EA039540B1 - Tigit-binding agents and uses thereof - Google Patents

Abstract

Agents that specifically bind TIGIT are disclosed. The TIGIT-binding agents may include polypeptides, antibodies, and/or bispecific agents. Also disclosed are methods of using the agents for enhancing the immune response and/or treatment of diseases such as cancer.

Description

filed May 28, 2015, U.S. Provisional Application No. 62/205279 filed August 14, 2015

and U.S. Provisional Application No. 62/313487, filed March 25, 2016, each of which is hereby incorporated by reference in its entirety.

Technical field

The invention relates generally to agents that bind TIGIT, in particular antibodies that specifically bind the extracellular domain of TIGIT, as well as methods of using said agents to modulate an immune response and/or to treat diseases such as cancer.

State of the art

The basis of immunotherapy is manipulation of the immune system and/or modulation of the immune system, including both the innate immune response and the adaptive immune response. The general goal of immunotherapy is to treat diseases by directing the immune response to a foreign agent, such as a pathogen or tumor cell. However, in some cases, immunotherapy is used to treat autoimmune diseases, which may result from an abnormal immune response against proteins, molecules and/or tissues present in a healthy body. Immunotherapy may include agents and methods for inducing or enhancing a specific immune response, or for inhibiting or reducing a specific immune response.

The immune system is a highly complex system composed of a significant number of cell types including, but not limited to, T cells, B cells, natural killer cells, antigen presenting cells, dendritic cells, monocytes, and macrophages. These cells have complex sensory systems to control their interactions and responses. These cells use both activating and inhibitory mechanisms and feedback loops to control responses and prevent the negative consequences of an uncontrolled immune response (eg, autoimmune diseases).

The concept of immunological surveillance in cancer is based on the theory that the immune system is able to recognize tumor cells, organize an immune response, and suppress tumor development and/or progression. However, it is clear that many cancer cells have developed mechanisms for escaping the immune system, which can ensure uninhibited tumor growth. Cancer/tumor immunotherapy is focused on the development of novel, hitherto unknown agents that can activate and/or further stimulate the immune system to provide a more effective attack against tumor cells resulting in increased tumor cell killing and/or inhibition of tumor growth.

Brief description of the invention

The invention provides various agents that bind a T cell immunoreceptor to Ig and ITIM (TIGIT) domains, including, but not limited to, antibodies that specifically bind to the extracellular domain of TIGIT. In some embodiments, said agent is a TIGIT antagonist. The present invention provides methods for using these agents. In some embodiments, the present invention provides methods of using said agents for immunotherapy. In some embodiments, the present invention provides methods of using said agents for cancer immunotherapy. In some embodiments, these agents are used in methods of inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing, or prolonging an immune response. In some embodiments, these agents are used in methods of inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing, or prolonging an immune response to a cancer and/or tumor. In some embodiments, these agents are used in methods for inhibiting the growth of a tumor or tumor cells. In some embodiments, these agents are used in methods of treating various types of cancer. In some embodiments, these methods include inhibiting the growth of cancer cells. In some embodiments, these agents are used in combination with at least one additional therapeutic agent.

The present invention also provides compositions, such as pharmaceutical compositions, containing the agents described herein. Also provided are polynucleotides and/or vectors encoding said agents and methods for producing said agents. Proposed cells containing or producing agents described in this document, as well as cells containing polynucleotides and/or vectors described in this document.

According to one aspect of the present invention, agents are provided that bind TIGIT. In some embodiments, said agent binds the mouse TIGIT. In some embodiments, said agent binds human TIGIT. In some embodiments, said agent binds mouse TIGIT and human TIGIT. In some embodiments, said agent is an antibody. According to some implementations

- 1 039540 said agent is an antibody that binds mouse TIGIT. In some embodiments, said agent is an antibody that binds human TIGIT.

In some embodiments, said agent is an antibody that binds mouse TIGIT and human TIGIT. In some embodiments, said agent is an antibody that binds human TIGIT and does not bind mouse TIGIT.

In some embodiments, said agent is an antibody specifically binding to the extracellular domain of TIGIT that contains a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEAQ ID NO: 9), and/or a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11) or RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16 ). In some embodiments, said agent is an antibody that contains a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9), and/or a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12) . In other embodiments, said agent is an antibody that comprises a heavy chain CDR1 containing GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9); and/or a light chain CDR1 containing QASQSNIYSDLAW (SEQ ID NO: 14), a light chain CDR2 containing RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHLVAWIYN (SEQ ID NO: 16). In other embodiments, said agent is an antibody that contains a heavy chain CDR1 containing GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SeQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9); and/or a light chain CDR1 containing QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHLVAWIYN (SEQ ID NO: 16).

In some embodiments, said agent is an antibody that specifically binds the extracellular domain of TIGIT, characterized in that said antibody contains a heavy chain variable region that differs by at least 90% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32; and/or a light chain variable region differing by at least 90% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said antibody comprises a heavy chain variable region differing by at least 95% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 32; and/or a light chain variable region differing by at least 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said antibody comprises a heavy chain variable region comprising SEQ ID NO: 17 and a variable region light chain containing SEQ ID NO: 18; a heavy chain variable region comprising SEQ ID NO: 19 and a light chain variable region comprising SEQ ID NO: 20; or a heavy chain variable region comprising SEQ ID NO: 32 and a light chain variable region comprising SEQ ID NO: 20.

In some embodiments, said agent is an antibody specifically binding to the extracellular domain of human TIGIT, which comprises a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), a heavy chain CDR2 containing YISYSGSTSYNPSLRS (SEQ ID NO: 58), and a heavy chain CDR3 chain containing ARRQVGLGFAY (SEQ ID NO: 59) and/or a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 containing SASYRYT (SEQ ID NO: 61) and a light chain CDR3 containing QQHYSTP (SEQ ID NO: 62).

In some embodiments, said agent is an antibody that specifically binds the extracellular domain of TIGIT, characterized in that said antibody contains a heavy chain variable region that shares at least 90% sequence identity with SEQ ID NO: 63 or SEQ ID NO: 67; and/or a light chain variable region having at least 90% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, the antibody comprises a heavy chain variable region having at least 95% sequence identity to SEQ ID NO: : 63 or SEQ ID NO: 67; and/or a light chain variable region having at least 95% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 63 and a light chain variable region chain containing SEQ ID NO: 64, or a heavy chain variable region containing SEQ ID NO: 67 and a light chain variable region containing SEQ ID NO: 68.

In some embodiments, said agent is an antibody, which is a monoclonal antibody, a humanized antibody, a human antibody, a recombinant antibody, a chimeric antibody, a bispecific antibody, an antibody fragment containing an antigen-binding site, an IgG antibody, an IgG1 antibody, an IgG2 antibody or an IgG4 antibody. In some embodiments, said antibody is monovalent. In some embodiments, said antibody is bivalent. In some embodiments, said antibody is monospecific. In some embodiments, said antibody is bispecific.

In some embodiments, said agent is an antibody that specifically binds TIGIT, characterized in that said antibody contains a heavy chain amino acid sequence selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29 , SEQ ID NO: 34 and SEQ ID NO: 56; and a light chain amino acid sequence selected from the group consisting of: SEQ ID NO: 28 and SEQ ID NO: 30. In some embodiments, said antibody comprises a heavy chain amino acid sequence as set forth in SEQ ID NO: 26 and a light chain amino acid sequence presented in SEQ ID NO: 28; the heavy chain amino acid sequence set forth in SEQ ID NO: 27 and the light chain amino acid sequence set forth in SEQ ID NO: 28; the heavy chain amino acid sequence set forth in SEQ ID NO: 29 and the light chain amino acid sequence set forth in SEQ ID NO: 30; the heavy chain amino acid sequence set forth in SEQ ID NO: 34 and the light chain amino acid sequence set forth in SEQ ID NO: 30; or the heavy chain amino acid sequence set forth in SEQ ID NO: 56 and the light chain amino acid sequence set forth in SEQ ID NO: 30.

In some embodiments, an antibody specifically binding to human TIGIT comprises a heavy chain amino acid sequence as set forth in SEQ ID NO: 70 and a light chain amino acid sequence as set forth in SEQ ID NO: 72.

In some embodiments, an antibody specifically binding to human TIGIT comprises a heavy chain amino acid sequence as set forth in SEQ ID NO: 82 and a light chain amino acid sequence as set forth in SEQ ID NO: 72.

