, the replicable expression vector may also contain a phenotype selection gene. Typical phenotype selection genes are those genes encoding proteins that confer antibiotic resistance to the host cell. Illustratively, an ampicillin resistance gene (amp), a tetracycline resistance gene (tet), or a carbenicillin resistance gene may be used.

Construction of a suitable vector containing the nucleic acid encoding the desired polypeptide is prepared using standard recombinant DNA procedures. The separate DNA fragments to be combined to form the vector are cut, customized, and ligated together in a particular order and orientation to produce the desired vector. In some embodiments, the DNA is cleaved using one or more suitable restriction enzymes in a suitable buffer. The appropriate buffer, DNA concentration, and incubation time and temperature are specified by the manufacturer of the restriction enzyme. Typically, an incubation time of about one or two hours at 37℃is sufficient, although several enzymes require higher temperatures. After incubation, enzymes and other contaminants were removed by extracting the digestion solution with a mixture of phenol and chloroform, and DNA was recovered from the aqueous fraction by precipitation with ethanol.

In order to ligate DNA fragments together to form a functional vector, the ends of the DNA fragments must be compatible with each other. In some cases, the ends will be directly compatible after endonuclease digestion. However, it may be desirable to first convert the cohesive ends typically produced by endonuclease digestion into blunt ends to make them suitable for ligation. To blunt the ends, the DNA is treated with 10 units of the Klenow fragment of DNA polymerase I (Klenow) in the presence of four deoxynucleotide triphosphates in a suitable buffer at 15 ℃ for at least 15 minutes. The DNA was then purified by phenol-chloroform extraction and ethanol precipitation.

The DNA fragments to be ligated together (digested beforehand with a suitable restriction enzyme such that the ends of each fragment to be ligated are compatible) are placed in solution. In some embodiments, the DNA fragments are provided in about equimolar amounts. In some embodiments, the solution will also contain ATP, ligase buffer, and a ligating such as T4 DNA ligase, such as at or about 10 units per 0.5 μg of DNA. If a DNA fragment is to be ligated into a vector, the vector is first linearized by cleavage with the appropriate restriction enzymes. The linearized vector is then treated with alkaline phosphatase or calf intestinal phosphatase. Phosphatase prevents self-ligation of the vector during the ligation step.

In some embodiments, a plurality of constructed replicable expression vectors are transformed into a suitable host cell. Suitable host cells include prokaryotic host cells. In some embodiments, the host cell used to express or produce the display library is an E.coli cell. Suitable prokaryotic host cells include E.coli strain JM101, E.coli K12 strain 294 (ATCC No. 31,446), E.coli strain W3110 (ATCC No. 27,325), E.coli X1776 (ATCC No. 31,537), E.coli XL-1Blue (stratagene) and E.coli B; however, many other strains of E.coli may also be used, such as HB101, NM522, NM538, NM539 and many other species and genera of prokaryotes. In addition to the E.coli strains listed above, bacillus species (such as Bacillus subtilis), other Enterobacteriaceae species (such as Salmonella typhimurium or Serratia marcescens), and various Pseudomonas species can be used as hosts. In some embodiments, the host cell is a protease deficient strain of E.coli. In some embodiments, the host cell is a TG 1-competent cell.

Transformation of prokaryotic cells can be readily accomplished using the calcium chloride method as described in Sambrook et al, supra, section 1.82. Alternatively, electroporation (Neumann et al, EMBO J.,1:841 1982) may be used to transform these cells.

In some embodiments, the method further comprises infecting the transformed host cell with a helper phage having a gene encoding a phage coat protein. In some embodiments, the method further comprises using a helper phage to promote adequate expression of the phagemid particles. In some embodiments, the helper phage is selected from the group consisting of M13K07, M13R408, M13-VCS, and Phi X174. In some embodiments, the helper phage is M13K07. The transformed infected host cell is then cultured under conditions suitable to form recombinant phagemid particles containing at least a portion of the plasmid and capable of transforming the host. Transformed cells are selected by growth on antibiotics such as tetracycline (tet) or ampicillin (amp), carbenicillin or other antibiotics, depending on the particular expression vector, to which the transformed cells are resistant due to the presence of the resistance gene on the vector.

After selection of transformed cells, these cells are grown in culture and plasmid DNA (or other vector into which the foreign gene has been inserted) is isolated. Plasmid DNA may be isolated using methods known in the art. The isolated DNA may be purified by methods known in the art. The purified plasmid DNA is then analyzed by restriction mapping and/or DNA sequencing.

1. Polypeptides for display

In some embodiments, the polypeptide for display comprises an ultralong CDR3. In bovine antibodies, the ultralong CDR3 sequences form structures in which subdomains with unusual architecture are formed by a "stem" consisting of two 12-residues, antiparallel β -strands (uplink and downlink), and a longer, e.g., 39-residue, disulfide-rich "knob" located at the top of the stem, distal to the canonical antibody paratope. The long antiparallel β -bands act as bridges to link the knob domain to the primary antibody scaffold. The unique "stem and knob" structure of ultralong CDR3 results in two antiparallel β chains (up and down stem chains) of the knob supporting disulfide bonding protruding out of the antibody surface to form a tiny antigen binding domain. In some embodiments, the ultralong CDR3 antibody comprises an upstream stem region, a knob region, and a downstream stem region in that order.

In some embodiments, the ultralong CDR-H3 comprises an upstream stem domain (stem a), a disulfide-rich knob region, and a downstream stem domain (stem B), wherein the knob region is located between the upstream stem domain and the downstream stem domain. In some embodiments, the sequence of the ultralong CDR-H3 provides the structure of an antiparallel β -chain protruding from the antibody, with a disulfide-rich knob region located at the tip of the antibody (fig. 1). Stem a contains mainly hydrophobic side chains and relatively conserved motifs at the bases, which initiate the uplink. This conserved motif is usually found after the first cysteine residue in the variable region sequence of various bovine or dairy cow sequences. In some embodiments, the base of stem A contains the residues CTTVHQ (SEQ ID NO: 98), CATVHQ (SEQ ID NO: 99), CAIVQQ (SEQ ID NO: 100) or CATVDQ (SEQ ID NO: 101) that stabilize the base by interacting with the residues of CDR-H1. Before the first conserved cysteine residues forming part of the disulfide-bonded knob region, stem a is linked by a variable number of residues (e.g., 2 to 8 amino acid residues). In some embodiments, the knob region includes a first conserved amino acid motif Cys-Pro (CP), wherein the starting cysteine residue is identical to the cysteine residue in the knob The acid residues form a first disulfide bond. The knob may include 2-12 cysteine residues capable of forming 2-6 disulfide bonds. The stems may have variable lengths and stem B may contain alternating aromatic compounds that form ladders by stacking interactions, which may contribute to the stability of the long solvent-exposed double-stranded β -strand (Wang et al cell.2013,153 (6): 1379-1393). In some embodiments, stem B contains a conserved pattern of alternating tyrosines, sometimes with motif YX supporting knob structure ₁ YX ₂ Y(SEQ ID NO:102)。

In some embodiments, the ultralong CDR3 comprises or is a peptide sequence of 25-70 amino acids. In some embodiments, the ultralong CDR3 is a peptide sequence of length between or between about 35 and 70 amino acids, 40 and 70 amino acids, 45 and 70 amino acids, 50 and 70 amino acids, 55 and 70 amino acids, or 60 and 70 amino acids.

In some embodiments, the ultralong CDR3 includes a cysteine motif. In some embodiments, the cysteine motif comprises 2-20 cysteine residues, e.g., between or about 2 and 18, 2 and 16, 2 and 14, 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 20, 4 and 18, 4 and 16, 4 and 14, 4 and 12, 4 and 10, 4 and 8, 4 and 6, 6 and 20, 6 and 18, 6 and 16, 6 and 14, 6 and 12, 6 and 10, 6 and 8, 8 and 20, 8 and 18, 8 and 16, 8 and 14, 8 and 12, 8 and 10, 10 and 20, 10 and 18, 10 and 16, 10 and 14, 10 and 12, 12 and 18, 12 and 16, 12 and 14, 14 and 20, 14 and 18, 14 and 16, 16 and 20, 16 and 18, or 18 and 20, each comprising a terminus. In some embodiments, the cysteine motif comprises 2-12 cysteine residues.

In some embodiments, the ultralong CDR3 knob comprises 1-10 disulfide bonds, e.g., between or about 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, 1 and 2, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, 2 and 3, 3 and 10, 3 and 9, 3 and 8, 3 and 7, 3 and 6, 3 and 5, 3 and 4, 4 and 10, 4 and 9, 4 and 8, 4 and 7, 4 and 6, 4 and 5, 5 and 10, 5 and 9, 5 and 8, 5 and 7, 5 and 6, 6 and 10, 6 and 8, 6 and 7, 7 and 10, 7 and 9, 7 and 8, 8 and 10, 8 and 9, or 9 and 10 disulfide bonds, each comprising an end value. In some embodiments, the ultralong CDR3 knob comprises 1-6 disulfide bonds.

In some embodiments, the ultralong CDR3 comprises an upstream stem domain. In some embodiments, the ultralong CDR3 comprises a downstream stem domain. In some embodiments, the cysteine motif is between the upstream stem domain and the downstream stem domain. In some embodiments, the upstream stem domain comprises the sequence CX ₂ TVX ₅ Q (SEQ ID NO: 103), wherein X ₂ And X ₅ Is any amino acid. In some embodiments, X ₂ Ser, thr, gly, asn, ala or Pro, and X ₅ His, gln, arg, lys, gly, thr, tyr, phe, trp, met, ile, val or Leu (SEQ ID NO: 104). In some embodiments, X ₂ Is Ser, ala or Thr, and X ₅ Is His or Tyr (SEQ ID NO: 105).

In other embodiments, the ultralong CDR3 does not include the upstream stem domain of the N-terminus of the cysteine motif. In some embodiments, the ultralong CDR3 does not include the downstream stem domain of the C-terminal end of the cysteine motif.

In some embodiments, the polypeptide for display (e.g., a polypeptide comprising ultralong CDR 3) is derived from a bovine antibody. In some embodiments, the polypeptide for display is produced by amplifying a sequence from a bovine complementary DNA (cDNA) library. In some embodiments, the cDNA template library is prepared from RNA isolated from bovine-derived Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, a cDNA template library is synthesized using a library of immunoglobulin specific primers. In some embodiments, cDNA template libraries are synthesized using IgM, igA, and IgG specific primer libraries. Exemplary primers used include those having the sequences shown in SEQ ID NO. 3 (IgG), SEQ ID NO. 4 (IgM), SEQ ID NO. 5 (IgA) and SEQ ID NO. 6 (IgG).

In some embodiments, the cattle are immunized with the target antigen. In some embodiments, the target antigen is a nontoxic bacterium, virus, viral protein, immunomodulatory protein (e.g., a checkpoint molecule), cancer antigen, human IgG, or recombinant protein thereof. In some embodiments, the target antigen is a viral protein. In some embodiments, the cattle are immunized with multiple target antigens (e.g., different viral antigens). In some embodiments, the different viral antigens are proteins associated with different variants, clades, or strains of the virus.

In some embodiments, the target antigen is a coronavirus, a coronavirus pseudovirus, or an antigen of such a virus, such as a recombinant coronavirus spike protein, or a Receptor Binding Domain (RBD) of a coronavirus spike protein. Coronaviruses may be from the subfamily of orthocoronaviruses, which is one of two subfamilies in the coronaviridae, the order of the mantle viridae and the riboviridae domains. There are four genera: alpha coronavirus, beta coronavirus, gamma coronavirus and delta coronavirus. SARS CoV2 is a beta coronavirus belonging to the subgenera of Sabovirus. In some embodiments, the coronavirus is selected from the group consisting of 229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV2. In some embodiments, the coronavirus is SARS-CoV2 selected from the group consisting of a Wuhan-Hu-1 isolate, a B.1.351south African variant, or a B.1.1.7UK variant. In some embodiments, the SARS CoV-2 specific antigen comprises an S-trimer polypeptide. In some embodiments, the SARS CoV-2 specific antigen comprises an S-monomer polypeptide. In some embodiments, the SARS CoV-2 specific antigen comprises a polynucleotide that encodes an S trimer or monomeric polypeptide. In some embodiments, the cattle are immunized with a plurality of target antigens associated with any combination of coronaviruses 229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV2. In some embodiments, the bovine is immunized with a plurality of target antigens associated with any combination of SARS-CoV2 variants selected from Wuhan-Hu-1 isolate, b.1.351south African variants, or b.1.1.7uk variants.

In some embodiments, the antigen is a cancer antigen. In some embodiments of the present invention, in some embodiments, the antigen is selected from ACTHR, endothelial cell Anxa-1, aminopeptidase N, anti-IL-6R, alpha-4-integrin, alpha-5-beta-3 integrin, alpha-5-beta-5 integrin, alpha Fetoprotein (AFP), ANPA, ANPB, APA, APN, APP, 1AR, 2AR, AT1, B2, BAGE1, BAGE2, B cell receptor BB1, BB2, BB4, calcitonin receptor, cancer antigen 125 (CA 125), CCK1, CCK2, CD5, CD10, CD11a, CD13, CD14, CD19, CD20, CD22, CD25, CD30, CD33, CD38, CD45, CD52, CD56, CD68, CD90, CD133, CD7, CD15, CD34, CD44, CD206, CD271, CEA (carcinoembryonic antigen), CGRP, chemokine receptor, cell membrane integrin-1, cell surface reticulin-1, cripto-1, CRLR, CD1, CD14, CD19, CD20, CD68 CXCR2, CXCR4, DCC, DLL3, E2 glycoprotein, EGFR, EGFRvIII, EMR, endosialin, EP2, EP4, epCAM, ephA2, ET receptor, fibronectin ED-B, FGFR, frizzled receptor, GAGE1, GAGE2, GAGE3, GAGE4, GAGE5, GAGE6, GLP-1 receptor, G-protein coupled receptor of family A (rhodopsin-like), G-protein coupled receptor of family B (secretin receptor-like), G-protein coupled receptor of family C (metabotropic glutamate receptor-like), GD2, GP100, GP120, glypican-3, hemagglutinin, heparin sulfate, HER1, HER2, HER3, HER4, HMFG, HPV 16/18 and E6/E7 antigens, hTERT, IL11-R, IL-13R, ITGAM, kalikrien-9, lewis Y, LH receptor, LHRH-R, LPA, MAC-1, MAGE 2, MAGE 3, MAGE 4, MART1, MC1R, mesothelin, MUC1, MUC16, neu (cell surface nucleolin), enkephalinase, neuropilin-1, neuropilin-2, NG2, NK1, NK2, NK3, NMB-R, notch-1, NY-ESO-1, OT-R, mutant P53, P97 melanoma antigen, NTR2, NTR3, P32 (P32/gC 1q-R/HABP 1), P75, PAC1, PAR1, patched (PTCH), PDGFR, PDFG receptor, PDT, protease-cleaved type IV collagen, protease 3, antiproliferative protein, protein tyrosine kinase 7, PSA, PSMA, purinergic P2X family (e.g., P2X 1-5) mutant Ras, RAMP1, RAMP2, RAMP3 Patched, RET receptor, plexin, smoothened, sst1, sst2A, sst2B, sst3, sst4, sst5, substance P, TEM, T cell CD3 receptor, TAG72, TGFBR1, TGFBR2, tie-1, tie-2, trk-A, trk-B, trk-C, TR1, TRPA, TRPC, TRPV, TRPM, TRPML, TRPP (e.g., TRPV1-6, TRPA1, TRPC1-7, TRPM1-8, TRPP1-5, TRPM 1-3), TSH receptor, VEGF receptor (VEGFR 1 or Flt-1, VEGFR2 or FLK-1/KDR, VEGF-3 or FLT-4), voltage-gated ion channel, VPAC1, VPAC2, nephroblastoma protein 1, Y2, Y4 and Y5.

In some embodiments of the present invention, in some embodiments, the antigen is HER1/EGFR, HER2/ERBB2, CD20, CD25 (IL-2 Rα receptor), CD33, CD52, CD133, CD206, CEA, CEACAM1, CEACAM3, CEACAM5, CEACAM6, cancer antigen 125 (CA 125), alpha Fetoprotein (AFP), lewis Y, TAG72, caprin-1, mesothelin, PDGF receptor, PD-1, PD-L1, CTLA-4, IL-2 receptor, vascular Endothelial Growth Factor (VEGF), CD30, epCAM, ephA2, glypican-3, gpA33, mucin, CAIX, PSMA, folic acid binding protein, gangliosides (such as GD2) GD3, GM1 and GM 2), VEGF receptor (VEGFR), integrin αvβ3, integrin α5β1, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, AFP, BCR complex, CD3, CD18, CD44, CTLA-4, gp72, HLA-DR 10 β, HLA-DR antigen, igE, MUC-1, nuC242, PEM antigen, metalloprotease, hepcidin receptor, hepaplatin ligand, HGF receptor, CXCR4, bombesin receptor and SK-1 antigen.

In some embodiments of the present invention, in some embodiments, the antigen is CD25, PD-1 (CD 279), PD-L1 (CD 274, B7-H1), PD-L2 (CD 273, B7-DC), CTLA-4, LAG3 (CD 223), TIM3 (HAVCR 2), 4-1BB (CD 137, TNFRSF 9), CXCR2, CXCR4 (CD 184), CD27, CEACAM1, galectin 9, BLTA, CD160, VISTA (PD 1 homolog), B7-H4 (VCTN 1), CD80 (B7-1), CD86 (B7-2), CD28, HHT 2 (B7-H7), CD28H, CD155, CD226, TIT, CD96, galectin 3, CD40L, CD, LIGHT (TNF 14), HVEM (TNFRSF 14), B7-H3 (CD 276), LIGHT Ox40L (TNFSF 4), CD137L (TNFSF 9, GITRL), B7RP1, ICOS (CD 278), ICOSL, KIR, GAL9, NKG2A (CD 94), GARP, TL1A, TNFRSF25, TMIGD2, BTNL2, the milk fat philin family, CD48, CD244, the Siglec family, CD30, CSF1R, MICA (MHC class I polypeptide related sequence a), MICB (MHC class I polypeptide related sequence B), the NKG2D, KIR family (killer cell immunoglobulin-like receptor), the LILR family (leukocyte immunoglobulin-like receptor, CD85, ILT, LIR), SIRPA (signal regulator α), CD47 (IAP), neuropilin 1 (NRP-1), VEGFR and VEGF.

In some embodiments, the antigen is an immunomodulatory protein (e.g., a checkpoint molecule). In some embodiments, the antigen is an immune checkpoint receptor ligand. Exemplary immune checkpoint molecules that can target blocking or suppression include, but are not limited to, PD1 (CD 279), PDL1 (CD 274, B7-H1), PDL2 (CD 273, B7-DC), CTLA-4, LAG3 (CD 223), TIM3, 4-1BB (CD 137), 4-1BBL (CD 137L), GITR (TNFRSF 18, AITR), CD40, ox40 (CD 134, TNFRSF 4), CXCR2, tumor Associated Antigen (TAA), B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules and expressed on all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY 55) and CGEN-15049. In some embodiments, the immune checkpoint molecule is CD25, PD-1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD, CD40L, OX, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, and VISTA.

In some embodiments, the polypeptide for display is synthetic. In some embodiments, the synthetic polypeptide comprises all or a portion of a bovine antibody, such as an ultralong CDR3 knob. In some embodiments, the synthetic polypeptide is a modified cyclic peptide. In some embodiments, the modified cyclic peptide comprises, for example, a bovine ultralong CDR3 knob sequence.

In some embodiments, the polypeptide for display comprises a heavy chain variable region comprising an ultralong CDR-H3 and a light chain variable region. Specific forms include single chain forms, such as single chain variable fragments (scFv). In other embodiments, the polypeptide for display is a smaller peptide of 25-70 amino acids (such as 40-70 amino acids), i.e., a knob peptide. Exemplary molecules for display and display libraries are described.

a. scFv peptides for display

In some embodiments, the polypeptide for display is a single chain variable fragment (scFv). In some embodiments, the scFv comprises a VH region with bovine ultralong CDRs 3. In some embodiments, the VH region is encoded by a sequence that has been amplified from a bovine cDNA template library, e.g., a cDNA template library prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle. In some embodiments, the amplification is performed by amplifying sequences encoding VH regions of a family of bovine antibodies known or suspected to contain ultralong CDR 3. In some embodiments, the sequences of the VH region of IgHV1-7 family are amplified to generate sequences encoding VH regions of scFv. In some embodiments, the VH region of the IgHV1-7 family is amplified with a forward primer comprising the sequence set forth in SEQ ID NO. 84 and a reverse primer comprising the sequence set forth in SEQ ID NO. 85. In some embodiments, the forward primer and/or the reverse primer further comprise sequences specific for restriction enzyme sites to facilitate cloning. In some embodiments, the VH region of the IgHV1-7 family is amplified with the forward primer set forth in SEQ ID NO. 12 and the reverse primer set forth in SEQ ID NO. 13.

In some embodiments, the preparation of the sequence of the VH region of the polypeptide for display further comprises a size separation step. In some embodiments, after amplification of VH region sequences of the IgHV1-7 family, such as from a bovine cDNA template library, the sequences encoding VH regions with ultralong CDRs 3 are separated from shorter sequences encoding VH regions without ultralong CDRs 3. In some embodiments, the size separation step further enriches for amplified sequences encoding VH regions with ultralong CDRs 3.

In some embodiments, the size separation step involves separating sequences of length, about or greater than 425, 450, 475, 500, 525, or 550 base pairs from sequences encoding a plurality of amplified VH regions, wherein sequences of length, about or greater than 425, 450, 475, 500, 525, or 550 base pairs comprise sequences encoding VH regions with ultralong CDR 3. In some embodiments, sequences of length, about or greater than 550 base pairs, are separated from the remainder of the sequence.

In some embodiments, size separation is performed by agarose gel electrophoresis. In some embodiments, 1.2%, 1.5% or 2% agarose gel is used. In some embodiments, a 2% agarose gel is used.

In some embodiments, the scFv comprises a VL region that is immobilized across the polypeptides of the display library. In some aspects, the use of an immobilized VL region improves selection and/or screening of scFv comprising a VH region with ultralong CDR 3. In some embodiments, the VL region is a lambda variable light chain (VL) region selected from the group consisting of BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8, and F18, or a humanized variant thereof. In some embodiments, the VL region is the BLV5B8 lambda VL region (SEQ ID NO: 110) or a humanized variant thereof. In some embodiments, the VL region is a BLV1H12 lambda VL region or a humanized variant thereof. In some embodiments, the BLV1H12 VL region is shown in SEQ ID NO. 2. In some embodiments, the humanized variant comprises one or more of the amino acid substitutions I29V and N32G and/or DNN to GDT in the CDR2 region based on the amino acid substitutions of Kabat numbering S2A, T5N, P8S, A12G, A S and amino acid substitutions in the P14L, CDR1 region. In some embodiments, the humanized variant of BLV1H12 comprises the sequence set forth in SEQ ID NO. 107.

In some embodiments, at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 30% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 40% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 50% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 60% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 70% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 80% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 90% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions. In some embodiments, at least or at least about 95% of the displayed scFvs comprise VH regions comprising ultralong CDR3 regions.

In some embodiments, the VH and VL regions of the scFv are directly joined. In some embodiments, the VH and VL regions of the scFv are joined indirectly, e.g., via a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the peptide linker is (Gly 4 Ser) 3 (SEQ ID NO: 94).

b. Knob peptides for display

In some embodiments, the polypeptide for display is an ultralong CDR3 knob, e.g., bovine ultralong CDR3. In some embodiments, the ultralong CDR3 knob is encoded by a sequence that has been amplified from a bovine cDNA template library, e.g., a cDNA template library prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

In some embodiments, the amplification is performed by amplifying the sequence encoding the ultralong CDR3 knob. In some embodiments, primers specific for the up-and down-stalk domains of the bovine ultralong CDR3 region are used to amplify sequences encoding the ultralong CDR3 knob. In some embodiments, the ultralong CDR3 knob comprises a portion of an upstream stem domain, such as 1, 2, 3, 4, 5, or 6 amino acids. In some embodiments, the ultralong CDR3 knob comprises a portion of a downstream stem domain, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids. In some embodiments, the upstream stem domain comprises the sequence CX ₂ TVX ₅ Q, wherein X ₂ And X ₅ Is any amino acid. In some embodiments, X ₂ Ser, thr, gly, asn, ala or Pro, and X ₅ Is His, gln, arg, lys, gly, thr, tyr, phe, trp, met, ile, val or Leu. In some embodiments, X ₂ Is Ser, ala or Thr, and X ₅ Is His or Tyr. In some embodiments, the primers used for amplification comprise or consist of the sequences set forth in SEQ ID NOS.7-11. In some embodiments, the primers used for amplification comprise or consist of the sequences set forth in SEQ ID NOS.8-11. In some embodiments, the primers used for amplification comprise or consist of the sequences set forth in SEQ ID NOS.121-130. In some embodiments, the primers used for amplification comprise or consist of the sequences set forth in SEQ ID NOS.123, 127 and 128.

In some embodiments, the primers used for amplification are different pools of primers specific for the upstream and downstream stem domains. In some embodiments, the pool of primers contains at least two, three, four, five, six, seven, eight, nine, or 10 different primers. In some embodiments, the pool of primers contains at least two, three, four, five, six, seven, eight, nine, or 10 primers that differ from the primers shown in SEQ ID NOS: 7-11 and 121-130. In some embodiments, the pool of primers contains at least two, three, four, five, six or seven primers different from the primers set forth in SEQ ID NOS: 8-11, 123, 127 and 128. In some embodiments, the pool of primers contains the primers shown in SEQ ID NOS.8-11. In some embodiments, the primer library contains the primers shown in SEQ ID NOS.123, 127 and 128. In some embodiments, the pool of primers contains the primers shown in SEQ ID NOS.8-11, 23, 27 and 28.

In some embodiments, the knob peptide is a peptide identified using a method as described in section ii.c. Once identified, the knob peptide sequence can be amplified using methods known to the skilled artisan. In other embodiments, the knob peptide may be synthetically produced. A variety of techniques may be employed, including recombinant methods, chemical synthesis, or combinations thereof. In some embodiments, the chemical synthesis method may include known chemical synthesis techniques, such as the phosphoramidite method. In some cases, recombinant or synthetic nucleic acids may be generated by Polymerase Chain Reaction (PCR).

c. Synthetic peptides for display

In some embodiments, the polypeptide for display is a synthetic peptide. In some embodiments, the synthetic peptide is a random sequence polypeptide having a cysteine motif and disulfide bonds as described herein, e.g., having 2-20 cysteine residues and 1-10 disulfide bonds. In some embodiments, the synthetic peptide has been selected from a library of random sequences having a cysteine motif and disulfide bonds as described herein, e.g., having 2-20 cysteine residues and 1-10 disulfide bonds. Methods for generating libraries of random sequences are known.

In some embodiments, the polypeptide for display is a semisynthetic ultralong CDR3 knob. In some embodiments, the semisynthetic ultralong CDR3 knob is derived from a bovine ultralong CDR3 knob that has been used as a modification scaffold. In some embodiments, the bovine ultralong CDR3 knob has been modified to include random mutations, e.g., while retaining the cysteine motif and disulfide structures as described herein, e.g., such that the semi-synthetic ultralong CDR3 knob still includes 2-20 cysteine residues and 1-10 disulfide bonds. In some embodiments, the bovine ultralong CDR3 knob has been modified to include an exogenous peptide sequence. In some embodiments, the bovine ultralong CDR3 knob has been modified to delete one or more peptide sequences therein, e.g., while retaining the cysteine motif and disulfide structure as described herein, e.g., such that the semisynthetic ultralong CDR3 knob still comprises 2-20 cysteine residues and 1-10 disulfide bonds.

In some embodiments, the polypeptide for display is a cyclic peptide. In some embodiments, the polypeptide for display is a modified cyclic peptide, e.g., has been modified to include an exogenous peptide sequence. In some embodiments, the modified cyclic peptide comprises an ultralong CDR3 knob sequence or portion thereof, including any one as described herein or identified according to the provided methods.

Cysteine-binding micro-proteins (cyclic peptides) include naturally occurring families of cysteine-binding micro-proteins or cyclic peptides found in various plant species. Cysteine-binding micro-proteins (cyclic peptides) are small peptides, typically consisting of about 30-40 amino acids, which may naturally occur in cyclic or linear forms, where the cyclic forms do not have a free N-or C-terminal amino or carboxyl terminus. They have a defined structure based on three intramolecular disulfide bonds and a small three-chain β -sheet (Craik et al 2001;Toxicon 39,43-60). Cyclic proteins exhibit conserved cysteine residues that define a structure referred to herein as a "cysteine knot". This family includes naturally occurring cyclic molecules and their linear derivatives and linear molecules that have undergone cyclization. These molecules can be used as molecular framework structures with enhanced stability compared to peptides of fewer structures. (Colgrave and Craik,2004;Biochemistry 43,5965-5975).

The main cyclic peptide features are due to the remarkable stability of cysteine knots, the small size that makes them easy to chemically synthesize, and the excellent tolerance to sequence variations. Cyclic peptide scaffolds exist in almost 30 different protein families, with conotoxins, spider toxins, pumpkin inhibitors, muzzle related proteins and plant cyclic peptides being the most populated families. Cyclic peptides from plants of Rubiaceae and Viola are mostly found as head-tail cyclic peptides (Craik et al 2010.Cell.Mol.Life Sci.67:9-16). However, in the family of pumpkin inhibitor cyclic peptides, cyclic and linear cyclic peptides have been identified from momordica cochinchinensis: cyclic trypsin inhibitors (MCoTI) -I and MCoTI-II and their linear counterparts MCoTI-III (Hernandez et al 2000.Biochemistry,39, 5722-5730). It is now clear that both cyclic and linear variants can exist in different cyclic peptide families, but the effect of cyclization is poorly understood. Cyclic peptides are expected to exhibit improved stability, better protease resistance and reduced flexibility when compared to their linear counterparts, thereby hopefully leading to enhanced biological activity. However, linear cyclic peptides have the advantage of being able to be more easily linked to other peptides or proteins.

For example, cyclic peptides are commonly found in plants. In aspects of the embodiments provided, the cyclic peptide is derived from linear or cyclic forms of cyclic peptides of Momordica cochinchinensis, rubiaceae and Viola species. In a preferred aspect, the cyclic peptides of the invention are derived from cyclic or linear forms of cyclic peptides of Momordica species including the pumpkin serine protease inhibitor family (Otlewski & Korowarsch Acta Biochim pol 1996;43 (3): 431-44), and in a more preferred aspect, from Momordica cochinchinensis trypsin inhibitors MCoTI-I [ SEQ ID NO:95] and MCoTI-II [ SEQ ID NO:96] (natural cyclic) and MCoTI-III (natural linear) [ SEQ ID NO:97].

Mcoti-I GGVCPKILQRCRRDSDSPGACICRGNGYCGSGSD[SEQ ID NO:95]

Mcoti-II GGVCPKILKKCRRDSDSPGACICRGNGYCGSGSD[SEQ ID NO:96]

Mcoti-III ERACPRILKKCRRDSDSPGACICRGNGYCG[SEQ ID NO:97]

In some embodiments, the cyclic peptide molecular framework comprises an amino acid sequence or analog thereof that forms a cysteine knot backbone, wherein the cysteine knot backbone comprises sufficient disulfide bonds or chemical equivalents thereof to impart a knotted topology on the three-dimensional structure of the cysteine knot backbone, and wherein at least one exposed amino acid residue is inserted or substituted (substituted) relative to the naturally occurring amino acid sequence, such as at one or more β -turns and/or within one or more loops. In some embodiments, the cyclic peptide is modified by insertion or substitution with an exogenous peptide sequence. Thus, a cyclic peptide described herein is a modified cyclic peptide as compared to a natural or wild-type unmodified cyclic peptide, wherein the modified cyclic peptide has one or more loops inserted into or substituted with one or more amino acid sequences (e.g., exogenous peptide sequences). In aspects of the provided embodiments, sufficient amino acid structure is incorporated for that modified cyclic peptide to provide high enzyme stability.

