RU2820576C2 - Stable compositions of anti-tigit antibodies, individually and in combination with antibodies against programmed death receptor 1 (pd-1), and methods of their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a stable composition containing from about 10 mg/ml to about 200 mg/ml of an anti-TIGIT antibody or an antigen-binding fragment thereof, from about 5 mm to about 20 mm of L-histidine buffer, from about 6 % to about 8% (w/v) of sucrose, about 0.01% to about 0.10% (w/v) of polysorbate 80, about 1 mM to about 20 mM of L-methionine, and from about 10 mg/ml to about 200 mg/ml of an anti-PD1 antibody or antigen binding fragment thereof, where the formulation is a co-formulation and where pH of the stable formulation is from 5.3 to 6.2.

EFFECT: invention is effective for the treatment of malignant tumors and for the treatment of chronic infections.

19 cl, 8 dwg, 6 tbl, 8 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The invention relates to therapeutic antibody compositions and their use for the treatment of various disorders. In one aspect, the invention relates to compositions containing antibodies or antigen-binding fragments thereof that bind to T-cell immunoreceptor with Ig and ITIM domains (TIGIT). In another aspect, such a composition further comprises an anti-programmed death receptor 1 (PD-1) antibody or an antigen-binding fragment thereof. The present invention also relates to methods for treating various malignant tumors and chronic infections using the compositions of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under U.S.S.N. 62/500278, filed May 2, 2017, the entire contents of which are incorporated herein by reference.

LINK TO LIST OF SEQUENCES ACCEPTED IN ELECTRONIC FORM

The sequence listing of this application has been accepted in electronic form via EFS-Web as a sequence listing in ASCII format, with the file name “24453WOPCT-SEQTXT-01MAY2018.TXT”, creation date May 1, 2018, and size 227 kb. This sequence listing received via EFS-Web is part of the specification and its entire contents are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Antibody drugs for use in humans may differ somewhat in the amino acid sequence of their constant domains or in their framework sequences within variable domains, however, they typically differ most significantly in their CDR sequences. Even antibodies that bind the same protein, the same polypeptide, or even potentially the same epitope may contain completely different CDR sequences. Therapeutic antibodies for use in humans can also be derived from human antibody germline sequences or from non-human (eg, rodent) antibody germline sequences, such as in humanized antibodies, resulting in additional diversity of potential CDR sequences. These sequence differences result in different stability in solution and different ability to respond to solution parameters. In addition, small changes in amino acid arrangement or changes in one or more amino acid residues can result in very different antibody stability and sensitivity to sequence-specific degradation pathways. Therefore, it is not currently possible to predict the solution conditions necessary to optimize antibody stability. Each antibody must be tested individually to determine the optimal composition of the solution. Bhambhani et al. (2012) J. Pharm. Sci. 101:1120.

Antibodies are also relatively high molecular weight proteins (~150,000 Da), for example, compared to other therapeutic proteins such as hormones and cytokines. Therefore, dosing with relatively high weight amounts of antibody drugs is often necessary to achieve the desired molar drug concentrations. In addition, subcutaneous administration of antibody drugs is often desirable because it allows self-administration. Self-administration eliminates the time and expense associated with visiting a medical facility for administration, such as intravenous administration. Subcutaneous delivery is limited by the volume of solution that can be practically delivered to the injection site in a single injection, which is typically approximately 1-1.5 ml. Subcutaneous self-administration is typically performed using a prefilled syringe or autoinjector filled with a liquid solution of the drug formulation rather than a lyophilized form to avoid the need for the patient to resuspend the drug before injection. Antibody drugs must be stable during storage to ensure efficacy and uniform dosing, so it is critical that any formulation chosen maintains the desired properties, such as high concentration, clarity and acceptable viscosity, and that it also maintains these properties and the effectiveness of the drug over an acceptably long shelf life under typical storage conditions.

TIGIT (T cell immunoreceptor with Ig and ITIM domains) is an immunomodulatory receptor expressed primarily on activated T cells and NK cells. TIGIT is also known as VSIG9; VSTM3; and WUCAM. Its structure shows one extracellular immunoglobulin domain, a type 1 transmembrane region, and two ITIM motifs. TIGIT forms part of a costimulatory network that consists of positive (CD226) and negative (TIGIT) immunomodulatory receptors on T cells, and ligands expressed on APCs (CD155 and CD112).

An important feature of TIGIT structure is the presence of an immunoreceptor tyrosine-binding inhibitory motif (ITIM) in its cytoplasmic tail domain. As with PD-1 and TIGIT, the ITIM domain in the cytoplasmic region of TIGIT is predicted to recruit tyrosine phosphatases such as SHP-1 and SHP-2, and this is followed by dephosphorylation of tyrosine residues within immunoreceptor tyrosine-based activating motifs (ITAMs) on the subunits T cell receptor (TCR). Thus, binding of TIGIT through the receptor ligands CD155 and CD112 expressed by tumor cells or TAMs may contribute to the suppression of TCR signaling and T cell activation, which are essential for maintaining effective antitumor immunity. Thus, a TIGIT-specific antagonist antibody can inhibit CD155- and CD112-induced suppression of T-cell responses and enhance antitumor immunity.

There is a need for stable anti-TIGIT antibody formulations for pharmaceutical use, for example, for the treatment of various cancers and infectious diseases, as well as stable anti-TIGIT antibody formulations co-formulated with anti-human PD-1 antibodies. Preferably, such formulations may have a long shelf life, be stable during storage and transportation, and may preferably be stable for several months to several years under conditions typical for storing self-administered drugs. those. at refrigerator temperature in the syringe, which leads to a long shelf life of the corresponding drug product.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an anti-TIGIT antibody composition or an antigen-binding fragment thereof, comprising (i) an anti-TIGIT antibody or an antigen-binding fragment thereof; (ii) buffer, (iii) non-reducing sugar; (iv) a non-ionic surfactant; and (v) antioxidant. In another embodiment, the formulation further comprises an anti-PD-1 antibody, such as pembrolizumab or nivolumab. In another embodiment, the composition further comprises a chelating agent.

In one embodiment, the formulation contains (i) from about 10 mg/ml to about 200 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof; (ii) about 5 mM to about 20 mM buffer; (iii) from about 6% to about 8% by weight/volume (w/v) non-reducing sugar; (iv) from about 0.01% to about 0.10% (w/v) nonionic surfactant; and (v) from about 1 mM to about 20 mM antioxidant. In one embodiment, the formulation further comprises an anti-PD-1 antibody, such as pembrolizumab or nivolumab. In another embodiment, the composition further comprises a chelating agent. In one embodiment, the composition has a pH between 4.5-6.5. In specific embodiments, the pH of the composition is from about pH 5.5 to about pH 6.2. In a further embodiment, the pH of the composition is from about pH 5.6 to about pH 6.0. In another embodiment, the pH of the composition is approximately 5.7. In another embodiment, the pH of the formulation is approximately 5.8. In another embodiment, the pH of the formulation is approximately 5.9. In another embodiment, the pH of the formulation is approximately 6.0. In another embodiment, the pH of the formulation is approximately 6.1. In another embodiment, the pH of the formulation is approximately 6.2.

In one embodiment of the formulation, the buffer is L-histidine buffer or sodium acetate, the non-reducing sugar is sucrose, the nonionic surfactant is polysorbate 80, and the antioxidant is methionine or a pharmaceutically acceptable salt thereof. In one embodiment, the antioxidant is L-methionine. In another embodiment, the antioxidant is a pharmaceutically acceptable salt of L-methionine, such as methionine HCl.

In another embodiment, the formulation contains (i) from about 10 mg/ml to about 200 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof; (ii) about 5 mM to about 20 mM L-histidine buffer or about 5 mM to about 20 mM sodium acetate buffer; (iii) from about 6% to about 8% w/v. sucrose; (iv) from about 0.01% to about 0.10% w/v. polysorbate 80; and (v) from about 1 mm to about 20 mm L-methionine. In another embodiment, the formulation further comprises an anti-PD-1 antibody, such as pembrolizumab or nivolumab. In one embodiment, the composition further comprises a chelating agent. In one embodiment, the chelating agent is present in an amount of from about 1 μM to about 50 μM. In one embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is EDTA. In one embodiment, the buffer is an L-histidine buffer. In one embodiment, the composition contains from about 5 mM to about 20 mM L-histidine buffer. In another embodiment, the composition contains from about 5 mM to about 10 mM L-methionine. In a further embodiment, the composition contains polysorbate 80 in a mass fraction of approximately 0.02% w/v. In one embodiment, the anti-TIGIT antibody formulation contains sucrose at a mass fraction of approximately 7% (w/v). In any of these embodiments, the methionine is L-methionine.

In embodiments of the formulation, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is from about 10 mg/ml to about 100 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen binding fragment thereof is approximately 10 mg/ml, 12.5 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 50 mg/ml, 75 mg/ml. ml or 100 mg/ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 20 mg/ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 25 mg/ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 50 mg/ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 75 mg/ml. In one embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 100 mg/ml.

In one aspect, the present invention provides a composition containing approximately 20 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one aspect, the present invention provides a composition containing approximately 25 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one aspect, the present invention provides a composition containing approximately 50 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one aspect, the present invention provides a composition containing approximately 75 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one aspect, the present invention provides a composition containing approximately 100 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one aspect of any of the above compositions, the composition has a pH of from about 5.4 to about 6.2. In another aspect, the composition has a pH of approximately 5.5-6.2. In another embodiment, the composition has a pH of approximately 5.8-6.1. In another embodiment, the pH is approximately 5.8. In one embodiment, the pH is 5.9. In another embodiment, the pH is 6.0. In a further embodiment, the pH is 6.1.

In one aspect of any of the above compositions, the composition comprises an anti-PD1 antibody or an antigen-binding fragment thereof. In one embodiment, the anti-PD1 antibody is pembrolizumab. In another aspect, the anti-PD1 antibody is nivolumab.

In another aspect, the composition may further comprise a chelating agent. In one embodiment, the chelating agent is DTPA. In one embodiment, the chelating agent is EDTA. In one aspect, the chelating agent is present in an amount of from about 1 μM to about 50 μM. In one embodiment, the composition contains approximately 5 μM chelating agent. In one embodiment, the composition contains approximately 10 μM chelating agent. In one embodiment, the composition contains approximately 15 μM chelating agent. In one embodiment, the composition contains approximately 20 μM chelating agent. In one embodiment, the composition contains approximately 25 μM chelating agent. In one embodiment, the composition contains approximately 30 μM chelating agent. In one embodiment, the composition contains approximately 35 μM chelating agent. In one embodiment, the composition contains approximately 40 μM chelating agent. In one embodiment, the composition contains approximately 45 μM chelating agent. In one embodiment, the composition contains approximately 50 μM chelating agent. In one embodiment, the chelating agent is DTPA, present in any of the amounts specified above. In another embodiment, the chelating agent is EDTA, present in any of the amounts specified above.

In one embodiment, the composition is contained in a glass vial. In another embodiment, the composition is contained in an injection device. In another embodiment, the composition is a liquid composition. In one aspect, the composition is frozen at at least below -70°C. In another embodiment, the composition is a solution reconstituted from a lyophilized composition.

In specific embodiments, the formulation is stable at refrigerator temperature (2-8°C) for at least 3 months, preferably 6 months, and more preferably 1 year, and even more preferably up to 2 years. In one embodiment of the formulation, after 12 months at 5°C, the % anti-TIGIT antibody monomer is ≥90%, as determined by size exclusion chromatography. In another embodiment of the formulation, after 12 months at 5°C the % of anti-TIGIT antibody heavy chain and light chain is ≥90% as determined by reducing CE-SDS. In another embodiment of the formulation, after 12 months at 5°C the % of anti-TIGIT antibody heavy chain and light chain is ≥95% as determined by reducing CE-SDS. In another embodiment of the formulation, after 12 months at 5°C the % of intact anti-TIGIT IgG antibody is ≥90% as determined by non-reducing CE-SDS. In another embodiment of the formulation, after 12 months at 5°C the % of intact anti-TIGIT IgG antibody is ≥95% as determined by non-reducing CE-SDS.

In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO:111 or a variant thereof, CDRL2 of SEQ ID NO:112 or a variant thereof, CDRL3 of SEQ ID NO:113 or a variant thereof, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO:108 or a variant thereof, CDRH2 of SEQ ID NO:154 or a variant thereof, and CDHR3 of SEQ ID NO:110 or its variant. In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: :112, CDRL3 of SEQ ID NO:113, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO:108, CDRH2 of SEQ ID NO:154, and CDHR3 of SEQ ID NO:110. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:148 or a variant thereof and a light chain variable region comprising SEQ ID NO:152 or a variant thereof. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:148 and a light chain variable region comprising SEQ ID NO:152. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:291 or a variant thereof, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 or its variant. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:291 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO:293. In another aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:292 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO:293. In another aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:292 or a variant thereof, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 293 or its variant.

In one aspect, the present invention provides a co-formulation of an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-human PD-1 antibody or an antigen-binding fragment thereof, comprising (i) an anti-TIGIT antibody or an antigen-binding fragment thereof; (ii) an anti-human PD-1 antibody, or an antigen-binding fragment thereof, (ii) a buffer, (iii) a non-reducing sugar; (iv) a non-ionic surfactant; and (v) antioxidant. In one embodiment, the joint formulation further comprises a chelating agent. In one embodiment, the chelating agent is EDTA. In another embodiment, the chelating agent is DTPA. In one embodiment of the co-formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1:2. In one embodiment of the co-formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1:1. In one embodiment of the co-formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 2:1.

In one embodiment of the invention, the combination composition contains (i) from about 1 mg/ml to about 200 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof; (ii) from about 1 mg/ml to about 200 mg/ml anti-human PD-1 antibody; (iii) about 5 mM to about 20 mM buffer; (iv) from about 6% to about 8% by weight/volume (w/v) non-reducing sugar; (v) from about 0.01% to about 0.10% (w/v) nonionic surfactant; and (vi) from about 1 mM to about 20 mM antioxidant. In one embodiment, the joint formulation further comprises a chelating agent. In one embodiment, the chelating agent is present in an amount of from about 1 μM to about 50 μM. In one embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is EDTA. In one embodiment of the co-formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1:2. In one embodiment of the co-formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 1:1. In one embodiment of the co-formulation, the ratio of anti-human PD-1 antibody to anti-TIGIT antibody is 2:1. In one embodiment, the co-formulation has a pH between 4.5 and 6.5. In specific embodiments, the pH of the composition is from about pH 5.5 to about pH 6.2. In a further embodiment, the pH of the composition is approximately pH 5.8-6.0.

In one embodiment of the co-formulation, the buffer is a histidine buffer or sodium acetate buffer, the non-reducing sugar is sucrose, the non-ionic surfactant is polysorbate 80, and the antioxidant is L-methionine. In another embodiment, the co-formulation contains (i) from about 1 mg/ml to about 100 mg/ml of an anti-TIGIT antibody or antigen-binding fragment thereof; (ii) from about 1 mg/ml to about 100 mg/ml of an antibody against human PD-1 or an antigen binding fragment thereof; (iii) about 5 mM to about 20 mM L-histidine or sodium acetate buffer from about 5 mM to about 20 mM; (iv) from about 6% to about 8% w/v. sucrose; (v) from about 0.01% to about 0.10% (w/v) polysorbate 80; and (vi) from about 1 mm to about 20 mm L-methionine. In one embodiment, the co-formulation optionally contains a chelating agent. In one embodiment, the chelating agent is present in an amount of from about 1 μM to about 50 μM. In one embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is EDTA. In one embodiment, the buffer is an L-histidine buffer. In one embodiment, the co-formulation contains from about 8 mM to about 12 mM L-histidine buffer. In another embodiment, the co-formulation contains from about 5 mM to about 10 mM L-methionine. In a further embodiment, the co-formulation contains polysorbate 80 at a mass fraction of approximately 0.02% w/v. In one embodiment, the co-formulation contains sucrose in a mass fraction of approximately 7% (w/v).

In embodiments of the co-formulation, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is from about 1 mg/ml to about 100 mg/ml. In embodiments of the co-formulation, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is from about 10 mg/ml to about 100 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 10 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 12.5 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 20 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 25 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 50 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 75 mg/ml. In another embodiment, the concentration of the anti-TIGIT antibody or antigen-binding fragment thereof is approximately 100 mg/ml.

In some embodiments of the co-formulation, the concentration of the anti-human PD-1 antibody is from about 1 mg/ml to about 100 mg/ml. In one embodiment of the co-formulation, the concentration of the anti-human PD-1 antibody is from about 10 mg/ml to about 100 mg/ml. In another embodiment, the concentration of the anti-human PD-1 antibody is 20 mg/ml. In another embodiment, the concentration of the anti-human PD-1 antibody is 25 mg/ml.

In one embodiment, the co-formulation contains approximately 20 mg/ml anti-PD1 antibody, approximately 20 mg/ml anti-TIGIT antibody, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one embodiment, the co-formulation contains approximately 25 mg/ml anti-PD1 antibody, approximately 25 mg/ml anti-TIGIT antibody, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one embodiment, the co-formulation contains approximately 50 mg/ml anti-PD1 antibody, approximately 50 mg/ml anti-TIGIT antibody, 10 mM L-histidine buffer, approximately 7% w/v. sucrose, approximately 0.02% w/v. polysorbate 80 and approximately 10 mM L-methionine.

In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO:111 or a variant thereof, CDRL2 of SEQ ID NO:112 or a variant thereof, CDRL3 of SEQ ID NO:113 or a variant thereof, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO:108 or a variant thereof, CDRH2 of SEQ ID NO:154 or a variant thereof, and CDHR3 of SEQ ID NO:110 or its variant. In one aspect of any of the formulations described above, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 111, CDRL2 of SEQ ID NO: :112, CDRL3 of SEQ ID NO:113, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO:108, CDRH2 of SEQ ID NO:154, and CDHR3 of SEQ ID NO:110. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:148 or a variant thereof and a light chain variable region comprising SEQ ID NO:152 or a variant thereof. In another aspect, the formulation comprises an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:148 and a light chain variable region comprising SEQ ID NO:152. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:291 or a variant thereof, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO:293 or its variant. In one aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:291 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO:293. In another aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:292 or a variant thereof, and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO:293 or its variant. In another aspect, the anti-TIGIT antibody or antigen binding fragment thereof further comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:292 and a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO:293.

In one aspect of any of the compositions described above, the anti-human PD-1 antibody or antigen binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO:1 or a variant thereof, CDRL2 of SEQ ID NO:2 or a variant thereof, CDRL3 of SEQ ID NO:3 or a variant thereof, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO:6 or a variant thereof, CDRH2 of SEQ ID NO:7 or a variant thereof, and CDHR3 of SEQ ID NO:8 or its variant. In one aspect of any of the compositions described above, the anti-human PD-1 antibody or antigen binding fragment thereof comprises three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO: 1, CDRL2 of SEQ ID NO: 2, CDRL3 of SEQ ID NO:3, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO:6, CDRH2 of SEQ ID NO:7, and CDHR3 of SEQ ID NO:8. In another aspect, the formulations comprise an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO:4 or a variant thereof and a heavy chain variable region comprising SEQ ID NO:9 or a variant thereof. In another aspect, the formulations comprise an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain variable region comprising SEQ ID NO:4 and a heavy chain variable region comprising SEQ ID NO:9. In another aspect, the formulations comprise an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO:5 and a heavy chain comprising SEQ ID NO:10. In another aspect, the formulations comprise an anti-human PD1 antibody or antigen binding fragment thereof comprising a light chain comprising SEQ ID NO:5 or a variant thereof and a heavy chain comprising SEQ ID NO:10 or a variant thereof. In one aspect of any of the formulations described above, the anti-human PD1 antibody or antigen binding fragment thereof is pembrolizumab. In another aspect, the anti-human PD1 antibody or antigen binding fragment thereof is nivolumab.

In one aspect of any of the co-formulations described above, the composition contains (i) an anti-TIGIT antibody or antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO:111, CDRL2 of SEQ ID NO:112, CDRL3 of SEQ ID NO:113, and the heavy chain CDRs comprise CDRH1 of SEQ ID NO:108, CDRH2 of SEQ ID NO:154, and CDHR3 of SEQ ID NO:110, and (ii) antibody against Human PD-1 or an antigen binding fragment thereof comprising three light chain CDRs and three heavy chain CDRs, wherein the light chain CDRs comprise CDRL1 of SEQ ID NO:1, CDRL2 of SEQ ID NO:2, CDRL3 of SEQ ID NO:3, and The heavy chain CDRs comprise CDRH1 from SEQ ID NO:6, CDRH2 from SEQ ID NO:7, and CDHR3 from SEQ ID NO:8.

In one aspect of any of the co-formulations described above, the composition comprises (i) an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:148 and a light chain variable region comprising SEQ ID NO:152 , and (ii) an anti-human PD1 antibody or antigen binding fragment thereof, comprising a light chain variable region comprising SEQ ID NO:4 and a heavy chain variable region comprising SEQ ID NO:9.

In another aspect of any of the co-formulations described above, the composition contains (i) an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:148, and further comprising a human IgG1 heavy chain constant domain containing the amino acid the sequence of SEQ ID NO:291, and a light chain variable region comprising SEQ ID NO:152, and further comprising a human kappa light chain constant domain containing the amino acid sequence of SEQ ID NO:293, and (ii) an anti-human PD1 antibody or an antigen binding fragment thereof, comprising a light chain comprising SEQ ID NO:5 and a heavy chain comprising SEQ ID NO:10.

In another aspect of any of the co-formulations described above, the composition contains (i) an anti-TIGIT antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:148, and further comprising a human IgG1 heavy chain constant domain containing the amino acid the sequence of SEQ ID NO:292, and a light chain variable region comprising SEQ ID NO:152, and further comprising a human kappa light chain constant domain containing the amino acid sequence of SEQ ID NO:293, and (ii) an anti-human PD1 antibody or an antigen binding fragment thereof, comprising a light chain comprising SEQ ID NO:5 and a heavy chain comprising SEQ ID NO:10.

In one embodiment of any of the formulations described above, the formulation is contained in a glass vial. In another embodiment, the composition is contained in an injection device. In another embodiment, the composition is a liquid composition. In one aspect, the composition is frozen at at least below -70°C. In another embodiment, the composition is a solution reconstituted from a lyophilized composition.

The present invention provides a method of treating a chronic infection or cancer in a mammalian subject (eg, a human) in need thereof, comprising: administering an effective amount of an anti-TIGIT antibody formulation or a co-formulation as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 shows the pH stability of the formulations over 9 months under various storage conditions.

FIGURE 2 shows the concentration stability of polysorbate 80 formulations over 9 months under various storage conditions.

FIGURE 3 shows ELISA potency stability data for the formulations over 9 months under various storage conditions.