In some embodiments, an antibody that specifically binds human TIGIT does not bind mouse TIGIT. In some embodiments, an antibody that specifically binds human TIGIT does not bind rat TIGIT. In some embodiments, an antibody that specifically binds human TIGIT does not bind rabbit TIGIT. In some embodiments, an antibody that specifically binds human TIGIT does not bind marmoset TIGIT. In some embodiments, an antibody that specifically binds human TIGIT does not bind dog TIGIT. In some embodiments, an antibody that specifically binds human TIGIT does not bind porcine TIGIT. In some embodiments, an antibody that specifically binds human TIGIT does not bind cynomolgus TIGIT. In some embodiments, an antibody that specifically binds human TIGIT does not bind rhesus monkey TIGIT.

In some embodiments, said agent is an antibody that specifically binds TIGIT, characterized in that said antibody comprises a heavy chain variable region and a light chain variable region from an antibody selected from the group consisting of: 313R11, 313R12, 313R14, 313R19, and 313R20. In some embodiments, said antibody is selected from the group consisting of: 313R11, 313R12, 313R14, 313R19, and 313R20. In some embodiments, said antibody is 313R19. In some embodiments, said antibody comprises a heavy chain variable region encoded by a plasmid deposited with ATCC (American Type Culture Collection) under PTA122180. In some embodiments, said antibody comprises a light chain variable region encoded by a plasmid deposited with ATCC under PTA-122181. In some embodiments, said antibody comprises a heavy chain containing a heavy chain variable region encoded by a plasmid deposited with ATCC under PTA-122180. In some embodiments, said antibody comprises a light chain containing a light chain variable region encoded by a plasmid deposited with ATCC under PTA122181. In some embodiments, said antibody comprises a heavy chain variable region encoded by plasmid deposited with ATCC under PTA-122180 and a light chain variable region encoded by plasmid deposited with ATCC under PTA-122181. In some embodiments, said antibody comprises a polypeptide encoded by plasmid deposited with ATCC under PTA-122180 and a polypeptide encoded by plasmid deposited with ATCC under PTA-122181.

According to another aspect of the present invention, there is provided an ATCC deposited plasmid assigned the number PTA-122180 and an ATCC deposited plasmid assigned the number PTA-122181.

In some embodiments, an antibody specifically binding to human TIGIT comprises a heavy chain variable region and a light chain variable region from the 313M32 antibody.

- 3 039540

In some embodiments, said antibody comprises a heavy chain variable region encoded by a plasmid deposited with ATCC under PTA-122346. In some embodiments, said antibody comprises a polypeptide containing a heavy chain variable region encoded by a plasmid deposited with ATCC under PTA-122346. In some embodiments, said antibody comprises a light chain variable region encoded by a plasmid deposited with ATCC under PTA-122347. In some embodiments, said antibody contains a light chain encoded by a plasmid deposited with ATCC under PTA-122347. In some embodiments, said antibody comprises a heavy chain variable region encoded by plasmid deposited with ATCC under PTA-122346 and a light chain variable region encoded by plasmid deposited with ATCC under PTA-122347. In some embodiments, said antibody comprises a polypeptide encoded by a plasmid deposited with ATCC under PTA-122346 and a polypeptide encoded by a plasmid deposited with ATCC under PTA-122347.

According to another aspect of the present invention, there is provided an ATCC deposited plasmid assigned the number PTA-122346 and an ATCC deposited plasmid assigned the number PTA-122347.

According to some embodiments of each of the above aspects and implementation options, as well as other aspects and implementation options described herein, the specified agent is monovalent. In some embodiments, said agent is bivalent. In some embodiments, said agent is monospecific. In some embodiments, said agent is bispecific. In some embodiments, said bispecific agent is a heterodimeric agent or a heterodimeric molecule. In some embodiments, the heterodimeric agent comprises an antibody described herein that specifically binds TIGIT.

According to another aspect of the present invention, an isolated antibody is provided that competes for specific binding to human TIGIT with an agent (eg, antibody) described herein. In some embodiments, the isolated antibody binds the same epitope on human TIGIT as the agent (eg, antibody) described herein. In some embodiments, an isolated antibody binds an epitope on human TIGIT that overlaps with an epitope on TIGIT that binds the agent (eg, antibody) described herein. In some embodiments, an antibody specifically binding to human TIGIT binds an epitope containing amino acids constituting the sequence of SEQ ID NO: 79. In some embodiments, an antibody specifically binding human TIGIT binds an epitope containing amino acids constituting the sequence of SEQ ID NO: 80. In some embodiments, an antibody that specifically binds human TIGIT binds an epitope containing the amino acids included in SEQ ID NO: 79 and SEQ ID NO: 80. In some embodiments, an antibody that specifically binds human TIGIT binds an epitope , containing amino acids Q62 and I109 of SEQ ID NO: 4. In some embodiments, an antibody that specifically binds human TIGIT binds an epitope containing amino acids Q62 and T119 of SEQ ID NO: 4. In some embodiments, an antibody that specifically binds a human TIGIT binds an epitope containing amino acids Q64 and I109 of SEQ ID NO: 4. In some embodiments, an antibody that specifically binds human TIGIT binds an epitope containing amino acids Q64 and T119 of SEQ ID NO: 4. In some embodiments, the antibody , which specifically binds human TIGIT, binds an epitope containing amino acids Q62, Q64, and I109 of SEQ ID NO: 4. In some embodiments, an antibody that specifically binds human TIGIT binds an epitope containing amino acids Q62, Q64, and T119 of SEQ ID NO: 4 In some embodiments, an antibody specifically binding to human TIGIT binds an epitope containing amino acids Q62, I109, and T119 of SEQ ID NO: 4. In some embodiments, an antibody specifically binding to human TIGIT binds an epitope containing amino acids Q64, I109, and T119 sequences S EQ ID NO: 4. In some embodiments, an antibody that specifically binds human TIGIT binds an epitope containing the amino acids Q62, Q64, I109, and T119 of SEQ ID NO: 4. In some embodiments, an antibody that specifically binds human TIGIT binds an epitope containing at least one amino acid selected from the group consisting of: N58, E60, Q62, Q64, L65, F107, I109, H111, T117, T119, G120 and R121 of the sequence SEQ ID NO: 4. In some embodiments, the specified epitope is a conformational epitope. In some embodiments, an antibody specifically binding to human TIGIT binds an epitope that does not contain the V100 amino acid of SEQ ID NO: 4.

In some embodiments of each of the above aspects and implementations, as well as other aspects and implementations described herein, an agent,

- 4 039540 which specifically binds TIGIT is an antibody, characterized in that said antibody is part of a bispecific agent. In some embodiments, the bispecific agent comprises a first arm that binds TIGIT and a second arm that binds a second target. In some embodiments, the bispecific agent comprises a first arm that specifically binds TIGIT and a second arm, said first arm comprising an anti-TIGIT antibody described herein. In some embodiments, the bispecific agent comprises a first arm that binds TIGIT and a second arm that contains an antigen-binding site from an antibody. In some embodiments, the bispecific agent comprises a first arm that binds TIGIT and a second arm that specifically binds PD-1, PD-L1, CTLA4, TIM-3, LAG-3, OX-40, or GITR. In some embodiments, the bispecific agent comprises a first arm that binds TIGIT and a second arm that specifically binds a tumor antigen. In some embodiments, the bispecific agent comprises a first arm that binds TIGIT and a second arm that contains an immune response stimulating agent. In some embodiments, said immune response stimulating agent is selected from the group consisting of: granulocyte macrophage colony stimulating factor (GMCSF), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (GCSF), interleukin 2 (IL-2), interleukin 3 ( IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), ligand 4-1BB, GITRL, OX-40L, antibodies against CD3, anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-GITR antibodies, anti-OX-40 antibodies, anti-LAG-3 antibodies, and anti-TIM-3 antibodies.

In some embodiments, said bispecific agent is a heterodimeric agent or a heterodimeric molecule. In some embodiments, said bispecific agent is a homodimeric agent or a homodimeric molecule. In some embodiments, the heterodimeric molecule contains a first arm that binds human TIGIT and a second arm that binds a second target. In some embodiments, the heterodimeric molecule comprises a first arm that specifically binds human TIGIT and a second arm, said first arm comprising the anti-TIGIT antibody described herein. In some embodiments, the heterodimeric molecule comprises a first arm that binds human TIGIT and a second arm that contains an antigen-binding site from an antibody that specifically binds a second target. In some embodiments, the heterodimeric molecule is a bispecific antibody. In some embodiments, the heterodimeric molecule contains a first arm that binds human TIGIT and a second arm that specifically binds a tumor antigen. In some embodiments, the heterodimeric molecule comprises a first arm that binds human TIGIT and a second arm that specifically binds PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, OX-40, 4-1BB, or GITR. In some embodiments, the heterodimeric molecule contains a first arm that binds TIGIT and a second arm that contains an immunotherapeutic agent. In some embodiments, said immunotherapeutic agent is selected from the group consisting of: granulocyte macrophage colony stimulating factor (GMCSF), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (GCSF), interleukin 2 (IL-2), interleukin 3 (IL- 3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (Cd86), ligand 4-1BB, GITRL, OX-40L, antibodies against Cd3, antibodies against CTLA-4, anti-PD-1, anti-PD-L1, anti-4-1BB, anti-GITR, anti-OX-40, anti-LAG-3, and anti-TIM-3.