In some embodiments, a modified cyclic peptide sequence may be defined as having a cysteine knot backbone moiety and an exogenous peptide sequence, the modified cyclic peptide comprising: i) An exogenous peptide sequence, wherein the sequence is about 2 to 50 amino acid residues; and ii) a cysteine knot backbone grafted onto the sequence of step I), wherein the cysteine knot backbone comprises structure (I):

wherein C is ₁ To C ₆ Is a cysteine residue; wherein C is ₁ And C ₄ 、C ₂ And C ₅ C ₃ And C ₆ Each linked by disulfide bonds to form a cysteine knot; wherein each X represents an amino acid residue in a ring, wherein the amino acid residues are the same or different; wherein d is about 1-2; wherein one or more of loops 1, 2, 3, 5 or 6 has an amino acid sequence comprising the sequence of clause i), wherein any loop comprising the sequence of clause i) comprises 2 to about 50 amino acids, and wherein a, b, c, e and f are the same or different and are each any number from 3 to 10 for any one of loops 1, 2, 3, 5 or 6 that does not comprise the sequence of clause i), and b, c, e and f are each any number from 1 to 20.

In some embodiments, the modified cyclic peptide sequence may be linear or cyclic.

In some embodiments, the modified cyclic peptide is derived from a linear or cyclic form of cyclic peptide of Momordica cochinchinensis, rubiaceae and Viola species. In some embodiments, the modified cyclic peptide is derived from a cyclic peptide of linear or cyclic form of a Momordica species including the pumpkin serine protease inhibitor family (Otlewski & Korowarsch Acta Biochim pol.1996;43 (3): 431-44). In some embodiments, the modified cyclic peptides are derived from the following Momordica cochinchinensis trypsin inhibitors MCoTI-I [ SEQ ID NO:95] and MCoTI-II [ SEQ ID NO:96] (natural cyclic) and MCoTI-III (natural linear) [ SEQ ID NO:97].

Mcoti-I GGVCPKILQRCRRDSDSPGACICRGNGYCGSGSD[SEQ ID

NO:95]

Mcoti-II GGVCPKILKKCRRDSDSPGACICRGNGYCGSGSD[SEQ ID

NO:96]

Mcoti-III ERACPRILKKCRRDSDSPGACICRGNGYCG[SEQ ID NO:

97]

For example, the unmodified or wild-type cyclic peptide may be a cyclic peptide as set forth in any one of SEQ ID NOs 95-97, with one or more loops inserted or substituted with one or more amino acid sequences (e.g., foreign peptide sequences). In a specific embodiment, the modified cyclic peptide is derived from a Mcoi-II (SEQ ID NO: 96) -based ring displacement library.

In some embodiments, the loop inserted or substituted by the exogenous peptide sequence is loop 1. In some embodiments, the loop inserted or substituted by the exogenous peptide sequence is loop 5. In some embodiments, the loop inserted or substituted by the exogenous peptide sequence is loop 6, such as a loop formed by cyclization.

In some embodiments, the foreign peptide sequence inserted into or substituted into the unmodified cyclic peptide, e.g., cyclic peptide Mcoi-II (SEQ ID NO: 96), is from 2 to 50 amino acid residues. In some embodiments, the exogenous peptide sequence is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, 40 to 40, or 50 amino acids. In some embodiments, the exogenous peptide sequence is 2 to 30 amino acids, such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids.

B. Display library

Also provided herein are libraries of display particles (e.g., phagemid particles), including any display particles produced by any of the provided methods.

Also provided herein is a phagemid comprising or being a replicable expression vector comprising a gene fusion encoding a fusion protein comprising: a first nucleic acid sequence encoding a single chain variable fragment having a bovine variable heavy chain (VH) region, the single chain variable fragment comprising an ultralong CDR3 joined to a lambda variable light chain (VL) region selected from the group consisting of BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8 and F18, or a humanized variant thereof; and a second nucleic acid sequence encoding at least a portion of a bacteriophage coat protein. In some embodiments, the VL region is the VL region of BLV1H 12.

Also provided herein is a phagemid comprising or being a replicable expression vector comprising a gene fusion encoding a fusion protein comprising a first nucleic acid sequence encoding a bovine ultralong CDR3 knob and a second nucleic acid sequence encoding at least a portion of a phage coat protein.

Also provided herein is a phagemid comprising or being a replicable expression vector comprising a gene fusion encoding a fusion protein comprising a first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming a conjugated disulfide bond and a second nucleic acid sequence encoding at least a part of a phage coat protein.

In some embodiments, also provided herein are libraries of display particles (e.g., phagemid particles) encoded by any of the phagemids described herein.

In some embodiments, the display particles comprise an ultralong CDR3 knob, e.g., any one as described herein.

In some embodiments, the display particles comprise a synthetic or semi-synthetic ultralong CDR3 knob, e.g., any one as described herein.

In some embodiments, the display particles comprise a cyclic peptide, e.g., any of the as described herein.

In some embodiments, the display particles comprise a modified cyclic peptide, e.g., any of the as described herein.

In some embodiments, the display particle comprises an scFv having a VH comprising an ultralong CDR3 region. In some embodiments, at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising a VH region with an ultralong CDR3 region. In some embodiments, at least or at least about 30% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 35% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 40% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 45% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 50% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 60% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 70% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 80% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 90% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions. In some embodiments, at least or at least about 95% of the display particles (e.g., phagemid particles) in the library comprise scFv comprising VH regions with ultralong CDR3 regions.

C. Library selection method

Also provided herein are methods for selecting an antibody binding protein specific for a target molecule from any of the display libraries described herein. These display libraries are then contacted with a target molecule and library members having the highest affinity for the target are separated from library members of lower affinity. These display libraries are then contacted with a target molecule and library members having the highest affinity for the target are separated from library members of lower affinity. The high affinity binding agent is then amplified by any suitable system. This process is repeated until a polypeptide having the desired affinity is obtained.

For example, a display library is a phage display library as described herein, wherein an ultralong CDR3scFv polypeptide or CDR 3-knob peptide is fused to a phage coat protein and is typically displayed on average as a single copy of each relevant polypeptide on the surface of a phagemid particle containing DNA encoding the polypeptide. These phagemid particles are then contacted with the target molecule and those particles with the highest affinity for the target are separated from those with lower affinity. The high affinity binding agent is then amplified by infecting the bacterial host and the competitive binding step is repeated. This process is repeated until a polypeptide having the desired affinity is obtained.

In some embodiments, provided methods include contacting any of the display libraries provided herein with a target molecule under conditions that allow binding of a display particle (e.g., a phagemid particle) to the target molecule. In some embodiments, the method further comprises separating the bound display particles (e.g., phagemid particles) from unbound display particles, thereby selecting display particles (e.g., phagemid particles) comprising antibody-binding proteins that bind to the target molecule. In some embodiments, the method comprises sequencing the fusion gene in the selected particle to identify the antibody binding protein.

The target molecules may be isolated from natural sources or prepared by recombinant methods by procedures known in the art. The purified target molecules can be attached to suitable substrates such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic acid copolymers, hydroxyalkyl methacrylate gels, polyacrylic and polymethacrylic acid copolymers, nylon, neutral and ionic carriers, and the like. Attachment of the target protein to the substrate may be accomplished by methods described in Methods in Enzymology,44 1976 or by other means known in the art.

After the target molecules are attached to the matrix, the immobilized target may be contacted with a library of display particles (e.g., phagemid particles) under conditions suitable for binding of at least a portion of the display particles to the immobilized target molecules. Typically, conditions including pH, ionic strength, temperature, etc., will mimic physiological conditions. Exemplary "contact" conditions may include incubation at 4 ℃ -37 ℃ (e.g., at room temperature) for 15 minutes to 4 hours, e.g., one hour. However, these may be appropriately changed depending on the nature of the interactive binding partner and the like. The mixture may be subjected to slight shaking, mixing or rotation. In addition, other suitable agents may be added, such as blocking agents that reduce non-specific binding. For example, 1% -4% BSA or other suitable blocking agent (e.g., milk) may be used. However, it should be understood that the person skilled in the art may vary and adjust the contact conditions according to the purpose of the screening method. For example, if the incubation temperature is, for example, room temperature or 37 ℃, this may increase the likelihood of identifying a binding agent that is stable under these conditions, for example, in the case of 37 ℃ incubation, stable under conditions found in humans. Such properties may be extremely advantageous if one or both of the binding partners are candidates for certain therapeutic applications, such as antibodies. Again, such adaptations to conditions are within the purview of those skilled in the art

Bound display particles ("binders") having a high affinity for the immobilized target molecule can be separated from those having a low affinity (and thus not bound to the target) by washing. The binding agent can be dissociated from the immobilized target molecules by a variety of methods. These methods include competitive dissociation using wild-type ligands, altering pH and/or ionic strength, and methods known in the art.

In some embodiments, the target molecule is a non-toxic bacterium, virus, viral protein, cancer antigen, human IgG, or recombinant protein thereof. In some embodiments, the target molecule is a viral protein. In some embodiments, the target molecule is a coronavirus, a coronavirus pseudovirus, a recombinant coronavirus spike protein, or a Receptor Binding Domain (RBD) of a coronavirus spike protein. In some embodiments, the coronavirus is selected from the group consisting of 229E, NL63, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV2. In some embodiments, the coronavirus is SARS-CoV2 selected from the group consisting of a Wuhan-Hu-1 isolate, a B.1.351south African variant, or a B.1.1.7UK variant.

In some embodiments, the method comprises a step wherein the previously selected display particles are re-expressed and subjected to a further selection step comprising using the same or a different target molecule. In some embodiments, the selecting step is repeated one or more times. In some embodiments, the further selection step comprises infecting a suitable host cell with a replicable expression vector encoding a previously selected display particle; collecting additional amplified display particles; and contacting the additional amplified display particles with the same or different target antigens. In some embodiments, different target molecules are associated with a target molecule and are the same type of pathogen, the same group of pathogens, or variants of a target molecule. In some embodiments, the target molecule and the different target molecule are associated with any combination of coronaviruses 229E, NL, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV2. In some embodiments, the target molecule and the different target molecule are associated with any combination of SARS-CoV2 variants selected from the group consisting of Wuhan-Hu-1 isolate, b.1.351south African variants, and b.1.1.7uk variants.

Once one or more sets of binding agents are selected or isolated according to the provided methods, they can be further analyzed. In some embodiments, further analysis involves isolating the binding agent by bacterial infection as an amplification step, isolating phage or phagemid DNA, and cloning the DNA sequence encoding the candidate binding agent contained in the phage or phagemid DNA into a suitable expression vector. Such an infection step may also allow for amplification of the binding agent. Alternatively, the binding agent may be amplified at this stage by other suitable methods, for example by PCR of the nucleic acid encoding the binding agent or transformation of the nucleic acid into a suitable host cell (in the case of a suitable expression vector).

Once the DNA encoding the binding agent is cloned into a suitable expression vector, the DNA encoding the binding agent may be sequenced or the protein may be expressed in soluble form, e.g., including according to the methods provided herein, and subjected to appropriate binding studies to further characterize the candidate at the protein level. Suitable binding studies will depend on the nature of the binding agent and include, but are not limited to ELISA, filter screen assays, FACS or immunofluorescent assays, biaCore affinity measurements or other methods of quantifying the binding constants, staining tissue slides or cells, and other immunohistochemical methods. One or more of these binding studies may be used to analyze the binding agent.

Also provided herein are methods of identifying an ultralong CDR H3 knob (such as a bovine CDR H3 knob) by including amino acid sequences from a sequence library. In some aspects, methods for identifying an ultralong CDR H3 knob include defining a region of a knob domain, such as by reference to the formulas described herein (e.g., shown below).

In some embodiments, the method for identifying an ultralong CDR H3 knob comprises defining the knob region N-terminal boundary as the first D in the "CPDG" motif _H Cysteine. In some embodiments, the method further comprises defining the C-terminal boundary as by subtracting the upstream stem from the framework 4 tryptophan positionThe number of residues. In some aspects, the method can be used to identify ultralong CDR H3 knobs from any antibody sequence. In specific embodiments, the antibody sequence is a bovine antibody, such as any of the antibodies described herein.

The expression of this embodiment of the method is as follows:

knob border position (C-terminal) =position-X of conserved framework 4 tryptophan; wherein X = the number of amino acids starting from the framework 3 canonical cysteine defining the upstream stem and ending at an amino acid preceding the conserved first D-region cysteine in the "CPDG" motif;

Number of residues in knob (K) =l-2X; wherein L = the number of amino acids covering the stem and knob domains, starting at canonical framework 3 cysteine and ending at canonical framework 4 tryptophan;

k position= (x+1) to (x+k)

Soluble peptide expression

In some embodiments, provided herein are also methods of producing a soluble disulfide bond containing peptide, including methods of producing any of the antibody binding proteins (also referred to as binders) identified by any of the methods described herein. The soluble peptides produced by the provided methods are peptides containing 2 or more cysteine residues (e.g., 25 to 70 amino acids in length) from which disulfide-bonded soluble proteins are desired to be produced. In some embodiments, provided methods include transforming a host cell, such as E.coli, with an expression vector encoding a soluble peptide. In some embodiments, the expression vector encodes a fusion protein comprising a soluble peptide and a chaperone protein (e.g., a bacterial chaperone protein). In some embodiments, the soluble peptide and chaperonin (e.g., bacterial chaperonin) are joined by a linker. In some embodiments, the linker is a cleavable linker.

In some embodiments, the fusion protein has increased solubility relative to the soluble protein alone. In some aspects, this increased solubility is conferred, at least in part, by the inclusion of a chaperonin (e.g., a bacterial chaperonin). In some aspects, inclusion of a chaperone protein (e.g., bacterial chaperone protein) promotes solubility of the fusion protein while allowing disulfide bond formation in the soluble peptide, including in a host cell environment that has been engineered or modified to promote disulfide bond formation. In some embodiments, the chaperonin (e.g., bacterial chaperonin) is thioredoxin a (TrxA).

In some embodiments, the provided methods further comprise culturing a host cell, e.g., a bacterium, such as e.coli, under conditions that allow expression of the fusion protein. In some embodiments, the provided methods further comprise, after culturing, isolating the expressed fusion protein from a supernatant of a lysate of a host cell (e.g., a bacterium, such as e.coli). In some embodiments, the provided methods further comprise cleaving the cleavable linker, thereby producing a soluble peptide that is free of the bacterial chaperone protein.

In some embodiments, the cleavable linker is an enterokinase cleavable tag. In some embodiments, the cleavable linker comprises the amino acid sequence DDDDK (SEQ ID NO: 106). In some embodiments, cleavage of the cleavable linker comprises addition of enterokinase. In some embodiments, enterokinase is added to the supernatant of the host cell lysate. In some embodiments, the provided methods further comprise removing enterokinase and/or bacterial chaperones from the solution containing the soluble peptide after cleavage of the cleavable linker.

In some embodiments, the soluble peptide is up to 70 amino acids in length. In some embodiments, the soluble peptide is 40 to 60 amino acids in length. In some embodiments, the soluble peptide is at least 42 amino acids in length. In some embodiments, the soluble peptide is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

In some embodiments, the soluble peptide is 25-70 amino acids. For example, in some embodiments, the soluble peptide is 35 amino acids or more in length, 40 amino acids or more in length, 45 amino acids or more in length, 50 amino acids or more in length, 55 amino acids or more in length, or 60 amino acids or more in length. In some embodiments, the soluble peptide is between or between about 35 and 70 amino acids, 40 and 70 amino acids, 45 and 70 amino acids, 50 and 70 amino acids, 55 and 70 amino acids, or 60 and 70 amino acids in length.

In some embodiments, the soluble peptide is 6 to 50 amino acids, 6 to 40 amino acids, 6 to 30 amino acids, 6 to 25 amino acids, 6 to 20 amino acids, 6 to 15 amino acids, 6 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids. In some embodiments, the soluble peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids, or 24 to 30 amino acids.

In some embodiments, the soluble peptide comprises a cysteine motif capable of forming a disulfide bond. In some embodiments, the cysteine motif comprises 2-20 cysteine residues, e.g., between or about 2 and 18, 2 and 16, 2 and 14, 2 and 12, 2 and 10, 2 and 8, 2 and 6, 2 and 4, 4 and 20, 4 and 18, 4 and 16, 4 and 14, 4 and 12, 4 and 10, 4 and 8, 4 and 6, 6 and 20, 6 and 18, 6 and 16, 6 and 14, 6 and 12, 6 and 10, 6 and 8, 8 and 20, 8 and 18, 8 and 16, 8 and 14, 8 and 12, 8 and 10, 10 and 20, 10 and 18, 10 and 16, 10 and 14, 10 and 12, 12 and 18, 12 and 16, 12 and 14, 14 and 20, 14 and 18, 14 and 16, 16 and 20, 16 and 18, or 18 and 20, each comprising a terminus. In some embodiments, the cysteine motif comprises 2-12 cysteine residues. In some embodiments, the soluble peptide comprises at least 4 Cys residues. In some embodiments, the soluble peptide contains 4 Cys residues. In some embodiments, the soluble peptide contains 6, 8, 10, or 12 Cys residues.

In some embodiments, the soluble peptide comprises 1-10 disulfide bonds, e.g., between or about 1 and 9, 1 and 8, 1 and 7, 1 and 6, 1 and 5, 1 and 4, 1 and 3, 1 and 2, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, 2 and 3, 3 and 10, 3 and 9, 3 and 8, 3 and 7, 3 and 6, 3 and 5, 3 and 4, 4 and 10, 4 and 9, 4 and 8, 4 and 7, 4 and 6, 4 and 5, 5 and 10, 5 and 9, 5 and 8, 5 and 7, 5 and 6, 6 and 10, 6 and 8, 6 and 7, 7 and 10, 7 and 9, 7 and 8, 8 and 10, 8 or 9 and 10, each comprising a terminus. In some embodiments, the soluble peptide comprises 1-6 disulfide bonds. In some embodiments, the soluble peptide contains 2-6 disulfide bonds. In some embodiments, the soluble peptide has at least 2 disulfide bonds. In some embodiments, the soluble peptide has 2 disulfide bonds. In some embodiments, the soluble peptide has 3, 4, or 5 disulfide bonds.

In some embodiments, the soluble peptide comprises 3-6 amino acids before the N-terminal most cysteine residue present in the soluble peptide. In some embodiments, the soluble peptide comprises 3, 4, 5, or 6 amino acids before the N-terminal most cysteine residue present in the soluble peptide.

In some embodiments, the soluble peptide comprises at least 6 amino acids after the most C-terminal cysteine residue present in the soluble peptide. In some embodiments, the soluble peptide comprises 6-9 amino acids after the most C-terminal cysteine residue present in the soluble peptide. In some embodiments, the soluble peptide comprises 6, 7, 8, or 9 amino acids after the most C-terminal cysteine residue present in the soluble peptide.

In some embodiments, the soluble peptide comprises a flexible linker. In some embodiments, a flexible linker is included at the N-terminus of the soluble peptide. In some embodiments, a flexible linker is present in addition to 3-6 amino acids preceding the N-terminal most cysteine residue present in the soluble peptide. In some embodiments, the flexible linker is included 3-6 amino acids before the N-terminal most cysteine residue present in the soluble peptide. In some embodiments, a flexible linker is included at the C-terminus of the soluble peptide. In some embodiments, a flexible linker is present in addition to at least 6 amino acids after the C-terminal most cysteine residue present in the soluble peptide. In some embodiments, the flexible linker is included at least 6 amino acids after the most C-terminal cysteine residue present in the soluble peptide.

In some embodiments, the flexible linker is GGGGAMGS (SEQ ID NO: 108). In some embodiments, the flexible linker is GGS (SEQ ID NO: 109). In some embodiments, a flexible linker (e.g., GGGGAMGS, SEQ ID NO: 108) allows cyclization of the soluble peptide. In some embodiments, cyclization is performed via chemical or enzymatic methods. In some embodiments, a flexible linker (e.g., GGGGAMGS, SEQ ID NO: 108) allows for sortase-mediated cyclization of soluble peptides. In some embodiments, the provided methods further comprise the step of cyclizing the soluble peptide, e.g., by chemical or enzymatic methods.

In some embodiments, the provided methods further comprise the step of enriching the soluble peptide. In some embodiments, the provided methods further comprise separating the soluble peptide from any soluble aggregates present in the solution (including soluble aggregates of the soluble peptide). In some embodiments, the separation involves separation of the active soluble peptide from its larger, inactive or less active soluble aggregates. In some embodiments, separation is achieved using chromatographic methods. In some embodiments, the enrichment or separation is performed by size exclusion chromatography. In some embodiments, the separation involves collecting one or more elution fractions containing the soluble peptide, but not its soluble aggregates, thereby producing an enriched or purified composition of the soluble peptide.

In some embodiments, the provided methods further comprise generating a multispecific binding molecule comprising a soluble peptide. In some embodiments, the multispecific binding molecule comprises multiple copies of a soluble peptide. In some embodiments, the multispecific binding molecules comprise different soluble peptides. In some embodiments, the multispecific binding molecule comprises a flexible linker (e.g., gly-Ser) between soluble peptides (e.g., between the C-terminus of one soluble peptide copy and the N-terminus of another soluble peptide copy). In some embodiments, one soluble peptide is present in the VH region expressed as IgG with the light chain, and a second soluble peptide is fused to the heavy chain constant region. In some embodiments, the multispecific binding molecule comprises two VH regions with the same soluble peptide. In some embodiments, the multispecific binding molecules comprise VH regions comprising different soluble peptides, e.g., using heavy chains with constant region mutations, such that only heterologous heavy chains effectively pair with each other to form dimers. In some embodiments, these mutations are "knob-in-hole" mutations, such as T22Y on one strand and Y86T on the other strand in the CH3 domain of Fc.

In some embodiments, the expression vector further comprises an inducible promoter sequence that controls expression of the fusion protein. As used herein, the term "promoter sequence" refers to a DNA sequence that is generally located upstream of a gene present in a DNA polymer and provides an initiation site for transcription of the gene into mRNA. Promoter sequences suitable for use in the present invention may be derived from viruses, phages, prokaryotic cells or eukaryotic cells, and may be constitutive promoters or inducible promoters.

In some embodiments, the inducible promoter sequence is operably linked to a sequence encoding a fusion protein. As used herein, the term "operably linked" refers to a first sequence being sufficiently close to a second sequence that the first sequence can affect the second sequence or a region under the control of the second sequence. For example, a promoter sequence may be operably linked to a gene sequence and is typically located at the 5' -end of the gene sequence such that expression of the gene sequence is under the control of the promoter sequence. In addition, regulatory sequences may be operably linked to the promoter sequence in order to enhance the ability of the promoter sequence to promote transcription. In this case, the regulatory sequence is usually located at the 5' -end of the promoter sequence.

Promoter sequences suitable for use in the present invention are preferably derived from any one of the following: viruses, bacterial cells, yeast cells, fungal cells, algal cells, plant cells, insect cells, animal cells, and human cells. For example, promoters useful in bacterial cells include, but are not limited to, tac promoter, T7A 1 promoter, lac promoter, trp promoter, trc promoter, araBAD promoter, and λPRPL promoter. Promoters useful in plant cells include, for example, 35S CaMV promoter, actin promoter, ubiquitin promoter, and the like; regulatory elements suitable for mammalian cells include the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter, CMV enhancer or SV40 enhancer.

Vectors suitable for use in the present invention include those commonly used in genetic engineering techniques, such as phages, plasmids, cosmids, viruses or retroviruses.

Vectors suitable for use in the present invention may also include other expression control elements such as transcription initiation sites, transcription termination sites, ribosome binding sites, RNA splice sites, polyadenylation sites, translation termination sites and the like. Vectors suitable for use in the present invention may also include additional regulatory elements such as transcription/translation enhancer sequences, and at least one marker gene or reporter gene that allows for screening of the vector under appropriate conditions. Marker genes suitable for use in the present invention include, for example, the dihydrofolate reductase gene and the G418 or neomycin resistance genes useful in eukaryotic cell culture, and the ampicillin, streptomycin, tetracycline or kanamycin resistance genes useful in E.coli and other bacterial culture. Vectors suitable for use in the present invention may also include nucleic acid sequences encoding secretion signals. These sequences are well known to those skilled in the art.

Depending on the vector and host cell system used, the recombinant gene product (protein) produced according to the invention may remain in the recombinant cell, be secreted into the culture medium, be secreted into the periplasm, or remain on the outer surface of the cell membrane. The recombinant gene product (protein) produced by the methods of the invention can be purified by using a variety of standard protein purification techniques including, but not limited to, affinity chromatography, ion exchange chromatography, gel filtration, electrophoresis, reverse phase chromatography, chromatography Jiao Ju, and the like. The recombinant gene product (protein) produced by the methods of the invention is preferably recovered in a "substantially pure" form. As used herein, the term "substantially pure" refers to the purity of a purified protein that allows for the effective use of the purified protein as a commercial product.

A. Host cells

The term "host cell" is used to refer to a cell that has been transformed, transfected or infected with a nucleic acid sequence, or that is capable of being transformed, transfected or infected with a nucleic acid sequence, and then expressing a selected gene of interest to recombinantly produce a protein of interest. The term includes progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent, as long as the selected gene or genetic modification is present.

The provided methods for producing a soluble peptide or fusion protein comprising a soluble peptide and a chaperone protein (e.g., a bacterial chaperone protein) may be performed using any host organism capable of expressing a heterologous polypeptide and capable of being genetically modified. The host organism is preferably a single-cell host organism, however, the use of multicellular organisms is also contemplated in the provided methods, provided that the organism can be modified as described herein and express the polypeptide of interest therein. For clarity, the term "host cell" will be used throughout this document, but it should be understood that a host organism may replace a host cell unless it is not feasible for technical reasons.

In some embodiments, the host cell is a prokaryotic cell, such as a bacterial cell. The host cell may be a gram positive bacterial cell (such as bacillus) or a gram negative bacterium (such as e.coli). The host organism may be an aerobic or anaerobic organism. In some embodiments, host cells are those having characteristics that favor expression of the polypeptide, e.g., host cells having fewer proteases than other types of cells. Suitable bacteria for this purpose include archaebacteria and eubacteria, for example of the enterobacteriaceae family. Other examples of useful bacteria include escherichia, enterobacter, azotobacter, erwinia, bacillus, pseudomonas, klebsiella, proteus, salmonella, serratia, shigella, rhizobium, vitreoscilla, and paracoccus. Other examples of useful bacteria include Corynebacterium, lactococcus, lactobacillus and Streptomyces species, in particular Corynebacterium glutamicum, lactococcus lactis, lactobacillus plantarum, streptomyces coelicolor, streptomyces lividans. Suitable E.coli hosts include E.coli DHB4, E.coli BL-21, which lack both lon (Phillips et al J. Bacteriol.159:283,1984) and ompT protease, E.coli AD494, E.coli W3110 (ATCC 27,325), E.coli 294 (ATCC 31,446), E.coli B and E.coli X1776 (ATCC 31,537). Other strains include E.coli B834, which is methionine-deficient and can therefore be used ³⁵ S-methionine or selenomethionine labels target proteins with high specific activity (Leahy et al Science 258:987, 1992). Other strains of interest include the BLR strain as well as the K-12 strains HMS174 and NovaBlue, which are recA-derivatives that improve plasmid monomer production and can help stabilize target plasmids containing repetitive sequences.

In some embodiments, the E.coli host cells used in the provided methods are engineered or modifiedTo improve the soluble expression of disulfide-bonded proteins in the cytosol of E.coli. In some embodiments, the cytoplasmic thiol-redox balance environment is altered via a change in a reduction pathway (such as thioredoxin reductase). In some embodiments, the E.coli host cell has an oxidative cytoplasm that allows disulfide bond formation. Various types of mutant strains lacking glutathione reductase Δgor, thioredoxin reductase and/or glutathione biosynthetic pathway, including SHuffle (New England Biolabs) and Origami ^TM (DE 3) (Novagen, germany) is commercially available. In some embodiments, the E.coli strain transformed as part of the provided methods is Origami ^TM (DE 3) (Novagen, germany) mutant strain.

Suitable bacillus strains include bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus brevis, bacillus alkalophilus, bacillus clausii, bacillus cereus, bacillus pumilus, bacillus thuringiensis, or bacillus alkalophilus. In the biotechnology industry, the gram positive bacterium bacillus subtilis is a preferred organism for secreted protein production. Its popularity is mainly based on the following facts: bacillus subtilis lacks an outer membrane that retains many proteins in the periplasm of gram-negative bacteria such as e. Thus, most of the bacillus subtilis proteins transported across the cytoplasmic membrane eventually go directly into the growth medium. In addition, the lack of the outer membrane means that the protein produced with bacillus subtilis is free of lipopolysaccharide (endotoxin). Other advantages of using bacillus subtilis as a host for protein production are its high genetic adaptability, availability of strains with mutations In almost all about 4100 genes, kits with strains and vectors for gene expression, and the fact that the bacteria are generally considered safe (Braun et al, curr. Opan. Biotechnol.10:376-381,1999; kobayashi et al, proc. Natl. Acad. Sci. U.S.A 100:4678-4683,2003; kunst et al Nature 390:249-256,1997; zeigler et al, in e. Goldman and l.green (editions), practical Handbook of microbiology.crc Press, boca Raton, fla., 2008).

In another embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a mammalian cell. Examples of mammalian cells include, but are not limited to, chinese hamster ovary Cells (CHO) (ATCC No. CCL 61), CHO DHFR-cells (Urlaub et al, proc.Natl. Acad. Sci.usa,97:4216-4220 (1980)), human Embryonic Kidney (HEK) 293 or 293T cells (ATCC No. CRL 1573), or 3T3 cells (ATCC No. CCL 92). The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, and product generation and purification are known in the art. Other suitable mammalian cell lines are monkey COS-1 (ATCC No. CRL 1650) and COS-7 cell line (ATCC No. CRL 1651) and CV-1 cell line (ATCC No. CCL 70). Additional exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell lines derived from in vitro culture of primary tissue, and primary explants are also suitable. Candidate cells may have a genotype defect in the selection gene or may contain a dominant acting selection gene. Other suitable mammalian cell lines include, but are not limited to, mouse neuroblastoma N2A cells, heLa, mouse L-929 cells, 3T3 lines derived from Swiss, balb-c or NIH mice, BHK or HaK hamster cell lines available from ATCC. Each of these cell lines is known and available to those skilled in the art of protein expression.

Many strains of yeast cells known to those skilled in the art can also be used as host cells for expressing the polypeptides described herein. Exemplary yeast cells include, for example, saccharomyces cerevisiae and Pichia pastoris. Fungi, such as aspergillus, may also be used as host cells for expression of the polypeptides described herein.

In addition, insect cell systems can be used in the provided methods when desired. Such cells are described, for example, in Kitts et al, biotechniques,14:810-817 (1993); lucklow, curr. Opin. Biotechnol.,4:564-572 (1993); and Lucklow et al (J.Virol., 67:4566-4579 (1993)) exemplary insect cells are Sf-9 and Hi5 (Invitrogen, carlsbad, calif.).

B. Soluble peptides

In some embodiments, the soluble peptide produced in the provided methods is a soluble ultralong CDR3 knob. In some embodiments, the soluble peptide produced in the provided methods is a soluble synthetic or semi-synthetic peptide. In some embodiments, the soluble peptide produced in the provided methods is a cyclic peptide. In some embodiments, the soluble peptide produced in the provided methods is a modified cyclic peptide. In some embodiments, the soluble peptides produced in the provided methods are semisynthetic or modified ultralong CDR3 knobs.

1. Soluble bovine ultralong CDR3 knob

In some embodiments, the soluble peptide produced in the provided methods is a soluble ultralong CDR3 knob. In some embodiments, the soluble ultralong CDR3 knob is a bovine ultralong CDR3. In some embodiments, the soluble ultralong CDR3 knob is encoded by a sequence that has been amplified from a bovine cDNA template library, e.g., a cDNA template library prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle. In some embodiments, the soluble ultralong CDR3 knob comprises all or a portion of a sequence that has been amplified from a bovine cDNA template library according to any of the methods provided herein (see, e.g., sections II-a-1-a and II-a-1-b). In some embodiments, the soluble ultralong CDR3 knob is any soluble ultralong CDR3 knob that has been identified or selected as a binding agent for a target molecule. In some embodiments, the soluble ultralong CDR3 knob is any ultralong CDR3 knob or portion thereof that has been identified or selected as a binding agent for a target molecule according to any of the methods provided herein (see, e.g., section II-C).

