EA045974B1 - ANTI-TUMOR ANTAGONISTS OF IMMUNE CHECKPOINT REGULATORS - Google Patents

Description

This application claims benefit from U.S. Provisional Patent Application No. 62/691,658, filed June 29, 2018, and U.S. Provisional Patent Application No. 62/823,989, filed March 26, 2019, the contents of which are specifically incorporated into this application by links.

Technical field

The present application relates generally to the treatment of cancer and, in particular, to bispecific inhibitors capable of modulating pathways associated with tumorigenesis and antitumor immunity.

State of the art

The inability of the host to destroy cancer cells remains an important problem. Although there are an increasing number of therapeutic monoclonal antibodies approved for the treatment of various types of cancer, the development of resistance to these antibodies is frequently observed given the many different molecular pathways underlying tumor growth and progression to metastasis. Although the immune system provides the primary mechanism for preventing cancer, cancer cells have the ability to antagonize immune surveillance. Natural control mechanisms have been identified that limit T cell activation to prevent collateral damage resulting from their unrestricted activity. This process is used by tumor cells to evade immune responses. Restoring the ability of effector cells of the immune system, in particular T lymphocytes, to recognize and eliminate cancer cells is the main goal of immunotherapy.

There is a need for improved therapeutic binding antagonists or antibodies and methods for treating cancer and chronic viral infections using such reagents.

Brief description of the invention

One aspect of the present invention relates to bispecific antitumor antagonists comprising a first targeting domain that specifically binds to an immune checkpoint regulator; a second scFv targeting domain that specifically binds TIGIT; and an immunoglobulin scaffold structurally linked to the first and second targeting domains, wherein said first targeting domain is located at the N-terminus of the antagonist and said second targeting domain is located at the C-terminus of the antagonist and is linked to the immunoglobulin scaffold by a linker. In some embodiments, the linker contains 4 or 6 copies of the G4S amino acid sequence (4xG4S and 6xG4S, respectively).

In some embodiments of the present invention, the first targeting domain specifically binds to PD-1, PD-L1, or LAG-3.

Another aspect of the present invention relates to humanized anti-LAG-3 antibodies that inhibit the binding of ligands to LAG-3.

Another aspect of the present invention relates to a method of treating a cell proliferative disorder. The method comprises administering to a subject in need thereof an effective amount of a bispecific antitumor antagonist or anti-LAG-3 antibody according to the present application.

Brief description of graphic materials

In fig. 1 shows the complementarity determining region (CDR) sequences of specific anti-TIGIT mAbs. Framework region (FR) sequences flanking the anti-TIGIT antibody CDR sequences are listed in FIG. 39A as the sequences of SEQ ID NO: 216-262.

In fig. 2A-2C show several embodiments of the anti-TIGIT antibody variable domain sequences.

In fig. 3 shows the CDR sequences of specific anti-PD-1 mAbs. The FR sequences flanking the anti-PD-1 antibody CDR sequences are listed in FIG. 39B as SEQ ID NO: 263-292.

In fig. 4A-4B show several embodiments of anti-PD-1 antibody variable domain sequences.

In fig. 5 shows the CDR sequences of specific anti-PD-L1 mAbs. The FR sequences flanking the anti-PD-L1 antibody CDR sequences are listed in FIG. 39C as SEQ ID NO: 293-315.

In fig. 6A-6C show several embodiments of the anti-PD-L1 antibody variable domain sequences.

In fig. 7A-7C depict three examples of bispecific antitumor antagonists, BiTPM-93 (Fig. 7A), Bi-TPM-94A (Fig. 7B) and BMRM-94B (Fig. 7C).

In fig. 8 shows a generalized order of the functional domains in the bispecific antagonists depicted in FIG. 7A-7C.

In fig. 9A-9B show the heavy chain (HC) and light chain (LC) amino acid sequences corresponding to the bispecific antagonists depicted in FIG. 7A-7C.

In fig. 10 depicts a blocking assay showing that Bi-TPM-94A provides more effective blocking of the interaction between PD-1 and its ligand PD-L1 (IC50=0.15 nM) compared to

- 1 045974 connection with Bi-TPM-93 (IC50=0.83 nM).

In fig. 11 shows the results of PAGE analysis under non-reducing conditions for Bi-TPM-94A and Bi-TPM-94B transiently expressed in the human embryonic kidney 293 (HEK) cell line.

In fig. 12 shows the results of Size Exclusion Liquid Chromatography (SE-UHPLC) analysis demonstrating the compositional heterogeneity of samples BiTPM-93 and Bi-TPM-94, which is eliminated by modifying the linker in Bi-TPM-94B.

In fig. 13A shows that the binding affinity of Bi-TPM-94A and Bi-TPM-94B to PD-1 exceeds the binding affinity of the reference anti-PD-1 antibody to PD-1. In fig. 13B shows that the binding affinity of Bi-TPM-94A and Bi-TPM-94B to TIGIT exceeds the binding affinity of the reference anti-TIGIT antibody to TIGIT.

In fig. 14A-14B show that Bi-TPM-94A and Bi-TPM-94B actively block both the binding of TIGIT to its ligand, human PVR (CD155) (Fig. 14A), and the binding of PD-1 to its ligand, PD-L1 (Fig. 14B).

In fig. 15 shows the results of an ELISA assay demonstrating simultaneous binding of BiTPM-94A and Bi-TPM-94B to PD-1 and TIGIT. In this assay, huPD-1-Fc-coated 96-well plates were incubated with serial dilutions of Bi-TPM-94A and Bi-TPM-94B samples followed by inhibition with the His-tagged huTIGIT protein, whereby bound molecules were detected using HRP-conjugated antibodies against His tag and TMB substrate.

In fig. 16A-16B show increased secretion of IFN-γ in human PBMC (donor 287, FIG. 16A; donor 401, FIG. 16B) in the presence of Bi-TPM-94B compared to individual parent anti-PD-1 and anti-TIGIT antibodies or a combination thereof , as well as negative controls.

In fig. 17A-17B show that Bi-TPM-94B enhances the proliferation of primary human T cells from PBMCs from donor 287 (FIG. 17A) and PBMCs from donor 401 (FIG. 17B) to a greater extent compared to individual parental anti-PD-1 and against TIGIT or a combination thereof, as well as negative controls.

In fig. 18 shows a pharmacokinetic profile showing that Bi-TPM-94A and BiTPM-94B have similar in vivo half-lives (T1/ ₂ ) after tail vein injection into 6-10 week old female CD1 mice. Bispecific antagonists were recovered from sera collected at various times after injection and analyzed by ELISA.

In fig. 19A shows the heavy chain CDR sequences corresponding to anti-LAG3 mAbs 2L2A.1, 2L2A.6, 2L27B and 3L1A. In fig. 19B shows the light chain CDR sequences corresponding to the anti-LAG-3 mAb 2L2A. 1, 2L2A.6, 2L27B and 3L1A. The FR sequences flanking the CDR sequences of anti-LAG-3 antibodies are listed in FIG. 39C as SEQ ID NO: 316-337.

In fig. 20 shows the VH and VL sequences of anti-LAG-3 mAbs 2L2A.1, 2L2A.6, 2L27B and 3L1A.

In fig. 21A-21B show assay results confirming the ability of anti-LAG-3 mAbs to block binding to LAG-3.

In fig. 22 shows the results of a cell-based blocking assay assessing the ability of anti-LAG-3 mAb 2L2A.1, reference (RM) anti-LAG-3 mAb, and fusion antibody 2L2A (SEQ ID NOs: 203 and 204) to block the interaction between LAG-3-muFc and its the main ligand, the major histocompatibility complex (MHC) antigen, expressed on Raji cells. The assay data were used to calculate the presented IC50 (nM) values.

In fig. 23A-23B show surface plasmon resonance (SPR) binding affinity analysis of anti-LAG-3 mAb 2L2A.1 to human LAG-3-His (FIG. 23A) or human LAG-3-mIgG2a (FIG. 23B), along with the corresponding binding affinity constants.

In fig. 24 shows the binding of a variant of the anti-LAG-3 mAb, 2L2A.1, or a reference antibody (RA) to LAG-3, including half-maximal effective concentrations (EC ₅₀ ), causing an effect intermediate between baseline and maximum levels.

In fig. 25 shows polyacrylamide gel electrophoresis (PAGE) analysis under non-denaturing conditions for the humanized anti-LAG-3 mAb 2L2A.1 transiently expressed in HEK293 cells. The positive control was HybPL1 (1PL11 CDRg-VH: 1PL25 CDRg-VL).

In fig. 26 shows FACS analysis showing coexpression of LAG-3 and PD-1 in activated human CD3+ T cells.

In fig. 27A-27B show IFN-γ production. PBMCs from two donors (donor 0105, Fig. 27A; donor 0817, Fig. 27B) were stimulated with staphylococcal enterotoxin B (SEB; lanes 2-4) or not stimulated with SEB (lane 1) in a 96-well plate. After stimulation of PBMCs, donors were incubated in the absence of antibodies (lanes 1, 2), in the presence of the reference anti-LAG-3 antibody (BM) (lane 3), or the anti-LAG-3 mAb, 2L2A. 1. The results of this analysis showed that 2L2A. 1 induce higher IFN-γ production in PBMCs from both donors compared to the reference anti-LAG-3 antibody.

- 2 045974

In fig. 28A-28C show increased IFN-γ production in human PBMCs from three donors (donor 223, Fig. 28A; donor 224, Fig. 28B; donor 225, Fig. 28C) stimulated with SEB (lanes 2-4) or not stimulated with SEB (track 1). In addition, donor PBMCs were incubated in the absence of antibodies (lanes 1, 2), in the presence of the reference (BM) anti-LAG-3 antibody (lane 3) or anti-LAG-3 mAb 2L2A. 1.

In fig. 29A-29C show that the anti-LAG-3 mAb is 2L2A. 1 enhances the proliferation of primary human T cells from donor 223 (FIG. 29A), donor 224 (FIG. 29B), and donor 225 (FIG. 29C) to a greater extent compared to the reference anti-LAG-3 antibody.

In fig. 30A-30B depict two examples of bispecific antitumor antagonists, BiLT-1 (FIG. 30A) and Bi-LT-3 (FIG. 30B).

In fig. 31 shows a generalized order of the functional domains in the bispecific antagonists depicted in FIG. 30A-30B.

In fig. 32 shows the heavy chain (HC) and light chain (LC) amino acid sequences corresponding to the bispecific antagonists depicted in FIG. 30A-30B.

In fig. 33A-33B show the results of a cell-based blocking assay that measured the ability of the bispecific antagonists LT-1 and LT-3 and the bispecific antagonist Bi-TPM-94B or the reference anti-LAG-3 mAb to block the interaction between LAG-3-muFc and its major ligand , a major histocompatibility complex (MHC) antigen expressed on Raji cells (FIG. 33A), or block the interaction between TIGIT and its ligand, human PVR (CD155) (FIG. 33B). The assay data were used to calculate the presented IC50 values (nM).

In fig. 34 shows the results of an ELISA assay demonstrating simultaneous binding of BiLT-1, Bi-LT-3, or parent anti-LAG-3 mAbs to LAG-3 and TIGIT. In this assay, LAG-3-muFc-coated 96-well plates were incubated with serial dilutions of Bi-LT-1, Bi-LT-3, or parent anti-LAG-3 mAb samples, followed by inhibition with His-tagged huTIGIT protein, due to to which bound molecules were detected using HRP-conjugated antibodies against the His tag and TMB substrate. The assay data were used to calculate the presented EC50 (nM) values.

In fig. 35A-35D depict pharmacokinetic profiles and in vivo half-lives (T _1/2 ) corresponding to the parent anti-LAG-3 mAb (Fig. 35A), the reference anti-LAG-3 mAb (Fig. 35B), Bi-LT-1 (Fig. 35C) or Bi-LT-3 (FIG. 35D) following tail vein injection into 6-10 week old female CD1 mice. Antibodies and bispecific antagonists were recovered from sera collected at various times after injection and analyzed by ELISA. T1 _/2 for parental mAb against LAG-3, Bi-LT-1 and Bi-LT-3 was five to six days, T1 _/2 for BM mAb against LAG-3 was either 2 days (mouse 3), or 7 days (mouse 4).

In fig. 36A shows a size exclusion chromatography (SEC) profile for Bi-LT-1 and Bi-LT-3 showing homogeneity and good stability at 4°C after 7 days. In fig. 36B shows the results of ultra-high performance liquid chromatography (SE-UHPLC) analysis illustrating the compositional homogeneity of Protein A purified Bi-LT-1 and Bi-LT-3, as evidenced by the low level of high molecular weight (HMW) products on days 0 and 7 (1.3%, 1.5%, respectively, for Bi-LT-1 and 1.3%, 1.4%, respectively, for Bi-LT-3) and low molecular weight (LMW) products on days 0 and 7 (0.2%, 0.2%, respectively, for Bi-LT-1 and 0.3%, 0.5%, respectively, for Bi-LT-3) compared to dimeric products on days 0 and 7 day (98.4%, 98.3%, respectively, for Bi-LT-1 and 98.4%, 98.1%, respectively, for Bi-LT-3).

In fig. 37A-37B show IFN-γ production in human PBMCs stimulated with staphylococcal enterotoxin B (SEB; lanes 2-8) or not stimulated with SEB (lane 1) in the presence of SHP-77 cells (Fig. 37A) or H358 cells (Fig. 37B ) followed by incubation in the absence of antibody (lanes 1, 2); in the presence of human IgG (lane 3); in the presence of parent anti-TIGIT mAb B21-35 (lane 4); in the presence of the parent anti-LAG-3 mAb 2L2A.1 (lane 5); in the presence of the parent anti-TIGIT mAb B21-35 and anti-LAG-3 mAb 2L2A. 1 (track 6); Bi-LT-1 (lane 7) and Bi-LT-3 (lane 8).

In fig. Figure 38 shows proliferation of CD4 T cells from human PBMCs stimulated with SEB in the presence of SHP-77 cells (lanes 2-8) and control human IgG (lane 3), anti-TIGIT mAb B21-35 (lane 4), anti-LAG-mAb 3 (lane 5), combinations of anti-TIGIT mAb and anti-LAG-3 (lane 6), Bi-LT-1 (lane 7), or Bi-LT-3 (lane 8) mAb.

In fig. 39A shows framework regions (FR) corresponding to the anti-TIGIT CDR in FIG. 1. In FIG. 39B shows FRs corresponding to anti-PD-1 CDRs. FIG. 3. In FIG. 39C shows FRs corresponding to the anti-PD-L1 CDRs in FIG. 5, and FRs corresponding to the anti-LAG-3 CDRs in FIG. 19A and 19B.

Detailed description

Definitions

Unless otherwise specified, all technical and scientific terms used in this application have the same meanings as commonly accepted by those skilled in the art to which

- 3 045974 refer to the described method and compositions. It should be noted that the use of subjects in the singular in this application and the attached claims includes the plural form of the specified subjects, unless otherwise clearly follows from the context. Thus, for example, a reference to a peptide includes one or more peptides or a plurality of such peptides. In accordance with the teachings of the present invention, any published patent or publication of a patent application described in this application is specifically incorporated herein by reference.

As used herein, the term TIGIT refers to any form of TIGIT and variants thereof that retain at least part of the activity of TIGIT. Unless otherwise indicated, for example by specific reference to human TIGIT, TIGIT includes native TIGIT sequences from all mammalian species, such as humans, dogs, cats, horses and cows.

As used herein, the term PD-1 refers to any form of PD-1 and variants thereof that retain, at least in part, the activity of PD-1. Unless otherwise indicated, for example by specific reference to human PD-1, PD-1 includes native PD-1 sequences from all mammalian species, such as humans, dogs, cats, horses and cows.

As used herein, the term PD-L1 refers to any form of PD-L1 and variants thereof that retain, at least in part, the activity of PD-L1. Unless otherwise indicated, for example by specific reference to human PD-L1, PD-L1 includes native PD-L1 sequences from all mammalian species, such as humans, dogs, cats, horses and cows.

The term agonist refers to a substance that promotes (ie, induces, stimulates, enhances or enhances) the biological activity or effect of another molecule. The term agonist includes substances that bind to a receptor, such as an antibody, and substances that promote the function of the receptor without binding to it (eg, by activating an associated protein).

The term antagonist or inhibitor refers to a substance that prevents, blocks, inhibits, neutralizes or reduces the biological activity or effect of another molecule, such as a receptor or ligand. The antagonist may be a monospecific antibody or a bispecific antibody.

As used herein, the term antibody refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen using one or more immunoglobulin variable regions. The antibody may be a full-length antibody, an antigen-binding fragment, or a single chain. The term antibody includes various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as alpha, delta, epsilon, gamma, and mu (or α, δ, ε, γ, and μ), with some having subclasses (eg, γ1-γ4). The nature of these chains determines the class of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Subclasses (isotypes) of immunoglobulins, such as IgG1, IgG2, IgG3, IgG4, etc., are well characterized and are known to be responsible for functional specialization. Modified versions of each of these classes and isotypes will readily be recognized by one skilled in the art in light of the present invention. Accordingly, these modified versions are within the scope of the present invention, and all classes of immunoglobulins are within the scope of the present invention. The following discussion will generally relate to the IgG class of immunoglobulin molecules.

Antibodies of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, bispecific, human, humanized, primatized, hybrid and single chain antibodies. The antibodies described herein can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, rat, donkey, rabbit, goat, guinea pig, camel, llama, horse or chicken. In some embodiments, the variable region may be derived from cartilaginous fish (eg, sharks).

The terms antibody fragment and antigen binding fragment are used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single chain Fv(scFv), single chain antibodies, disulfide linked Fv fragments (sdFv), VL or VH domain-containing fragments, Fab expression library-derived fragments, and anti-idiotypic antibodies (anti-Id). Regardless of the structure, the antibody fragment binds to the same antigen that is recognized by the intact antibody. The term antibody fragment includes DART and diabodies. The term antibody fragment also includes any synthetic or genetically engineered proteins containing immunoglobulin variable regions that act as an antibody by binding to a specific antigen to form a complex. Single chain variable fragment, or scFv, refers to a fusion protein between the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins. In some aspects, the regions are linked by a short linker peptide of ten to about 25 amino acids. The linker may be enriched with glycine to impart flexibility, as well as serine or threonine to increase solubility, and may connect the N-terminus of the VH to the C-terminus of the VL or vice versa. This protein retains the specificity of the original immunoglobulin, without

- 4 045974 looking at the removal of constant regions and the introduction of a linker. In the case of IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000 daltons. The four chains are typically linked by disulfide bonds in a Y configuration, where the connection of the light chains to the heavy chains begins at the fork of the Y structure and continues through the variable region.

Both light and heavy chains are divided into regions of structural and functional homology. The terms constant and variable are used in a functional sense. In this regard, it should be understood that the variable domains of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. In contrast, the light chain (CL) and heavy chain constant domains (CH1, CH2 or CH3) confer important biological properties such as secretion, transplacental motility, Fc receptor binding, complement fixation, etc. Traditionally, the numbering of constant region domains in conventional antibodies increases as they move away from the antigen-binding site or amino terminus of the antibody. In conventional antibodies, the N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually contain the carboxy terminus of the heavy and light chains, respectively.

As stated above, the variable region allows the antibody to selectively recognize and specifically bind to epitopes on antigens. That is, the VL domain and the VH domain or set of complementarity determining regions (CDRs) of an antibody combine to form a variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms an antigen-binding site present at the end of each arm of the Y structure. More specifically, the antigen binding site is defined by three CDRs on each of the VH and VL chains (ie, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3). In some cases, for example, certain immunoglobulin molecules are derived from camelid species or are constructed from camelid immunoglobulins. Alternatively, the immunoglobulin molecule may consist of only heavy chains with no light chains, or only light chains with no heavy chains.

In natural antibodies, the six CDRs present in each antigen-binding domain are short, non-contiguous sequences of amino acids that have a specific arrangement resulting in the formation of an antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domains, called framework regions, have less pronounced intermolecular variability. The framework regions largely adopt a β-sheet conformation, and the CDRs form loops that connect and in some cases form part of the β-sheet structure. Thus, the framework regions form a framework that ensures the correct orientation of the CDRs through interchain non-covalent interactions. The antigen-binding domain, formed by positioned CDRs, defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. One skilled in the art will readily be able to identify the amino acids comprising the CDRs and framework regions, respectively, for any given heavy or light chain variable region since they have been precisely defined.

As used herein, the terms VH1 and VH2 refer to immunoglobulin heavy chain variable domains corresponding to two different binding specificities. Likewise, the terms VL1 and VL2 refer to light chain variable domains corresponding to two different binding specificities. It should be understood that the VH1 and VL1 regions, when used together, determine the overall binding specificity, and the VH2 and VL2 domains determine the second binding specificity.

As used herein, the term framework region (FR) refers to variable domain residues other than CDR residues. Each variable domain typically contains four FRs flanking the corresponding CDRs. For example, the VH domain typically has four HFRs: HFR1, HFR2, HFR3, and HFR4, flanking three HCDRs in the configuration HFR1-HCDR1-HFR2-HCDR2HFR3-HCDR3-HFR4. Likewise, the LH domain typically has four LFRs flanking three LCDRs in the configuration: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4. Examples of FRs that can be used in the antagonists described herein are summarized in FIG. 39A-39C.

Light chains are classified as kappa or lambda (K, λ). Each heavy chain class can be coupled to a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the tail portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when immunoglobulins are created using hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from the N-terminus at the branched ends of the Y structure to the C-terminus at the bottom of each chain.

When used herein, the terms light chain constant region or CL are used interchangeably with reference to amino acid sequences derived from the

- 5 045974 chain of antibody. Preferably, the light chain constant region comprises at least one of a kappa constant domain or a lambda constant domain.

As used herein, the term heavy chain constant region includes amino acid sequences derived from the immunoglobulin heavy chain. A heavy chain constant region-containing polypeptide contains at least one of a CH1 domain, a hinge domain (eg, an upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen binding polypeptide for use according to the invention may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain containing a CH1 domain, at least part of a hinge domain and a CH2 domain; a polypeptide chain containing a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the polypeptide of the invention comprises a polypeptide chain comprising a CH3 domain. In addition, an antibody for use according to the invention may lack at least a portion of the CH2 domain (eg, all or part of the CH2 domain). It should be understood that the heavy chain constant region may be modified such that its amino acid sequence differs from the native immunoglobulin molecule. For example, we have discovered that the Fc loop in the CH3 domain can be resistant to the introduction of large insertions or can accommodate large insertions (eg, more than 100 amino acids).

The heavy chain constant region of the antibody described herein can be derived from various immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion may comprise a hybrid hinge region derived in part from an IgG1 molecule and in part from an IgG4 molecule.

A light chain-heavy chain pair refers to a set of a light chain and a heavy chain that can form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

The subunit structures and three-dimensional configurations of the constant regions of various classes of immunoglobulins are well known. As used herein, the term VH domain includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term CH1 domain includes the first (closest to the amino terminus) domain of the immunoglobulin heavy chain constant region. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

As used herein, the term CH2 domain includes the portion of the heavy chain molecule that extends, for example, from about residue 244 to residue 360 of an antibody according to conventional numbering schemes (residues 244-360, Kabat numbering system; and residues 231-340, EU numbering system). The CH2 domain is unique in that it is not tightly paired with another domain. Rather, two N-linked branched carbohydrate chains are located between the two CH2 domains of the intact native IgG molecule. The CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.

As used herein, the term hinge region includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge regions can be divided into three distinct domains: the upper, middle, and lower hinge domains.

As used herein, the term disulfide bond includes a covalent bond formed between two sulfur atoms. The amino acid cysteine contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most natural IgG molecules, the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, using the EU numbering system).