In some embodiments, the heterodimeric molecule described herein comprises a first arm containing a first CH3 domain and a second arm containing a second CH3 domain, with each CH3 domain modified to promote the formation of heterodimers. In some embodiments, said CH3 domains are modified based on electrostatic effects. In some embodiments, said CH3 domains are modified in a ridge-to-trough pattern.

In some embodiments, the bispecific agent described herein comprises a first arm containing a first CH3 domain and a second arm containing a second CH3 domain, with each CH3 domain modified to promote the formation of heterodimers. In some embodiments, said CH3 domains are modified based on electrostatic effects. In some embodiments, said CH3 domains are modified in a ridge-to-trough pattern.

In some embodiments of each of the above aspects and embodiments, as well as other aspects and embodiments described herein, the agent described herein specifically binds TIGIT and inhibits the binding of TIGIT to the poliovirus receptor (PVR). In some embodiments, the agent specifically binds TIGIT and inhibits or blocks the interaction between TIGIT and PVR. According to some variations

- 5 039540 antam implementation agent specifically binds TIGIT and inhibits the binding of TIGIT to PVR-L2. In some embodiments, the agent specifically binds TIGIT and inhibits or blocks the interaction between TIGIT and PVR-L2. In some embodiments, the agent specifically binds TIGIT and inhibits TIGIT binding to PVR-L3. In some embodiments, the agent specifically binds TIGIT and inhibits or blocks the interaction between TIGIT and PVR-L3. In some embodiments, said agent is a TIGIT antagonist. In some embodiments, the agent specifically binds TIGIT and inhibits TIGIT signaling. In some embodiments, the agent specifically binds TIGIT and is a TIGIT-mediated signaling antagonist. In some embodiments, the agent specifically binds TIGIT and inhibits TIGIT activation. In some embodiments, the agent specifically binds TIGIT and inhibits TIGIT phosphorylation. In some embodiments, the agent specifically binds TIGIT and reduces the expression of TIGIT on the cell surface.

In some embodiments of each of the above aspects and embodiments, as well as other aspects and embodiments described herein, the agent described herein specifically binds TIGIT and induces an immune response, activates an immune response, stimulates an immune response, increases, enhances the immune response and/or prolongs the duration of the immune response. In some embodiments, said immune response is directed to a tumor or tumor cell. In some embodiments, said immune response is directed to a virus or a virus-infected cell. In some embodiments, said agent enhances cellular immunity. In some embodiments, said agent increases T cell activity. In some embodiments, said agent increases cytolytic T-cell (CTL) activity. In some embodiments, said agent increases natural killer (NK) cell activity. In some embodiments, said agent increases IL-2 production and/or the number of IL-2 producing cells. In some embodiments, said agent increases IFN-γ production and/or the number of IFN-γ producing cells. In some embodiments, said agent enhances a Th1-type immune response. In some embodiments, said agent reduces IL-4 production and/or the number of IL-4 producing cells. In some embodiments, said agent reduces IL-10 and/or the number of IL-10 producing cells. In some embodiments, said agent reduces IL-6 production and/or the number of IL-6 producing cells. In some embodiments, said agent reduces IL-5 production and/or the number of IL-5 producing cells. In some embodiments, said agent reduces a Th2-type immune response. In some embodiments, said agent reduces the number of Treg cells. In some embodiments, said agent reduces Treg activity. In some embodiments, said agent inhibits and/or reduces the suppressor activity of Treg cells. In some embodiments, said agent reduces the number of SCMP cells. In some embodiments, said agent inhibits and/or reduces the suppressor activity of myeloid-derived suppressor cells (MSC cells).

In some embodiments of each of the above aspects and embodiments, as well as other aspects and embodiments described herein, the agent described herein specifically binds TIGIT and inhibits tumor growth. In some embodiments, said agent reduces tumor growth. In some embodiments, said agent reduces tumor growth to an undetectable size. In some embodiments, said agent induces long-term antitumor immunity.

According to another aspect of the present invention proposed compositions containing the agent described in this document. Methods for using a composition containing an agent described herein are also provided.

According to another aspect of the present invention, pharmaceutical compositions are provided comprising an agent described herein and a pharmaceutically acceptable carrier. Also provided are methods for treating cancer and/or inhibiting tumor growth in a subject (eg, a human) comprising administering to said subject an effective amount of a composition containing an agent described herein. Also provided are methods for treating a viral infection in a subject (eg, a human) comprising administering to said subject an effective amount of a composition containing an agent described herein.

According to certain embodiments of each of the above aspects, as well as other aspects and/or implementation options described in various sections of this document, the specified agent is selected. In some embodiments, said agent is substantially pure.

According to another aspect of the present invention, polynucleotides are provided that contain a polynucleotide that encodes for an agent described herein. According to some variants

- 6 039540 there implementation of the specified polynucleotide is selected. In some embodiments, the present invention provides vectors that contain said polynucleotides, as well as cells that contain said vectors and/or said polynucleotides. In some embodiments, the present invention also provides cells containing or producing the agent described herein. In some embodiments, said cell is a monoclonal cell line.

According to another aspect of the present invention, methods are provided for modulating an immune response in a subject. In some embodiments, said method for modulating an immune response comprises a method for inducing an immune response, activating an immune response, stimulating an immune response, increasing, enhancing, or prolonging an immune response in a subject. In some embodiments, a method for inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing, or prolonging an immune response in a subject comprises administering a therapeutically effective amount of an antibody, bispecific agent, or polypeptide described herein. In some embodiments, a method for inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing or prolonging an immune response in a subject comprises administering a therapeutically effective amount of a heterodimeric bispecific agent or a homodimeric bispecific agent as described herein. In some embodiments, a method of inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing or prolonging an immune response in a subject comprises administering a therapeutically effective amount of an antibody specifically binding TIGIT described herein. In some embodiments, a method for inducing an immune response in a subject comprises administering an agent described herein. In some embodiments, a method for activating an immune response in a subject comprises administering an agent described herein. In some embodiments, a method for stimulating an immune response in a subject comprises administering an agent described herein. In some embodiments, a method for enhancing an immune response in a subject comprises administering an agent described herein. In some embodiments, a method for enhancing an immune response in a subject comprises administering an agent described herein. In some embodiments, a method for increasing the duration of an immune response in a subject comprises administering an agent described herein. In some embodiments, said immune response is caused by antigenic stimulation. In some embodiments, said antigenic challenge is a tumor or tumor cell. In some embodiments, said antigenic challenge is a pathogen. In some embodiments, said antigenic challenge is a virus. In some embodiments, said antigenic challenge is a virus-infected cell. In some embodiments, said immune response is directed against a tumor or cancer.

In some embodiments, the present invention provides methods for increasing the activity of immune cells. In some embodiments, a method for increasing the activity of immune cells comprises contacting said cells with an effective amount of an agent described herein. In some embodiments, said immune cells are T cells, NK cells, monocytes, macrophages, cells of myeloid origin, antigen presenting cells (APC cells), and/or B cells. In some embodiments, a method for increasing NK cell activity in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for increasing T cell activity in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for increasing T cell and/or NK cell activation in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for enhancing a T cell response in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for increasing CTL cell activity in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for inhibiting Treg cell activity in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for inhibiting the suppressor activity of Treg cells in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for inhibiting SPCM cell activity in a subject comprises administering to said subject a therapeutically effective amount of an agent described herein. According to some options

- 7 039540 implementing a method for inhibiting the suppressor activity of SKMP cells in a subject includes administering to said subject a therapeutically effective amount of an agent described herein.

In some embodiments, the present invention provides methods for inducing an immune response, activating an immune response, stimulating an immune response, increasing, enhancing or prolonging an immune response in a subject, comprising administering to said subject a therapeutically effective amount of an agent that binds human TIGIT. In some embodiments, a method of inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing or prolonging an immune response in a subject comprises administering to said subject a therapeutically effective amount of an agent that inhibits or reduces TIGIT activity. In some embodiments, a method of inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing or prolonging an immune response in a subject comprises administering to said subject a therapeutically effective amount of an agent that inhibits or reduces TIGIT signaling. In some embodiments, said immune response is directed against a tumor cell, tumor, or cancer. In some embodiments, said immune response is directed against a viral infection, a viral antigen, or a virus-infected cell.