2. Soluble synthetic peptides

In some embodiments, the soluble peptide produced in the provided methods is a soluble synthetic or semi-synthetic peptide. In some embodiments, the soluble peptides produced in the provided methods are semisynthetic or modified ultralong CDR3 knobs. In some embodiments, the soluble peptide produced in the provided methods is a cyclic peptide. In some embodiments, the soluble peptide produced in the provided methods is a modified cyclic peptide.

a. Soluble synthetic ultralong CDR3 knob

In some embodiments, the soluble peptide is a semisynthetic ultralong CDR3 knob. In some embodiments, the semisynthetic ultralong CDR3 knob is derived from a bovine ultralong CDR3 knob that has been used as a modification scaffold. In some embodiments, the bovine ultralong CDR3 knob is encoded by a sequence that has been amplified from a bovine cDNA template library, e.g., a cDNA template library prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle. In some embodiments, the bovine ultralong CDR3 knob comprises all or a portion of a sequence that has been amplified from a bovine cDNA template library according to any of the methods provided herein (see, e.g., sections II-a-1-a and II-a-1-b). In some embodiments, the bovine ultralong CDR3 knob is any bovine ultralong CDR3 knob that has been identified or selected as a binding agent for a target molecule. In some embodiments, the bovine ultralong CDR3 knob is any ultralong CDR3 knob or portion thereof that has been identified or selected as a binding agent for a target molecule according to any of the methods provided herein (see, e.g., section II-C).

In some embodiments, the bovine ultralong CDR3 knob has been modified to include random mutations, e.g., while retaining the cysteine motif and disulfide structures as described herein, e.g., such that the semi-synthetic ultralong CDR3 knob still includes 2-20 cysteine residues and 1-10 disulfide bonds. In some embodiments, the bovine ultralong CDR3 knob has been modified to include an exogenous peptide sequence. In some embodiments, the bovine ultralong CDR3 knob has been modified to delete one or more peptide sequences therein, e.g., while retaining the cysteine motif and disulfide structure as described herein, e.g., such that the semisynthetic ultralong CDR3 knob still comprises 2-20 cysteine residues and 1-10 disulfide bonds.

b. Soluble cyclic peptides

In some embodiments, the soluble peptide produced in the provided methods is a soluble cyclic peptide. In some embodiments, the cyclic peptide is a cyclic peptide that has been modified to include an exogenous peptide sequence.

Mcoti-I GGVCPKILQRCRRDSDSPGACICRGNGYCGSGSD[SEQ ID NO:95]

Mcoti-II GGVCPKILKKCRRDSDSPGACICRGNGYCGSGSD[SEQ ID NO:96]

Mcoti-III ERACPRILKKCRRDSDSPGACICRGNGYCG[SEQ ID NO:97]

Mcoti-I GGVCPKILQRCRRDSDSPGACICRGNGYCGSGSD[SEQ ID NO:95]

Mcoti-II GGVCPKILKKCRRDSDSPGACICRGNGYCGSGSD[SEQ ID NO:96]

Mcoti-III ERACPRILKKCRRDSDSPGACICRGNGYCG[SEQ ID NO:97]

Antibodies comprising peptides

In some embodiments, provided herein are also methods comprising producing full length IgG or Fab. In some embodiments, full length IgG or Fab is produced from an antibody binding protein or peptide selected according to any of the methods provided herein. In some embodiments, full length IgG or Fab is produced from the soluble peptides produced according to any of the methods provided herein.

In some embodiments, the antibody binding protein is an scFv, and the method comprises constructing a heavy chain or portion thereof comprising ligating a VH region of the scFv with a constant region or portion thereof.

In some embodiments, the method comprises constructing a humanized VH region by replacing the knob region of the ultralong CDR3 region of the humanized bovine VH region with the ultralong CDR3 region of the selected antibody binding protein. In some embodiments, the ultralong CDR3 region of the selected antibody binding protein is replaced between the uplink and downlink stem chains of a humanized bovine VH region. In some embodiments, the VH region comprises the formula V1-X-V2, wherein the V1 region of the heavy chain comprises the sequence set forth in SEQ ID NO. 111; the X region comprises the ultralong CDR3 of the selected antibody; and the V2 region comprises the sequence set forth in SEQ ID NO. 112.

In some embodiments, the method further comprises constructing a heavy chain or portion thereof comprising ligating the humanized VH region to the constant region or portion thereof. In some embodiments, the heavy chain or portion thereof is a human IgG1 heavy chain or portion thereof. In some embodiments, the method further comprises coexpression of the heavy chain or portion thereof with the light chain.

In some embodiments, the light chain is a bovine light chain of BLVH12, BLV5D3, BLV8C11, BF1H1, BLV5B8, or F18, or a humanized variant thereof. In some embodiments, the light chain is the BLV1H12 light chain (SEQ ID NO: 113) or a humanized variant thereof. In some embodiments, the light chain is a humanized light chain as set forth in SEQ ID NO. 114. In some embodiments, the light chain is the BLV5B8 light chain (SEQ ID NO: 115) or a humanized variant thereof. In some embodiments, the light chain is a human light chain. In some embodiments, the light chain is selected from the group consisting of VL1-47, VL1-40, VL1-51 and VL 2-18. In some embodiments, the light chain is shown in any one of SEQ ID NOS 116-120.

In some embodiments, the antibody binding proteins or peptides selected or produced by these methods are formed as multi-specific binding proteins comprising any of a variety of provided peptides, such as knob peptides. In some embodiments, the plurality of peptides (such as knob peptides) are paratopes. In some embodiments, the plurality of peptides (such as knob peptides) is 2, 3, or 4 peptides. An exemplary format for generating a multispecific polypeptide is depicted in fig. 12.

In some embodiments, one or more peptides, such as knob peptides, are linked in tandem in a single polypeptide chain separated by a flexible linker (e.g., a GGGS or other similar flexible linker, including longer linkers of (GGGS) n, where n is 1-3). In some embodiments, tandem single polypeptides may include 2, 3, 4, or more peptides, such as knob peptides, to produce bivalent, trivalent, tetravalent, or other multivalent molecules.

In some embodiments, a peptide (such as a knob peptide) is reformatted by replacing the knob region of an ultralong CDR-H3 scaffold (including any of the humanized ultralong heavy chain molecules described herein). The heavy chain may be complexed with a light chain (such as any of the light chain molecules described herein). In some embodiments, when produced in a cell, the double-stranded polypeptide is formed by dimerization resulting from disulfide bond formation between two heavy chain molecules. In some embodiments, the modified immunoglobulin containing a peptide (such as a knob peptide) is a homodimer containing a peptide (e.g., a knob peptide). In other embodiments, two different heavy chains may be co-expressed in a cell using a knob-in-hole engineering strategy or other strategy to produce a heterodimer, wherein two different heavy chains each carrying a different peptide (e.g., knob peptide) may interact to form a heterodimer. In some embodiments, residues of the constant chain are modified by amino acid substitutions to promote heterodimer formation. In some of any of the embodiments, the one or more amino acid modifications are selected from the group consisting of knob-into-hole modifications and charge mutations that reduce or prevent self-association due to charge repulsion. Heterodimers can be formed by transforming a first nucleic acid molecule encoding a first polypeptide subunit and a second nucleic acid molecule encoding a second, different polypeptide subunit into a cell. In some aspects, heterodimers are produced upon expression and secretion from cells due to covalent or non-covalent interactions between residues of two polypeptide subunits to mediate dimer formation. In such processes, a mixture of dimer molecules is typically formed, including homodimers and heterodimers. For heterodimer formation, an additional purification step may be necessary. For example, the first polypeptide and the second polypeptide may be engineered to include tags with metal chelates or other epitopes, wherein the tags are different. The labeled domains can be used for rapid purification by metal chelate chromatography and/or by antibodies to allow detection by western blot, immunoprecipitation, or activity depletion/blocking in bioassays. Methods include those described in U.S. patent No. 10995127. In some embodiments, the human IgG1 comprises a T22Y amino acid substitution in the CH3 domain and the second IgG1 heavy chain comprises a Y86T amino acid substitution in the heavy chain.

V. immunization

In some embodiments, provided methods include using or amplifying a cDNA template library prepared from RNA isolated from an immunized cow. In some embodiments, the method further comprises immunizing the bovine with the target antigen.

In some embodiments, the target antigen is a avirulent bacterium, virus, viral protein, cancer antigen, human IgG, or recombinant protein thereof. In some embodiments, the target antigen is a virus or viral protein, e.g., a virus or viral protein associated with a coronavirus such as SARS CoV-2.

In some embodiments, the bovine is immunized by administering at least one dose of an antigen composition comprising a target antigen or a set of related target antigens (e.g., antigens associated with viral variants). In some embodiments, the antigen composition further comprises an adjuvant. The skilled person is familiar with many potentially useful adjuvants, such as Freund's complete adjuvant, alum and squalene. See, for example, U.S. patent application publication No. 20150361160, which is incorporated by reference herein in its entirety for all purposes. Adjuvants useful in the compositions of the present invention include, but are not limited to, oil emulsion compositions (oil-in-water emulsions and water-in-oil emulsions), complete Freund's Adjuvant (CFA), and Incomplete Freund's Adjuvant (IFA). In one embodiment, the adjuvant comprises RIBI, iscomatrix or ENABL CI (VaxLiant). Adjuvants suitable for use in the present invention include bacterial or microbial derivatives such as derivatives of enterobacterial Lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.

Methods of immunizing cattle, such as cattle, to produce, for example, high titer colostrum, milk, serum, or immune tissue (e.g., PBMCs) are known in the art. Such methods are disclosed, for example, in U.S. patent application publication nos. US20070053917 and US20130022619, each of which is incorporated by reference herein in its entirety for all purposes.

In some embodiments, immunization comprises administration of a priming dose and at least one boosting dose of an antigen composition. In some embodiments, immunization comprises administering more than one booster dose of the antigen composition. In one embodiment, the priming dose and the at least one boosting dose comprise the same antigen composition. In some embodiments, more than one booster dose comprises the same antigen composition. The immunogenic composition may be administered to the animal at intervals over a period of days, weeks or months. At the end of the immunization protocol, hyperimmune material such as blood, milk or colostrum is harvested. In one embodiment, the hyperimmune material is collected less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 9 months, or less than 12 months after administration of the priming dose. In one embodiment, the hyperimmune material is collected between about 3 months and about 6 months after administration of the priming dose. In one embodiment, the hyperimmune material is collected between about 3 months and about 9 months after administration of the priming dose. In some embodiments, the hyperimmune material is collected between about 3 months and about 12 months after administration of the priming dose. In one embodiment, the hyperimmune material is collected between about 6 months and about 12 months after administration of the priming dose.

In some embodiments, the methods further comprise isolating the biological sample from a bovine. In some embodiments, the biological sample is milk, blood, serum, colostrum, or Peripheral Blood Mononuclear Cells (PBMCs). In one embodiment, the biological sample is collected less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 9 months, or less than 12 months after administration of the priming dose. In one embodiment, the biological sample is collected between about 3 months and about 6 months after administration of the priming dose. In some embodiments, the biological sample is collected between about 3 months and about 9 months after administration of the priming dose. In some embodiments, the biological sample is collected between about 3 months and about 12 months after administration of the priming dose. In some embodiments, the biological sample is collected between about 6 months and about 12 months after administration of the priming dose.

In some embodiments, the methods further comprise isolating Peripheral Blood Mononuclear Cells (PBMCs) from the cattle and cloning the polynucleotide encoding the candidate binding peptide (e.g., containing ultralong CDR 3). In one embodiment, cloning the polynucleotide comprises performing single cell RT-PCR amplification.

VI compositions and formulations

Also provided are compositions, including pharmaceutical compositions and formulations, comprising the binding polypeptides (such as antibodies or antigen binding fragments or knob peptides) described herein. In one embodiment, the composition comprises a soluble peptide produced as described herein. In one embodiment, the composition comprises a fusion protein comprising a soluble peptide produced as described herein. In one embodiment, the composition comprises a soluble peptide identified for its ability to bind to a target molecule (e.g., identified as described herein). In some embodiments, the composition comprises a knob polypeptide or synthetic peptide comprising an ultralong CDR 3. The pharmaceutical compositions and formulations generally comprise one or more optional pharmaceutically acceptable carriers or excipients.

The term "pharmaceutical formulation" refers to a preparation in a form that allows for the biological activity of the active ingredient contained therein to be effective, and which does not contain additional components that have unacceptable toxicity to the subject to whom the formulation is to be administered.

By "pharmaceutically acceptable carrier" is meant an ingredient of the pharmaceutical formulation that is non-toxic to the subject other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

In some aspects, the choice of carrier is determined in part by the particular cell, binding molecule and/or antibody and/or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. The vehicle is described in Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Edit (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); a low molecular weight (less than about 10 residues) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

In some aspects, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, remington, the Science and Practice of Pharmacy, lippincott Williams & Wilkins; 21 st edition (2005, 5 months, 1 day).

Formulations of the antibodies described herein may include lyophilized formulations and aqueous solutions.

In some embodiments, the antibodies described herein may be administered in unit dosage form in a pharmaceutically acceptable diluent, carrier or excipient. Conventional pharmaceutical practice can be employed to provide a suitable formulation or composition for administration to an individual being treated for SARS CoV-2 infection. In some embodiments, the administration is prophylactic. Any suitable route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository, oral administration or via inhalation.

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual or suppository administration. In some embodiments, the cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic and intraperitoneal administration.

In some embodiments, the composition is provided as a sterile liquid preparation (e.g., an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition) that may be buffered to a selected pH in some aspects. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, the liquid composition is somewhat more convenient to administer, particularly by injection. On the other hand, the adhesive composition may be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may comprise a carrier, which may be a solvent or dispersion medium comprising, for example, water, brine, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions may be prepared by incorporating the binding molecules in a solvent, such as with a suitable carrier, diluent or excipient, such as sterile water, physiological saline, dextrose and the like. The composition may also be lyophilized. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents and the like, depending on the route of administration and the desired preparation. In some aspects, reference may be made to standard text for the preparation of suitable preparations.

Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable compositions can be brought about by the use of delayed absorbers (for example, aluminum monostearate and gelatin).

The pharmaceutical composition according to the invention may be in the form of, for example, a unit dosage form, such as an ampoule, a vial, a suppository, a tablet, a pill or a capsule. The formulation may be administered to a human individual in a therapeutically or prophylactically effective amount (e.g., an amount that prevents, eliminates, or alleviates a pathological condition) to provide treatment for the disease or condition. The preferred dosage of the therapeutic agent to be administered may depend on variables such as the type and extent of the disorder, the general health of the particular patient, the formulation of the compound excipients, and the route of administration thereof.

In certain embodiments, the compositions described herein can be formulated for pulmonary administration, and in certain embodiments, the compositions are formulated for administration via inhalation (e.g., intrabronchial, intranasal or buccal inhalation, intranasal instillation). The composition may be administered using a nebulizer, inhaler, nebulizer, aerosolizer, mist, dry powder inhaler, metered dose nebulizer, metered dose mist, metered dose nebulizer, or other suitable delivery device.

In some embodiments, the composition is a lyophilized composition. In some embodiments, the composition is formulated for aerosol administration, and in certain embodiments, the composition is formulated for oral administration or administration via inhalation.

The pharmaceutical compositions described herein are prepared in a manner known per se, for example by conventional dissolution, lyophilization, mixing, granulation or shaping processes. Pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., remington: the Science and Practice of Pharmacy (21 st edition), editions A.R.Gennaro,2005,Lippincott Williams&Wilkins,Philadelphia,PA, and Encyclopedia of Pharmaceutical Technology, editions j.swarbrick and J.C.Boylan,2013,Marcel Dekker,New York,NY).

Where aerosol administration is appropriate, squalamine or its derivatives may be formulated into an aerosol using standard methods. The term "aerosol" includes any airborne suspension of squalamine or its derivatives, which is capable of being inhaled into the bronchioles or nasal passages, and includes dry powders and aqueous aerosols, as well as pulmonary and nasal aerosols. In particular, the aerosol comprises an airborne suspension of droplets of squalamine or its derivatives, as may be produced in a metered dose inhaler or nebulizer or in a nebulizer. Aerosols also include dry powder compositions of the compounds of the invention suspended in air or other carrier gases, which may be delivered by, for example, blowing from an inhaler device. See Ganderton & Jones, drug Delivery to the Respiratory Tract (Ellis Horwood, 1987); gonda, critical Reviews in therapeutic Drug Carrier Systems,6:273-313 (1990); and Raeburn et al Pharmacol. Toxicol. Methods,27:143-159 (1992).

Formulations for in vivo administration are typically sterile. Preparing an injectable composition under sterile conditions in a conventional manner; the same applies to introducing the composition into an ampoule or vial and sealing the container. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

In some aspects, the pharmaceutical compositions may employ a timed release, delayed release, and sustained release delivery system such that delivery of the composition occurs prior to sensitization of the site to be treated and for a time sufficient to cause sensitization of the site. Many types of release delivery systems are available and known. Such systems can avoid repeated administration of the composition, thereby increasing the convenience of the subject and physician.

In some embodiments, the pharmaceutical composition contains an amount (such as a therapeutically effective or prophylactically effective amount) of a binding polypeptide, such as an antibody or antigen binding fragment, effective to treat or prevent a disease or disorder. In some embodiments, the treatment or prevention efficacy is monitored by periodic assessment of the subject being treated. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and may be determined. The desired dose may be delivered by a single bolus administration of the composition, by multiple bolus administration of the composition, or by continuous infusion administration of the composition

VII methods of use

Provided herein are therapeutic methods and uses for treating a disease or disorder in a subject. In some embodiments, the methods and uses comprise administering a provided binding polypeptide, such as an antibody or antigen binding fragment or knob peptide, to a subject (e.g., a human). In some embodiments, the binding polypeptide or composition comprising the same is administered to the subject by parenteral administration. In some embodiments, the binding polypeptide or composition comprising the same is administered intramuscularly, subcutaneously, intravenously, topically, orally, or by inhalation. In particular embodiments, particularly for delivery knob peptides, administration is by inhalation. In some embodiments, the provided binding polypeptides (such as knob peptides) can be administered by aerosol administration, for example, by delivery using an inhaler or nebulizer.

In some embodiments, provided embodiments relate to methods for treating or preventing cancer or a proliferative disease in a subject. In some embodiments, the provided embodiments relate to methods for treating or preventing a coronavirus infection in a subject. In some embodiments, these methods are used to prophylactically treat a viral infection in a subject at risk for the viral infection. In some embodiments, these methods are used to treat a subject known or suspected to have a viral infection. In some embodiments, these methods can prevent a viral infection, such as a coronavirus infection, in a subject. In some embodiments, the methods can reduce the signs of symptoms of a coronavirus infection in a subject, such as reducing the presence or severity of one or more signs or symptoms. In some embodiments, a binding molecule (such as an antibody or antigen binding fragment or knob peptide) is administered to a subject in an effective amount to effect treatment of an infection. Also provided herein are uses of binding polypeptides (such as antibodies or antigen binding fragments or knob peptides) in such methods and treatments, as well as in the manufacture of medicaments for use in the practice of such methods of treatment. In some embodiments, the methods are performed by administering a binding polypeptide or composition comprising the same to a subject having, already having, or suspected of having a disease or disorder. In some embodiments, the methods thereby treat a disease or condition or disorder in a subject. Also provided herein is the use of any of the compositions (such as the pharmaceutical compositions provided herein) for treating a disease or disorder associated with a coronavirus infection (e.g., due to SARS-CoV-2).

In some embodiments, the provided binding polypeptides (such as antibodies or antigen binding fragments or knob peptides) are administered to a subject in an effective amount or a therapeutically effective amount. An effective amount or therapeutically effective dose of a provided binding polypeptide (such as an antibody or antigen binding fragment or knob peptide) for use in treating or preventing a viral infection is an amount sufficient to reduce one or more signs and/or symptoms of the infection in the subject being treated, whether by inducing regression or elimination of such signs and/or symptoms or by inhibiting progression of such signs and/or symptoms. The amount administered may vary depending on the age and size of the subject to be administered, the target disorder, condition, route of administration, and the like. In one embodiment, an effective amount or therapeutically effective dose of a provided binding polypeptide (such as an antibody or antigen binding fragment thereof or knob peptide) for treating or preventing a viral infection, such as an adult subject, is about 0.001mg/kg to about 200mg/kg, such as 0.01mg/kg to 200mg/kg or 0.1mg/kg to 200mg/kg. The frequency and duration of treatment may be adjusted depending on the severity of the infection.

The methods and uses provided include methods and uses for treating a viral infection in a subject. For example, the method of treatment comprises administering a provided binding polypeptide, such as an antibody or antigen binding fragment or knob peptide, to a subject having one or more signs or symptoms of a disease or infection (e.g., viral infection) in an effective or therapeutically effective amount or dose.

In some embodiments, the methods and uses provided include prophylactic methods and uses. In some embodiments, provided herein are methods for prophylactic administration of provided binding polypeptides (such as antibodies or antigen binding fragments or knob peptides) to subjects at risk of viral infection in order to prevent such infection. In some embodiments, the amount administered is an effective or therapeutically effective amount or dose. In some embodiments, the provided methods and uses prevent viral infection in a subject. In some embodiments, preventing a viral infection by the provided methods involves administering a provided binding polypeptide (such as an antibody or antigen binding fragment or knob peptide) to a subject to inhibit the manifestation of a disease or infection (e.g., a viral infection) in the subject. In some embodiments, the methods reduce one or more signs or symptoms of a viral infection.

Exemplary embodiments

The provided embodiments include:

1. a method of making a bovine ultralong CDR3 antibody display library, the method comprising:

(a) Amplifying sequences encoding various variable heavy chain (VH) regions of the IgHV1-7 family from a library of bovine antibody VH chain complementary DNA (cDNA) templates;

(b) Constructing a plurality of replicable expression vectors for said plurality of VH regions, wherein each replicable expression vector comprises a first nucleic acid sequence encoding a single chain variable fragment (scFv) comprising an amplified VH region that is joined to a lambda VL region selected from the group consisting of variable light chain (VL) regions of BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8 and F18, or a humanized variant thereof;

(c) Transforming a suitable host cell with the plurality of replicable expression vectors under conditions suitable for producing amplified display particles; and

(d) Collecting the amplified display particles, wherein the amplified display particles comprise display particles displaying a fusion protein comprising an scFv.

2. The method of embodiment 1, wherein the VL region is a BLV1H12 VL region.

3. A method of making a bovine ultralong CDR3 antibody display library, the method comprising:

(b) Constructing a plurality of replicable expression vectors for said plurality of VH regions, wherein each replicable expression vector comprises a first nucleic acid sequence encoding a single chain variable fragment (scFv) comprising an amplified VH region that is ligated to a BLV1H12 lambda variable light chain (VL) region or a humanized variant thereof;

4. The method of any one of embodiments 1-3, wherein the cDNA template library is prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

5. The method of any one of embodiments 1-3, further comprising preparing the cDNA template library from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

6. The method of embodiment 4 or embodiment 5, further comprising immunizing the bovine with a target antigen.

7. The method of any one of embodiments 1-6, wherein the amplified display particles comprise bacterial display, yeast display, mammalian display, phage display, mRNA display, ribosome display, or DNA display particles.

8. The method of any one of embodiments 1-7, wherein the amplified display particles are phage display particles.

9. The method of any one of embodiments 1-8, wherein the amplified display particles are phagemid particles.

10. The method of embodiment 9, wherein each replicable expression vector further comprises a second nucleic acid encoding at least a portion of a phage coat protein, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce the phagemid particles, whereby the fusion protein comprises the at least a portion of a phage coat protein.

11. A method of making a bovine ultralong CDR3 antibody phage display library, the method comprising:

(a) Immunizing a bovine with a target antigen;

(b) Preparing a library of antibody Variable Heavy (VH) strand complementary DNA (cDNA) templates from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from the immunized cattle;

(c) Amplifying sequences encoding a plurality of VH regions of the IgHV1-7 family from the cDNA template library;

(d) Constructing a plurality of replicable expression vectors for said plurality of VH regions, wherein each replicable expression vector comprises: (1) A first nucleic acid sequence encoding a single-chain variable fragment (scFv) comprising an amplified VH region joined to a BLV1H12 lambda variable light chain (VL) region or a humanized variant thereof, and (2) a second nucleic acid encoding at least a portion of a phage coat protein;

(e) Transforming a suitable host cell with the plurality of replicable expression vectors;

(f) Infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce amplified phagemid particles; and

(g) Collecting the amplified phagemid particles, wherein the amplified phagemid particles comprise phagemid particles displaying a fusion protein comprising the at least part of the phage coat protein and the scFv.

12. The method of any one of embodiments 1-11, wherein the BLV1H12 lambda VL region is set forth in SEQ ID No. 2.

13. The method of any one of embodiments 1-11, wherein the BLV1H12 lambda VL region is a humanized variant of the lambda VL region of BLV1H 12.

14. The method of embodiment 13, wherein the humanized variant comprises one or more of the amino acid substitutions I29V and N32G and/or DNN to GDT in the CDR2 regions based on the amino acid substitutions of Kabat numbering S2A, T5N, P S, A12G, A S and amino acid substitutions in the P14L, CDR1 regions.

15. The method of any one of embodiment 13 or embodiment 14, wherein the humanized variant comprises the sequence set forth in SEQ ID No. 107.

16. The method of any one of embodiments 2-15, wherein the amplified VH region is indirectly joined to the BLV1H12 lambda VL region via a peptide linker.

17. The method of embodiment 16, wherein the peptide linker is (Gly ₄ Ser) ₃ (SEQ ID NO:94)。

18. The method of any one of embodiments 1-17, wherein the plurality of VH regions of the IgHV1-7 family are amplified from the cDNA template library with a forward primer comprising the sequence set forth in SEQ ID No. 84 and a reverse primer comprising the sequence set forth in SEQ ID No. 85.

19. The method of any one of embodiments 1-18, wherein prior to said constructing, the method further comprises size separating the sequences encoding the plurality of amplified VH regions to enrich for VH regions with ultralong CDRs 3.

20. The method of embodiment 19, wherein the size separation is performed by gel electrophoresis.

21. The method of embodiment 20, wherein the gel electrophoresis is performed using 1.2%, 1.5% or 2% agarose gel, optionally using 2% agarose gel.

22. The method of any one of embodiments 19-21, wherein the size separation comprises separating sequences of about, or greater than 550 base pairs in length from the sequences encoding the plurality of amplified VH regions, wherein the sequences of about, or greater than 550 base pairs in length comprise sequences encoding VH regions with ultralong CDR 3.

23. The method of any one of embodiments 1-22, wherein at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

24. The method of any one of embodiments 1-23, wherein at least or at least about 30% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

25. The method of any one of embodiments 1-24, wherein at least or at least about 40% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

26. The method of any one of embodiments 1-25, wherein at least or at least about 50% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

27. The method according to any one of embodiments 1-26, wherein the ultralong CDR3 is a 25-70 amino acid peptide sequence comprising a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

28. The method of any one of embodiments 1-27, wherein the ultralong CDR3 is 40 to 60 amino acids in length.

29. The method of any one of embodiments 1-28, wherein the ultralong CDR3 is at least 42 amino acids in length.

30. The method according to any one of embodiments 1-29, wherein the ultralong CDR3 is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

31. The method of any one of embodiments 1-30, wherein the ultralong CDR3 comprises at least 4 cysteine residues.

32. The method of any one of embodiments 1-31, wherein the ultralong CDR3 comprises 4 cysteine residues.

33. The method of any one of embodiments 1-31, wherein the ultralong CDR3 comprises 6, 8, 10, or 12 cysteine residues.

34. The method of any of embodiments 1-33, wherein the ultralong CDR3 has at least 2 disulfide bonds.

35. The method of any of embodiments 1-34, wherein the ultralong CDR3 has 2 disulfide bonds.

36. The method of any of embodiments 1-34, wherein the ultralong CDR3 has 3, 4, or 5 disulfide bonds.

37. A method of preparing an ultralong CDR 3-knob display library, the method comprising:

(a) Amplifying sequences encoding a plurality of CDR 3-only knob antibodies from a library of bovine antibody Variable Heavy (VH) chain complementary DNA (cDNA) templates with forward and reverse primers specific for the up-and down-stem domains of the bovine ultralong CDR3 region;

(b) Constructing a plurality of replicable expression vectors for the plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises a first nucleic acid sequence encoding an amplified CDR3 knob;

(d) Collecting the amplified display particles, wherein the amplified display particles comprise display particles displaying a fusion protein comprising an amplified CDR3 knob.

38. The method of embodiment 37, wherein the cDNA template library is prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

39. The method of embodiment 37, further comprising preparing the cDNA template library from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

40. The method of embodiment 38 or embodiment 39, further comprising immunizing the cow with a target antigen.

41. The method of any one of embodiments 37-40, wherein the amplified display particles comprise bacterial display, yeast display, mammalian display, phage display, mRNA display, ribosome display, or DNA display particles.

The method of any one of embodiments 37-41, wherein the amplified display particles are phage display particles.

43. The method of any one of embodiments 37-42, wherein the amplified display particles are phagemid particles.

44. The method of embodiment 43, wherein each replicable expression vector further comprises a second nucleic acid encoding at least a portion of a phage coat protein, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce the phagemid particles, whereby the fusion protein comprises the at least a portion of a phage coat protein.

45. A method of making an ultralong CDR 3-knob phage display library, the method comprising:

(a) Immunizing a bovine with a target antigen;

(c) Amplifying sequences encoding a plurality of CDR 3-only knob antibodies from the cDNA template library with forward and reverse primers specific for the upstream and downstream stem domains of the bovine ultralong CDR3 region;

(d) Constructing a plurality of replicable expression vectors for said plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises (1) a first nucleic acid sequence encoding an amplified CDR3 knob and (2) a second nucleic acid encoding at least a portion of a phage coat protein;

(g) Collecting the amplified phagemid particles, wherein the amplified phagemid particles comprise phagemid particles displaying a fusion protein comprising the at least part of the phage coat protein and an amplified CDR3 knob.

46. The method according to any one of embodiments 37-45, wherein the primer comprises or consists of any one of the sequences set forth in SEQ ID NOS: 7-11.

47. The method of any one of embodiments 37-46, wherein each of the plurality of CDR 3-only knob antibodies comprises a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

48. The method of embodiment 47, wherein the peptide sequence is 40 to 60 amino acids in length.

49. The method of embodiment 47 or embodiment 48, wherein the peptide sequence is at least 42 amino acids in length.

50. The method of any one of embodiments 47-49, wherein the peptide sequence is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

51. The method of any one of embodiments 47-50, wherein the peptide sequence comprises at least 4 cysteine residues.

52. The method of any one of embodiments 47-51, wherein the peptide sequence comprises 4 cysteine residues.

53. The method of any one of embodiments 47-51, wherein the peptide sequence comprises 6, 8, 10 or 12 cysteine residues.

54. The method of any one of embodiments 47-53, wherein the peptide sequence has at least 2 disulfide bonds.

55. The method of any one of embodiments 47-54, wherein the peptide sequence has 2 disulfide bonds.

56. The method of any one of embodiments 47-54, wherein the peptide sequence has 3, 4, or 5 disulfide bonds.

57. The method of any of embodiments 6-36 and 40-56, wherein the target antigen is a non-toxic bacterium, virus, viral protein, immunomodulatory protein (e.g., a checkpoint molecule), cancer antigen, human IgG, or recombinant protein thereof.

58. The method according to any one of embodiments 1-57, wherein the cDNA template library is synthesized using IgM (SEQ ID NO: 4), igA (SEQ ID NO: 5) and IgG specific (SEQ ID NO:3 and 6) primer libraries.

59. A method of preparing an ultralong CDR 3-knob display library, the method comprising:

(a) Constructing a plurality of replicable expression vectors for a plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises a first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds;

(b) Transforming a suitable host cell with the plurality of replicable expression vectors under conditions suitable for producing amplified display particles; and

(c) Collecting the amplified display particles, wherein the amplified display particles comprise display particles displaying a fusion protein comprising a CDR3 knob.