FIGURE 4 shows UP-SEC monomer stability (%) data for formulations over 9 months under various storage conditions.

FIGURE 5 shows high molecular weight (HMW) UP-SEC stability data for the formulations over 9 months under various storage conditions.

FIGURE 6 shows UP-SEC low molecular weight (LMW) stability data for formulations over 9 months under various storage conditions.

FIGURE 7 shows the stability data of heavy chain+light chain purity (%) by reducing CE-SDS for the formulations over 9 months under various storage conditions.

FIGURE 8 shows non-reducing CE-SDS intact IgG purity stability data (%) for formulations over 9 months under various storage conditions.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to compositions containing anti-TIGIT antibodies and antigen-binding fragments thereof, containing methionine. The present invention also relates to co-formulations of an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-human PD-1 antibody or an antigen-binding fragment thereof containing methionine. In each case, the formulation and co-formulation optionally contain a chelating agent.

I. Definition and abbreviations

As used throughout the description and the accompanying claims, the following abbreviations are adopted:

API active pharmaceutical ingredient

CDR complementarity determining region in immunoglobulin variable regions, defined using the Kabat numbering system unless otherwise noted

CHO Chinese hamster ovaries

CI confidence interval

DTPA diethylenetriaminepentaacetic acid

EC50 concentration resulting in 50% efficacy or binding

ELISA enzyme-linked immunosorbent assay

FFPE formaldehyde fixed, paraffin embedded

FR frame area

HRP horseradish peroxidase

HNSCC squamous cell carcinoma of the head and neck

IC50 concentration resulting in 50% inhibition

IgG immunoglobulin G

IHC immunohistochemistry or immunohistochemistry

mAb monoclonal antibody

MES 2-(N-morpholino)ethanesulfonic acid

NCBI National Center for Biotechnology Information

NSCLC non-small cell lung cancer

PCR polymerase chain reaction

PD-1 programmed death protein 1 (also called programmed cell death protein-1 and programmed death receptor 1)

PD-L1 programmed cell death receptor ligand 1

PD-L2 programmed cell death receptor ligand 2

PS80 polysorbate 80

TNBC triple negative breast cancer

V _H immunoglobulin heavy chain variable region

VK immunoglobulin kappa light chain variable region

V _L immunoglobulin light chain variable region

v/v volume per volume

WFI water for injection

w/v mass per volume

To make the invention easier to understand, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this specification, all other technical and scientific terms used herein have the meaning that is commonly accepted by one skilled in the art to which this invention relates.

As used throughout the specification and the appended claims, the unspecified and qualified singular forms include plural references unless the context clearly requires otherwise.

A reference to "or" denotes either or both possibilities unless the context clearly requires one of the specified possibilities. In some cases, "and/or" is used to highlight either or both possibilities.

"Treat" or "treat" a cancer, as used herein, means administering a composition of the invention to a subject who has an immune condition or a cancer condition, or who has been diagnosed with a cancer or pathogenic infection (eg, viral, bacterial, fungal), to achieve at least one beneficial therapeutic effect, such as, for example, a reduction in the number of cancer cells, a decrease in tumor size, a decrease in the rate of infiltration of cancer cells into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth. “Treatment” may include one or more of the following: inducing/increasing an antitumor immune response, stimulating an immune response to a pathogen, toxin, and/or self-antigen, stimulating an immune response to a viral infection, reducing one or more tumor markers, inhibiting growth or survival tumor cells, killing or reducing the size of one or more malignant lesions or tumors, reducing the level of one or more tumor markers, relieving, reducing the severity or duration of a malignant tumor, prolonging the survival of a patient relative to the expected survival for a similar untreated patient.

"Immune condition" or "immune disorder" includes, for example, pathological inflammation, inflammatory disorder and autoimmune disorder or disease. “Immune condition” also refers to infection, persistent infection, and proliferative conditions such as cancer, tumors, and angiogenesis, including infections, tumors, and malignancies that are resistant to destruction by the immune system. "Malignant condition" includes, for example, malignant tumor, malignant tumor cells, tumors, angiogenesis, and premalignant conditions such as dysplasia.

Beneficial therapeutic effects in cancer can be measured in a number of ways (See, WA Weber, J. Nucl. Med . 50:1S-10S (2009)). For example, for tumor growth inhibition, according to NCI standards, a T/C of ≤42% represents the minimum level of antitumor activity. A T/C < 10% is considered a high level of antitumor activity, where T/C (%) = median tumor volume after treatment/median tumor volume control × 100. In some embodiments, the treatment achieved by administering a composition of the invention is any of progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS). PFS, also referred to as “time to tumor progression,” measures the length of time during and after treatment that the cancer does not grow, and includes the amount of time patients experience a complete response or partial response, as well as the amount of time patients are experiencing stable disease. DFS refers to the length of time during and after treatment that a patient remains disease-free. OS refers to the extension of life expectancy compared to naïve or untreated individuals or patients. While an embodiment of the compositions, treatments and uses of the present invention may not be effective in achieving a beneficial therapeutic effect in every patient, it should be effective in a statistically significant number of subjects, as determined by any statistical test known in the art, such as Student's t-test, ^Chi2 test, Mann and Whitney U-test, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test and Wilcoxon test.

The term “patient” (alternatively referred to as “subject” or “individual” as used herein) refers to a mammal (eg, rat, mouse, dog, cat, rabbit) that can be treated with the compositions of the invention, most preferably a human. In some embodiments, the patient is an adult patient. In other embodiments, the patient is a pediatric patient.

The term "antibody" refers to any form of antibody that has a desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, humanized, fully human antibodies and chimeric antibodies. “Primary antibodies” are antibodies produced by exposing the immune system to an antigen before modifying the antibody for its intended use, for example, humanizing the antibody for use as a human therapeutic antibody.

Typically, the basic structural unit of an antibody contains a tetramer. Each tetramer contains two identical pairs of polypeptide chains, with each pair having one "light" (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition. The variable regions of each light/heavy chain pair form the binding region of the antibody. Thus, as a rule, an intact antibody has two binding sites. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Generally, human light chains are classified as kappa and lambda light chains. Additionally, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and they define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a “J” region of about 12 or more amino acids, where the heavy chain also includes a “D” region of about 10 or more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989).

Typically, the variable domains of both heavy and light chains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). CDRs are typically aligned through framework regions to allow binding to a specific epitope. Generally, from N-terminus to C-terminus, the variable domains of both light and heavy chains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of an amino acid to each domain is generally carried out in accordance with the definitions from Sequences of Proteins of Immunological Interest , Kabat, et al .; National Institutes of Health, Bethesda, Md. ; 5 ^th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem . 32:1-75; Kabat, et al ., (1977) J. Biol. Chem . 252:6609-6616; Chothia, et al ., (1987) J Mol. Biol . 196:901-917 or Chothia, et al ., (1989) Nature 342:878-883.

An antibody that "specifically binds" to a specified target protein is an antibody that is shown to bind preferentially to that target over other proteins, but this specificity does not require absolute binding specificity. An antibody is considered “specific” for its intended target if its binding detects the presence of the target protein in the sample, for example, without producing undesirable results such as false positives. Antibodies or binding fragments thereof that can be used in the present invention can bind to the target protein with an affinity of at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater , and most preferably at least 100 times greater than the affinity for non-target proteins. For the purposes of the invention, an antibody is said to specifically bind to a polypeptide containing a given amino acid sequence, for example, the amino acid sequence of a mature human TIGIT molecule or human PD-1, if it binds to polypeptides containing that sequence, but does not bind to proteins lacking that sequence. sequences.

"Chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in an antibody derived from a particular species (eg, human) or belonging to a particular class or subclass of the antibody, while the remainder chain(s) is identical or homologous to corresponding sequences in an antibody derived from another species (eg, mouse) or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided they have the desired biological activity.

“Co-formulated” or “co-formulated” or “co-formulated” or “co-formulated”, as used herein, refers to at least two different antibodies or antigen-binding fragments thereof that are formulated together and stored in the form of a combination product in a single vial or container (eg, injection device), rather than being formulated and stored individually and then mixed before administration or administered separately. In one embodiment, the co-formulation contains two different antibodies or antigen-binding fragments thereof.

The term “pharmaceutically effective amount” or “effective amount” means an amount based on which a sufficient therapeutic composition or formulation is administered to a patient to treat a disease or condition. One skilled in the art will recognize that this level can be varied according to patient characteristics such as age, weight, etc.

The term “about” when defining the amount (e.g., mM, or M) of a substance or composition, the percentage (v/v or w/v) of a component of a composition, the pH of a solution/formulation, or the value of a parameter characterizing a process step, or etc. refers to a numerical quantity variation that may arise, for example, during typical measurement, manipulation and sampling techniques involved in the preparation, characterization and/or use of a substance or composition; due to experimental error in these methods; due to differences in manufacture, source or purity of ingredients used to prepare or use the compositions or carry out the methods; and so on. In specific embodiments, “about” may mean a change of ±0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10%.

For the purposes of the invention, "x% (w/v)" is equivalent to x g/100 ml (eg, 5% w/v is equivalent to 50 mg/ml).

The compositions of the present invention include antibodies and fragments thereof that are biologically active when reconstituted or in liquid form.

The terms "malignant tumor", "cancerous" or "malignant" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of malignant tumors include, but are not limited to, carcinoma, lymphoma, leukemia, blastoma and sarcoma. More specific examples of such malignancies include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, gastrointestinal malignancy (gastrointestinal cancer), kidney cancer, ovarian cancer, liver cancer , lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, malignant brain tumor, stomach cancer, urinary cancer bladder, hepatoma, breast cancer, colon carcinoma and head and neck cancer.

"Chothia" refers to the antibody numbering system described in Al-Lazikani et al. JMB 273 : 927-948 (1997).

"Kabat", as used herein, refers to the immunoglobulin alignment and numbering system first presented in Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.).

"Growth inhibitory agent", as used herein, refers to a compound or composition that inhibits the growth of a cell, especially a cancer cell, expressing any of the genes identified herein, either in vitro or in vivo . Thus, a growth inhibitory agent is an agent that significantly reduces the percentage of cells overexpressing such genes in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a point other than S phase), such as agents that induce G1-phase arrest and M-phase arrest. Classic M-phase blockers include the vinca alkaloids (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, and etoposide. G1-phase arresting agents also extend to S-phase arresting agents, such as DNA alkylating agents such as decarbazine, mechlorethamine, and cisplatin. Additional information can be found in The Molecular Basis of Cancer , Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogens, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995).

The terms "TIGIT binding fragment", "antigen binding fragment thereof", "binding fragment thereof" or "fragment thereof" include a fragment or derivative of an antibody that still substantially retains its biological activity of binding antigen (human TIGIT) and inhibiting its activity (e.g. blocking the binding of human TIGIT to its native ligands). Thus, the term “antibody fragment” or TIGIT binding fragment refers to a portion of a full-length antibody, typically its antigen binding or variable region. Examples of anti-TIGIT antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments. Typically, the binding moiety or derivative retains at least 10% of its TIGIT inhibitory activity. In some embodiments, the binding moiety or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (or more) of its TIGIT inhibition activity, although it may use any binding moiety with sufficient affinity to produce the desired biological effect. In some embodiments, the antigen binding moiety binds its antigen with an affinity of at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity for unrelated antigens. In one embodiment, the antibody has an affinity greater than about 10 ⁹ liters/mol, as determined, for example, by the Scatchard assay. Munsen et al. (1980) Analyt. Biochem . 107:220-239. It is also contemplated that the TIGIT binding fragment may include variants having conservative amino acid substitutions that do not substantially alter its biological activity.

The terms “PD-1 binding fragment,” “antigen binding fragment thereof,” “binding fragment thereof,” or “fragment thereof” include an antibody fragment or derivative that still substantially retains its biological activity of binding and inhibiting antigen (human PD-1). activity (eg, blocking PD-1 binding to PDL1 and PDL2). Thus, the term “antibody fragment” or PD-1 binding fragment refers to a portion of a full-length antibody, typically its antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments. Typically, the binding fragment or derivative retains at least 10% of its PD-1 inhibitory activity. In some embodiments, the binding fragment or derivative retains at least 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (or more) of its PD-1 inhibitory activity, although any binding moiety with sufficient affinity to produce the desired biological effect can be used. In some embodiments, the antigen binding moiety binds its antigen with an affinity of at least two times greater, preferably at least ten times greater, more preferably at least 20 times greater, and most preferably at least 100 times greater than the affinity for unrelated antigens. In one embodiment, the antibody has an affinity greater than about 10 ⁹ liters/mol, as determined, for example, by the Scatchard assay. Munsen et al. (1980) Analyt. Biochem . 107:220-239. It is also contemplated that the PD-1 binding fragment may include variants having conservative amino acid substitutions that do not substantially alter its biological activity.

"Human antibody" refers to an antibody containing only human immunoglobulin protein sequences. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Likewise, “mouse antibody” or “rat antibody” refers to an antibody containing only mouse or rat immunoglobulin sequences, respectively.

"Humanized antibody" refers to forms of antibodies containing sequences from non-human (eg, murine) antibodies, as well as from human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. Typically, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to hypervariable loops from a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to hypervariable loops from a non-human immunoglobulin, and all or substantially all of all of the FR regions are regions from human immunoglobulin sequences. The humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically from human immunoglobulin. Humanized forms of rodent antibodies may generally contain the same CDR sequences from the parent rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

Antibodies of the present invention also include antibodies with modified (or blocked) Fc regions to provide altered effector functions. See , for example, US Patent No. 5624821; WO2003/086310; WO2005/120571; WO2006/0057702; Presta (2006) Adv. Drug Delivery Rev. 58:640-656. Such modifications can be used to enhance or suppress various immune system responses, with possible beneficial effects for diagnosis and therapy. Changes in the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fcs. Changes in Fc may also alter the antibody half-life of therapeutic antibodies, and longer half-lives may result in less frequent dosing, with a concomitant increase in convenience and decreased material utilization. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

"Fully human antibody" refers to an antibody containing only human immunoglobulin protein sequences. A fully human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Likewise, “mouse antibody” refers to an antibody containing only murine immunoglobulin sequences. A fully human antibody can be produced in humans, in a transgenic animal having human immunoglobulin germline sequences, through phage display or other molecular biological methods.

"Hypervariable region" refers to the amino acid residues of an antibody responsible for antigen binding. The hypervariable region contains amino acid residues from the “complementarity determining region” or “CDR” (e.g. residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain, and residues 31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in heavy chain variable domain, as defined by the Kabat numbering system (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), and/or such residues from the “hypervariable loop” (i.e. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3 ) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917). As used herein, the term “framework” or “FR” residues refers to variable domain residues other than hypervariable region residues, defined herein as CDR residues. CDR and FR residues are determined according to standard Kabat sequencing. Kabat et al. (1987) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda Md.

"Conservatively modified variants" or "conservative substitutions" referring to amino acid substitutions are known to one of ordinary skill in the art and can be implemented substantially without altering the biological activity of the resulting molecule, even in desired regions of the polypeptide. Such illustrative substitutions are preferably made in accordance with the substitutions specified in Table 1 as follows:

Table 1. Illustrative Conservative Amino Acid Substitutions

Original balance Conservative substitution Ala (A) Gly; Ser Arg(R) Lys, His Asn(N) Gln; His Asp (D) Glu; Asn Cys(C) Ser; Ala Gln(Q) Asn Glu (E) Asp; Gln Gly (G) Ala His(H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met(M) Leu; Ile; Tyr Phe(F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser Trp(W) Tyr; Phe Tyr (Y) Trp; Phe Val(V) Ile; Leu

In addition, one skilled in the art will know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not significantly alter biological activity. See , for example, Watson et al. (1987) Molecular Biology of the Gene , The Benjamin/Cummings Pub. Co., p. 224 (4th Edition).

The phrase “essentially consists of,” or variations such as “essentially consists of” or “substantially consisting of,” as used throughout the specification and claims, indicate the inclusion of any listed elements or groups of elements, and the optional inclusion of others elements, similar or different in nature, in relation to the listed elements, which do not significantly change the basic or novel properties of the specified dosage regimen, method or composition. By way of non-limiting example, a binding compound that is substantially composed of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues that do not significantly affect the properties of the binding compound.

“Comprising” or variants such as “comprises”, “contains” or “is contained” is used throughout the description and claims in the inclusive sense, i.e., to indicate the presence of specified features, but not to exclude the presence or add additional features that may substantially improve the method or utility of any embodiment of the invention, unless the context otherwise requires by express or necessary implication.

"Isolated antibody" and "isolated antibody fragment" refer to purification status and in such context means that the molecule is substantially free from other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growing medium. In general, the term “isolated” is not intended to mean the complete absence of such material or the absence of water, buffers or salts unless they are present in amounts that would significantly interfere with the experimental or therapeutic use of the binding compound as described herein.

"Monoclonal antibody" or "mAb" or "Mab", as used herein, refers to a population of essentially homogeneous antibodies, i.e., the antibody molecules contained in the population are identical in amino acid sequence, except for possible natural mutations, which may be present in small quantities. In contrast, conventional (polyclonal) antibody preparations typically include many different antibodies having different amino acid sequences in their variable domains, particularly in their CDRs, which are often specific for different epitopes. The term “monoclonal” refers to the nature of the antibody as being derived from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be produced by the hybridoma method first described in Kohler et al. (1975) Nature 256: 495, or they can be produced by recombinant DNA methods (see , for example, US Patent No. 4816567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the methods described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol . 222: 581-597, for example. See also Presta (2005) J. Allergy Clin. Immunol . 116:731.

“Tumor,” when referring to a subject diagnosed with or suspected of having a malignant tumor, refers to a malignant or potentially malignant neoplasia or mass of tissue of any size, and includes primary tumors and secondary neoplasias. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or fluid areas. The different types of solid tumors are named according to the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemias (malignant tumors of the blood) do not usually form solid tumors (US National Cancer Institute, Dictionary of Cancer Terms).

The term "tumor size" refers to the overall size of the tumor, which can be measured as the length and width of the tumor. Tumor size can be determined in a variety of ways known in the art, such as by taking measurements of the tumor(s) after removal from a subject, such as using a caliper, or while in the body, using imaging techniques, such as bone scans, ultrasound, CT scan or MRI.

"Variable regions" or "V region", as used herein, refers to a fragment of IgG chains that differs in sequence between different antibodies. It extends to Kabat residue 109 in the light chain and 113 in the heavy chain.

The term "buffer" includes means that maintain the pH of the solution of the compositions of the invention within an acceptable range, or, for lyophilized compositions of the invention, provide an acceptable pH of the solution before lyophilization.

The terms "lyophilization", "lyophilized" and "sublimated" refer to the process by which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation under vacuum conditions. Excipients can be included in formulations prior to lyophilization to increase the storage stability of the lyophilized product.

The term "pharmaceutical composition" refers to preparations that are in such a form as to allow the active ingredients to be effective and that do not contain additional components that are toxic to the subjects to whom the composition will be administered. The terms "composition" and "pharmaceutical composition" are used interchangeably throughout the description.

"Pharmaceutically acceptable" refers to excipients (carriers, additives) and compositions that can be conveniently administered to a subject to provide an effective dose of the active ingredient used and that are "considered to be substantially safe" e.g., that are physiologically tolerable and generally do not result in allergic or similar adverse reaction such as stomach upset and the like when administered to a human. In another embodiment, the term refers to molecules and compositions approved by a federal or state government regulatory agency, or listed in the US Pharmacopoeia or other generally accepted pharmacopoeia for use in animals, and more particularly, in humans.

A “diluted” formulation is a formulation prepared by diluting a lyophilized protein formulation in a diluent such that the protein is distributed in the diluted formulation. The diluted formulation is suitable for administration, e.g. parenteral administration) and may optionally be suitable for subcutaneous administration.

"Reconstitution time" is the time required to rehydrate the lyophilized formulation using the solution to a particle-free clarified solution.

A “stable” formulation is one in which the protein substantially retains its physical stability and/or chemical stability and/or biological activity upon storage. Various analytical methods for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. For example, in one embodiment, a stable formulation is a formulation without significant changes observed at refrigerator temperature (2-8°C) for at least 12 months. In another embodiment, a stable formulation is a formulation without significant changes observed at refrigerator temperature (2-8°C) for at least 18 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 3 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 6 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 12 months. In another embodiment, a stable formulation is a formulation with no significant changes observed at room temperature (23-27°C) for at least 18 months. The stability criteria for the antibody composition are as follows. Typically, no more than 10%, preferably 5%, of the antibody monomer is degraded, as measured by SEC-HPLC. Typically, the composition is colorless, or transparent to slightly opalescent by visual analysis. Typically, the concentration, pH and osmolality of the formulation do not change by more than +/-10%. The activity is typically in the range of 60-140%, preferably 80-120%, of the control or reference. Typically, no more than 10%, preferably 5%, truncated antibodies, ie, % small molecules, as determined, for example, by HP-SEC, are observed. Typically, no more than 10%, preferably no more than 5%, of antibody aggregation is observed, i.e. % high molecular weight molecules, as determined, for example, by HP-SEC.

An antibody “maintains its physical stability” in a pharmaceutical formulation if it does not show a significant increase in aggregation, precipitation and/or denaturation by visual inspection of color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering. Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines the tertiary structure of the protein, and by FTIR spectroscopy, which determines the secondary structure of the protein.

An antibody "retains its chemical stability" in a pharmaceutical formulation unless it is shown to undergo significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by techniques such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by techniques such as peptide mapping coupled with mass spectroscopy or MALDI/TOF/MS) , deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement) and isomerization (assessed by isoaspartic acid measurement, peptide mapping, etc.).

An antibody "retains its biological activity" in a pharmaceutical composition if the biological activity of the antibody at a given time is within the predetermined range of biological activity indicated at the time the pharmaceutical composition is prepared. The biological activity of an antibody can be determined, for example, by an antigen binding assay.

The term "isotonic" means that the composition of interest has essentially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of approximately 270-328 mOsm. Slightly hypotonic pressure is 250-269, and slightly hypertonic pressure is 328-350 mOsm. Osmotic pressure can be measured, for example, using a steam or freezing point type osmometer.

II. Compositions and co-formulations according to the invention.

In one aspect, the invention relates to stable biological compositions containing anti-TIGIT antibodies or antigen-binding fragments thereof that specifically bind to human TIGIT as an active pharmaceutical ingredient. Inclusion of methionine in such formulations reduces the oxidation of methionine residues present in the Fc region of the anti-TIGIT antibody and, in the example of the anti-TIGIT antibody containing CDRH3 of SEQ ID NO:110, tryptophan. Such formulations may further contain a chelating agent such as DTPA, which may further reduce oxidation.