According to another aspect of the present invention, methods are provided for inhibiting the growth of tumor cells or a tumor, comprising contacting said tumor or tumor cell with an effective amount of an agent described herein. In some embodiments, a method for inhibiting tumor growth comprises contacting the tumor or tumor cell with an effective amount of an agent that binds human TIGIT.

According to another aspect of the present invention, methods are provided for inhibiting tumor growth in a subject, comprising administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method for inhibiting tumor growth in a subject comprises administering to said subject a therapeutically effective amount of an agent that binds human TIGIT. In some embodiments, a method for inhibiting tumor growth in a subject comprises administering to said subject a therapeutically effective amount of an antibody that binds human TIGIT. In some embodiments, a method for inhibiting tumor growth in a subject comprises administering to said subject a therapeutically effective amount of a bispecific agent that binds human TIGIT. In some embodiments, said tumor is selected from the group consisting of colon and rectal tumor, colon tumor, ovarian tumor, pancreatic tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor. tract, melanomas, cervical tumors, bladder tumors, glioblastomas and tumors of the head and neck.

According to another aspect of the invention, methods are provided for treating cancer in a subject, comprising administering to said subject a therapeutically effective amount of an agent described herein. In some embodiments, a method of treating cancer in a subject comprises administering to said subject a therapeutically effective amount of an agent that binds TIGIT. In some embodiments, a method of treating cancer in a subject comprises administering to said subject a therapeutically effective amount of an antibody that binds human TIGIT. In some embodiments, a method of treating cancer in a subject comprises administering to said subject a therapeutically effective amount of a bispecific agent that binds human TIGIT. In some embodiments, said cancer is selected from the group consisting of colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer. uterus, bladder cancer, glioblastoma, and head and neck cancer.

According to another aspect of the present invention, methods are provided for stimulating and/or inducing long-term antitumor immunity in a subject, comprising administering to said subject a therapeutically effective amount of an agent described herein.

According to another aspect of the invention, methods of stimulating a protective response in a subject are provided, comprising administering to said subject a therapeutically effective amount of an agent described herein in combination with an antigen of interest. In some embodiments, said antigen of interest is a tumor antigen. In some embodiments, said antigen of interest is a tumor cell biomarker. In some embodiments, said antigen of interest is a tumor stem cell marker.

According to some implementation options of each of the above aspects and implementation options, as well as other aspects and implementation options described in this document, the specified

- 8 039540 methods include administering to said subject an immune response stimulating agent. In some embodiments, said immune response stimulating agent is selected from the group consisting of GM-CSF, MCSF, GCSF, IL-3, IL-12, IL-1, IL-2, B7-1 (CD80), B7-2 ( CD86), anti-CD3, anti-CTLA-4, anti-CD28, anti-PD-L1, and anti-PD1.

In some embodiments of each of the above aspects and embodiments, as well as other aspects and embodiments described herein, the method further comprises administering at least one additional therapeutic agent. In some embodiments, said additional therapeutic agent is a chemotherapeutic agent. In some embodiments, said additional therapeutic agent is an antibody. In some embodiments, said additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, or an anti-TIM-3 antibody. In some embodiments, said additional therapeutic agent is an inhibitor of the Notch pathway, the Wnt pathway, or the RSPO/LGR pathway.

In some embodiments, said additional therapeutic agent is an immunotherapeutic agent. As used herein, the term immunotherapeutic agent is used in the broadest possible sense and refers to a substance that directly or indirectly affects or modulates the immune system. In some embodiments, an immunotherapeutic agent is an agent that directly or indirectly stimulates the immune system by inducing activation or upregulation of any components of the immune system. Since TIGIT binding agents are considered immunotherapeutic agents, said additional immunotherapeutic agent may be considered a second immunotherapeutic agent. In some embodiments, said second immunotherapeutic agent is selected from the group consisting of: GM-CSF, MCSF, GCSF, IL-2, IL-3, IL-12, IL-15, B7-1 (CD80), B7-2 (CD86) , 4-1BB ligand, GITRL, OX-40 ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-CD28 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-4-1BB antibody, anti-GITR antibody, antibody against OX-40, antibodies against LAG-3 and antibodies against TIM-3. In some embodiments, said second immunotherapeutic agent is a fusion protein containing: GM-CSF, MCSF, GCSF, IL-2, IL-3, IL-12, IL-15, B7-1 (CD80), B7-2 (CD86) , 4-1BB ligand, GITRL, OX-40 ligand, or fragment thereof. In some embodiments, said second immunotherapeutic agent is a fusion protein containing at least one copy of the extracellular domain of GITRL, an OX40 ligand, or a 4-1BB ligand.

In some embodiments of each of the above aspects and embodiments, as well as other aspects and embodiments described herein, said subject is a human. In some embodiments, the subject has at least partially removed a tumor or cancer.

In some embodiments of each of the above aspects and embodiments, as well as other aspects and embodiments described herein, said tumor or said cancer expresses PD-L1. In some embodiments, the method further comprises the step of determining the expression level of PD-L1 in said tumor or cancer. In some embodiments, determination of the level of PD-L1 expression is performed prior to treatment or contact with an agent described herein. In some embodiments, if said tumor or cancer is overexpressed by PD-L1, the agent described herein is administered to said subject. In some embodiments, if said tumor or cancer is overexpressed by PD-L1, said tumor or cancer is contacted with an agent described herein.

In the event that aspects or embodiments of the present invention are described in terms of the Markush group or other group of alternatives, the present invention covers not only the entire specified group as a whole, but also each representative of the specified group individually, and all possible subgroups of the main group, as well as a specified major group that lacks one or more members of the group. The present invention may also provide for the express exclusion of one or more members of the group in the claimed invention.

Brief description of the drawings

Fig. 1A and 1B - FACS analysis of rabbit antibodies produced in response to mouse TIGIT. (A) HEK-293T cells were transiently transfected with an expression cDNA vector encoding mouse TIGIT ECD-CD4TM-GFP (green fluorescent protein) or human ECD TIGIT-CD4TM-GFP. Transfected cells were incubated with rabbit antibodies and analyzed by flow cytometry. Specific binding is indicated by the presence of a diagonal signal on each FACS plot. (B) HEK-293T cells were transiently transfected with an expression cDNA vector encoding mouse TIGIT-CD4TM-GFP. Transfected cells were incubated with soluble mouse PVR/Fc fusion protein with or without rabbit antibodies produced in response to mouse TIGIT.

- 9 039540 antibodies, and analyzed by flow cytometry. Specific binding is indicated by the presence of a diagonal signal on each FACS plot. Binding blockage is shown as the loss of specific binding and is circled on the FACS plot;

fig. 2A and 2B Western blot analysis of protein phosphorylation after TIGIT-PVR interaction. Human Jurkat T cells were stably transduced with FLAG-labeled mouse TIGIT/GFP and E.G7-OVA cells were stably transduced with mouse PVR/GFP. (A) Phosphorylation of TIGIT. (B) Phosphorylation of SHP1 and Erk1/2;

fig. 3 Western blot analysis of TIGIT phosphorylation after TIGITPVR interaction in the absence or presence of anti-TIGIT antibodies;

fig. 4 Western blot analysis of SHP1 and Erk1/2 phosphorylation after TIGIT-PVR interaction in the absence or presence of anti-TIGIT antibodies;

fig. 5A and 5B - TIGIT-mediated inhibition of cytokine production. (A) IL2 secretion in B3Z T cells and B3Z T cells expressing TIGIT. (B) Secretion of IL-2 in B3Z T cells and B3Z T cells expressing TIGIT after pretreatment with anti-TIGIT antibodies;

fig. 6A and 6B - TIGIT-mediated inhibition of natural killer cell activity. (A) Cytotoxicity of original NK-92 cells and NK-92 cells expressing TIGIT. (B) Cytotoxicity of native NK-92 cells and TIGIT-expressing NK-92 cells after pretreatment with anti-TIGIT antibodies;

fig. 7A and 7B show inhibition of tumor growth by anti-TIGIT antibodies. CT26.WT colon tumor cells were implanted subcutaneously in Balb/c mice (n=10 mice/group). Mice were injected on days 10, 15, 18, 22, 25 and 29 with 0.25 mg/mouse of anti-TIGIT 313R11 antibody, anti-TIGIT 313R12 antibody, control mouse IgG1 antibody, and control mouse IgG2 antibody. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points. Data are given as tumor volumes (mm ³ ) for days after injection. (A) Figures show mean±SEM values for each group. (B) Additional study with anti-TIGIT 313R12 antibody and control antibody;