As used herein, the term variant of an antibody, antibody fragment, or antibody domain refers to an antibody, antibody fragment, or antibody domain that is (1) at least 80%, at least 85%, at least 90% consistent, is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the parent antibody, antibody fragment, or antibody domain, and (2) which binds specifically to the same target to which the parent antibody, antibody fragment, or antibody domain specifically binds. It should be understood that if the measure of sequence identity is described in the phrase at least x% identical or at least x% identical, such embodiment includes any and all percentage values represented by

- 6 045974 as an integer equal to or greater than the lower limit. In addition, it should be understood that when an amino acid sequence is provided herein, it is considered to further describe or include amino acid sequences that are at least 80%, at least 85%, at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% identity with the specified amino acid sequence.

As used herein, the phrase humanized antibody refers to an antibody derived from a non-human antibody, typically a murine monoclonal antibody. Alternatively, a humanized antibody may be derived from a hybrid antibody that retains or substantially retains the antigen-binding properties of the parent non-human antibody, but which has reduced immunogenicity compared to the parent antibody when administered to humans.

As used herein, the phrase hybrid antibody refers to an antibody in which the immunoreactive region or site is derived from or derived from the first species and the constant region (which may be intact, part of the constant region, or a modified constant region in accordance with the present invention) is derived from the second type. In some embodiments, the target binding region or site is from a non-human source (eg, mouse or primate) and the constant region is of human origin.

The multispecific antibodies of the present invention encompass a variety of compositions and techniques, including asymmetric IgG-like antibodies (eg, triomab/quadroma, Trion Pharma/Fresenius Biotech); ridge-to-groove antibodies (Genentech); CrossMAb (Roche); electrostatically coupled antibodies (AMGEN); LUZ-Y (Genentech); strand exchange engineered domain proteins (SEEDBodies, EMD Serono; biolonic, Merus); Fab replacement antibodies (Genmab), symmetric IgG-like antibodies (e.g. dual-targeting (DT) Ig (GSK/Domantis); two-in-one antibodies (Genentech); cross-linked MAbs (Karmanos Cancer Center), mAb2 (F -star); Cov X-body (Cov X/Pfizer); double variable domain (DVD) and Ig fusion proteins (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab (Medimmune/AZ); BsAb (ZymoGenetics) ; HERCULES (Biogen Idec, TvAb, Roche); scFv/Fc fusion proteins; SCORPION (Emergent BioSolutions/Trubion, ZymoGenetics/BMS); dual affinity antibody retargeting technology (Fc-DART), MacroGenics; (scFv)2-Fab dual action (National Research Center for Antibody Medicine); F(ab)2 fusion proteins (Medarex/AMGEN); Dual-acting Fab or Bis-Fab (Genentech); Dock-and-Lock (DNL, ImmunoMedics); Fab-Fv (UCB -Celltech); scFv-based antibodies and diabodies (e.g., bispecific T-cell recruiters (BiTEs, Micromet); tandem diabodies (Tandab, Affimed); DART (MacroGenics); single-chain diabodies; TCR-like antibodies (AIT, Receptor Logics); human serum albumin fusion protein scFv (Merrimack); COMBODIES (Epigen Biotech) and IgG/re-IgG fusion proteins, such as immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics).

The term specifically binds or has specificity generally implies that the antibody binds to an epitope via its antigen binding domain, and that the binding involves some complementarity between the antigen binding domain and the epitope. By this definition, an antibody is said to bind specifically to an epitope if it binds to said epitope through its antigen-binding domain more readily than it would bind to a random unrelated epitope. The term specificity is used herein to define the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be considered to have a higher specificity for a given epitope compared to antibody B, or antibody A may be considered to bind epitope C with a higher specificity compared to its specificity for the related epitope D. According to some In embodiments of the invention, an antibody or antibody fragment has specificity for an antigen if said antibody or antibody fragment forms a complex with an antigen with a dissociation constant (K _d ) of 10 ⁶ M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, or 10 ^-10 M or less.

As used herein, the phrase hybrid antibody refers to an antibody in which the immunoreactive region or site is derived from or derived from the first species, and the constant region (which may be intact, part of the constant region, or a modified constant region in accordance with the present invention), obtained from the second species. In some embodiments, the target binding region or site is of non-human origin (eg, mouse or primate) and the constant region is of human origin.

The term antagonist antibody refers to an antibody that binds to a target and prevents or reduces its biological effect. In some embodiments, the term may refer to an antibody that prevents the biological function of the target to which it binds, such as TIGIT.

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As used herein, the term anti-PD-1 antagonist antibody refers to an antibody that is capable of inhibiting the biological activity of PD-1 and/or subsequent PD-1-mediated event(s). Anti-PD-1 antagonist antibodies include antibodies that block, antagonize, inhibit or reduce (to any extent, including significant) the biological activity of PD-1, including downstream events mediated by PD-1 such as PD-1 binding and subsequent signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10 and inhibition of antitumor immune responses. It is clearly understood that for purposes of the present invention, the term anti-PD-1 antagonist antibody (used interchangeably with the term PD-1 antagonist antibody, anti-PD-1 antagonist antibody, or anti-PD-1 antagonist antibody) includes all previously defined terms , names, functional states and characteristics whereby PD-1 itself, its biological activity or the consequences of its biological activity are substantially eliminated, reduced or neutralized to any significant extent. In some embodiments, the anti-PD-1 antagonist antibody binds to PD-1 and enhances the antitumor immune response.

As used herein, the term anti-PD-L1 antagonist antibody refers to an antibody that is capable of inhibiting the biological activity of PD-L1 and/or subsequent PD-L1-mediated event(s). PD-L1 antagonist antibodies include antibodies that block, antagonize, inhibit or reduce (to any extent, including significant) the biological activity of PD-L1, including downstream events mediated by PD-L1 such as PD-L1 binding and subsequent signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10 and inhibition of antitumor immune responses. It should be clearly understood that for purposes of the present invention, the term anti-PD-L1 antagonist antibody (used interchangeably with the term PD-L1 antagonist antibody, anti-PD-L1 antagonist antibody, or anti-PD-L1 antagonist antibody) includes all previously defined terms, names , functional states and characteristics whereby PD-L1 itself, its biological activity, or the consequences of its biological activity are substantially eliminated, reduced, or neutralized to any significant extent. In some embodiments, the anti-PD-L1 antagonist antibody binds to PD-L1 and enhances the antitumor immune response.

The phrase immune checkpoint regulator refers to a functional class of agents that inhibit or stimulate immune checkpoint signaling. Immune checkpoint regulators include receptors and their associated ligands, which together provide ways to inhibit or stimulate signaling pathways that would otherwise lead to T cell activation. Examples of immune checkpoint regulators include, but are not limited to, TIGIT and its CD155 ligand, PVR; PD-1 and its ligands PD-L1 and PD-L2; CTLA-4 and its ligands B7-1 and B7-2; TIM-3 and its ligand galectin-9; LAG-3 and its ligands, including liver sinusoidal endothelial cell lectin (LSECtin) and galectin-3; CD122 and its ligand CD122R; CD70, B7H3, B and T lymphocyte attenuator (BTLA) and VISTA.

The phrases checkpoint regulator antagonist, immune checkpoint binding antagonist, and immune checkpoint antagonist are used interchangeably herein to refer to a class of agents that interfere with the activity (or inhibit the activity) of an immune checkpoint regulator in a manner that results in binding to said checkpoint regulator or by its ligand, signaling through the checkpoint regulator receptor is blocked or inhibited. Inhibition of this signaling may result in a reversal of immunosuppression such that T cell immunity against cancer cells can be restored or enhanced. Immune checkpoint regulator antagonists include antibody fragments, peptide inhibitors, dominant negative peptides and small molecule drugs, presented alone or as part of a fusion protein or conjugate.

The phrases immune checkpoint binding agonist and immune checkpoint agonist are used interchangeably herein to refer to a class of agents that stimulate the activity of an immune checkpoint regulator such that, as a result of binding to said checkpoint regulator or a ligand thereof, signaling through the checkpoint regulator receptor is stimulated. points. Stimulation of this signaling may lead to the restoration or enhancement of T-cell immunity against cancer cells. Examples of immune checkpoint regulator agonists include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily such as CD27, CD40, OX40 (CD 134), glucocorticoid-induced TNFR family-related protein (GITR), and 4- 1BB (CD137) and their ligands. Additional agonists of checkpoint regulators belong to the B7-CD28 superfamily, including CD28 and ICOS.

The phrases dominant negative protein or dominant negative peptide refer to a protein

- 8 045974 or a peptide derived from a wild-type protein that has been genetically modified by mutation and/or deletion in such a way that the modified protein or peptide interferes with the function of the endogenous wild-type protein from which it is derived.

The phrase small molecule drug refers to a molecular compound, often organic or organometallic, that is not a polymer, has medicinal activity, and has a molecular weight of less than about 2 kDa, less than about 1 kDa, less than about 900 Da, less than about 800 Da, or less than about 700 Da. The term includes most drug compounds, called drugs, other than protein or nucleic acids, where a small peptide or nucleic acid analog can be considered a small molecule drug. Examples include chemotherapeutic anticancer drugs and enzyme inhibitors. Low molecular weight drugs can be obtained synthetically, semi-synthetically (i.e. from natural precursors) or biologically.

It should be understood that when describing the order of polypeptide domains with hyphens between individual domains (for example, CH2-CH3), the listed domains are arranged in order from the amino terminus to the carboxy terminus.

The phrases specifically bind or have specificity generally imply that the antibody binds to an epitope via its antigen binding domain and that said binding involves some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to bind specifically to an epitope when it binds to said epitope through its antigen binding domain more readily than it could bind to a random unrelated epitope. The term specificity is used herein to define the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody A may be considered to have higher specificity for a given epitope compared to antibody B, or antibody A may be considered to bind to epitope C with higher specificity than its specificity to the related epitope D.

The term immunoconjugate refers to an antibody that is covalently fused to an inhibitory peptide or small molecule drug. The peptide or small molecule drug may be linked to the C-terminus of a heavy chain constant region or the N-terminus of a light and/or heavy chain variable region.

The linker can be used to stably covalently link a peptide or small molecule drug, such as a maytansinoid, to antitumor antagonists. Linkers may be sensitive or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioester groups, acid-labile groups, photolabile groups, peptidase-labile groups and esterase-labile groups. Linkers also include charged linkers and hydrophilic forms thereof, as described herein and known in the art. The immunoconjugate may further comprise a flexible peptide of 3-15 amino acids (or a spacer) between the antitumor antagonist and the peptide and/or small molecule drug.

As used herein, the term immunoglobulin scaffold refers to any polymer of amino acids that exhibits properties desired to support antagonist function, including adding antibody specificity, enhancing antibody function, or maintaining antibody structure and stability. The immunoglobulin framework may comprise one or more immunoglobulin constant regions, including CH1, CH2 and/or CH3 regions of the immunoglobulin heavy chain and/or CL regions of the immunoglobulin light chain. Binding domains of a donor polypeptide may be grafted onto the immunoglobulin scaffold to impart binding specificity to the scaffold for the donor polypeptide.

As used herein, the phrase multispecific inhibitor refers to a molecule containing at least two targeting domains with different binding specificities. In some embodiments, a multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen-binding domains targeting different antigens or epitopes. According to specific embodiments of the invention, the multispecific inhibitor is a bispecific antibody.

As used herein, the term bispecific refers to a molecule containing at least two targeting domains with different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and inhibiting the biological function of said target molecule upon binding thereto. In some embodiments, the bispecific checkpoint regulator antagonist is

- 9 045974 polymer molecule containing two or more peptides. In some embodiments, the targeting domain comprises an antigen binding domain or CDR of the antibody. In some embodiments, the bispecific inhibitor is a bispecific antibody.

The terms bispecific antibody and bispecific antagonist are used interchangeably herein to refer to an antibody that can specifically bind to two different antigens (or epitopes). In some embodiments, a bispecific antibody is a full-length antibody that binds to one antigen (or epitope) on one of its two binding fragments (one HC/LC pair) and binds to another antigen (or epitope) on its second fragment ( another HC/LC pair). In these embodiments, the bispecific antibody contains two distinct antigen binding moieties (both in specificity and CDR sequences) and is monovalent for each antigen to which it binds.

In other embodiments, a bispecific antibody is a full-length antibody that can bind to two different antigens (or epitopes) in each of its two binding moieties (two HC/LC pairs). According to these embodiments, the bispecific antibody contains two identical antigen-binding fragments having the same specificity and identical CDR sequences, and is bivalent for each antigen to which it binds.

Examples of bispecific antibodies may include asymmetric IgG-like antibodies (eg, triomab/quadroma, Trion Pharma/Fresenius Biotech); ridge-to-groove antibodies (Genentech); CrossMAb (Roche); electrostatically coupled antibodies (AMGEN); LUZ-Y (Genentech); strand exchange engineered domain proteins (SEEDBodies, EMD Serono; biolonic, Merus); Fab replacement antibodies (Genmab), symmetric IgG-like antibodies (e.g. dual-targeting (DT) Ig (GSK/Domantis); two-in-one antibodies (Genentech); cross-linked MAbs (Karmanos Cancer Center), mAb2 (F -star); Cov X-body (Cov X/Pfizer); double variable domain (DVD) and Ig fusion proteins (Abbott); IgG-like bispecific antibodies (Eli Lilly); Ts2Ab (Medimmune/AZ); BsAb (ZymoGenetics) ; HERCULES (Biogen Idec, TvAb, Roche); scFv/Fc fusion proteins; SCORPION (Emergent BioSolutions/Trubion, ZymoGenetics/BMS); dual affinity retargeting technology (Fc-DART), MacroGenics; dual (scFv)2-Fab ( National Research Center for Antibody Medicine); F(ab)2 fusion proteins (Medarex/AMGEN); Dual-acting Fab or Bis-Fab (Genentech); Dockand-Lock (DNL, ImmunoMedics); Fab-Fv (UCB-Celltech); scFv-based antibodies and diabodies (eg, bispecific T-cell recruiters (BiTEs, Micromet); tandem diabodies (Tandab, Affimed); DART (MacroGenics); single-chain diabodies; TCR-like antibodies (AIT, Receptor Logics); human serum albumin fusion protein scFv (Merrimack); COMBODIES (Epigen Biotech) and IgG/re-IgG fusion proteins (e.g. immunocytokines (EMDSerono, Philogen, ImmunGene, ImmunoMedics).

The terms treat and treatment refer to the relief of one or more symptoms associated with a cell proliferative disorder; preventing or delaying the onset of one or more symptoms of a cell proliferative disorder and/or reducing the severity or frequency of one or more symptoms of a cell proliferative disorder.

The phrases patient in need, patient in need of treatment, or subject in need of treatment include subjects, such as mammalian subjects, that will benefit from administration of an antitumor antagonist according to the present invention for the treatment of a cell proliferative disorder.

The terms therapeutically effective amount, pharmacologically effective amount, and physiologically effective amount are used interchangeably to refer to the amount of antitumor antagonist that is necessary to achieve a threshold level of active antagonist agents in the bloodstream or target tissue. The exact amount will depend on a variety of factors, for example, the specific active agent, the components and physical characteristics of the composition, the intended patient population, the patient's problems, etc., and can be readily determined by one of ordinary skill in the art based on the information provided herein. application or otherwise available in the relevant literature.

The terms improve, increase, or decrease, as used herein, indicate values or parameters relative to a baseline measurement, such as a measurement in the same subject prior to the initiation of treatment described herein, or a measurement in a control subject (or multiple control subjects), not receiving the treatment described in this application.

A control subject is a subject suffering from the same cell proliferative disorder as the treated subject who is approximately the same age as the treated subject (to ensure that the disease stages of the treated subject , and control subjects are comparable). The individual being treated (also referred to as a patient or subject) may be a fetus, infant, child, adolescent, or adult suffering from a cell proliferative disorder.

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The term cell proliferative disorder refers to a disorder characterized by abnormal cell proliferation; a proliferative disorder does not imply any limitation in the rate of cell growth, but only indicates a loss of normal control that affects cell growth and division. Thus, according to some embodiments of the invention, in a cell proliferative disorder, cells can divide at the same rate as normal cells, but do not respond to signals that limit such growth. The term cell proliferative disorder includes a neoplasm, cancer or tumor.

The term cancer or tumor refers to any of various malignancies characterized by a proliferation of cells that have the ability to invade surrounding tissues and/or metastasize to new sites of colonization, and includes leukemia, lymphoma, carcinoma, melanoma, sarcoma, germinal cell tumor and blastoma. Examples of cancers to be treated by the methods of the present invention include brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, non-small cell lung cancer, mesothelioma, ovarian cancer, cancer prostate, stomach and uterine cancer, leukemia and medulloblastoma.

The term leukemia refers to a progressive malignant disease of the hematopoietic organs and is generally characterized by altered proliferation and development of leukocytes and their precursors in the blood and bone marrow. Examples of leukemias include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocythaemic leukemia, basophilic leukemia, blast cell leukemia, leukemia cattle, chronic myelocytic leukemia, cutaneous leukemia, embryonal leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymph regional leukemia, lymphocytic leukemia, lymphogenous leukemia , lymphoid leukemia, lymphosarcoma leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Nägeli leukemia, leukemia h plasma cells, plasmacytic leukemia, promyelocytic leukemia, Reader cell leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia and undifferentiated cell leukemia.

The term carcinoma refers to the malignant growth of epithelial cells that tend to infiltrate surrounding tissues and give rise to metastases. Examples of carcinomas include, for example, acinar cell carcinoma, adenocarcinoma, adenoid cystic carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenal cortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basocellular carcinoma, basaloid carcinoma, basospinocellular carcinoma, bronchoalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, mucinous carcinoma, comedonal carcinoma, uterine carcinoma, cribriform carcinoma, advanced pleural carcinoma, cutaneous carcinoma, columnar carcinoma, columnar cell carcinoma, ductal carcinoma, solid carcinoma, embryonal carcinoma , encephaloid carcinoma, epinoid carcinoma, epithelial adenoid carcinoma, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, gelatinous carcinoma, colloid carcinoma, giant cell carcinoma, giant cell carcinoma, glandular carcinoma, granulosa cell carcinoma, basal cell carcinoma, hematoid carcinoma, hepatitis taphole carcinoma, Hurtle cell carcinoma, hyaline carcinoma, hypomephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher carcinoma, Kulczycki cell carcinoma, large cell carcinoma, lenticular carcinoma, lenticular carcinoma, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanotic carcinoma, Molle carcinoma, mucinous carcinoma, mucoid carcinoma, mucous cell carcinoma, mucoepidermoid carcinoma, mucosal carcinoma, mucinous carcinoma, myxomatodal carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid nue carcinoma, papillary carcinoma , periportal carcinoma, preinvasive carcinoma, spiny cell carcinoma, renal cell carcinoma of the kidney, reserve cell carcinoma, sarcoma-like carcinoma, Schneiderian carcinoma, scrotal carcinoma, signet ring cell carcinoma, simple carcinoma, small cell carcinoma, solanoid carcinoma, spherical cell carcinoma, spindle cell carcinoma carcinoma, spongiform carcinoma, squamous cell carcinoma, squamous cell carcinoma, string carcinoma, telangiectatic carcinoma, smooth muscle fiber and vascular tissue carcinoma, transitional cell carcinoma, tuberous carcinoma, tuberous carcinoma, verrucous carcinoma and villous carcinoma.

The term sarcoma refers to a tumor consisting of a substance similar to an embryonic compound

- 11 045974 filament tissue, which generally consists of densely packed cells enclosed in a fibrillar or homogeneous substance. Examples of sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemeti's sarcoma, adipose tissue sarcoma, liposarcoma, alveolar soft tissue sarcoma, ameloblastic sarcoma, botryoid sarcoma, granulocytic sarcoma, choriocarcinoma, embryonic sarcoma arcoma, Wilms' sarcoma, sarcoma Endometry, stromal sarcoma, sarcoma of Ying, fascial sarcoma, fibroblast sarcoma, gigantic cell sarcoma, granulocytic sarcoma, Hodgkinskaya sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, for example, immunoblastic sarcoma of B-cells, lymphomas (for example, Nekokhodzhinsky lymphoma), immune. Noblastic sarcoma T cells , Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, foliate cytosarcoma, synovital sarcoma and telangiectatic sarcoma.

The term melanoma refers to a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman melanoma, S91 melanoma, Harding-Passy melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, and superficial spreading melanoma.

Additional cancers include, for example, Hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant insulinoma pancreatic cancer, malignant carcinoid, precancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer and adrenal cancer.

I. Checkpoint regulator antagonists

In one aspect, the present invention provides an antitumor antagonist comprising an immunoglobulin scaffold with (1) a pair of fragments containing variable domain regions that specifically bind to a first immune checkpoint regulator, and (2) a single chain fragment (scFv) that specifically binds to a second immune checkpoint regulator.

In another aspect, the present invention provides an antitumor antagonist comprising an immunoglobulin scaffold structurally related to a first immune checkpoint regulator antagonist and a second immune checkpoint regulator antagonist in the form of a scFv.

In both aspects, the immunoglobulin framework may comprise one or more immunoglobulin constant regions, for example CH1, CH2 and/or CH3 IgG. According to specific embodiments of the invention, the immunoglobulin framework is an Fc fragment (CH2-CH3 hinge).

According to some embodiments of the present invention, an antitumor antagonist comprises an immunoglobulin scaffold, wherein the N-terminus of said antagonist contains a first immune checkpoint regulator antagonist structurally related thereto, wherein said first immune checkpoint regulator antagonist specifically binds to PD-1, PD- L1, LAG-3, TIGIT, and a second antagonist of immune checkpoint regulators is located at the C-terminus of the antagonist in the form of a scFv that specifically binds to PD-1, PD-L1, LAG-3 or TIGIT.

According to some embodiments of the present invention, the scFv contains a linker connecting the heavy chain variable regions to the light chain variable regions. According to one embodiment of the invention, the linker contains an amino acid sequence containing between 3, 4, 5, 6, 7, 8, 9 or 10 copies of the G4S amino acid sequence. According to another embodiment of the invention, the linker contains the amino acid sequence shown in any of the sequences of SEQ ID NO: 188-191. According to a more specific embodiment of the invention, the linker contains the amino acid sequence of SEQ ID NO: 191.

In one embodiment, the anti-TIGIT scFv comprises one or more heavy chain CDRs selected from SEQ ID NOs: 1-25 and one or more light chain CDRs selected from SEQ ID NOs: 26-47.

According to another embodiment of the invention, the anti-TIGIT scFv contains heavy chain/light chain variable regions, wherein the scFv contains a heavy chain variable region (HCVR) that has at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66, and the lung variable region chain (LCVR) that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with LCVR, which has an amino acid sequence selected from the group consisting of from SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, 63 and 67.

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In a more specific embodiment, the anti-TIGIT scFv comprises an HCVR that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 66, and an LCVR that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an LCVR that has the amino acid sequence SEQ ID NO: 67.

According to another embodiment of the invention, the anti-TIGIT scFv contains: HCVR, which contains (1) HCDR1 with the sequence SEQ ID NO: 23, HCDR2 with the sequence SEQ ID NO: 24 and HCDR 3 with the sequence SEQ ID NO:25 in combination with (2 ) HFR1, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 260, HFR2, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 247 , HFR3 that has at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 261, and HFR4 that has at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 236; and further comprises an immunoglobulin LCVR that contains (1) LCDR1 of the sequence SEQ ID NO: 45, LCDR2 of the sequence SEQ ID NO: 46 and LCDR3 of the sequence SEQ ID NO: 47 in combination with (2) LFR1, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 220, LFR2, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 228, LFR3, which has at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 234, LFR4, which has at least 80%, 85%, or 90% identity with the amino acid sequence of SEQ ID NO: 262.

According to another embodiment of the invention, the first targeting domain comprises one or more variable regions from an anti-PD-1 antibody, and the second targeting domain comprises an anti-TIGIT scFv, as described above.

In one embodiment, the anti-PD-1 targeting domain comprises one or more heavy chain CDRs selected from SEQ ID NOs: 68-81 and/or one or more light chain CDRs selected from SEQ ID NOs: 82- 95.