60. The method of embodiment 59, wherein the amplified display particles comprise bacterial display, yeast display, mammalian display, phage display, mRNA display, ribosome display, or DNA display particles.

61. The method of embodiment 59 or embodiment 60, wherein the amplified display particles are phage display particles.

62. The method of any one of embodiments 59-61, wherein the amplified display particles are phagemid particles.

63. The method of embodiment 62, wherein each replicable expression vector further comprises a second nucleic acid encoding at least a portion of a phage coat protein, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce the phagemid particles, whereby the fusion protein comprises the at least a portion of a phage coat protein.

64. A method of making an ultralong CDR 3-knob phage display library, the method comprising:

(a) Constructing a plurality of replicable expression vectors for a plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises: (1) A first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds, and (2) a second nucleic acid encoding at least a portion of a bacteriophage coat protein;

(b) Transforming a suitable host cell with a plurality of replicable expression vectors;

(c) Infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein sufficient to produce amplified phagemid particles; and

(d) Collecting the amplified phagemid particles, wherein the amplified phagemid particles comprise phagemid particles displaying a fusion protein comprising the at least part of the phage coat protein and a CDR3 knob.

65. The method of any one of embodiments 27-36 and 47-64, wherein the peptide sequence comprises an upstream stem domain and a downstream stem domain, wherein the cysteine motif is between the upstream stem domain and the downstream stem domain.

66. The method of embodiment 64 or embodiment 65, wherein the peptide sequence is amplified from DNA from a bovine immunized with the target antigen.

67. The method of embodiment 66, wherein the peptide sequence is amplified from a variable heavy chain cDNA library from the immunized cow using primers specific for either side of the stem domain of the bovine ultralong CDR3 region.

68. The method of any one of embodiments 27-36, 47-64, 66 and 67, wherein the peptide sequence does not comprise an upstream stem domain of the N-terminus of the cysteine motif.

69. The method according to any one of embodiments 27-36, 47-64 and 66-68, wherein the peptide sequence does not comprise the downstream stem domain of the C-terminus of the cysteine motif.

70. The method of any one of embodiments 65-67 and 69, wherein the upstream stem domain comprises the sequence CX ₂ TVX ₅ Q, wherein X ₂ And X ₅ Is any amino acid.

71. The method of embodiment 70, wherein X ₂ Ser, thr, gly, asn, ala or Pro, and X ₅ Is His, gln, arg, lys, gly, thr, tyr, Phe. Trp, met, ile, val or Leu.

72. The method of embodiment 70 or embodiment 71, wherein X ₂ Is Ser, ala or Thr, and X ₅ Is His or Tyr.

73. The method of any one of embodiments 64, 65 and 68-72, wherein the peptide sequence is a synthetic CDR 3-knob.

74. The method of any one of embodiments 64, 65 and 68-73, wherein the peptide sequence is a cyclic peptide or a modified cyclic peptide.

75. The method of any one of embodiments 64, 65 and 68-73, wherein the peptide sequence is a semisynthetic CDR 3-knob derived from a bovine CDR 3-knob.

76. The method of any one of embodiments 64-75, wherein the peptide sequence is 40 to 60 amino acids in length.

77. The method of any one of embodiments 64-76, wherein the peptide sequence is at least 42 amino acids in length.

78. The method of any one of embodiments 64-77, wherein the peptide sequence is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

79. The method according to any one of embodiments 64-78, wherein the peptide sequence comprises at least 4 cysteine residues.

80. The method according to any one of embodiments 64-79, wherein the peptide sequence comprises 4 cysteine residues.

81. The method of any one of embodiments 64-79, wherein the peptide sequence comprises 6, 8, 10, or 12 cysteine residues.

82. The method of any one of embodiments 64-81, wherein the peptide sequence has at least 2 disulfide bonds.

83. The method of any one of embodiments 64-82, wherein the peptide sequence has 2 disulfide bonds.

84. The method of any one of embodiments 64-82, wherein the peptide sequence has 3, 4, or 5 disulfide bonds.

85. The method of any of embodiments 64, 65 and 68-84, wherein the plurality of CDR3 knobs are mutated at one or more selected positions within the nucleic acid sequence encoding the peptide sequence, wherein the plurality of replicable expression vectors are a family of mutated vectors.

86. The method of any one of embodiments 1-85, wherein the expression vector further comprises a secretion signal sequence.

87. The method of embodiment 86, wherein the secretion signal sequence is a pelB signal sequence.

88. The method of any one of embodiments 1-87, wherein the suitable host cell is an e.

89. The method of any one of embodiments 1-88, wherein the suitable host cell is a TG1 inducible competent cell.

90. The method of any one of embodiments 9-36, 43-58, and 62-89, wherein said phagemid particle is derived from M13 phage.

91. The method according to any one of embodiments 10-36, 44-58 and 63-90, wherein the coat protein is the M13 phage gene III coat protein (pIII).

92. The method of any one of embodiments 10-36, 44-58, and 63-91, wherein the helper phage is selected from the group consisting of M13K07, M13R408, M13-VCS, and Phi X174.

93. The method of any one of embodiments 10-36, 44-58, and 63-92, wherein the helper phage is M13K07.

94. The method of any one of embodiments 1-93, wherein the display particle displays one copy of the fusion protein on average on the surface of the particle.

95. A library of display particles produced by the method according to any one of embodiments 1-94.

96. A replicable expression vector comprising a gene fusion encoding a fusion protein comprising a first nucleic acid sequence encoding a single chain variable fragment comprising a bovine variable heavy chain (VH) region comprising an ultralong CDR3 joined to a lambda VL region of a variable light chain (VL) region selected from the group consisting of BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8 and F18, or a humanized variant thereof.

97. A replicable expression vector comprising a gene fusion encoding a fusion protein, said gene fusion comprising a first nucleic acid sequence encoding a single chain variable fragment comprising a bovine variable heavy chain (VH) region comprising an ultralong CDR3 joined to a BLV1H12 λ variable light chain (VL) region or a humanized variant thereof.

98. The replicable expression vector of embodiment 96 or embodiment 97, further comprising a second nucleic acid sequence encoding at least a portion of a phage coat protein.

99. A display particle encoded by the replicable expression vector of any of embodiments 96-98.

100. A library of display particles comprising a plurality of display particles according to embodiment 95 or 99.

101. The library of embodiment 100, wherein at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region.

102. The library of embodiment 100 or embodiment 101, wherein the library is one of

At least or at least about 30% of the display particles comprise an scFv comprising a VH region comprising an ultralong CDR3 region.

103. The library according to any one of embodiments 100-102, wherein any one of the library

Less or at least about 40% of the display particles comprise an scFv comprising a VH region comprising an ultralong CDR3 region.

104. The library according to any one of embodiments 100-103, wherein any one of the library

Less or at least about 50% of the display particles comprise an scFv comprising a VH region comprising an ultralong CDR3 region.

105. A replicable expression vector comprising a gene fusion encoding a fusion protein, said gene fusion comprising a first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming a disulfide bond.

106. The replicable expression vector of embodiment 105, further comprising a second nucleic acid sequence encoding at least a portion of a bacteriophage coat protein.

107. A display particle encoded by the replicable expression vector of embodiment 105 or embodiment 106.

108. A library of display particles comprising a plurality of display particles according to embodiment 107.

109. The library according to any one of embodiments 95, 100-104 and 108, wherein the display particles are phage display particles.

110. The library according to any one of embodiments 95, 100-104, 108 and 109, wherein the display particles are phagemid particles.

111. A method for selecting an antibody binding protein, the method comprising:

(1) Contacting the library of display particles according to any one of embodiments 95, 100-104 and 108-110 with a target molecule under conditions that allow binding of the display particles to the target molecule; and

(2) Separating the bound display particles from unbound display particles, thereby selecting display particles comprising antibody binding proteins that bind the target molecule.

112. The method of embodiment 111, wherein the display particle is a phage display particle.

113. The method of embodiment 111 or embodiment 112, wherein the display particle is a phagemid particle.

114. The method of any one of embodiments 111-113, wherein the target molecule is a non-toxic bacterium, virus, viral protein, immunomodulatory protein (e.g., a checkpoint molecule), cancer antigen, human IgG, or recombinant protein thereof.

115. The method of any one of embodiments 111-114, wherein the target molecule is a coronavirus, a coronavirus pseudovirus, a recombinant coronavirus spike protein, or a Receptor Binding Domain (RBD) of a coronavirus spike protein.

116. The method of embodiment 115, wherein the coronavirus is selected from the group consisting of 229E, NL, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV2.

117. The method of embodiment 115 or embodiment 116, wherein the coronavirus is SARS-CoV2 selected from the group consisting of a Wuhan-Hu-1 isolate, a B.1.351south African variant or a B.1.1.7UK variant.

118. The method of any of embodiments 111-117, further comprising:

(i) Infecting a suitable host cell with a replicable expression vector encoding the selected display particles bound in (2);

(ii) Collecting the amplified display particles; and

(iii) Repeating steps (1) and (2) using the amplified display particles as a library of the display particles.

119. The method of embodiment 118, wherein the display particle is a phagemid particle, and the method further comprises infecting the transformed host cell with an amount of helper phage having a gene encoding the phage coat protein sufficient to produce an amplified phagemid particle.

120. The method of embodiment 118 or embodiment 119, wherein the steps are repeated one or more times.

121. The method of any of embodiments 118-120, wherein the steps are repeated with the same target molecule or different target molecules.

122. The method of embodiment 121, wherein the steps are repeated with a different target molecule, and the different target molecule is associated with the target molecule.

123. The method of embodiment 121 or embodiment 122, wherein the different target molecule is the same type of pathogen, the same group of pathogens, or a variant of the target molecule as the target molecule.

124. The method of any one of embodiments 111-123, further comprising sequencing the fusion gene in the selected display particle to identify the antibody binding protein.

125. The method of embodiment 124, further comprising producing full length IgG or Fab from the selected antibody binding proteins.

126. The method of embodiment 124 or embodiment 125, wherein the antibody binding protein is an scFv, and the method comprises constructing a heavy chain or portion thereof comprising conjugating the VH region of the scFv to a constant region or portion thereof.

127. The method of embodiment 124 or embodiment 125, wherein the method comprises constructing a humanized VH region by replacing the knob region of the ultralong CDR3 region of the humanized bovine VH region with the ultralong CDR3 region of the selected antibody binding protein.

128. The method of embodiment 127, wherein the ultralong CDR3 region of the selected antibody binding protein is replaced between the uplink and downlink stem chains of a humanized bovine VH region.

129. The method of embodiment 128, wherein the VH region comprises the formula V1-X-V2, wherein the V1 region of the heavy chain comprises the sequence set forth in SEQ ID No. 111; the X region comprises the ultralong CDR3 of the selected antibody; and the V2 region comprises the sequence set forth in SEQ ID NO. 112.

130. The method of any one of embodiments 127-129, wherein the method further comprises constructing a heavy chain or portion thereof comprising ligating the humanized VH region with a constant region or portion thereof.

131. The method of embodiment 126 or embodiment 130, wherein the heavy chain or portion thereof is a human IgG1 heavy chain or portion thereof.

132. The method of any one of embodiments 126, 130, and 131, further comprising coexpression of the heavy chain or portion thereof with a light chain.

133. The method of embodiment 132, wherein the light chain is a bovine light chain of BLVH12, BLV5D3, BLV8C11, BF1H1, BLV5B8, or F18, or a humanized variant thereof

134. The method of embodiment 132 or embodiment 133, wherein the light chain is the BLV1H12 light chain (SEQ ID NO: 113) or a humanized variant thereof.

135. The method of any one of embodiments 131-134, wherein the light chain is a humanized light chain as set forth in SEQ ID No. 114.

136. The method of embodiment 132 or embodiment 133, wherein the light chain is the BLV5B8 light chain (SEQ ID NO: 115) or a humanized variant thereof.

137. The method of embodiment 132, wherein the light chain is a human light chain.

138. The method of embodiment 132 or embodiment 137, wherein the light chain is selected from the group consisting of VL1-47, VL1-40, VL1-51, and VL 2-18.

139. The method according to any one of embodiments 132, 137 and 138, wherein the light chain is set forth in any one of SEQ ID NOs 116-120.

140. A method for producing a soluble ultralong CDR3 knob, the method comprising:

(a) Transforming E.coli with an expression vector encoding a fusion protein comprising an ultralong CDR3 knob and a bacterial chaperone joined by a cleavable linker, wherein the ultralong CDR3 knob is a 25-70 amino acid peptide sequence having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds;

(b) Culturing the bacterium under conditions allowing expression of the fusion protein;

(c) Isolating the fusion protein from the supernatant of the bacterial cell lysate; and

(d) Cleaving the cleavable linker of the fusion protein, thereby producing a soluble ultralong CDR3 knob comprising 1-6 disulfide bonds that is free of the bacterial chaperone protein.

141. The method of embodiment 140, wherein the ultralong CDR3 knob is an antibody binding protein identified by the method according to any one of embodiments 111-124.

142. The method of embodiment 140 or embodiment 141, wherein the fusion protein has increased solubility relative to the ultralong CDR3 knob alone.

143. The method of any one of embodiments 140-142, wherein the bacterial chaperonin is thioredoxin a (TrxA).

144. The method according to any of embodiments 140-143, wherein the cleavable linker is an enterokinase cleavable tag having the amino acid sequence DDDDK (SEQ ID NO: 106).

145. The method of any of embodiments 140-144, wherein cleaving the cleavable linker comprises adding enterokinase to the supernatant.

146. The method of any of embodiments 140-145, wherein the soluble ultralong CDR3 knob comprises an additional linker to allow cyclization of the soluble ultralong CDR3 knob via a chemical or enzymatic method, optionally wherein the additional linker allows for sortase-mediated cyclization.

147. The method of embodiment 146, further comprising cyclizing the soluble ultralong CDR3 knob.

148. The method of any one of embodiments 140-147, further comprising (e) removing the enterokinase and/or the bacterial chaperone protein from a solution comprising the soluble ultralong CDR3 knob.

149. The method of any of embodiments 140-148, further comprising enriching the soluble ultralong CDR3 knob from a solution comprising the soluble ultralong CDR3 knob, optionally wherein the enriching comprises size exclusion chromatography.

150. The method of any one of embodiments 140-149, further comprising generating a multispecific binding molecule comprising the soluble ultralong CDR3 knob.

151. The method of any of embodiments 140-150, wherein the ultralong CDR3 knob is 3-8kDa or 4-5kDa in size.

152. A fusion protein comprising an ultralong CDR3 knob and a bacterial chaperone joined by a cleavable linker, wherein the ultralong CDR3 knob is a 25-70 amino acid peptide sequence having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

153. The fusion protein according to embodiment 152, wherein the bacterial chaperone protein is thioredoxin a (TrxA).

154. The fusion protein according to embodiment 152 or embodiment 153, wherein the cleavable linker is an enterokinase cleavable tag having the amino acid sequence DDDDK (SEQ ID NO: 106).

155. The fusion protein according to any one of embodiments 152-154, wherein the ultralong CDR3 knob comprises 1-6 disulfide bonds.

156. A composition comprising the fusion protein according to any one of embodiments 152-155.

157. A purified soluble ultralong CDR3 knob produced by the method of any one of embodiments 140-151, wherein the soluble ultralong CDR3 is 25-75 amino acids in length and comprises 1-6 disulfide bonds.

158. The purified soluble ultralong CDR3 knob of embodiment 157, wherein the ultralong CDR3 knob is 3-8kDa in size.

159. The purified soluble ultralong CDR3 knob of embodiment 157 or embodiment 158, wherein the ultralong CDR3 knob is of a size of 4-5kDa.

160. A composition comprising the purified soluble ultralong CDR3 of any one of embodiments 157-159.

161. The composition of embodiment 160, further comprising a pharmaceutically acceptable carrier.

162. The composition of embodiment 160 or embodiment 161, formulated for parenteral administration.

163. The composition of any of embodiments 160-162, formulated for intravenous, intramuscular, topical, otic, conjunctival, nasal, inhalation, or subcutaneous administration.

164. The composition of any one of embodiments 160-163, formulated for administration by inhalation.

Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: generation of anti-SARS CoV-2 antibodies

Cattle were immunized with SARS CoV-2 spike protein or Receptor Binding Domain (RBD) portion thereof and serum was collected to assess binding activity.

A. Spike protein and receptor binding domain expression and purification

SARS CoV-2 spike trimer proteins from the parent Wuhan-Hu-1 isolate (NCBI YP 009724390.1) or b.1.351"South African" variants with mutations E484K (and K417N and N501Y) or parent Receptor Binding Domain (RBD) proteins (amino acids 319 to 541 of spike proteins) were produced by transfection of HEK293 cells. Will be about 120 x 10 ⁶ HEK293 Freestyle cells were combined with 293fectin (Invitrogen) and 120 μg of pCAGGS-based vector containing (1) a gene encoding a spike protein with the furin cleavage site removed and having K986P and V987P stabilizing mutations The extracellular domain of (2) a T4-fibritin trimerization domain and a c-terminal 6 xHis-tag, or (2) a spike RBD domain (amino acids 319 to 541 of spike protein) having a c-terminal 6 xHis-tag.

Cells were shaken with 8% CO2 at 37℃for 4 days, and 150. Mu.l TCM-ProteaseArrest tissue culture protease inhibitor (G-Biosciences) was added on day 3. Supernatant containing secreted spike or RBD proteins was clarified from the supernatant by centrifugation at 4000RPM for 5 minutes followed by filtration through a 0.45 μm PES filter. The supernatant was concentrated and buffer exchanged into PBS at 4 ℃ using an Amicon ultracentrifugation filtration unit (mwco=50,000 for protein S preparation, mwco=10,000 for RBD protein) (EMD-Millipore). The concentrated supernatant was then purified with a TALON cobalt metal affinity resin (Takara Bio) according to the manufacturer's protocol, except that 50mM, 100mM, 200mM, 300mM and 400mM imidazole gradient elution fractions (1 column volume each) were collected. The eluted fractions were resolved on SDS-PAGE gels stained with InstantBuue Coomassie protein dye (Abcam). Fractions containing a single spike protein band or a single RBD band were pooled, buffer exchanged into PBS as described above, and protein concentration was quantified based on extinction coefficient and molecular weight of spike protein or RBD protein, respectively, using Nanodrop One (Thermo Scientific).

B. Immunization protocol

Two calves were immunized with 200 μg/dose of purified Wuhan-Hu-1 spike protein or RBD protein variants distributed at 5 neck positions and boosted according to the disclosed methods (Sok et al Nature 2017,548 (7665):108-111; wang et al Cell 2013,153 (6): 1379-1393). Serum was collected and IgG ELISA was performed on RBD domains from SARS-CoV-2 spike on serum from RBD immunized calves at serum dilutions of 1:100 to 1:10,000. Spike protein reactivity was observed 7-21 days after immunization. As shown in fig. 2A, the binding activity to RBD domains was significant after the first immunization.

Serum IgG neutralization of spike proteins and viruses was also assessed using the Plaque Reduction and Neutralization Test (PRNT). In this in vitro assay, the virus and serum IgG are pre-incubated together prior to simultaneous application to the allowed cells, such that the virus successfully bound by the antibody is no longer able to penetrate the cells and/or is no longer able to propagate the infection further. As a result, when a cell monolayer is stained, lesions of infection and cellular injury, known as "plaques," appear to be smaller in size and/or number.

The percent neutralization of IgG in serum from both parental Spike protein and RBD immunized cattle in Vero6 cells was determined using pseudoviruses expressing SARS CoV-2 Spike protein as model viruses. Pseudoviruses can be treated with BSL-2 at high titers compared to natural viruses and can infect cells only in a single round. As shown in fig. 2B, igG obtained from cattle in either immunization regimen was able to successfully neutralize pseudoviruses in a dose-dependent manner. At higher concentrations, serum IgG (ng/mL) from RBD-only immunized cattle was observed to neutralize 100% of pseudovirus.

Together, these results support that the immunized bovine serum and the antibodies contained therein can neutralize SARS-CoV-2.

Example 2: ultralong CDR3 scFv antibodies or CDR 3-knob-only phage display libraries for antibody discovery Generating

Peripheral Blood Mononuclear Cells (PBMCs) were collected from immunized cattle as described in example 1 and RNA was extracted for use in generating two phage display libraries as described below. Specifically, about 1 to 5X 10 was collected 14 to 64 days after immunization ⁷ PBMCs were used and stored prior to RNA extraction and cDNA synthesis.

Two library strategies were employed, using a peptide ((Gly) with a through flexible linker ₄ Ser) ₃ 15 amino acid linker, SEQ ID NO: 94) or an antibody in the form of an scFv of a variable heavy chain (VH) and a variable light chain (VL) fragment, or using a separate CDR 3-knob. In both methods, the scFv or CDR 3-knob is fused to pIII via a flexible Gly4Ser linker. FIG. 3A depicts pIII fusion constructs in each display library. The generation of the display library is summarized below.

Construction of ScFv library

In the first strategy, an immunized bovine-derived VH DNA fragment was combined with an immobilized light chain BLV1H12 (Stanfield et al Science immunology 2016,1 (1): aaf 7962.). RBD and full length spike protein immune libraries were constructed for different immunization time points.

From 5X 10 using RNAeasy kit (Qiagen) ⁶ -10 ⁷ RNA was isolated from individual bovine PBMCs. Immune bovine antibody VH repertoires were obtained by cDNA synthesis from 5 μg total RNA using Superscript IV first strand cDNA synthesis kit (thermospher, # 18091050) followed by PCR amplification. To generate a VH template library, cDNA templates for VH were synthesized using IgM (SEQ ID NO: 4), igA (SEQ ID NO: 5) and IgG specific (SEQ ID NO:3 and 6) primer pools.

In these hybridization libraries, full length donor ultralong VH was amplified from VH template libraries with VH family specific primer pairs. Specifically, the two VH regions were amplified with FR1 and FR4 primers specific to the bovine IgHV1-7 family (SEQ ID NOS: 12 and 13, respectively) in order to enrich the VH region with an ultralong CDR3 region. The amplified product was combined with the linker-BLV 1H12 lambda light chain variable region (BLV 1H12 light chain shown in SEQ ID NO:2 and encoded by the DNA sequence shown in 1) by cloning into a pre-cloned pTAU1 pIII fusion phage display vector (pTAU 1-BLV1H12 (-VH)) (see FIG. 3C). The amplified product was digested with NcoI and XhoI (NEB) for 2 hours and subcloned into pTAU1-BLV1H12 (-VH) as NcoI-XhoI fragment for isolation of the peptide ((Gly) by flexible linker ₄ Ser) ₃ SEQ ID NO: 94) linked VH and VL. In a further step, some of the extra long VH fragments were additionally enriched by separation from shorter VH fragments using agarose gel electrophoresis prior to digestion with NcoI and XhoI restriction enzymes. As shown in fig. 3D, 2% agarose gel achieved maximum separation between the ultralong VH fragment (about 550 base pairs in length) and the shorter VH fragment without the ultralong CDR3 region (about 400 base pairs in length).

Then, it was ligated overnight at 16℃with T4 DNA ligase. The final library was obtained by electroporation of competent TG1 cells (Lucigen) with the purified ligation product. There is a minimum of 10 per library ⁷ Individual clones, wherein>90% have inserts.

CDR 3-knob library construction

In a second strategy, a library of VH templates is generated substantially as described in the first strategy. Then, a CRD 3-knob (also referred to as "CDR 3-knob only") library of ultralong VH only, immunobovine origin was constructed by amplifying stem-knob CDRs from a VH template library using conserved primers and cloning as pIII fusion into a pTAU1 phage display pIII fusion vector.

Specifically, RNAeasy kit (Qiagen) was used from 5×10 ⁶ -10 ⁷ RNA was isolated from individual bovine PBMCs. Immune bovine antibody CDR 3-knob repertoires were obtained by cDNA synthesis from 5 μg total RNA using Superscript IV first strand cDNA synthesis kit (thermosbottle) followed by PCR amplification. To generate a VH template library, CDNA templates for CDR 3-knobs were synthesized using IgM (SEQ ID NO: 4), igA (SEQ ID NO: 5) and IgG specific (SEQ ID NO:3 and 6) primer libraries.

Primary stem-knob CDR3 was amplified from the 1 st chain cDNA using IgHV1-7 family specific primers (SEQ ID NOS: 7-11) specific for either side of the stem domain of the CDR3 region. They were then cloned as NcoI-NotI fragments into pTAU1 phage vector after digestion with NcoI and NotI (NEB) for 2 hours and ligation overnight at 16℃with T4 DNA ligase (see FIG. 3B). The final library was obtained by electroporation of competent TG1 cells (Lucigen) with the purified ligation product. There is a minimum of 10 per library ⁷ Individual clones, wherein>90% have inserts.

Example 3: screening of phage display libraries and ultralong VH or CDR 3-knob domains directed against SARSCOV-2 Selection of (3)

The VH ultralong CDR3 scFv or CDR-only knob library generated as described in example 2 was subjected to two-five rounds of phage display selection against SARS CoV-2 target protein (both the parent Wuhan-Hu-1 or "South African" b.1.351 variant spike protein or the parent Wuhan Hu-1 RBD). Spike protein from the viral isolate or parent RBD was coated on NUNC immune tubes overnight at 4℃with 1mL of 10. Mu.g/mL PBS solution of target protein. The tube was then blocked on a blood mixer with 3-4ml of 2% milk powder dissolved in PBS for 1 hour at room temperature and washed 3 times with PBS.

For each selection, about 10 from a different immunized scFv or CDR3 knob library generated as described in example 2 ¹² Individual phage particles were added to 1mL of 4% milk powder dissolved in PBS and made up to a total volume of 2mL with PBS, then added to the tube with target protein and incubated on a blood mixer for 2 hours at room temperature. Tubes were then washed with 10xPBS/0.1% Tween 20 and 10 xPBS.

The bound phage were recovered on a blood mixer with 1mL fresh 0.1M triethylamine for 10 min and neutralized with 0.5mL 1M tris (pH 7.0) on ice. Log phase TG1 phage-component ^TM Cells were infected with eluted phage at 37℃at 200rpm for 1 hour and then grown overnight at 30℃on 2xTY agar supplemented with 2% glucose/50. Mu.g/mL carbenicillin.

After each selection round described above, TG1 bacteria were scraped from the master plate into 20mL of 2xTY medium supplemented with 20% glycerol/2% glucose/50. Mu.g/mL carbenicillin. About 4-5mL of this solution was added to 20mL of 2xTY medium supplemented with 2% glucose/50 μg/mL carbenicillin containing 100 μ l M K07 helper phage (moi=10). The suspension was incubated at 37℃at 200rpm for 1 hour and added to 200mL of 2xTY/0.2M sucrose/50. Mu.g/mL of carbenicillin/25. Mu.g/mL of kanamycin/20. Mu.m IPTG followed by incubation at 30℃at 200rpm overnight. After 1 hour incubation on ice, the amplified phage were precipitated from the clarified culture supernatant with 1/5 volume of 2.5M NaCl, 20% PEG 8000 in a 250mL Oakridge centrifuge tube. Phage-containing material was pelleted in a Sorvall centrifuge at 14,000g for 20 minutes, resuspended in 2mL PBS, and 1mL was reserved for the next round of selection. Each library was subjected to 2-5 rounds of selection, with phage ELISA performed on each round starting from round 2.

From each selection, individual colonies were picked into 600. Mu.L of 2xTY medium supplemented with 50. Mu.g/mL carbenicillin and 2% w/v glucose in 96-deep well culture plates and incubated overnight at 37℃with shaking at 200 rpm. For each culture, 50. Mu.L was transferred to a fresh 96-deep well plate containing 200. Mu.L/well of the same medium and grown for 3 hours. Will be about 10 ⁸ Kanamycin resistance unit (k.r.u.)M13K07 kanamycin resistant helper phage was added to each well and the plates incubated for 1 hour at 37 ℃. Expression medium (800. Mu.L/well 2xTY medium supplemented with 0.2M sucrose, 100. Mu.g/mL carbenicillin, 25. Mu.g/mL kanamycin, and 20. Mu.M IPTG) was added to each well and amplification continued overnight at 30 ℃.

The plates were centrifuged at 2000g for 10 min at 4℃and 25. Mu.L of culture supernatant per well was used for ELISA. Half-area Costar ELISA plates were coated with 1. Mu.g/mL spike target protein in 50. Mu.L/well RBD or PBS overnight at 4℃and blocked with 100. Mu.L/Kong Rongjie of 2% milk powder in PBS for 1 hour at room temperature and then washed with 2X 100. Mu.L/well PBS. Approximately 25 μl of phage culture supernatant per well was added to each target or negative control plate containing 25 μl/well of 4% milk powder/PBS and allowed to bind for 1 hour at room temperature. Each plate was washed twice with 200 μl/well PBS containing 0.1% Tween 20, then twice with 200 μl/well PBS. Bound phage were detected with 50. Mu.L/well in 2% milk powder/PBS, 1:5000 dilution of anti-M13-HRP conjugates (Sinobiologics) for 1 hour at room temperature. Plates were washed and developed with 50. Mu.L/well TMB (3, 3', 5' -tetramethylbenzidine) substrate buffer (Thermofiser) for 5-10 min at room temperature. According to the manufacturer's protocol, 100. Mu.L/well 0.5. 0.5N H ₂ SO ₄ The reaction was terminated and the optical density was read at 450 nm.

Positive clones from the screened scFv library were sequenced and both short and extra long VH sequences were transferred into pFUSE human IgG1 Fc heavy chain expression vectors for co-expression with chimeric BLV1H12 lambda light chain-human lambda light chain constant regions in mammalian HEK293 cells. Positive clones from the screening knob-only CDR3 library were synthesized as complete VH gene fragments and cloned into pFUSE human IgG1 Fc vector and expressed similarly to chimeric BLV1H12 lambda light chain as described above. Specifically, in a 50. Mu.L reaction, 2X Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Scientific) and pair V are used _H Frame 1 specific primers (Forward) and pair J _H Frame 4 specific primers (reverse) PCR amplification of each V from 10ng phage plasmid miniprep (Qiagen) _H . Cloning of PCR-generated inserts into pFUSE mammalian expression vectors 5'EcoRI and 3 on the 5' end of the human IgG1 Fc geneThe' NheI site. Mixing with a feed containing bovine V _L Sequence (BLV 1H 12) and human λC _L The second pFUSE plasmid pair of sequences was used to transfect HEK 293F cells. The cells were mixed at 1X 10 ⁶ The individual cells/mL were seeded in 30-60mL Freestyle 293 expression medium (Gibco) and then at 37℃and 8% CO ₂ Is incubated in a humid environment. The heavy and light chain plasmids 1:1 were pooled to a total of 1 μg DNA/mL 293F culture and then diluted in Opti MEM I medium (Gibco) to a final volume of 1mL/30mL 293F culture. For every 30mL of 293F culture, approximately 60. Mu.L of 293fectin transfection reagent (Gibco) and 940. Mu.L of Opti MEM I were combined, then gently mixed and incubated for 5 minutes at room temperature before being added to the diluted DNA. The mixture was incubated at room temperature for 30 minutes and then transferred to 293F cultures.

Media was harvested 5 days after transfection and the expressed chimeric bovine human IgG1 antibody was purified by immobilized protein a Sepharose (Cytiva Life Sciences) chromatography and then tested for antigen binding and neutralization of live and pseudoviruses.

Candidate antibodies selected from the library screen were identified and sequenced (table E1). Many selected antibodies contain ultralong CDR3 domains. Thus, although ultralong CDR3 antibodies account for only about 10% of naturally occurring bovine antibodies, the candidate antibodies from the immunity described in example 1 generated and screened by the phage display method described above are highly enriched for bovine antibodies with ultralong CDR3 (i.e., more than 40% of the candidates are characterized by CDR3 having at least 50 amino acids).

Exemplary antibodies SA-R2C3 and SA-R2D9 antibodies were derived from an ultralong scFv library (immunized with parent Wuhan-Hu 1S protein) and identified by a screen involving selection on South African variant spike proteins. Exemplary SKM and SKD antibodies were identified from a screen of phage libraries directly derived from the CDR 3-knob library.