fig. 8 - inhibition of tumor growth by anti-TIGIT antibodies. CT26.WT colon tumor cells were implanted subcutaneously in Balb/c mice (n=10 mice/group). Mice were injected on days 10, 14, 17 and 21 with 0.25 mg/mouse of anti-TIGIT 313R12 antibody, anti-TIGIT 313R13 antibody, and control mouse IgG2 antibody. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points. Data are given as tumor volumes (mm ³ ) for days after injection. The figures show mean ± SEM values for each group;

fig. 9A-9D - ELISpot assays for IFN-γ, IL-2, IL-4 and IL-10. Spleen cells were harvested from mice bearing CT26.WT tumors treated with anti-TIGIT 313R12 antibody or an isotype matched control antibody. Cells were incubated in the presence or absence of AH-1 peptide and then analyzed using ELISpot kits. (A) The total optical density (TOD) of cells producing IFN-γ is shown. (B) TOD of cells producing IL-2. (C) The TOD of cells producing IL-4 is shown. (B) TOD of cells producing IL-10;

fig. 10 - activity of natural killer cells in a population of splenocytes of mice treated with anti-TIGIT antibody;

fig. 11 - Treg activity in a splenocyte population of mice treated with anti-TIGIT antibody;

fig. 12A-12E are FACS analysis of splenocytes from mice treated with anti-TIGIT antibody. (A) Percentage of CD3+ cells in total living cells. (B) Percentage of CD4+ cells in total living cells. (C) Percentage of CD8+ cells of total living cells. (D) Percentage of central memory CD4+ T cells in the total CD4+ population. (E) Percentage of CD8+ central memory T cells of the total CD8+ population;

fig. 13A-13D are FACS analysis of TIGIT positive or negative Treg cells in a splenocyte population of mice treated with an anti-TIGIT antibody. (A) Percentage of TIGIT-positive cells out of total living cells. (B) Percentage of FoxP3+ cells of total CD4+ cells (Treg identification). (C) Percentage of TIGIT positive cells out of total Treg cells. (D) Percentage of TIGIT-negative cells of total Treg cells;

fig. 14A-14D are FACS analysis of SCMP cells in a splenocyte population of mice treated with an anti-TIGIT antibody. (A) Percentage of CD11b+ cells out of total living cells. (B) Percentage of SCMP cells of the total number of CD11b+ cells. (C) Percentage of HSCMP cells of the total number of CD11b+ cells. (D) Percentage of MSCMP cells of total CD11b+ cells;

fig. 15 - inhibition of tumor growth by anti-TIGIT antibodies. Renca cells (murine renal adenocarcinoma) were implanted subcutaneously in Balb/c mice (n=10 mice/group). Mice were injected on days 7, 10, 14, 17, 21 and 24 with 0.25 mg/mouse of anti-TIGIT 313R12 antibody and control mouse IgG2 antibody. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at specified time points. Data are given as tumor volumes (mm ³ ) for days after injection. The figures show mean ± SEM values for each group;

fig. 16 Tumor Growth Inhibition with Anti-TIGIT 313R12 Dose Study. CT26.WT mouse colon tumor cells were implanted subcutaneously in Balb/c mice (n=10 mice/group). Mice were treated with 0.5, 1, 3, 5, 10, 15, or 30 mg/kg of anti-TIGIT 313R12 antibody or control antibody. Mice were dosed by intraperitoneal injection twice a week for a total of 6 doses. Tumor growth was monitored and tumor volumes were measured with an electronic caliper;

fig. 17A-17F - In vivo tumor growth inhibition by anti-TIGIT 313R12 antibody and anti-PD-L1 antibody. CT26.WT mouse colon tumor cells were implanted subcutaneously (30,000 cells/mouse) in Balb/c mice. Mice were treated with 0.25 mg/mouse of anti-TIGIT 313R12 antibody, anti-PD-L1 antibody, combination of 313R12 and anti-PD-L1 antibody, or control antibody (n=10 per group). Mice were injected with antibodies twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points. (A) Tumor volumes in individual mice in the control antibody treated group. (B) Tumor volumes in individual mice in the anti-TIGIT 313R12 treated group. (C) Tumor volumes in individual mice in the anti-PDL1 antibody treatment group. (D) Tumor volumes in individual mice in the anti-TIGIT 313R12 and anti-PD-L1 treated group. (E) Mean rate of tumor growth for the four experimental groups. (F) Survival curve;

fig. 18A-18E - In vivo inhibition of tumor growth by anti-TIGIT 313R12 antibody and anti-PD-1 antibody. CT26.WT mouse colon tumor cells were implanted subcutaneously (30,000 cells/mouse) in Balb/c mice. Mice were treated with 0.25 mg/mouse of anti-TIGIT 313R12 antibody, anti-PD-1 antibody, combination of 313R12 and anti-PD-1 antibody, or control antibody (n=15 per group). Mice were injected with antibodies twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points. (A) Tumor volumes in individual mice in the control antibody treated group. (B) Tumor volumes in individual mice in the anti-TIGIT 313R12 treated group. (C) Tumor volumes in individual mice in the anti-PD1 antibody treated group. (D) Tumor volumes in individual mice in the anti-TIGIT 313R12 and anti-PD-1 treated group. (E) Mean rate of tumor growth for the four experimental groups;

fig. 19A and 19B FACS analysis of blocking by anti-TIGIT antibody of human TIGIT binding to PVR. (A) HEK-293T cells were transiently transfected with an expression cDNA vector encoding human CD4TM-GFP TIGIT ECD or human CD4TM-GFP TIGIT ECD. Transfected cells were incubated with soluble human PVR/Fc fusion protein in the presence of antibodies produced in response to TIGIT (313R19, 313M26, or 313M32) at concentrations of 10, 2, or 0.4 μg/mL, or without antibody, and analyzed with flow cytometry. Specific binding is indicated by the presence of a diagonal signal on the FACS plot. Binding blockage is shown as the loss of specific binding in the dark box on the FACS plot. (B) HEK-293T cells were transiently transfected with an expression cDNA vector encoding human TIGIT ECD-CD4TM-GFP (green fluorescent protein) or human TIGIT-CD4TM-GFP ECD. Transfected cells were incubated with soluble PVR human/rabbit Fc fusion protein in the presence of antibodies produced in response to TIGIT (313R19 or 313M32) at concentrations of 10, 5, 2.5, or 1.25 μg/mL, or without antibody, and analyzed by flow cytometry. Specific binding is indicated by the presence of a diagonal signal on the FACS plot. Binding blockage is shown as the loss of specific binding in the dark box on the FACS plot;

fig. 20 Western blot analysis of TIGIT phosphorylation after TIGIT-PVR interaction in the absence or presence of anti-TIGIT antibodies 313R19, 313M26 and 313M32;

fig. 21 - sequence alignment of human TIGIT (SEQ ID NO: 4), cynomolgus monkey TIGIT (SEQ ID NO: 77) and rhesus monkey TIGIT (SEQ ID NO: 78);

fig. 22 Epitope mapping by FACS analysis of anti-TIGIT antibody binding to human TIGIT protein variants containing specific amino acid substitutions. hTIGIT variants are represented by (1) E36K and I41V; (2) T51M, (3) Q62H and Q64H, (4) D72E, (5) S78Y and S80A, (6) V100M, (7) I109T and T119R, and (8) G135S;

fig. 23 is a diagram showing PVR associated with TIGIT. The human TIGIT structure is represented as a sphere and the PVR structure as a ribbon. Amino acids containing at least a portion of the 313M32 epitope are depicted as black spheres. Amino acids containing at least a portion of the 313M34 epitope are depicted as gray spheres;

fig. 24A and 24B show competitive binding of anti-TIGIT antibodies. (A) Competition study for 313R19 and 313M26. (C) Competitive Investigation of 313M34 and 313R19 or 313M34 and

- 11 039540

313M32;

fig. 25 Reversal of PVR-mediated inhibition of cytokine secretion by anti-TIGIT antibodies;

fig. 26 - analysis of antibody-dependent cellular cytotoxicity;

fig. 27 - Inhibition of tumor growth by anti-TIGIT antibodies 313R19 and 313M32 in a humanized mouse model. Humanized mice were injected subcutaneously with patient-derived melanoma tumor cells (OMP-M9, 75,000 cells/mouse). The tumors were allowed to grow for 19 days until an average volume of approximately 50 mm ³ was reached. Tumor-bearing mice were randomized into groups (n=8 mice per group). Tumor-bearing mice were treated with either a control antibody, an anti-TIGIT 313R19 antibody, or an anti-TIGIT 313M32 antibody. Mice were dosed at 1 or 5 mg/kg every 5 days. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points.