In another embodiment, the anti-PD-1 targeting domain comprises: an HCVR that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 96, 98, 100, 102, 104 and 106; An LCVR that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with an LCVR that has an amino acid sequence selected from the group consisting of SEQ ID NO: 97, 99, 101, 103, 105 and 107; or both.

In another embodiment, the anti-PD-1 targeting domain comprises: HCVR, which comprises (1) HCDR1 of SEQ ID NO:79, HCDR2 of SEQ ID NO:80, and HCDR3 of SEQ ID NO:81 in combination with (2) HFR1, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 283, HFR2, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 277, HFR3, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 288, HFR4, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 274; An immunoglobulin LCVR that contains (1) LCDR1 of the sequence SEQ ID NO: 93, LCDR2 of the sequence SEQ ID NO: 94 and LCDR 3 of the sequence SEQ ID NO: 95 in combination with (2) LFR1, which has at least 80% , 85% or 90% identity with the amino acid sequence of SEQ ID NO: 289, LFR2, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 290, LFR3, which has at least 80% identity , 85% or 90% identity with the amino acid sequence of SEQ ID NO: 291, and LFR4, which has at least 80%, 85% or 90% identity with the amino acid sequence of SEQ ID NO: 292; or both.

In another embodiment, the anti-PD-1 targeting domain comprises an HCVR that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity SEQ ID NO: 106; LCVR that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence of SEQ ID NO: 107; or both of these areas.

In a specific embodiment, the bispecific PD-1/TIGIT antagonist comprises a first targeting domain that comprises HCVR, which has the amino acid sequence of SEQ ID NO: 106, and/or LCVR, which has the amino acid sequence of SEQ ID NO: 107, in combination with scFv against TIGIT, which contains HCVR, which has the amino acid sequence of SEQ ID NO: 66, and LCVR, which has the amino acid sequence

- 13 045974 item SEQ ID NO: 67.

In a more specific embodiment, the bispecific PD-1/TIGIT antagonist scFv comprises a linker connecting heavy chain variable regions to light chain variable regions in a second targeting domain, wherein said linker comprises the amino acid sequence of SEQ ID NO: 191.

According to another embodiment of the invention, the bispecific PD-1/TIGIT antagonist comprises: an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 160; an immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 161; or both of these circuits.

According to another embodiment of the invention, the bispecific PD-1/TIGIT antagonist comprises: an immunoglobulin heavy chain containing the amino acid sequence of SEQ ID NO: 162; an immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 161; or both of these circuits.

In another embodiment, the bispecific PD-1/TIGIT antagonist comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 160 and an immunoglobulin light chain comprising the amino acid sequence of SEQ ID NO: 161.

According to another embodiment of the invention, the first targeting domain comprises one or more variable regions from an anti-PD-L1 antibody, and the second targeting domain comprises an anti-TIGIT scFv, as described above.

In one embodiment, the anti-PD-L1 targeting domain comprises one or more heavy chain CDRs selected from SEQ ID NOs: 108-122 and/or one or more light chain CDRs selected from SEQ ID NOs: 123- 138.

In another embodiment, the anti-PD-L1 targeting domain comprises an HCVR that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical amino acid sequence selected from the group consisting of SEQ ID NO: 139, 141, 143, 145, 147, 149, 151 and 153, and/or LCVR, at least 80%, at least 85%, at least at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 140, 142, 144, 146, 148, 152 and 154.

In another embodiment, the anti-PD-L1 targeting domain comprises an HCVR that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical amino acid sequence of SEQ ID NO: 153, and/or LCDR at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to LCVR, containing the amino acid sequence SEQ ID NO: 154.

In another embodiment, the anti-PD-L1 targeting domain comprises: an immunoglobulin HCVR, which comprises (1) HCDR1 of SEQ ID NO: 111, HCDR2 of SEQ ID NO: 114, and HCDR 3 of SEQ ID NO: 115. combinations with (2) HFR1 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 283, HFR2 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 277 , HFR3 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 300, and HFR4 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 274; An immunoglobulin LCVR that contains (1) LCDR1 of SEQ ID NO: 123, LCDR2 of SEQ ID NO: 124 and LCDR 3 of SEQ ID NO: 125 in combination with (2) LFR1, at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 294, LFR2, at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 295, LFR3, at least 80%, 85% or 90 % identical to the amino acid sequence of SEQ ID NO: 296, and LFR4 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 276; or both.

In a specific embodiment, the bispecific PD-L1/TIGIT antagonist comprises a first targeting domain that comprises: HCVR, which has the amino acid sequence of SEQ ID NO: 153, and/or LCVR, which has the amino acid sequence of SEQ ID NO: 154, in combination with a second targeting domain as an anti-TIGIT scFv that contains an HCVR that has the amino acid sequence of SEQ ID NO: 66, and an LCVR that has the amino acid sequence of SEQ ID NO: 67. According to a more specific embodiment of the invention, the scFv in a bispecific anti-PD antagonist -L1/TIGIT contains a linker connecting HCVR anti-TIGIT with LCVR anti-TIGIT in a second targeting domain, wherein said linker contains the amino acid sequence of SEQ ID NO: 191.

According to another embodiment of the invention, the first targeting domain comprises one or more variable regions from an anti-LAG-3 antibody, and the second targeting domain comprises

- 14 045974 scFv against TIGIT as described above.

In one embodiment, the anti-LAG-3 targeting domain comprises one or more immunoglobulin heavy chain CDRs selected from SEQ ID NOs: 163-171 and/or one or more immunoglobulin light chain CDRs selected from SEQ ID NOs: 172- 178.

In another embodiment, the anti-LAG-3 targeting domain comprises an HCVR that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical amino acid sequence selected from the group consisting of SEQ ID NO: 180, 182, 184 and 186, and/or LCVR, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to an LCVR comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 181, 183, 185 and 187.

In another embodiment, the anti-LAG-3 targeting domain comprises: an immunoglobulin HCVR comprising (1) HCDR1 of SEQ ID NO: 163, HCDR2 of SEQ ID NO: 164, and HCDR 3 of SEQ ID NO: 165 in combination with (2) HFR1 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 316, HFR2 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 317, HFR3 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 318, and HFR4 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 319; An immunoglobulin LCVR comprising (1) LCDR1 of SEQ ID NO: 172, LCDR2 of SEQ ID NO: 173 and LCDR 3 of SEQ ID NO: 174 in combination with (2) LFR1, at least 80% 85 % or 90% identical to the amino acid sequence of SEQ ID NO: 320, LFR2, at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 321, LFR3, at least 80%, 85% or 90% identical identical to the amino acid sequence of SEQ ID NO: 322, and LFR4 at least 80%, 85% or 90% identical to the amino acid sequence of SEQ ID NO: 323; or both.

In another embodiment, the anti-LAG-3 targeting domain comprises an immunoglobulin HCVR that has an amino acid sequence that is 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 180, and/or an immunoglobulin LCVR that has an amino acid sequence that is 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 181.

According to a specific embodiment of the invention, the bispecific LAG-3/TIGIT antagonist comprises a first targeting domain that contains an HCVR that has the amino acid sequence of SEQ ID NO: 180, and/or an LCVR that has the amino acid sequence of SEQ ID NO: 181, in combination with a scFv anti-TIGIT containing HCVR, which has the amino acid sequence of SEQ ID NO: 66, and LCVR, containing the sequence of SEQ ID NO: 67. In a more specific embodiment of the invention, the scFv in the bispecific anti-LAG3/TIGIT antagonist contains a linker connecting the HCVR anti-TIGIT to LCVR against TIGIT in the second targeting domain, wherein said linker contains the amino acid sequence of SEQ ID NO: 189 or SEQ ID NO: 191.

According to one embodiment of the invention, the bispecific LAG-3/TIGIT antagonist comprises an immunoglobulin heavy chain comprising the amino acid sequence of SEQ ID NO: 192 or 193; an immunoglobulin light chain containing the amino acid sequence of SEQ ID NO: 181, or both of these chains.

Antibodies against LAG-3 and their antigen-binding fragments

According to another aspect, the present invention provides antibodies, including antigen-binding portions thereof, that specifically bind to LAG-3. In fig. 19A shows the heavy chain CDR1, CDR2 and CDR3 sequences corresponding to anti-LAG-3 mAbs 2L2A.1, 2L2A.6, 2L27B and 3L1A. In fig. 19B shows the light chain CDR1, CDR2 and CDR3 sequences corresponding to the anti-LAG-3 mAb 2L2A. 1, 2L2A.6, 2L27B and 3L1A. In fig. 20 shows the VH and VL sequences of anti-LAG-3 mAbs 2L2A.1, 2L2A.6, 2L27B and 3L1A.

In one embodiment, the anti-LAG-3 antibody or antigen-binding portion thereof comprises: an immunoglobulin heavy chain CDR1 (HCDR1) sequence that is at least 80%, at least 85%, or at least 90% identical to the amino acid sequence of HCDR1 selected from the group consisting of SEQ ID NOs: 163, 166 and 169; an immunoglobulin heavy chain CDR2 (HCDR2) sequence that is at least 80%, at least 85%, or at least 90% identical to the amino acid sequence of HCDR2 selected from the group consisting of SEQ ID NOs: 164, 167 and 170; An immunoglobulin heavy chain CDR3 (HCDR3) that is at least 80%, at least 85%, or at least 90% identical in amino acid sequence to HCDR3 selected from the group consisting of SEQ ID NOs: 165, 168 and 171; An immunoglobulin light chain CDR1 (LCDR1) that is at least 80%, at least 85%, or at least 90% identical in amino acid sequence to LCDR1 is selected

- 15 045974 from the group consisting of SEQ ID NOs: 172, 175 and 177; An immunoglobulin light chain CDR2 (LCDR2) that is at least 80%, at least 85%, or at least 90% identical to the amino acid sequence of LCDR2 selected from the group consisting of SEQ ID NOs: 173 and 178; and an immunoglobulin light chain CDR3 (LCDR3) that is at least 80%, at least 85%, or at least 90% identical to the amino acid sequence of LCDR3 selected from the group consisting of SEQ ID NOs: 174, 176 and 179.

According to another embodiment of the invention, the anti-LAG-3 antibody or antigen-binding portion thereof comprises: an immunoglobulin HCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 163, 166 and 169; an HCDR2 immunoglobulin amino acid sequence selected from the group consisting of SEQ ID NOs: 164, 167 and 170; an immunoglobulin HCDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 165, 168 and 171; an immunoglobulin LCDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 172, 175 and 177; an immunoglobulin LCDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 173 and 178; and an immunoglobulin LCDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 174, 176 and 179.

According to another embodiment of the invention, the anti-LAG-3 antibody or antigen-binding portion thereof comprises: an immunoglobulin HCVR that has at least 80%, 85%, 90%, 95%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 180, 182, 184 and 186; An immunoglobulin LCVR that has at least 80%, 85%, 90%, 95%, or 99% identity with an LCVR that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 181, 183, 185, and 187; or both.

According to another embodiment of the invention, the anti-LAG-3 antibody or antigen-binding portion thereof comprises: an immunoglobulin HCVR that has an amino acid sequence selected from the group consisting of SEQ ID NOs: 180, 182, 184 and 186; An immunoglobulin LCVR which has an amino acid sequence selected from the group consisting of SEQ ID NOs: 181, 183, 185 and 187; or both.

According to another embodiment of the invention, the anti-LAG-3 antibody or antigen-binding portion thereof comprises: at least 80%, at least 85%, at least 90%, at least 95% immunoglobulin heavy chain sequence, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO: 180; an immunoglobulin light chain sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 181; or both of these sequences.

According to a more specific embodiment of the invention, the anti-LAG-3 antibody or antigen-binding portion thereof comprises: an immunoglobulin heavy chain sequence SEQ ID NO: 180; immunoglobulin light chain sequence SEQ ID NO: 181; or both of these sequences.

According to another aspect, the present invention provides one or more nucleic acids encoding any anti-LAG-3 antibody or any antigen-binding portion thereof, as described herein.

According to another aspect, the present invention provides one or more expression vectors containing one or more nucleic acids encoding any anti-LAG-3 antibody or any antigen-binding portion thereof, as described herein.

In another aspect, the present invention provides a host cell transformed with one or more nucleic acids or one or more expression vectors encoding any anti-LAG-3 antibody or any antigen binding moiety as described herein.

According to another aspect, the present invention provides a bispecific antitumor antagonist comprising a first targeting domain that specifically binds to LAG-3; and a second targeting domain specifically binding to PD-1, PD-L1 or TIGIT, wherein said first targeting domain comprises any of the LAG-3 binding moieties described above. Preferably, the bispecific antitumor LAG-3 antagonist comprises an immunoglobulin framework comprising one or more IgG constant regions, eg CH1, CH2, CH3 and/or CL.

In some embodiments of the present invention, the first targeting domain is located at the N-terminus and the second targeting domain is located at the C-terminus. In other embodiments, the first targeting domain is located at the C-terminus and the second targeting domain is located at the N-terminus. In other embodiments, the second targeting domain is inserted into a loop region of, for example, a CH3 domain.

In one embodiment, the bispecific antitumor antagonist comprises a first targeting domain that specifically binds to LAG-3 and a second targeting domain that specifically binds to PD-1, wherein said first targeting domain comprises any of the LAG-3 binding moieties described above. , and the specified second one is aimed

- 16 045974 This domain contains any of the PD-1 binding fragments described below. For example, according to one embodiment of the invention, the first targeting domain comprises the amino acid sequence HCVR SEQ ID NO: 180 in combination with LCVR, which has the amino acid sequence SEQ ID NO: 181, and the second targeting domain comprises HCVR, which has the amino acid sequence SEQ ID NO: 181. 106, in combination with LCVR, which has the amino acid sequence of SEQ ID NO: 107. Alternatively, the second domain may be in the form of a PD-1 ECD.

According to another embodiment of the invention, the bispecific antitumor antagonist comprises a first targeting domain that specifically binds to LAG-3 and a second targeting domain that specifically binds to PD-L1, wherein said first targeting domain comprises any of the LAG-3 binding moieties described above and wherein said second targeting domain comprises any of the PD-L1 binding moieties described below. For example, according to one embodiment of the invention, the first targeting domain comprises HCVR, which has the amino acid sequence of SEQ ID NO: 180, in combination with LCVR, which has the amino acid sequence of SEQ ID NO: 181, and the second targeting domain contains HCVR, which has the amino acid sequence SEQ ID NO: 153, in combination with LCVR, which has the amino acid sequence of SEQ ID NO: 154.

According to another embodiment of the invention, the bispecific antitumor antagonist comprises a first targeting domain that specifically binds to LAG-3 and a second targeting domain that specifically binds to TIGIT, wherein said first targeting domain comprises any of the LAG-3 binding moieties described above, and said second targeting domain comprises any of the TIGIT binding moieties described below. For example, according to one embodiment of the invention, the first targeting domain comprises HCVR, which has the amino acid sequence of SEQ ID NO: 180, in combination with LCVR, which has the amino acid sequence of SEQ ID NO: 181, and the second targeting domain contains HCVR, which has the amino acid sequence SEQ ID NO: 66, in combination with LCVR, which has the amino acid sequence of SEQ ID NO: 67. Alternatively, the second domain may be in the form of a TIGIT ECD.

Examples of immunoglobulin frameworks include, for example, a full-length CH1-CH2CH3 segment as set forth in SEQ ID NOs: 155-157 and 205-215, or an Fc fragment (CH2-CH3 hinge) containing the amino acid sequence set forth in any of SEQ ID NOs: NO: 195-202.

Anti-TIGIT antibody and anti-TIGIT antibody fragments

In some embodiments of the present invention, the checkpoint regulatory antagonist comprises an anti-TIGIT antibody or antigen-binding fragment(s) thereof. In fig. 1 shows the CDR sequences of the anti-TIGIT mAb, while FIG. 2A-2B show several embodiments of anti-TIGIT antibody variable domain sequences for use in accordance with the present invention.

In one embodiment, an anti-TIGIT antibody or antigen-binding fragment(s) thereof comprises: (1) an immunoglobulin HCVR comprising three complementarity determining regions (HCDR): HCDR1, HCDR2 and HCDR3, wherein HCDR1 has an amino acid sequence of at least 80 %, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 6 , 11, 15, 17, 20 and 23, wherein HCDR2 has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99 % or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 7, 9, 12, 13, 16, 18, 21 and 24, and wherein HCDR3 has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 5, 8, 10, 14, 19, 22 and 25; and (2) an immunoglobulin LCVR comprising the sequences LCDR1, LCDR2 and LCDR3, wherein LCDR1 has at least 80%, at least 85%, at least 90%, at least 95% of the amino acid sequence at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 26, 29, 31, 33, 35, 39, 42 and 45, where LCDR2 has an amino acid sequence of at least 80 %, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 27, 30 , 36, 37, 40, 43 and 46, and wherein LCDR3 has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 28, 32, 34, 38, 41, 44 and 47; wherein said antibody or antigen-binding portion thereof specifically binds to human TIGIT. According to another embodiment

- 17 045974 bretium, anti-TIGIT antibody or antigen-binding fragment(s) thereof contains: (1) an immunoglobulin HCVR that has an amino acid sequence of at least 80%, at least 85%, at least 90%, according to at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 48, 50, 52, 54, 56, 58, 60, 62, 64 and 66; and (2) an immunoglobulin LCVR that has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 49, 51, 53, 55, 57, 59, 61, 63, 65 and 67; wherein said antibody or antigen-binding portion thereof specifically binds to human TIGIT.

Antibodies against PD-1 and their antigen-binding fragments

In some embodiments of the present invention, the checkpoint regulatory antagonist comprises an anti-PD-1 antibody or antigen-binding fragment(s) thereof. In fig. 3 shows the CDR sequences of the anti-PD-1 mAb, and FIG. 4A-4C show several embodiments of anti-PD-1 antibody variable domain sequences for use in accordance with the present invention.

In one embodiment, the anti-PD-1 antibody or antigen-binding fragment(s) thereof comprises: (1) an immunoglobulin HCVR comprising the sequences HCDR1, HCDR2 and HCDR3, wherein HCDR1 is at least 80% amino acid in sequence 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 68, 71, 74, 76 and 79, wherein HCDR2 has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequence, selected from the group consisting of SEQ ID NOs: 69, 72, 77 and 80, and wherein HCDR3 has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 70, 73, 75, 78 and 81; and (2) an immunoglobulin LCVR comprising the sequences LCDR1, LCDR2 and LCDR3, wherein LCDR1 has at least 80%, at least 85%, at least 90%, at least 95% of the amino acid sequence at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 82, 85, 88, 89, 90 and 93, wherein LCDR2 has an amino acid sequence that is at least 80% identical to at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 83, 86, 91 and 94 and wherein LCDR3 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 84, 87, 92, and 95, wherein the antibody or antigen-binding portion thereof specifically binds to human PD-1.

In some embodiments of the present invention, an anti-PD-1 antibody or antigen-binding fragment(s) thereof comprises: (1) an immunoglobulin HCVR that has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 96, 98, 100, 102, 104 and 106; and (2) an immunoglobulin LCVR that has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 97, 99, 101, 103, 105 and 107, wherein the antibody, or an antigen-binding portion thereof, specifically binds to human PD-1.

Antibodies against PD-L1 and their antigen binding fragment

In some embodiments of the present invention, the checkpoint regulatory antagonist comprises an anti-PD-L1 antibody or antigen-binding fragment(s) thereof. In fig. 5 shows the CDR sequences of the anti-PD-L1 mAb, and FIG. 6A-6C show several embodiments of anti-PD-L1 antibody variable domain sequences for use in accordance with the present invention.

In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment(s) thereof comprises: (1) an immunoglobulin HCVR comprising the sequences HCDR1, HCDR2 and HCDR3, wherein HCDR1 is at least 80% amino acid in sequence 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 108, 111, 117 and 120, wherein HCDR2 has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from group consisting of SEQ ID NOs: 109, 112, 114, 116, 118 and 121, where HCDR3 has the amino acid sequence

- 18 045974 at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 110, 113, 115, 119 and 122; and (2) an immunoglobulin LCVR, wherein the light chain variable region contains three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, wherein LCDR1 has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 123, 126, 130, 133 and 136, wherein LCDR2 has the amino acid sequence , at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 124, 127, 131, 134 and 137, and wherein LCDR3 has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least at least 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 125, 128, 129, 132, 135 and 138, wherein the antibody or antigen binding portion thereof specifically binds to human PD-L1.

In some embodiments of the present invention, an anti-PD-L1 antibody or antigen binding fragment(s) thereof comprises: (1) an immunoglobulin HCVR that has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 139, 141, 143, 145, 147, 149, 151 and 153; and (2) an immunoglobulin LCVR that has an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 140, 142, 144, 146, 148, 150, 152 and 154, wherein the antibody or antigen-binding portion thereof specifically binds to human PD-L1.

II. Other implementation options

HCVR and LCVR described herein may be associated with an immunoglobulin scaffold. In some embodiments, the immunoglobulin scaffold is in the form of IgG1, IgG2, or IgG4. The immunoglobulin scaffold may contain CH1-CH2-CH3 regions, or may contain a naturally occurring Fc fragment or a non-naturally occurring or mutated Fc fragment, such as a non-effector or substantially non-effector Fc fragment (eg, IgG2 or human IgG4) or, alternatively, an Fc having enhanced binding to one or more activating Fc receptors (FcyRI, FcYRIIa or FcYRIIIa) to enhance the depletion of the Treg cell population in the tumor environment. Accordingly, in some embodiments, the anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, HCVR and LCVR antibodies described herein may be linked to an Fc containing one or more modifications , typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.

In one embodiment, an immunoglobulin framework for use in the present invention comprises a CH1-CH2-CH3 region comprising the amino acid sequence set forth in SEQ ID NOs: 155-157 and 205-215. According to another embodiment of the invention, the immunoglobulin framework contains or essentially consists of an Fc receptor, such as an Fc receptor containing the amino acid sequence set forth in any of SEQ ID NOs: 195-202.

Moreover, the antibody described herein may be modified chemically (for example, one or more chemical moieties may be attached to the antibody), or the antibody may be modified to alter its degree of glycosylation, to alter one or more of its functionalities. properties. More specifically, in some embodiments, the antibodies herein may contain modifications in the Fc region to produce an Fc variant having (a) increased or decreased antibody-dependent cellular cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC) ), (c) increased or decreased affinity for C1c.| and/or (d) increased or decreased affinity for the Fc receptor compared to the parent Fc. Such Fc region variants generally contain at least one amino acid modification in the Fc region. A combination of amino acid modifications is considered particularly desirable. For example, a variant Fc region may contain two, three, four, five, etc. substitutions, for example, at certain positions of the specified Fc region identified in this application.

In applications where effector function must be avoided entirely, such as when antigen binding alone is sufficient to achieve the desired therapeutic effect and effector function only results in (or increases the risk of) unwanted side effects

- 19 045974 occurrence), IgG4 antibodies can be used, or antibodies or fragments that lack the Fc region or a significant portion thereof can be developed, or mutations can be introduced into the Fc fragment to completely eliminate glycosylation (eg, N297A). Alternatively, a hybrid construct of human IgG2 (CH1 domain and hinge region) and human IgG4 (CH2 and CH3 domains) can be created that lacks effector function, lacks the ability to bind FcyR (eg, IgG2) and activate complement (eg, IgG4). When using an IgG4 constant domain, it is generally preferable to include the S228P substitution, which mimics the hinge sequence in IgG1 and thereby stabilizes the IgG4 molecules by reducing the exchange of Fab fragments between the therapeutic antibody and endogenous IgG4 in the patient undergoing treatment.

According to preferred embodiments of the invention, the first and second targeting domains are presented in a humanized immunoglobulin framework. In addition, the IgG framework may contain the amino acid substitution N297A or K447A.