Exemplary ultralong antibodies SKD (SEQ ID NO: 68), SKM (SEQ ID NO: 69), R4C1 (SEQ ID NO: 70), R5C1 (SEQ ID NO: 71), SR3A3 (SEQ ID NO: 72), R2F12 (SEQ ID NO: 73), and R2G3 (SEQ ID NO: 74) are shown in FIG. 4 with alignment of the germline reference sequences (SEQ ID NO: 75). The length of CDR3 and the number of cysteine residues are also shown each.

Example 4: assessment of binding to spike protein and RBD

Selected clones expressed and purified as chimeric bovine-human IgG1 antibodies as described in example 3 were then assayed for their ability to bind RBD and spike protein.

A.SARS CoV-2

RBD and spike binding of chimeric bovine-human IgG1 antibodies were assessed by ELISA. About 50. Mu. LRBD or spike protein (1. Mu.g/mL in PBS) was added to each well of a half-area Costar ELISA plate (Corning) and coated overnight at 4 ℃. Plates were blocked with 180 μl/well of 2% milk powder/TBS/0.1% Tween20 for 2 hours at room temperature. Purified chimeric bovine-human IgG1 antibodies were diluted 5-fold from 20nM-0.00129nM in 2% milk powder/TBS/0.1% Tween20 and 50 μl/well of each dilution was added to coated/uncoated wells in duplicate. Plates were incubated for 1 hour at room temperature, then washed four times with 180 μl TBS/0.1% Tween20, and bound IgG was detected for 30 minutes at room temperature with 50 μl/well of anti-human Fc-HRP (Jackson ImmunoResearch Laboratories, inc.) diluted 1:5000 in 2% milk powder/TBS/0.1% Tween 20. Plates were then washed five times with 180 μl TBS/0.1% Tween20, followed by 50 μl/well of TMB (3, 3', 5' -tetramethylbenzidine) substrate buffer (Thermo Scientific). After 1-2 minutes at room temperature, 50. Mu.L/well 1N H ₂ SO ₄ The reaction was terminated and OD 450nm values were recorded.

Representative results of three clones tested are shown in fig. 5A and 5B. As shown in fig. 5A, the purified chimeric bovine-human IgG1 antibodies (R2G 3, R2F12, and R4C 1) each showed binding to spike protein. Unrelated bovine-human IgG1 (136S IgG) did not show binding to spike proteins. As shown in FIG. 5B, V with clones R2G3 and R2F12 _H Shows binding to RBD. Unrelated bovine-human IgG1 (136S IgG) and V with clone R4C1 _H Chimeric antibodies of (C) do not show binding to RBinding of BD protein. These results are consistent with the following findings: the antibody R4C1 binds to a non-RBD epitope in the spike protein, while R2G3 and R2F12 bind to RBD epitopes.

SKM	Is that	0.24	Is that	0.19
					R2G3	Is that	0.056	Is that	0.032
R2F12	Is that	0.085	Is that	0.050
					SR3A3	Is that	-	Is that	0.037

Variants of SARS CoV-2

RBD and spike binding of chimeric bovine-human IgG1 antibodies was assessed by ELISA against further isolates of SARS CoV-2 (including variants from the β, δ and omnikow lineages, and SARS CoV-1 virus). Approximately 50. Mu.L of RBD or spike protein (1. Mu.g/ml in PBS) was added to each well and coated overnight at 4℃as described in example 4. The plates were closed at room temperature for 2 hours. Purified chimeric bovine-human IgG1 antibodies were diluted 5-fold from 20nM-0.00129nM and 50 μl/well of each dilution was added to coated/uncoated wells in duplicate. Plates were incubated for 1 hour at room temperature, then washed four times, and bound IgG was detected with anti-human Fc-HRP (Jackson ImmunoResearch Laboratories, inc.). The plates were then washed five times, then TMB substrate buffer was added. After 1-2 minutes at room temperature, the reaction was stopped with H2SO4 and the OD 450nm was recorded.

FIG. 5C shows ELISA binding of IgG antibodies to recombinant stabilized spike proteins derived from wild-type (WT) Wuhan-Hu-1 strain, beta strain (previously described as South African strain) or delta strain. Exemplary antibodies SKD and SKM were observed to appear to lose detectable binding to β, but maintained binding to WT and δsars CoV-2. Other antibodies showed binding in the concentration ranges tested for each S protein.

In a set of supplemental experiments with RBD, figure 5D shows ELISA binding curves for selected IgG antibodies against either omigram Rong Bianti RBD (left) or recombinant stabilized spike trimer (right). In the exemplary RBD binders tested, only R2D9 was observed to remain bound to the armstrong variant spike RBD. It was also observed that R4C1, R5C1 and R2D9 bind to full length omnikom spikes with EC50 in the sub nanomolar range.

FIG. 5E reflects exemplary ELISA data for R4C1 and R2D9 versus SARS-CoV-2 versus SARS-CoV-1. P1B4, also known as NC-Cowl, was used as a negative control, see Sok et al Nature 2017. These data show that R4C1 retains full binding activity to SARS-CoV-1, while alternative exemplary antibody R2D9 loses >10x binding. However, it was observed that R2D9 remained in the low nanomolar range with some binding activity to SARS-CoV-1.

Finally, FIG. 5F shows ELISA binding activity (top) for three different exemplary antibody knob candidates against WT (Wuhan) SARS CoV-2 spike protein. For this experiment, each exemplary knob was expressed with a DO1 epitope tag that was detected with anti-DO 1 antibodies reflected on the X-axis. Fig. 5G further depicts the improved western blot. Here, the exemplary antibody knob shown was heated to 70 ℃ in the presence of SDS, then resolved by SDS-PAGE, then transferred to nitrocellulose membrane and detected with biotinylated RBD. RBDs were biotinylated using EZ-Link NHS-LC-LC-biotin (Thermo Fisher). NHS-LC-LC-biotin was reconstituted in DMF and combined with purified RBD at a molar ratio of 1:5 (RBD: biotin) and then incubated for 30 minutes at room temperature. The reaction was then applied to a Pierce polyacrylamide rotary desalting column 7K MWCO equilibrated in PBS. Aprotonin was chosen as a similar size control. It was observed that the R2G3 knob remained bound to the RBD despite the heat and SDS treatment.

Example 5: virus neutralization

In some aspects, the binding of the antibody to the viral antigen protein is insufficient to reduce cell entry or infectious reproduction. While some antibodies, known as neutralizing antibodies, have the ability to inhibit viruses in vitro and/or in vivo and are therefore considered more relevant for therapeutic applications. Thus, the candidate antibodies described above were tested for their ability to neutralize SARS CoV-3 pseudovirus (a model virus) infected cells to determine the neutralizing ability of the candidate antibodies. Pseudoviruses may be considered to be treated with high titers of BSL-2 compared to naturally occurring SARS virus isolates, and are therefore suitable for screening, such as in a pseudoviral luciferase assay (PVLA).

The pseudovirus of SARS CoV-2S protein, which expresses the parent Wuhan-Hu-1 spike protein sequence in its vial envelope, was engineered so that the gene for luciferase expression was carried as its cargo. Upon successful penetration into the cell, the luciferase is expressed such that the rate of pseudoviral neutralization inhibition is inversely proportional to the luciferase activity expressed in Relative Light Units (RLU). These pseudotyped viruses were used for neutralization assays in CRFK-hACE2 cells. ACE2 overexpression is considered a mechanism by which cell lines exhibiting "high infectivity" can be generated as a receptor for SARS-CoV-2 entry. In contrast, cell lines with minimal or low ACE2 expression may be considered to exhibit "low infectivity".

Specifically, mock medium or serial dilutions (5-fold) of antibody Fab were mixed with the same amount of pseudotyped virus carrying the Wild Type (WT) of SARS-CoV-2 and incubated for 1 hour at 37 ℃. The mixture was then transduced into CRFK-hACE or CRFK-hDDP4 cells in the presence of polybrene (Santa Cruz Biotech, santa Cruz, calif.) (10. Mu.g/mL). After incubation of the transduced cells at 37 ℃ for 48 hours, lysis buffer was added and RLU was measured.

Summary of pseudovirus neutralization of the identified antibodies is shown in table E3. Niu Chaochang CDR3 antibodies are highly potent and neutralise variant strains, with half maximal inhibition for some antibodies at concentrations below 1-5 ng/mL. In general, ultralong CDR3 antibodies exhibit more efficient neutralization than antibodies with standard CDR3 lengths.

Example 6: bacterial expression and purification of CDR 3-knob-only antibodies

A system was developed to express and purify CDR 3-knob, which is a small peptide sequence of 25-50 amino acids with 1-6 disulfide bonds derived from an ultralong CDR3 bovine antibody as described above. The expression system comprises fusion with the bacterial chaperone protein TrxA. CDR 3-knob and trxA-CDR-knob fusion were tested for spike and RBD binding.

Trxa-CDR 3-knob fusion and CDR 3-knob expression and purification

CDR 3-knob from the candidate ultralong CDR3 antibodies described in examples 2-5 was cloned as a KpnI-XhoI (or NcoI-XhoI, as appropriate) (fig. 6A) fragment into pET32b vector (EMD-Millipore) and transformed into Origami 2DE3 bacteria and expressed as described below. These CDR 3-knobs have the sequences shown in SEQ ID NOS: 60-67 and are encoded by the DNA sequences shown in SEQ ID NOS: 52-60, respectively.

trxA-CDR 3-knob fusion clones were grown overnight at 37℃in 20mL 2 xTY/50. Mu.g/mL carbenicillin/10. Mu.g/mL tetracycline/2% glucose, transferred to 200mL of the same medium and grown at 37℃to an OD600nm of about 1.0, after which the bacteria were centrifuged and resuspended in 200mL2 xTY/50. Mu.g/mL carbenicillin/0.5 mM IPTG and grown overnight at 22 ℃. Bacteria were reprecipitated, resuspended in 10mL Bugbuster HT (EMD-Millipore), spun at room temperature for 30 minutes, and the residue was precipitated at 14,000g for 20 minutes at 4 ℃. The supernatant was added to an equilibrated Talon resin column (1 mL resin TaKaRa), spun at 4 ℃ for 2 hours, washed with five column volumes of wash buffer (5 mM imidazole), then with 1 column volume of wash buffer (10 mM imidazole), eluted with 2.5mL of 300mM imidazole elution buffer, then exchanged with PD10 spin column (GE Healthcare) buffer into PBS/saline. trxA-CDR 3-knob was adjusted to 50mM Tris pH7.4, 150mM NaCl and 2.5mM CaCl2 (1 Xenterokinase (EK) reaction buffer), and 400u of recombinant his-tagged enterokinase (Genscript) was added and incubated overnight at room temperature. Digested trxA and enterokinase were removed by incubation on a freshly equilibrated Talon resin column (1.2 mL resin) for 2 hours at 4 ℃ and the purified CDR-knob was collected in the flow-through. The sample buffer was again exchanged into saline/PBS. In some cases, endotoxin removal may be performed by anion exchange chromatography prior to use or testing (such as testing in virus neutralization assays). CDR 3-knobs cloned and expressed as independent domains in E.coli are shown in SEQ ID NOS 60-67.

The stepwise purification is depicted in fig. 6B. As shown in FIG. 6C, stepwise purification as monitored by SDS-PAGE was effective to purify trxA-CDR 3-knob fusion proteins as well as soluble CDR 3-knobs from E.coli lysates. FIG. 6D depicts an exemplary SDS-PAGE gel of several purified ultralong CDR H3 knob peptides. The sample is treated with a reducing agent DTT, which in some aspects is sufficient to break disulfide bonds. A similarly sized aprotinin was included as a size control.

IMAC purified trxA-CDR 3-knob fusion spike or RBD binding

To assess CDR 3-knob binding as trxA fusion, half-area Costar ELISA plates were coated overnight at 4 ℃ with serial dilutions of IMAC purified trxA-knob fusion from 25 μl trxA fusion in 50 μl/well PBS prior to cleavage of enterokinase from trxA. RBD binding clones R2G3, R2F12, SKM and SKD (nucleotide sequences are shown in SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:57, respectively; and amino acid sequences are shown in SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64 and SEQ ID NO:65, respectively) and spike binding clone R4C1 (nucleic acid sequence is shown in SEQ ID NO:55 and amino acid sequence is shown in SEQ ID NO: 63) were tested.

Plates were then blocked with 100. Mu.L/well of 2% milk powder/PBS for 1 hour at room temperature, and then washed twice with 100. Mu.L/well of PBS. About 50. Mu.L/well of 1. Mu.g/mL Wuhan-Hu-1 spike protein was incubated in 2% milk powder/PBS for 1 hour, and the wells were washed three times with 100. Mu.L/well of PBS. To detect bound spike protein, 1 μg/mL of full length IgG chimeric ultralong CDR3, i.e. anti RBD R2G3 IgG1 (for R4C 1) or anti R4C1 IgG1 antibody (for R2F12, R2G3, SKD and SKM fusions) in 2% milk powder/PBS was added, incubated for 1 hour, and then wells were washed three times with 100 μl/well of PBS. Bound IgG was then detected as follows: wells were washed three times with 100 μl/well of PBS by incubation with 1:5000 dilution of anti-human IgG-Fc-HRP conjugate in 2% milk powder/PBS for 1 hour. The plates were then washed and developed with 50. Mu.L/well TMB (3, 3', 5' -tetramethylbenzidine) substrate buffer (Thermofiser) for 5-10 minutes at room temperature. With 100. Mu.L/well of 0.5. 0.5N H ₂ SO ₄ The reaction was terminated and read at 450 nm.

As shown in FIG. 7A (where R2F12 is denoted "F12" and R2G3 is denoted "G3"), the trxA-knob fusion proteins tested showed spike protein binding. Control conditions in which fusion proteins R3C1 and R2G3 were incubated in the absence of spike protein (denoted as "R3C1 no spike" and "G3 no spike") did not show binding. Binding to the uncoated plate TrxA-R2G3 fusion protein is also shown in figure 7B, respectively.

B. Purified R2G3 CDR 3-knob binds to Wuhan-Hu-1 RBD

The purified R2G3 CDR 3-knob (after cleavage of enterokinase from trxA as described above) was evaluated for binding to RBD by ELISA. The nucleic acid sequence encoding the R2G3 CDR 3-knob is shown in SEQ ID NO:52 and the amino acid sequence is shown in SEQ ID NO: 60.

Wells in half area Costar ELISA plates (Corning) were coated with 50 μl/well of purified CDR 3-knob (2-fold dilution from 84-0.082031nM in PBS) in duplicate. Plates were incubated for 1 hour at 37℃and then blocked with 180. Mu.L/well of 2% milk powder/TBS/0.1% Tween20 for 2 hours at room temperature. The biotinylated RBD was then diluted to 0.5 ng/. Mu.L in 2% milk/TBS/0.1% Tween20 and added to the coated/uncoated wells at 50. Mu.L/well. After 1 hour at room temperature, wells were washed four times with 180 μl/well of TBS/0.1% Tween20, and bound biotinylated RBD was detected for 30 minutes at room temperature with 50 μl/well of streptavidin-HRP (Invitrogen) diluted 1:5000 in 2% milk/TBS/0.1% Tween 20. Wells were then washed five times with 180 μl/well TBS/0.1% Tween20, then 50 μl/well TMB (3, 3', 5' -tetramethylbenzidine) substrate buffer (Thermo Scientific) was added. After 1-2 minutes at room temperature, 50. Mu.L/well 1N H ₂ SO ₄ The reaction was terminated and OD450 nm values were recorded. The average OD450 of the uncoated wells was subtracted from the OD450 of each coated well. Background-subtracted OD450 values were plotted against Log (CDR 3-knob nM) in GraphPad Prism (GraphPad Software LLC).

As shown in fig. 8A, the soluble R2G3 knob is shown to bind RBD. As shown in fig. 8B, soluble R2G3 knob binding was increased relative to the binding of the reference anti-spike protein antibody, CR 3022.

C. Binding of truncated R2G3CDR 3-knob to Wuhan-Hu-1RBD

The truncated R2G3CDR 3-knob was cloned and generated as described above using the pET32b vector encoding the R2G3 truncated mutant followed by the enterokinase cleavage site. The amino acid sequence of the truncated R2G3 mutant is shown in fig. 8C. As shown in FIG. 8D, truncations 1-3 showed tight bands after enterokinase cleavage and gel electrophoresis (0.75. Mu.g truncated knob protein/lane, 250mM DTT).

RBD binding of truncated R2G3CDR 3-knob was also tested as described above. As shown in FIG. 8E, truncations 1-3 retain RBD binding capacity, while truncations 4 and 5 lack RBD binding.

D. Defining minimum CDR 3-knob C-terminal requirements

To determine the C-terminal requirement (i.e., C-terminal minimal sequence) of the prototype CDR 3-knob, a series of R2G3 truncations were cloned into pET32b and expressed and purified as described in example 6 above. These truncations are shown in table E4 below.

The quality of the expression material was assessed by SDS-PAGE and RBD ELISA as described in example 6D above. It was observed that only truncations 4 (G3 trunk 4) and 5 (G3 trunk 5) did not exhibit RBD binding capacity. Truncations 3A (G3 trunk 3A) and 3B (G3 trunk 3B) showed reduced binding in ELISA and increased band diffusion in SDS-PAGE, as depicted in fig. 8A. As shown in FIG. 8B, ELISA with truncations 1-3 did not observe loss of binding activity relative to the parental R2G3 CDR 3-knob. These data support that a minimum of at least 9 amino acids are required after the last non-canonical Cys residue for R2G3 binding.

E. CDR 3-knob purification by size exclusion chromatography

Size Exclusion Chromatography (SEC) was used to resolve whether the purified soluble CDR 3-knob was present in multiple forms after bacterial expression. Soluble R4C1 and R2G3 knobs were prepared as described above and subjected to SEC.

As shown in fig. 9A, SEC revealed at least two different elution fractions (fractions A4 and A7) for the purified R4C1 knob, indicating that the purified R4C1 knob was present in multiple forms after bacterial expression. Fractions A4 and A7 were subjected to gel electrophoresis. As shown in fig. 9B, fraction A4 contained larger soluble aggregates and smaller active soluble CDR 3-knobs. Fraction A7 contained only a small active soluble CDR 3-knob.

As shown in fig. 9C, SEC revealed only one different eluted fraction (fraction A6) for the purified R2G3 knob (fraction A6). This result was confirmed by gel electrophoresis of fraction A6 (fig. 9D).

Example 7: SARS-CoV2 Virus neutralization comparison of chimeric Fab ultralong CDR3 and CDR 3-knob

To assess virus neutralization of only CDR 3-knob antibodies, assays to assess neutralization of pseudoviruses or live WT SARS-CoV2 viruses were performed. In this example, purified R2G3CDR 3-knobs ("G3-knobs") or Fab of chimeric R2G3 ultralong CDR3 antibodies ("G3-Fab") or full length IgG chimeric R2G3 ultralong CDR3 antibodies ("G3") were tested as shown.

A pseudoviral luciferase assay (PLSA) substantially as described in example 5 was performed. Viral neutralization was assessed against pseudotyped viruses carrying SARS-CoV-2 (Wuhan-Hu-1) wild-type (WT) spike protein or S variant (E484K/N507Y; B.1.1.7 or "UK" variants; and K417N/E484K/N501Y; B.1.351 or "SA" variants). Mock medium or serial dilutions (5-fold) of antibody G3-knob, G3-Fab or G3 were mixed with the same amount of pseudotyped virus carrying SARS-CoV-2 wild-type (WT), S variants (484K, B.1.1.7 and B.1.351) and incubated for 1 hour at 37 ℃. The mixture was then transduced into CRFK-hACE or CRFK-hDDP4 cells in the presence of polybrene (Santa Cruz Biotech, santa Cruz, calif.) (10. Mu.g/mL). ACE2 overexpression is considered a mechanism by which cell lines exhibiting "high infectivity" can be generated as a receptor for SARS-CoV-2 entry. In contrast, cell lines with minimal or low ACE2 expression may be considered to exhibit "low infectivity".

After incubation of the transduced cells at 37 ℃ for 48 hours, lysis buffer was added and RLU was measured. Serial dilutions of each antibody G3-Fab or G3-knob were generated and the 50% effective concentration (EC 50) values were determined by GraphPad Prism software using variable slope (GraphPad, la Jolla, CA). The results are summarized in table E5.

To evaluate the neutralizing activity against live SARS-CoV-2, the neutralizing activity of selected G3, G3-Fab or G3-knob antibodies against SARS-CoV-2 or B.1.17 or B.1.351 variant replication in Vero E6 cells was studied. Briefly, 50-100 plaque forming units of SARS-CoV-2hCoV/USA-WA1/2020 (wild type), SARS-CoV-2hCoV-19/England/204820464/2020 (B.1.1.7 variant) or SARS-CoV-2hCoV-19/South African/KRIP-EC-K005321/2020 (B.1.351 variant) were mixed with either mock medium or serial dilutions (5-fold) of G3-Fab or G3-knob. After 1 hour incubation at 37 ℃, the mixtures were seeded into confluent Vero E6 cells in 24-well plates. After 2 hours incubation, medium containing agar (1% final concentration) and neutral red was added to the cells. After 48-72 hours, plaques in each well were counted. EC50 values were determined as described above and are shown in table E5 below.

In summary, the results shown in table E5 demonstrate that the standard IgG Fab format or exemplary bovine ultralong CDR 3R 2G3, which is a CDR 3-only knob format, exhibits potent neutralizing activity against WT SARS-CoV-2 as well as the tested variants. Niu Chaochang CDR3 antibodies are highly potent and neutralise variant strains, with half maximal inhibition at concentrations below 1-5ng/mL, depending on the antibody format. Notably, only CDR 3-knob antibodies maintained sub-nanomolar potency despite being short sequences of only 51 amino acids in length. Due to the small size of the CDR 3-knob antibody, this example supports the utility of CDR 3-knob antibodies as novel therapeutic antibody candidates for inhalation formulations for respiratory targets (including other viruses, bacteria, other infectious diseases, asthma, or lung cancer).

SARS CoV-2 variants

In a further evaluation of virus neutralization of ultralong CDR3 antibodies, assays were performed to evaluate neutralization of live WT SARS-CoV2 virus or several variants SARS CoV-2 virus. In this example, full length IgG chimeric ultralong CDR3 antibodies F12, G3, SKD, and SKM were tested as shown.

A pseudoviral luciferase assay (PLSA) substantially as described in example 5 was performed. Viral neutralization was assessed against pseudoviruses carrying SARS-CoV-2 (Wuhan-Hu-1) wild-type (WT) spike protein, S variants (E484K/N507Y; B.1.1.7 or "UK" variants; and K417N/E484K/N501Y; B.1.351 or "SA" variants) or 484K. The mock medium or serial dilutions (5-fold) of the antibody were mixed with the same amount of pseudotyped virus carrying SARS-CoV-2 Wild Type (WT), S variants (484K, b.1.1.7 and b.1.351) and incubated for 1 hour at 37 ℃. The mixture was then transduced into CRFK-hACE or CRFK-hDDP4 cells in the presence of polybrene (Santa Cruz Biotech, santa Cruz, calif.) (10. Mu.g/mL). ACE2 overexpression is considered a mechanism by which cell lines exhibiting "high infectivity" can be generated as a receptor for SARS-CoV-2 entry. In contrast, cell lines with minimal or low ACE2 expression may be considered to exhibit "low infectivity".

After incubation of the transduced cells at 37 ℃ for 48 hours, lysis buffer was added and RLU was measured. As shown in fig. 10A-10D, each exemplary ultralong CDR3 antibody exhibits activity against more than one variant SARS CoV-2S protein. Serial dilutions of each antibody were generated and the 50% effective concentration (EC 50) values were determined by GraphPad Prism software using variable slope (GraphPad, la Jolla, CA). The results are summarized in table E6.

In summary, the results shown in Table E5 demonstrate that the exemplary bovine ultralong CDR3 antibodies, F12, G3, SKD and SKM, exhibit potent neutralizing activity against WT SARS-CoV-2 as well as the variants tested. Niu Chaochang CDR3 antibodies are highly potent and neutralise variant strains, with half maximal inhibition at concentrations below 1-5ng/mL, depending on the antibody format. Notably, only CDR 3-knob antibodies maintained sub-nanomolar potency despite being short sequences of only 51 amino acids in length. Due to the small size of the CDR 3-knob antibody, this example supports the utility of CDR 3-knob antibodies as novel therapeutic antibody candidates for inhalation formulations for respiratory targets (including other viruses, bacteria, other infectious diseases, asthma, or lung cancer).

Example 8: SARS CoV-1 cross-reactivity

To assess the possible cross-reactivity and broad neutralization of exemplary ultralong CDR3 antibodies, assays to assess pseudovirus neutralization were performed. In this example, exemplary R4C1 and R2D9 ultralong CDR3 antibodies were tested as shown.

A pseudoviral luciferase assay (PLSA) substantially as described in example 5 was performed. Viral neutralization was assessed against pseudotyped viruses carrying SARS-CoV-2 (Wuhan-Hu-1) wild-type (WT) spike protein, S protein of SARS-CoV-1 virus or VSV-G control. The mock medium or serial dilutions (5-fold) of antibody G3-knob, G3-Fab or G3 were mixed with the same amount of pseudotyped virus carrying SARS-CoV-2 wild-type (WT), SARS-CoV-1 wild-type or VSV-G and incubated for 1 hour at 37 ℃. The mixture was then transduced into cells in the presence of polybrene (Santa Cruz Biotech, santa Cruz, calif.) (10. Mu.g/mL).

After incubation of the transduced cells at 37 ℃ for 48 hours, lysis buffer was added and the percent neutralization was measured. Serial dilutions of each antibody were generated against inhibition curves of the simulated treatment, and the percent maximum neutralization (MPN), i.e. the percent at which the neutralization curves of those viruses that were neutralized tended to plateau, was determined by GraphPad Prism software using variable slope (GraphPad, la Jolla, CA).

Fig. 11A shows IC50 values of different IgG antibodies against pseudoviruses from various coronavirus strains. Note that R4C1 and R2D9 retain activity against the omnikow variant of SARS-CoV-2. All antibodies showed sub-nanomolar potency, several of which are in the low picomolar range.

Example 9: neutralization of live variant viruses

To assess additional cross-reactivity and potential broad neutralization of exemplary antibodies, assays to assess pseudovirus neutralization in addition to live virus were performed. In this example, the examples SKM, SKD, R C1 (IgG, fab and knob), G3 (IgG, fab and knob) and R2D9 (IgG and knob) as described above were tested as shown.

A pseudoviral luciferase assay (PLSA) substantially as described in example 5 was performed. Virus neutralization was assessed against pseudotyped viruses carrying SARS-CoV-2 (Wuhan-Hu-1) wild-type (WT) spike protein, S protein of SARS-CoV-2. Beta. Lineage virus or SARS-CoV-2. Delta. Lineage virus. The mock medium or serial dilutions (5-fold) of the antibody, knob or fab, were mixed with the same amount of pseudotyped virus carrying SARS-CoV-2 spike protein and incubated for 1 hour at 37 ℃. The mixture was then transduced into cells in the presence of polybrene (Santa Cruz Biotech, santa Cruz, calif.) (10. Mu.g/ml). After incubation of the transduced cells at 37 ℃ for 48 hours, lysis buffer was added and RLU was measured.

Neutralization was also determined using live virus under BSL-3 conditions. Serial dilutions (5-fold) of the antibody, knob or fab, were mixed with the same amount of wild-type SARS-CoV-2 virus (Wuhan-Hu-1) or alpha (United Kingdom) or beta (South Africa) lineage variants and incubated for 1 hour at 37 ℃ similar to that described above. Cells were washed and then after incubation at 37 ℃ for 48 hours, plaque Forming Units (PFU) were measured.

In experiments using pseudoviruses or live viruses, the percent neutralization was measured. Serial dilutions of each antibody were generated against inhibition curves of the simulated treatment, and the percent maximum neutralization (MPN), i.e. the percent at which the neutralization curves of those viruses that were neutralized tended to plateau, was determined by GraphPad Prism software using variable slope (GraphPad, la Jolla, CA). For example, the results of exemplary antibody candidates R2G3 (IgG, fab, and knob) are shown in fig. 11B. The results are summarized in Table E7 in ng/mL, with the standard deviation of three independent replicates on the right.

Example 10: bispecific and multispecific antibodies with ultralong CDR3

Knobs derived from bovine ultralong CDRH3 antibodies are expressed as fusion proteins or as part of dimeric or multimeric molecules, resulting in bivalent, bispecific, multivalent or multispecific proteins (fig. 12). Two or more knobs are expressed as fusion proteins, e.g., with a flexible linker (e.g., gly-Gly-Gly-Ser, etc.) between the C-terminus of one knob and the N-terminus of the other knob. In addition, bispecific molecules were prepared in which one knob was in its wild-type conformation, such as a bovine or humanized bovine VH region, and expressed as IgG along with the light chain, while the second knob was fused to the C-terminus of the heavy chain constant region. In this case, the two VH regions are identical and have the specificity of knob 1, but the C-terminus has the new specificity as determined by knob 2.

In another approach, a "knob-in-hole" technique is employed in which two heavy chains are co-expressed, one heavy chain having a VH region with one knob (knob 1) within its CDRH3 and the second heavy chain having a VH region with a second knob (knob 2) within its CDRH 3. The two heavy chains also differ in having constant region mutations such that only heterologous heavy chains effectively pair with each other to form dimers. In this case, homodimers did not form to a significant extent. Such "knob-to-hole" mutations include T22Y (on one strand) and Y86T (on the other strand) in the CH3 domain of Fc.

DNA vectors encoding such molecules are generated by standard molecular biology techniques and expressed and purified as described in the previous examples above. In addition, small molecule linkers or polyethylene glycol (PEG) linkers, including heterobifunctional or heteromultifunctional linkers (e.g., pierce), are used to chemically covalently link together individual knobs. In this case, the individual knob is expressed and purified and then added together in the presence of the linker and appropriate reaction conditions to covalently couple the linker to the knob protein. Amine, carboxyl, maleimide, NHS ester and hydrazide chemistries are typically used in these crosslinking methods. In addition, a knob is used in the case of nanoparticles to provide specificity or activity to the nanoparticles. In this regard, the nanoparticles may be protein-based nanoparticles, including particles formed from viral proteins, albumin nanoparticles, and the like. Nanoparticles may also be derived from non-protein molecules, including lipids (e.g., lipid particles), carbohydrates, and the like.

Example 11: bioinformatic identification of bovine ultralong CDR H3 knob domain ends

Algorithms were developed to identify bovine ultralong CDR H3 knob domain boundaries by amino acid sequence. By sequence, the bovine ultralong CDR H3 region ranges from "the third residue following a conserved cysteine in frame 3 to the residue immediately before a conserved tryptophan in frame 4" (Wang et al Cell 2013,153 (6): 1379-1393). Structurally, knob domains are defined as small disulfide-rich domains located on the distal end of antiparallel β -charged stem domains (fig. 13A and 13B).

The crystal structure of the exemplary bovine ultralong antibodies was analyzed in combination with the sequence (table E8) (fig. 14) to explicitly define knob boundaries by both sequence and structure. In analysis, the first residue of the knob domain is defined as the first conserved D before the conserved "PDG" motif _H Cysteine, or in rare exceptional cases other residues at this position, such as a01. In order to locate the last knob domain residues, then also the stem domain is defined. Symmetry of the upstream and downstream stem β -band lengths was observed by crystal structure analysis. The conserved framework 3 cysteine at the 3 amino acid position preceding the first CDR H3 residue (Wang et al 2013) is located near the base of the uplink and is located directly opposite the conserved framework 4 tryptophan, which is one residue downstream of the last CDR H3 residue (Wang et al 2013). In the analysis, the first upstream stem residue was defined as the conserved framework 3 cysteine and the last downstream stem residue was defined as the conserved framework 4 tryptophan. The C-terminal knob border position was located by subtracting the number of ascending stem residues from the frame 4 tryptophan position (table E8).