In one aspect, the invention also provides a co-formulation of an anti-TIGIT antibody with an anti-PD-1 antibody. The major degradation pathways of pembrolizumab include oxidation of methionine 105 (Met105) in the heavy chain CDR3 (eg, M105 of SEQ ID NO:10) upon peroxide stress, and oxidation of Met105 and methionine Fc residues upon exposure to light. Pembrolizumab maintained its bioactivity under most of the stress conditions for the degradation levels tested. however, a decrease in affinity for PD-1 was observed for peroxide-stressed samples via surface plasmon resonance (SPR). An exposed methionine residue or methionine residue in the CDR of an antibody has the potential to affect the biological activity of the antibody through oxidation. The addition of methionine may reduce the oxidation of Met105 in the pembrolizumab heavy chain CDR.

Antibodies against PD-1 and their antigen-binding fragments

In one aspect, the invention relates to stable biological compositions containing anti-TIGIT antibodies or antigen-binding fragments thereof co-formulated with anti-human PD-1 antibodies or antigen-binding fragments thereof that specifically bind to human PD-1 (e.g., a human or humanized anti-PD-1 antibody). PD-1) as an active pharmaceutical ingredient (API PD-1), as well as methods of using the compositions of the invention. Any anti-PD-1 antibody or antigen-binding fragment thereof can be used in the combined compositions and methods of the invention. In specific embodiments, the PD-1 API is an anti-PD-1 antibody selected from pembrolizumab and nivolumab. In specific embodiments, the anti-PD-1 antibody is pembrolizumab. In alternative embodiments, the anti-PD-1 antibody is nivolumab. Table 2 shows the amino acid sequences of exemplary anti-human PD-1 antibodies pembrolizumab and nivolumab. Alternative anti-PD-1 antibodies and antigen binding fragments that can be used in the combined compositions and methods of the invention are shown in Table 3.

In some embodiments, an anti-human PD-1 antibody or antigen binding fragment thereof for use in the co-formulations of the invention comprises three light chain CDRs of CDRL1, CDRL2 and CDRL3, and/or three heavy chain CDRs of CDRH1, CDRH2 and CDRH3.

In one embodiment of the invention, CDRL1 is SEQ ID NO:1 or a variant of SEQ ID NO:1, CDRL2 is SEQ ID NO:2 or a variant of SEQ ID NO:2, and CDRL3 is SEQ ID NO:3 or a variant of SEQ ID NO:3.

In one embodiment, CDRH1 is SEQ ID NO:6 or a variant of SEQ ID NO:6, CDRH2 is SEQ ID NO:7 or a variant of SEQ ID NO:7, and CDRH3 is SEQ ID NO:8 or a variant of SEQ ID NO:8.

In one embodiment, the three light chain CDRs are SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

In an alternative embodiment of the invention, CDRL1 is SEQ ID NO:11 or a variant of SEQ ID NO:11, CDRL2 is SEQ ID NO:12 or a variant of SEQ ID NO:12, and CDRL3 is SEQ ID NO:13 or a variant of SEQ ID NO:13.

In one embodiment, CDRH1 is SEQ ID NO:16 or a variant of SEQ ID NO:16, CDRH2 is SEQ ID NO:17 or a variant of SEQ ID NO:17, and CDRH3 is SEQ ID NO:18 or a variant of SEQ ID NO:18.

In one embodiment, the three light chain CDRs are SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the three heavy chain CDRs are SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

In an alternative embodiment, the three light chain CDRs are SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13, and the three heavy chain CDRs are SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

In a further embodiment of the invention, CDRL1 is SEQ ID NO:21 or a variant of SEQ ID NO:21, CDRL2 is SEQ ID NO:22 or a variant of SEQ ID NO:22, and CDRL3 is SEQ ID NO:23 or a variant of SEQ ID NO:23.

In another embodiment, CDRH1 is SEQ ID NO:24 or a variant of SEQ ID NO:24, CDRH2 is SEQ ID NO:25 or a variant of SEQ ID NO:25, and CDRH3 is SEQ ID NO:26 or a variant of SEQ ID NO:26.

In another embodiment, the three light chain CDRs are SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, and the three heavy chain CDRs are SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26.

Some anti-human PD-1 antibodies and antigen binding fragments of the invention contain a light chain variable region and a heavy chain variable region. In some embodiments, the light chain variable region contains SEQ ID NO:4 or a variant of SEQ ID NO:4, and the heavy chain variable region contains SEQ ID NO:9 or a variant of SEQ ID NO:9. In further embodiments, the light chain variable region comprises SEQ ID NO:14 or a variant of SEQ ID NO:14, and the heavy chain variable region comprises SEQ ID NO:19 or a variant of SEQ ID NO:19. In further embodiments, the heavy chain variable region comprises SEQ ID NO:27 or a variant of SEQ ID NO:27 and the light chain variable region comprises SEQ ID NO:28 or a variant of SEQ ID NO:28, SEQ ID NO:29 or a variant of SEQ ID NO:29, or SEQ ID NO:30, or a variant of SEQ ID NO:30. In such embodiments, the light chain or heavy chain variable region sequence variant is identical to the control sequence except for the presence of one, two, three, four, or five amino acid substitutions. In some embodiments, the replacements are in the framework region (ie, outside the CDR). In some embodiments, one, two, three, four, or five of the amino acid substitutions are conservative substitutions.

In one embodiment of the co-formulations of the invention, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO:4 and a heavy chain variable region containing or consisting of SEQ ID NO:9. In a further embodiment, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO:14 and a heavy chain variable region comprising or consisting of SEQ ID NO:19. In one embodiment of the compositions of the invention, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO:28 and a heavy chain variable region containing or consisting of SEQ ID NO:27. In a further embodiment, the anti-human PD-1 antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO:29 and a heavy chain variable region comprising or consisting of SEQ ID NO:27. In another embodiment, the antibody or antigen binding fragment comprises a light chain variable region comprising or consisting of SEQ ID NO:30 and a heavy chain variable region containing or consisting of SEQ ID NO:27.

In another embodiment, the co-formulations of the invention comprise an anti-human PD-1 antibody or antigen binding protein having a V domain_Land/or domain V_H with at least 95%, 90%, 85%, 80%, 75% or 50% sequence homology with one of the V domains_Lor V domains_H, described above, and having specific binding to PD-1. In another embodiment, the anti-human PD-1 antibody or antigen binding protein of the co-formulations of the invention comprises V domains_L and V_Hhaving up to 1, 2, 3, 4 or 5 or more amino acid substitutions, and has specific binding to PD-1.

In any of the embodiments above, the PD-1 API may be a full-length anti-PD-1 antibody or an antigen-binding fragment thereof that specifically binds to human PD-1. In specific embodiments, the PD-1 API is a full-length anti-PD-1 antibody selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE. Preferably, the antibody is an IgG antibody. Any IgG isotype may be used, including IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ . Various constant domains can be attached to the V _L and V _H regions presented herein. For example, if the particular intended use of an antibody (or fragment) of the present invention involves altered effector functions, a non-IgG1 heavy chain constant domain may be used. Although IgG1 antibodies provide long half-life and effector functions such as complement activation and antibody-dependent cellular cytotoxicity, such activities may not be desirable for all applications of the antibody. In such cases, the IgG4 constant domain can be used, for example.

In embodiments of the invention, the PD-1 API is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:5 and a heavy chain containing or consisting of a sequence of amino acid residues , as set forth in SEQ ID NO:10. In alternative embodiments, the PD-1 API is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:15 and a heavy chain containing or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:20. In further embodiments, the PD-1 API is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:32 and a heavy chain containing or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:31. In further embodiments, the PD-1 API is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:33 and a heavy chain containing or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:31. In further embodiments, the PD-1 API is an anti-PD-1 antibody comprising a light chain comprising or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:34 and a heavy chain containing or consisting of a sequence of amino acid residues as set forth in SEQ ID NO:31. In some co-formulations of the invention, the PD-1 API is pembrolizumab or a biosimilar pembrolizumab. In some co-formulations of the invention, the PD-1 API is nivolumab or a biosimilar nivolumab.

Typically, amino acid sequence variants of anti-PD-1 antibodies and antigen binding fragments of the invention or anti-TIGIT antibodies and antigen binding fragments of the invention may have an amino acid sequence having at least 75% amino acid sequence identity with the amino acid sequence of a reference antibody or antigen binding fragment (e.g., severe chain, light chain, _VH , _VL or humanized sequence), more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95 , 98 or 99%. Sequence identity or homology is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to those of the anti-PD-1 antibody, after sequence alignment and gapping as necessary to achieve maximum percentage sequence identity, and not considering no conservative substitutions as part of sequence identity. None of the N-terminal, C-terminal or internal extensions, deletions or insertions in the antibody sequence should be construed as affecting sequence identity or homology.

Sequence identity refers to the degree to which amino acids from two polypeptides are the same at equivalent positions when the two sequences are optimally aligned. Sequence identity can be determined using the BLAST algorithm, where algorithm parameters are selected to obtain the closest match between corresponding sequences over the full length of the corresponding reference sequences. The following references refer to BLAST algorithms commonly used for sequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al ., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al ., (1993) Nature Genet. 3:266-272; Madden, T. L., et al. , (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., et al. , (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al. , (1997) Genome Res. 7:649-656; Wootton, J.C., et al. , (1993) Comput. Chem. 17:149-163; Hancock, JM et al. , (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al. , “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R. M., et al. , "Matrices for detecting distant relationships." in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3." M. O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, DJ, et al. , (1991) Methods 3:66-70; Henikoff, S., et al. , (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al. , (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al. , (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al. , (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al. , (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F. "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

Likewise, any class of light chains may be used in the compositions and methods herein. Specifically, kappa, lambda, or variants thereof may be used in the present compositions and methods.

Table 2. Exemplary anti-PD-1 antibody sequences

Antibody sign Amino acid sequence SEQ ID NO. Pembrolizumab light chain CDR1 RASKGVSTSGYSYLH 1 CDR2 LASYLES 2 CDR3 QHSRDLPLT 3 Variable region EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK 4 Light chain EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 5 Pembrolizumab heavy chain CDR1 NYYMY 6 CDR2 GINPSNGGTNFNEKFKN 7 CDR3 RDYRFDMGFDY 8 Variable region QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS 9 Heavy chain QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 10 Nivolumab light chain CDR1 RASQSVSSYLA eleven CDR2 DASNRAT 12 CDR3 QQSSNWPRT 13 Variable region EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK 14 Light chain EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 15 Nivolumab heavy chain CDR1 NSGMH 16 CDR2 VIWYDGSKRYYADSVKG 17 CDR3 NDDY 18 Variable region QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS 19 Heavy chain QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 20

Table 3. Additional anti-PD-1 antibodies and antigen binding fragments that can be used in the combined compositions, methods and applications of the invention.

A. Antibodies and antigen binding fragments containing hPD-1.08A light and heavy chain CDRs in WO2008/156712 CDRL1 SEQ ID NO:21 CDRL2 SEQ ID NO:22 CDRL3 SEQ ID NO:23 CDRH1 SEQ ID NO:24 CDRH2 SEQ ID NO:25 CDRH3 SEQ ID NO:26 C. Antibodies and antigen-binding fragments containing a mature h109A heavy chain variable region and one of the mature K09A light chain variable regions in WO 2008/156712 VR heavy chain SEQ ID NO:27 VR light chain SEQ ID NO:28 or SEQ ID NO:29 or SEQ ID NO:30 D. Antibodies and antigen-binding fragments containing a mature 409 heavy chain and one of the mature K09A light chains in WO 2008/156712 Heavy chain SEQ ID NO:31 Light chain SEQ ID NO:32 or SEQ ID NO:33 or SEQ ID NO:34

In some embodiments of the co-formulation of the invention, the PD-1 API (ie, an anti-PD-1 antibody or antigen binding fragment thereof) is present at a concentration of from about 25 mg/ml to about 100 mg/ml. In alternative embodiments, the API is present at a concentration of about 10 mg/ml, about 25 mg/ml, about 50 mg/ml, about 75 mg/ml, or about 100 mg/ml.

Antibodies against TIGIT and their antigen binding fragments

In one aspect, the invention relates to stable biological compositions containing anti-TIGIT antibodies or antigen-binding fragments thereof that specifically bind to human TIGIT (e.g., human or humanized anti-TIGIT antibody), as an active pharmaceutical ingredient (TIGIT API), as well as to methods of using the compositions of the invention.

In another aspect, the invention also provides biological co-formulations comprising (i) an anti-TIGIT antibody or antigen binding fragment thereof that specifically binds to human TIGIT (e.g., a human or humanized anti-TIGIT antibody) and (ii) an anti-PD-1 antibody a human or an antigen-binding fragment thereof that specifically binds to human PD-1. Any anti-TIGIT antibody or antigen-binding fragment thereof can be used in the composition, including co-formulation, and methods of the invention. Exemplary anti-TIGIT antibody sequences are listed below in Tables 4 and 5.

In some embodiments, the anti-TIGIT antibody or antigen binding fragment thereof comprises three light chain CDRs of CDRL1, CDRL2 and CDRL3, and/or three heavy chain CDRs of CDRH1, CDRH2 and CDRH3.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises CDRH1 comprising SEQ ID NO:35, CDRH2 comprising SEQ ID NO:36, CDRH3 comprising any of SEQ ID NO:37, 103, 104, 105, 106 , 107 or 160, CDRL1 containing SEQ ID NO:38, CDRL2 containing any of SEQ ID NO:39, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 69, and CDRL3 containing any from SEQ ID NO:40, 98, 99, 100, 101, 102 or 162.

In another embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises CDRH1 comprising SEQ ID NO:81, CDRH2 comprising SEQ ID NO:82, CDRH3 comprising SEQ ID NO:83, CDRL1 comprising SEQ ID NO:84, CDRL2 containing SEQ ID NO:85, and CDRL3 containing SEQ ID NO:86.

In another embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises CDRH1 comprising SEQ ID NO:108, CDRH2 comprising any of SEQ ID NO:109, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 154, 155 or 167, CDRH3 containing any of SEQ ID NO: 110, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186 or 187, CDRL1 containing SEQ ID NO:111, CDRL2 containing any of SEQ ID NO:112, 132, 133, 134, 135, 136, 137, 138, 139, 140 , 141, 142 or 168, and CDRL3 containing the amino acid sequence of SEQ ID NO:113.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises CDRH1 comprising SEQ ID NO:35, CDRH2 comprising SEQ ID NO:36, CDRH3 comprising SEQ ID NO:37, CDRL1 comprising SEQ ID NO:38 , CDRL2 containing SEQ ID NO:39, and CDRL3 containing the amino acid sequence of SEQ ID NO:40.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises CDRH1 containing SEQ ID NO:108, CDRH2 containing any of SEQ ID NO:109, 154 or 145, CDRH3 containing SEQ ID NO:110, CDRL1 containing SEQ ID NO:111, CDRL2 containing SEQ ID NO:112, and CDRL3 containing the amino acid sequence of SEQ ID NO:113.

In another embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:108, CDRH2 containing SEQ ID NO:154, CDRH3 containing SEQ ID NO:110, CDRL1 containing SEQ ID NO: :111, CDRL2 containing SEQ ID NO:112, and CDRL3 containing SEQ ID NO:113.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region. In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:41 and a light chain variable region comprising SEQ ID NO:42.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:87 and a light chain variable region comprising SEQ ID NO:88.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:114 and a light chain variable region comprising SEQ ID NO:115.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising any of SEQ ID NOs:43-58, 65-75 and 87, and a light chain variable region comprising any of SEQ ID NO:59 -64, 76-80 and 88.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising any of SEQ ID NOs:144-149 and a light chain variable region comprising any of SEQ ID NOs:150-153.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising a heavy chain variable region comprising SEQ ID NO:148 and a light chain variable region comprising SEQ ID NO:152.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:147 and a light chain variable region comprising SEQ ID NO:150.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:148 and a light chain variable region comprising SEQ ID NO:153.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:163 and a light chain variable region comprising SEQ ID NO:165.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:169 and a light chain variable region comprising SEQ ID NO:171.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:164 and a light chain variable region comprising SEQ ID NO:166.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:170 and a light chain variable region comprising SEQ ID NO:172.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment comprises CDRH1 containing SEQ ID NO:188, CDRH2 containing SEQ ID NO:189, CDRH3 containing any of SEQ ID NO:190, 220, 221 or 222, CDRL1, containing SEQ ID NO:191, CDRL2 containing SEQ ID NO:192, and CDRL3 containing any of SEQ ID NO:193, 232, 233, 234, 235, 236 or 237.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises CDRH1 comprising SEQ ID NO:204, CDRH2 comprising any of SEQ ID NO:205, 256, 257, 258, 259, 260, 261, 262 or 263, CDRH3 containing SEQ ID NO:206, CDRL1 containing SEQ ID NO:207, CDRL2 containing SEQ ID NO:208, and CDRL3 containing SEQ ID NO:209.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region. In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:194 and a light chain variable region comprising SEQ ID NO:195.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:196 and a light chain variable region comprising SEQ ID NO:200.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:210 and a light chain variable region comprising SEQ ID NO:211.

In one embodiment, an anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:212 and a light chain variable region comprising SEQ ID NO:216.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising any of SEQ ID NOs: 197, 198, 199, 223, 224, 225, 226, 227, 228, 229, 230 and 231, and a light chain variable region comprising any of SEQ ID NO:201, 202, 203, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 , 254 and 255.

In one embodiment, the anti-TIGIT antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising any of SEQ ID NOs: 213, 214, 215, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285 and 286, and a light chain variable region comprising any of SEQ ID NOs: 217, 218 and 219.

Additional anti-TIGIT antibodies that can be used in the formulations described herein include those described, for example, in PCT International Application No. WO 2016/106302; WO 2016/011264; and WO 2009/126688.

TABLE 6: Exemplary Heavy Chain Sequences

Heavy chain constant domain -IgG1 291 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain constant domain - IgG4 S228P 292 TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Kappa light chain constant domain 293 VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In any of the above embodiments, the anti-TIGIT antibody or antigen binding fragment thereof is an antibody comprising any of the variable heavy chains described above and any human heavy chain constant domain. In one embodiment, the antibody or antigen binding fragment thereof of the invention is of the IgG isotype and contains a human IgG1, IgG2, IgG3 or IgG4 heavy chain constant domain. In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human IgG1 heavy chain constant domain (SEQ ID NO:291) or a variant thereof, wherein the variant contains up to 20 modified amino acid substitutions. In one embodiment, an antibody or antigen binding fragment thereof of the invention is an antibody comprising a human IgG1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:291. In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human IgG1 heavy chain constant domain, wherein the IgG1 constant domain is afucosylated. In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human IgG4 heavy chain constant domain or a variant thereof, wherein the variant contains up to 20 modified amino acid substitutions. In another embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human IgG4 heavy chain constant domain wherein the amino acid at position 228 (using the EU numbering scheme) is changed from Ser to Pro. In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO:292.

In any of the above embodiments, the anti-TIGIT antibody or antigen binding fragment thereof may comprise any of the variable light chains described above and a human light chain constant domain. In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human kappa light chain constant domain or a variant thereof, wherein the variant contains up to 20 modified amino acid substitutions. In another embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human lambda light chain constant domain or a variant thereof, wherein the variant contains up to 20 modified amino acid substitutions. In one embodiment, an antibody or antigen binding fragment thereof of the invention comprises a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO:293.

Compositions

The compositions of the invention minimize the formation of antibody aggregates (high molecular weight molecules) and particles, high and low molecular weight molecules, minimize the oxidation of methionine residues, and ensure that the antibody retains biological activity over time.

In one aspect, the invention relates to various compositions of an anti-TIGIT antibody or antigen-binding fragment thereof. For example, the present invention relates to compositions containing (i) an anti-TIGIT antibody or antigen binding fragment thereof, (ii) a buffer (eg, L-histidine or acetate), (iii) a non-reducing sugar (eg, sucrose); (iv) a non-ionic surfactant (eg polysorbate 80); and (v) an antioxidant (eg L-methionine). In one embodiment, the composition further comprises an anti-PD-1 antibody. In one aspect, the composition further comprises a chelating agent. In one embodiment, the chelating agent is present in an amount of from about 1 μM to about 50 μM. In one embodiment, the chelating agent is diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelating agent is EDTA.

In another aspect, the present invention also provides various co-formulations of an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-human PD-1 antibody or an antigen-binding fragment thereof. In one embodiment, the composition contains (i) an anti-TIGIT antibody or antigen-binding fragment thereof, (ii) an anti-human PD-1 antibody or antigen-binding fragment thereof, (iii) a buffer (e.g., L-histidine or acetate), (iv) a non-reducing a sugar (eg, sucrose), (v) a nonionic surfactant (eg, polysorbate 80), and (vi) an antioxidant (eg, L-methionine). In one embodiment, the composition further comprises a chelating agent. In one embodiment, the chelating agent is present in an amount of from about 1 μM to about 50 μM. In one embodiment, the chelating agent is diethylenetriaminepentaacetic acid (DTPA). In another embodiment, the chelating agent is EDTA.

The pharmaceutical compositions of the present invention may include buffers.

Buffers that can be used in the pharmaceutical compositions and methods of the invention include succinate (sodium or potassium), L-histidine, phosphate (sodium or potassium), tris (tris(hydroxymethyl)aminomethane), diethanolamine, citrate (sodium), acetate ( sodium) etc. In one embodiment of the invention, the buffer is present in the composition at a concentration of approximately 1-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 mM). In specific embodiments of the invention, the buffer is a histidine, for example, L-histidine buffer.

The buffer of this invention has a pH ranging from about 4.5 to about 6.5; approximately 5.0-6.2; approximately 5.5-6.0; and preferably has a pH of approximately 5.8. In establishing the exemplary composition, histidine and acetate buffers in the pH range of 5.0-6.0 were examined as appropriate. When a range of pH values is specified, for example, “pH between pH 5.5 and 6.0,” the range is intended to include the specified values. For example, the range from about 5.0 to about 6.0 includes 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0. Unless otherwise indicated, pH refers to the pH after reconstitution of the lyophilized formulations of the present invention. pH is typically measured at 25°C using a standard pH meter with a glass electrode. For the purposes of the invention, a solution containing “histidine buffer at pH X” refers to a solution at pH X and containing a histidine buffer, i.e. pH is intended to indicate the pH of a solution.