In specific embodiments, an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or fragments thereof may be modified to increase its biological half-life. Various approaches can be used, including, for example, those that increase the binding affinity of the Fc region to FcRn. In one embodiment, the antibody is modified within the CH1 or CL region to contain a salvage receptor binding epitope derived from two loops of the CH2 domain of the IgG Fc region, as described in U.S. Patent Nos. 5,869,046 and 6,121,022. The numbering of residues in the Fc region corresponds to the EU index. Sequence variants described herein are given by reference to the residue number followed by the designation of the amino acid replacing the naturally occurring amino acid, and optionally preceded by the designation of the naturally occurring residue at that position. If multiple amino acids may be present at a given position, for example if the sequences differ between naturally occurring isotypes or if multiple mutations may be introduced at that position, then they are separated by a slash (for example, X/Y/Z).

Examples of Fc variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 305A, 307A, 311A, 312A, 378Q, 380A, 382A, 434A (Shields et al. (2001) J. Biol. Chem., 276(9): 6591-6604), 252F, 252Y, 252W, 254T, 256Q, 256E, 256D, 433R, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H (Dall'Acqua et al. (2002) J. Immunol. , 169:5171-5180, Dall'Acqua et al (2006) J Biol Chem 281:23514-23524 and US Patent No. 8,367,805.

Modification of specific conserved residues in the Fc fragment of IgG (1253, H310, Q311, H433, N434), such as the N434A variant (Yeung et al. (2009) J. Immunol. 182: 7663), has been proposed as a way to increase affinity in relation to FcRn, thus increasing the half-life of the antibody from the bloodstream (WO 98/023289). A combination Fc variant containing M428L and N434S has been shown to increase binding to FcRn and increase serum half-life by up to five-fold (Zalevsky et al. (2010) Nat. Biotechnol. 28: 157). The combined Fc variant, containing modifications T307A, E380A and N434A, also increases the half-life of IgG1 antibodies (Petkova et al. (2006) Int. Immunol. 18: 1759). In addition, combination Fc variants containing the M252Y-M428L, M428L-N434H, M428L-N434F, M428L-N434Y, M428L-N434A, M428L-N434M and M428L-N434S variants have been shown to increase the half-life (US Patent 2006/173170 ). In addition, a combination Fc variant containing M252Y, S254T and T256E has been reported to increase the half-life by almost 4-fold (Dall'Acqua et al. (2006) J. Biol. Chem. 281:23514).

The bispecific antitumor antagonists of the present invention can be constructed with an IgG backbone. More specifically, any of the bispecific antagonists of the present invention can be constructed with an IgG1 or IgG4 backbone. The use of an IgG1 scaffold is preferred for cancer treatment when the target is present on antigen-presenting cells that can mediate antibody-dependent cellular cytotoxicity (ADCC). The use of an IgG4 scaffold enables antigen targeting in cases where antigen binding alone is sufficient to achieve the desired therapeutic benefits. IgG4-based antagonists prevent unwanted effector functions associated with, for example, IgG1 antibodies, including FcyR binding and complement activation.

Homodimers and heterodimers

One of the problems associated with the efficient preparation of bispecific antibodies concerns the mispairing of heavy and light chains when chains with different binding specificities are coexpressed. The table lists several options for amino acid substitutions to prevent mispairing between heavy chains having different binding specificities that enhance or preferentially promote correct association between the desired heavy chains. To obtain bispecific antitumor anta

- 20 045974 gonists in accordance with the present invention, any approach can be used to prevent or reduce mismatching between heavy chains.

The knob-to-hole (KiH) approach is based on modifications at the contact region between two CH3 domains, where most interactions occur. Typically, a bulky residue is introduced into the CH3 domain of one heavy chain of an antibody, which acts similarly to a key. A depression is formed in another heavy chain that can accommodate this bulky residue, simulating a lock. The resulting heterodimeric Fc moiety can be further stabilized using artificial disulfide bridges.

An alternative approach is based on the use of charged residues with ionic interactions or steric complementarity. This approach involves changing the charge polarity at the CH3 interaction region such that coexpression of electrostatically coupled Fc domains supports favorable attractive interactions and heterodimer formation while maintaining a hydrophobic core, while unfavorable repulsive charge interactions suppress homodimerization, see table. The amino acid numbering in the table follows the Kabat numbering scheme and can be applied to the heavy chain amino acid sequences of the antibodies described herein.

In some embodiments of the present invention, the immunoglobulin scaffold may be replaced by another dimeric structure containing, for example, leucine zipper (LZ) domains. The leucine zipper is a common three-dimensional structural motif in proteins, typically as part of the DNA-binding domain in various transcription factors. One LZ typically contains 4-5 leucine residues spaced about 7 residues apart, which form an amphipathic alpha helix with a hydrophobic region running along one side. In a particular embodiment, the heterodimeric protein scaffold comprises an LZ from the c-jun transcription factor linked to an LZ from the c-fos transcription factor. Although cjun is known to form jun-jun homodimers and c-fos is not known to form homodimers, the formation of jun-fos heterodimers greatly predominates over the formation of jun-jun homodimers.

The leucine zipper domain can be inserted in place of the CH2-CH3 sequences in the protein backbone, or it can be placed at the carboxy terminus of the two heavy chains in a bispecific antitumor antagonist. In the latter case, a furin cleavage site can be introduced between the carboxy terminus of CH3 and the amino terminus of the leucine zipper, thereby facilitating furin-mediated cleavage of the leucine zipper after the heterodimerization step when coexpressing the heavy and light chains of a bispecific antitumor antagonist in an appropriate mammalian cell expression system (see Wranik et al ., J. Biol. Chem., 287 (5): 43331-43339, 2012).

Type NS1 NS2 Protrusion into depression Y349C, T366S, L368A, Y407V S354C, T366W Ionic, electrostatic S183E, E356K, E357K, D399K S183K, K370E, K409D, K439E Ionic, electrostatic K392D, K409D E356K, D399K HA-TF replacement S364H, F405A Y349T, T394F HF-ΤΑ replacement S364H, T394F Y349T, F405A Leucine zipper heterodimer Human c-Jun Leucine Fastener Human leucine c-fos fastener

The amino acid numbering in the table follows the Kabat numbering scheme and can be applied to the heavy chain amino acid sequences of the antibodies described herein. The mutations described in the table can be applied to the sequence (published or not) of any IgG1 immunoglobulin heavy chain, as well as to other classes of immunoglobulins and their subclasses (or isotypes).

When heavy and light chains of monospecific, bispecific antibodies are coexpressed, a light chain with one binding specificity can also mispair with a heavy chain with a different binding specificity. Therefore, in some embodiments, portions of the heavy chain, light chain, or both chains may be modified relative to the wild-type antibody chains from which they are derived to prevent or reduce mismatching of both heavy chain constant regions with each other, as well as mismatching constant regions of light chains with their heavy chain equivalents.

The problem of light chain mispairing can be solved in several ways. Co

- 21 045974 According to some embodiments of the invention, sterically complementary mutations and/or disulfide bridges can be included in the two VL/VH interaction regions. In other embodiments, mutations based on ionic or electrostatic interactions may be included. In some embodiments, light chain mispairing can be prevented or reduced by using a first fragment with the S183E mutation in the heavy chain CH1 domain and the S176K mutation in the light chain CL domain. The second fragment may contain the S183K mutation in the CH1 domain of the heavy chain and the S176E mutation in the CL domain of the light chain. In other embodiments, a CrossMab approach is used where one fragment of a bispecific antitumor antagonist (eg, a Fab fragment) remains intact, but another fragment having a different binding specificity exchanges one or more light chain domains with one or more heavy chain domains. chains at the heavy chain:light chain contact boundary.

Methods for immunoglobulin domain sequences, including specific mutations to prevent mismatching of heavy and light chains as described above, are further described in US Patent Application Publications No. 2014/0243505, 2013/0022601.

Conjugates

In specific embodiments, the antitumor antagonists of the present application are chemically conjugated to one or more peptides and/or small molecule drugs. The peptides or small molecule drugs may be the same or different. Peptides or small molecule drugs can be attached, for example, to reduced SH groups and/or to carbohydrate side chains. Methods for preparing covalent or non-covalent conjugates of peptides or small molecule drugs with antibodies are known in the art, and any such known method can be used.

According to some embodiments of the present invention, the peptide or small molecule drug is attached to the hinge region of the reduced antibody component through the formation of a disulfide bond. Alternatively, such agents can be linked using heterobifunctional cross-linkers such as N-succinyl-3-(2-pyridyldithio) propionate (SPDP). General methods for such conjugation are well known in the art. In some embodiments of the present invention, the peptide or small molecule drug is conjugated via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same agent that is bound to the thiol group, or the carbohydrate group can be used to bind another therapeutic or diagnostic agent. Methods for conjugating peptide inhibitors or small molecule drugs to antibodies via carbohydrate moieties of the antibody are well known to those skilled in the art. For example, according to one embodiment of the invention, the method includes reacting an antibody component containing an oxidized carbohydrate moiety with a carrier polymer that contains at least one free amino functional group. This reaction produces a primary Schiff base (imine) bond, which can be stabilized by reduction to a secondary amine to form the final conjugate. Examples of methods for conjugating small molecule drugs and peptides to antibodies are described in US Patent Application Publication No. 2014/0356385.

Preferably, the antitumor antagonists of the present invention retain certain desirable characteristics and pharmacokinetic properties of the antibodies, including desired in vitro and in vivo stability (e.g., single half-life and storage stability), efficient delivery to the desired target cells, increased affinity for binding partners, desired antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, as well as reduced renal clearance or excretion. Accordingly, when developing antitumor antagonists, careful attention may be paid to the size and need for specific effector functions of the constant region.

The size of TIGIT, PD-1 and PD-L1 inhibitors, including monospecific, bispecific antitumor antagonists derived from them, can vary from 50 kDa to 300 kDa, from 50 kDa to 250 kDa, from 60 kDa to 250 kDa, from 80 kDa to 250 kDa, from 100 kDa to 250 kDa, from 125 kDa to 250 kDa, from 150 kDa to 250 kDa, from 60 kDa to 225 kDa, from 75 kDa to 225 kDa, from 100 kDa to 225 kDa, from 125 kDa to 225 kDa , from 150 kDa to 225 kDa, from 60 kDa to 200 kDa, from 75 kDa to 200 kDa, from 100 kDa to 125 kDa to 200 kDa, from 150 kDa to 200 kDa, from 60 kDa to 150 kDa, from 75 kDa to 150 kDa, 100 kDa to 150 kDa, 60 kDa to 125 kDa, 75 kDa to 125 kDa, 75 kDa to 100 kDa, or be in any range that is covered by any combination of the integers listed in the above ranges, or any ranges specified by any combination of integers between any of the above ranges.

Sets

The present application further provides a kit containing any one or more checkpoint regulator antagonists or antitumor antagonists according to the present invention.

- 22 045974 shaving. In some embodiments, the kit also contains additional components, including syringes and needles for administration, as well as reagents, including secondary detection antibodies and additional human antibodies described herein for use in combination therapies. The kit typically contains a label and/or instructions indicating the intended use of the kit's contents. Said label or instruction may contain any textual or written material provided on the surface of the kit or along with said kit or otherwise accompanying the kit.

III. Methods of using antitumor antagonists

The antitumor antagonists of the present invention have a variety of in vitro and in vivo applications, including, for example, enhancing immune responses and treating cancers, infectious diseases or autoimmune diseases.

According to specific embodiments, the present invention provides a method of treating a cell proliferative disorder; a method for reducing or depleting a population of regulatory T cells in a tumor; a method of treating a microbial infection or a method of treating an immunological disorder, wherein the method comprises administering to a subject in need thereof an effective amount of an antitumor antagonist according to the present application.

In some embodiments of the present invention, antitumor antagonists of the present invention are administered to cells in culture in vitro or ex vivo or to human subjects, for example, in vivo, to enhance immunity in various diseases. Accordingly, this application provides methods for modifying the immune response in a subject, comprising administering to the subject an antibody or an antigen-binding fragment thereof, as described herein, such that the immune response in the subject is enhanced, stimulated or enhanced. Preferred subjects include patients who are individuals for whom an enhanced immune response is desired. The methods are particularly useful for treating patients who are individuals suffering from a disorder that can be treated by enhancing an immune response (eg, a T cell mediated immune response). The methods are particularly useful for treating cancer or chronic infections in vivo. For example, compositions of anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-LAG-3 antibodies may be administered together with the antigen of interest, or the antigen may already be present in the body of the subject being treated (eg, a subject containing a tumor or virus), to enhance antigen-specific immunity. When administering anti-TIGIT antibodies together with another agent, they may be administered separately or simultaneously.

In some embodiments of the present invention, the checkpoint regulator antagonist used in accordance with the method described above is an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, a fragment thereof, or a combination thereof. In some embodiments of the present invention, the checkpoint regulator antagonist is a monospecific or bispecific antibody.

In some embodiments of the present invention, the checkpoint regulatory antagonist or antitumor antagonist is provided as an antibody or antibody fragment. In some embodiments of the present invention, the antibodies described herein are human antibodies or humanized antibodies.

Also included are methods for detecting the presence and/or measuring human TIGIT, human PD-1, human PD-L1 or human LAG3 in a sample, comprising contacting the sample and control sample with a human monoclonal antibody or antigen binding fragment thereof that specifically binds human TIGIT, human PD-1 or human PD-L1 under conditions allowing the formation of a complex between the antibody or fragment thereof and human TIGIT, human PD-1 or human PD-L1. Complex formation is then detected, with the difference in complex formation between the sample and the control sample indicating the presence of human TIGIT antigen in the sample.

Given the ability of anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies and anti-LAG-3 antibodies to block inhibition or co-inhibition of T cell responses, such as antigen-specific T cell responses, the present invention provides in vitro methods and in vivo use of the antibodies described herein to stimulate, enhance or positively regulate antigen-specific T cell responses, for example, antitumor T cell responses. In some embodiments, CD3 stimulation is also provided (eg, by co-incubation with a cell expressing membrane CD3), which stimulation may occur simultaneously, before or after treatment with an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody or anti-LAG-3 antibody. For example, the present invention provides a method of enhancing an antigen-specific T cell response, comprising contacting a T cell with an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-LAG-3 antibody described herein, and optionally CD3 in such a way that there is an enhancement of the antigen-specific T-cell response to

- 23 045974 example, by removing the inhibitory effect mediated by TIGIT, PD-1, PD-L1 or LAG-3. To measure the antigen-specific T-cell response, any suitable indicator of the antigen-specific T-cell response can be used. Non-limiting examples of such suitable indicators include increased proliferation of T cells in the presence of an antibody and/or increased production of cytokines in the presence of an antibody. According to a preferred embodiment of the invention, the production of interleukin-2 and/or interferon-gamma by antigen-specific T cells is enhanced.

Also included are methods of enhancing an immune response (e.g., an antigen-specific T cell response) in a subject, comprising administering to said subject an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist described in of the present application such that there is an enhancement of the immune response (eg, antigen-specific T-cell response) in the specified subject. According to a preferred embodiment of the invention, the subject is a subject containing a tumor, and the immune response against said tumor is enhanced. The tumor may be a solid tumor or a liquid tissue tumor, such as a hematologic malignancy. In some embodiments, the tumor is an immunogenic tumor. According to other embodiments of the invention, the tumor is not immunogenic. In some embodiments, the tumor is PD-L1 positive. In other embodiments, the tumor is PD-L1 negative. The subject may also be a subject containing a virus in which the immune response against said virus is enhanced by administration of an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, a monospecific antitumor antagonist, or a bispecific antitumor antagonist, as described in this application.

In one embodiment, a method of inhibiting the growth of tumor cells in a subject comprises administering to the subject an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist described herein, thereby that tumor growth is inhibited in said subject. Also provided are methods of treating a chronic viral infection in a subject, comprising administering to said subject an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist as described herein, such that the subject is being treated for a chronic viral infection.

The present invention also includes methods for causing depletion of a population of Treg cells in a tumor microenvironment in a subject containing a tumor, such as a cancer, comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an LAG-3 or a bispecific antitumor antagonist described herein, containing an Fc fragment that stimulates the depletion of the Treg cell population in the tumor microenvironment. The Fc may be, for example, an Fc having an effector function or enhanced effector function, eg, an Fc that binds or potentiates binding to one or more activating Fc receptors.

In a preferred embodiment, depletion of the Treg cell population occurs without significantly depleting or inhibiting the Ten cell population within the tumor microenvironment, and without significantly depleting or inhibiting _Teff and T _hed cells outside the tumor microenvironment. In some embodiments, the subject has higher levels of TIGIT on Treg cells compared to Teff cells, for example, in the tumor microenvironment. In some embodiments, anti-TIGIT antibodies or TIGIT antagonists can cause depletion of Treg cells in tumors and/or Treg cells in tumor-infiltrating lymphocytes (TILs). For example, in the CT26 tumor model, anti-mouse TIGIT antibody in the form of mouse IgG2a (which exhibits effector function) led to partial depletion of both Treg cells and CD8+ T cells, but did not deplete the CD4+ T cell population. An equivalent non-effector anti-TIGIT antibody in the form of mouse IgG1 D265A did not deplete the T cell population.

When deciding whether to use an Fc fragment that has effector function or a non-effector anti-TIGIT antibody, careful consideration must be given to the balance between Treg cell depletion, which may enhance the antitumor immune response, and CD8+ T cell depletion, which results in the elimination of some of the cells necessary to actually kill tumor cells. Although _Treg cell depletion would be expected to enhance antitumor activity, recent studies have shown that TIGIT binding to TIGIT' Treg cells promotes Treg _- mediated suppression of Teff cell proliferation (Joller et al. (2014) Immunity 40: 569), suggesting that blocking TIGIT signaling (eg, using a TIGIT antagonist antibody of the present invention) may also enhance antitumor activity. Accordingly, it may be most effective to use a TIGIT antagonist antibody that does not have effector function, which: i) blocks

- 24 045974 TIGIT signaling in T _cells , thus reducing their immunosuppressive activity; ii) activates antitumor CD8+ T cells by blocking the inhibitory effects of TIGIT while avoiding effector-mediated depletion of their population; and iii) enhances DNAM-mediated activation by allowing DNAM to bind to PVR (CD155, a TIGIT ligand) that would otherwise be bound by TIGIT (and reducing direct TIGIT-DNAM interactions) (Johnston et al. (2014) Cancer Cell 26: 923 ). The same applies to the use of anti-PD1 antibodies, anti-PD-L1 antibodies or bispecific antitumor antagonists.

In specific embodiments, an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist described herein is provided to a subject as an adjuvant therapy. Treatment of a cancer patient with an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist according to the present application may result in a long-term sustained response compared to the current standard of care ; long-term survival of at least 1, 2, 3, 4, 5, 10 or more years, disease-free survival of at least 1, 2, 3, 4, 5 or 10 or more years. In specific embodiments of the invention, treating a patient suffering from cancer with an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist prevents cancer recurrence or delays cancer recurrence, e.g. 1, 2, 3, 4, 5 or 10 or more years. Treatment with anti-TIGIT, anti-PD-1, anti-PD-L1 and/or anti-LAG-3 antibodies may be used as first or second line therapy.

In specific preferred embodiments, the subject suffers from a cell proliferative disorder or cancer. Blocking PVR/nectin2 signaling through TIGIT with anti-TIGIT antibodies may result in an enhanced immune response to cancer cells in the patient. The present application provides methods of treating a subject suffering from cancer, comprising administering to the subject an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist of these antigens as described herein, such in such a way that the subject is treated, for example, in such a way that the growth of cancerous tumors is suppressed or reduced and/or regression of tumors occurs. Anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG3 antibodies, or a bispecific antitumor antagonist of these antigens, as described herein, can be used alone to inhibit the growth of cancerous tumors. Alternatively, any of these antitumor antagonists can be used in combination with another agent, for example, other anticancer targets, immunogenic agents, standard cancer treatments, or other antibodies, as described below.

Accordingly, this application provides methods for treating cancer, for example, by inhibiting the growth of tumor cells in a subject, comprising administering to said subject a therapeutically effective amount of a TIGIT, PD-1, PD-L1, or LAG-3 antagonist or a bispecific antitumor antagonist as described herein. application. Preferably, the antibody is a human anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, or anti-LAG-3 antibody comprising the HCVR and LCVR of said anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, or anti-LAG-3 antibody. LAG-3 described herein, or said antibody may be a hybrid, humanized, or non-human anti-hu TIGIT, anti-hu PD-1, anti-PD-L1, or anti-LAG-3 antibody, such as a hybrid, humanized, or non-human anti-TIGIT antibody , an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-LAG-3 antibody that competes for binding or binds to the same epitope as at least one of the anti-TIGIT, anti-PD-1, or anti-PD-L1 antibodies or against LAG-3 described in this application.

Cancers that can be inhibited by the antibodies of the invention generally include cancers that respond to immunotherapy. Non-limiting examples of cancers that may be treated include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell non-small cell lung cancer (NSCLC), non-NSCLC, glioma, gastrointestinal cancer, kidney cancer (eg, clear cell cancer), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, renal cancer (eg, renal cell carcinoma (RCC)), prostate cancer (eg, hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cancer cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer (or carcinoma), gastric cancer, germinal cell tumor, childhood sarcoma, sinonasal natural killer cell tumor, melanoma (eg, metastatic malignant melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, cancer small intestine, endocrine system cancer, parathyroid cancer

- 25 045974 prominent gland, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureter cancer, renal pelvis cancer, central nervous system (CNS) neopoasma, primary CNS lymphoma, tumor angiogenesis, spinal cord tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmental cancers, including asbestos-related, virus-related cancers (eg, human papillomavirus-related tumor ( HPV)), and hematological malignancies originating from any of the two main blood cell lines, i.e. myeloid cell lineage (from which granulocytes, red blood cells, platelets, macrophages and mast cells originate) or lymphoid cell lineage (from which B cells, T cells, NK cells and plasma cells originate), such as all types of leukemia, lymphoma and myeloma, for example, acute, chronic, lymphocytic and/or myelogenous leukemia, such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), undifferentiated AML (AML) , myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (variant M3 or M3 [M3V]), myelomonocytic leukemia (variant M4 or M4 with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M5), erythroleukemia), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma; lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NEIL), B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphastoma, mucosal lymphoid tissue (MALT) lymphoma, anaplastic (eg, Ki 1 ⁺ ) large cell lymphoma, adult T-cell lymphoma/leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, T-lymphoblastic progenitor cell lymphoma, T -lymphoblastic lymphoma and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disease, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of the lymphoid cell line, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, B-lymphoblastic progenitor cell lymphomas, cutaneous T-cell lymphoma (T - CLC, also called mycosis fungoides or Sézary syndrome) and lymphoplasmacytoid lymphoma (LPL) with Waldenström's macroglobulinemia; myelomas such as IgG secreting myeloma, light chain secreting myeloma, non-secreting myeloma, smoldering myeloma (also called low-grade myeloma), solitary plasmacytoma and multiple myelomas, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid origin; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous system, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including, but not limited to, T-cell disorders , such as T-prolymphocytic leukemia (T-PLL), including small cell and medullary cell type; large granular lymphocyte leukemia (LGL), preferably T-cell type; a/d T-cell NHL of the liver and spleen; peripheral/postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head or neck cancer, kidney cancer, colorectal cancer, thyroid cancer; acute myeloid lymphoma, as well as any combination of these types of cancer. The methods described herein can also be used to treat metastatic cancer, resistant cancer (eg, cancer resistant to prior immunotherapy, eg, anti-CTLA-4 or anti-PD-1 blocking antibody), and recurrent cancer.

An anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist may be administered alone, in combination with another antitumor antagonist, or simultaneously with another antitumor antagonist. An anti-TIGIT antibody, an anti-PD-1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist may also be administered in combination or concomitantly with an immunogenic agent such as cancer cells, tumor vaccines, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules ), cells transfected with genes encoding immunostimulatory cytokines as part of a cancer vaccination strategy (He et al. (2004) J. Immunol. 173: 4919-28), or with an oncolytic virus.