In summary, our algorithm (below) defines the knob region N-terminal limit as the first D in the "CPDG" motif _H Cysteine, and defines the C-terminal boundary as the position located by subtracting the number of upstream stem residues from the tryptophan position in frame 4(FIG. 15). This algorithm serves as a general rule applicable to bovine ultralong CDR H3 antibody sequences.

In summary, our algorithm (below) defines the knob region N-terminal limit as the first D in the "CPDG" motif _H Cysteine, and the C-terminal boundary was defined as the position located by subtracting the number of upstream stem residues from the tryptophan position of frame 4 (fig. 15). This algorithm serves as a general rule applicable to bovine ultralong CDR H3 antibody sequences.

The algorithm is described as follows: l = number of amino acids covering stem and knob domains, starting at canonical framework 3 cysteine and ending at canonical framework 4 tryptophan. X = the number of amino acids starting from the framework 3 canonical cysteine defining the upstream stem and ending at an amino acid preceding the conserved first D-region cysteine in the "CPDG" motif.

Position-x= knob border position (C-terminal) of conserved framework 4 tryptophan; number of residues in knob (K) =l-2X; k position= (x+1) to (x+k)

Bovine ultralong antibodies with the disclosed crystal structure were analyzed, with X amino acids in the uplink and downlink. The total number of amino acids (L) comprising the stem and knob domains and the total number of amino acids (K) comprising only the knob domains for each antibody are also recorded.

Example 12: defining a minimum CDR 3-knob C-terminus and a minimum CDR 3-knob N-terminus

The algorithm described in example 11 was experimentally verified by expressing and testing the C-terminal truncation (section a below) and the N-terminal truncation (section B below) from stem and knob regions of antibodies with unknown structure. In some cases, 1, 2, 3, 4, or 5 amino acids may be added to the knob end to improve expression or stability.

A. Defining a minimum CDR 3-knob C-terminus

To determine the C-terminal requirement of the prototype CDR 3-knob, a series of R2G3 truncations were cloned into pET32b and expressed as described in example 6 above. The quality of the expression material was also assessed by SDS-PAGE and RBD ELISA as described in example 6. Exemplary tested R2G3 truncations are shown in table E9 below, each truncations being performed with a reduced terminal linker.

As shown in fig. 16A, only truncations 4 and 5 resulted in no RBD binding being observed. Truncations 3A and 3B showed reduced binding in ELISA and increased band diffusion in SDS-PAGE (fig. 16B). Truncations 1-3 did not lose binding activity relative to the parent R2G3 CDR 3-knob. Taken together, these results support a minimum of 9 amino acids after the last non-canonical Cys residue used for R2G3 binding.

B. Defining the minimum CDR 3-knob N-terminus

A series of R2G3 truncations were cloned into pET32b to define the N-terminal requirement of the prototype CDR 3-knob, similarly as described in example 11, and expressed as described in example 6 above. The quality of the expression material was assessed by SDS-PAGE and RBD ELISA as described in example 6. Exemplary R2G3 truncations tested are shown in table E10 below.

Each exemplary N-terminal truncate tested was observed by ELISA to show a similar binding profile to biotinylated RBD, and band diffusion was observed in SDS-PAGE (FIGS. 17A and 17B, respectively). It was noted that truncate 5 produced two bands via SDS-PAGE, however this was not associated with any decrease in binding activity. These results suggest that none of the amino acids deleted in these exemplary truncated R2G3 sequences are part of the knob domain.

Example 13: selective amplification of ultralong CDR 3-knob domains

The ultralong CDR 3-knob domain was selectively amplified from a bovine VH template library. A bovine VH template library was prepared substantially as described in example 2.

Primary stem-knob CDR3 was amplified from the 1 st chain cDNA using IgHV1-7 family specific primers specific to either side of the stem domain of the CDR3 region. The primary stem-knob CDR3 was amplified using a pool of primers containing all of the primers shown in SEQ ID NOS: 8-11 and one of the primers shown in SEQ ID NOS: 122-130. Gel electrophoresis was then performed using a 2% agarose gel to analyze the amplified sequences for the presence of the ultralong CDR 3-knob domain.

The alignment of the primers shown in SEQ ID NOS.122-130 (primers p1-p 9) with the sequences of an exemplary standard short CDR3 antibody (antibodies 028-030) and an ultralong CDR3 antibody (antibodies 01-026) is shown in FIG. 18A. The sequence identifiers (SEQ ID NOs) for the sequences shown in FIG. 18A are shown in Table E11.

The results of gel electrophoresis showed that amplification with the primer pools containing the primers shown in SEQ ID NOs 123, 127 and 128 resulted in enrichment of the ultralong CDR 3-knob domain (FIG. 18B), especially annealing between 65℃and 68 ℃. Specifically, while two bands are apparent for the PCR product obtained using some primers, indicating amplification of the standard short and ultralong CDR 3-knob domains, only one band corresponding to the sequence of the ultralong CDR 3-knob domain was obtained using the primers shown in SEQ ID NOS: 123, 127 and 128 (expected PCR product size is about 300-350 bp).

A stem-knob CDR3 library was constructed from DNA amplified using the primers shown in SEQ ID NOS: 8-11, 123, 127 and 128. The library was constructed essentially as described in example 2 and two rounds of selection were performed for spike proteins as described in example 3. More than 90% of the screened clones were spike-binding clones, and all binding clones were ultralong CDR3 antibodies.

These results indicate that ultralong CDR 3-knob domains can be selectively amplified from VH template libraries using specific primers specific for the stem domain of the CDR3 region.

The present invention is not intended to be limited in scope to the specifically disclosed embodiments, which are provided to illustrate various aspects of the invention. Various modifications to these compositions and methods will be apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the disclosure.

Sequence(s)

Sequence listing

<110> applied biomedical science research institute (Applied Biomedical Science Institute)

<120> screening and expression methods of disulfide-bonded binding polypeptides

<130> 165772000440

<140> not yet allocated

<141> along with the submission

<150> US 63/187,931

<151> 2021-05-12

<150> US 63/288,992

<151> 2021-12-13

<160> 154

<170> FastSEQ 4.0 version for Windows

<210> 1

<211> 330

<212> DNA

<213> artificial sequence

<220>

<223> BLV1H12 light chain DNA

<400> 1

caggccgtcc tgaaccagcc aagcagcgtc tccgggtctc tggggcagcg ggtctcaatc 60

acctgtagcg ggtcttcctc caatgtcggc aacggctacg tgtcttggta tcagctgatc 120

cctggcagtg ccccacgaac cctgatctac ggcgacacat ccagagcttc tggggtcccc 180

gatcggttct cagggagcag atccggaaac acagctactc tgaccatcag ctccctccag 240

gctgaggacg aagcagatta tttctgcgcg tctgccgagg actctagttc aaatgccgtg 300

tttggaagcg gcaccacact gacagtccta 330

<210> 2

<211> 110

<212> PRT

<213> artificial sequence

<220>

<223> BLV1H12 variable light chain

<400> 2

Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Gly

20 25 30

Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu

35 40 45

Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser

85 90 95

Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu

100 105 110

<210> 3

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> igGCDNA2REV

<400> 3

ccgctcttca gggcacccga gttcc 25

<210> 4

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> igMCDNA2REV

<400> 4

ctgactgtgc tgttgttgaa cttcc 25

<210> 5

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> igACDNA2REV

<400> 5

gacacgctgt cgccattctg gttcc 25

<210> 6

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> igGCDNA1.7REV

<400> 6

cgggcacggt caccatgctg ctgagagagt ag 32

<210> 7

<211> 56

<212> DNA

<213> artificial sequence

<220>

<223> BOVVHFR4REV

<400> 7

ttacctgcgg ccgctgagga gacggtgacc aggagtccaa ctggagctcc atcaag 56

<210> 8

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> BOVSTALKFOR1

<400> 8

cagccggcca tggccacata ctacagtact actgtacacc 40

<210> 9

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> BOVSTALKFOR2

<400> 9

cagccggcca tggccacata ctacagtact actgtatacc 40

<210> 10

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> BOVSTALKFOR3

<400> 10

cagccggcca tggccacata ctacagtact actgtgctcc 40

<210> 11

<211> 40

<212> DNA

<213> artificial sequence

<220>

<223> BOVSTALKFOR4

<400> 11

cagccggcca tggccacata ctacagtggt actgtgcacc 40

<210> 12

<211> 37

<212> DNA

<213> artificial sequence

<220>

<223> BOVVHNCOFOR2

<400> 12

aaaaagccat ggtgcaggtg cagctgcggg agtcggg 37

<210> 13

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> BOVVHFR4XHOREV

<400> 13

ttacctctcg agtgaggaga cggtgaccag gagtcc 36

<210> 14

<211> 507

<212> DNA

<213> artificial sequence

<220>

<223> R2G3

<400> 14

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctcgctc 60

acctgcgcgg cctctggatt ctcattgagc gacaaggctg taggctgggt ccgccgggct 120

ccagggaagg cgctggagtg gctcggtagt atagacactg gtggaagcac aggctataac 180

ccaggcctga aatcccggct cagcatcacc aaggacaact ccaagagcca agtctccctg 240

tcaattagca gcgtaacgtc tgaggactcg gccacatact actgtgcaac tgtacaccag 300

aaaacagctg aaggagacaa aacgtgtcct gatggttacg agcatacttg tggttgcatt 360

gggggttgtg gttgcaaaag gtctgcctgt ataggtgcac tttgttgcca agcgtcgttg 420

ggtggttggc ttagtgacgg tgaaacctac acttacgagt tccacgtcga tacctggggc 480

caaggactcg tggtcaccgt ctcctca 507

<210> 15

<211> 507

<212> DNA

<213> artificial sequence

<220>

<223> SR3A3

<400> 15

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacaa tctctggatt ctcattgagt agctatgctg tactctgggt ccgccaggct 120

ccagggaagc cgctggagtg gctcggtagt atagacactg cggaaaacac aggctataac 180

ccaggcctga aatcccggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

tcagtgagca gcgtgacaac cgaggactcg gccacatact actgtgctac tgtacaccag 300

aaaacgcgaa aagaaaaaaa ttgtcctgat ggctatatct atagttctaa tatcactagc 360

ggttttgatt gtggtgtctg gatttgtcgt cgcgtcggta gtgccttctg tagtcgtact 420

ggtgattata ctagtcctac tgaacttgac atttacgagt tctacgtcga agggtggggc 480

cagggagtcc cggtcaccgt ctcctca 507

<210> 16

<211> 498

<212> DNA

<213> artificial sequence

<220>

<223> R2F12

<400> 16

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacgg tctctggatt ctcattgagc gacaaggctg taggctgggt ccgccgggct 120

ccagggaagg cgctggagtg gctcggtagt atagacactg gtggaatgac aggctataac 180

ccaggcctga aatcccggct cagcatcacc aaggacaact ccaaaagcca agtctctcta 240

tcagtgaata gcgtgacaac tgaggactcg gccacgtact actgtgccac tgtagaccag 300

aaaacgaaaa atgcttgccc tgatgatttc gattatcgtt gttcgtgtat cggtggttgt 360

ggctgcgccc gtaaaggatg cgttggtcct ctttgttgtc gttctgattt gggtggctat 420

cttactgata gtcctgctta catttacgaa tggtatattg atctttgggg ccaaggactc 480

ctggtcaccg tctcctca 498

<210> 17

<211> 405

<212> DNA

<213> artificial sequence

<220>

<223> R5A3

<400> 17

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cgtcacagac cctctcgctc 60

acctgcacgg cctctggatt ctcattgagc gacaaggctg taggctgggt ccgccaggct 120

ccagggaagg cgctggagtg gctcggtagt atagacactg gtggaagcac aggctataac 180

ccaggcctga aatcccggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

tcagtgagca gcgtgacaac tgaggactcg gccacatact actgtactac tgtgcactgt 300

agtgatggtg gttatgttga ggcgggtttt ggttgttggc cttgggatta tggttatcct 360

tacgtcgatg cctggggcca aggactcctg gtcaccgtct cctca 405

<210> 18

<211> 396

<212> DNA

<213> artificial sequence

<220>

<223> R4G11

<400> 18

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacgg tctctggatt ctcattgagc agctatggta taacctgggt ccgccaggct 120

ccagggaagg cgctggagtg cctcggtagt ataagcagtg gtggaaccac agactacaac 180

ccagccctga aatcccggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

tcagtgagca gcgtgacacc tgaggacacg gccacatact actgttcgaa gtggaattta 300

gaatatactt ggggtggtgt tggttgcgct agttttgctg atgaggacac ccacgttgat 360

gcctggggcc aaggactcct ggtcaccgtc tcctca 396

<210> 19

<211> 417

<212> DNA

<213> artificial sequence

<220>

<223> R4G3

<400> 19

caggtgcagc tgcgggagtc gggccccagc ctgatgaagc cctcacagac cctctccctc 60

acctgcacgg tctctgggtt ctcattgagc gactatgctg taggctgggt ccgccaggcc 120

ccagggaagg cgctggagtg gctcggtggt atagacactg gtggaagcac aggctataac 180

ccaggcctgg aatcccggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

tcagtgagca gcgtgacaac tgaggactcg gccacatact actgtactac tgtggtcctt 300

tgttatttta attatgttgt tcgtcgttat aattgtggtg gtcttggtta tgggcatggc 360

tttaatagtt tctacgtcga tgcctggggc caaggactcc tggtcaccgt ctcctca 417

<210> 20

<211> 417

<212> DNA

<213> artificial sequence

<220>

<223> R4E5

<400> 20

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacga cctctggatt ctcactgaga aactatgctg taggctgggt ccgccaggct 120

ccggggaagg cgctggagtg gctcggtggt atagacactg gtggaagcac aggctataac 180

ccaggcctgg aatcccggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

tcagtgagca gcgtgacaac tgaggactcg gccacatact actgtactac tgtggtcctt 300

tgttatttta attatgttgt tcgtcgttat aattgtggtg gtcttggtta tgggcatggc 360

tttaatagtt tctacgtcga tgcctggggc caaggactcc tggtcaccgt ctcctca 417

<210> 21

<211> 507

<212> DNA

<213> artificial sequence

<220>

<223> R4C1

<400> 21

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cgtcacagac cctctcgctc 60

acctgcacgg cctctggatt ctcattgagc gataaggctg taggctgggt ccgccaggct 120

ccagggaagc cgctggagtg gctcggtagt atagacactg cggaaaacac aggctataac 180

ccaggcctga aatctcggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

tcagtgagca gcgtgacaac tgaggactcg gccacatact actgtgctac tgtacaccag 300

aaaacgcgaa aagaaaaaaa ttgtcctgat ggctatatct atagttctaa taccgccagc 360

ggttatgatt gtggtgtctg gatttgtcgt cgcgtcggta gtgccttctg tagtcgtact 420

ggtgattata ctagtcctag tgaatttgac atttacgagt tctacgtcga agggtggggc 480

cagggactcc tggtcaccgt ctcctca 507

<210> 22

<211> 417

<212> DNA

<213> artificial sequence

<220>

<223> R4A10

<400> 22

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacga cctctggatt ctcattgagc gactatgctg taggctgggt ccgccaggct 120

ccagggaagg cgctggagtg gctcggtggt atagacactg gtggaagcac aggctataac 180

ccaggcctga aatcccggct cagcatcacc aaggacaact ccaagagtca agtctctctg 240

tcagtgagca gcgtgacaac tgaggattcg gccacatact actgtactgc cgtggtcctc 300

tgttattaca atcgggttgt gcgtcgtaat aattgtggtg ggcttggtta tgattatggt 360

tttgatcatt tctacgtcga tgcctggggc caaggactcc tggtcaccgt ctcctca 417

<210> 23

<211> 384

<212> DNA

<213> artificial sequence

<220>

<223> R2G1

<400> 23

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacgg tctctggatt ctcattgagc aactatgctg taggctgggt ccgccaggct 120

ccagggaagg cgctggagtg cctcggtgat gtagacagta gtggaggcac agcctataac 180

ccagccctga aatcccggtt catcatcgcc aaggacaact ccaagaacca agtctctctg 240

tcagtccgca gcgtgacacc tgaggacacg gccacatact actgtgcgaa gtttgctaag 300

ggtactacga gtgctggtgc ttgtgattat tcagaaagct acgtcgatgc ctggggccag 360

ggactcctgg tcaccgtctc ctca 384

<210> 24

<211> 399

<212> DNA

<213> artificial sequence

<220>

<223> R2D6

<400> 24

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacga cctctggatt ctcactgagc agctatgctg taggctgggt ccgccaggct 120

ccggggaagg cgctggagtg ggttggtgat atagattatg tcggaaacac agactataac 180

ccagccctga aatcccggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

gtagtgagca gcgtgacagc tgaggacgcg gccacatact actgtgcgaa atattccggt 300

gcttatgctt atgctgcttg caattattat ggttggcgtt gtgcttggga aagctacatc 360

gatgcctggg gccaaggact cctggtcacc gtctcctca 399

<210> 25

<211> 399

<212> DNA

<213> artificial sequence

<220>

<223> R2B1

<400> 25

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacgg tctctggatt ttcattaagc gataataatg taggctgggt ccgccaggct 120

ccaggaaagg cgctggagtg gctcggtgta atgcataatg atgggaacaa aggctataac 180

ccagccctga aatcccggct cagcatcacc aaggacagct ccaagagcca agtctctcta 240

tcactaagca gcgtgacaag tgaggacacg gccacatact actgtacaag agacaatgca 300

cgttgtgata gttggacgta tgacagctgt gatacttggt atcgcaattc gtggcacgtt 360

gatgcctggg gccaaggact cctggtcacc gtctcctca 399

<210> 26

<211> 504

<212> DNA

<213> artificial sequence

<220>

<223> SKM-BLV1H12

<400> 26

caggtgcagc tgcgcgagtc gggccccagc ctggtgaagc cgtcacagac cctctcgctc 60

acctgcacgg cctctggatt ctcattgagc gacaaggctg taggctgggt ccgccaggct 120

ccagggaagg cgctggagtg gctcggtagt atagacactg gtggaaacac aggctataac 180

ccaggcctga aatcccggct cagcatcacc aaggacaact ccaagagtca agtctctctg 240

tcagtgagca gcgtgacaac tgaggactcg gccacatact actgtactac tgtgcaccaa 300

gagaccttac gtagttgtcc tgatggttat attgataatt ctggatgcac ggctgattgg 360

ggttgtgcag ctcttgattg ttggcggcgt cgttttggtt accacagcac tgatccttct 420

cattatactg gtgcgacgta tatttacacg tacagcttgc acatcgatgc ctggggccaa 480

ggactcctgg tcaccgtctc ctca 504

<210> 27

<211> 507

<212> DNA

<213> artificial sequence

<220>

<223> SKD-BLV1H12

<400> 27

caggtgcagc tgcgcgagtc gggccccagc ctggtgaagc cgtcacagac cctctcgctc 60

acctgcacgg cctctggatt ctcattgagc gacaaggctg taggctgggt ccgccaggct 120

ccagggaagg cgctggagtg gctcggtagt atagacactg gtggaaacac aggctataac 180

ccaggcctga aatcccggct cagcatcacc aaggacaact ccaagagtca agtctctctg 240

tcagtgagca gcgtgacaac tgaggactcg gccacatact actgtactac tgtgcaccag 300

cgtacaagcg aaaaaagaag ttgtcctggc ggtagtagta gacgttatcc tagtggcgcc 360

agttgtgacg ttagtggggg cgcttgtgcg tgttatgttt ctaattgtag aggcgttttg 420

tgtcctactc ttaacgaaat cgttgcttat acctacgaat ggcacgtcga cgcctggggc 480

caaggactcc tggtcaccgt ctcctca 507

<210> 28

<211> 378

<212> DNA

<213> artificial sequence

<220>

<223> RBD F4

<400> 28

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacgg tctctggatt ctcattgagc agcaatggtg tggtctgggt ccgccaggct 120

ccagggaagg cgctggagtg gctcggtgat atatgcagta ctggaggcac aagctttaac 180

ccagccctga aatcccggct cagcatcgcc aaggacaact ccaagagcca agtctctctg 240

tcagtgagaa gcgtgacacc tgaggacacg gccacatatt actgtgcaag aagtcgtggt 300

tatgattgtt atgctaatgt ggatgctttg gactacgtcg atgcctgggg ccaaggactc 360

ctggtcaccg tctcctca 378

<210> 29

<211> 378

<212> DNA

<213> artificial sequence

<220>

<223> RBD C6

<400> 29

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cctcacagac cctctccctc 60

acctgcacgg tctctggatt ctcattgagc agcaatggtg tagtctgggt ccgccaggct 120

ccagggagac cactggagtg gctcggtgat atatgcagta atggaggcac aagctttaac 180

ccagccctga aatcccggct cagcatcgcc aaggacaact ccgagagcca agtctctctg 240

accgtgagaa gcgtgacacc tgaggacaca gccacatatt actgtgcaag aagtcgtggt 300

tatgattgtt atgcttatgt ttatgctttg gacaccgtcg atgcctgggg ccaaggactc 360

ctggtcaccg tctcctca 378

<210> 30

<211> 378

<212> DNA

<213> artificial sequence

<220>

<223> RBD A2

<400> 30

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc ccctacagat cctctccctc 60

acctgcacgg tctctggatt ctcattgagc agcaatggtg tggtctgggt ccgccaggct 120

ccagggaagg cgctggagtg gctcggtgat atatgcagta ctggaggcac aagctttaac 180

ccagccctga aatcccggct cagcatcgcc aaggacaact ccaagagcca agtctctctg 240

tcagtgagaa gcgtgacacc tgaggacacg gccacatatt actgtgcaag aagtcgtggt 300

tatgattgtt atgctaatgt ggatgctttg gactacgtcg atgcctgggg ccaaggactc 360

ctggtcaccg tctcctca 378

<210> 31

<211> 507

<212> DNA

<213> artificial sequence

<220>

<223> SA-R2C3

<400> 31

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cgtcacagac cctctcgctc 60

acctgcacgg cctctggatt ctcattgagc gataagcctg taggctgggt ccgccaggct 120

ccagggaagc cactggagtg gctcggtagt atagacactg cggaaaacac aggctataac 180

ccaggcctga aatctcggct cagcatcacc aaggacaact ccaagagcca agtctctctg 240

tcactgagca gcgtgacgac tgaggactcg gccacatact actgtgctac tgtacaccag 300

aaaacgcgga aggaaaaaag ttgtcctgat ggctatctct atagttctaa taccggccgc 360

ggttatgatt gtggtgtctg gacttgtcgt cgcgtcggtg gtgaattctg tagtgctact 420

ggtgattgga ctagtcctag tgaagaagac ttttacgaat tctacgtcga tacgtggggc 480

cagggagccc cggtcaccgt ctcctca 507

<210> 32

<211> 501

<212> DNA

<213> artificial sequence

<220>

<223> SA-R2D9

<400> 32

caggtgcagc tgcgggagtc gggccccagc ctggtgaagc cgtcacagac cctctcgctc 60

acctgcacgg cctctggatt ctcattaagc gacaaggcta ttggctgggt ccgccaggct 120

ccagggaagg cgctggagtg gctcggtagt atagacaccc gtggaaacac aggctataac 180

ccaggcctga aatcccgact cagcatcacc aaggacagct ccaagagcca agtctctctg 240

tcagtgaaca gcgtgacaac tgaagactcg gccacgtacc tctgtgctat tgtgcagcag 300

atcacacaca aaacttgtcc taatggttac aattggtttg atcgttgttg ttcttgggat 360

ggtacctgtg gtgatggttg ttgcagtaat cgtgcttggc ctagtggtaa tggtagagcc 420

gacagtagta ttggtgaaac ttatggttac gaatttcacg tggctgcctg gggccaagga 480

ctcctggtca ccgtctcctc a 501

<210> 33

<211> 169

<212> PRT

<213> artificial sequence

<220>

<223> R2G3

<400> 33

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ala Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Val Gly Trp Val Arg Arg Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Ile Ser Ser Val Thr Ser Glu Asp Ser Ala Thr Tyr Tyr Cys Ala

85 90 95

Thr Val His Gln Lys Thr Ala Glu Gly Asp Lys Thr Cys Pro Asp Gly

100 105 110

Tyr Glu His Thr Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser

115 120 125

Ala Cys Ile Gly Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu

130 135 140

Ser Asp Gly Glu Thr Tyr Thr Tyr Glu Phe His Val Asp Thr Trp Gly

145 150 155 160

Gln Gly Leu Val Val Thr Val Ser Ser

165

<210> 34

<211> 169

<212> PRT

<213> artificial sequence

<220>

<223> SR3A3

<400> 34

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Ile Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Ala Val Leu Trp Val Arg Gln Ala Pro Gly Lys Pro Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Ala Glu Asn Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala

85 90 95

Thr Val His Gln Lys Thr Arg Lys Glu Lys Asn Cys Pro Asp Gly Tyr

100 105 110

Ile Tyr Ser Ser Asn Ile Thr Ser Gly Phe Asp Cys Gly Val Trp Ile

115 120 125

Cys Arg Arg Val Gly Ser Ala Phe Cys Ser Arg Thr Gly Asp Tyr Thr

130 135 140

Ser Pro Thr Glu Leu Asp Ile Tyr Glu Phe Tyr Val Glu Gly Trp Gly

145 150 155 160

Gln Gly Val Pro Val Thr Val Ser Ser

165

<210> 35

<211> 166

<212> PRT

<213> artificial sequence

<220>

<223> R2F12

<400> 35

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Val Gly Trp Val Arg Arg Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Gly Gly Met Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Asn Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala

85 90 95

Thr Val Asp Gln Lys Thr Lys Asn Ala Cys Pro Asp Asp Phe Asp Tyr

100 105 110

Arg Cys Ser Cys Ile Gly Gly Cys Gly Cys Ala Arg Lys Gly Cys Val

115 120 125

Gly Pro Leu Cys Cys Arg Ser Asp Leu Gly Gly Tyr Leu Thr Asp Ser

130 135 140

Pro Ala Tyr Ile Tyr Glu Trp Tyr Ile Asp Leu Trp Gly Gln Gly Leu

145 150 155 160

Leu Val Thr Val Ser Ser

165

<210> 36

<211> 135

<212> PRT

<213> artificial sequence

<220>

<223> R5A3

<400> 36

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr

85 90 95

Thr Val His Cys Ser Asp Gly Gly Tyr Val Glu Ala Gly Phe Gly Cys

100 105 110

Trp Pro Trp Asp Tyr Gly Tyr Pro Tyr Val Asp Ala Trp Gly Gln Gly

115 120 125

Leu Leu Val Thr Val Ser Ser

130 135

<210> 37

<211> 132

<212> PRT

<213> artificial sequence

<220>

<223> R4G11

<400> 37

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Cys Leu

35 40 45

Gly Ser Ile Ser Ser Gly Gly Thr Thr Asp Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ser

85 90 95

Lys Trp Asn Leu Glu Tyr Thr Trp Gly Gly Val Gly Cys Ala Ser Phe

100 105 110

Ala Asp Glu Asp Thr His Val Asp Ala Trp Gly Gln Gly Leu Leu Val

115 120 125

Thr Val Ser Ser

130

<210> 38

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> R4G3

<400> 38

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Met Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Gly Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu Glu

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr

85 90 95

Thr Val Val Leu Cys Tyr Phe Asn Tyr Val Val Arg Arg Tyr Asn Cys

100 105 110

Gly Gly Leu Gly Tyr Gly His Gly Phe Asn Ser Phe Tyr Val Asp Ala

115 120 125

Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

130 135

<210> 39

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> R4E5

<400> 39

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Thr Ser Gly Phe Ser Leu Arg Asn Tyr

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Gly Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu Glu

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr

85 90 95

Thr Val Val Leu Cys Tyr Phe Asn Tyr Val Val Arg Arg Tyr Asn Cys

100 105 110

Gly Gly Leu Gly Tyr Gly His Gly Phe Asn Ser Phe Tyr Val Asp Ala

115 120 125

Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

130 135

<210> 40

<211> 169

<212> PRT

<213> artificial sequence

<220>

<223> R4C1

<400> 40

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Pro Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Ala Glu Asn Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala

85 90 95

Thr Val His Gln Lys Thr Arg Lys Glu Lys Asn Cys Pro Asp Gly Tyr

100 105 110

Ile Tyr Ser Ser Asn Thr Ala Ser Gly Tyr Asp Cys Gly Val Trp Ile

115 120 125

Cys Arg Arg Val Gly Ser Ala Phe Cys Ser Arg Thr Gly Asp Tyr Thr

130 135 140

Ser Pro Ser Glu Phe Asp Ile Tyr Glu Phe Tyr Val Glu Gly Trp Gly

145 150 155 160

Gln Gly Leu Leu Val Thr Val Ser Ser

165

<210> 41

<211> 139

<212> PRT

<213> artificial sequence

<220>

<223> R4A10

<400> 41

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Thr Ser Gly Phe Ser Leu Ser Asp Tyr

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Gly Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr

85 90 95

Ala Val Val Leu Cys Tyr Tyr Asn Arg Val Val Arg Arg Asn Asn Cys

100 105 110

Gly Gly Leu Gly Tyr Asp Tyr Gly Phe Asp His Phe Tyr Val Asp Ala

115 120 125

Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

130 135

<210> 42

<211> 128

<212> PRT

<213> artificial sequence

<220>

<223> R2G1

<400> 42

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Cys Leu

35 40 45

Gly Asp Val Asp Ser Ser Gly Gly Thr Ala Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Phe Ile Ile Ala Lys Asp Asn Ser Lys Asn Gln Val Ser Leu

65 70 75 80

Ser Val Arg Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys Phe Ala Lys Gly Thr Thr Ser Ala Gly Ala Cys Asp Tyr Ser Glu

100 105 110

Ser Tyr Val Asp Ala Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

115 120 125

<210> 43

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> R2D6

<400> 43

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Thr Ser Gly Phe Ser Leu Ser Ser Tyr

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Val

35 40 45

Gly Asp Ile Asp Tyr Val Gly Asn Thr Asp Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Val Val Ser Ser Val Thr Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Ala

85 90 95

Lys Tyr Ser Gly Ala Tyr Ala Tyr Ala Ala Cys Asn Tyr Tyr Gly Trp

100 105 110

Arg Cys Ala Trp Glu Ser Tyr Ile Asp Ala Trp Gly Gln Gly Leu Leu

115 120 125

Val Thr Val Ser Ser

130

<210> 44

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> R2B1

<400> 44

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Asn

20 25 30

Asn Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Val Met His Asn Asp Gly Asn Lys Gly Tyr Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Ser Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Leu Ser Ser Val Thr Ser Glu Asp Thr Ala Thr Tyr Tyr Cys Thr

85 90 95

Arg Asp Asn Ala Arg Cys Asp Ser Trp Thr Tyr Asp Ser Cys Asp Thr

100 105 110

Trp Tyr Arg Asn Ser Trp His Val Asp Ala Trp Gly Gln Gly Leu Leu

115 120 125

Val Thr Val Ser Ser

130

<210> 45

<211> 168

<212> PRT

<213> artificial sequence

<220>

<223> SKM-BLV1H12

<400> 45

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr

85 90 95

Thr Val His Gln Glu Thr Leu Arg Ser Cys Pro Asp Gly Tyr Ile Asp

100 105 110

Asn Ser Gly Cys Thr Ala Asp Trp Gly Cys Ala Ala Leu Asp Cys Trp

115 120 125

Arg Arg Arg Phe Gly Tyr His Ser Thr Asp Pro Ser His Tyr Thr Gly

130 135 140

Ala Thr Tyr Ile Tyr Thr Tyr Ser Leu His Ile Asp Ala Trp Gly Gln

145 150 155 160

Gly Leu Leu Val Thr Val Ser Ser

165

<210> 46

<211> 169

<212> PRT

<213> artificial sequence

<220>

<223> SKD-BLV1H12

<400> 46

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Gly Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr

85 90 95

Thr Val His Gln Arg Thr Ser Glu Lys Arg Ser Cys Pro Gly Gly Ser

100 105 110

Ser Arg Arg Tyr Pro Ser Gly Ala Ser Cys Asp Val Ser Gly Gly Ala

115 120 125

Cys Ala Cys Tyr Val Ser Asn Cys Arg Gly Val Leu Cys Pro Thr Leu

130 135 140

Asn Glu Ile Val Ala Tyr Thr Tyr Glu Trp His Val Asp Ala Trp Gly

145 150 155 160

Gln Gly Leu Leu Val Thr Val Ser Ser

165

<210> 47

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> RBD F4

<400> 47

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Asn

20 25 30

Gly Val Val Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Asp Ile Cys Ser Thr Gly Gly Thr Ser Phe Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ala Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Arg Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Ser Arg Gly Tyr Asp Cys Tyr Ala Asn Val Asp Ala Leu Asp Tyr