In one embodiment of the invention, the anti-TIGIT antibody composition, and the combined anti-TIGIT antibody and anti-human PD-1 antibody composition, contain a non-reducing sugar. For the purposes of the invention, a “non-reducing sugar” is a sugar that is unable to act as a reducing agent because it does not contain or cannot be converted into a form containing a free aldehyde group or a free ketone group. Examples of non-reducing sugars include, but are not limited to, disaccharides such as sucrose and trehalose. In one embodiment, the non-reducing sugar is present in an amount of about 1-10% (w/v) (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%). In another embodiment, the non-reducing sugar is present in an amount of from about 6% to about 8% (w/v) (6, 7 or 8%). In a further embodiment, the non-reducing sugar is present in an amount of approximately 6% (w/v). In a further embodiment, the non-reducing sugar is present in an amount of approximately 7% (w/v). In a further embodiment, the non-reducing sugar is present in an amount of approximately 8% (w/v). In one embodiment, the non-reducing sugar is sucrose, trehalose or raffinose. In another embodiment, the non-reducing sugar is sucrose. In a further embodiment, sucrose is present at 6-8% w/v. In one embodiment, sucrose is present at approximately 6% (w/v). In one embodiment, sucrose is present at approximately 7% (w/v). In one embodiment, sucrose is present at approximately 8% (w/v).

The compositions of the invention also contain a surfactant. Within the scope of the invention, a surfactant is a surfactant that is amphipathic in nature. Surfactants can be added to the formulations herein to provide stability, reduce and/or prevent aggregation, or to prevent and/or inhibit protein damage during processing conditions such as purification, filtration, lyophilization, transportation, storage and delivery. In the present invention, a surfactant can be used to provide additional stability to the active ingredient(s).

Nonionic surfactants that can be used in the formulations, including co-formulations, of the invention include, but are not limited to, polyoxyethylene sorbitan fatty acid esters (polysorbates sold under the trade name Tween® (Uniquema Americas LLC, Wilmington, DE)) including polysorbate-20 (polyoxyethylene sorbitan monolaurate), polysorbate-40 (polyoxyethylene sorbitan monopalmitate), polysorbate-60 (polyoxyethylene sorbitan monostearate) and polysorbate-80 (polyoxyethylene sorbitan monooleate); polyoxyethylene alkyl esters such as Brij® 58 (Uniquema Americas LLC, Wilmington, DE) and Brij® 35; poloxamers (eg poloxamer 188); Triton® X-100 (Union Carbide Corp., Houston, TX) and Triton® X-114; NP40; Span 20, Span 40, Span 60, Span 65, Span 80 and Span 85; copolymers of ethylene and propylene glycol (e.g. Pluronic® series of nonionic surfactants such as Pluronic® F68, Pluronic® 10R5, Pluronic® F108, Pluronic® F127, Pluronic® F38, Pluronic® L44, Pluronic® L62 (BASF Corp., Ludwigshafen, Germany); and sodium dodecyl sulfate (SDS). In one embodiment, the nonionic surfactant is polysorbate 80 or polysorbate 20. In one embodiment, the nonionic surfactant is polysorbate 20. In another embodiment, The nonionic surfactant is polysorbate 80.

The amount of nonionic surfactant to be included in the compositions of the invention is an amount sufficient to perform the desired function, i.e. the minimum amount required to stabilize the active pharmaceutical ingredient (i.e., an anti-TIGIT antibody or an antigen-binding fragment thereof, or both an anti-TIGIT antibody or an antigen-binding fragment thereof and an anti-human PD-1 antibody or an antigen-binding fragment thereof) in a formulation. All percentages of nonionic surfactant are given as % w/v. Typically, the surfactant is present at a concentration of from about 0.008% to about 0.1% w/v. In some embodiments of this aspect of the invention, the surfactant is present in the composition in an amount of from about 0.01% to about 0.1%; from about 0.01% to about 0.09%; from about 0.01% to about 0.08%; from about 0.01% to about 0.07%; from about 0.01% to about 0.06%; from about 0.01% to about 0.05%; from about 0.01% to about 0.04%; from about 0.01% to about 0.03%, from about 0.01% to about 0.02%, from about 0.015% to about 0.04%; from about 0.015% to about 0.03%, from about 0.015% to about 0.02%, from about 0.02% to about 0.04%, from about 0.02% to about 0.035%, or from about 0. 02% to approximately 0.03%. In specific embodiments, the surfactant is present in an amount of approximately 0.02%. In alternative embodiments, the surfactant is present in an amount of about 0.01%, about 0.015%, about 0.025%, about 0.03%, about 0.035%, or about 0.04%.

In illustrative embodiments of the invention, the surfactant is a nonionic surfactant selected from the group consisting of: polysorbate 20 and polysorbate 80. In preferred embodiments, the surfactant is polysorbate 80.

In specific embodiments, the compositions, including co-formulations, of the invention contain from about 0.01% to about 0.04% w/v. polysorbate 80. In further embodiments, the compositions described herein contain polysorbate 80 in an amount of about 0.008% w/v, about 0.01% w/v. In one embodiment, the amount of polysorbate 80 is approximately 0.015 w/v%. In another embodiment, the amount of polysorbate 80 is approximately 0.02% w/v. In a further embodiment, the amount of polysorbate 80 is approximately 0.025% w/v. In another embodiment, the amount of polysorbate 80 is approximately 0.03% w/v. In a further embodiment, the amount of polysorbate 80 is approximately 0.035% w/v. In another embodiment, the amount of polysorbate 80 is approximately 0.04% w/v. In a further embodiment, the amount of polysorbate 80 is approximately 0.045% w/v. In specific embodiments, the compositions of the invention contain approximately 0.02% w/v. polysorbate 80.

The compositions, including co-formulations, of the present invention also contain methionine, or a pharmaceutically acceptable salt thereof, as an antioxidant. In one embodiment of the invention, the methionine is L-methionine. In another embodiment, methionine is a pharmaceutically acceptable salt of L-methionine, such as methionine HCl. In one embodiment, methionine is present in the composition at a concentration of approximately 1-20 mM (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 and 20 mM). In another embodiment, methionine is present from about 5 mM to about 10 mM (5, 6, 7, 8, 9 and 10 mM). In another embodiment, methionine is present at approximately 10 mM.

The compositions, including co-formulations, of the present invention may also further contain a chelating agent. In one embodiment of the invention, the chelating agent is present in the composition at a concentration of approximately 5-30 μM (eg, 5, 10, 15, 20, 25, or 30 μM). In one embodiment, the chelating agent is DTPA. In another embodiment, the chelating agent is EDTA.

Lyophilized pharmaceutical compositions

Lyophilized therapeutic protein formulations provide several advantages. Lyophilized formulations generally provide better chemical stability than solution formulations and thus increase half-life. The lyophilized formulation may also be reconstituted in varying concentrations, depending on clinical factors such as route of administration or dosage. For example, the lyophilized formulation can be reconstituted at a high concentration (ie, in a small volume), if necessary, for subcutaneous administration, or at a lower concentration, if necessary, for intravenous administration. High concentrations may also be necessary if high doses are required for a particular subject, particularly for subcutaneous administration where the volume of injection must be minimized. One such lyophilized antibody formulation is described in US Patent No. 6267958, the entire contents of which are therefore incorporated by reference. Lyophilized formulations of another therapeutic protein are described in US Patent No. 7247707, the entire contents of which are therefore incorporated by reference.

Typically, the lyophilized formulation is prepared by diluting at a high concentration of drug product (DP, in one exemplary embodiment, the humanized anti-PD-1 antibody pembrolizumab or an antigen binding fragment thereof), i.e., diluting in a low volume of water. Subsequent dilution with water or isotonic buffer can then be easily used to dilute the DP to a lower concentration. Typically, excipients are included in the lyophilized formulation of the present invention at levels that provide approximately isotonic composition when diluted at a high DP concentration, e.g. for subcutaneous administration. Dilution in a larger volume of water to obtain a lower DP concentration inevitably reduces the tonicity of the diluted solution, however, such a reduction may not be of great importance in non-subcutaneous applications, e.g. intravenous administration. If isotonicity is desired at a lower DP concentration, the lyophilized powder can be reconstituted in a standard low volume of water, and then further reconstituted with an isotonic diluent such as 0.9% sodium chloride.

The lyophilized compositions of the present invention are prepared by lyophilizing (sublimation) a solution prepared for lyophilization. Sublimation is carried out by freezing the composition and subsequent sublimation of water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic point or collapse temperature of the composition. Typically, primary drying rack temperatures range from about -30 to 25°C (assuming the product remains frozen during primary drying) with a suitable pressure typically ranging from about 50 to 250 mTorr ( from 6.7 to 33.3 Pa). The composition, size and type of container containing the sample (eg, glass vial), and volume of liquid determine the time required for drying, which can range from several hours to several days (eg, 40-60 hours). The secondary drying step can be carried out at approximately 0-40°C, depending primarily on the type and size of the container, and the type of protein used. The secondary drying time is determined by the desired level of residual moisture in the product and typically takes at least approximately 5 hours. Typically, the moisture content of the lyophilized formulation is less than about 5%, and preferably less than about 3%. The pressure may be the same as the pressure used in the primary drying step. Sublimation conditions can be changed depending on the composition and size of the bottle.

In some cases, it may be desirable to lyophilize the protein composition in a container in which the protein must be reconstituted to avoid a transfer step. The container in this case may, for example, be a 3, 5, 10, 20, 50 or 100 cm ³ bottle.

The lyophilized formulations of the present invention are reconstituted before administration. The protein can be diluted to concentrations of approximately 10, 15, 20, 25, 30, 40, 50, 60, 75, 80, 90 or 100 mg/ml, or higher concentrations such as 150 mg/ml, 200 mg/ml, 250 mg/ml or 300 mg/ml, up to approximately 500 mg/ml. In one embodiment, the protein concentration after dilution is approximately 10-300 mg/ml. In one embodiment, the protein concentration after dilution is approximately 20-250 mg/ml. In one embodiment, the protein concentration after dilution is approximately 150-250 mg/ml. In one embodiment, the protein concentration after dilution is approximately 180-220 mg/ml. In one embodiment, the protein concentration after dilution is approximately 50-150 mg/ml. In one embodiment, the protein concentration after dilution is approximately 100 mg/ml. In one embodiment, the protein concentration after dilution is approximately 75 mg/ml. In one embodiment, the protein concentration after dilution is approximately 50 mg/ml. In one embodiment, the protein concentration after dilution is approximately 25 mg/ml. High protein concentrations are particularly useful when subcutaneous delivery of a diluted formulation is intended. However, for other routes of administration, such as intravenous administration, lower protein concentrations may be desirable (e.g. approximately 5-50 mg/ml).

Dilution is typically carried out at approximately 25°C to ensure complete hydration, although other temperatures can be used as desired. The time required for dilution depends, for example, on the type of diluent, amount of excipient(s) and protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffering solution (eg, phosphate-buffered saline), sterile saline, Ringer's solution, or dextrose solution.

Liquid pharmaceutical compositions

A liquid antibody formulation can be prepared by taking a drug (eg, a humanized anti-PD-1 antibody) that is in liquid form (eg, pembrolizumab in an aqueous pharmaceutical formulation) and replacing its buffer with the desired buffer in the last step of the purification process. The lyophilization step is not present in this embodiment. The drug substance in the final buffer is concentrated to the desired concentration. Excipients such as sucrose and polysorbate 80 are added to the drug substance and diluted using a suitable buffer to the final protein concentration. The final formulation of the drug substance is filtered using 0.22 µm filters and filled into the final container (e.g. glass bottles).

III. Methods of application

The invention also relates to a method of treating a malignant tumor in a subject, comprising administering an effective amount of any of the compositions of the invention; i.e., any composition described herein, to a subject. In some specific embodiments of this method, the composition is administered to the subject via intravenous administration. In other embodiments, the composition is administered to the subject via subcutaneous administration. In one embodiment, the invention relates to a method of treating a cancer in a human patient, comprising administering any composition of the invention to the patient.

In any of the methods of the invention, the cancer may be selected from the group consisting of: melanoma, lung cancer, head and neck cancer, bladder cancer, breast cancer, gastrointestinal cancer, multiple myeloma, hepatocellular carcinoma, lymphoma , kidney cancer, mesothelioma, ovarian cancer, esophageal cancer, anal cancer, bile duct cancer, colorectal cancer, cervical cancer, thyroid cancer, salivary gland cancer, prostate cancer (eg, hormone-refractory prostate adenocarcinoma ), pancreatic cancer, colon cancer, esophageal cancer, liver cancer, thyroid cancer, glioblastoma, glioma and other neoplastic malignancies.

In some embodiments, the lung cancer is non-small cell lung cancer.

In alternative embodiments, the lung cancer is small cell lung cancer.

In some embodiments, the lymphoma is Hodgkin's lymphoma.

In other embodiments, the lymphoma is non-Hodgkin's lymphoma. In specific embodiments, the lymphoma is a mediastinal large B-cell lymphoma.

In some embodiments, the breast cancer is triple negative breast cancer.

In further embodiments, the breast cancer is ER+/HER2- breast cancer.

In some embodiments, the bladder cancer is a urothelial malignancy.

In some embodiments, the head and neck cancer is a nasopharyngeal malignancy. In some embodiments, the cancer is thyroid cancer. In other embodiments, the cancer is a salivary gland cancer. In other embodiments, the cancer is a squamous cell carcinoma of the head and neck.

In one embodiment, the invention relates to a method of treating metastatic non-small cell lung cancer (NSCLC) in a human patient, comprising administering a composition of the invention to the patient. In specific embodiments, the patient has a tumor with high PD-L1 expression [(tumor proportion score (TPS) ≥50%)] and has not previously been treated with platinum-containing chemotherapy. In other embodiments, the patient has a tumor that expresses PD-L1 (TPS ≥1%) and has previously been treated with platinum-containing chemotherapy. In other embodiments, the patient has a PD-L1 expressing tumor (TPS ≥1%) and has not previously been treated with platinum-containing chemotherapy. In specific embodiments, the patient has had disease progression during or after chemotherapy with platinum-containing agents. In specific embodiments, PD-L1 TPS is determined through FDA-approved testing. In specific embodiments, the patient's tumor does not have EGFR or ALK genomic aberrations. In specific embodiments, the patient's tumor has an EGFR or ALK genomic aberration, and the patient had disease progression during or after treatment for the EGFR or ALK aberration(s), prior to administration of an anti-PD-1 antibody or antigen-binding fragment thereof.

In some embodiments, the cancer is a metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

In some embodiments, the cancer is a metastatic colorectal cancer with high levels of microsatellite instability (MSI-H).

In some embodiments, the cancer is a microsatellite instability-high (MSI-H) solid tumor.

In some embodiments, the cancer is a solid tumor with a high mutation load.

In some embodiments, the malignancy is selected from the group consisting of: melanoma, non-small cell lung cancer, relapsed or refractory classical Hodgkin lymphoma, head and neck squamous cell carcinoma, urothelial malignancy, esophageal cancer, gastric cancer, and hepatic cell cancer.

In other embodiments of the above treatment methods, the cancer is a hematologic malignancy. In specific embodiments, the hematologic malignancy is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), positive for EBV DLBCL, primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), malignant tumor expressing myeloid cell leukemia protein-1 (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or small lymphocytic lymphoma (SLL).

Malignancies for which improved disease-free and overall survival has been demonstrated based on the presence of tumor-infiltrating lymphocytes in biopsy or surgical specimens, such as melanoma, colorectal, liver, kidney, gastric/esophageal, breast, pancreatic, and ovarian cancers , are covered by the methods and treatments described herein. It is known that such subtypes of malignant tumors are sensitive to immune control by T lymphocytes. Also covered are refractory or recurrent malignancies whose growth can be inhibited using the antibodies described herein.

Additional malignancies that may benefit from treatment using the formulations described herein include those associated with persistent infection with viruses such as human immunodeficiency viruses, hepatitis viruses class A, B and C, Epstein-Barr virus, Human papillomaviruses are known to have a causal relationship with, for example, Kaposi's sarcoma, liver cancer, nasopharyngeal cancer, lymphoma, cervical, vulvar, anal, penile and oral cavity cancers.

The compositions can also be used to prevent or treat infection and infectious disease. Thus, the invention relates to a method of treating a chronic infection in a mammalian subject, comprising administering an effective amount of a composition of the invention to the subject. In some specific embodiments of this method, the composition is administered to the subject via intravenous administration. In other embodiments, the composition is administered to the subject via subcutaneous administration.

These agents can be used alone or in combination with vaccines to stimulate an immune response to pathogens, toxins, and self-antigens. Antibodies or antigen-binding fragments thereof can be used to stimulate an immune response to viruses infectious to humans, including, but not limited to: human immunodeficiency viruses, hepatitis viruses class A, B and C, Epstein-Barr virus, human cytomegalovirus, human papillomaviruses and viruses herpes. Antagonistic anti-PD-1 antibodies or antibody fragments can be used to stimulate an immune response to infection by bacterial or fungal parasites and other pathogens. Viral infections with hepatitis B and C and HIV are among the infections considered to be chronic viral infections.

The compositions of the invention can be administered to a patient in combination with one or more "additional drugs". The additional drug may be a biological drug (including, but not limited to, antibodies against VEGF, EGFR, Her2/neu, VEGF receptors, other growth factor receptors, CD20, CD40, CD-40L, OX-40, 4-1BB and ICOS), immunogenic agent (eg, attenuated cancer cells, tumor antigens, antigen presenting cells such as dendritic cells sensitized with tumor-derived antigen or nucleic acids, immunostimulatory cytokines (eg, IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines such as, but not limited to, GM-CSF).

As noted above, in some embodiments of the methods of the invention, the method further includes administering an additional drug. In specific embodiments, the additional drug is an anti-LAG3 antibody or an antigen-binding fragment thereof, an anti-GITR antibody or an antigen-binding fragment thereof, an anti-CTL4 antibody or an antigen-binding fragment thereof, an anti-CD27 antibody or an antigen-binding fragment thereof. In one embodiment, the additional drug is a Newcastle disease virus vector expressing IL-12. In a further embodiment, the additional drug is dinaciclib. In additional embodiments, the additional drug is a STING agonist.

Suitable routes of administration may, for example, include parenteral delivery, including intramuscular, subcutaneous, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal. Drugs can be administered by a variety of conventional routes, such as intraperitoneal, parenteral, intra-arterial or intravenous injection. Methods of administration in which the volume of solution must be limited (for example, subcutaneous administration) require a lyophilized formulation to allow reconstitution in high concentrations.

The choice of dosage of the adjuvant drug depends on several factors, including the turnover rate of the molecule in the serum or tissue, the level of symptoms, the immunogenicity of the molecule, and the availability of the target cells, tissue, or organ in the individual being treated. The dose of the additional drug should be an amount that provides an acceptable level of side effects. Accordingly, the dose level and dosing frequency of each additional drug (eg, biotherapeutic or chemotherapeutic agent) may depend, in part, on the specific drug, the severity of the cancer being treated, and patient characteristics. Guidelines are available for selecting appropriate doses of antibodies, cytokines, and small molecules. See , for example, Wawrzynczak (1996) Antibody Therapy , Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis , Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases , Marcel Dekker, New York, NY; Baert et al . (2003) New Engl. J. Med. 348:601-608; Milgrom et al . (1999) New Engl. J. Med. 341:1966-1973; Slamon et al . (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al . (2000) New Engl. J. Med. 342:613-619; Ghosh et al . (2003) New Engl. J. Med. 348:24-32; Lipsky et al . (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Ed); Medical Economics Company; ISBN: 1563634457; 57th edition (November 2002). Determination of an appropriate dosage regimen may be made by the clinician, for example, using parameters or factors known or suspected in the art to influence or predicted to influence treatment, and the determination may depend, for example, on the patient's clinical history (eg , previous therapy), type and stage of malignancy being treated, and biomarkers of response to one or more of the drugs in combination therapy.

Numerous literature sources are available to facilitate the selection of pharmaceutically acceptable carriers or excipients for an additional drug. See , for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary , Mack Publishing Company, Easton, PA (1984); Hardman et al . (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics , McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy , Lippincott, Williams, and Wilkins, New York, NY; Avis et al . (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications , Marcel Dekker, NY; Lieberman, et al . (eds.) (1990) Pharmaceutical Dosage Forms: Tablets , Marcel Dekker, NY; Lieberman et al . (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems , Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety , Marcel Dekker, Inc., New York, NY.

The antibody pharmaceutical composition can be administered by continuous infusion, or in doses at intervals, for example, one day, 1-7 times a week, one week, two weeks, three weeks, once a month, once every two months, etc. . The preferred dosing method is one that involves a maximum dosage or dosing frequency that avoids significant undesirable side effects. The total weekly dose is typically at least 0.05 mcg/kg, 0.2 mcg/kg, 0.5 mcg/kg, 1 mcg/kg, 10 mcg/kg, 100 mcg/kg, 0.2 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 10 mg/kg, 25 mg/kg, 50 mg/kg body weight or more. See , for example, Yang et al. (2003) New Engl. J. Med. 349:427-434; Herold et al. (2002) New Engl. J. Med. 346:1692-1698; Liu et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al . (20003) Cancer Immunol. Immunother. 52:133-144. The desired dose of a small molecule drug, such as a peptide mimetic, natural product, or organic chemical compound, is approximately the same as that for an antibody or polypeptide, on a mole/kg basis.

Embodiments of the invention also include one or more of the biological compositions described herein (i) for use in, (ii) for use as a drug or composition for, or (iii) for use in the preparation of a drug for: (a) therapy (for example, the human body); (b) medicine; (c) inducing or increasing an antitumor immune response; (d) decreasing the amount of one or more tumor markers in a patient; (e) stopping or slowing the growth of a tumor or malignant tumor of the blood cells; (f) stopping or slowing the progression of PD-1 related disease or anti-TIGIT antibody related disease; (g) stopping or slowing the progression of a malignant tumor; (h) stabilizing PD-1 related disease or anti-TIGIT antibody related disease; (i) inhibiting the growth or survival of tumor cells; (j) destruction or reduction in size of one or more malignant lesions or tumors; (k) reducing the progression, onset or severity of PD-1 related disease or anti-TIGIT antibody related disease; (l) reducing the severity or duration of clinical symptoms of a PD-1-related or anti-TIGIT antibody-related disease, such as a malignancy, (m) prolonging patient survival relative to the expected survival for a similar untreated patient, n) inducing complete or partial remission a malignant condition or other PD-1 related or anti-TIGIT antibody related disease, o) treatment of a malignant tumor; or p) treatment of chronic infections.

GENERAL METHODS

Standard molecular biology techniques are described in Sambrook, Fritsch and Maniatis (1982 & 1989 ^2nd Edition, 2001 ^3rd Edition) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ^ed ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA , Vol. 217, Academic Press, San Diego, CA). Standard methods are also presented in Ausbel, et al . (2001) Current Protocols in Molecular Biology, Vols.1-4 , John Wiley and Sons, Inc. New York, NY, which covers bacterial cloning and DNA mutagenesis (Volume 1), cloning in mammalian cells and yeast (Volume 2), glycoconjugates and protein expression (Volume 3), and bioinformatics (Volume 4).