Many experimental tumor vaccination strategies have been developed. According to one such strategy, a vaccine is prepared using autologous or allogeneic tumor cells. Some of these cell-based vaccines have been shown to be most effective when the tumor cells are transduced to express granulocytic

- 26 045974 macrophage colony stimulating factor (granulocyte-macrophage colony stimulating facto, GMCSF). GM-CSF has been shown to be a potent activator of antigen presentation for antitumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3539-43). Cancer vaccines have been shown to enhance tumor infiltration by effector T cells in preclinical models. The main types of cancer vaccines include peptide vaccines, vectored antigen-specific vaccines, whole cell vaccines, and dendritic cell vaccines. All vaccine-based therapies are designed to deliver one or multiple antigenic epitopes or whole-cell antigens to patients and stimulate tumor-specific effector T cells. Thus, vaccine-based therapy may be the most effective way to induce tumor infiltration by T cells.

Gene expression studies and large-scale analysis of gene expression patterns in various tumors have led to the identification of so-called tumor-specific antigens (Rosenberg, S. A. (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in the cell from which the tumor arose, for example melanocyte antigens gp100, MAGE and Trp-2 antigens. Even more important is the ability to show that many of these antigens are targets of tumor-specific T cells found in the host.

Inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 can be used in combination with a set of recombinant proteins and/or peptides expressed in the tumor to induce an immune response to these proteins. The immune system may perceive such proteins as self-antigens and, therefore, be tolerant to them. The tumor antigen may include the protein telomerase, which is required for the synthesis of chromosomal telomeres and which is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994) Science 266: 2011-2013). Tumor antigens may also be neoantigens expressed by cancer cells as a result of somatic mutations that result in a change in protein sequence or the creation of fusion proteins between two unrelated sequences (eg, bcr-abl in the Philadelphia chromosome), or an idiotype from B-cell tumors.

Non-limiting examples of cancer vaccines include sipuleucel-T (Provenge®), an FDA-approved tumor vaccine for the treatment of metastatic prostate cancer; tumor cells transfected to express the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), such as whole-cell GM-CSF-secreting irradiated pancreatic cancer cell-based allogeneic vaccine (GVAX; Johns Hopkins); a multipeptide vaccine consisting of immunogenic peptides derived from the mammary cancer antigens, neu, legumain and β-catenin, which provided an extension of vaccine-induced progression-free survival in mammary tumor-bearing mice when administered in combination with an anti-PD-1 antibody (Karyampudi L. et al. (2014) Cancer Res 74: 2974-2985); melanoma antigen peptides such as gp100 peptides, MAGE, Trp-2, MARTI and/or tyrosinase antigens. Other cancer vaccines include proteins from viruses implicated in human cancers, such as human papillomaviruses (HPV) (eg, Gardasil®, Gardasil 9® and Cervarix®); hepatitis B virus (eg, Engerix-B and Recombivax HB); hepatitis C virus (HCV), Kaposi's sarcoma-associated herpes virus (KASV). Another form of tumor-specific antigen that can be used in combination with TIGIT inhibition is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of tumor cell proteins and are highly effective when delivered to antigen-presenting cells to induce antitumor immunity. Talimogene laherparepvec (TVEC, or Imlygic®) is an FDA-approved oncolytic virus for the treatment of certain patients with metastatic melanoma that cannot be removed by surgery.

Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime the immune system to produce antigen-specific responses. DCs can be generated ex vivo and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DCs have also been directly fused to tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6: 332-336). As a vaccination method, DC immunization can be effectively combined with TIGIT blockade to activate (restore) more potent antitumor responses.

Inhibition of TIGIT, PD-1, PD-L1, and/or LAG-3 can also be combined with standard cancer treatments (eg, surgery, radiation, and chemotherapy). In particular, inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 can be effectively combined with chemotherapeutic regimens. In these examples, the dose of the chemotherapeutic reagent administered may be reduced (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination

- 27 045974 is the use of an antitumor antagonist in combination with decarbazine for the treatment of melanoma. Another example of such a combination is the use of a checkpoint regulator antagonist or an antitumor antagonist in combination with interleukin-2 (IL-2) for the treatment of melanoma. For example, the scientific rationale for the combined use of TIGIT, PD-1, PD-L1 and/or LAG-3 inhibition and chemotherapy to promote cell death is the cytotoxic effect of most chemotherapeutic compounds, which should lead to increased levels of tumor antigen in the antigen presentation pathway. Other combination therapies that may produce a synergistic effect with inhibition of TIGIT, PD-1, PD-L1, and/or LAG-3 through cell death are radiation therapy, surgery, and hormonal deprivation. Each of these protocols provides a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3. Inhibition of angiogenesis results in the death of tumor cells, which can provide tumor antigen to the host antigen presentation pathway.

The anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, and bispecific antitumor antagonists described herein can also be used in combination with bispecific antibodies that target Fca- or Fcy receptor-expressing effector cells to tumor cells (see, for example, US patents No. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, bispecific antibodies against Fc receptor/tumor antigen (eg, Her-2/neu) have been used to target macrophages to the tumor site. Such targeting may result in more efficient activation of tumor-specific responses. Inhibition of TIGIT, PD-1, PD-L1, and/or LAG-3 could lead to an enhancement of the T cell component of these responses. Alternatively, the antigen can be delivered directly to DCs using bispecific antibodies that bind to the tumor antigen and a dendritic cell-specific cell surface marker.

Evasion of tumors from the host immune surveillance is achieved through a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by tumors. These include TGF-P, IL-10 and Fas ligand, among others. Antibodies to each of these compounds can be used in combination with the antitumor antagonists described herein to counteract the effects of the immunosuppressive agent and promote host immune antitumor responses.

Other antibodies that activate host immunological reactivity can be used in combination with the antitumor antagonists described herein. These antibodies include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Antibodies against CD40 can effectively replace the activity of helper T cells (Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunction with antibodies against TIGIT. Activating antibodies to T cell costimulatory molecules such as OX-40 (Weinberg et al. (2000) Immunol 164: 2160-2169), CD137/4-1BB (Melero et al. (1997) Nature Medicine 3: 682-685 (1997) and ICOS (Hutloff et al. (1999) Nature 397: 262-266), may also provide increased levels of T cell activation. In addition, inhibitors of other immune checkpoint regulators may also be used in combination with other antitumor antagonists, described herein, as further described below.

Bone marrow transplantation is currently used to treat various tumors of hematopoietic origin. While the consequence of this treatment is graft-versus-host disease, TIGIT inhibition can be used to improve the effectiveness of donor transplanted tumor-specific T cells by reducing graft-versus-tumor disease.

Ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients to stimulate antigen-specific T cells against cancer or viral infections in the presence of anti-TIGIT antibodies may result in increased frequency of transfer and activity of adoptively transferred T cells.

There are also several experimental treatment protocols that involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients to stimulate antigen-specific T cells directed against the tumor (Greenberg & Riddell (1999) Science 285: 546- 51). These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-TIGIT antibodies can increase the transfer frequency and activity of adoptively transferred T cells.

According to competitive embodiments of the invention, an antitumor antagonist described herein can be administered to a subject suffering from an infectious disease, in particular chronic infections. In this case, as in the case of use for the treatment of cancer, antibody-mediated inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 can be used alone or as an adjuvant in combination with vaccines to enhance the immune response to pathogens, toxins and autoantigens. Examples of pathogens for which this therapy can be used

- 28 045974 peutic approach include, but are not limited to, HIV, hepatitis (A, B and C), influenza, herpes, giardia, Plasmodium falciparum, leishmania, Staphylococcus aureus and Pseudomonas aeruginosa. Inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 is particularly useful for the treatment of established infections caused by agents such as HIV that present new or changed antigens during the course of infections. Administration of anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, or bispecific antitumor antagonists can ensure that these antigens are recognized as foreign agents to induce an appropriate T cell response.

Other pathogenic viruses that cause infections that can be treated using the methods described herein include HIV, hepatitis (A, B or C), herpesvirus infections (for example, VZV, HSV-1, HAV-6, HSV-II and CMV , Epstein-Barr virus), as well as infections caused by adenovirus, influenza virus, flavivirus, echoviruses, rhinoviruses, Coxsackie viruses, coronaviruses, respiratory syncytial viruses, mumps viruses, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, virus HTLV, Dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, or a combination thereof.

Examples of pathogenic bacteria or diseases caused by such bacteria that can be treated using the methods described herein include Chlamydia, Rickettsia, Mycobacteria, Staphylococci, Streptococci, Pneumonococci, Meningococci and Gonococci, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Diphtheria , Salmonella, Bacilli, Cholera, Leptospirosis tetanus, botulism, anthrax, plague and Lyme disease.

Examples of pathogenic fungi causing infections that can be treated using the methods described herein include Candida (e.g. albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (e.g. fumigatus, niger, etc. etc.), Mucorales (e.g. mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

Examples of pathogenic parasites causing infections that can be treated using the methods described herein include Entamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoeba sp., Giardia Zambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei , Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis.

In all of the above methods, inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 can be combined with other types of immunotherapy, such as cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2) or bispecific antibody therapy using two different binding specificities to provide enhanced presentation of tumor antigens.

The anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, and bispecific antitumor antagonists described herein can be used to enhance antigen-specific immune responses by administering one or more of these antibodies together with the antigen of interest (eg, vaccine). Accordingly, this application provides methods for enhancing an immune response to an antigen in a subject, comprising administering to said subject: (i) an antigen and (ii) an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody or a bispecific antitumor antagonist or a combination thereof such that the immune response to the antigen is enhanced in the specified subject. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the sections above, such as tumor antigens (or tumor vaccines) discussed above, or antigens from viruses, bacteria or other pathogens described above.

In specific embodiments, a peptide or fusion protein containing an epitope to which an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist binds can be used as a vaccine instead of or in addition to the antitumor antagonist(s).

Suitable methods for in vivo and in vitro administration of antibody compositions (eg, human monoclonal antibodies, multispecific antibodies, or antagonists and immunoconjugates) described herein are well known in the art and can be selected by one skilled in the art. For example, antibody compositions can be administered by injection (eg, intravenous or subcutaneous). Suitable dosages of molecules used will depend on the age and body weight of the subject and the concentration and/or dosage form of the antibody composition.

Combination therapies

In another aspect, the present invention provides combination therapies for enhancing an antigen-specific T cell response in a subject. In one embodiment, the method comprises contacting a T cell with an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, a fragment of said antibody, or a bispecific antitumor antagonist in combination with a second antibody, an antibody fragment ,

- 29 045974 antagonist or drug in such a way that the antigen-specific T-cell response or apoptotic pathway is enhanced. For example, in some embodiments of the present invention, the first antibody or antibody fragment specifically binds to TIGIT, and the second antibody or antibody fragment specifically binds to PD-1, PD-L1, or LAG3.

In a related aspect, a method of reducing or depleting a population of regulatory T cells in a tumor of a subject in need thereof comprises administering an effective amount of an antibody or antibody fragment in combination with a second antibody, antibody fragment, antagonist, or drug such that the number of regulatory T cells in the patient is reduced. the specified subject.

In some embodiments of the present invention, the subject suffers from a cell proliferative disease or cancer, as described herein.

In other embodiments, the subject suffers from a chronic viral infection, inflammatory disease, or autoimmune disease, as described herein.

Providing two distinct signals to T cells is a common lymphocyte activation model for activation of resting T cells by antigen presenting cells (APCs). This model also provides for the distinction between self and non-self and immune tolerance. The primary, or antigen-specific signal, is transmitted through the T-cell receptor (TCR) after recognition of a foreign antigenic peptide presented along with a major histocompatibility complex (MHC) molecule. The second, or costimulatory, signal is delivered to T cells by costimulatory molecules expressed on antigen presenting cells (APCs). This signal causes T cells to promote clonal expansion, cytokine secretion, and effector function. In the absence of costimulation, T cells may become refractory to antigen stimulation, resulting in a tolerogenic response to foreign or endogenous antigens.

In the two-signal model, T cells receive both positive co-stimulatory and negative co-inhibitory signals. Regulating such positive and negative signals is critical to maximizing protective host immune responses while maintaining immune tolerance and preventing autoimmune reactions. Negative signals appear to be necessary for the induction of T cell tolerance, whereas positive signals promote T cell activation. Both co-stimulatory and co-inhibitory signals are provided to antigen-exposed T cells, and the interaction between these co-stimulatory and co-inhibitory signals is important in controlling the strength of the immune response. In addition, the signals transmitted to T cells change as an infection or immune system challenge is cleared, intensified, or persisted, and these changes greatly influence the responding T cells and alter the immune response.

The costimulation mechanism is of therapeutic interest because manipulation of costimulatory signals has been shown to provide a way to enhance or stop the cellular immune response. It has recently been discovered that T cell dysfunction or tolerance can occur concomitantly with the induced and sustained expression of immune checkpoint regulators such as programmed cell death polypeptide 1 (PD-1) and its ligands, PD-L1 and PD-L2. Overexpression of PD-L1 is observed in many cancers and is often associated with poor prognosis (Thompson RH et al., Cancer Res 2006, 66(7):3381). In addition, most tumor-infiltrating T lymphocytes preferentially express PD-1, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, indicating that PD-1 stimulation on tumor-reactive T cells may contribute to impaired antitumor immune responses (Blood 2009 114(8):1537). This may be due to the use of PDL1 signaling mediated by PD-L1-expressing tumor cells interacting with PD-1-expressing T cells, resulting in attenuated T cell activation and immune evasion. Inhibition of the PD-L1/PD-1 interaction provides a way to enhance T-cell immunity, including CD8' T cell-mediated killing of cancer cells and tumors. A similar enhancement of T cell immunity was observed when PDL1 binding to its binding partner B7-1 was inhibited. Therefore, therapeutic targeting of PD-1 and other immune checkpoint regulators is an area of increased interest.

Combining inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 signaling with other signaling pathways that are dysregulated in tumor cells may provide ways to improve treatment efficacy. In recent years, a number of immune checkpoint regulators have been identified in the form of receptors and their ligands. One important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, which includes CTLA-4 and its ligands, B7-1 and B7-2; PD-1 and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC); B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA) and B7-H6. Additional antagonisms

- 30 045974 immune checkpoints include, but are not limited to, TIM-3 and its ligand, galectin-9; LAG-3 and its ligands, including liver sinusoidal endothelial cell lectin (LSECtin) and galectin-3; CD122 and its ligand CD122R; Cd70, B7H3, B and T lymphocyte attenuator (BTLA) and VISTA (Le Mercier et al. (2015) Front. Immunol., (6), Article 418). In addition, a number of checkpoint regulatory antagonists have been identified and tested in various clinical and preclinical models and/or approved by the US Food and Drug Administration (FDA) (Kyi et al., FEBS Letters, 588: 368-376 ( 2014) (Kyi et al., FEBS Letters, 588:368-376 (2014). The concept of inhibitory receptor blockade, also known as immune checkpoint blockade, has been established, for example, based on the FDA approval of the PD-1 inhibitors, nivolumab and pembrolizumab; and the anti-CTLA-4 antibody, ipilimumab, for the treatment of metastatic melanoma.

An immune checkpoint antagonist modulates or interferes with the activity of an immune checkpoint regulator such that, by binding to said checkpoint regulator or a ligand thereof, signaling through the checkpoint regulator receptor is blocked or inhibited. By inhibiting this signaling, immunosuppression can be reversed such that T cell immunity directed against cancer cells can be restored or enhanced. In contrast, an immune checkpoint agonist (eg, a costimulatory molecule) stimulates the activity of an immune checkpoint regulator such that, by binding to said checkpoint regulator or a ligand thereof, it stimulates signaling through the checkpoint regulator receptor. Stimulation of this signaling can lead to the restoration or enhancement of T-cell immunity directed against cancer cells.

Accordingly, according to one embodiment of the invention, a method of stimulating an immune response in a subject comprises administering to said subject an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, fragment(s) of said antibody (e.g., HCVR and/or LCVR anti-TIGIT antibody) or a bispecific antitumor antagonist described herein, in combination with another immune checkpoint regulator described herein above, such that an immune response is stimulated in the subject, for example, to inhibit growth tumors or to stimulate an antiviral response.

In one embodiment, an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, fragment(s) of said antibody, or a bispecific antitumor antagonist according to the present application is administered in combination with another immune regulator checkpoints, either as single antibodies or as multispecific antibodies that have specificity for binding to multiple products. Typically, an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-LAG-3 antibody, or a bispecific antitumor antagonist described herein can be combined to stimulate an immune response with (i) an antagonist of the IgSF family of proteins. B7 or TNF family that inhibit T cell activation, or a cytokine antagonist that inhibits T cell activation (eg, IL-6, IL-10, TGF-β, VEGF or other immunosuppressive cytokines), and or (ii) an agonist stimulating receptors of the IgSF family, B7 family or TNF family or cytokines to stimulate T cell activation to stimulate an immune response.

In one embodiment, the subject is administered an anti-TIGIT antibody or HCVR and/or LCVR fragments thereof in combination with an anti-PD-1 antibody or a PD-1 antagonist. In another embodiment, the subject is administered an anti-TIGIT antibody or HCVR and/or LCVR fragments thereof in combination with an anti-PD-L1 antibody or a PD-L1 antagonist. In another embodiment, the subject is administered an anti-TIGIT antibody or HCVR and/or LCVR fragments thereof in combination with an anti-CTLA-4 antibody or a CTLA-4 antagonist.

In specific embodiments of the invention, combination therapy with an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, and/or an anti-LAG-3 antibody, a fragment thereof, or any of the bispecific antagonists of the present invention exclusively selects subjects suffering from cancer, which have high levels of immune checkpoint regulator ligand expression. As an example, according to one embodiment of the invention, a subject suffering from cancer that has high expression of PVR (CD155) and/or nectin-2 (CD112) and/or low expression of PD-L1 may be selected for monotherapy anti-TIGIT antibodies, fragments thereof or TIGIT antagonists according to the present invention or combination therapy together with a PD-1 antagonist or other immune checkpoint regulator.

The anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody may be administered separately from the second antibody, antibody fragment, or antagonist, or a multispecific antibody or antagonist may be administered having at least one binding specificity for TIGIT and a second binding specificity for in relation to another target product. In addition, the antibody

- 31 045974 anti-TIGIT, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody or bispecific antagonist according to this application can be administered together with one or more additional agents, for example, antibodies, antagonists or drugs in amount(s) effective to stimulate the immune response and/or apoptosis, to further enhance, stimulate or activate the immune response and/or apoptosis in the subject.

In some embodiments, an anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibody or fragment(s) thereof is administered following treatment with another antitumor antagonist. For example, in one embodiment, anti-TIGIT, anti-PD-1, anti-PD-L1, or anti-LAG-3 antibodies may be administered only after treatment with a PD-1/PD-L1 antagonist has failed and resulted in an incomplete therapeutic response. , or there was a recurrence of the tumor or relapse (or failure of PDF'). In some embodiments, cancers experiencing such treatment failure may be screened for expression of, for example, PVR and/or nectin-2, and only if high levels of expression are present will treatment with an anti-TIGIT antibody, an anti-PD antibody -1, anti-PD-L1 antibody or anti-LAG-3 antibody, fragment or antagonist according to the present invention.

In one embodiment, an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-LAG-3 antibody or fragment(s) thereof is administered in combination with a PD-1, PD-L1, PD-L2 antagonist, TIGIT or LAG-3.

Other anti-PD-1 antibodies include, but are not limited to, nivolumab (BMS936558, MDX-1106, OPDIVO™), a humanized immunoglobulin G4 (IgG4) mAb (BristolMyers Squibb); pembrolizumab (MK-3475, lambrolizumab, KEYTRUDA™) (Merck); pidilizumab (CT011) (Medivation) and AMP-224 (Merck). Antibodies against PD-1 are commercially available, for example, from ABCAM (AB137132), BIOLEGEND™ (EH12.2H7, RMP1-14) and AFFYMETRIX EBIOSCIENCE (J105, J116, MIH4).

Other anti-PD-L1 antibodies include atezolizumab (MPDL3280A, RG7446), a fully human IgG4-based mAb (Genentech/Roche); BMS-936559 (MDX-1105), a fully humanized IgG4-based mAb (Bristol-Myers Squibb); MEDI4736, humanized IgG antibody (Medimmune/AstraZeneca); and fully human IgG4-based monoclonal antibody MSB0010718C (Merck, EMD Serono).

Examples of anti-CTLA-4 antibodies for use in accordance with the methods of the present invention include ipilimumab, trevilizumab and tremelimumab.

In specific embodiments, the antitumor antagonist is a dominant negative protein directed against an immune checkpoint regulator. In specific embodiments, the dominant negative protein comprises an extracellular domain derived from a member selected from the group consisting of PD-L1, PD-L2, PD-1, B7-1, B7-2, B7H3, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA, VISTA, CD70, and their combinations. In certain specific embodiments, said extracellular domains are fused to the immunoglobulin constant region or Fc receptor of the antibodies described herein. Such mutants can bind to the endogenous receptor in such a way that a complex is formed in which reduced signaling is observed. In some embodiments, the extracellular domain is fused to an immunoglobulin constant region or Fc fragment or monomer in an oligomeric protein complex.

In certain embodiments, the dominant negative PDL1 antagonist comprises a PD-1 extracellular domain. Examples of a dominant negative protein are AMP-224 (jointly developed by Glaxo Smith Kline and Amplimmune), a recombinant fusion protein containing the extracellular domain of PD-L2 and the Fc portion of human IgG. In another embodiment, the dominant negative PD-L1 antagonist contains one or more mutation(s) in PD-1 that prevent its ability to bind to PD-L1.

Examples of immune checkpoint regulator agonists include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily, such as CD27, CD40, OX40, GITR, and 4-1BB (CD137), and their ligands or members B7CD28 superfamily, including CD28 and ICOS (CD278). Additional checkpoint regulator agonists include CD2, CDS, ICAM-1, LFA-1 (CD11a/CD18), CD30, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83 ligand. Immune checkpoint agonists may include antibodies or soluble fusion protein agonists containing one or more co-stimulatory domains. Agonist antibodies include, but are not limited to, anti-CD40 mAbs such as CP-870893, lucatumumab and dacetuzumab; anti-CD137 mAbs such as urelumab BMS-663513 and PF-05082566; mAb against OX40; anti-GITR mAbs such as TRX518; anti-CD27 mAbs such as CDX-1127; and mAb against ICOS.

Examples of GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (on

- 32 045974 example, divalent antibodies against GITR), such as, for example, the GITR fusion protein described in US patents No. 6,111,090 and 8,586,023; European patent No. 090505B1, US patent publications under the PCT: WO 2010/003118 and 2011/090754. Antibodies against GITR are described, for example, in US patent No. 7,025,962, 7,618,632, 7,812,135, 8,388,967 and 8,591,886; European patents No. 1947183B1 and 1866339; publications in accordance with PCT WO 2011/028683, WO 2013/039954, WO 2005/007190, WO2007/133822, WO2005/055808, WO99/40196, WO2001/03720, WO99/20758, WO2006/0832 89, WO2005/115451, WO2011/051726. An example of an anti-GITR antibody is TRX518.

Another family of membrane-bound ligands that bind to co-stimulatory or coinhibitory receptors is the TNF family of molecules, which bind to cognate members of the TNF receptor family, including CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4 -1BBL, CD137/4-1BB, TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA , LTpR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α/TNF γ, TNFR2, TNFa, LTeR 1, lymphotoxin a (32, FAS, FASL, RELT , DR6, TROY, NGFR (see, for example, Tansey, MG et al. (2009) Drug Discovery Today, 14 (23-24): 1082-1088).

Immune checkpoint agonists or co-stimulatory molecules include cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response and include, but are not limited to, MHC class I molecules, MHC class II molecules, TNF receptor proteins, immunoglobulin -like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1 , LFA-1 (CD11a/CD18), 4-1ВВ (CD137), B7-Н3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d , CD11d, , CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226) , SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), SEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a and a ligand that specifically binds CD83.