100 105 110

Val Asp Ala Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

115 120 125

<210> 48

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> RBD C6

<400> 48

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Asn

20 25 30

Gly Val Val Trp Val Arg Gln Ala Pro Gly Arg Pro Leu Glu Trp Leu

35 40 45

Gly Asp Ile Cys Ser Asn Gly Gly Thr Ser Phe Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ala Lys Asp Asn Ser Glu Ser Gln Val Ser Leu

65 70 75 80

Thr Val Arg Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Ser Arg Gly Tyr Asp Cys Tyr Ala Tyr Val Tyr Ala Leu Asp Thr

100 105 110

Val Asp Ala Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

115 120 125

<210> 49

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> RBD A2

<400> 49

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Leu Gln

1 5 10 15

Ile Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Asn

20 25 30

Gly Val Val Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Asp Ile Cys Ser Thr Gly Gly Thr Ser Phe Asn Pro Ala Leu Lys

50 55 60

Ser Arg Leu Ser Ile Ala Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Arg Ser Val Thr Pro Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Ser Arg Gly Tyr Asp Cys Tyr Ala Asn Val Asp Ala Leu Asp Tyr

100 105 110

Val Asp Ala Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

115 120 125

<210> 50

<211> 169

<212> PRT

<213> artificial sequence

<220>

<223> SA-R2C3

<400> 50

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Pro Val Gly Trp Val Arg Gln Ala Pro Gly Lys Pro Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Ala Glu Asn Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Leu Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Ala

85 90 95

Thr Val His Gln Lys Thr Arg Lys Glu Lys Ser Cys Pro Asp Gly Tyr

100 105 110

Leu Tyr Ser Ser Asn Thr Gly Arg Gly Tyr Asp Cys Gly Val Trp Thr

115 120 125

Cys Arg Arg Val Gly Gly Glu Phe Cys Ser Ala Thr Gly Asp Trp Thr

130 135 140

Ser Pro Ser Glu Glu Asp Phe Tyr Glu Phe Tyr Val Asp Thr Trp Gly

145 150 155 160

Gln Gly Ala Pro Val Thr Val Ser Ser

165

<210> 51

<211> 167

<212> PRT

<213> artificial sequence

<220>

<223> SA-R2D9

<400> 51

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Ser Ile Asp Thr Arg Gly Asn Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Ser Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Asn Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Leu Cys Ala

85 90 95

Ile Val Gln Gln Ile Thr His Lys Thr Cys Pro Asn Gly Tyr Asn Trp

100 105 110

Phe Asp Arg Cys Cys Ser Trp Asp Gly Thr Cys Gly Asp Gly Cys Cys

115 120 125

Ser Asn Arg Ala Trp Pro Ser Gly Asn Gly Arg Ala Asp Ser Ser Ile

130 135 140

Gly Glu Thr Tyr Gly Tyr Glu Phe His Val Ala Ala Trp Gly Gln Gly

145 150 155 160

Leu Leu Val Thr Val Ser Ser

165

<210> 52

<211> 144

<212> DNA

<213> artificial sequence

<220>

<223> R2G3

<400> 52

gaaggagaca aaacgtgtcc tgatggttac gagcatactt gtggttgcat tgggggttgt 60

ggttgcaaaa ggtctgcctg tataggtgca ctttgttgcc aagcgtcgtt gggtggttgg 120

cttagtgacg gtgaaaccta cact 144

<210> 53

<211> 153

<212> DNA

<213> artificial sequence

<220>

<223> SR3A3

<400> 53

aaagaaaaaa attgtcctga tggctatatc tatagttcta atatcactag cggttttgat 60

tgtggtgtct ggatttgtcg tcgcgtcggt agtgccttct gtagtcgtac tggtgattat 120

actagtccta ctgaacttga catttacgag ttc 153

<210> 54

<211> 150

<212> DNA

<213> artificial sequence

<220>

<223> R2F12

<400> 54

aaaacgaaaa atgcttgccc tgatgatttc gattatcgtt gttcgtgtat cggtggttgt 60

ggctgcgccc gtaaaggatg cgttggtcct ctttgttgtc gttctgattt gggtggctat 120

cttactgata gtcctgctta catttacgaa 150

<210> 55

<211> 147

<212> DNA

<213> artificial sequence

<220>

<223> R4C1

<400> 55

aaagaaaaaa attgtcctga tggctatatc tatagttcta ataccgccag cggttatgat 60

tgtggtgtct ggatttgtcg tcgcgtcggt agtgccttct gtagtcgtac tggtgattat 120

actagtccta gtgaatttga catttac 147

<210> 56

<211> 147

<212> DNA

<213> artificial sequence

<220>

<223> SKM-BLV1H12

<400> 56

ctgcgtagtt gtcctgatgg ttatattgat aattctggat gcacggctga ttggggttgt 60

gcagctcttg attgttggcg gcgtcgtttt ggttaccaca gcactgatcc ttctcattat 120

actggtgcga cgtatattta cacgtac 147

<210> 57

<211> 150

<212> DNA

<213> artificial sequence

<220>

<223> SKD-BLV1H12

<400> 57

agcgaaaaaa gaagttgtcc tggcggtagt agtagacgtt atcctagtgg cgccagttgt 60

gacgttagtg ggggcgcttg tgcgtgttat gtttctaatt gtagaggcgt tttgtgtcct 120

actcttaacg aaatcgttgc ttatacctac 150

<210> 58

<211> 156

<212> DNA

<213> artificial sequence

<220>

<223> SA-R2C3

<400> 58

cggaaggaaa aaagttgtcc tgatggctat ctctatagtt ctaataccgg ccgcggttat 60

gattgtggtg tctggacttg tcgtcgcgtc ggtggtgaat tctgtagtgc tactggtgat 120

tggactagtc ctagtgaaga agacttttac gaattc 156

<210> 59

<211> 156

<212> DNA

<213> artificial sequence

<220>

<223> SA-R2D9

<400> 59

atcacacaca aaacttgtcc taatggttac aattggtttg atcgttgttg ttcttgggat 60

ggtacctgtg gtgatggttg ttgcagtaat cgtgcttggc ctagtggtaa tggtagagcc 120

gacagtagta ttggtgaaac ttatggttac gaattt 156

<210> 60

<211> 51

<212> PRT

<213> artificial sequence

<220>

<223> R2G3

<400> 60

Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr Cys Gly Cys

1 5 10 15

Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly Ala Leu Cys

20 25 30

Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp Gly Glu Thr Tyr Thr

35 40 45

Tyr Glu Phe

50

<210> 61

<211> 51

<212> PRT

<213> artificial sequence

<220>

<223> SR3A3

<400> 61

Lys Glu Lys Asn Cys Pro Asp Gly Tyr Ile Tyr Ser Ser Asn Ile Thr

1 5 10 15

Ser Gly Phe Asp Cys Gly Val Trp Ile Cys Arg Arg Val Gly Ser Ala

20 25 30

Phe Cys Ser Arg Thr Gly Asp Tyr Thr Ser Pro Thr Glu Leu Asp Ile

35 40 45

Tyr Glu Phe

50

<210> 62

<211> 50

<212> PRT

<213> artificial sequence

<220>

<223> R2F12

<400> 62

Lys Thr Lys Asn Ala Cys Pro Asp Asp Phe Asp Tyr Arg Cys Ser Cys

1 5 10 15

Ile Gly Gly Cys Gly Cys Ala Arg Lys Gly Cys Val Gly Pro Leu Cys

20 25 30

Cys Arg Ser Asp Leu Gly Gly Tyr Leu Thr Asp Ser Pro Ala Tyr Ile

35 40 45

Tyr Glu

50

<210> 63

<211> 50

<212> PRT

<213> artificial sequence

<220>

<223> R4C1

<400> 63

Arg Lys Glu Lys Asn Cys Pro Asp Gly Tyr Ile Tyr Ser Ser Asn Thr

1 5 10 15

Ala Ser Gly Tyr Asp Cys Gly Val Trp Ile Cys Arg Arg Val Gly Ser

20 25 30

Ala Phe Cys Ser Arg Thr Gly Asp Tyr Thr Ser Pro Ser Glu Phe Asp

35 40 45

Ile Tyr

50

<210> 64

<211> 49

<212> PRT

<213> artificial sequence

<220>

<223> SKM-BLV1H12

<400> 64

Leu Arg Ser Cys Pro Asp Gly Tyr Ile Asp Asn Ser Gly Cys Thr Ala

1 5 10 15

Asp Trp Gly Cys Ala Ala Leu Asp Cys Trp Arg Arg Arg Phe Gly Tyr

20 25 30

His Ser Thr Asp Pro Ser His Tyr Thr Gly Ala Thr Tyr Ile Tyr Thr

35 40 45

Tyr

<210> 65

<211> 50

<212> PRT

<213> artificial sequence

<220>

<223> SKD-BLV1H12

<400> 65

Ser Glu Lys Arg Ser Cys Pro Gly Gly Ser Ser Arg Arg Tyr Pro Ser

1 5 10 15

Gly Ala Ser Cys Asp Val Ser Gly Gly Ala Cys Ala Cys Tyr Val Ser

20 25 30

Asn Cys Arg Gly Val Leu Cys Pro Thr Leu Asn Glu Ile Val Ala Tyr

35 40 45

Thr Tyr

50

<210> 66

<211> 52

<212> PRT

<213> artificial sequence

<220>

<223> SA-R2C3

<400> 66

Arg Lys Glu Lys Ser Cys Pro Asp Gly Tyr Leu Tyr Ser Ser Asn Thr

1 5 10 15

Gly Arg Gly Tyr Asp Cys Gly Val Trp Thr Cys Arg Arg Val Gly Gly

20 25 30

Glu Phe Cys Ser Ala Thr Gly Asp Trp Thr Ser Pro Ser Glu Glu Asp

35 40 45

Phe Tyr Glu Phe

50

<210> 67

<211> 52

<212> PRT

<213> artificial sequence

<220>

<223> SA-R2D9

<400> 67

Ile Thr His Lys Thr Cys Pro Asn Gly Tyr Asn Trp Phe Asp Arg Cys

1 5 10 15

Cys Ser Trp Asp Gly Thr Cys Gly Asp Gly Cys Cys Ser Asn Arg Ala

20 25 30

Trp Pro Ser Gly Asn Gly Arg Ala Asp Ser Ser Ile Gly Glu Thr Tyr

35 40 45

Gly Tyr Glu Phe

50

<210> 68

<211> 75

<212> PRT

<213> artificial sequence

<220>

<223> SKD

<400> 68

Cys Thr Thr Val His Gln Arg Thr Ser Glu Lys Arg Ser Cys Pro Gly

1 5 10 15

Gly Ser Ser Arg Arg Tyr Pro Ser Gly Ala Ser Cys Asp Val Ser Gly

20 25 30

Gly Ala Cys Ala Cys Tyr Val Ser Asn Cys Arg Gly Val Leu Cys Pro

35 40 45

Thr Leu Asn Glu Ile Val Ala Tyr Thr Tyr Glu Trp His Val Asp Ala

50 55 60

Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

65 70 75

<210> 69

<211> 74

<212> PRT

<213> artificial sequence

<220>

<223> SKM

<400> 69

Cys Thr Thr Val His Gln Glu Thr Leu Arg Ser Cys Pro Asp Gly Tyr

1 5 10 15

Ile Asp Asn Ser Gly Cys Thr Ala Asp Trp Gly Cys Ala Ala Leu Asp

20 25 30

Cys Trp Arg Arg Arg Phe Gly Tyr His Ser Thr Asp Pro Ser His Tyr

35 40 45

Thr Gly Ala Thr Tyr Ile Tyr Thr Tyr Ser Leu His Ile Asp Ala Trp

50 55 60

Gly Gln Gly Leu Leu Val Thr Val Ser Ser

65 70

<210> 70

<211> 75

<212> PRT

<213> artificial sequence

<220>

<223> R4C1

<400> 70

Cys Ala Thr Val His Gln Lys Thr Arg Lys Glu Lys Asn Cys Pro Asp

1 5 10 15

Gly Tyr Ile Tyr Ser Ser Asn Thr Ala Ser Gly Tyr Asp Cys Gly Val

20 25 30

Trp Ile Cys Arg Arg Val Gly Ser Ala Phe Cys Ser Arg Thr Gly Asp

35 40 45

Tyr Thr Ser Pro Ser Glu Phe Asp Ile Tyr Glu Phe Tyr Val Glu Gly

50 55 60

Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

65 70 75

<210> 71

<211> 75

<212> PRT

<213> artificial sequence

<220>

<223> R5C1

<400> 71

Cys Ala Thr Val His Gln Lys Thr Arg Lys Glu Lys Ser Cys Pro Asp

1 5 10 15

Gly Tyr Leu Tyr Ser Ser Asn Thr Gly Arg Gly Tyr Asp Cys Gly Val

20 25 30

Trp Thr Cys Arg Arg Val Gly Gly Glu Phe Cys Ser Ala Thr Gly Asp

35 40 45

Trp Thr Ser Pro Ser Glu Glu Asp Phe Tyr Glu Phe Tyr Val Asp Thr

50 55 60

Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

65 70 75

<210> 72

<211> 75

<212> PRT

<213> artificial sequence

<220>

<223> SR3A3

<400> 72

Cys Ala Thr Val His Gln Lys Thr Arg Lys Glu Lys Asn Cys Pro Asp

1 5 10 15

Gly Tyr Ile Tyr Ser Ser Asn Ile Thr Ser Gly Phe Asp Cys Gly Val

20 25 30

Trp Ile Cys Arg Arg Val Gly Ser Ala Phe Cys Ser Arg Thr Gly Asp

35 40 45

Tyr Thr Ser Pro Thr Glu Leu Asp Ile Tyr Glu Phe Tyr Val Glu Gly

50 55 60

Trp Gly Gln Gly Val Pro Val Thr Val Ser Ser

65 70 75

<210> 73

<211> 72

<212> PRT

<213> artificial sequence

<220>

<223> RR2F12

<400> 73

Cys Ala Thr Val Asp Gln Lys Thr Lys Asn Ala Cys Pro Asp Asp Phe

1 5 10 15

Asp Tyr Arg Cys Ser Cys Ile Gly Gly Cys Gly Cys Ala Arg Lys Gly

20 25 30

Cys Val Gly Pro Leu Cys Cys Arg Ser Asp Leu Gly Gly Tyr Leu Thr

35 40 45

Asp Ser Pro Ala Tyr Ile Tyr Glu Trp Tyr Ile Asp Leu Trp Gly Gln

50 55 60

Gly Leu Leu Val Thr Val Ser Ser

65 70

<210> 74

<211> 75

<212> PRT

<213> artificial sequence

<220>

<223> RR2G3

<400> 74

Cys Ala Thr Val His Gln Lys Thr Ala Glu Gly Asp Lys Thr Cys Pro

1 5 10 15

Asp Gly Tyr Glu His Thr Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys

20 25 30

Arg Ser Ala Cys Ile Gly Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly

35 40 45

Trp Leu Ser Asp Gly Glu Thr Tyr Thr Tyr Glu Phe His Val Asp Thr

50 55 60

Trp Gly Gln Gly Leu Val Val Thr Val Ser Ser

65 70 75

<210> 75

<211> 69

<212> PRT

<213> artificial sequence

<220>

<223> germ line

<400> 75

Cys Thr Thr Val His Gln Ser Cys Pro Asp Gly Tyr Ser Tyr Gly Tyr

1 5 10 15

Gly Cys Gly Tyr Gly Tyr Gly Cys Ser Gly Tyr Asp Cys Tyr Gly Tyr

20 25 30

Gly Gly Tyr Gly Gly Tyr Gly Gly Tyr Gly Tyr Ser Ser Tyr Ser Tyr

35 40 45

Ser Tyr Thr Tyr Glu Tyr Tyr Val Asp Ala Trp Gly Gln Gly Leu Leu

50 55 60

Val Thr Val Ser Ser

65

<210> 76

<211> 1272

<212> PRT

<213> artificial sequence

<220>

<223> WT Wuhan-Hu-1S protein (NCBI reference sequence:

YP_009724390.1）

<400> 76

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

1185 1190 1195 1200

Gln Glu Leu Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp

1205 1210 1215

Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser

1235 1240 1245

Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu

1250 1255 1260

Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 77

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> Wuhan-Hu-1S protein (NCBI reference sequence:

YP_009724390.1）RBD AA 319-541

<400> 77

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

<210> 78

<211> 1229

<212> PRT

<213> artificial sequence

<220>

<223> removal of furin site

(AA 685-686) and Wuhan-Hu-1S protein with K986P and V987P stabilizing mutations (NCBI reference sequence:

YP 009724390.1), only extracellular domain

<400> 78

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Val Ala Ser Gln

675 680 685

Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala

690 695 700

Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val

705 710 715 720

Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys

725 730 735

Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu

740 745 750

Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile

755 760 765

Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys

770 775 780

Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe

785 790 795 800

Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile

805 810 815

Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile

820 825 830

Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile

835 840 845

Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr

850 855 860

Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile

865 870 875 880

Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe

885 890 895

Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn

900 905 910

Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala

915 920 925

Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly

930 935 940

Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu

945 950 955 960

Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn

965 970 975

Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp

980 985 990

Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln

995 1000 1005

Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala

1010 1015 1020

Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe

1025 1030 1035 1040

Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His

1045 1050 1055

Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn

1060 1065 1070

Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro

1075 1080 1085

Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln

1090 1095 1100

Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val

1105 1110 1115 1120

Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr

1125 1130 1135

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1140 1145 1150

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser

1155 1160 1165

Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1170 1175 1180

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu

1185 1190 1195 1200

Leu Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly

1205 1210 1215

Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile

1220 1225

<210> 79

<211> 1288

<212> PRT

<213> artificial sequence

<220>

<223> 7LYN South African (B.1.351) SARS-CoV-2 spike protein variant (S-GSAS-B.1.351)

<400> 79

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Ala

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Val Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

1185 1190 1195 1200

Gln Glu Leu Gly Lys Tyr Glu Gln Gly Ser Gly Tyr Ile Pro Glu Ala

1205 1210 1215

Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu

1220 1225 1230

Leu Ser Thr Phe Leu Gly Arg Ser Leu Glu Val Leu Phe Gln Gly Pro

1235 1240 1245

Gly His His His His His His His His Ser Ala Trp Ser His Pro Gln

1250 1255 1260

Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ala

1265 1270 1275 1280

Trp Ser His Pro Gln Phe Glu Lys

1285

<210> 80

<211> 1288

<212> PRT

<213> artificial sequence

<220>

<223> furin site was removed and had K986P and V987P stabilizing mutations

7LYN South African (B.1.351) SARS-CoV-2 spike protein variant (S-GSAS-B.1.351),

Extracellular domain only

<400> 80

Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Phe Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Ala

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Ile Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Gly Ser Ala Ser Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Val Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

1185 1190 1195 1200

Gln Glu Leu Gly Lys Tyr Glu Gln Gly Ser Gly Tyr Ile Pro Glu Ala

1205 1210 1215

Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu

1220 1225 1230

Leu Ser Thr Phe Leu Gly Arg Ser Leu Glu Val Leu Phe Gln Gly Pro

1235 1240 1245

Gly His His His His His His His His Ser Ala Trp Ser His Pro Gln

1250 1255 1260

Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Ala

1265 1270 1275 1280

Trp Ser His Pro Gln Phe Glu Lys

1285

<210> 81

<211> 100

<212> PRT

<213> artificial sequence

<220>

<223> IgHV 1-7V Gene

<400> 81

Gln Val Gln Leu Arg Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Asp Lys

20 25 30

Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Gly Ile Asp Thr Gly Gly Ser Thr Gly Tyr Asn Pro Gly Leu Lys

50 55 60

Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val Ser Leu

65 70 75 80

Ser Val Ser Ser Val Thr Thr Glu Asp Ser Ala Thr Tyr Tyr Cys Thr

85 90 95

Thr Val His Gln

100

<210> 82

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> IDHD8-2

<400> 82

Ser Cys Pro Asp Gly Tyr Ser Tyr Gly Tyr Gly Cys Gly Tyr Gly Tyr

1 5 10 15

Gly Cys Ser Gly Tyr Asp Cys Tyr Gly Tyr Gly Gly Tyr Gly Gly Tyr

20 25 30

Gly Gly Tyr Gly Tyr Ser Ser Tyr Ser Tyr Ser Tyr Thr Tyr Glu Tyr

35 40 45

<210> 83

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> IGHJ2-4

<400> 83

Tyr Val Asp Ala Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

1 5 10 15

<210> 84

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> minimum BOVVHNCOFOR2 primer

<400> 84

tgcaggtgca gctgcgggag tcggg 25

<210> 85

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> minimum BOVVHFR4 XHOEV primer

<400> 85

tgaggagacg gtgaccagga gtcc 24

<210> 86

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 parent

<400> 86

Gly Gly Gly Gly Ala Met Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp

1 5 10 15

Gly Tyr Glu His Thr Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg

20 25 30

Ser Ala Cys Ile Gly Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp

35 40 45

Leu Ser Asp Gly Glu Thr Tyr Thr

50 55

<210> 87

<211> 51

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 TRUNC1

<400> 87

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp Gly Glu

35 40 45

Thr Tyr Thr

50

<210> 88

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 TRUNC2

<400> 88

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp Gly Glu

35 40 45

<210> 89

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 TRUNC3

<400> 89

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser

35 40 45

<210> 90

<211> 44

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 TRUNC3A

<400> 90

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu

35 40

<210> 91

<211> 43

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 TRUNC3B

<400> 91

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp

35 40

<210> 92

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 TRUNC4

<400> 92

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly

35 40

<210> 93

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> R2G3 TRUNC5

<400> 93

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser

35

<210> 94

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Flexible Joint

<400> 94

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 95

<211> 34

<212> PRT

<213> artificial sequence

<220>

<223> Mcoti-I

<400> 95

Gly Gly Val Cys Pro Lys Ile Leu Gln Arg Cys Arg Arg Asp Ser Asp

1 5 10 15

Ser Pro Gly Ala Cys Ile Cys Arg Gly Asn Gly Tyr Cys Gly Ser Gly

20 25 30

Ser Asp

<210> 96

<211> 34

<212> PRT

<213> artificial sequence

<220>

<223> Mcoti-II

<400> 96

Gly Gly Val Cys Pro Lys Ile Leu Lys Lys Cys Arg Arg Asp Ser Asp

1 5 10 15

Ser Pro Gly Ala Cys Ile Cys Arg Gly Asn Gly Tyr Cys Gly Ser Gly

20 25 30

Ser Asp

<210> 97

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> Mcoti-III

<400> 97

Glu Arg Ala Cys Pro Arg Ile Leu Lys Lys Cys Arg Arg Asp Ser Asp

1 5 10 15

Ser Pro Gly Ala Cys Ile Cys Arg Gly Asn Gly Tyr Cys Gly

20 25 30

<210> 98

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> base of stem A

<400> 98

Cys Thr Thr Val His Gln

1 5

<210> 99

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> base of stem A

<400> 99

Cys Ala Thr Val His Gln

1 5

<210> 100

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> base of stem A

<400> 100

Cys Ala Ile Val Gln Gln

1 5

<210> 101

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> base of stem A

<400> 101

Cys Ala Thr Val Asp Gln

1 5

<210> 102

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> stem B

<220>

<221> variant

<222> 2

<223> Xaa is any amino acid

<220>

<221> variant

<222> 4

<223> Xaa is any amino acid

<400> 102

Tyr Xaa Tyr Xaa Tyr

1 5

<210> 103

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> upstream Stem Domain

<220>

<221> variant

<222> 2

<223> Xaa is any amino acid

<220>

<221> variant

<222> 5

<223> Xaa is any amino acid

<400> 103

Cys Xaa Thr Val Xaa Gln

1 5

<210> 104

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> upstream Stem Domain

<220>

<221> variant

<222> 2

<223> Xaa is Ser, thr, gly, asn, ala or Pro

<220>

<221> variant

<222> 5

<223> Xaa is His, gln, arg, lys, gly, thr, tyr, phe,

Trp, met, ile, val or Leu

<400> 104

Cys Xaa Thr Val Xaa Gln

1 5

<210> 105

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> upstream Stem Domain

<220>

<221> variant

<222> 2

<223> Xaa is Ser, ala or Thr

<220>

<221> variant

<222> 5

<223> Xaa is His or Tyr

<400> 105

Cys Xaa Thr Val Xaa Gln

1 5

<210> 106

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> enterokinase cleavable tag

<400> 106

Asp Asp Asp Asp Lys

1 5

<210> 107

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> humanized BLV1H12 variable light chain

<400> 107

Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asp Thr Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Ala Glu Asp Ser Ser

85 90 95

Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln

100 105 110

Pro

<210> 108

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Flexible Joint

<400> 108

Gly Gly Gly Gly Ala Met Gly Ser

1 5

<210> 109

<211> 3

<212> PRT

<213> artificial sequence

<220>

<223> Flexible Joint

<400> 109

Gly Gly Ser

1

<210> 110

<211> 113

<212> PRT

<213> artificial sequence

<220>

<223> BLV5B8 variable light chain region

<400> 110

Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Gly

20 25 30

Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu

35 40 45

Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser

85 90 95

Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln

100 105 110

Pro

<210> 111

<211> 94

<212> PRT

<213> artificial sequence

<220>

<223> humanized V1 region

<400> 111

Gln Val Gln Leu Arg Glu Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Asp Lys

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Ser Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90

<210> 112

<211> 11

<212> PRT

<213> artificial sequence

<220>

<223> V2 region

<400> 112

Trp Gly Gln Gly Leu Leu Val Thr Val Ser Ser

1 5 10

<210> 113

<211> 216

<212> PRT

<213> artificial sequence

<220>

<223> BLV1H12 light chain

<400> 113

Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Gly

20 25 30

Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu

35 40 45

Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser

85 90 95

Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ser Pro Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu

115 120 125

Leu Asn Gly Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ser Val Thr Val Val Trp Lys Ala Asp Gly Ser Thr Ile Thr

145 150 155 160

Arg Asn Val Glu Thr Thr Arg Ala Ser Lys Gln Ser Asn Ser Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser Lys

180 185 190

Gly Ser Tyr Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys

195 200 205

Thr Val Lys Pro Ser Glu Cys Ser

210 215

<210> 114

<211> 216

<212> PRT

<213> artificial sequence

<220>

<223> B15 humanized light chain

<400> 114

Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Gly Asp Thr Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Ala Glu Asp Ser Ser

85 90 95

Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 115

<211> 216

<212> PRT

<213> artificial sequence

<220>

<223> BLV5B8 light chain

<400> 115

Gln Ala Val Leu Asn Gln Pro Ser Ser Val Ser Gly Ser Leu Gly Gln

1 5 10 15

Arg Val Ser Ile Thr Cys Ser Gly Ser Ser Ser Asn Val Gly Asn Gly

20 25 30

Tyr Val Ser Trp Tyr Gln Leu Ile Pro Gly Ser Ala Pro Arg Thr Leu

35 40 45

Ile Tyr Gly Asp Thr Ser Arg Ala Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Arg Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Ser Leu Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Phe Cys Ala Ser Ala Glu Asp Ser Ser

85 90 95

Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ser Pro Pro Ser Val Thr Leu Phe Pro Pro Ser Thr Glu Glu

115 120 125

Leu Asn Gly Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ser Val Thr Val Val Trp Lys Ala Asp Gly Ser Thr Ile Thr

145 150 155 160

Arg Asn Val Glu Thr Thr Arg Ala Ser Lys Gln Ser Asn Ser Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Ser Ser Asp Trp Lys Ser Lys

180 185 190

Gly Ser Tyr Ser Cys Glu Val Thr His Glu Gly Ser Thr Val Thr Lys

195 200 205

Thr Val Lys Pro Ser Glu Cys Ser

210 215

<210> 116

<211> 216

<212> PRT

<213> artificial sequence

<220>

<223> human VL1-51

<400> 116

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Ala Glu Asp Ser Ser

85 90 95

Ser Asn Ala Val Phe Gly Ser Gly Thr Thr Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 117

<211> 98

<212> PRT

<213> artificial sequence

<220>

<223> human germline light chain variable region sequence VL1-47

<400> 117

Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn

20 25 30

Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Ser Gly

<210> 118

<211> 99

<212> PRT

<213> artificial sequence

<220>

<223> human germline light chain variable region sequences VL1-40 x 1

<400> 118

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser

85 90 95

Leu Ser Gly

<210> 119

<211> 98

<212> PRT

<213> artificial sequence

<220>

<223> human germline light chain variable region sequence VL1-51 x 01

<400> 119

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala

<210> 120

<211> 99

<212> PRT

<213> artificial sequence

<220>

<223> human germline light chain variable region sequence VL2-18 x 02

<400> 120

Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr

20 25 30

Asn Arg Val Ser Trp Tyr Gln Gln Pro Pro Gly Thr Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Phe