Methods for protein purification have been described, including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization (Coligan, et al . (2000) Current Protocols in Protein Science, Vol. 1 , John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, protein glycosylation are described (see, for example, Coligan, et al . (2000) Current Protocols in Protein Science, Vol. 2 , John Wiley and Sons, Inc., New York ;Ausubel, et al . (2001) Current Protocols in Molecular Biology, Vol. 3 , John Wiley and Sons, NY, NY, pp. 16.0.5-16.22.17; (2001) Products for Life Science Research , St. Louis, MO pp. 45-89; Amersham Pharmacia Biotech , Piscataway, NJ, pp. 384-391). The production, purification and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al . (2001) Current Protocols in Immunology, Vol. 1 , John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard methods for characterizing ligand/receptor interactions are available (see, for example, Coligan, et al . (2001) Current Protocols in Immunology, Vol. 4 , John Wiley, Inc., New York).

Monoclonal, polyclonal and humanized antibodies can be produced (see, for example, Sheperd and Dean (eds.) (2000) Monoclonal Antibodies , Oxford Univ. Press, New York, NY; Kontermann and Dubel (eds.) (2001) Antibody Engineering , Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY, pp. 139-243 ; 6205; He, et al. (1998) J. Chem . 160:1029 ; 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. 224:487-499;

An alternative to humanization is the use of human antibody libraries displayed on phages or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring. Harbor, New York; Kay et al . (1996) Phage Display of Peptides and Proteins: A Laboratory Manual , Academic Press, San Diego, CA (1999) Nature Biotechnol 17:397-399).

Antigen purification is not necessary to obtain antibodies. Animals can be immunized with cells carrying the antigen of interest. Splenocytes can then be isolated from immunized animals, and the splenocytes can be fused with a myeloma cell line to produce a hybridoma (see, for example, Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., higher; Kaithamana et al. (1999) J. Immunol.163:5157-5164).

Antibodies can be conjugated, for example, to small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapy, diagnosis, kits or other purposes, and include antibodies attached, for example, to dyes, radioactive isotopes, enzymes or metals, for example colloidal gold (see, for example, Le Doussal et al. (1991) J Immunol 146:169-175; J Immunol 160 : 3891-3898; J Immunol 162 : 2804-2811; Immunol 168:883-889).

Flow cytometry techniques are available, including fluorescence-activated cell sorting (FACS) (see, for example, Owens, et al . (1994) Flow Cytometry Principles for Clinical Laboratory Practice , John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry , 2nd ^ed .; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry , John Wiley and Sons, Hoboken, NJ). Fluorescent reagents suitable for modifying nucleic acids are available, including nucleic acid primers and probes, polypeptides and antibodies, for use, for example, as diagnostic reagents (Molecular Probesy (2003) Catalog , Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalog , St. Louis, MO).

Standard techniques for the histology of the immune system are described (see, for example, Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology , Springer Verlag, New York, NY; Hiatt, et al . (2000) Color Atlas of Histology . Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al . (2002) Basic Histology: Text and Atlas , McGraw-Hill, New York, NY).

Software packages and databases are available to determine, for example, antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments (see, for example, GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); Menne, et al . (2000) ; ( 2000) Bioinformatics Note 16:741-742; Comput . Methods Biomed 68 :177-181; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

Analytical methods

Analytical methods useful for assessing product stability include size exclusion chromatography (SEC), dynamic light scattering (DLS), differential scanning calorimetry (DSC), iso-asp quantification, activity assay, UV analysis at 340 nm, UV spectroscopy and FTIR . SEC (J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm Bio. Anal., 14:1133-1140 (1986)) measures the percentage of monomer in the product and provides information on the amount of soluble aggregates. DSC (Pharm. Res., 15:200 (1998); Pharm. Res., 9:109 (1982)) provides information on the protein denaturation temperature and glass transition temperature. DLS (American Lab., November (1991)) measures the average diffusion coefficient and provides information on the number of soluble and insoluble aggregates. UV analysis at 340 nm measures the intensity of scattered light at 340 nm and provides information about the approximate amounts of soluble and insoluble aggregates. UV spectroscopy measures absorbance at 278 nm and provides information about protein concentration. FTIR (Eur. J. Pharm. Biopharm., 45:231 (1998); Pharm. Res., 12:1250 (1995); J. Pharm. Scien., 85:1290 (1996); J. Pharm. Scien. , 87:1069 (1998)) measures the IR spectrum in the amide one region, and provides information about the secondary structure of the protein.

The iso-asp content of the samples was measured using an Isoquant isoaspartate detection system (Promega). The kit uses the protein isoaspartyl methyltransferase (PIMT) enzyme to specifically detect the presence of isoaspartic acid residues in a target protein. PIMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to isoaspartic acid at the alpha-carboxyl position, producing S-adenosyl-L-homocysteine (SAH) in the process. It is a relatively small molecule and can usually be isolated and quantified by reverse phase HPLC using the HPLC SAH standards provided in the kit.

The activity or bioidentity of an antibody can be measured by its ability to bind to its antigen. The specific binding of an antibody to its antigen can be quantified by any method known to one skilled in the art, for example, an immunoassay such as an ELISA (enzyme-linked immunosorbent assay).

The contents of all publications mentioned herein are incorporated by reference for the purpose of describing and disclosing methods and materials that can be used in combination with the present invention.

While various embodiments of the invention have been described herein with reference to the accompanying drawings, it is to be understood that the invention is not limited to these precise embodiments and that various changes and modifications thereto can be made by one skilled in the art without departing from the scope or content of the invention. , as defined in the accompanying claims.

EXAMPLES

EXAMPLE 1

Anti-TIGIT Antibody Formulation Buffer Screening

A large-scale formulation development study was conducted for three anti-TIGIT antibodies, where each anti-TIGIT antibody has the following CDRs: HCDR1 from SEQ ID NO:108, HCDR2 from SEQ ID NO:154, HCDR3 from SEQ ID NO:110, LCDR1 from SEQ ID NO: :111, LCDR2 from SEQ ID NO:112 and LCDR3 from SEQ ID NO:113, to assess (1) biophysical/biochemical lability; (2) conditions for obtaining the preformulation (pH, salt and buffer) and (3) compatibility with the platform among the compositions. Samples were analyzed using turbidity UV/Vis spectrophotometry (A350) as a surrogate for larger aggregates, size exclusion chromatography (UP-SEC) to detect high molecular weight molecules, capillary isoelectric focusing (cIEF) to measure the effect of stress on the charge distribution on the surface of a molecule, reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS) for detection of proteolytic cleavage of heavy or light chains, analysis of particles not visible to the naked eye for detection of aggregates not visible to the naked eye.

The large-scale formulation screening included 1 mg/mL anti-TIGIT antibody formulated in a sample of three buffers: Acetate buffer with pH values ranging from 5.0 to 6.2, Citrate buffer with pH values ranging from 5.6 to 6.8 and L-histidine buffer with pH values ranging from 5.0 to 6.8. Thus, pH values ranging from 5.0 to 6.8 and ionic strengths of 0–150 mM NaCl were tested. Samples were stressed at 50°C for 10 days and analyzed for thermal stability using differential scanning fluorimetry (DSF), colloidal stability using size exclusion chromatography (UP-SEC), aggregation susceptibility using Guava (Non Visible Particle Characterization Assay). based on flow cytometry), turbidity (A350 measurements), charge variant profile (cIEF) and fragmentation profile using Caliper.

Based on the results obtained in the study, the formulation that provided maximum protein stability was 10 mM L-histidine in the pH range of 5.6 to 6.2. For 10 mM L-histidine, pH range 5.6 to 6.2 showed minimal aggregation as monitored by UP-SEC, with ΔSEC of the main peak ranging between -1.63 and -1.85% (compared to SEC of the main peaks ranging from -2.0 to -5.0% under different buffer and pH conditions) (data not shown). The cIEF profile showed that there was a decrease in the relative peak area for the main peak and basic molecules, and an increase in the relative peak area of the acidic variants for samples after 10 days at 50°C (cIEF of the main peak ranging between -9.0 and -11.3% ) (data not shown). A decrease in the number of basic variants and an increase in the number of acidic variants upon exposure to elevated temperature is common for mAbs. The addition of salt decreased protein stability among the compositions tested.

EXAMPLE 2

pH Range Study for Anti-TIGIT Antibody Formulation

In this study, an anti-TIGIT antibody having the following CDRs: HCDR1 from SEQ ID NO:108, HCDR2 from SEQ ID NO:154, HCDR3 from SEQ ID NO:110, LCDR1 from SEQ ID NO:111, LCDR2 from SEQ ID NO: :112 and LCDR3 from SEQ ID NO:113 were tested at a concentration of 50 mg/ml in 10 mM L-histidine buffer. 7% (w/v) sucrose was added to the formulation to increase the volumetric stability (as a stabilizer and nonionic tonicity modifier) of the molecule. Anti-TIGIT antibody was formulated in 10 mM L-histidine buffer, 7% sucrose at pH 5.5, pH 6.0 and pH 6.5. The stability of such compositions was assessed as follows:

(1) The stability of the molecule was monitored by thermal stability and storage stability under accelerated aging conditions (5°C, 25°C, and 40°C for up to 6 months), protected from light.

(2) Stability studies were also conducted for freeze-thaw stress and shaking stress.

(3) Shake studies were performed on formulations containing various concentrations of polysorbate 80 (PS-80) to evaluate the concentration of PS-80 in the formulation.

(4) A light stress study was conducted to evaluate the need for L-methionine in the formulations.

Materials and methods

Thermal stability study (3 months)

50 mg/ml anti-TIGIT antibody was formulated in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml polysorbate 80 at pH 5.5, pH 6.0, or pH 6.5. The resulting compositions were sterilized by filtration and filled into 2R vials, closed with chlorobutyl stoppers and protected with aluminum sealing rims. A stepwise stability study was conducted at 5°C (ambient humidity), 25°C (60% relative humidity) and 40°C/(75% relative humidity). Samples were analyzed using UP-SEC, HP-IEX, cIEF (selected samples), MFI, CE-SDS (non-reducing “NR” and reducing “R”), reducing peptide mapping (MFI and reducing peptide mapping were performed at selected time points) .

A 1-month thermostability study was designed for 25 mg/ml anti-TIGIT antibody formulated in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml PS-80, pH 6.0. The resulting compositions were sterilized by filtration and filled into 2R vials, closed with chlorobutyl stoppers and protected with aluminum sealing rims. A staggered stability study was conducted at 5°C (ambient humidity), 25°C (60% relative humidity) and 40°C/(75% relative humidity) for one month. Samples were analyzed using UP-SEC, cIEF and CE-SDS (NR and R).

Shaking Stability Study

50 mg/ml anti-TIGIT antibody was formulated in 10 mM L-histidine buffer, 7% sucrose, pH 6.0 with various concentrations of polysorbate 80 (0, 0.1, and 0.2 mg/ml). The resulting compositions were sterilized by filtration and filled into 2R vials (filling volume 1.2 ml), closed with chlorobutyl stoppers and protected with aluminum sealing rims. Samples were shaken horizontally at 300 rpm for up to 7 days at 18-22°C. The samples were analyzed using UP-SEC, MFI, CE-SDS (NR and R).

Freeze-thaw stability

50 mg/ml anti-TIGIT antibody was formulated in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml polysorbate 80, pH 6.0. The resulting compositions were sterilized by filtration and filled into 2R vials, closed with chlorobutyl stoppers and protected with aluminum sealing rims. Samples were subjected to 5 freeze-thaw cycles from -80°C to 18-22°C (at least 24 hours in freezing conditions and at room temperature until completely thawed). Samples were analyzed using UP-SEC, MFI, CE-SDS (NR and R).

Stability study under light stress

Early development studies indicated the presence of an exposed tryptophan residue, as well as several methionine residues susceptible to oxidation under light stress. Studies were designed under visible light ICH light stress conditions (CWF, 0.1x ICH, 0.2x ICH, 0.5x ICH, 1x ICH) in formulations in the presence and absence of L-methionine (composition 1: 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml polysorbate 80, pH 6.0, and composition 2: 10 mM L-histidine buffer, 10 mM L-methionine, 7% sucrose, 0.2 mg/ml polysorbate 80, pH 6.0). Samples were analyzed using UP-SEC, cIEF, CE-SDS (NR and R), and restorative peptide mapping.

results

Results for Thermal Stability

Formulation at 50 mg/ml: For all stability assays tested, no significant changes were observed at 5°C at all pH values after 3 months (data not shown). At 25°C and 40°C, pH 5.5 and pH 6.0 showed similar stability, and these conditions resulted in greater stability than pH 6.5 (data not shown). Degradation rates at pH 6.5 for the UP-SEC, CE-SDS (NR) and cIEF assays at 25°C and 40°C were relatively higher than those observed at pH 5.5 and pH 6.0, as well as compared to a reference molecule. Thus, a pH range of 5.5 to 6.0 (pI 8.7) was considered acceptable. No oxidation, deamidation or isomerization was observed for the anti-TIGIT antibody after 3 months at all temperatures and pH values.

Formulation at 25 mg/ml: Degradation was observed at 25°C and 40°C for all assays tested. Degradation rates were found to be similar to those under 50 mg/ml conditions (see above).

Shaking Stability Study

For formulations not containing polysorbate 80, visible particles are shown at the end of 7 days. Analysis of particles not visible to the naked eye showed that the number of particles of 10 μm or greater was significantly reduced in the formulation containing polysorbate 80 at a concentration of 0.2 mg/ml. No significant differences between samples were observed using other assays.

Freeze-thaw stability

No changes in the stability of the molecule were observed after 5 freeze-thaw cycles in all assays tested.

Stability study under light stress

Degradation was observed when samples of both formulations were exposed to light stress at 0.5x ICH or higher when tested by UP-SEC, cIEF, CE-SDS (NR and R). When conditions were set below 0.5x ICH, no significant degradation was shown for either formulation. Reductive peptide mapping data under light stress conditions at 0.5x and above showed oxidation of tryptophan and methionine residues. 10 mM L-methionine in the formulation reduced the levels of oxidation of methionine residues, but did not affect the levels of tryptophan oxidation.

Conclusion

Based on the above, 10 mM L-histidine buffer, 10 mM L-methionine, 7% sucrose, 0.2 mg/ml polysorbate 80, pH 5.5-6.0, were considered adequate to impart stability to support refrigerated storage .

EXAMPLE 3

Additional pH studies

An anti-TIGIT antibody having the following CDRs: HCDR1 from SEQ ID NO:108, HCDR2 from SEQ ID NO:154, HCDR3 from SEQ ID NO:110, LCDR1 from SEQ ID NO:111, LCDR2 from SEQ ID NO:112, and LCDR3 from SEQ ID NO:113, and on the IgG1 backbone, were formed in six 10 mM histidine buffers with different pH (ranging from 5.0 to 6.5). Thermal stability of various formulations was tested at 2~8°C, 25°C and 40°C for 8 weeks.

Compound Anti-TIGIT antibody Buffer pH Sucrose (% w/v) Polysorbate 80 (% w/v) 1 50 mg/ml 10 mM L-histidine pH=5.0 7% 0.02% 2 50 mg/ml 10 mM L-histidine pH=5.3 7% 0.02% 3 50 mg/ml 10 mM L-histidine pH=5.6 7% 0.02% 4 50 mg/ml 10 mM L-histidine pH=5.9 7% 0.02% 5 50 mg/ml 10 mM L-histidine pH=6.2 7% 0.02% 6 50 mg/ml 10 mM L-histidine pH=6.5 7% 0.02%

Histidine buffers of different pH (5.0~6.5) were prepared by titrating 10 mM L-histidine buffer to 10 mM L-histidine-HCl buffer. The anti-TIGIT antibody buffer was changed to six different histidine buffers with different pHs through four to five cycles of ultrafiltration using a centrifugation device at 4°C and 4500 rpm~5000 rpm (105~260 min in each cycle ). After buffer exchange, a specified amount of sucrose and polysorbate 80 storage solution (1%, w/w) were added to the different pH solutions to achieve the target amount, and an appropriate amount of the appropriate histidine buffer was added so as to bring the antibody concentration to approximately 50 mg/ml.

The formulations were then aseptically filtered using a 0.22 μm membrane filter. 3 ml of each sample was aseptically filled into 6 ml glass vials for thermal stability studies at T0, 4 weeks (4H) and 8 weeks (8H). 1 ml of each sample was aseptically filled into 6 ml glass vials for thermal stability testing for two weeks (2H) thermal stability testing. The filled vials were capped and hermetically sealed immediately after filling. All of the above stages were carried out in a laminar flow hood. These vials were placed in closed boxes and stored at different temperatures for thermal stability studies.

Results and discussion

The appearance of all samples remained the same for four weeks under all conditions.

However, after 8 weeks, samples at 2~8°C and 25°C showed a slight yellowish coloration, and samples at 40°C showed a deeper yellowish coloration. All samples were slightly opalescent and free of visible particles throughout the study period. No significant protein concentration measurements were observed in all samples throughout the study.

The colloidal stability of the samples was assessed by purity by size exclusion chromatography (SEC), in which the percentage of monomer was determined, as well as the percentages of high molecular weight molecules (HMW) and late elution peaks (LMW molecules). Analysis was performed using an Agilent 1260 Infinity system with a TSKGel G3000SWXL size exclusion chromatography column (300 × 7.8 mm, 5 µm). The mobile phase was 50 mM PB, 300 mM NaCl, pH 7.0 ± 0.2, and the flow rate was set to 1.0 mL/min. Samples were diluted to 10 mg/mL for injection and detected at 280 nm using a UV detector.

UPSEC details are given in the table below:

2-8 ° C 25 ° C 40 ° C pH T0 2H 4H 8H 2H 4H 8H 2H 4H 8H Main peak, % 5 97.7 97.5 97.4 97.5 97.7 97.4 97 97.2 96 94.5 5.3 97.7 97.5 97.7 97.4 97.6 97.2 96.8 96.5 96.2 94.6 5.6 97.6 97.4 97.6 97.3 97.1 97.1 96.8 97 96.1 94.8 5.9 97.5 97.3 97.5 97.3 97.3 97 96.5 96.2 96 94.7 6.2 97.3 97.2 97.5 97.1 97.1 96.8 96.5 96.4 95.7 94.3 6.5 97.3 97.1 97.4 97.1 96.9 96.6 96.2 95.8 95.5 94.6 %HMW 5 2.3 2.5 2.6 2.5 2.3 2.4 2.8 2.4 2.8 3.4 5.3 2.3 2.5 2.3 2.6 2.4 2.5 2.9 2.8 2.9 3.7 5.6 2.4 2.6 2.4 2.7 2.7 2.7 3 2.8 3.2 3.9 5.9 2.5 2.7 2.5 2.7 2.7 2.8 3.2 3.3 3.3 4.1 6.2 2.7 2.8 2.5 2.9 2.9 3 3.3 3.3 3.6 4.6 6.5 2.7 2.9 2.6 2.9 3.1 3.2 3.5 3.7 3.9 4.3 %LMW 5 N.D. N.D. N.D. N.D. N.D. 0.2 0.3 0.4 1.3 2.1 5.3 N.D. N.D. N.D. N.D. N.D. 0.2 0.2 0.6 0.8 1.7 5.6 N.D. N.D. N.D. N.D. 0.2 0.2 0.2 0.2 0.7 1.3 5.9 N.D. N.D. N.D. N.D. N.D. 0.2 0.3 0.5 0.7 1.2 6.2 N.D. N.D. N.D. N.D. N.D. 0.2 0.2 0.3 0.7 1.1 6.5 N.D. N.D. N.D. N.D. N.D. 0.2 0.2 0.5 0.7 1.1

As shown in the table above, the SEC main peak % was stable at 2~8°C in all samples, however, at 25°C and 40°C, a significant decrease in the main peak % was observed. The rate of decrease in % of the main peak was higher in samples at 40°C than at 25°C. At 40°C for eight weeks, %HMW was greater in samples at pH 6.2 and 6.5, while %LMW was greater in samples at pH 5.0 and 5.3.

To assess the chemical stability of the formulations, capillary isoelectric focusing (cIEF) was performed to assess chemical stability and to monitor the change in charge variant profile over time. Briefly, 20 μL (2.0 mg/mL) of reference standard or sample was mixed with 0.5 μL of pI 5.85 marker, 0.5 μL of pI 9.77 marker, 1 μL of Farmalite 3-10, 0.5 µl of farmalit 5-8, 0.5 µl of farmalit 8-10.5, 35 µl of 1% methylcellulose, 37.5 µl of 8 M urea. Purified water was added to bring the final volume to 100 μL. The mixture was then analyzed using an iCE-3 capillary isoelectric focusing analyzer equipped with a fluorocarbon coated detector along the entire length of the capillary. Focusing was carried out in two stages: (1) 1.5 kV for 1 min and (2) 3 kV for 8 min. Throughout the experiment, the autosampler rack was maintained at 5°C.

The cIEF data for assessing the levels of acidic variants, % of the main peak and % of the main variants are shown in the table below.

2-8 ° C 25 ° C 40 ° C pH T0 2H 4H 8H 2H 4H 8H 2H 4H 8H Main peak, % 5 62.2 63 60 62.6 62.8 57.6 58.4 50.2 37.5 29.1 5.3 62.2 63 59.9 62.4 62.8 57.7 58.6 50.6 39.1 30.2 5.6 62.3 62.8 59.9 62.8 62.6 58.1 58.7 51.9 41.4 31 5.9 62 62.9 59.7 62.5 62.8 58.1 58.4 51.7 41.2 32.4 6.2 62.1 62.8 59.6 62.2 62.5 57.5 58.6 51.7 41.8 31.8 6.5 62.2 62.9 59.4 62.5 61.9 56.1 57.4 51.1 40.4 33.2 Sour, % 5 31.7 30.3 32.7 30.2 30.5 33.5 33.9 39.7 49.2 58.2 5.3 31.8 30.6 32.3 30.6 30.5 34 34.1 40.3 49.5 59.4 5.6 31.4 30.8 32.6 30.3 30.8 34.2 34.3 39.8 48.9 59.5 5.9 31.9 30.7 33.1 30.6 30.9 34.3 34.8 40.7 48.8 59.1 6.2 32.1 30.8 33.5 31.2 31.3 34.5 34.6 41 49 59.7 6.5 31.5 31 33.3 31 31.9 35.5 35.8 41.5 50.3 58.5 Basic, % 5 6 6.7 7.3 7.2 6.8 8.9 7.7 10 13.3 12.6 5.3 6 6.4 7.7 7 6.7 8.3 7.3 9.1 11.3 10.3 5.6 6.2 6.5 7.5 6.8 6.5 7.6 7.2 8.2 9.6 9.5 5.9 6.1 6.4 7.3 6.9 6.3 7.5 6.7 7.6 9.9 8.4 6.2 5.9 6.4 6.9 6.6 6.3 8 6.8 7.2 9.3 8.6 6.5 6.3 6.2 7.2 6.4 6.2 8.4 6.8 7.3 9.4 8.3

As can be seen in the table above, at 2~8°C, the % of main peak, % of acidic peak and % of main peak by cIEF were relatively stable and comparable in all samples.