In one aspect, T cell responses can be stimulated by a combination of an anti-TIGIT mAb, an anti-PD-1 mAb, an anti-PD-L1 mAb, or an anti-LAG-3 mAb according to the present invention and one or more of (i) a protein antagonist that inhibits T activation -cells (eg, immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, galectin 9, CEACAM-1, BTLA, CD69, galectin- 1, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD-1H, LAIR1, TIM-1, CD96 and TIM-4, and (ii) a protein agonist that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, CD40, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3 and CD28H.

Examples of agents that modulate one of the above proteins and can be combined with anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies and/or anti-LAG-3 antibodies according to the present invention for the treatment of cancer include: YERVOY™/ ipilimumab or tremelimumab (for CTLA-4), galiximab (for B7.1), OPDIVO™/nivolumab/BMS-936558 (for PD-1), pidilizumab/CT-011 (for PD-1), KEYTRUDA™/pembrolizumab/ MK-3475 (for PD-1), AMP224 (for В7DC/PD-L2), BMS-936559 (for В7-Н1), MPDL3280A (for В7-Н1), MEDI-570 (for ICOS), AMG557 (for В7Н2 ), MGA271 (for B7H3), IMP321 (for LAG-3), urelumab/BMS-663513 and PF-05082566 (for CD137/41BB), CDX-1127 (for CD27), MEDI-6383 and MEDI-6469 (for OX40 ), RG-7888 (for OX40L), atacicept (for TACI), CP-870893 (for CD40), lucatumumab (for CD40), dacetuzumab (for CD40), and muromonab-CD3 (for CD3).

Other molecules that can be combined with the antitumor antagonists described herein for the treatment of cancer include inhibitory NK cell receptor antagonists or activating NK cell receptor agonists. For example, TIGIT, PD-1, and/or PD-L1 antagonist antibodies can be combined with KIR antagonists (e.g., lirilumab, CSF-1R antagonists such as RG7155.

Evasion of tumors from the immune surveillance of the host organism occurs through a large number of mechanisms. Many of these mechanisms can be overcome by inactivating immunosuppressive proteins expressed by tumors. These include, but are not limited to, TGF-β, IL10, and Fas ligand. Antibodies to each of these compounds can be used in combination with the antitumor antagonists described herein to counteract the effects of the immunosuppressive agent and promote host immune antitumor responses.

Other antibodies that activate the host immune response can be used in combination with the antitumor antagonists described herein. Antibodies indicated

- 33 045974 include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Antibodies against CD40 are capable of effectively replacing the activity of helper T cells and can be used in combination with the antitumor antagonists described herein. Activating antibodies for T cell co-stimulatory molecules such as OX-40, CD137/4-1BB, and ICOS may also provide increased levels of T cell activation.

In certain embodiments, the antitumor antagonists described herein may be coadministered with one or more other therapeutic agents, such as an anticancer agent, a radiotoxic agent, or an immunosuppressive agent. Such co-administration may overcome problems associated with the development of drug resistance, changes in tumor cell antigenicity leading to antibody resistance, and toxicity (by administering lower doses of one or more agents).

The antitumor antagonists described herein may be associated with an agent (as an immunocomplex) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the agent, or may be administered in conjunction with other known therapies, such as anticancer therapy, such as radiation therapy. The antitumor antagonists described herein can be co-administered with one or more anticancer agents so as to provide synergistic action of the two anticancer agents through different mechanisms to achieve a cytotoxic effect on human cancer cells.

The antitumor antagonists described herein can be combined with an anticancer agent such as an alkylating agent; anthracycline antibiotic; antimetabolite; detoxifying agent; interferon; polyclonal or monoclonal antibody; EGFR inhibitor; HER2 inhibitor; histone deacetylase inhibitor; hormone; mitosis inhibitor; phosphatidylinositol 3-kinase (PI3K) inhibitor; Akt inhibitor; mammalian target of rapamycin (mTOR) inhibitor; proteasome inhibitor; poly(ADP-ribose) polymerase (PARP) inhibitor; Ras/MAPK pathway inhibitor; centrosome declustering agent; multikinase inhibitor; serine/threonine kinase inhibitor; tyrosine kinase inhibitor; VEGF/VEGFR inhibitor; a taxane or taxane derivative, an aromatase inhibitor, an anthracycline, a microtubule-targeting drug, a topoisomerase poison drug, a molecular target or enzyme inhibitor (eg, a kinase or protein methyltransferase), a cytidine analogue, or a combination thereof.

Examples of alkylating agents include, but are not limited to, cyclophosphamide (Cytoxan; Neosar); chlorambucil (Leukeran); melphalan (Alkeran); carmustine (BiCNU); busulfan (Busulfex); lomustine (CeeNU); dacarbazine (DTIC-Dome); oxaliplatin (Eloxatin); carmustine (Gliadel); ifosfamide (Ifex); mechlorethamine (Mustargen); busulfan (Myleran); carboplatin (Paraplatin); cisplatin (CDDP; Platinol); temozolomide (Temodar); thiotepa (Thioplex); bendamustine (Treanda) or streptozocin (Zanosar).

Examples of anthracycline antibiotics include, but are not limited to, doxorubicin (Adriamycin); liposomal doxorubicin (Doxil); mitoxantrone (Novantrone); bleomycin (Blenoxane); daunorubicin (Cembidine); liposomal daunorubicin (DaunoXome); dactinomycin (Cosmegen); epirubicin (Ellence); idarubicin (Idamycin); plicamycin (Mithracin); mitomycin (Mutamycin); pentostatin (Nipent) or valrubicin (Valstar).

Examples of antimetabolites include, but are not limited to, fluorouracil (Adrucil); capecitabine (Xeloda); hydroxyurea (Hydrea); mercaptopurine (Purinethol); pemetrexed (Alimta); fludarabine (Fludara); nelarabine (Arranon); cladribine (Cladribine Novaplus); clofarabine (Clolar); cytarabine (Cytosar-U); decitabine (Dacogen); liposomal cytarabine (DepoCyt); hydroxyurea (Droxia); pralatrexate (Folotyn); floxuridine (FUDR); gemcitabine (Gemzar); cladribine (Leustatin); fludarabine (Oforta); methotrexate (MTX; Rheumatrex); methotrexate (Trexall); thioguanine (Tabloid); TS-1 or cytarabine (Tarabine PFS).

Examples of detoxifying agents include, but are not limited to, amifostine (Ethyol) or mesna (Mesnex).

Examples of interferons include, but are not limited to, interferon alpha (Intron A) or interferon alpha-2a (Roferon-A).

Examples of polyclonal or monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin); ofatumumab (Arzerra); bevacizumab (Avastin); rituximab (Rituxan); cetuximab (Erbitux); panitumumab (Vectibix); tositumomab/iodine 131 tositumomab (Bexxar); alemtuzumab (Campath); ibritumomab (Zevalin; In-111; Y-90 Zevalin); gemtuzumab (Mylotarg); eculizumab ordenosumab (Soliris).

Examples of EGFR inhibitors include, but are not limited to, gefitinib (Iressa); lapatinib (Tykerb); cetuximab (Erbitux); erlotinib (Tarceva); panitumumab (Vectibix); PKI -166; canertinib (CI-1033); matuzumab (Emd7200) or EKB-569.

Examples of HER2 inhibitors include, but are not limited to, trastuzumab (Herceptin); lapatinib (Tykerb) or AC-480.

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Examples of histone deacetylase inhibitors include, but are not limited to, vorinostat (Zolinza), valproic acid, romidepsin, entinostat, abexinostat, givinostat and mocetinostat.

Examples of hormones include, but are not limited to, tamoxifen (Soltamox; Nolvadex); raloxifene (Evista); megestrol (Megace); leuprolide (Lupron; Lupron Depot; Eligard; Viadur); fulvestrant (Faslodex); letrozole (Femara); triptorelin (Trelstar LA; Trelstar Depot); exemestane (Aromasin); goserelin (Zoladex); bicalutamide (Casodex); anastrozole (Arimidex); fluoxymesterone (Androxy; Halotestin); medroxyprogesterone (Provera; Depo-Provera); estramustine (Emcyt); flutamide (Eulexin); toremifene (Fareston); degarelix (Firmagon); nilutamide (Nilandron); abarelix (Plenaxis); or testolactone (Teslac).

Examples of mitosis inhibitors include, but are not limited to, paclitaxel (Taxol; Onxol; Abraxane); docetaxel (Taxotere); vincristine (Oncovin; Vincasar PFS); vinblastine (Velban); etoposide (Toposar; Etopophos; VePesid); teniposide (Vumon); ixabepilone (Ixempra); nocodazole; epothilone; vinorelbine (Navelbine); camptothecin (CPT); irinotecan (Camptosar); topotecan (Hycamtin); amsacrine or lamellarin D (LAM-D).

Examples of phosphatidylinositol 3-kinase (PI3K) inhibitors include wortmannin, an irreversible PI3K inhibitor, demethoxyviridin, a wortmannin derivative LY294002, a reversible PI3K inhibitor; VKM120 (Buparlisib); idelalisib (PI3K delta inhibitor); duvelisib (IPI-145, PI3K delta and gamma inhibitor); alpelisib (BYL719), an alpha-specific PI3K inhibitor; TGR 1202 (formerly known as RP5264), an oral PΡΚ^λ^ή inhibitor and copanlisib (BAY 80-6946), a PI3Ka inhibitor, a predominant inhibitor of the δ isoform.

Examples of Akt inhibitors include, but are not limited to, miltefosine, AZD5363, GDC-0068, MK2206, perifosine, RX-0201, PBI-05204, GSK2141795 and SR13668.

Examples of MTOR inhibitors include, but are not limited to, everolimus (Afinitor) or temsirolimus (Torisel); rapamune, ridaforolimus; deforolimus (AP23573), AZD8055 (AstraZeneca), OSI-027 (OSI), INK-128, BEZ235, PI-103, Torinl, PP242, PP30, Ku-0063794, WAY-600, WYE-687, WYE354 and CC-223 .

Examples of proteasome inhibitors include, but are not limited to, bortezomib (PS-341), ixazomib (MLN 2238), MLN 9708, delanzomib (CEP-18770), carfilzomib (PR-171), YU101, oprosomib (ONX-0912), marizomib (NPI-0052) and disufiram.

Examples of PARP inhibitors include, but are not limited to, olaparib, iniciarib, velaparib, BMN-673, BSI-201, AG014699, ABT-888, GPI21016, MK4827, INO-1001, CEP-9722, PJ-34, TiqA, Phen, PF-01367338 and their combinations.

Examples of Ras/MAPK pathway inhibitors include, but are not limited to, trametinib, selumetinib, cobimetinib, CI-1040, PD0325901, AS703026, RO4987655, RO5068760, AZD6244, GSK1120212, TAK-733, U0126, MEK162 and G DC-0973.

Examples of centrosome declustering agents include, but are not limited to, griseofulvin; noscapine, noscapine derivatives such as brominated noscapine (eg, 9bromonoscapine), reduced bromonoscapine (RBN), N(3-bromobenzyl)noscapine, aminonoscapine and water-soluble derivatives thereof; CW069; phenanthridene-derived poly(ADFribose) polymerase inhibitor, PJ-34; N-(3-pyridylmethyl)-5-nitro-2-furamide, N-(2-thienylmethyl)-5-nitro-2-furamide and N-benzyl-5-nitro-2-furamide.

Examples of multikinase inhibitors include, but are not limited to, regorafenib; sorafenib (Nexavar); sunitinib (Sutent); BIBW 2992; E7080; Zd6474; PKC-412; motesanib or AP24534.

Examples of serine/threonine kinase inhibitors include, but are not limited to, ruboxistaurine; eryl/easudyl hydrochloride; flavopiridol; seliciclib (CYC202; Roscovitrine); SNS-032 (BMS387032); Pkc412; bryostatin; KAI-9803; SF1126; VX-680; Azd1152; Arry-142886 (AZD-6244); SCIO-469; GW681323; CC-401; CEP-1347 or PD 332991.

Examples of tyrosine kinase inhibitors include, but are not limited to, erlotinib (Tarceva); gefitinib (Iressa); imatinib (Gleevec); sorafenib (Nexavar); sunitinib (Sutent); trastuzumab (Herceptin); bevacizumab (Avastin); rituximab (Rituxan); lapatinib (Tykerb); cetuximab (Erbitux); panitumumab (Vectibix); everolimus (Afinitor); alemtuzumab (Campath); gemtuzumab (Mylotarg); temsirolimus (Torisel); pazopanib (Votrient); dasatinib (Sprycel); nilotinib (Tasigna); vatalanib (Ptk787; ZK222584); SER-701; SU5614; MLN518; XL999; VX-322; Azd0530; BMS-354825; SKI-606 SR-690; AG490; WHI-P154; WHI-P131; AC-220 or AMG888.

Examples of VEGF/VEGFR inhibitors include, but are not limited to, bevacizumab (Avastin); sorafenib (Nexavar); sunitinib (Sutent); ranibizumab; pegaptanib or vandetinib.

Examples of microtubule-targeting drugs include, but are not limited to, paclitaxel, docetaxel, vincristine, vinblastine, nocodazole, epothilones, and navelbine.

Examples of topoisomerase poison drugs include, but are not limited to, teniposide, etoposide, adriamycin, camptothecin, daunorubicin, dactinomycin, mitoxantrone, amsacrine, epirubicin, and idarubicin.

- 35 045974

Examples of taxanes or taxane derivatives include, but are not limited to, paclitaxel and docetaxol.

Examples of common chemotherapeutic, antineoplastic, antiproliferative agents include, but are not limited to, altretamine (Hexalen); isotretinoin (Accutane; Amnesteem; Claravis; Sotret); tretinoin (Vesanoid); azacitidine (Vidaza); bortezomib (Velcade), asparaginase (Elspar); levamisole (Ergamisol); mitotane (Lysodren); procarbazine (Matulane); pegaspargase (Oncaspar); denileukin diftitox (Ontak); porfimer (Photofrin); aldesleukin (Proleukin); lenalidomide (Revlimid); bexarotene (Targretin); thalidomide (Thalomid); temsirolimus (Torisel); arsenic trioxide (Trisenox); verteporfin (Visudyne); mimosine (Leucenol); (1M tegafur-0.4M 5-chloro-2,4-dihydroxypyrimidine-1M potassium oxonate) or lovastatin.

In specific embodiments, inhibition of TIGIT, PD-1, PD-L1, and/or LAG-3 is combined with standard cancer treatments (eg, surgery, radiation, and chemotherapy). Inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 can be effectively combined with chemotherapeutic regimens. In these cases, it is possible to reduce the dose of the administered chemotherapy reagent. An example of such a combination is the use of an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-LAG-3 antibody in combination with decarbazine for the treatment of melanoma. Another example of such a combination is the use of an anti-TIGIT antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-LAG-3 antibody in combination with interleukin-2 (TL-2) for the treatment of melanoma. It is believed that inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3 in combination with chemotherapy may enhance apoptosis and increase tumor antigen presentation to a cytotoxic immune response. Other synergistic combination therapies include inhibition of TIGIT, PD-1, PD-L1, and/or LAG-3 through cell death when used in combination with radiation, surgery, or hormonal deprivation. Each of these protocols creates a source of tumor antigen in the host body.

In specific embodiments, the checkpoint regulatory antagonists described herein can be used in multispecific antagonists or in combination with bispecific antibodies that direct Fca or FcY receptor expressing effector cells to tumor cells (see, for example, US Pat. No. 5 922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, bispecific antibodies against Fc receptor/tumor antigen (eg, Her-2/neu) have been used to target macrophages to cancer cells or tumors. Such targeting may result in more efficient activation of tumor-specific responses. T-cell tumor-specific responses can be enhanced by inhibition of TIGIT, PD-1, PD-L1 and/or LAG-3. Alternatively, antigen can be delivered directly to DCs using bispecific antibodies that bind to the tumor antigen and a dendritic cell-specific cell surface marker.

IV. Nucleic acids and host cells for checkpoint regulator expression

According to another aspect, the present invention provides nucleic acids encoding the antitumor antagonists of the present invention, including heavy and light chains, as well as expression vectors containing such nucleic acids. In particular, the nucleic acids encode one or more HCDR, LCDR, HCVR and/or LCVR corresponding to any of the antibodies, antagonists or fragments described herein.

Thus, according to one aspect, the present invention provides one or more nucleic acids encoding any antitumor antagonists, antibodies, or antigen-binding portions thereof, as described herein.

According to another aspect, the present invention provides one or more expression vectors containing one or more nucleic acids encoding any antitumor antagonists, antibodies, or antigen-binding portions thereof, as described herein.

According to another aspect, the present invention provides a host cell transformed with one or more expression vectors containing one or more nucleic acids encoding any antitumor antagonists, antibodies, or antigen-binding portions thereof, as described herein.

DNA molecule(s) encoding antigen-binding sites can be isolated and sequenced from the monoclonal antibody produced in hybridoma cells using standard procedures (for example, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of monoclonal antibodies ).

Alternatively, amino acid sequences from immunoglobulins of interest can be determined by direct protein sequencing, and suitable coding nucleotide sequences can be designed according to a universal codon table. In other cases, nucleotide and amino acid sequences of antigen binding sites or other immunoglobulin sequences, including constant regions, hinge regions, and the like,

- 36 045974 can be obtained from published sources well known in the art.

Expression vectors encoding a particular monospecific or bispecific antitumor antagonist can be used to synthesize the antitumor antagonists of the present invention in cultured cells in vitro, or the expression vectors can be directly administered to a patient to express the antitumor antagonist in vivo or ex vivo. As used herein, the term expression vector refers to a viral or non-viral vector containing a polynucleotide encoding one or more polypeptide chains corresponding to the monospecific or bispecific antitumor antagonists of the present invention in a form suitable for expression from the polynucleotide(s) in a host cell for for the production of antibodies or for direct administration as a therapeutic agent.

A nucleic acid sequence is operably linked to another nucleic acid sequence when the first sequence is in a functional relationship with the latter. For example, presequence or signal peptide DNA is operably linked to polypeptide DNA if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to the coding sequence if it is located in such a way that it facilitates the translation process. In general, the term operably linked means that the DNA sequences being linked are contiguous, and in the case of a signal peptide, contiguous and in the same reading frame. However, enhancers do not have to be adjacent. Linking is accomplished by ligation at convenient restriction sites. If such sites are not available, synthetic oligonucleotide adapters or linkers can be used in accordance with conventional practice.

Nucleic acid sequences for the expression of antitumor antagonists typically contain an amino-terminal signal peptide sequence that is removed from the mature protein. Since signal peptide sequences can influence expression levels, polynucleotides can encode any of different N-terminal signal peptide sequences. Those skilled in the art will appreciate that the design of an expression vector may depend on factors such as the choice of host cell for transformation, the desired level of protein expression, and the like.

The regulatory sequences described above refer to DNA sequences necessary for the expression of an operably linked coding sequence in one or more host organisms. The term regulatory sequences is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence in many types of host cells, or sequences that direct expression of a nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). Expression vectors generally contain transcription termination sequences and may additionally contain one or more elements that positively affect mRNA stability.

The expression vector contains one or more transcriptional regulatory elements, including promoters and/or enhancers, for directed expression of antitumor antagonists. A promoter contains a DNA sequence that initiates transcription at a relatively fixed position relative to the transcription start site. The promoter contains core elements necessary for the basic interaction of RNA polymerase and transcription factors, and can work in conjunction with other upstream and response elements.

It should be understood that as used herein, the term promoter is used in its broadest sense to include transcription regulatory elements (TREs) from genomic genes or hybrid TREs derived therefrom, including a TATA box or initiation element for the precise initiation of transcription in the presence or absence of additional TREs (i.e., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that regulate the activation or repression of functionally related genes in response to developmental and/or environmental stimuli, and trans-acting regulatory proteins or nucleic acids. A promoter may contain a genomic fragment or a hybrid of one or more TREs combined together.

Preferred promoters are promoters capable of driving high level expression in the target cell of interest. Promoters may include constitutive promoters (eg, HCMV, SV40, elongation factor 1α (EF-1a)) or promoters that are preferentially expressed in the particular cell type of interest. Enhancers generally refer to DNA sequences that function beyond the transcription start site and can be located 5' or 3' to the transcription unit. In addition, enhancers can

- 37 045974 walk within the intron, as well as within the coding sequence. They usually range from 10 to 300 bp in length. and function in cis position. Enhancers increase and/or regulate transcription from nearby promoters. Preferred enhancers are enhancers that direct high levels of expression in the antibody-producing cell. Cell- or tissue-specific transcription regulatory elements (TREs) can be inserted into expression vectors to restrict expression to the desired cell types. Pol III promoters (H1 or U6) are particularly useful for the expression of shRNAs from which miRNAs are expressed. An expression vector may be designed to facilitate expression of an antitumor antagonist in one or more cell types.

In specific embodiments, one or more expression vectors can be designed to express both an antitumor antagonist and one or more siRNAs targeting the Tie2 pathway, the VEGF pathway, or an immune checkpoint regulator.

Small interfering RNA (siRNA) is a double-stranded RNA that can be engineered to induce sequence-specific post-transcriptional gene silencing of mRNA molecules. Synthetically produced miRNAs structurally mimic the types of miRNAs that are normally processed by the Dicer enzyme in cells. When expressed from a vector, the expression vector is constructed to transcribe a short double-stranded hairpin-like RNA (shRNA), which is processed to produce the target siRNA within the cell. Synthetic siRNAs and shRNAs can be designed using well-known algorithms and can be synthesized using a standard DNA/RNA synthesizer.

For coexpression of individual antitumor antagonist chains, suitable splice donor and acceptor sequences can be inserted to express both products. Alternatively, an internal ribosome binding sequence (IRES) or 2A peptide sequence can be used to express multiple products from a single promoter. The IRES provides a structure to which the ribosome can bind, which does not have to be at the 5' end of the mRNA. Therefore, an IRES can cause the ribosome to initiate translation at the second start codon within an mRNA, allowing the production of more than one polypeptide from a single mRNA. The 2A peptide contains short sequences that mediate cotranslational self-cleavage of peptides located upstream and downstream of the 2A site, allowing the production of two different proteins from a single transcript in equimolar quantities. CHYSEL is a non-limiting example of a 2A peptide that promotes the release by the eukaryotic ribosome during translation of the growing polypeptide chain it synthesizes without dissociation from the mRNA. The ribosome continues translation, thus producing a second polypeptide.

The expression vector may include a viral vector or a non-viral vector. Viral vectors can be derived from adeno-associated virus (AAV), adenovirus, herpes virus, vaccinia virus, poliovirus, poxvirus, retrovirus (including lentivirus such as HIV-1 and HIV-2), Sindbis virus and other RNAs -viruses, alphavirus, astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, togavirus, etc. A non-viral vector is simply a naked expression vector that is not packaged with viral components (eg, capsids and/or envelopes).

In specific cases, these vectors can be designed to target specific diseases or cell populations by using the targeting properties inherent in the viral vector or created in the specified viral vector. Specific cells can be targets for polynucleotide delivery as well as expression. Thus, the term targeting in this case may be based on the use of endogenous or heterologous binding agents in the form of capsids, envelope proteins, antibodies for delivery to specific cells, the use of tissue-specific regulatory elements to restrict expression to a specific subset(s) of cells, or the use of both.

According to some embodiments of the present invention, the expression of the antibody chains is under the control of a regulatory element, such as a tissue-specific or universal promoter. In some embodiments of the present invention, a generic promoter, such as a CMV promoter, a chicken CMV-beta-actin (CAG) fusion promoter, a tissue-specific promoter, or a tumor-specific promoter may be used to control the expression of a particular heavy or light chain of an antibody or a single chain derivative thereof.

Nonviral expression vectors can be used to transfer nonviral genes either by direct injection of naked DNA or by encapsulating polynucleotides encoding an antitumor antagonist into liposomes, microparticles, microcapsules, virus-like particles, or anucleate red blood cells. Such compositions can be further linked through chemical conjugation to targeting domains to facilitate targeting and/or penetration of nucleic acids into the desired cells of interest. In addition, plasmid vectors can be incubated with synthetic gene transfer molecules, such as polymeric DNA-binding cations such as polylysine, protamine and albumin, and linked to cell-targeting ligands,

- 38 045974 such as asialoorosomucoid, insulin, galactose, lactose or transferrin.