<210> 121

<211> 38

<212> DNA

<213> artificial sequence

<220>

<223> BOVVHFR4REV

<400> 121

ttacctgcgg ccgctgagga gacggtgacc aggagtcc 38

<210> 122

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp1

<400> 122

ttttttgcgg ccgcccaggc gctgacgtac cattc 35

<210> 123

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp2

<400> 123

ttttttgcgg ccgcccaggc atcgacgtag aattc 35

<210> 124

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp3

<400> 124

ttttttgcgg ccgcccagac atcgacgaaa aattc 35

<210> 125

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp4

<400> 125

ttttttgcgg ccgcccaggc atggacgtaa aattg 35

<210> 126

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp5

<400> 126

ttttttgcgg ccgcccaagt ctcgacataa aattc 35

<210> 127

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp6

<400> 127

ttttttgcgg ccgcccaggc atcgacgagc cattg 35

<210> 128

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp7

<400> 128

ttttttgcgg ccgcccaggc atcgacgtgc cattc 35

<210> 129

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp8

<400> 129

ttttttgcgg ccgcccaggc atcgacgtgg aattc 35

<210> 130

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> ULp9

<400> 130

ttttttgcgg ccgcccaggc atcgacgtgg aagct 35

<210> 131

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> G3 parent

<400> 131

Gly Gly Ser Glu Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr

1 5 10 15

Cys Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly

20 25 30

Ala Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp

35 40 45

<210> 132

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> G3 NTRUNC1

<400> 132

Gly Gly Ser Gly Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr Cys

1 5 10 15

Gly Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly Ala

20 25 30

Leu Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp

35 40 45

<210> 133

<211> 44

<212> PRT

<213> artificial sequence

<220>

<223> G3 NTRUNC2

<400> 133

Gly Gly Ser Asp Lys Thr Cys Pro Asp Gly Tyr Glu His Thr Cys Gly

1 5 10 15

Cys Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly Ala Leu

20 25 30

Cys Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp

35 40

<210> 134

<211> 43

<212> PRT

<213> artificial sequence

<220>

<223> G3 NTRUNC3

<400> 134

Gly Gly Ser Lys Thr Cys Pro Asp Gly Tyr Glu His Thr Cys Gly Cys

1 5 10 15

Ile Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly Ala Leu Cys

20 25 30

Cys Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp

35 40

<210> 135

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> G3 NTRUNC4

<400> 135

Gly Gly Ser Thr Cys Pro Asp Gly Tyr Glu His Thr Cys Gly Cys Ile

1 5 10 15

Gly Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly Ala Leu Cys Cys

20 25 30

Gln Ala Ser Leu Gly Gly Trp Leu Ser Asp

35 40

<210> 136

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> G3 NTRUNC5

<400> 136

Gly Gly Ser Cys Pro Asp Gly Tyr Glu His Thr Cys Gly Cys Ile Gly

1 5 10 15

Gly Cys Gly Cys Lys Arg Ser Ala Cys Ile Gly Ala Leu Cys Cys Gln

20 25 30

Ala Ser Leu Gly Gly Trp Leu Ser Asp

35 40

<210> 137

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 014

<400> 137

tttgttgcca agcgtcgttg ggtggttggc ttagtgacgg tgaaacctac acttacgagt 60

tccacgtcga tacctggggc caaggactcg tggtcaccgt ctcctca 107

<210> 138

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultra-long CD3 antibody 015

<400> 138

gtgccttctg tagtcgtact ggtgattata ctagtcctac tgaacttgac atttacgagt 60

tctacgtcga agggtggggc cagggagtcc cggtcaccgt ctcctca 107

<210> 139

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultra-long CD3 antibody 032

<400> 139

cttggcctag tggtaatggt agagccgaca gtagtattgg tgaaacttat ggttacgaat 60

ttcacgtggc tgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 140

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 016

<400> 140

tttgttgtcg ttctgatttg ggtggctatc ttactgatag tcctgcttac atttacgaat 60

ggtatattga tctttggggc caaggactcc tggtcaccgt ctcctca 107

<210> 141

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultra-long CD3 antibody 031

<400> 141

gtgaattctg tagtgctact ggtgattgga ctagtcctag tgaagaagac ttttacgaat 60

tctacgtcga tacgtggggc cagggagccc cggtcaccgt ctcctca 107

<210> 142

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 027

<400> 142

ctaattgtag aggcgttttg tgtcctactc ttaacgaaat cgttgcttat acctacgaat 60

ggcacgtcga cgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 143

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultra-long CD3 antibody 021

<400> 143

gtgccttctg tagtcgtact ggtgattata ctagtcctag tgaatttgac atttacgagt 60

tctacgtcga agggtggggc cagggactcc tggtcaccgt ctcctca 107

<210> 144

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 026

<400> 144

gttaccacag cactgatcct tctcattata ctggtgcgac gtatatttac acgtacagct 60

tgcacatcga tgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 145

<211> 93

<212> DNA

<213> artificial sequence

<220>

<223> Standard short CDR3 antibody 028

<400> 145

gcaagaagtc gtggttatga ttgttatgct aatgtggatg ctttggacta cgtcgatgcc 60

tggggccaag gactcctggt caccgtctcc tca 93

<210> 146

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 018

<400> 146

agtggaattt agaatatact tggggtggtg ttggttgcgc tagttttgct gatgaggaca 60

cccacgttga tgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 147

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 019

<400> 147

attatgttgt tcgtcgttat aattgtggtg gtcttggtta tgggcatggc tttaatagtt 60

tctacgtcga tgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 148

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultra-long CD3 antibody 020

<400> 148

attatgttgt tcgtcgttat aattgtggtg gtcttggtta tgggcatggc tttaatagtt 60

tctacgtcga tgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 149

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultra-long CD3 antibody 022

<400> 149

atcgggttgt gcgtcgtaat aattgtggtg ggcttggtta tgattatggt tttgatcatt 60

tctacgtcga tgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 150

<211> 99

<212> DNA

<213> artificial sequence

<220>

<223> ultra-long CD3 antibody 023

<400> 150

gcgaagtttg ctaagggtac tacgagtgct ggtgcttgtg attattcaga aagctacgtc 60

gatgcctggg gccagggact cctggtcacc gtctcctca 99

<210> 151

<211> 100

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 024

<400> 151

attccggtgc ttatgcttat gctgcttgca attattatgg ttggcgttgt gcttgggaaa 60

gctacatcga tgcctggggc caaggactcc tggtcaccgt 100

<210> 152

<211> 107

<212> DNA

<213> artificial sequence

<220>

<223> ultralong CD3 antibody 025

<400> 152

acaatgcacg ttgtgatagt tggacgtatg acagctgtga tacttggtat cgcaattcgt 60

ggcacgttga tgcctggggc caaggactcc tggtcaccgt ctcctca 107

<210> 153

<211> 93

<212> DNA

<213> artificial sequence

<220>

<223> Standard short CDR3 antibody 029

<400> 153

gcaagaagtc gtggttatga ttgttatgct tatgtttatg ctttggacac cgtcgatgcc 60

tggggccaag gactcctggt caccgtctcc tca 93

<210> 154

<211> 93

<212> DNA

<213> artificial sequence

<220>

<223> Standard short CDR3 antibody 030

<400> 154

gcaagaagtc gtggttatga ttgttatgct aatgtggatg ctttggacta cgtcgatgcc 60

tggggccaag gactcctggt caccgtctcc tca 93

Claims

(b) Constructing a plurality of replicable expression vectors for said plurality of VH regions, wherein each replicable expression vector comprises a nucleic acid sequence encoding a single chain variable fragment (scFv) comprising an amplified VH region joined to a lambda VL region selected from the group consisting of variable light chain (VL) regions of BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8 and F18, or a humanized variant thereof;

2. The method of claim 1, wherein the VL region is a BLV1H12 VL region.

(b) Constructing a plurality of replicable expression vectors for said plurality of VH regions, wherein each replicable expression vector comprises a nucleic acid sequence encoding a single chain variable fragment (scFv) comprising an amplified VH region that is joined to said BLV1H12 lambda variable light chain (VL) region or a humanized variant thereof;

4. The method of any one of claims 1-3, wherein the cDNA template library is prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

5. The method of any one of claims 1-4, further comprising preparing the cDNA template library from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

6. The method of claim 4 or claim 5, further comprising immunizing the cow with a target antigen.

7. The method of any one of claims 1-6, wherein the amplified display particles comprise bacterial display, yeast display, mammalian display, phage display, mRNA display, ribosome display, or DNA display particles.

8. The method of any one of claims 1-7, wherein the amplified display particles are phage display particles.

9. The method of any one of claims 1-8, wherein the amplified display particles are phagemid particles.

10. The method of claim 9, wherein the nucleic acid sequence is a first nucleic acid sequence, each replicable expression vector further comprises a second nucleic acid sequence encoding at least a portion of a phage coat protein, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce the phagemid particles, whereby the fusion protein comprises the at least a portion of a phage coat protein.

(a) Immunizing a bovine with a target antigen;

(d) Constructing a plurality of replicable expression vectors for said plurality of VH regions, wherein each replicable expression vector comprises: (1) A first nucleic acid sequence encoding a single-chain variable fragment (scFv) comprising an amplified VH region joined to the BLV1H12 lambda variable light chain (VL) region or a humanized variant thereof, and (2) a second nucleic acid sequence encoding at least a portion of a phage coat protein;

12. The method of any one of claims 1-11, wherein the BLV1H12 lambda VL region is set forth in SEQ ID No. 2.

13. The method of any one of claims 1-11, wherein the BLV1H12 lambda VL region is a humanized variant of the lambda VL region of BLV1H 12.

14. The method of claim 13, wherein the humanized variant comprises one or more of the amino acid substitutions I29V and N32G and/or DNN to GDT in the CDR2 regions based on the amino acid substitutions of Kabat numbering S2A, T5N, P S, A12G, A S and amino acid substitutions in the P14L, CDR1 regions.

15. The method of any one of claim 13 or claim 14, wherein the humanized variant comprises the sequence set forth in SEQ ID No. 107.

16. The method of any one of claims 1-15, wherein the amplified VH region is indirectly joined to the BLV1H12 lambda VL region via a peptide linker.

17. The method of claim 16, wherein the peptide linker is (Gly ₄ Ser) ₃ (SEQ ID NO:94)。

18. The method of any one of claims 1-17, wherein the plurality of VH regions of the IgHV1-7 family are amplified from the cDNA template library with a forward primer comprising the sequence set forth in SEQ ID No. 84 and a reverse primer comprising the sequence set forth in SEQ ID No. 85.

19. The method of any one of claims 1-18, wherein prior to the constructing, the method further comprises size separating the sequences encoding the plurality of amplified VH regions to enrich for VH regions with ultralong CDRs 3.

20. The method of claim 19, wherein the size separation is performed by gel electrophoresis.

21. The method of claim 20, wherein the gel electrophoresis is performed using 1.2%, 1.5% or 2% agarose gel, optionally using 2% agarose gel.

22. The method of any one of claims 19-21, wherein the size separation comprises separating sequences of length, about or greater than 550 base pairs from the sequences encoding the plurality of amplified VH regions, wherein the sequences of length, about or greater than 550 base pairs comprise sequences encoding VH regions with ultralong CDR 3.

23. The method of any one of claims 1-22, wherein at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

24. The method of any one of claims 1-23, wherein at least or at least about 30% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

25. The method of any one of claims 1-24, wherein at least or at least about 40% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

26. The method of any one of claims 1-25, wherein at least or at least about 50% of the amplified particles display scFv comprising a VH region comprising an ultralong CDR3 region.

27. The method of any one of claims 1-26, wherein the ultralong CDR3 is a 25-70 amino acid peptide sequence comprising a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

28. The method of any one of claims 1-27, wherein the ultralong CDR3 is 40 to 60 amino acids in length.

29. The method of any one of claims 1-28, wherein the ultralong CDR3 is at least 42 amino acids in length.

30. The method of any one of claims 1-29, wherein the ultralong CDR3 is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

31. The method of any one of claims 1-30, wherein the ultralong CDR3 comprises at least 4 cysteine residues.

32. The method of any one of claims 1-31, wherein the ultralong CDR3 comprises 4 cysteine residues.

33. The method of any one of claims 1-31, wherein the ultralong CDR3 comprises 6, 8, 10, or 12 cysteine residues.

34. The method of any one of claims 1-33, wherein the ultralong CDR3 has at least 2 disulfide bonds.

35. The method of any one of claims 1-34, wherein the ultralong CDR3 has 2 disulfide bonds.

36. The method of any one of claims 1-34, wherein the ultralong CDR3 has 3, 4, or 5 disulfide bonds.

37. The method of any one of claims 1-36, wherein the method further comprises identifying CDR 3-knob sequences in the scFv sequence.

38. A method of preparing an ultralong CDR 3-knob display library, the method comprising:

(b) Constructing a plurality of replicable expression vectors for said plurality of CDR 3-only knob antibodies,

wherein each replicable expression vector comprises a nucleic acid sequence encoding an amplified CDR3 knob;

39. The method of claim 38, wherein the cDNA template library is prepared from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

40. The method of claim 38 or claim 39, further comprising preparing the cDNA template library from RNA isolated from Peripheral Blood Mononuclear Cells (PBMCs) from immunized cattle.

41. The method of claim 39 or claim 40, further comprising immunizing the cow with a target antigen.

42. The method of any one of claims 38-41, wherein the amplified display particles comprise bacterial display, yeast display, mammalian display, phage display, mRNA display, ribosome display, or DNA display particles.

43. The method of any one of claims 38-42, wherein the amplified display particles are phage display particles.

44. The method of any one of claims 38-43, wherein the amplified display particles are phagemid particles.

45. The method of claim 44, wherein the nucleic acid sequence is a first nucleic acid sequence, each replicable expression vector further comprises a second nucleic acid sequence encoding at least a portion of a phage coat protein, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce the phagemid particles, whereby the fusion protein comprises the at least a portion of a phage coat protein.

46. A method of making an ultralong CDR 3-knob phage display library, the method comprising:

(a) Immunizing a bovine with a target antigen;

(d) Constructing a plurality of replicable expression vectors for said plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises (1) a first nucleic acid sequence encoding an amplified CDR3 knob and (2) a second nucleic acid sequence encoding at least a portion of a phage coat protein;

47. The method according to any one of claims 38 to 46, wherein the primer comprises or consists of any one of the sequences set forth in SEQ ID NOs 7 to 11 and 121 to 130, optionally any one of the sequences set forth in SEQ ID NOs 123, 127 and 128.

48. The method of any one of claims 38-47, wherein the method further comprises identifying the CDR 3-knob from the bovine antibody Variable Heavy (VH) chain template sequence.

49. The method of claim 37 or claim 48, wherein the CDR 3-knob is identified from an antibody sequence by an algorithm comprising:

identifying a conserved cysteine in frame 3 and a conserved tryptophan in frame 4; and

determining the sequence of the CDR-3 knob, wherein:

the CDR-3 knob has an amino acid sequence length K;

the sequence starts at position x+1 and ends at x+k; and is also provided with

K＝L-2X；

Wherein L is the number of amino acids in the amino acid sequence starting from the conserved cysteine in frame 3 and ending in the conserved tryptophan in frame 4, and X is from the first cysteine in frame 3 to D in CDR H3 _H The region encodes the amino acid number of the first conserved cysteine.

50. The antibody of claim 49, wherein the antibody sequence is a bovine antibody.

51. The method of claim 49 or 50, wherein the identified CDR 3-knob is extended by one, two, three, four or five amino acids at the N-and/or C-terminus as compared to the identified sequence.

52. The method of any one of claims 38-51, wherein each of the plurality of CDR 3-only knob antibodies comprises a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

53. The method of claim 52, wherein the peptide sequence is 40 to 60 amino acids in length.

54. The method of claim 52 or claim 53, wherein the peptide sequence is at least 42 amino acids in length.

55. The method of any one of claims 52-54, wherein the peptide sequence is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

56. The method of any one of claims 52-55, wherein the peptide sequence comprises at least 4 cysteine residues.

57. The method of any one of claims 52-56, wherein the peptide sequence comprises 4 cysteine residues.

58. The method of any one of claims 52-56, wherein the peptide sequence comprises 6, 8, 10, or 12 cysteine residues.

59. The method of any one of claims 52-58, wherein the peptide sequence has at least 2 disulfide bonds.

60. The method of any one of claims 52-59, wherein the peptide sequence has 2 disulfide bonds.

61. The method of any one of claims 52-59, wherein the peptide sequence has 3, 4, or 5 disulfide bonds.

62. The method of any one of claims 6-37 and 41-61, wherein the target antigen is a non-toxic bacterium, virus, viral protein, immunomodulatory protein, cancer antigen, human IgG, or recombinant protein thereof.

63. The method of any one of claims 1-62, wherein the library of cDNA templates is synthesized using a pool of IgM, igA, and IgG-specific primers comprising or consisting of the sequences shown in SEQ ID No. 4, primers comprising or consisting of the sequences shown in SEQ ID No. 5, primers comprising or consisting of the sequences shown in SEQ ID No. 3, and primers comprising or consisting of the sequences shown in SEQ ID No. 6.

64. A method of preparing an ultralong CDR 3-knob display library, the method comprising:

(a) Constructing a plurality of replicable expression vectors for a plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises a nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds;

65. The method of claim 64, wherein the amplified display particles comprise bacterial display, yeast display, mammalian display, phage display, mRNA display, ribosome display, or DNA display particles.

66. The method of claim 64 or claim 65, wherein the amplified display particles are phage display particles.

67. The method of any one of claims 64-66, wherein the amplified display particles are phagemid particles.

68. The method of claim 67, wherein the nucleic acid sequence is a first nucleic acid sequence, each replicable expression vector further comprises a second nucleic acid sequence encoding at least a portion of a phage coat protein, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce the phagemid particles, whereby the fusion protein comprises the at least a portion of a phage coat protein.

69. A method of making an ultralong CDR 3-knob phage display library, the method comprising:

(a) Constructing a plurality of replicable expression vectors for a plurality of CDR 3-only knob antibodies, wherein each replicable expression vector comprises: (1) A first nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds, and (2) a second nucleic acid sequence encoding at least a portion of a bacteriophage coat protein;

70. The method of any one of claims 64-69, wherein at least one of the plurality of CDR 3-knob antibodies is identified from an antibody sequence by an algorithm comprising:

identifying said conserved cysteines in frame 3 and said conserved tryptophan in frame 4; and

determining the sequence of the CDR-3 knob, wherein:

the CDR-3 knob has an amino acid sequence length K;

the sequence starts at position x+1 and ends at x+k; and is also provided with

K＝L-2X；

Wherein L is the number of amino acids in the amino acid sequence starting from said conserved cysteine in frame 3 and ending in said conserved tryptophan in frame 4, and X is from the first cysteine in frame 3 to said D in CDR H3 _H The region encodes the amino acid number of the first conserved cysteine.

71. The antibody of claim 70, wherein the antibody sequence is a bovine antibody.

72. The method of claim 70 or claim 71, wherein the at least one CDR 3-knob antibody has a sequence that extends one, two, three, four, or five amino acids at the N-and/or C-terminus as compared to the identified sequence.

73. The method of any one of claims 27-37 and 52-72, wherein the peptide sequence comprises an upstream stem domain and a downstream stem domain, wherein the cysteine motif is between the upstream stem domain and the downstream stem domain.

74. The method of any one of claims 64-73, wherein the peptide sequence is amplified from DNA from a bovine immunized with a target antigen.

75. The method according to claim 74, wherein the peptide sequence is amplified from a variable heavy chain cDNA library from the immunized cow using primers specific for either side of the stem domain of the bovine ultralong CDR3 region.

76. The method of any one of claims 27-37, 52-72, 74 and 75, wherein the peptide sequence does not comprise an upstream stem domain of the N-terminus of the cysteine motif.

77. The method of any one of claims 27-37, 52-72, and 74-76, wherein the peptide sequence does not comprise the downstream stem domain of the C-terminus of the cysteine motif.

78. The method of any one of claims 73-75 and 77, wherein the upstream stem domain comprises the sequence CX ₂ TVX ₅ Q, wherein X ₂ And X ₅ Is any amino acid.

79. The method of claim 78, wherein X ₂ Ser, thr, gly, asn, ala or Pro, and X ₅ Is His, gln, arg, lys, gly, thr, tyr, phe, trp, met, ile, val or Leu.

80. The method of claim 78 or claim 79, wherein X ₂ Is Ser, ala or Thr, and X ₅ Is His or Tyr.

81. The method of any one of claims 64-73 and 76-80, wherein the peptide sequence is a synthetic CDR 3-knob.

82. The method of any one of claims 64-73 and 76-81, wherein the peptide sequence is a cyclic peptide or a modified cyclic peptide.

83. The method of any one of claims 64-73 and 76-81, wherein the peptide sequence is a semisynthetic CDR 3-knob derived from a bovine CDR 3-knob.

84. The method of any one of claims 64-83, wherein the peptide sequence is 40 to 60 amino acids in length.

85. The method of any one of claims 64-84, wherein the peptide sequence is at least 42 amino acids in length.

86. The method of any one of claims 64-85, wherein the peptide sequence is 42 amino acids, 43 amino acids, 44 amino acids, 45 amino acids, 46 amino acids, 47 amino acids, 48 amino acids, 49 amino acids, 50 amino acids, 51 amino acids, 52 amino acids, 53 amino acids, 54 amino acids, 55 amino acids, 56 amino acids, 57 amino acids, 58 amino acids, 59 amino acids, or 60 amino acids in length.

87. The method of any one of claims 64-86, wherein the peptide sequence comprises at least 4 cysteine residues.

88. The method of any one of claims 64-87, wherein the peptide sequence comprises 4 cysteine residues.

89. The method of any one of claims 64-87, wherein the peptide sequence comprises 6, 8, 10, or 12 cysteine residues.

90. The method of any one of claims 64-89, wherein the peptide sequence has at least 2 disulfide bonds.

91. The method of any one of claims 64-90, wherein the peptide sequence has 2 disulfide bonds.

92. The method of any one of claims 64-90, wherein the peptide sequence has 3, 4, or 5 disulfide bonds.

93. The method of any of claims 64-73 and 76-92, wherein the plurality of CDR3 knobs are mutated at one or more selected positions within the nucleic acid sequence encoding the peptide sequence, wherein the plurality of replicable expression vectors are a family of mutated vectors.

94. The method of any one of claims 1-93, wherein the expression vector further comprises a secretion signal sequence.

95. The method of claim 94, wherein the secretion signal sequence is a pelB signal sequence.

96. The method of any one of claims 1-95, wherein the suitable host cell is an e.

97. The method of any one of claims 1-96, wherein the suitable host cell is a TG1 inducible competent cell.

98. The method of any one of claims 9-37, 44-63, and 67-97, wherein the phagemid particle is derived from M13 phage.

99. The method of any one of claims 10-37, 45-63, and 68-98, wherein the coat protein is M13 phage gene III coat protein (pIII).

100. The method of any one of claims 10-37, 45-63, and 68-99, wherein the helper phage is selected from the group consisting of M13K07, M13R408, M13-VCS, and Phi X174.

101. The method of any one of claims 10-37, 45-63, and 68-100, wherein the helper phage is M13K07.

102. The method of any one of claims 1-101, wherein the display particle displays one copy of the fusion protein on average on the surface of the particle.

103. A library of display particles produced by the method of any one of claims 1-102.

104. A replicable expression vector comprising a gene fusion encoding a fusion protein, said gene fusion comprising a nucleic acid sequence encoding a single-chain variable fragment comprising a bovine variable heavy chain (VH) region comprising an ultralong CDR3 joined to a lambda VL region of a variable light chain (VL) region selected from the group consisting of BLV1H12, BLV5D3, BLV8C11, BF1H1, BLV5B8 and F18, or a humanized variant thereof.

105. A replicable expression vector comprising a gene fusion encoding a fusion protein, said gene fusion comprising a nucleic acid sequence encoding a single-chain variable fragment comprising a bovine variable heavy chain (VH) region comprising an ultralong CDR3 joined to a BLV1H12 λ variable light chain (VL) region or a humanized variant thereof.

106. The replicable expression vector of claim 104 or claim 105, wherein said nucleic acid sequence is a first nucleic acid sequence and said replicable expression further comprises a second nucleic acid sequence encoding at least a portion of a bacteriophage coat protein.

107. A display particle encoded by the replicable expression vector of any of claims 104-106.

108. A library of display particles, the library comprising a plurality of display particles according to claim 107.

109. The library of claim 103 or claim 108, wherein at least or at least about 20%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region.

110. The library of any one of claims 103, 108 and 109, wherein at least or at least about 30% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region.

111. The library of any one of claims 103 and 108-110, wherein at least or at least about 40% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region.

112. The library of any one of claims 103 and 108-111, wherein at least or at least about 50% of the display particles in the library comprise scFv comprising a VH region comprising an ultralong CDR3 region.

113. A replicable expression vector comprising a gene fusion encoding a fusion protein, said gene fusion comprising a nucleic acid sequence encoding a peptide sequence of 25-70 amino acids having a cysteine motif comprising 2-12 cysteine residues capable of forming a disulfide bond.

114. The replicable expression vector of claim 113, wherein said nucleic acid sequence is a first nucleic acid sequence and said replicable expression vector further comprises a second nucleic acid sequence encoding at least a portion of a bacteriophage coat protein.

115. A display particle encoded by the replicable expression vector of claim 113 or claim 114.

116. A library of display particles, the library comprising a plurality of display particles according to claim 115.

117. The library of any one of claims 103, 108-112 and 116, wherein the display particles are phage display particles.

118. The library of any one of claims 103, 108-112, 116 and 117, wherein the display particles are phagemid particles.

119. A method for selecting an antibody binding protein, the method comprising:

(1) Contacting the library of display particles of any one of claims 103, 108-112 and 116-118 with a target molecule under conditions that allow binding of the display particles to the target molecule; and

120. The method of claim 119, wherein the display particle is a phage display particle.

121. The method of claim 119 or claim 120, wherein the display particle is a phagemid particle.

122. The method of any one of claims 119-121, wherein the target molecule is a non-toxic bacterium, virus, viral protein, immunomodulatory protein, cancer antigen, human IgG, or recombinant protein thereof.

123. The method of any one of claims 119-122, wherein the target molecule is a coronavirus, a coronavirus pseudovirus, a recombinant coronavirus spike protein, or a Receptor Binding Domain (RBD) of a coronavirus spike protein.

124. The method of claim 123, wherein the coronavirus is selected from the group consisting of 229E, NL, OC43, HKU1, MERS-CoV, SARS-CoV, and SARS-CoV2.

125. The method of claim 123 or claim 124, wherein the coronavirus is SARS-CoV2 selected from the group consisting of Wuhan-Hu-1 isolate, b.1.351south African variant, or b.1.1.7uk variant.

126. The method of any one of claims 119-125, further comprising:

(ii) Collecting the amplified display particles; and

127. The method of claim 126, wherein the display particle is a phagemid particle, and the method further comprises infecting the transformed host cell with a helper phage having a gene encoding the phage coat protein in an amount sufficient to produce an amplified phagemid particle.

128. The method of claim 126 or claim 127, wherein the steps are repeated one or more times.

129. The method of any one of claims 126-128, wherein the steps are repeated with the same target molecule or a different target molecule.

130. The method of claim 129, wherein the steps are repeated with a different target molecule and the different target molecule is associated with the target molecule.

131. The method of claim 129 or claim 130, wherein the different target molecule is the same type of pathogen as the target molecule, is in the same group of pathogens as the target molecule, or is a variant of the target molecule.

132. The method of any one of claims 119-131, further comprising sequencing the fusion gene in the selected display particle to identify the antibody binding protein.

133. The method of claim 132, further comprising producing full length IgG or Fab from the selected antibody binding proteins.

134. The method of claim 132 or claim 133, wherein the antibody binding protein is an scFv and the method comprises constructing a heavy chain or portion thereof comprising conjugating the VH region of the scFv to a constant region or portion thereof.

135. The method of claim 132 or claim 133, wherein the method comprises constructing a humanized VH region by replacing a knob region of an ultralong CDR3 region of a humanized bovine VH region with an ultralong CDR3 region of a selected antibody binding protein.

136. The method of claim 135, wherein the ultralong CDR3 region of the selected antibody binding protein is replaced between an upstream and a downstream stem chain of a humanized bovine VH region.

137. The method of claim 136, wherein the VH region comprises the formula V1-X-V2, wherein the V1 region of the heavy chain comprises the sequence set forth in SEQ ID No. 111; said X region comprising said ultralong CDR3 of the selected antibody binding protein; and the V2 region comprises the sequence set forth in SEQ ID NO. 112.

138. The method of any one of claims 135-137, wherein the method further comprises constructing a heavy chain or portion thereof comprising ligating the humanized VH region with a constant region or portion thereof.

139. The method of claim 134 or claim 138, wherein the heavy chain or portion thereof is a human IgG1 heavy chain or portion thereof.

140. The method of any one of claims 134, 138 and 139, further comprising coexpression of the heavy chain or portion thereof with a light chain.

141. The method of claim 140, wherein the light chain is a bovine light chain of BLVH12, BLV5D3, BLV8C11, BF1H1, BLV5B8, or F18, or a humanized variant thereof.

142. The method of claim 140 or claim 141, wherein the light chain is a BLV1H12 light chain comprising the sequence set forth in SEQ ID No. 113 or a humanized variant thereof.

143. The method according to any one of claims 140-142, wherein the light chain is a humanized light chain as set forth in seq id No. 114.

144. The method of claim 140 or claim 141, wherein the light chain is a BLV5B8 light chain comprising the sequence set forth in SEQ ID No. 115 or a humanized variant thereof.

145. The method of claim 140, wherein the light chain is a human light chain.

146. The method of claim 140 or claim 145, wherein the light chain is selected from the group consisting of VL1-47, VL1-40, VL1-51, and VL 2-18.

147. The method of any one of claims 140, 145 and 146, wherein the light chain is set forth in any one of SEQ ID NOs 116-120.

148. A method for producing a soluble ultralong CDR3 knob, the method comprising:

149. The method of claim 148, wherein the ultralong CDR3 knob is an antibody binding protein selected by the method of any one of claims 119-132.

150. The method of claim 148 or claim 149, wherein the fusion protein has increased solubility relative to the ultralong CDR3 knob alone.

151. The method of any of claims 148-150, wherein the bacterial chaperonin is thioredoxin a (TrxA).

152. The method of any one of claims 148-151, wherein the cleavable linker is an enterokinase cleavable tag having the amino acid sequence DDDDK (SEQ ID NO: 106).

153. The method of any one of claims 148-152, wherein cleaving the cleavable linker comprises adding enterokinase to the supernatant.

154. The method of any of claims 148-153, wherein the soluble ultralong CDR3 knob comprises an additional linker to allow cyclization of the soluble ultralong CDR3 knob via a chemical or enzymatic method, optionally wherein the additional linker allows for sortase-mediated cyclization.

155. The method of claim 154, further comprising cyclizing the soluble ultralong CDR3 knob.

156. The method of any of claims 148-155, further comprising (e) removing the enterokinase and/or the bacterial chaperone protein from a solution comprising the soluble ultralong CDR3 knob.

157. The method of any of claims 148-156, further comprising enriching the soluble ultralong CDR3 knob from a solution comprising the soluble ultralong CDR3 knob, optionally wherein the enriching comprises size exclusion chromatography.

158. The method of any one of claims 148-157, further comprising generating a multispecific binding molecule comprising the soluble ultralong CDR3 knob.

159. The method of any of claims 148-158, wherein the ultralong CDR3 knob is 3-8kDa or 4-5kDa in size.

160. A fusion protein comprising an ultralong CDR3 knob and a bacterial chaperone joined by a cleavable linker, wherein the ultralong CDR3 knob is a 25-70 amino acid peptide sequence having a cysteine motif comprising 2-12 cysteine residues capable of forming 1-6 disulfide bonds.

161. The fusion protein of claim 160, wherein the bacterial chaperone protein is thioredoxin a (TrxA).

162. The fusion protein according to claim 160 or claim 161, wherein the cleavable linker is an enterokinase cleavable tag having the amino acid sequence DDDDK (SEQ ID NO: 106).

163. The fusion protein according to any one of claims 160-162, wherein the ultralong CDR3 knob comprises 1-6 disulfide bonds.

164. A composition comprising the fusion protein of any one of claims 160-163.

165. A method of identifying CDR3 knob sequences from antibody sequences, the method comprising:

determining the sequence of the CDR-3 knob, wherein:

the CDR-3 knob has an amino acid sequence length K;

the sequence starts at position x+1 and ends at x+k; and is also provided with

K＝L-2X；

166. The antibody of claim 165, wherein the antibody sequence is a bovine antibody.

167. The method of claim 165 or claim 166, wherein the CDR 3-knob antibody has a sequence that extends one, two, three, four, or five amino acids at the N-and/or C-terminus as compared to the identified sequence.

168. A purified soluble ultralong CDR3 knob produced by the method of any one of claims 148-159, wherein the soluble ultralong CDR3 is 25-75 amino acids in length and comprises 1-6 disulfide bonds.

169. The purified soluble ultralong CDR3 knob of claim 168, wherein the ultralong CDR3 knob is 3-8kDa in size.

170. The purified soluble ultralong CDR3 knob of claim 168 or claim 169, wherein the ultralong CDR3 knob is 4-5kDa in size.

171. The purified soluble ultralong CDR3 knob of any one of claims 168-170, wherein:

the knob has an amino acid sequence length K;

the sequence starts at position x+1 and ends at x+k; and is also provided with

K＝L-2X；

Wherein L is the conserved cysteine in the amino acid sequence of the antibody starting in frame 3 and endingThe amino acid number of said conserved tryptophan in frame 4 and X is from the first cysteine in frame 3 to said D in CDRH3 _H The region encodes the amino acid number of the first conserved cysteine.

172. The purified soluble ultralong CDR3 knob of claim 171, wherein the antibody sequence is a bovine antibody.

173. The purified soluble ultralong CDR3 knob of claim 171 or claim 172, wherein the knob sequence has a sequence further extended by one, two, three, four, or five amino acids at the N and/or C terminus.

174. A peptide knob sequence of length K, wherein:

the knob has an amino acid sequence length K;

the sequence starts at position x+1 and ends at x+k; and is also provided with

K＝L-2X；

Wherein L is the number of amino acids in the amino acid sequence of the antibody starting at said conserved cysteine in frame 3 and ending at said conserved tryptophan in frame 4, and X is from the first cysteine in frame 3 to said D in CDR H3 _H The region encodes the amino acid number of the first conserved cysteine.

175. The peptide knob sequence of claim 174, wherein the antibody sequence is a bovine antibody.

176. The peptide knob sequence of claim 174 or claim 175, wherein the knob sequence has a sequence further extended by one, two, three, four or five amino acids at the N and/or C terminus.

177. A composition comprising the purified soluble ultralong CDR3 of any one of claims 168-173.

178. The composition of claim 177, further comprising a pharmaceutically acceptable carrier.

179. The composition of claim 177 or claim 178, formulated for parenteral administration.

180. The composition of any one of claims 177-179, formulated for intravenous, intramuscular, topical, otic, conjunctival, nasal, inhalation, or subcutaneous administration.

181. The composition of any one of claims 177-180, formulated for administration by inhalation.