At 2~8°C, the cIEF % of the main peak also increased; the increase in samples at buffer pH 5.0 and 5.3 was higher than in other samples. At 25°C, % main peak, % acidic peak and % basic peak were stable during the first two weeks, but changed slightly after four weeks, where % main peak decreased while % acidic peak increased respectively). The rate of change in different compositions was comparable. At 40°C, a significant decrease in the % of the main peak and a noticeable increase in the % of the acid peak were found in all formulations even after two weeks, however, the degree of change was similar in each formulation. The % of the main peak also increased; the increase in samples in pH 5.0 and 5.3 buffer was greater than in other samples.

To assess the purity of the formulations, a non-reducing Caliper assay was performed. Briefly, purchased sample buffer was mixed with 10% sodium dodecyl sulfate (SDS) solution in a volume ratio of 20 to 1, and 100 mM N-ethylmaleimide solution was added to the mixed solution in a volume ratio of 0.7 to 20 (relative to the sample denaturation solution). The standard or sample was first diluted to 1 mg/mL, and 2 μL of the diluted standard or sample was mixed with 7 μL of sample denaturation solution. The mixture was incubated at 70°C for 10 min. 35 μL of purified water was added to the incubation solution, and 42 μL of the mixed solution was transferred to a 96-well plate for analysis. The sample plate was analyzed on a LabChip GX II HT using the HT Antibody Analysis 200.

The Caliper non-reducing assay data for estimating % purity is shown in the table below:

2-8 ° C 25 ° C 40 ° C pH T0 2H 4H 8H 2H 4H 8H 2H 4H 8H Non-regenerating Caliper Purity, % 5 96 95.2 94.6 94.9 94.9 93.6 94.1 91.4 87.8 84.4 5.3 96 95 94.7 94.9 94.8 93.8 94.1 92.7 89.5 86.4 5.6 96.1 95 94.6 94.9 94.8 93.9 94.1 93 90.7 88.5 5.9 96.3 94.9 94.5 94.9 94.8 93.9 94.2 93.4 91.1 89.3 6.2 96.2 94.9 94.5 95 94.8 93.7 94.5 93.3 91.3 88.7 6.5 96 95.1 94.4 94.9 94.7 93.4 94.1 93.2 90.7 89.3

As shown in the table above - at 2~8°C for eight weeks, the non-reducing Caliper purity of each formulation was relatively stable. At 25°C for eight weeks, the purity of each formulation decreased slightly. At 40°C, the purity decreased significantly, especially when the samples were in pH 5.0 and 5.3 buffer, the decrease was much faster than for the others. The molecular size was stable throughout the study (data not shown).

To further evaluate the purity of the formulations, Caliper reduction analysis was also performed. Briefly, purchased sample buffer was mixed with 10% SDS solution in a volume ratio of 20 to 1, and 1 M dithiothreitol solution was added to the mixed solution in a volume ratio of 0.7 to 20 (relative to the sample denaturation solution). The standard or sample was first diluted to 1 mg/mL, and 2 μL of the diluted standard or sample was mixed with 7 μL of sample denaturation solution. The mixture was incubated at 70°C for 10 min. 35 μL of purified water was added to the incubation solution, and 42 μL of the mixed solution was transferred to a 96-well plate for analysis. The sample plate was analyzed on a LabChip GX II HT using the HT Antibody Analysis 200.

The Caliper recovery assay data for estimating % purity is shown in the table below:

2-8 ° C 25 ° C 40 ° C pH T0 2H 4H 8H 2H 4H 8H 2H 4H 8H Purity, % (Caliper_recovery) 5 99.4 99.4 99.3 99.2 99.3 99.1 98.9 98 97.1 91.3 5.3 99.4 99.4 99.2 99.2 99.3 99 99 98.5 97.7 94.9 5.6 99.4 99.4 99.3 99.1 99.3 99.2 98.8 98.7 98.2 96.8 5.9 99.4 99.4 99.2 99.1 99.2 99.2 98.8 99.1 98.6 96.8 6.2 99.3 99.3 99.3 99.2 99.3 99.2 98.9 98.9 98.7 96.9 6.5 99.3 99.4 99.2 99.1 99.3 99.2 98.8 98.9 98.5 97.5

As can be seen above, at 2~8°C and 25°C for eight weeks, the Reducing Caliper purity of each formulation was relatively stable. At 40°C, a clear decrease in purity according to Caliper_recovery. found in all compositions. The purity of samples in pH 5.0 buffer decreased the most, followed by samples in pH 5.3 buffer. The rate of decrease in buffers pH 5.6, 5.9 and 6.2 was comparable but slower. The purity of samples in pH 6.5 buffer decreased most slowly. The decrease in purity was likely due to a decrease in heavy chain (HC) %, while light chain (LC) % was stable in the study. The size of the light chain and heavy chain of the antibody was stable in all samples over 8H.

EXAMPLE 4

Composition of anti-TIGIT antibody without methionine

An anti-TIGIT antibody having the following CDRs: HCDR1 from SEQ ID NO:108, HCDR2 from SEQ ID NO:154, HCDR3 from SEQ ID NO:110, LCDR1 from SEQ ID NO:111, LCDR2 from SEQ ID NO:112, and LCDR3 from SEQ ID NO:113, tested at a concentration of 50 mg/ml in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml PS-80 with a pH ranging from 5.0 to 6.5. The stability of the molecule was monitored by thermal stability and stability during storage under accelerated aging conditions, protected from light. In addition to thermal stability, freeze-thaw stability, shaking stability, and light stress stability were also studied. Stability testing included UP-SEC, cIEF, CE-SDS, MFI, and restorative peptide mapping.

results

Thermal stability study (8 weeks)

For all stability assays tested, the anti-TIGIT antibody was stable at 5°C for all labilities tested. Degradation rates as observed using UP-SEC, Caliper CE-SDS, cIEF, MFI were higher at 40°C than at 25°C. At 25°C and 40°C, the following results were noted:

UP-SEC: A decrease in % monomer was observed for all pH values from 5.0 to 6.5. At lower pH values (5.0 and 5.3), the decrease in the main peak was primarily due to an increase in % low molecular weight (LMW) molecules, while the decrease in % monomer at pH 6.5 was primarily due to % LMW ( HMW) molecules. The decrease in % monomer was highest at pH 6.5 after 8 weeks of stability analysis under accelerated aging conditions. (data not provided)

cIEF: A decrease in the main cIEF peak was observed for all pH values from 5.0 to 6.5 at 25°C and 40°C. At higher pH values (6.3-6.5), the decrease in the main peak was mainly due to an increase in the content of acidic variants, while at pH 5.0 and 5.3, the decrease in the main peak was due to an increase in the content of both acidic variants. , and the main options. (data not provided)

CE-SDS (Caliper): With non-reducing CE-SDS, a decrease in the main peak was mainly observed at 40°C, due to the presence of fragmented molecules. Formulations at lower pH values (5.0 and 5.3) showed relatively higher fragmentation rates than the other pH values.

CE-SDS reductive analysis (Caliper) revealed that at 5°C and 25°C for eight weeks, the purity of each formulation was relatively stable. At 40°C, a clear decrease in purity was found in all formulations. The purity of samples in pH 5.0 buffer decreased the most, followed by samples in pH 5.3 buffer.

MFI: An increase in particles not visible to the naked eye was observed for all formulations at 40°C. At pH 6.3 and 6.5 there was the highest increase in particles not visible to the naked eye relative to the other buffers.

Reductive Peptide Mapping: Among the identified labilities, only M254 showed a relative increase in oxidation after 8 weeks at 40°C compared to the parent samples.

Conclusion: 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml PS-80, pH 5.6-6.3 adequately maintained storage stability of anti-TIGIT antibody for 8 weeks.

Shaking Stability Study

No changes were observed in the content of soluble aggregates, charged variants, fragmentation, or particles not visible to the naked eye when formulated with 50 mg/ml anti-TIGIT antibody (in 10 mM L-histidine buffer, 7% sucrose, pH 5.8, and any of 0.0 1, 0.2 and 0.3 mg/ml PS-80) were subjected to moderate shaking for up to 7 days at 100 rpm at 18-22°C.

Freeze-thaw stability

No changes in soluble aggregates, charged variants, fragmentation, or particles not visible to the naked eye were observed when 50 mg/ml anti-TIGIT antibody (in 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml PS-80, pH 5,8) were subjected to 5 freeze/thaw cycles (freezing at -80°C for 2 hours and thawing at room temperature for 1 hour).

Stability study under light stress

The formulation at 50 mg/ml was subjected to visible light stress (5000 lux) for 48 hours. Under these conditions, there was minimal change in soluble aggregates, charged variants, particles not visible to the naked eye, pH, concentration, fragmentation, and oxidation under ~0.2x ICH conditions (12 hour light exposure). Under ~1x conditions (48 hour light exposure), there was an increase in soluble aggregates, acidic variants, fragmentation, particles not visible to the naked eye, and methionine oxidation.

Conclusion

Based on these studies, 10 mM L-histidine buffer, 7% sucrose, 0.2 mg/ml PS-80 pH 5.3-6.3 were able to maintain the stability of the anti-TIGIT antibody. Methionine oxidation was observed upon exposure to severe light stress. As noted in Example 2, the addition of 10 mM L-methionine reduces the oxidation of methionine residues.

EXAMPLE 5

Polysorbate 80 screening

For an anti-TIGIT antibody having the following CDRs: HCDR1 from SEQ ID NO:108, HCDR2 from SEQ ID NO:154, HCDR3 from SEQ ID NO:110, LCDR1 from SEQ ID NO:111, LCDR2 from SEQ ID NO:112 and LCDR3 from SEQ ID NO:113, four (4) formulations were prepared at pH 5.8, in 10 mM L-histidine buffer, and with various concentrations of PS-80 (as shown below). The stability of the protein in various formulations was studied under conditions with or without shaking for a period of 7 days at 20°C.

Compound Anti-TIGIT antibody Buffer pH Sucrose (% w/v) Polysorbate 80 (mg/ml) 1 50 mg/ml 10 mM L-histidine pH=5.8 7% 0 mg/ml 2 50 mg/ml 10 mM L-histidine pH=5.8 7% 0.1 mg/ml 3 50 mg/ml 10 mM L-histidine pH=5.8 7% 0.2 mg/ml 4 50 mg/ml 10 mM L-histidine pH=5.8 7% 0.3 mg/ml

Formulations were prepared in 10 mM L-histidine buffer at pH 5.8 using a laboratory scale TFF buffer exchange system. Proteins formulated with varying levels of polysorbate 80 were then aseptically filtered using a 0.22-μm membrane filter. 2 ml of each sample was aseptically filled into 6 ml glass vials. Filled vials were capped and crimped immediately after filling. The samples were divided into shaking group and non-shaking group. In the shaking group, these vials were transferred into closed boxes and then placed in a thermostatic shaker and shaken at 100 rpm, 20°C for up to 7 days. In the no-shaking group, these vials were transferred into closed boxes and placed in a thermostatic shaker, but the shaker was not turned on at 20°C for up to 7 days.

The stability of the antibody in various formulations in the presence or absence of shaking was studied after 3 and 7 days.

UPSEC data for assessing High Molecular Molecular Weight (HMW or Aggregates), % Monomer and LMW (Low Molecular Weight) levels are presented in the table below:

3 days 7 days Compound T0 No shaking 100 rpm No shaking 100 rpm Main peak, % 1 97.1 97.2 97.2 97.1 97.2 2 97 97.1 97.1 97 97 3 97 97.1 97.1 96.9 96.9 4 97.1 97.1 97.1 96.9 96.9 High molecular weight (HMW), % 1 2.9 2.8 2.8 2.9 2.8 2 3 2.9 2.9 3 3 3 3 2.9 2.9 3.1 3.1 4 2.9 2.9 2.9 3.1 3.1 Low molecular weight (LMW), % 1 N.D. N.D. N.D. N.D. N.D. 2 N.D. N.D. N.D. N.D. N.D. 3 N.D. N.D. N.D. N.D. N.D. 4 N.D. N.D. N.D. N.D. N.D.

As can be seen, polysorbate 80 content did not have a significant effect on SEC purity under shaking or non-shaking conditions. Polysorbate 80 content had no significant effect on the pI, percentage of major peak, acidic peak, and major peak in the cIEF assay, with or without shaking, up to 7 days.

3 days 7 days T0 No shaking 100 rpm No shaking 100 rpm Main peak, % 1 61.5 61.4 61.4 62.4 62.3 2 61.5 61.5 61.9 61.9 62.1 3 61.2 60.8 60.5 61.5 61.6 4 61.9 61.4 61.5 62 61.8 Sour, % 1 30.9 31.1 30.5 31.1 30.6 2 31.2 31.6 30.5 31.3 31.1 3 31.5 32.2 32.3 31.5 31.6 4 31.1 31.8 31.4 31.4 31.5 Basic, % 1 7.6 7.5 8.1 6.5 7.1 2 7.3 6.9 7.6 6.8 6.7 3 7.3 7 7.2 6.9 6.8 4 7 6.7 7 6.7 6.7

Polysorbate 80 content had no significant effect on non-reducing Caliper purity, with or without shaking, up to 7 days, as shown in the table below.

3 days 7 days Compound T0 No shaking 100 rpm No shaking 100 rpm Non-regenerating Caliper Purity, % 1 94.9 94.8 95 94.4 94.5 2 94.9 94.8 94.6 94.7 94.4 3 94.7 94.8 94.8 94.3 94.4 4 94.9 94.8 94.6 94.3 94.3

Caliper reduction analysis was also performed. PS-80 content had no significant effect on reducing Caliper purity, with or without shaking, over 7 days.

3 days 7 days Compound T0 No shaking 100 rpm No shaking 100 rpm Recovery Caliper Purity, % 1 99.2 99.2 99.2 99.2 99.2 2 99.2 99.3 99.2 99.2 99.2 3 99.1 99 99.2 99.2 99.2 4 99.2 99.1 99.2 99.2 99.1

To measure particles not visible to the naked eye, approximately 1500 μL of each sample was removed from the glass vial container and tested by micro-flow imaging (MFI), according to the user manual. Particle concentrations in various size ranges have been recorded, including 1~2 µm, 2~5 µm, 5~10 µm, 10~25 µm and >25 µm (see below). Polysorbate 80 content had no significant effect on particle concentration in the presence or absence of shaking up to 7 days.

3 days 7 days Compositions T0 No shaking 100 rpm No shaking 100 rpm 1 µm≤ ECD < 2 µm 1 8318 N.A. 9080 1205 2046 2 25396 10831 4429 7668 5898 3 8048 2660 15877 2952 4932 4 8867 2590 13115 9214 2728 2 µm≤ ECD < 5 µm 1 1792 4516 2700 140 574 2 6068 4215 711 1137 806 3 818 363 3950 927 735 4 1321 297 3476 2418 310 5 µm ≤ ECD < 10 µm 1 369 N.A. 486 27 52 2 770 647 79 237 63 63 3 121 63 379 121 94 4 104 80 253 167 61 10 µm ≤ ECD < 25 µm 1 78 N.A. 103 2 22 2 135 73 7 59 5 3 39 13 66 20 8 4 14 25 34 16 8 ECD ≥ 25 µm 1 0 N.A. 6 0 2 2 7 14 3 21 15 3 10 10 10 2 2 4 2 6 12 0 12

EXAMPLE 6

Adding a chelating agent

This study compared the stability of an anti-TIGIT antibody having the following CDRs: HCDR1 from SEQ ID NO:108, HCDR2 from SEQ ID NO:154, HCDR3 from SEQ ID NO:110, LCDR1 from SEQ ID NO:111, LCDR2 from SEQ ID NO: :112 and LCDR3 from SEQ ID NO:113, in 10 mM L-histidine buffer (pH=5.8), 0.02% (w/v) polysorbate 80, 10 mM L-methionine (“L-Met”) "), 7% w/v. sucrose in the presence or absence of 20 μM or 50 μM DTPA.

The three formulations were filled into vials and staged stability studies were conducted at 5°C (ambient humidity), 25°C (60% relative humidity), and 40°C (75% relative humidity) for eighteen weeks protected from light.

Compound Anti-TIGIT antibody L-histidine buffer (pH 5.8) Sucrose % (wt./vol.) L-Met Polysorbate 80% (w/v) DTPA 1 50 mg/ml 10 mM 7% 10 mM 0.02% 0 2 50 mg/ml 10 mM 7% 10 mM 0.02% 20 µM DTPA 3 50 mg/ml 10 mM 7% 10 mM 0.02% 50 µM DTPA

The colloidal stability of the samples was assessed by purity by size exclusion chromatography (SEC), in which the percentage of monomer was determined, as well as the percentages of high molecular weight molecules (HMW) and late elution peaks (LMW molecules). UPSEC data for estimating the levels of %HMW (aggregates), %monomer and %LMW are presented in the table below:

5 ° C 25 ° C 40 ° C Compound T0 4H 8H 18N 4H 8H 18N 2H 4H 8H 18N %HMW 1 1.54 N/A 1.57 1.57 1.53 1.57 1.64 1.51 1.61 1.72 2.2 2 1.57 1.59 1.59 1.57 1.56 1.57 1.59 1.51 1.56 1.65 1.94 3 1.61 1.61 1.61 1.58 1.56 1.57 1.59 1.53 1.57 1.63 1.91 % monomer 1 98.2 N/A 98.2 98.2 98.1 98.0 97.8 97.9 97.6 97.2 95.7 2 98.2 98.1 98.1 98.2 98.1 98.0 97.9 98.0 97.7 97.3 96.1 3 98.1 98.1 98.1 98.2 98.1 98.0 97.9 98.0 97.7 97.4 96.2 %LMW 1 0.30 N/A 0.28 0.22 0.36 0.42 0.54 0.55 0.82 1.11 2.07 2 0.28 0.28 0.27 0.22 0.35 0.40 0.55 0.53 0.77 1.02 1.94 3 0.25 0.27 0.26 0.23 0.35 0.41 0.56 0.51 0.77 1.02 1.92

As shown in the table above, at 5°C, 25°C and 40°C, all three formulations showed a trend towards an increase in % HMW peak and % LMW peak (and therefore a decrease in monomer peak %), up to time point 18 weeks At 25°C, both formulations showed similar trends but smaller changes compared to 40°C. At 5°C, no significant changes were observed. Formulation 1 showed a greater increase in %HMW and %LMW compared to Formulation 2 (20 μM DTPA) and Formulation 3 (50 μM DTPA). In addition, formulation 1 showed a greater reduction in % monomer compared to formulations 2 and 3. Similar results were observed in the HP-IEX analysis (data not shown).

To evaluate whether DTPA could protect the formulations from oxidative stress, three formulations were filled into vials and exposed to light (0.5X ICH and 1X ICH). As can be seen in the table below, formulation 1 showed a greater increase in % oxidation of M254, M430 and W104 (oxidation-sensitive methionine and tryptophan residues) compared to formulation 2 (20 µM DTPA) and formulation 3 (µM DTPA). Thus, DTPA may further improve the stability of the anti-TIGIT antibody formulation.

Composition 1 Composition 1 Composition 2 Composition 3 Control in the dark 0.5X ICH 1X ICH 0.5X ICH 1X ICH 0.5X ICH 1X ICH LC_M4 0.2 0.2 0.2 0.2 0.2 0.2 0.2 HC_M34 0.3 0.3 0.3 0.3 0.3 0.3 0.4 HC_M81 0.2 0.2 0.2 0.2 0.2 0.2 0.3 HC_M254 3.6 16.3 30.2 13.7 27.5 15.9 21.5 HC_M430 1 10.9 19.2 9.3 18.3 8.6 16.2 HC_W104 0.6 8.8 17.7 7.3 16.7 6.9 12.1 5 ° C 25 ° C 40 ° C Compound T0 4H 8H 18N 4H 8H 18N 2H 4H 8H 18N Conc. PS 80 1 0.24 0.23 0.23 0.22 0.20 0.18 0.16 0.19 0.17 0.16 0.13 2 0.24 0.23 0.23 0.21 0.20 0.18 0.16 0.19 0.16 0.16 0.14 3 0.22 0.22 0.22 0.21 0.19 0.17 0.15 0.18 0.16 0.15 0.13

EXAMPLE 7

Long-term stability of anti-TIGIT antibody formulations

This example describes long-term stability data for an anti-TIGIT antibody formulated in L-histidine buffer, L-methionine, sucrose, polysorbate 80 and water for injection as follows:

Ingredients Concentration (mg/ml) Quantity (mg/vial) Active Anti-TIGIT antibody 50.0 100.0 Inactive (excipients) L-histidine 0.465 0.930 L-histidine monohydrochloride monohydrate 1.47 2.94 L-Methionine 1.49 2.98 Sucrose 70 140.0 Polysorbate 80 0.20 0.40 Water for injection N.A. Quantity sufficient for 2.0ml

The formulations were filled into USP Type 1 glass vials with an elastomeric stopper and an aluminum seal rim. The vials were then incubated under three different storage conditions: 5°C (ambient humidity), 25°C (60% relative humidity), and 40°C (75% relative humidity). Data were collected at time zero, 1 month, 3 months, 6 months for all storage conditions, 9 months (for storage conditions at 5°C and 25°C), 12 months (for storage conditions at 5°C and 25°C) , 18 months (for storage conditions at 5°C), 24 months (for storage conditions at 5°C) and 36 months (for storage conditions at 5°C).

results

The results show the overall physical and chemical stability of the anti-TIGIT antibody when stored under recommended long-term storage conditions at 5°C for 18 months. No measurable loss of activity was observed and purity was within specifications under recommended storage conditions. The results are shown in the following tables:

5 ° C/ambient humidity Measured characteristic Time point (month) Originally 1 3 6 9 12 18 Biological activity by binding ELISA 96 92 100 94 101 92 99 Purity according to UPSEC, % High molecular weight molecules (%) 1.33 1.48 1.40 1.63 1.65 1.66 1.77 Monomer (%) 98.7 98.5 98.6 98.3 98.3 98.3 * Low molecular weight molecules (%) <QL <QL <QL <QL <QL <QL <QL Charge options according to HP-IEX, % Sour options 21.46 21.54 22.04 22.51 22.49 22.48 22.92 Total number of main peak 68.8 68.4 67.9 67.3 67.1 67.3 67.1 Main options 9.70 10.11 10.06 10.15 10.38 10.20 9.95 Purity according to non-reducing CE-SDS, % 96.4 96.4 96.3 96.2 96.0 95.9 95.7 Purity according to reducing CE-SDS, % 98.1 98.2 98.0 98.1 98.0 98.1 97.7 pH 6.1 6.1 6.1 6.0 6.0 6.0 6.0 Protein concentration 51.7 51.4 51.2 51.0 51.5 50.9 51.5 UV A350 0.150 0.142 0.138 0.144 0.146 0.145 0.150 QL=Quantification Limit (0.4%)
* value is 98.2 25 ° C/60% relative humidity Measured characteristic Time point (month) Originally 1 3 6 9 12 Biological activity by binding ELISA 96 90 94 94 98 95 Purity according to UPSEC, % High molecular weight molecules (%) 1.33 1.57 1.48 1.72 1.77 1.83 Monomer (%) 98.7 98.4 98.3 97.9 97.7 97.4 Low molecular weight molecules (%) <QL <QL <QL <QL 0.57 0.77 Charge options according to HP-IEX, % Sour options 21.46 23.19 28.74 34.16 39.86 44.04 Total number of main peak 68.8 65.8 59.7 53.8 47.6 43.5 Basic options 9.70 11.06 11.51 12.02 12.55 12.44 Purity according to non-reducing CE-SDS, % 96.4 96.0 95.2 94.4 93.2 92.1 Purity according to reducing CE-SDS, % 98.1 98.0 97.8 97.4 96.7 96.1 pH 6.1 6.1 6.1 6.0 6.0 6.0 Protein concentration 51.7 51.1 51.1 50.9 51.5 50.9 UV A350 0.150 0.150 0.163 0.179 0.196 0.209 QL=Quantification Limit (0.4%) 40 ° C/75% relative humidity Measured characteristic Time point (month) Originally 1 3 6 ^s 9 12 Biological activity by binding ELISA 96 96 99 94 ND ND Purity according to UPSEC, % High molecular weight molecules (%) 1.33 1.64 1.78 2.63 ND ND Monomer (%) 98.7 97.9 96.9 93.8 ND ND Low molecular weight molecules (%) <QL 0.43 1.28 3.53 ND ND Charge options according to HP-IEX, % Sour options 21.46 38.03 61.62 80.02 ND ND Total number of main peak 68.8 47.8 25.1 10.0 ND ND Basic options 9.70 14.17 13.29 9.95 ND ND Purity according to non-reducing CE-SDS, % 96.4 93.6 88.9 79.6 ND ND Purity according to reducing CE-SDS, % 98.1 96.9 94.2 87.7 ND ND pH 6.1 6.1 6.1 6.0 ND ND Protein concentration 51.7 52.0 51.2 51.4 ND ND UV A350 0.150 0.188 0.251 0.453 ND ND QL=Quantification Limit (0.10%)

Protein concentration

Protein concentration stability data for all time points and conditions did not show any significant changes as a function of time or storage conditions, and all results were within the acceptance criteria of 45-55 mg/mL.

pH

There were no significant changes in pH for the 5 C, 25 C, and 40 C conditions. Figure 1 shows pH data for time points from 0 to 9 months.