Alternatively, naked DNA may be used. Naked DNA capture efficiency can be improved by compaction or the use of biodegradable latex beads. Such delivery can be further improved by treating the beads to increase their hydrophobicity and thus facilitate endosome disruption and release of DNA into the cytoplasm.

V. Methods for producing monospecific or multispecific antibodies

According to another aspect, the present invention provides host cells transformed with nucleic acids or expression vectors encoding HCVR and/or LCVR of anti-TIGIT antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies and/or anti-LAG-3 antibodies, or nucleic acids acids/expression vectors encoding a monospecific or bispecific antitumor antagonist according to the present invention. The host cell may be any bacterial or eukaryotic cell capable of expressing nucleic acids or expression vectors encoding HCVR and/or LCVR anti-TIGIT, anti-PD-1, anti-PD-L1 and/or anti-LAG-3 antibodies, or any other co-administered antibodies or antagonists described herein.

According to another aspect, a method for producing an antitumor antagonist includes culturing a host cell transformed with one or more nucleic acids or expression vectors encoding HCVR and/or LCVR anti-TIGIT, anti-PD-1, anti-PD-L1 and/or anti-LAG-3, under conditions that allow the production of an antibody or fragment thereof and the purification of said antibody from the cell.

According to a further aspect, the present invention provides a method for producing an antibody, comprising culturing a cell transiently or stably expressing one or more constructs encoding one or more polypeptide chains of the antibody; and purifying said antibody from the cultured cells. Any cell capable of producing a functional antibody can be used. In preferred embodiments, the cell expressing the antibody is a eukaryotic cell or is of mammalian origin, preferably a human cell. Cells from various cell types of tissues can be used to express antibodies. In other embodiments, the cell is a yeast cell, an insect cell, or a bacterial cell. Preferably, the antibody-producing cell is stably transformed with a vector expressing the antibody.

One or more expression vectors encoding antibody heavy or light chains can be introduced into a cell using any standard method, such as naked DNA, cationic lipid-mediated transfection, polymer-mediated transfection, peptide-mediated transfection, virus-mediated infection, the use of physical or chemical agents or treatments, electroporation, etc. In addition, cells can be transfected with one or more expression vectors to express the antibody along with a selectable marker to facilitate the selection of stably transformed clones expressing the antibody. Antibodies produced by such cells can be collected and/or purified according to methods known in the art, such as centrifugation, chromatography, etc.

Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analogue G418, hydromycin and puromycin. When such selectable markers are successfully transferred into a host mammal cell, the transformed host mammal cell can survive when placed under selective pressure. There are two widely used different categories of selection regimes. The first category is based on cellular metabolism and the use of a mutant cell line that lacks the ability to grow independently of supplemented media. Two examples are DHFR CHO cells and mouse LTK cells. These cells are unable to grow without the addition of nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes required for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are placed in a supplemented medium. An alternative to supplementing the medium is to introduce an intact DHFR or TK gene into cells that lack the corresponding genes, thereby altering their nutrient requirements for growth. Individual cells that have not been transformed with the DHFR or TK gene will be unable to survive in unsupplemented media.

The second category is dominant selection, which refers to a selection scheme used for any cell type and does not require the use of a mutant cell line. Such selection schemes typically use a drug to stop the growth of the host cell. Cells that contain the new gene will express the drug resistance protein and survive selection. Examples of such dominant selection include the drugs neomycin, mycophenolic acid or hygromycin. These three examples use bacterial genes under eukaryotic control to confer resistance to a suitable drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Other drugs include neomycin analogue G418 and puromi

- 39 045974 cin.

Examples of antibody-expressing cells include human Jurkat cells, human embryonic kidney 293 (HEK) cells, Chinese hamster ovary (CHO) cells, WEHI mouse fibrosarcoma cells, as well as single-celled protozoan species such as Leishmania tarentolae. In addition, stably transformed antibody-producing cell lines can be generated using primary cells immortalized with c-myc or other immortalizing agents.

According to one embodiment of the invention, the cell line comprises a stably transformed Leishmania cell line, such as Leishmania tarentolae. Leishmania are known to provide a viable, fast-growing single-celled host for high-level expression of eukaryotic proteins that have mammalian-type glycosylation patterns. A commercially available eukaryotic protein expression kit in Leishmania is available from Jena Bioscience GmbH, Jena, Germany.

In some embodiments of the present invention, the cell line expresses at least 1 mg, at least 2 mg, at least 5 mg, at least 10 mg, at least 20 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, or at least 500 mg antibody/liter of culture.

Antibodies of the present invention can be isolated from antibody-expressing cells after they have been cultured and maintained in any suitable culture medium such as RPMI, DMEM and AIM V®. Antibodies can be purified using standard protein purification techniques (e.g., affinity purification, chromatography, etc.), including the use of protein A or protein G immunoaffinity purification. In some embodiments of the present invention, antibodies are engineered to be secreted into culture supernatants with subsequent isolation from these supernatants.

VI. Pharmaceutical compositions and methods of treatment

Another aspect of the present invention relates to pharmaceutical compositions and methods for treating a cell proliferative disorder such as cancer, chronic infections or immune compromised conditions. According to one embodiment of the invention, the pharmaceutical composition contains one or more antitumor antagonists according to the present invention. In some embodiments of the present invention, the antitumor antagonist(s) comprise one or more checkpoint regulatory antagonists, such as anti-T cell Ig and ITIM domain inhibitors (TIGITs), PD-1 inhibitors, PD-L1 inhibitors, and LAG-3 inhibitors . The antagonist(s) are provided together with a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention may contain one or more different antibodies, one or more multispecific antibodies, one or more immunoconjugates, or a combination thereof, as described herein.

As described above, methods of using the pharmaceutical compositions described herein include administering to a subject in need thereof an effective amount of a pharmaceutical composition according to the present invention.

Any suitable route or method of administration may be used to provide a therapeutically or prophylactically effective dose of an antibody or antagonist to a patient. Examples of routes or methods of administration include parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intratumoral), oral, topical (nasal, transdermal, intradermal, or intraocular), mucosal (e.g., nasal, sublingual, buccal, rectal, vaginal), inhalation, intralymphatic, spinal, intracranial, intraperitoneal, intratracheal, intravesical, intrathecal, intestinal, intrapulmonary, intralymphatic, intracavitary, intraocular, intraarticular and transurethral administration, as well as local delivery using a catheter or stent.

The pharmaceutical composition containing the antibody or antagonist of the present invention may be presented in any pharmaceutically acceptable carrier(s) or excipient(s). As used herein, the term pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retarding agents, etc., that are physiologically compatible. The pharmaceutical compositions may contain suitable solid or gel carriers or excipients. Examples of carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the compositions. Pharmaceutically acceptable carriers may additionally contain minimal amounts of excipients, such as wetting or emulsifying agents, preservatives or buffers, which enhance shelf life.

- 40 045974 storage or effectiveness of therapeutic agents.

The antitumor antagonist may be included in a pharmaceutical composition suitable for parenteral administration. Suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for liquid dosage form). For the lyophilized dosage form, cryoprotectants may be included, primarily 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents may be included in the lyophilized dosage form, primarily 1-10% mannitol (optimally 2-4%). In both liquid and lyophilized dosage forms, stabilizers can be used, mainly 1-50 mM L-methionine (optimally 5-10 mM). Other suitable bulking agents include glycine and arginine. Substances such as 0-0.05% polysorbate-80 (optimally 0.005-0.01%) may also be included. Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants.

Therapeutic anticancer antagonist drugs can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, the preservative benzyl alcohol) or sterile water before injection. The pharmaceutical composition may be presented in a form for parenteral administration by injection, for example, by bolus injection or continuous infusion.

The therapeutic agents in the pharmaceutical compositions may be presented in a therapeutically effective amount or a prophylactically effective amount. A therapeutically effective amount refers to an amount that is effective at the doses and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount of the recombinant vector may vary depending on the condition being treated, the severity and course of the condition, the route of administration, the preventive or therapeutic purposes for administering the antibody or agent, the bioavailability of the particular agent(s), the ability of the antitumor antagonist to induce the desired response in the subject, the prior therapy, age, weight and gender of the patient, the patient's clinical history and response to the antibody, the type of antitumor antagonist used, the decision of the treating physician, etc. A therapeutically effective amount is also an amount of recombinant vector such that the therapeutically beneficial effects outweigh any toxic or harmful effects. A prophylactically effective amount refers to an amount effective at the doses and for periods of time necessary to achieve the desired prophylactic result.

Preferably, the polypeptide domains in the antitumor antagonist are derived from the same host to which they are intended to be administered to reduce inflammatory responses against the administered therapeutic agents.

The antitumor antagonist is suitably administered to the patient in a single dose or over multiple treatment procedures and may be administered to the patient at any time from the time of diagnosis. The antitumor antagonist may be administered as a sole treatment or in conjunction with other drugs or therapies useful in treating the condition of interest.

As a general rule, a therapeutically effective amount or a prophylactically effective amount of an antitumor antagonist is administered in the range of from about 1 ng/kg body weight per day to about 100 mg/kg body weight per day in one or more administrations. In a specific embodiment, each antitumor antagonist is administered in a range of about 1 ng/kg body weight per day to about 10 mg/kg body weight per day, from about 1 ng/kg body weight per day to about 1 mg/kg body weight body weight per day, from about 1 ng/kg body weight per day to about 100 μg/kg body weight per day, from about 1 ng/kg body weight per day to about 10 μg/kg body weight per day, from about 1 ng /kg body weight per day to about 1 μg/kg body weight per day, from about 1 ng/kg body weight per day to about 100 ng/kg body weight per day, from about 1 ng/kg body weight per day to about 10 ng/kg body weight per day, from about 10 ng/kg body weight per day to about 100 mg/kg body weight per day, from about 10 ng/kg body weight per day to about 10 mg/kg body weight per day , from about 10 ng/kg body weight per day to about 1 mg/kg body weight per day, from about 10 ng/kg body weight per day to about 100 μg/kg body weight per day, from about 10 ng/kg body weight of body weight per day to about 10 μg/kg body weight per day, from about 10 ng/kg body weight per day to about 1 μg/kg body weight per day, from 10 ng/kg body weight per day to about 100 ng/kg body weight per day, from about 100 ng/kg body weight per day to about 100 mg/kg body weight per day, from about 100 ng/kg body weight per day to about 10 mg/kg body weight per day, from about 100 ng/kg body weight per day to about 1 mg/kg body weight per day, from about 100 ng/kg body weight per day to about 100 μg/kg body weight per day, from about 100 ng/kg body weight per day to about 10 μg/kg body weight per day, from about 100 ng/kg body weight per day to about 1 μg/kg body weight per day, from about 1 μg/kg body weight per day to about 100 mg/kg body weight per day day, from about 1 μg/kg body weight per day to about 10 mg/kg body weight per day, from about 1 μg/kg body weight per day to about 1 mg/kg body weight per day, from about 41 045974 1 mcg/kg body weight per day to about 100 mcg/kg body weight per day, from about 1 mcg/kg body weight per day to about 10 mcg/kg body weight per day, from about 10 mcg/kg body weight per day up to about 100 mg/kg body weight per day, from about 10 μg/kg body weight per day to about 10 mg/kg body weight per day, from about 10 μg/kg body weight per day to about 1 mg/kg body weight per day, from about 10 mcg/kg body weight per day to about 100 mcg/kg body weight per day, from about 100 mcg/kg body weight per day to about 100 mg/kg body weight per day, from about 100 mcg/day kg body weight per day to about 10 mg/kg body weight per day, from about 100 μg/kg body weight per day to about 1 mg/kg body weight per day, from about 1 mg/kg body weight per day to about 100 mg/kg body weight per day, from about 1 mg/kg body weight per day to about 10 mg/kg body weight per day, from about 10 mg/kg body weight per day to about 100 mg/kg body weight per day.

In other embodiments, the antitumor antagonist is administered at a dose of 500 μg to 20 g every three days or at a dose of 25 mg/kg body weight every three days.

In other embodiments, each antitumor antagonist is administered in a range from about 10 ng to about 100 ng per individual administration, from about 10 ng to about 1 μg per individual administration, from about 10 ng to about 10 μg per individual administration, from about 10 ng to about 100 mcg per single administration, from about 10 ng to about 1 mg per single administration, from about 10 ng to about 10 mg per single administration, from about 10 ng to about 100 mg per single administration, from about 10 ng to about 1000 mg per injection, about 10 ng to about 10,000 mg per single administration, about 100 ng to about 1 mcg per single administration, about 100 ng to about 10 mcg per single administration, about 100 ng to about 100 mcg per single administration administration, about 100 ng to about 1 mg per single administration, about 100 ng to about 10 mg per single administration, about 100 ng to about 100 mg per single administration, about 100 ng to about 1000 mg per injection, about 100 ng to about 10,000 mg per single administration, about 1 mcg to about 10 mcg per single administration, about 1 mcg to about 100 mcg per single administration, about 1 mcg to about 1 mg per single administration, about 1 mcg to about 10 mg per single administration, about 1 mcg to about 100 mg per single administration, about 1 mcg to about 1000 mg per injection, about 1 mcg to about 10,000 mg per single administration, about 10 mcg to about 100 mcg per single administration, about 10 mcg to about 1 mg per single administration, about 10 mcg to about 10 mg per single injection, about 10 mcg to about 100 mg per single injection, about 10 mcg to about 1,000 mg per injection, about 10 mcg to about 10,000 mg per single administration, about 100 mcg up to about 1 mg per single administration, about 100 mcg to about 10 mg per single administration, about 100 mcg to about 100 mg per single administration, about 100 mcg to about 1000 mg per injection, about 100 mcg to about 10,000 mg per single administration , about 1 mg to about 10 mg per single administration, about 1 mg to about 100 mg per single administration, about 1 mg to about 1000 mg per injection, about 1 mg to about 10,000 mg per single administration, about 10 mg to about 100 mg per single administration, about 10 mg to about 1000 mg per injection, about 10 mg to about 10,000 mg per single administration, about 100 mg to about 1000 mg per injection, about 100 mg to about 10,000 mg per single administration, and about 1000 mg up to approximately 10,000 mg per single administration. The antitumor antagonist can be administered every day, every 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3 or 4 weeks.

In other specific embodiments, the antitumor antagonist can be administered at a dose of about 0.0006 mg/day, 0.001 mg/day, 0.003 mg/day, 0.006 mg/day, 0.01 mg/day, 0.03 mg/day , 0.06 mg/day, 0.1 mg/day, 0.3 mg/day, 0.6 mg/day, 1 mg/day, 3 mg/day, 6 mg/day, 10 mg/day, 30 mg/day, 60 mg/day, 100 mg/day, 300 mg/day, 600 mg/day, 1000 mg/day, 2000 mg/day, 5000 mg/day, or 10,000 mg/day. The dose is expected to depend on the disease, body weight, age and health status of the patient.

In specific embodiments, coding sequences for an antitumor antagonist are included in a suitable expression vector (eg, a viral or non-viral vector) for expressing an effective amount of said antitumor antagonist in a patient suffering from a cell proliferative disorder. According to specific embodiments of the invention, including the administration of, for example, one or more recombinant AAV viruses (rAAV), the pharmaceutical composition may contain rAAV in an amount including at least 10 ¹⁰ , at least 10 ¹¹ , at least 10 ¹² , at least at least 10 ¹³ or at least 10 ¹⁴ genomic copies (GC) or recombinant viral particles per kg or any range thereof. According to specific embodiments of the invention, the pharmaceutical composition contains an effective amount of a recombinant virus, such as rAAV, in an amount containing at least 10 ¹⁰ , at least 10 ¹¹ , at least 10 ¹² , at least 10 ¹³ , at least 10 ¹⁴ at least 10 ¹⁵ genomic copies or genomic copies of recombinant viral particles per subject, or any range thereof.

Doses can be tested in several animal models accepted in the art,

- 42 045974 suitable for any specific cell proliferative disorder.

Delivery methods may also include the use of polycationic condensed DNA bound or unlinked to killed viruses, linked to DNA ligand, liposomes, carrier cells for delivery into eukaryotic cells, deposition of photopolymerized hydrogel materials, hand-held gene gun for gene transfer, ionizing radiation, charge neutralization nucleic acids or fusion with cell membranes, particle-mediated gene transfer, etc.

The present invention is further illustrated by the following examples, which do not limit the present invention. The contents of all references, patents and published patent applications cited throughout this application, as well as figures and tables, are incorporated herein by reference.

Examples

Example 1: Creation of Monoclonal Antibodies

Monoclonal antibodies (mAbs) according to the present invention were generated and screened using methods well known in the art, see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York. Antigen-specific hybridoma mAbs were cloned, sequenced and constructed using methods well known in the art, see, for example, Lo. V.K.C Methods in Molecular Biology™. Volume 248 2004. Antibody Engineering.

In fig. 1 shows the CDR sequences of the anti-TIGIT mAb. In fig. 2A-2B show several embodiments of anti-TIGIT antibody variable domain sequences. In fig. Figure 3 shows the CDR sequences of the anti-PD-1 mAb. In fig. 4A-4C show several embodiments of anti-PD-1 antibody variable domain sequences. In fig. anti-PD-L1 mAb CDR sequences are shown. In fig. 6A-6C show several embodiments of anti-PD-L1 antibody variable domain sequences.

Example 2: Design of Anti-PD-1 Bispecific Antibodies with Anti-TIGIT scFv

In fig. 7A-7C show three examples of bispecific antitumor antagonists, Bi-TPM93 (Fig. 7A), Bi-TPM-94A (Fig. 7B) and Bi-TPM-94B (Fig. 7C). These antagonists contain an anti-PD-1 antibody backbone (PD-01) (Fig. 7A) or an anti-PD-1 antibody backbone (PD-06/2P17) (Fig. 7A, 7B), as well as anti-TIGIT scFv with severe variable regions chain and light chain from anti-TIGIT mAb T-10/B21, separated by a 3xG4S linker (Fig. 7A, 7B) or a 6xG4S linker (Fig. 7C).

In fig. 8 shows the functional domain sequences present in the bispecific antibody depicted in FIG. 7A-7C.

In fig. 9A-9B show examples of heavy chain (HC) and light chain (LC) sequences corresponding to the bispecific antibodies depicted in FIG. 7A-7C.

Example 3: Expression and functional characterization of anti-PD1/TIGIT bispecific antibodies

To evaluate the ability of Bi-TPM-93 and Bi-TPM-94A to block PD-1, a PD-1 IC50 assay was performed in which serial dilutions of bispecific mAbs were incubated at 4°C for 30 min with PD-1-transfected CHOK1 cells human, and 7 μg/ml FITC-labeled human PD-L1Fc protein, followed by washing and fixation of cells before analysis using the iQue intellicyt system. The results of the above analysis in FIG. 10 show that Bi-TPM-94. containing VH and VL sequences from PD-06/2P17, blocks the interaction between PD-1 and its ligand PD-L1 better (IC50 = 0.15 nM) than Bi-TPM-93 containing VH and VL sequences from PD-01 (IC50 = 0.83 nM).

In fig. 11 shows PAGE analysis under non-reducing conditions demonstrating active transient expression of both Bi-TPM-94A and Bi-TPM-94B in human embryonic kidney 293 (HEK) cells.

To assess the degree of compositional homogeneity of the antagonist samples corresponding to Bi-TPM93, Bi-TPM-94A and Bi-TPM-94B, these samples were purified and analyzed by size exclusion liquid chromatography (SE-UHPLC). Sample purification was performed as follows: first, collected cell culture fluids (HCCF) were filtered through a 0.2-μm pore size filter, followed by affinity purification using Hitrap Protein A HP chromatography (GE Healthcare) at a rate of 1 mL/min. After affinity purification, the material was subjected to cation exchange chromatography (CEX) using Sepax Proteomix® SCX-NP5 columns with gradient elution at 0.8 ml/min. The amount of aggregated (high molecular weight, HMW), dimeric and low molecular weight (LMW) fragments was determined by SE-UPLC using Tosoh TSKgel UP-G3000SWXL columns.

The results of this analysis (Fig. 12) unexpectedly revealed some degree of heterogeneity in the composition of samples Bi-TPM 93 and Bi-TPM-94A, which was eliminated by modifying the linker in Bi-TPM-94B, that is, increasing the linker length from 3xG4S to 6xG4S.

Biolayer interferometry with the

- 43 045974 using the Octet RED96 (ForteBio) system. Briefly, 20 nM bispecific antagonist was loaded onto anti-human IgG capture biosensors. Association of the analyte (His-tagged human PD-1 protein or His-tagged human TIGIT protein) was monitored by placing the biosensors in wells containing His-tagged PD-1 or His-tagged TIGIT in serial dilutions for 5 minutes. Dissociation was measured after transferring the biosensors into pure kinetic buffer and monitoring the interferometric signal for 10 minutes. Observed association and dissociation rates ( _Ka and Kd) were normalized using a general 1:1 binding model including at least 5 concentrations of interest, followed by calculation of the equilibrium binding constant _KD .

The results of the above analysis in FIG. 13A show that the binding affinity of Bi-TPM-94A and Bi-TPM-94B to PD-1 exceeds the binding affinity of the reference anti-PD-1 antibody to PD-1. Likewise, FIG. 13B shows that the binding affinity of Bi-TPM-94A and Bi-TPM-94A to TIGIT exceeds the binding affinity of the reference anti-TIGIT antibody to TIGIT.

A blocking assay using CHO cells stably expressing TIGIT and 1 μg/ml biotinylated PVR-muFc was used to compare the ability of Bi-TPM-94A and BiTPM-94B to block the binding of TIGIT to the TIGIT ligand PVR. Briefly, cells were incubated with biotinylated PVR-Fc and Bi-TPM molecules, washed, and bound PVR-muFc was detected with PE-conjugated streptavidin using the iQue Intellicyt system. In fig. 14A shows that both molecules similarly block the binding of TIGIT and PVR. Likewise, both molecules can block the binding of PD-1 to its ligand PD-L1 (Fig. 14B). The results of this analysis also showed that the IC50 values for BnTPM-94A and Bi-TPM-94B were slightly better than those for the reference antibodies (RM) against TIGIT and PD-1.

To evaluate the ability of Bi-TPM-94A and Bi-TPM-94B to simultaneously bind PD-1 and TIGIT, 96-well ELISA plates coated with huPD-1-Fc (5 μg/ml) were blocked with 1% bovine serum albumin (BSA) in phosphate. -buffered saline (PBS) and incubated for 2 hours with bispecific antibodies against PD-1/TIGIT in serial dilutions, followed by the addition of His-tagged huTIGIT protein and incubation for 2 hours. After washing, HRP-conjugated anti-His tag antibodies and TMB substrate were added as detection agents and quantified using a Perkin Elmer multimode plate reader. The results of this analysis are shown in FIG. 15 show simultaneous binding of PD-1 and TIGIT by the antagonists Bi-TPM-94A and BiTPM-94B.

To evaluate the ability of Bi-TPM-94B to induce IFN-γ production, 250,000 human PBMCs obtained from donors screened for reactivity to CMV antigen, i.e. donor 287 (Fig. 16A) and donor 401 (Fig. 16B), stimulated with 0.1 μg/ml CMV-infected cell lysates to stimulate CMV-reactive T cells (lanes 2-7) or not stimulated with CMV-infected cell lysates (track 1). Shp-77 cells were cocultured with PBMCs to provide an immune suppressive environment and then incubated with human IgG (lane 3), parent anti-TIGIT mAb B21-35 (lane 4), parent anti-PD-1 mAb 2P17 (lane 5), parent Anti-TIGIT mAb B21-35 in combination with the parent anti-PD-1 mAb 2P17 (lane 6) or BiTPM-94B (lane 7). After 5 days, IFN-γ production in cell culture supernatants was examined using ELISA.