Detailed description of the invention

The invention provides novel agents, including but not limited to polypeptides, antibodies, and heterodimeric molecules that modulate an immune response. These agents include polypeptides, antibodies and heterodimeric molecules that specifically bind TIGIT and agents that modulate TIGIT activation and/or signaling. These agents include polypeptides, antibodies, and heterodimeric molecules that inhibit TIGIT activation and/or signaling, thus enhancing the immune response. Also proposed are related polypeptides and polynucleotides, compositions containing these agents, and methods for obtaining these agents. Screening methods are provided to identify agents that modulate the immune response. Methods of using said novel agents are provided, for example, methods for inhibiting tumor growth and/or methods for treating cancer. Methods for treating viral infections are also provided. Additionally, methods of using said novel agents are provided, for example, methods of activating an immune response, methods of stimulating an immune response, methods of stimulating an immune response, methods of enhancing an immune response, methods of activating natural killer (NK) and/or T cells, methods of increasing the activity of NK cells and/or T cells, methods for stimulating NK and/or T cell activity, methods for reducing and/or inhibiting suppressor T cells, and/or methods for reducing and/or inhibiting suppressor cells of myeloid origin. In addition, the novel agents described herein may be used in methods for inhibiting an immune response, methods for suppressing an immune response, methods for reducing T cell activity, and/or methods for treating autoimmune diseases.

I. Definitions.

To facilitate understanding of the present invention, the following are definitions of certain terms and expressions.

The terms agonist and agonist as used herein refer to or describe an agent capable of directly or indirectly substantially inducing a biological activity, activating a biological activity, stimulating a biological activity, increasing or enhancing the biological activity of a target and/or pathway. The term agonist as used herein includes any agent that partially or completely induces the activity of a protein, activates a protein, stimulates the activity of a protein, increases or enhances the activity of a protein.

The terms antagonist and antagonist as used herein refer to or describe an agent capable of directly or indirectly, partially or completely blocking, inhibiting, reducing or neutralizing the biological activity of a target and/or pathway. The term antagonist herein includes any agent that partially or completely blocks, inhibits, reduces or neutralizes the activity of a protein.

The terms modulation and modulate as used herein refer to a change or change in biological activity. Modulation includes stimulation of activity or inhibition of activity, but is not limited to them. Modulation may be an increase or decrease in activity, a change in binding characteristics, or any other change in biological, functional, or immunological properties associated with the activity of a protein, pathway, system, or other biological target of interest.

The term antibody as used herein refers to an immunoglobulin molecule that recognizes and specifically binds a target through at least one antigen-binding site. Said target may be a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the above. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab') ₂ and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies. , monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antibody antigen-binding site, and any other modified immunoglobulin molecule containing an antigen-binding site, provided that said antibodies exhibit the desired biological asset

- 12 039540 An antibody can belong to any of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on their identity to their constant heavy chain domains, called α, δ, ε, γ and μ, respectively. Different classes of immunoglobulins contain different well-known subunit structures and three-dimensional configurations. Antibodies can be free or conjugated to other molecules, including but not limited to toxins and radioisotopes.

The term antibody fragment refers to a portion of an intact antibody and, in general, to the antigen-determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab-, Fab'-, F(ab') ₂ - and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed by antibody fragments. An antibody fragment herein includes an antigen binding site or an epitope binding site.

The term antibody variable region refers to an antibody light chain variable region or an antibody heavy chain variable region, alone or in combination. In general, a heavy chain or light chain variable region consists of four framework regions connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in close proximity to each other by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site(s) of the antibody. There are at least two methods for determining CDR regions: (1) an approach based on interspecies sequence variability (i.e. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda MD. ), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition, combinations of the two approaches are sometimes used in the art to define CDRs.

The term monoclonal antibody as used herein refers to a homogeneous population of antibodies involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include a mixture of different antibodies that recognize different antigenic determinants. The term monoclonal antibody encompasses both intact and full-length monoclonal antibodies and antibody fragments (e.g. Fab-, Fab'-, F(ab') ₂ -, Fv-), single-chain (scFv) antibodies, fusion proteins containing an antibody fragment, and any other modified immunoglobulin molecule containing an antigen binding site. In addition, a monoclonal antibody refers to such antibodies obtained using any number of techniques, including, but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.

The term humanized antibody as used herein refers to antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which amino acid residues from CDR regions are replaced with amino acid residues from CDR regions of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding potential. In some instances, the amino acid residues of the framework variable region of a human immunoglobulin may be substituted for the corresponding amino acid residues in the antibody of the non-human species. Said humanized antibody may be further modified by substituting additional amino acid residues within the framework variable region and/or within the substituted non-human amino acid residues to further refine and optimize the specificity, affinity and/or binding potential of the antibody. Said humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to a non-human immunoglobulin, while all or substantially all of the framework variable regions are represented by human immunoglobulin sequence regions. In some embodiments, said variable domains comprise framework regions of a human immunoglobulin sequence. In some embodiments, said variable domains comprise human immunoglobulin consensus sequence framework regions. Said humanized antibody may also comprise at least a portion of an immunoglobulin (Fc) constant region or domain, typically a human immunoglobulin.

The term human antibody as used herein refers to a human-produced antibody or an antibody having an amino acid sequence corresponding to a human-produced antibody produced using any of the techniques known in the art.

The term chimeric antibody as used herein refers to an antibody characterized in that the amino acid sequence of an immunoglobulin molecule is from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody derived from a single mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding potential, while the homologous constant regions - 13 039540 sequences of an antibody originating from another species. The constant regions are usually human regions to avoid the occurrence of an immune response to the antibody.

The terms epitope and antigenic determinant are used interchangeably herein and refer to the part of an antigen or target that can be recognized and, in particular, binds a particular antibody. If the antigen or target is a polypeptide, epitopes can be formed by either a continuous or non-contiguous sequence of amino acids, the convergence of which is provided by the tertiary folding of the protein. Epitopes formed by a contiguous sequence of amino acids (also called linear epitopes) are generally retained when proteins are denatured, while epitopes formed by tertiary folding (also called conformational epitopes) are usually lost when proteins are denatured. An epitope typically comprises at least 3, more often at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The terms selectively bind or specifically bind means that an agent interacts with an epitope, protein, or target molecule more frequently, faster, longer, with greater affinity, or with some combination of these features, than with alternative substances, including related and unrelated proteins. In certain embodiments, specifically binds means, for example, that the agent binds a protein or target with a KD of about 0.1 mM or less, but more often less than about 1 μM. In some embodiments, binds specifically means that the agent binds the target to a KD of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. Due to the sequence identity of homologous proteins from different species, specific binding may involve an agent that recognizes a protein or target from more than one species (eg, mouse TIGIT and human TIGIT). Similarly, due to the presence of homology in certain regions of the polypeptide sequences of different proteins, specific binding may involve an agent that recognizes more than one protein or target. It should be understood that, in some embodiments, an agent that specifically binds a first target may or may not specifically bind a second target. Accordingly, specific binding does not necessarily imply (although it may include) exclusive binding, ie. binding to a single target. Accordingly, an agent may, in some embodiments, specifically bind to more than one target. In some embodiments, multiple targets may be present that bind to the same antigen binding site on said agent. For example, an antibody in some cases may contain two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In some other alternative embodiments, the antibody may be bispecific and contain at least two antigen-binding sites with different specificities. Usually, but not necessarily, when referring to binding, specific binding is meant.

The terms polypeptide, peptide, and protein are used interchangeably herein and refer to polymers of amino acids of any length. Said polymer may be linear or branched, may contain modified amino acids, and may contain non-amino acid interrupting amino acid sequence components. These terms also cover a polymer of amino acids that has been modified naturally or through intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, naturally occurring amino acids), as well as other modifications known in the art. It should be understood that since the polypeptides of the present invention may be based on antibodies or other members of the immunoglobulin superfamily, in some embodiments the polypeptide may be produced in single chain form or as two or more associated chains.

The terms polynucleotide and nucleic acid and nucleic acid molecule are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. Said nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase.