Polysorbate 80

Polysorbate 80 content at the recommended storage conditions of 5°C decreased slightly to 0.13 mg/mL at 9 months and 18 months (18 month data not shown). A decreasing trend in polysorbate 80 content was observed at 25°C (under accelerated aging conditions) and 40°C (under stress conditions). At 40°C, the concentration of polysorbate 80 decreased to 0.06 mg/ml after 6 months, and at 25°C, the concentration of polysorbate 80 decreased to 0.07 mg/ml after 9 months. Polysorbate 80 concentration data for up to 9 months are shown in Figure 2.

ELISA Binding Activity

There was no clear trend at any time point or condition for the ELISA results obtained. Activity data for up to 9 months is shown in Figure 3.

Purity according to UP-SEC

UP-SEC purity data is illustrated below in Figure 4 for % monomer, in Figure 5 for % high molecular weight molecules, and in figure 6 for % low molecular weight molecules, up to 9 months.

Under recommended storage conditions of 5°C, there is a slight decrease in % monomer with a corresponding small increase in % high molecular weight molecules over the course of an 18 month stability study. % of small molecules from baseline to 18 months are below the limit of quantitation (<QL), which is 0.4%. At 25°C conditions, monomer % decreased from baseline to 12 months with a corresponding increase in high molecular weight %. After 9 and 12 months, % LMWs above QL were recorded.

Under stress conditions at 40°C, monomer % decreased from 98.7% to 93.8% with a corresponding increase in high molecular weight molecules from 1.33% to 2.63% and low molecular weight molecules from <QL to 3.53%. This result was not unexpected given the nature of the storage conditions.

Restoring and non-restoring CD-SDS

Figures 7 and 8 show purity data up to 9 months as determined by reducing and non-reducing CD-SDS. There were no significant trends in reducing (% heavy and light chain) or non-reducing (% intact IgG) CE-SDS under long-term storage conditions at 5°C, and the results were within the drug product GMP acceptance criteria of ≥ 90.0%. Under accelerated aging conditions at 25°C, a decreasing trend was observed for non-reducing conditions. For CE-SDS under reducing conditions, a decreasing trend was also observed. For both reducing and non-reducing CE-SDS, all results up to the 9-month time point were within GMP acceptance criteria. Under stress conditions at 40°C, after 6 months, both reducing and non-reducing CD-SDS results were below the ≥ 90.0% acceptance criteria established by GMP for the drug product. The non-reducing CE-SDS result fell out of specification at 3 months with a score of 88.9%, and then further decreased at 6 months to 79.6%. For reducing CE-SDS, % heavy chain and % light chain decreased from 94.2% after 3 months to 87.7% after 6 months. This decrease was not unexpected at 40°C, given the nature of these conditions.

HP-IEX charging options

At 5°C (long-term storage), there is a slight increase in % acidic variants from the initial 21.46% to 22.49% after 9 months, with a corresponding slight decrease in the total amount of the main peak from 68.8% to 67.1% after 9 months. The % of the main variants begins to increase slightly after 9 months, with an increase from 10.15% after 6 months to 10.38% after 9 months. At 25°C (accelerated aging conditions), the total amount of the main peak decreased from 68.8% at the initial time point to 47.6% after 9 months. Along with the decrease in the total amount of the main peak, a corresponding increase in the content of acidic variants was observed from 21.46% to 39.86% and a slight increase in the content of basic variants from 9.70% to 11.51%. At 40°C (under stress conditions), there was a decrease in the total amount of the main peak to 10.1% after 6 months, together with a corresponding significant increase in the content of acidic variants to 80.02% and basic variants to 9.95%.

Particles

Particles were measured by mHIAC. Results at 5°C conditions were well below the acceptance criteria of ≤ 6000 particles per container for ≥ 10 µm and ≤ 600 particles per container for ≥ 25 µm from baseline to 9 months. At 25°C, particle content >= 10 µm was recorded to increase from 13 particles per container at the baseline time point to 460 particles per container after 9 months. There was a reduction in particle content for particles >= 25 µm with a result of 3 particles per container after 9 months. Data for all time points at 25C were within the analytical acceptance criteria of both >=10 µm and >=25 µm. Data at 40°C conditions showed a very strong increase in >=10 µm particles from 8258 particles per container after 9 months. This result was outside the acceptance criteria of <=600 particles per container. The result for particles >=25 µm increased at the 9 month stability analysis time point to 124 particles per container, meeting the acceptance criteria of >=25 µm (<=600 particles per container).

Turbidity

Turbidity was determined by spectrophotometric absorbance at 350 nm. With longer storage at 5°C, there was no noticeable change until time point 9. At 25C, there was a small increase after 3 months with a result of 0.163 AU, it continued to increase until 9 months with a result of 0.196 AU. At 40C, there is a more pronounced increase, starting at 1 month with 0.188 AU, and then a strong increase to 0.453 AU after 9 months.

Conclusions

Based on these data, at the time of testing of 18 months, no significant changes or trends were observed in 5 C storage conditions during stability studies for pH, protein concentration, appearance and visible particles (data not shown), and activity and particles (data not shown). presented). With the exception of a slight increase in color and a decrease in PS-80 to 0.13 mg/ml, no significant changes or trends were observed in any stability test at 5°C.

Based on long-term stability data at 5C, the anti-TIGIT antibody formulation containing L-histidine buffer, sucrose, polysorbate 80 and L-methionine has an expected shelf life of 30 months. For formulations additionally containing a chelating agent, a reduction in the degradation of polysorbate 80 that was observed is expected.

EXAMPLE 8

Co-formulation of anti-TIGIT antibody and anti-PD-1 antibody.

Co-formulation of the two antibodies into one formulation is more convenient for patients and increases compliance by dosing the two antibodies together. Co-formulation of the two antibodies into one formulation is more convenient for patients and increases compliance by dosing the two antibodies together. An anti-TIGIT antibody having the following CDRs: HCDR1 from SEQ ID NO:108, HCDR2 from SEQ ID NO:154, HCDR3 from SEQ ID NO:110, LCDR1 from SEQ ID NO:111, LCDR2 from SEQ ID NO:112, and LCDR3 from SEQ ID NO:113, on an IgG1 backbone, co-formulated with pembrolizumab. Based on protein-protein interactions (shown below), the co-formulation (shown below) is found to be stable at pH 5.0-6.0. Therefore, the co-formulation (P1T1) at pH 5.0, 5.5 and 6.0 is selected and placed for additional thermostability studies at 5°C, 25°C and 40°C, along with two control formulations (anti-PD1 antibodies and anti-TIGIT antibodies).

Joint lineups Pembrolizumab/anti-TIGIT antibody ratio (wt/wt) Pembrolizumab Anti-TIGIT antibody Total concentration P1T1 1:1 20 mg/ml 20 mg/ml 40 mg/ml P1 (control) 1:0 20 mg/ml No 20 mg/ml T1 (control) 0:1 No 20 mg/ml 10 mg/ml

The compositions were prepared in the form of liquid compositions as follows:

Compound Buffer pH Cryoprotector/tonicity modifier Surface-active substance Antioxidant P1T1 L-histidine (10 mM) 5, 5.5, 6 Sucrose (7%) PS-80 (0.02%) 10 mM L-Met P1 (control) L-histidine (10 mM) 5, 5.5, 6 Sucrose (7%) PS-80 (0.02%) 10 mM L-Met T1 (control) L-histidine (10 mM) 5, 5.5, 6 Sucrose (7%) PS-80 (0.02%) 10 mM L-Met

1 ml of each composition was filled into 2R bottles. Stability was measured by visual inspection, protein concentration, microflow imaging (MFI) (particle assessment), mixed-mode size exclusion chromatography (SEC) (aggregation assessment), IEX (charge variant assessment) and UP-SEC (aggregation assessment). The method for testing thermal stability is as follows:

T0 1 month 2 months 3 months 5 months Additionally 5°C
(ambient air humidity) 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 25°C
(60% relative humidity) 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 40°C
(75% relative humidity) 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions 1 combination + 2 mono-compositions

Protein-protein interactions, indicative of colloidality and thermal stability, of various co-formulations were measured. Repulsive protein-protein interactions, as indicated by a positive value of the diffusion interaction parameter (K _D ) K _D >0, indicate a stable composition with a low susceptibility to aggregation. The Kd for co-composition was found to have a positive K _D value, which is indicative of repulsive and stabilizing protein-protein interactions, which may indicate a lower aggregation susceptibility and a stable co-composition.

Based on a positive diffusion interaction parameter (K _D ) or K _D >0, it is expected that co-formulated antibodies may have good co-formulation behavior similar to single antibody formulations.

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LIST OF SEQUENCES

<110> NARASIMHAN, Chakravarti Nachu

DE, Arnab

<120> STABLE COMPOSITIONS OF ANTI-TIGIT ANTIBODIES, INDIVIDUALLY AND IN

COMBINATIONS WITH ANTIBODIES AGAINST PROGRAMMED DEATH RECEPTOR 1

(PD-1), AND METHODS OF THEIR APPLICATION

<130> 24453

<150> 62/500,278

<151> 2017-05-02

<160> 293

<170> PatentIn version 3.5

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<223> protein

<400> 6

Asn Tyr Tyr Met Tyr

15

<210> 7

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 7

Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 8

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 8

Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr

1 5 10

<210> 9

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 9

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 10

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 10

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 11

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 11

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 12

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 12

Asp Ala Ser Asn Arg Ala Thr

15

<210> 13

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 13

Gln Gln Ser Ser Asn Trp Pro Arg Thr

15

<210> 14

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 14

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 15

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 15

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 16

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 16

Asn Ser Gly Met His

15

<210> 17

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 17

Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 18

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 18

Asn Asp Asp Tyr

1

<210> 19

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Ser

<210> 20

<211> 440

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 20

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 21

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 21

Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Phe Ser Tyr Leu His

1 5 10 15

<210> 22

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 22

Leu Ala Ser Asn Leu Glu Ser

15

<210> 23

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 23

Gln His Ser Trp Glu Leu Pro Leu Thr

15

<210> 24

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 24

Ser Tyr Tyr Leu Tyr

15

<210> 25

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 25

Gly Val Asn Pro Ser Asn Gly Gly Thr Asn Phe Ser Glu Lys Phe Lys

1 5 10 15

Ser

<210> 26

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 26

Arg Asp Ser Asn Tyr Asp Gly Gly Phe Asp Tyr

1 5 10

<210> 27

<211> 120

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 27

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 28

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 28

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 29

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 29

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 30

<211> 111

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 30

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 31

<211> 447

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 31

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

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

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 32

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 32

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 33

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 33

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 34

<211> 218

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 34

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Asp

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile Ser

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 35

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 35

Ser Asp Tyr Trp Gly

15

<210> 36

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 36

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

1 5 10 15

<210> 37

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 37

Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly Tyr

1 5 10 15

Phe Asp Phe

<210> 38

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 38

Lys Ala Ser Gln Ser Ile His Lys Asn Leu Ala

1 5 10

<210> 39

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 39

Tyr Ala Asn Ser Leu Gln Thr

15

<210> 40

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 40

Gln Gln Tyr Tyr Ser Gly Trp Thr

15

<210> 41

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 41

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

Gln Leu His Ser Val Thr Thr Asp Asp Thr Ala Thr Tyr Ser Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

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

115 120 125

<210> 42

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 42

Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Asn Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Leu Gly Glu Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Leu Lys

100 105

<210> 43

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 43

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 44

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 44

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 45

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 45

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Ile Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 46

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 46

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Ser Arg Ile Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 47

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 47

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 48

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 48

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 49

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 49

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 50

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 50

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 51

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 51

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

1 5 10 15

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

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 52

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 52

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

1 5 10 15

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

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 53

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 53

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

1 5 10 15

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

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Ile Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 54

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 54

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Ser Arg Ile Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 55

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 55

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

1 5 10 15

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

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 56

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 56

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

1 5 10 15

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

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 57

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 57

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 58

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 58

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Ser Ser Ile Ser Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

Lys Leu His Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 59

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 60

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 60

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 61

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 61

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 62

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 62

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 63

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 63

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 64

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 64

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 65

<211> 129

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 65

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala

115 120 125

Ser

<210> 66

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 66

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 67

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 67

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 68

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 68

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Lys Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 69

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 69

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Met Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 70

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 70

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Pro Pro Gly Lys Lys Met Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 71

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 71

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Phe Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 72

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 72

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Lys Pro Pro Gly Lys Lys Met Glu Trp Ile

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 73

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 73

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Arg 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 74

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 74

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp

20 25 30

Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 75

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 75

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Ser Ser Ile Ala Ser Asp

20 25 30

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

35 40 45

Gly Phe Ile Thr Tyr Ser Gly Ser Thr Ser 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 Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Trp Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120 125

<210> 76

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 76

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 77

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 77

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 78

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 78

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 79

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 79

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 80

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 80

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Asn Ser Leu Gln Thr Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 81

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 81

Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn

1 5 10

<210> 82

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 82

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

1 5 10 15

<210> 83

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 83

Leu Ile Tyr Tyr Asp Tyr Gly Gly Ala Met Asn Phe

1 5 10

<210> 84

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 84

Lys Ala Ser Gln Gly Val Ser Thr Thr Val Ala

1 5 10

<210> 85

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 85

Ser Ala Ser Tyr Arg Tyr Thr

15

<210> 86

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 86

Gln His Tyr Tyr Ser Thr Pro Trp Thr

15

<210> 87

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 87

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

1 5 10 15

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

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Ser Asn Ser Gly Ser Ala Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

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

85 90 95

Ala Thr Leu Ile Tyr Tyr Asp Tyr Gly Gly Ala Met Asn Phe Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 88

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 88

Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Gly Val Ser Thr Thr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 89

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 89

Tyr Ala Ser Asn Leu Gln Thr

15

<210> 90

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 90

Tyr Ala Ser Ser Leu Gln Thr

15

<210> 91

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 91

Tyr Ala Ser Thr Leu Gln Thr

15

<210> 92

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 92

Tyr Ala Thr Thr Leu Gln Thr

15

<210> 93

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 93

Tyr Ala Ser Tyr Leu Gln Thr

15

<210> 94

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 94

Tyr Ala Asn Gln Leu Gln Thr

15

<210> 95

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 95

Tyr Ala Gly Ser Leu Gln Thr

15

<210> 96

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 96

Tyr Ala Ser Gln Leu Gln Thr

15

<210> 97

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 97

Tyr Ala Asp Ser Leu Gln Thr

15

<210> 98

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 98

Gln Gln Tyr Tyr Ser Gly Phe Thr

15

<210> 99

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 99

Gln Gln Tyr Tyr Ser Gly Tyr Thr

15

<210> 100

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 100

Gln Gln Tyr Tyr Ser Gly Ile Thr

15

<210> 101

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 101

Gln Gln Tyr Tyr Ser Gly Val Thr

15

<210> 102

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 102

Gln Gln Tyr Tyr Ser Gly Leu Thr

15

<210> 103

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 103

Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Phe Glu Gly Tyr

1 5 10 15

Phe Asp Phe

<210> 104

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 104

Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Tyr Glu Gly Tyr

1 5 10 15

Phe Asp Phe

<210> 105

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 105

Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Ile Glu Gly Tyr

1 5 10 15

Phe Asp Phe

<210> 106

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 106

Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Val Glu Gly Tyr

1 5 10 15

Phe Asp Phe

<210> 107

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 107

Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Leu Glu Gly Tyr

1 5 10 15

Phe Asp Phe

<210> 108

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 108

Ser Tyr Val Met His

15

<210> 109

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 109

Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 110

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 110

Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val

1 5 10

<210> 111

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 111

Arg Ala Ser Glu His Ile Tyr Ser Tyr Leu Ser

1 5 10

<210> 112

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 112

Asn Ala Lys Thr Leu Ala Glu

15

<210> 113

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 113

Gln His His Phe Gly Ser Pro Leu Thr

15

<210> 114

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 114

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Gly Gly Pro Tyr Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 115

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 115

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Thr Leu Glu Leu Lys

100 105

<210> 116

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 116

Tyr Ile Asp Pro Tyr Asn Arg Gly Ala Lys Tyr Asn Glu Lys Phe Gly

1 5 10 15

<210> 117

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 117

Tyr Ile Asp Pro Tyr Asn Leu Gly Ala Lys Tyr Asn Glu Lys Phe Gly

1 5 10 15

<210> 118

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 118

Tyr Ile Asp Pro Tyr Asn Lys Gly Ala Lys Tyr Asn Glu Lys Phe Gly

1 5 10 15

<210> 119

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 119

Tyr Ile Asp Pro Tyr Asn Phe Gly Ala Lys Tyr Asn Glu Lys Phe Gly

1 5 10 15

<210> 120

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 120

Tyr Ile Asp Pro Tyr Asn Ser Gly Ala Lys Tyr Asn Glu Lys Phe Gly

1 5 10 15

<210> 121

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 121

Tyr Ile Asp Pro Tyr Asn Tyr Gly Ala Lys Tyr Asn Glu Lys Phe Gly

1 5 10 15

<210> 122

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 122

Tyr Ile Asp Pro Tyr Asn Val Gly Ala Lys Tyr Asn Glu Lys Phe Gly

1 5 10 15

<210> 123

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 123

Tyr Ile Asp Pro Tyr Asn Asp Arg Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 124

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 124

Tyr Ile Asp Pro Tyr Asn Asp Asn Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 125

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 125

Tyr Ile Asp Pro Tyr Asn Asp Gln Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 126

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 126

Tyr Ile Asp Pro Tyr Asn Asp Glu Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 127

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 127

Tyr Ile Asp Pro Tyr Asn Asp Leu Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 128

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 128

Tyr Ile Asp Pro Tyr Asn Asp Lys Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 129

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 129

Tyr Ile Asp Pro Tyr Asn Asp Ser Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 130

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 130

Tyr Ile Asp Pro Tyr Asn Asp Tyr Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 131

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 131

Tyr Ile Asp Pro Tyr Asn Asp Val Ala Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 132

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 132

Ala Ala Lys Thr Leu Ala Glu

15

<210> 133

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 133

Tyr Ala Lys Thr Leu Ala Glu

15

<210> 134

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 134

Trp Ala Lys Thr Leu Ala Glu

15

<210> 135

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 135

Ser Ala Lys Thr Leu Ala Glu

15

<210> 136

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 136

Thr Ala Lys Thr Leu Ala Glu

15

<210> 137

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 137

Ile Ala Lys Thr Leu Ala Glu

15

<210> 138

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 138

Val Ala Lys Thr Leu Ala Glu

15

<210> 139

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 139

Asn Asn Lys Thr Leu Ala Glu

15

<210> 140

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 140

Asn Ile Lys Thr Leu Ala Glu

15

<210> 141

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 141

Asn Leu Leu Thr Leu Ala Glu

15

<210> 142

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 142

Asn Thr Lys Thr Leu Ala Glu

15

<210> 143

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 143

Asn Val Lys Thr Leu Ala Glu

15

<210> 144

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 144

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 145

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 145

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ala Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 146

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 146

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 147

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 147

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 148

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 148

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 149

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 149

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Leu Thr Ser Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Val Val Tyr Tyr Cys

85 90 95

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

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 150

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 150

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 151

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 151

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 152

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 152

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 153

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 153

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 154

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 154

Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 155

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 155

Tyr Ile Asp Pro Tyr Asn Asp Gly Ala Lys Tyr Ser Gln Lys Phe Gln

1 5 10 15

Gly

<210> 156

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 156

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

1 5 10 15

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

20 25 30

Thr Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Glu Asp Thr Ala Ile Tyr Phe Cys Thr

85 90 95

Lys Ser Gly Trp Ala Phe Phe Asp Tyr Trp Gly Gln Gly Val Met Val

100 105 110

Thr Val Ser Ser

115

<210> 157

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 157

Asp Ile Gln Met Thr Gln Ser Pro Ser Leu Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Leu Asn Cys Ile Ala Ser Gln Asn Ile Tyr Lys Ser