The results of the above analysis in FIG. 16A-16B demonstrate increased secretion of IFN-γ in human PBMC (donor 287, Fig. 16A; donor 401, Fig. 16B) in the presence of Bi-TPM-94B compared to monospecific parent mAbs or a combination of monospecific parent antibodies against PD1 and anti-TIGIT , as well as negative controls.

To evaluate the ability of Bi-TPM-94B to induce T cell proliferation, 250,000 human PBMCs from donor 287 (Figure 17A) and donor 401 (Figure 17B) were stimulated with 0.1 μg/ml CMV-infected cell lysates for 2 days to stimulate CMV-reactive T cells and then labeled with carboxyfluorescein succinimidyl ester (CFSE). CFSE-labeled PBMCs were then cocultured with Shp-77 cells to provide an immune suppressive environment and further incubated with human IgG (lane 1), parental anti-TIGIT mAb B21-35 (lane 2), parental anti-PD-1 mAb 2P17 ( lane 3), parental anti-TIGIT mAb B21-35 in combination with parental anti-PD-1 mAb 2P17 (lane 4) or Bi-TPM-94B (lane 5). After 5 days, the CSFE signal on CD3+ T cells was analyzed using the iQue intellicyt system and a proliferation index was calculated based on the loss of CFSE signal.

The results of this analysis are shown in FIG. 17A-17B show that Bi-TPM-94B enhanced the proliferation of primary human T cells from PBMCs from donor 287 (FIG. 17A) and PBMCs from donor 401 (FIG. 17B) to a greater extent compared to the use of individual anti-PD-1 antibodies and against TIGIT or combinations thereof, as well as negative controls.

To evaluate the pharmacokinetic properties of Bi-TPM-94A and Bi-TPM-94B in vivo, a pharmacokinetic profile was obtained. Briefly, 10 mg/kg Bi-TMP-94A or VnTPM-94B was administered intravenously into the tail.

- 44 045974 stomatal vein to female CD1 mice aged 6-10 weeks (n=2 mice). Serum was collected at 3 minutes, 3 hours, 1 day, 3 days, 7 days and 10 days after injection. To detect antibodies in serum, 96-well ELISA plates were coated with P(ab')2-fragment of goat anti-human IgG (Fc-specific) antibodies (Sigma, #SAB3701274) at a concentration of 5 μg/ml, and then blocked with 5% milk in PBS. After the blocking step, mouse serum and purified Bi-TPM-94A or Bi-TPM-94B molecules (as standards) were serially diluted in 5% milk and then added to the plate and incubated for 2 hours. After incubation, the wells were washed and then incubated in the presence of AffiniPure peroxidase-conjugated mouse anti-human IgG antibody specific for the FcY fragment (Jackson ImmunoResearch #209-035-098) and TMB ELISA substrate solution (Thermo Scientific #34029) and quantitation was performed. estimation based on the OD650 signal using a Perkin Elmer multi-mode tablet reader.

The results of this analysis are shown in FIG. 18 showed that the half-life (T1/2) of the bispecific antagonists Bi-TPM-94A and Bi-TMP-94B was 7–10 days. Thus, the Bi-TPM-94A and Bi-TPM-94B molecules have improved binding properties with higher affinity compared to Bi-TPM-93, and Bi-TPM-94B with an extended G4S linker in the scFv fragment of the anti-TIGIT antibody has additional advantage compared to Bi-TPM-93 and Bi-TPM-94A in terms of homogeneity.

Example 4: Identification and functional characterization of anti-LAG-3 monoclonal antibodies

In another aspect, the present application relates to the screening and characterization of monoclonal antibodies or antigen-binding portions thereof that specifically bind to the human immune checkpoint regulator, LAG-3. In fig. 19A shows the heavy chain CDR1, CD2 and CDR3 sequences corresponding to the isolated anti-LAG-3 mAbs 2L2A.1, 2L2A.6, 2L27B and 3L1A. In fig. 19B shows the light chain CDR1, CD2 and CDR3 sequences corresponding to anti-LAG-3 mAbs 2L2A.1, 2L2A.6, 2L27B and 3L1A. In fig. 20 shows the VH and VL sequences of anti-LAG-3 mAb 2L2A. 1, 2L2A.6, 2L27B and 3L1A.

To show that mAbs 2L2A and 2L27B (Fig. 21A) and 2L37A and 3L1A (Fig. 21B) block the interaction between human LAG-3 and its major ligand, the major histocompatibility complex (MHC II) class II antigen expressed on Raji cells, blocking assay was performed. Briefly, 2-fold serial dilutions of mAbs 2L2A and 2L27B (Fig. 21A) and 2L37A and 3L1A (Fig. 21B) were prepared. Serial dilutions of the mAb and human LAG-3-huFc were incubated for 30 minutes at room temperature and then added to Raji cells and incubated for an additional 30 minutes on ice. Raji cells were then washed and LAG-3-huFc binding to Raji cells was detected using anti-human IgG PE antibodies. Cells were fixed before analysis using the iQue Intellicyt system. The results of the above analysis in FIG. 21A-21B confirm the ability of mouse anti-LAG-3 mAbs to block the interaction between LAG-3 and its major ligand, major histocompatibility complex (MHC II) class II antigen, to a degree comparable to the reference anti-LAG-3 mAb (BM).

The blocking assay described above was further used to calculate IC50 values reflecting the inhibition by anti-LAG-3 antibodies of LAG-3 binding to MHC II, particularly the humanized variant of the anti-LAG-3 antibody, 2L2A. 1, a reference anti-LAG-3 antibody (BM, BMS-986016, Bristol-Myers Squibb) and a fusion antibody 2L2A containing the mouse 2L2A CDR in a human Ig.

The results of the above analysis in FIG. 22 show that the humanized mAb 2L2A. 1 anti-LAG-3 is a more effective blocker than the BM mAb and the fusion antibody 2L2A containing the mouse 2L2A CDR in the human Ig, as evidenced by the lower IC50 value obtained.

To assess the binding affinity and kinetics of the anti-LAG-3 mAb 2L2A. 1 with His-tagged human LAG-3, biolayer interferometry was performed using the Octet RED96 system (ForteBio) essentially as described above in Example 3 with reference to FIG. 13A-13B, as determined by surface plasmon resonance (SPR), along with corresponding affinity constants. In fig. 23A shows the binding affinity of the humanized antibody 2L2A.1 to His-tagged human LAG-3. Likewise, FIG. 23B shows the binding affinity constants for binding of the humanized 2L2A antibody. 1 with human LAG-3 fused to mouse IgG2a (huLAG-3-mIgG2a).

For additional assessment of 2L2A ability. 1 bind to human LAG-3, 2L2A was added to Cno-Κι cells (20,000 cells/well) expressing human LAG-3. 1 or reference antibody (RA) against LAG-3 in serial dilutions. The mixtures were incubated at 4°C for 20 min, washed 3 times and stained with secondary antibodies - PE-labeled F(ab')2 fragments of goat antibodies against the Fc fragment of human IgG (Thermo Scientific # H10104) - by incubating at 4°C C for 20 min. Cells were washed and resuspended in 7AAD and fixed in 10% neutral buffered formalin for 15 minutes before analysis using the iQue Intellicyt system. Corresponding EC50 values were also determined, reflecting the half-maximal effective concentrations (EC50) that produce an effect corresponding to the average

- 45 045974 relationship between baseline and maximum effect on human LAG3 binding. As shown in FIG. 24, the results demonstrate that 2L2A.1 has higher binding affinity to human LAG-3 compared to the BM antibody.

In fig. 25 shows polyacrylamide gel electrophoresis (PAGE) analysis under non-denaturing conditions demonstrating high level expression of the humanized anti-LAG-3 mAb variant 2L2A. 1 in human embryonic kidney (HEK) 293 cells with transient expression compared to control (ctr1) antibody.

To assess the coexpression of LAG-3 and PD-1 in activated human CD3+ T cells, FACS analysis was performed on donor PBMCs activated with anti-human CD3/CD28 antibodies. The results of this analysis, shown in FIG. 26 confirmed the coexpression of LAG-3 and PD-1 in human PBMCs activated by staphylococcal enterotoxin B (SEB).

To evaluate the ability of 2L2A.1 to induce IFN-γ production, 100,000 human PBMCs from two donors, i.e. donor 0105 (Fig. 27A) and donor 0817 (Fig. 27B) were plated in a 96-well ELISA plate. PBMCs were either unstimulated (lane 1) or stimulated with 0.5 μg/ml staphylococcal enterotoxin B (SEB; lanes 2–4). The stimulated cells were supplemented with reference anti-LAG-3 mAb (BM) (lane 3), mAb 2L2A.1 (lane 4), or isotype-matched control antibodies (lane 2) and incubated at 37°C for 5 days. IFN-γ production was then examined in cell culture supernatants using ELISA. The results in Fig. 27A-27B demonstrate increased IFN-γ secretion in human PBMCs from both donors in the presence of 2L2A. 1 versus VM versus LAG-3.

A similar analysis was performed on PBMCs from 3 additional donors—donor 223 (FIG. 28A), donor 224 (FIG. 28B), and donor 225 (FIG. 28C)—in which PBMC proliferation was further assessed. The results in Fig. 28A-28B further demonstrate increased IFN-γ secretion in human PBMCs from donors in the presence of 2L2A.1 compared to anti-LAG-3 BM. To assess proliferation, PBMCs from 3 donors were labeled with CSFE, stimulated with 100 ng/ml SEB, and supplemented with reference anti-LAG-3 mAb (BM) (lane 3), mAb 2L2A.1 (lane 4), or isotype-matched control antibodies (lane 2). PBMC mixtures were incubated at 37°C for 5 days and loss of CFSE signal on CD4 T cells was quantified by FACS to obtain a proliferation index. The results shown in FIG. 29A and 29B demonstrate that 2L2A.1 can induce higher proliferation of primary T cells compared to the reference anti-LAG-3 mAb (BM).

Example 5: Design and functional characterization of anti-LAG-3/scFv anti-TIGIT bispecific antagonist

Based on the design and characterization of the anti-PD-1/scFv anti-TIGIT bispecific antagonist described in Example 1, it was interesting to evaluate the possibility of leveraging the manufacturability and functionality advantages of this design in a similar anti-LAG-3/scFv anti-TIGIT bispecific antibody design. In fig. 30A-30B show two examples of bispecific antitumor antagonists - Bi-LT-1 with an extended 4xG4S linker in the scFv (Fig. 30A) and BiLT-3 with an extended 6xG4S linker in the scFv (Fig. 30B). In fig. 31 shows a generalized order of the functional domains in the bispecific antagonists depicted in FIG. 30A30B. In fig. 32 shows the heavy chain (HC) and light chain (LC) amino acid sequences corresponding to the bispecific antagonists depicted in FIG. 30A-30B.

To evaluate the ability of the bispecific antitumor antagonists Bi-LT-1 and Bi-LT-3 to block the interaction between TIGIT and its ligand, human PVR (CD155), and additionally block the interaction between LAG-3 and its major ligand, major histocompatibility complex type II antigen (MHC II), cell blocking assays were performed as follows.

Briefly, to show that Bi-LT-1 and Bi-LT-3 can block the interaction between TIGIT and its ligand, human PVR (CD155), a cell-based blocking assay was performed in which Bi-LT1, Bi-LT-3, or Bi-TPM-94B (described in Example 1 above) in serial dilutions was incubated with CHOK1 cells transfected with human TIGIT and CD155/PVR-mouse IgG2a complex for 30 minutes on ice. Binding of the CD155/PVR-mouse IgG2a complex to CHOK1 cells was detected using PE-labeled anti-mouse IgG antibodies. Cells were fixed before analysis using the iQue intellicyt system. The results of this analysis are presented in FIG. 33A and demonstrate that Bi-LT-1 and Bi-LT-3 with extended linkers in scFv retain their bioactivity towards TIGIT.

To show that the bispecific antitumor antagonists Bi-LT-1 and Bi-LT-3 can also block the interaction between LAG-3 and its major ligand, major histocompatibility complex (MHC II) class II antigen, a cell-based blocking assay was performed in which Bi Serial dilutions of -LT1, Bi-LT-3, or anti-LAG-3 reference antibody (BM) were incubated with LAG-3-mouse IgG2a complex for 30 minutes at room temperature, added to Raji cells, and incubated for an additional 30 minutes at ice. Raji cells were then washed, and the binding of LAG-3-mouse IgG2a to Raji cells was detected using PE-conjugated antibodies pro

- 46 045974 mouse IgG tive. Cells were fixed before analysis using the iQue intellicyt system. The results of this analysis are presented in FIG. 33B and indicate that Bi-LT-1 and Bi-LT-1 retain their bioactivity against LAG-3, similar to the reference anti-LAG3 antibody. The assay data were further used to calculate the reported IC50 values (nM), which were comparable to the IC50 value of the reference anti-LAG-3 antibody (BM). The results in Fig. 33A and 33B together demonstrate that Bi-LT-1 and Bi-LT-3 retain their biological activity against both TIGIT and LAG-3.

To evaluate the ability of Bi-LT-1 and Bi-LT-3 to simultaneously bind to LAG-3 and TIGIT, LAG-3-mIgG was applied to an ELISA plate at a concentration of 5 μg/ml and incubated at 4°C overnight, and then blocked before adding LT-1, LT-3, or the original anti-LAG-3 mAb at serial dilutions. After incubation for 1 hour at room temperature, the plate was washed, 500 ng/ml His-tagged HuTIGIT was added, and incubated for 1 hour at room temperature. Plate-bound His-tagged HuTIGIT was then detected using HRP-conjugated antibodies against the His tag and TMB substrate. The results of the above analysis in FIG. 34 demonstrate that Bi-LT-1 and Bi-LT-3 can simultaneously bind to LAG-3 and TIGIT.

In fig. 35A-35D show the pharmacokinetic profiles and in vivo half-life (T1/2) corresponding to the parent anti-LAG-3 mAb (Fig. 35A), the reference anti-LAG-3 mAb (Fig. 35B), Bi-LT-1 (Fig. 35C) or Bi-LT-3 (Fig. 35D) after tail vein injection into female CD1 mice aged 6-10 weeks. Antibodies and bispecific antagonists were recovered from sera obtained at various times after injection and analyzed by ELISA. The results show that BiLT-1 and Bi-LT-3 have pharmacokinetic properties similar to the parent anti-LAG3 antibody and the reference antibody, with a half-life (T1/2) of 5-6 days.

To assess the compositional homogeneity and stability of Protein A purified Bi-LT-1 and BiLT-3, size exclusion chromatography (SEC) profiles were obtained for Bi-LT-1 and Bi-LT-3 as described above. The results of the above analysis in FIG. 36A are consistent with data for both extended scFv linkers, providing homogeneity in composition and good stability after 7 days at 4°C. In fig. 36B shows size exclusion liquid chromatography (SE-UHPLC) analysis performed as described above. The results of this analysis are consistent with the compositional homogeneity observed in FIG. 36A, as can be seen from the low level of high molecular weight (HMW) products on days 0 and 7 (1.3%, 1.5%, respectively for Bi-LT-1 and 1.3%, 1.4%, respectively for Bi-LT-3) and low molecular weight (LMW) products on days 0 and 7 (0.2%, 0.2%, respectively for Bi-LT-1 and 0.3%, 0.5%, respectively for Bi-LT-3) compared to dimeric products on days 0 and 7 (98.4%, 98.3%, respectively, for Bi-LT-1 and 98.4%, 98.1%, respectively, for Bi-LT- 3).

To evaluate the ability of Bi-LT-1 and Bi-LT-3 to induce IFN-γ production, human PBMCs were stimulated with SEB at a concentration of 100 ng/ml in the presence of SHP-77 cells (Fig. 37A) or H358 cells (Fig. 37B) with receiving an immunosuppressive signal. Human control IgG (lane 3), anti-TIGIT mAb (B21-35) alone (lane 4), anti-LAG-3 mAb alone (lane 5), combination of anti-TIGIT mAb and anti-LAG-3 mAb (lane 6) were then added. Bi-LT-1 (lane 7) or Bi-LT-3 (lane 8) to restore T cell function (Fig. 37A, 37B). After 4 days, cell culture supernatants were collected and their IFN-γ levels were tested using ELISA. The results of these analyzes are shown in FIG. 37A and 37B show that LT-1 and LT-3 are more active than the combination of parent antibodies against TIGIT and LAG-3.

In fig. 38 shows an assessment of the ability of Bi-LT-1 and Bi-LT-3 to induce proliferation of CD4 T cells. Human PBMCs were labeled with CSFE and then stimulated with SEB at 100 ng/ml in the presence of SHP-77 cells to produce an immunosuppressive signal. 128 nM control human IgG (lane 3), anti-TIGIT mAb B21-35 (lane 4), anti-LAG-3 mAb (lane 5), combinations of anti-TIGIT mAb and anti-LAG-3 mAb (lane 6), Bi-LT- 1 (lane 7) or Bi-LT-3 (lane 8) was used to restore T cell function. After 5 days, loss of CFSE signal on CD4 T cells was quantified by FACS to determine the proliferation index. Similar to the increased stimulation of IFNy compared to the parent antibodies and the combination of the two parent antibodies, both Bi-LT-1 and Bi-LT-3 had an increased ability to stimulate the proliferation of human CD4 T cells.

The above description is intended to explain to those skilled in the art how to practice the present invention, and is not intended to describe in detail all those obvious modifications and variations that will be apparent to one skilled in the art upon reading the description. However, all such obvious modifications and variations are intended to be included within the scope of the present invention, which is defined by the following claims. The claims are intended to include the claimed components and steps in any order that is effective to achieve the intended purposes, unless the context specifically requires otherwise.

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Claims (16)

CLAIM 1. Antitumor antagonist containing: a first targeting domain that specifically binds PD-1 or LAG-3; and a second targeting domain comprising a scFv that specifically binds TIGIT, said scFv comprising: (1) An immunoglobulin HCVR comprising three complementarity determining regions (HCDR): HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 23, and HCDR2 contains the amino acid sequence of SEQ ID NO: 24, and HCDR3 contains the amino acid sequence of SEQ ID NO: 25; and (2) an immunoglobulin LCVR comprising three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains the amino acid sequence of SEQ ID NO: 45, wherein LCDR2 contains the amino acid sequence of SEQ ID NO: 46, and wherein LCDR3 contains the amino acid sequence SEQ ID NO: 47; wherein said first targeting domain that specifically binds PD-1 comprises: (a) an immunoglobulin HCVR comprising three complementarity determining regions (HCDR): HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 79, and HCDR2 contains the amino acid sequence of SEQ ID NO: 80, and HCDR3 contains the amino acid sequence of SEQ ID NO: 81; and (b) an immunoglobulin LCVR comprising three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains the amino acid sequence of SEQ ID NO: 93, wherein LCDR2 contains the amino acid sequence of SEQ ID NO: 94, and wherein LCDR3 contains the amino acid sequence SEQ ID NO: 95; and wherein said first targeting domain, which specifically binds to LAG-3, comprises: (i) an immunoglobulin HCVR comprising three complementarity determining regions (HCDR): HCDR1, HCDR2 and HCDR3, wherein HCDR1 contains the amino acid sequence of SEQ ID NO: 163, and HCDR2 contains the amino acid sequence of SEQ ID NO: 164, and HCDR3 contains the amino acid sequence of SEQ ID NO: 165; and (ii) an immunoglobulin LCVR comprising three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, wherein LCDR1 contains the amino acid sequence of SEQ ID NO: 172, wherein LCDR2 contains the amino acid sequence of SEQ ID NO: 173, and wherein LCDR3 contains the amino acid sequence SEQ ID NO: 174. 2. Antitumor antagonist according to claim 1, characterized in that said first targeting domain specifically binds to PD-1 and contains: (1) An immunoglobulin HCVR containing three complementarity determining regions (HCDR): HCDR1, HCDR2 and HCDR3, where HCDR1 contains the amino acid sequence SEQ ID NO: 79, where HCDR2 contains the amino acid sequence SEQ ID NO: 80, where HCDR3 contains the amino acid sequence SEQ ID NO: 81; and (2) an immunoglobulin LCVR comprising three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, where LCDR1 contains the amino acid sequence of SEQ ID NO: 93, where LCDR2 contains the amino acid sequence of SEQ ID NO: 94, and where LCDR3 contains the amino acid sequence SEQ ID NO: 95. 3. Antitumor antagonist according to claim 2, characterized in that said first targeting domain contains: HCVR immunoglobulin that has at least 90% identity with SEQ ID NO: 106; and/or An immunoglobulin LCVR that has at least 90% identity with SEQ ID NO: 107. 4. Antitumor antagonist according to claim 3, characterized in that said first targeting domain contains: HCVR immunoglobulin containing the amino acid sequence SEQ ID NO: 106; and/or An immunoglobulin LCVR containing the amino acid sequence of SEQ ID NO: 107. - 48 045974 5. Antitumor antagonist according to claim 1, characterized in that said first targeting domain specifically binds to LAG-3 and contains: (1) An immunoglobulin HCVR containing three complementarity determining regions (HCDR): HCDR1, HCDR2 and HCDR3, where HCDR1 contains the amino acid sequence of SEQ ID NO: 163, where HCDR2 contains the amino acid sequence of SEQ ID NO: 164, where HCDR3 contains the amino acid sequence of SEQ ID NO: 165; and (2) an immunoglobulin LCVR comprising three complementarity determining regions (LCDR): LCDR1, LCDR2 and LCDR3, where LCDR1 contains the amino acid sequence of SEQ ID NO: 172, where LCDR2 contains the amino acid sequence of SEQ ID NO: 173, and where LCDR3 contains the amino acid sequence SEQ ID NO: 174. 6. Antitumor antagonist according to claim 5, characterized in that said first targeting domain contains: An immunoglobulin HCVR that is at least 90% identical to the amino acid sequence of SEQ ID NO: 180; and/or An immunoglobulin LCVR that is at least 90% identical to the amino acid sequence of SEQ ID NO: 181. 7. Antitumor antagonist according to claim 6, characterized in that said first targeting domain contains: HCVR immunoglobulin having the amino acid sequence SEQ ID NO: 180; and/or An immunoglobulin LCVR having the amino acid sequence of SEQ ID NO: 181. 8. Antitumor antagonist according to any one of claims 1-7, characterized in that said scFv contains: An HCVR immunoglobulin that has at least 90% identity with the amino acid sequence of SEQ ID NO: 66; and/or An immunoglobulin LCVR that has at least 90% identity with the amino acid sequence of SEQ ID NO: 67. 9. Antitumor antagonist according to claim 8, characterized in that said scFv contains: HCVR immunoglobulin containing the amino acid sequence SEQ ID NO: 66; and/or An immunoglobulin LCVR containing the amino acid sequence of SEQ ID NO: 67. 10. The antitumor antagonist according to any one of claims 1 to 9, further comprising an immunoglobulin scaffold having an amino terminus and a carboxy terminus, wherein said first targeting domain is linked to the amino terminus of said immunoglobulin scaffold and said second targeting domain is linked to the carboxy terminus of said immunoglobulin scaffold. using a peptide linker, and wherein said peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 188-191. 11. Antitumor antagonist according to any one of claims 2-4, which contains: an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO: 160 or 162; and/or an immunoglobulin light chain that has the amino acid sequence of SEQ ID NO: 161. 12. Antitumor antagonist according to any one of claims 5-7, which contains: an immunoglobulin heavy chain having the amino acid sequence of SEQ ID NO: 192 or 193; and/or an immunoglobulin light chain that has the amino acid sequence of SEQ ID NO: 194. 13. Nucleic acid encoding one or more antitumor antagonist domains according to any one of claims 1 to 12. 14. An expression vector containing one or more nucleic acids according to claim 13. 15. A method of treating a cell proliferative disorder in a subject, comprising administering to the subject in need thereof an effective amount of an antitumor antagonist according to any one of claims 1 to 12, a nucleic acid according to claim 13, or an expression vector according to claim 14. 16. The method of treatment according to claim 15, characterized in that the cell proliferative disorder is a tumor or cancer. -