The terms identical or percent identity, in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or contain a given percentage of the same nucleotides or amino acid residues when compared or aligned (with gaps if necessary) with maximum match, without regard to any conservative amino acid substitutions that are included in the definition of sequence identity. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to perform amino acid or nucleotide sequence alignment are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package and variations thereof. In some embodiments, two nucleic acids or two polypeptides of the present invention are substantially identical, meaning that they differ by at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned with maximum match, as assessed using a sequence comparison algorithm or by visual examination. In some embodiments, identity is observed over a region of sequences that is at least about 10, at least about 20, at least about 4060 nucleotides or amino acid residues, at least about 60-80 nucleotides or amino acid residues, or any integer value between the specified values. In some embodiments, identity is observed over a region that is greater than 60-80 nucleotides or amino acid residues, such as at least about 80-100 nucleotides or amino acid residues, and in some embodiments, said sequences are substantially identical throughout the compared sequences, for example, the coding region of a nucleotide sequence.

A conservative amino acid substitution is one in which one amino acid residue replaces another amino acid residue with a similar side chain. The art has generally characterized families of amino acid residues with similar side chains, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains ( e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). For example, a phenylalanine substitution for tyrosine is considered a conservative substitution. Typically, conservative substitutions in the sequences of the polypeptides and/or antibodies of the present invention do not eliminate the binding of the polypeptide or antibody containing the indicated amino acid sequence to the target binding site. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate binding are well known in the art.

The term vector as used herein means a construct that is capable of delivering and generally expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, deproteinized DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and encapsulated DNA or RNA expression vectors. into liposomes.

A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is isolated is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature . Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, the polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition that is isolated is substantially pure.

The term substantially pure as used herein refers to at least 50% pure (i.e. free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure. or at least 99% pure material.

The term immune response as used herein includes response from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune response. It includes, but is not limited to, both T-cell and B-cell responses, as well as responses mediated by other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, and the like.

The terms cancer and cancerous as used herein refer to a physiological condition or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and blood cancers such as lymphoma and leukemia.

The terms tumor and neoplasm as used herein refer to any tissue mass that is the result of cell overgrowth or proliferation, either benign (noncancerous) or malignant (cancerous), including precancerous lesions.

The term metastasis, as used herein, refers to the process by which a cancer spreads or is transferred from its site of origin to other areas of the body to develop a similar cancerous lesion in a new location. Typically, a metastatic or metastasizing cell refers to a cell that loses adhesive contacts with neighboring cells and migrates through the bloodstream or lymph from the primary site of the disease to secondary sites throughout the body.

The terms cancer stem cell, CSC, tumor stem cell, and tumor originating cell are used interchangeably herein and refer to cancer or tumor cells that: (1) have a high proliferative capacity; (2) capable of asymmetric cell division to form differentiated progeny cells of one or more types, said differentiated cells having reduced proliferative or differentiation potential; and (3) capable of symmetrical cell division for self-renewal or self-sustainment. These properties confer on cancer stem cells the ability to form or form a tumor or cancer when transplanted sequentially into a suitable host (eg mouse), unlike most tumor cells which are unable to form tumors. Self-renewal or differentiation of cancer stem cells occurs in a chaotic manner, with the formation of tumors containing abnormal cell types that can change over time as mutations occur.

The terms cancer cell and tumor cell refer to the entire population of cells derived from a cancer or tumor or precancerous lesion, including both nontumorogenic cells, which form the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells). In this document, the terms cancer cell or tumor cell are modified by the term non-tumorogenic solely to describe cells that are unable to renew and differentiate to distinguish said tumor cells from cancer stem cells.

The term tumorigenic in this document refers to the functional features of a cancer stem cell, including the properties of self-renewal (resulting in additional tumorigenic cancer stem cells) and proliferation to form all other tumor cells (resulting in differentiated and therefore non-tumorogenic tumor cells). cells).

The term tumorigenicity as used herein refers to the ability of a random sample of tumor cells to form palpable tumors when sequentially transplanted into suitable hosts (eg, mice).

The term subject refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, canines, felines, rabbits, rodents, and the like, that serves as the recipient of a particular treatment. Generally, the terms subject and patient are used interchangeably herein to refer to a human subject.

The term pharmaceutically acceptable refers to a substance that is or may be approved by a regulatory agency of the federal government or the state government, or listed in the USP or other generally accepted pharmacopoeias, as being suitable for use in animals, including humans.

The terms pharmaceutically acceptable excipient, carrier or adjuvant or acceptable pharmaceutical carrier refers to an excipient, carrier or adjuvant that can be administered to a subject in conjunction with at least one agent according to the present description, which does not abolish the pharmacological activity of the latter and is non-toxic when administered in doses, sufficient to provide a therapeutic effect. In general, according to the judgment of those skilled in the art and the US Food and Drug Administration (FDA), a pharmaceutically acceptable excipient, carrier, or adjuvant is the inactive ingredient of any formulation.

The terms effective amount or therapeutically effective amount or therapeutic effect refers to the amount of an agent, antibody, polypeptide, polynucleotide, small organic molecule, or other drug described herein that is effective in treating a disease or disorder in a subject, e.g., a mammal. In the case of a cancer or tumor, a therapeutically effective amount of an agent (e.g., a polypeptide or antibody) has a therapeutic effect, and thus may enhance or further stimulate an immune response, enhance or further stimulate an antitumor response, increase the cytolytic activity of immune cells, increase the killing of tumor cells , increase the killing of tumor cells by immune cells, reduce the number of tumor cells; reduce tumorigenicity, incidence of tumorigenicity, or tumorigenic potential; reduce the number or frequency of cancer stem cells; reduce the size of the tumor; reduce the cancer population

- 16 039540 cells; inhibit or stop the infiltration of cancer cells into peripheral organs, including, for example, the spread of cancer into soft tissues and bones; inhibit and stop the metastasis of a tumor or cancer cells; inhibit and stop the growth of a tumor or cancer cells; alleviate to some extent one or more of the symptoms associated with cancer; reduce morbidity and mortality; improve the quality of life; or provide a combination of such effects.

The terms treatment, or treatment, or treat; or alleviate or mitigate refers to both (1) therapeutic measures that provide for the cure, slowing, amelioration of symptoms, and/or arrest of the progression of a diagnosed pathological condition or disorder, and (2) prophylactic or preventive measures that prevent or slow the development of the target pathological condition or disorder. Accordingly, those in need of treatment include those who already have said disorder; subject to said disorder; and those in whom the disorder is to be prevented. In the case of cancer or tumor, treatment of a subject according to the methods of the present invention is successful if said patient exhibits one or more of the following: increased immune response, increased anti-tumor response, increased cytolytic activity of immune cells, increased killing tumor cells, increased killing of tumor cells by immune cells, a decrease in the number or complete absence of cancer cells; reduction in tumor size; inhibition or absence of infiltration by cancer cells of peripheral organs, including the spread of cancer cells into soft tissues and bones; inhibition or absence of tumor or cancer cell metastasis; inhibition or absence of cancer growth; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improving the quality of life; reduction of tumorigenicity; a decrease in the number or frequency of occurrence of cancer stem cells; or some combination of effects.

In the present description and claims, terms in the singular, including in combination with the definition of the specified(th, th), include the corresponding terms in the plural, unless otherwise clearly follows from the context.

It should be understood that herein, descriptions of embodiments using the term comprising also include otherwise similar implementations described using the terms consisting of and/or consisting essentially of. It should also be understood that, throughout this document, descriptions of embodiments using the term consisting essentially of also include otherwise similar embodiments described using the term consisting of.

As used herein, the term approximately or approximately in relation to a value or measure includes (and describes) embodiments directed to the specified value or specified measure. For example, a description referring to approximately X includes a description of X.

The term and/or in terms such as A and/or B herein includes both A and B; A or B; A (separately); and B (separately). Similarly, the term and/or in terms such as A, B and/or C encompasses each of the following implementations: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separately); and C (separately).

II. TIGIT binding agents.

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is a type I transmembrane glycoprotein that contains the immunoglobulin variable domain (IgV). TIGIT belongs to the poliovirus receptor (PVR) family and binds to the poliovirus receptor (PVR; CD155) with high affinity, and to PVRL-2 (CD112) and PVRL-3 (CD113) with lower affinity. TIGIT is expressed on T cells, including regulatory and memory T cells, as well as on NK cells, and is stimulated upon activation of naive CD4+ T cells. The full length amino acid (AA) sequences of mouse TIGIT (UniProtKB No. P86176) and human TIGIT (UniProtKB No. Q495A1) are known in the art and are provided herein as SEQ ID NO: 1 and SEQ ID NO: 4, respectively. In this document, when referring to amino acid positions, the numbering used corresponds to the full-length amino acid sequences, including the signal sequence.