20 25 30

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

35 40 45

Tyr Asn Ala Asn Ser Leu Gln Ala Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

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

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 158

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 158

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

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

35 40 45

Ala Ser Ile Ser Tyr Glu Gly Ser Arg Thr His Tyr Gly Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Gly Ser Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg His Thr Gly Thr Leu Asp Trp Leu Val Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Ile Val Ser Ser

115

<210> 159

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 159

Asn Ile Val Met Ala Gln Ser Pro Lys Ser Met Ser Ile Ser Ala Gly

1 5 10 15

Asp Arg Val Thr Met Asn Cys Lys Ala Ser Gln Asn Val Asp Asn Asn

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Lys Ser Val Gln Ala

65 70 75 80

Glu Asp Ala Ala Phe Tyr Tyr Cys Gln Arg Ile Tyr Asn Phe Pro Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 160

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (13)..(13)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (13)..(13)

<223> protein

<400> 160

Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Xaa Glu Gly Tyr

1 5 10 15

Phe Asp Phe

<210> 161

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (3)..(3)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (4)..(4)

<223> protein

<400> 161

Tyr Ala Xaa Xaa Leu Gln Thr

15

<210> 162

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (7)..(7)

<223> protein

<400> 162

Gln Gln Tyr Tyr Ser Gly Xaa Thr

15

<210> 163

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (6)..(6)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (9)..(9)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (12)..(12)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (15)..(15)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (16)..(16)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (17)..(17)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (23)..(23)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (25)..(25)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (27)..(27)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (30)..(30)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (37)..(37)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (39)..(39)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (40)..(40)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (43)..(43)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (44)..(44)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (45)..(45)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (48)..(48)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (67)..(67)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (68)..(68)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (70)..(70)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (71)..(71)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (79)..(79)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (81)..(81)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (83)..(83)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (87)..(87)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (88)..(88)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (92)..(92)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (94)..(94)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (110)..(110)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (119)..(119)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (122)..(122)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (123)..(123)

<223> protein

<400> 163

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

1 5 10 15

Xaa Leu Ser Leu Thr Cys Xaa Val Xaa Gly Xaa Ser Ile Xaa Ser Asp

20 25 30

Tyr Trp Gly Trp Xaa Arg Xaa Xaa Pro Gly Xaa Xaa Xaa Glu Trp Xaa

35 40 45

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

50 55 60

Ser Arg Xaa Xaa Ile Xaa Xaa Asp Thr Ser Lys Asn Gln Phe Xaa Leu

65 70 75 80

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

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Xaa Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Xaa Gly Thr Xaa Xaa Thr Val Ser Ser

115 120 125

<210> 164

<211> 127

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (6)..(6)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (9)..(9)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (12)..(12)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (15)..(15)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (16)..(16)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (23)..(23)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (25)..(25)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (27)..(27)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (30)..(30)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (37)..(37)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (39)..(39)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (40)..(40)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (44)..(44)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (45)..(45)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (48)..(48)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (67)..(67)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (71)..(71)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (83)..(83)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (88)..(88)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (110)..(110)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (122)..(122)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (123)..(123)

<223> protein

<400> 164

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Xaa Val Xaa Gly Xaa Ser Ile Xaa Ser Asp

20 25 30

Tyr Trp Gly Trp Xaa Arg Xaa Xaa Pro Gly Lys Xaa Xaa Glu Trp Xaa

35 40 45

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

50 55 60

Ser Arg Xaa Thr Ile Ser Xaa Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Xaa Ser Val Thr Ala Xaa Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Met Pro Ser Phe Ile Thr Leu Ala Ser Leu Ser Thr Xaa Glu Gly

100 105 110

Tyr Phe Asp Phe Trp Gly Gln Gly Thr Xaa Xaa Thr Val Ser Ser

115 120 125

<210> 165

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (10)..(10)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (21)..(21)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (22)..(22)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (40)..(40)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (42)..(42)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (43)..(43)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (46)..(46)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (52)..(52)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (53)..(53)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (58)..(58)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (77)..(77)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (83)..(83)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (87)..(87)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (95)..(95)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (105)..(105)

<223> protein

<400> 165

Asp Ile Gln Met Thr Gln Ser Pro Ser Xaa Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Xaa Xaa Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Xaa Gly Xaa Xaa Pro Lys Xaa Leu Ile

35 40 45

Tyr Tyr Ala Xaa Xaa Leu Gln Thr Gly Xaa Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Xaa Leu Gln Pro

65 70 75 80

Glu Asp Xaa Ala Thr Tyr Xaa Cys Gln Gln Tyr Tyr Ser Gly Xaa Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Xaa Lys

100 105

<210> 166

<211> 106

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> undefined sign

<222> (43)..(43)

<223> protein

<220>

<221> undefined sign

<222> (46)..(46)

<223> protein

<220>

<221> undefined sign

<222> (52)..(52)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (53)..(53)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (56)..(56)

<223> protein

<220>

<221> undefined sign

<222> (58)..(58)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (62)..(62)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (63)..(63)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (68)..(68)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (83)..(83)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (95)..(95)

<223> protein

<400> 166

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Ile His Lys Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Xaa Pro Lys Xaa Leu Ile

35 40 45

Tyr Tyr Ala Xaa Xaa Leu Gln Thr Gly Xaa Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Xaa Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Gly Xaa Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 167

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (7)..(7)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (8)..(8)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (12)..(12)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (13)..(13)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (16)..(16)

<223> protein

<400> 167

Tyr Ile Asp Pro Tyr Asn Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe Xaa

1 5 10 15

Gly

<210> 168

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> undefined sign

<222> (1)..(1)

<223> protein

<220>

<221> undefined sign

<222> (2)..(2)

<223> protein

<400> 168

Xaa Xaa Lys Thr Leu Ala Glu

15

<210> 169

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (5)..(5)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (9)..(9)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (11)..(11)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (12)..(12)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (16)..(16)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (20)..(20)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (38)..(38)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (40)..(40)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (44)..(44)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (56)..(56)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (57)..(57)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (61)..(61)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (62)..(62)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (65)..(65)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (67)..(67)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (68)..(68)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (72)..(72)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (74)..(74)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (76)..(76)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (79)..(79)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (85)..(85)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (87)..(87)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (89)..(89)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (91)..(91)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (92)..(92)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (104)..(104)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (111)..(111)

<223> protein

<400> 169

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

1 5 10 15

Ser Val Lys Xaa Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Xaa Gln Xaa Pro Gly Gln Xaa Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe

50 55 60

Xaa Gly Xaa Xaa Thr Leu Thr Xaa Asp Xaa Ser Xaa Ser Thr Xaa Tyr

65 70 75 80

Met Glu Leu Ser Xaa Leu Xaa Ser Xaa Asp Xaa Xaa Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val Trp Gly Xaa Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 170

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (16)..(16)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (44)..(44)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (56)..(56)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (57)..(57)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (61)..(61)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (62)..(62)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (65)..(65)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (72)..(72)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (74)..(74)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (76)..(76)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (79)..(79)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (85)..(85)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (89)..(89)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (92)..(92)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (104)..(104)

<223> protein

<400> 170

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Xaa

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Gln Xaa Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Tyr Asn Xaa Xaa Ala Lys Tyr Xaa Xaa Lys Phe

50 55 60

Xaa Gly Arg Val Thr Leu Thr Xaa Asp Xaa Ser Xaa Ser Thr Xaa Tyr

65 70 75 80

Met Glu Leu Ser Xaa Leu Arg Ser Xaa Asp Thr Xaa Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 171

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (9)..(9)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (17)..(17)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (18)..(18)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (40)..(40)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (43)..(43)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (45)..(45)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (48)..(48)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (50)..(50)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (51)..(41)

<223> protein

<220>

<221> undefined sign

<222> (51)..(51)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (70)..(70)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (72)..(72)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (74)..(74)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (76)..(76)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (83)..(83)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (84)..(84)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (100)..(100)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (103)..(103)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (106)..(106)

<223> protein

<400> 171

Asp Ile Gln Met Thr Gln Ser Pro Xaa Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Xaa Xaa Val Thr Ile Thr Cys Arg Ala Ser Glu His Ile Tyr Ser Tyr

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Xaa Gly Lys Xaa Pro Xaa Leu Leu Xaa

35 40 45

Tyr Xaa Xaa Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Xaa Phe Xaa Leu Xaa Ile Xaa Ser Leu Gln Pro

65 70 75 80

Glu Asp Xaa Xaa Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Xaa Gly Thr Xaa Leu Glu Xaa Lys

100 105

<210> 172

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (43)..(43)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (50)..(50)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (51)..(51)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (70)..(70)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (83)..(83)

<223> protein

<400> 172

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

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

20 25 30

Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Xaa Pro Lys Leu Leu Ile

35 40 45

Tyr Xaa Xaa Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Xaa Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Xaa Ala Thr Tyr Tyr Cys Gln His His Phe Gly Ser Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 173

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (6)..(6)

<223> protein

<400> 173

Gly Gly Pro Tyr Gly Xaa Tyr Phe Asp Val

1 5 10

<210> 174

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 174

Gly Gly Pro Tyr Gly Ala Tyr Phe Asp Val

1 5 10

<210> 175

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 175

Gly Gly Pro Tyr Gly Asp Tyr Phe Asp Val

1 5 10

<210> 176

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 176

Gly Gly Pro Tyr Gly Glu Tyr Phe Asp Val

1 5 10

<210> 177

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 177

Gly Gly Pro Tyr Gly Phe Tyr Phe Asp Val

1 5 10

<210> 178

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 178

Gly Gly Pro Tyr Gly Gly Tyr Phe Asp Val

1 5 10

<210> 179

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 179

Gly Gly Pro Tyr Gly Ile Tyr Phe Asp Val

1 5 10

<210> 180

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 180

Gly Gly Pro Tyr Gly Lys Tyr Phe Asp Val

1 5 10

<210> 181

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 181

Gly Gly Pro Tyr Gly Asn Tyr Phe Asp Val

1 5 10

<210> 182

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 182

Gly Gly Pro Tyr Gly Gln Tyr Phe Asp Val

1 5 10

<210> 183

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 183

Gly Gly Pro Tyr Gly Arg Tyr Phe Asp Val

1 5 10

<210> 184

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 184

Gly Gly Pro Tyr Gly Ser Tyr Phe Asp Val

1 5 10

<210> 185

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 185

Gly Gly Pro Tyr Gly Thr Tyr Phe Asp Val

1 5 10

<210> 186

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 186

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

1 5 10 15

<210> 187

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 187

Gly Gly Pro Tyr Gly Tyr Tyr Phe Asp Val

1 5 10

<210> 188

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 188

Gly Ala Trp Met Asp

15

<210> 189

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 189

Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu Ser

1 5 10 15

Val Lys Gly

<210> 190

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 190

Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr

1 5 10

<210> 191

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 191

Ser Ala Ser Ser Ser Val Ser Ser Gly Tyr Leu Tyr

1 5 10

<210> 192

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 192

Gly Thr Ser Thr Leu Ala Ser

15

<210> 193

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 193

His Gln Trp Ser Ser Phe Pro Tyr Thr

15

<210> 194

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 194

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

1 5 10 15

Ser Met Lys Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Ser Gly Ala

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Ser Gly Ile Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 195

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 195

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Asn Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp

35 40 45

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

50 55 60

Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Asn Met Glu

65 70 75 80

Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Met Lys

100 105

<210> 196

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (33)..(33)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (34)..(34)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (76)..(76)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (79)..(79)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (80)..(80)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (85)..(85)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (86)..(86)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (87)..(87)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (93)..(93)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (104)..(104)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (105)..(105)

<223> protein

<400> 196

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Xaa Ser Lys Xaa Xaa

65 70 75 80

Val Tyr Leu Gln Xaa Xaa Xaa Leu Arg Ala Glu Asp Xaa Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Xaa Xaa Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 197

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 197

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 198

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 198

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 199

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 199

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Gly Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 200

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (20)..(20)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (46)..(46)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (48)..(48)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (59)..(59)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (79)..(79)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (84)..(84)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (92)..(92)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (107)..(107)

<223> protein

<400> 200

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Xaa Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Xaa Leu Xaa

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Xaa Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Xaa Glu

65 70 75 80

Pro Glu Asp Xaa Ala Val Tyr Tyr Cys His Gln Xaa Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Xaa Lys

100 105

<210> 201

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 201

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 202

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 202

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 203

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 203

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Trp Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 204

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 204

Glu Phe Thr Met His

15

<210> 205

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 205

Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 206

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 206

Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr

1 5 10

<210> 207

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 207

Lys Ala Ser Gln Asp Val Lys Thr Ala Val Ala

1 5 10

<210> 208

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 208

Ser Ala Ser Tyr Arg Asn Thr

15

<210> 209

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 209

Gln Gln His Tyr Ser Thr Pro Phe Thr

15

<210> 210

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 210

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 211

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 211

Asp Ile Val Leu Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Ser Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Asp Ser Val Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Thr Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 212

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (11)..(11)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (12)..(12)

<223> protein

<220>

<221> undefined sign

<222> (24)..(24)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (30)..(30)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (33)..(33)

<223> protein

<220>

<221> undefined sign

<222> (34)..(34)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (37)..(37)

<223> protein

<220>

<221> undefined sign

<222> (38)..(38)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (47)..(47)

<223> protein

<220>

<221> undefined sign

<222> (55)..(56)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (61)..(61)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (62)..(62)

<223> protein

<220>

<221> undefined sign

<222> (74)..(74)

<223> protein

<220>

<221> undefined sign

<222> (77)..(77)

<223> protein

<220>

<221> undefined sign

<222> (81)..(81)

<223> protein

<220>

<221> undefined sign

<222> (91)..(91)

<223> protein

<220>

<221> undefined sign

<222> (105)..(106)

<223> protein

<400> 212

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Xaa Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Xaa His Trp Val Xaa Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Xaa Xaa Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Ala Thr Leu Thr Val Asp Xaa Ser Thr Xaa Thr Ala Tyr

65 70 75 80

Xaa Glu Leu Ser Ser Leu Arg Ser Glu Asp Xaa Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Xaa Xaa Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 213

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 213

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 214

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 214

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 215

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 215

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asn Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 216

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (55)..(55)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (56)..(56)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (60)..(60)

<223> protein

<220>

<221> UNDEFINED SIGN

<222> (73)..(73)

<223> protein

<400> 216

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Xaa Xaa Gly Val Pro Xaa Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Xaa Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 217

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 217

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

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

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 218

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 218

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 219

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 219

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Lys Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Asn Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 220

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 220

Ala Leu Tyr Glu Gly Phe Tyr Phe Asp Tyr

1 5 10

<210> 221

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 221

Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr

1 5 10

<210> 222

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 222

Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr

1 5 10

<210> 223

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 223

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Glu Gly Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 224

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 224

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 225

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 225

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 226

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 226

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Glu Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 227

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 227

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 228

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 228

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Ser Ser

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 229

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 229

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Glu Gly Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 230

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 230

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Ala Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 231

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 231

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ala Glu Ile Arg Thr Lys Val Asn Asn His Ala Thr Asn Tyr Gly Glu

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Arg Gly Ala Leu Tyr Asp Ser Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 232

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 232

His Gln Ala Ser Ser Phe Pro Tyr Thr

15

<210> 233

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 233

His Gln Asp Ser Ser Phe Pro Tyr Thr

15

<210> 234

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 234

His Gln Glu Ser Ser Phe Pro Tyr Thr

15

<210> 235

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 235

His Gln Phe Ser Ser Phe Pro Tyr Thr

15

<210> 236

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 236

His Gln Gly Ser Ser Phe Pro Tyr Thr

15

<210> 237

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 237

His Gln His Ser Ser Phe Pro Tyr Thr

15

<210> 238

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 238

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ala Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 239

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 239

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Asp Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 240

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 240

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Glu Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 241

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 241

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Phe Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 242

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 242

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Gly Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 243

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 243

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Lys Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 244

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 244

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ala Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 245

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 245

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Asp Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 246

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 246

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Glu Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 247

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 247

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Phe Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 248

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 248

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Gly Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 249

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 249

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 250

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 250

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Ala Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 251

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 251

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Asp Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 252

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 252

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Glu Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 253

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 253

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Phe Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 254

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 254

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln Gly Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 255

<211> 108

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 255

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Ser Gly

20 25 30

Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Trp

35 40 45

Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys His Gln His Ser Ser Phe Pro

85 90 95

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 256

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 256

Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 257

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 257

Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 258

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 258

Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 259

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 259

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

1 5 10 15

Gly

<210> 260

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 260

Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 261

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 261

Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 262

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 262

Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 263

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 263

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

1 5 10 15

Gly

<210> 264

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 264

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 265

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 265

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 266

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 266

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 267

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 267

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 268

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 268

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 269

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 269

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 270

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 270

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 271

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 271

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Thr Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 272

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 272

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 273

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 273

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 274

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 274

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 275

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 275

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 276

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 276

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 277

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 277

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 278

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 278

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 279

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 279

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 280

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 280

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Gln Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 281

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 281

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asp Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 282

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 282

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asn Ala Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 283

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 283

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 284

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 284

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Ser Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 285

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 285

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Gly Gly Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 286

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 286

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

Gly Gly Leu Lys Pro Asp Asn Ser Gly Ile Ser Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 287

<211> 119

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 287

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ala Tyr Tyr Arg Tyr Asp Ala Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 288

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 288

Ser Ser Thr Thr Ala Gln Val Asn Trp Glu Gln Gln Asp Gln Leu

1 5 10 15

<210> 289

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 289

Ile Tyr His Thr Tyr Pro Asp Gly Thr

15

<210> 290

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 290

Gly Arg Ile Phe Leu

15

<210> 291

<211> 330

<212> Protein

<213> Artificial sequence

<220>

<223> protein

IgG1

<400> 291

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 292

<211> 325

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 292

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

1 5 10 15

Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

20 25 30

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

35 40 45

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

50 55 60

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr

65 70 75 80

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

85 90 95

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

100 105 110

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

115 120 125

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

130 135 140

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

145 150 155 160

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

165 170 175

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

180 185 190

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

195 200 205

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

210 215 220

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

225 230 235 240

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

245 250 255

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

260 265 270

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

275 280 285

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

290 295 300

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

305 310 315 320

Leu Ser Leu Gly Lys

325

<210> 293

<211> 105

<212> Protein

<213> Artificial sequence

<220>

<223> protein

<400> 293

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

1 5 10 15

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

20 25 30

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

35 40 45

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

50 55 60

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

65 70 75 80

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

85 90 95

Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<---

Claims (48)

1. A stable composition for the treatment of a malignant tumor in a patient, where the stable composition contains: (i) from about 10 mg/ml to about 200 mg/ml anti-TIGIT antibody or antigen binding fragment thereof, wherein the anti-TIGIT antibody or antigen binding fragment thereof comprises: three light chain CDRs containing CDRL1 from SEQ ID NO: 111, CDRL2 from SEQ ID NO: 112 and CDRL3 from SEQ ID NO: 113, and three heavy chain CDRs containing CDRH1 with SEQ ID NO: 108, CDRH2 with SEQ ID NO: 154 and CDRH3 with SEQ ID NO: 110; (ii) about 5 mM to about 20 mM L-histidine buffer; (iii) about 6% to about 8% (w/v) sucrose; (iv) about 0.01% to about 0.10% (w/v) polysorbate 80; (v) about 1 mM to about 20 mM L-methionine; And (vi) about 10 mg/ml to about 200 mg/ml anti-PD1 antibody or antigen binding fragment thereof, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises: three light chain CDRs with SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and three heavy chain CDRs with SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, where the composition is the joint composition and where the pH of a stable composition is from 5.3 to 6.2. 2. The composition according to claim 1, where the anti-PD–1 antibody or its antigen-binding fragment contains a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 9. 3. The composition according to any one of claims 1, 2, where the composition contains an anti-PD-1 antibody, which is pembrolizumab. 4. Composition according to any one of claims 1-3, containing about 10 mg/ml anti-PD1 antibody and about 10 mg/ml anti-TIGIT antibody. 5. Composition according to any one of claims 1 to 4, additionally containing a chelating agent. 6. The composition according to claim 5, where the chelating agent is DTPA. 7. The composition according to any one of claims 1 to 6, where the composition is contained in a glass vial or in an injection device. 8. The composition according to any one of claims 1 to 7, where the composition is a liquid composition, the composition is frozen at least below -70°C, or the composition is a solution reconstituted from a lyophilized composition. 9. Composition according to any one of claims 1 to 8, where after 12 months at 5°C: (i) the % monomer of the anti-TIGIT antibody is ≥95%, as determined by size exclusion chromatography; (ii) the % of heavy chain and light chain of the anti-TIGIT antibody is ≥90%, as measured by reducing CE–SDS; (iii) the % of heavy chain and light chain of the anti-TIGIT antibody is ≥95%, as measured by reducing CE–SDS; (iv) % intact IgG anti-TIGIT antibody is ≥ 90% as measured by non-reducing CE–SDS; and/or (v) % intact IgG anti-TIGIT antibody is ≥ 95% as measured by non-reducing CE–SDS. 10. Composition according to claims 1-9, where the anti-TIGIT antibody or antigen-binding fragment contains a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 148, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 152. 11. Composition according to claims 1-9, where the anti-TIGIT antibody contains (i) a human IgG1 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 291, and a human kappa light chain constant domain containing the amino acid sequence of SEQ ID NO: 293; or (ii) a human IgG4 heavy chain constant domain comprising the amino acid sequence of SEQ ID NO: 292, and human kappa light chain constant domain containing the amino acid sequence of SEQ ID NO: 293. 12. Composition according to claims 1-11, where the pH of the composition is from 5.5 to 6.3. 13. Composition according to claims 1-12, containing from about 8 mm to about 12 mm L-histidine buffer; and/or containing about 0.02% (w/v) polysorbate 80. 14. The composition of claims 1 to 13, containing from about 10 mg/ml to about 100 mg/ml of an anti-TIGIT antibody or an antigen-binding fragment thereof, or wherein the concentration of an anti-TIGIT antibody or an antigen-binding fragment thereof is about 10 mg/ml, 12 .5 mg/ml, 25 mg/ml, 50 mg/ml, 75 mg/ml or 100 mg/ml. 15. The composition according to claim 14, where the ratio of anti-PD-1 antibody and anti-TIGIT is 1:1. 16. The composition according to claims 1-15, where the composition is a liquid composition, the composition is frozen to at least a temperature below -70°C, or the composition is a solution reconstituted from a lyophilized composition. 17. A method of treating a malignant tumor in a human patient, comprising administering an effective amount of a composition according to any one of claims 1 to 16. 18. A method of treating a chronic infection in a human patient, comprising administering an effective amount of a composition according to any one of claims 1 to 16. 19. Use of a stable composition according to any one of claims 1 to 16 for the production of a medicinal product for the treatment of a malignant tumor.