The present invention provides agents that specifically bind TIGIT. These agents are referred to herein as TIGIT binding agents. In some embodiments, said TIGIT binding agent is an antibody. In some embodiments, said TIGIT binding agent is a polypeptide. In some embodiments, said TIGIT binding agent binds the mouse TIGIT. In some embodiments, said TIGIT binding agent binds human TIGIT. In some embodiments, said TIGIT binding agent binds mouse TIGIT and human TIGIT. In some embodiments, said TIGIT binding agent binds human TIGIT and does not bind mouse TIGIT.

In some embodiments, the agent binds TIGIT and interferes with the interaction of TIGIT with a second protein. In some embodiments, the agent binds the TIGIT and interferes with the interaction of the TIGIT with the PVR. In some embodiments, the agent binds TIGIT and interferes with the interaction of TIGIT with PVRL-2. In some embodiments, the agent binds TIGIT and interferes with the interaction of TIGIT with PVRL-3. In some embodiments, the agent specifically binds TIGIT and said agent disrupts TIGIT binding to PVR and/or disrupts PVR activation of TIGIT signaling.

In some embodiments, said TIGIT binding agent is an antibody that specifically binds the extracellular domain of TIGIT, or a fragment thereof. In some embodiments, said TIGIT binding agent is an antibody that specifically binds the extracellular domain of mouse TIGIT, or a fragment thereof. In some embodiments, said TIGIT binding agent is an antibody that specifically binds to the extracellular domain of human TIGIT, or a fragment thereof. In some embodiments, said TIGIT binding agent is an antibody that specifically binds the extracellular domain of mouse TIGIT and human TIGIT, or a fragment thereof. In some embodiments, said TIGIT binding agent is an antibody that specifically binds an Ig-like domain of TIGIT. In some embodiments, said TIGIT binding agent is an antibody that specifically binds a TIGIT IgV domain. In some embodiments, said TIGIT binding agent is an antibody that binds within amino acids 22-141 of human TIGIT and/or amino acids 29-148 of mouse TIGIT. In some embodiments, said TIGIT binding agent is an antibody that binds within amino acids 22-141 of SEQ ID NO: 4 and/or amino acids 29-148 of SEQ ID NO: 1. In some embodiments, binding of said agent occurs at within amino acids 22-124 of human TIGIT and/or amino acids 29-127 of mouse TIGIT. In some embodiments, binding of said agent occurs within amino acids 22-124 of SEQ ID NO: 4 and/or amino acids 29-127 of SEQ ID NO: 1. In some embodiments, binding of said TIGIT binding agent occurs within SEQ ID NO: 3 or its fragment. In some embodiments, binding of said TIGIT binding agent occurs within SEQ ID NO: 6 or a fragment thereof. In some embodiments, said TIGIT binding agent is an antibody that binds within amino acids 22-141 of human TIGIT. In some embodiments, said TIGIT binding agent is an antibody that binds within amino acids 22-141 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds between amino acids 50-124 of TIGIT person. In some embodiments, said TIGIT binding agent is an antibody that binds within amino acids 50-124 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent binds within SEQ ID NO: 6 or a fragment thereof.

In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing amino acids included in SEQ ID NO: 79. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing amino acids included in the composition of the sequence of SEQ ID NO: 80. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing the amino acids included in the sequences of SEQ ID NO: 79 and SEQ ID NO: 80. In some embodiments, said TIGIT binding agent the agent is an antibody that binds an epitope containing amino acids Q62 and I109 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing amino acids Q62 and T119 after sequence of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing amino acids Q64 and I109 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing amino acids Q64 and T119 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing amino acids Q62, Q64, and I109 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent the agent is an antibody that binds an epitope containing amino acids Q62, Q64 and T119 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing amino acids Q62, I109 and T119 pos SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing an amino acid

- 18 039540 you Q64, I109 and T119 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope containing the amino acids Q62, Q64, I109, and T119 of SEQ ID NO: 4. In some embodiments, said TIGIT binding agent in embodiments, said TIGIT binding agent is an antibody that binds an epitope containing at least one amino acid selected from the group consisting of: N58, E60, Q62, Q64, L65, F107, I109, H111, T117, T119, G120, and R121 of SEQ ID NO: 4. In some embodiments, said epitope is a conformational epitope. In some embodiments, said TIGIT binding agent is an antibody that binds an epitope that does not contain the V100 amino acid of SEQ ID NO: 4.

In some embodiments, said TIGIT binding agent (e.g., antibody) binds TIGIT with a dissociation constant (K _D ) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. In some embodiments, the TIGIT binding agent binds TIGIT to a K _D of about 20 nM or less. In some embodiments, the TIGIT binding agent binds TIGIT to a K _D of about 10 nM or less. In some embodiments, the TIGIT binding agent binds TIGIT to a K _D of about 1 nM or less. In some embodiments, the TIGIT binding agent binds TIGIT to a K _D of about 0.5 nM or less. In some embodiments, the TIGIT binding agent binds TIGIT with a K _D of about 0.1 nM or less. In some embodiments, the TIGIT binding agent binds TIGIT to a KD of about 50 pM or less. In some embodiments, the TIGIT binding agent binds TIGIT to a KD of about 25 pM or less. In some embodiments, the TIGIT binding agent binds TIGIT to a KD of about 10 pM or less. In some embodiments, the TIGIT binding agent binds TIGIT with a K _D of about 1 pM or less. In some embodiments, said TIGIT binding agent binds both human TIGIT and mouse TIGIT with a K _D of about 10 nM or less. In some embodiments, the TIGIT binding agent binds both human TIGIT and mouse TIGIT with a K _D of about 1 nM or less. In some embodiments, the TIGIT binding agent binds both human TIGIT and mouse TIGIT with a K _D of about 0.1 nM or less. In some embodiments, said dissociation constant of a binding agent (eg, antibody) with TIGIT is a dissociation constant determined using a TIGIT fusion protein containing at least a portion of the extracellular domain of the TIGIT protein immobilized on a Biacore chip. In some embodiments, the dissociation constant of the binding agent (eg, antibody) with TIGIT is the dissociation constant determined using the binding agent captured by the anti-human IgG antibody on the Biacore chip and the soluble TIGIT protein.

In some embodiments, the TIGIT binding agent comprises a first antigen binding site that specifically binds TIGIT and a second antigen binding site that specifically binds a second target. In some embodiments, the TIGIT binding agent is a bispecific agent that contains a first antigen-binding site that specifically binds TIGIT and a second antigen-binding site that specifically binds a second target. In some embodiments, the TIGIT binding agent binds both TIGIT and the second target to a KD of about 100 nM or less. In some embodiments, the TIGIT binding agent binds both TIGIT and the second target with a K _D of about 50 nM or less. In some embodiments, the TIGIT binding agent binds both TIGIT and the second target to a KD of about 20 nM or less. In some embodiments, the TIGIT binding agent binds both TIGIT and the second target with a K _D of about 10 nM or less. In some embodiments, the TIGIT binding agent binds both TIGIT and the second target with a K _D of about 1 nM or less. In some embodiments, the affinity of one of the antigen-binding sites may be weaker than the affinity of the other antigen-binding site. For example, the K _D of one antigen binding site may be approximately 1 nM and the K D of the second antigen binding site may be approximately 10 nM. In some embodiments, said difference in affinity between two antigen binding sites can be about 2-fold or more, about 3-fold or more, about 5-fold or more, about 8-fold or more, about 10-fold or more, about 15-fold. times or more, about 20 times or more, about 30 times or more, about 50 times or more, or about 100 times or more. Modulation of the affinities of the two antigen-binding sites can affect the biological activity of the bispecific antibody. For example, reducing the affinity of the TIGIT antigen binding site or the second target may have the desired effect, such as reduced binding agent toxicity and/or increased therapeutic index.

In some embodiments, said TIGIT binding agent (e.g., antibody) binds TIGIT at a half maximum effective concentration (EC ₅₀ ) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less; or about 0.1 nM or less. In some embodiments, the TIGIT binding agent binds to human TIGIT at a half maximum effective concentration (EC ₅₀ ) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less , about 1 nM or less; or about 0.1 nM or less. In some embodiments, the TIGIT binding agent binds mouse TIGIT and/or human TIGIT with an EC ₅₀ of about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

In some embodiments, said TIGIT binding agent is an antibody. In some embodiments, said antibody is a recombinant antibody. In some embodiments, said antibody is a monoclonal antibody. In some embodiments, said antibody is a chimeric antibody. In some embodiments, said antibody is a humanized antibody. In some embodiments, said antibody is a human antibody. In some embodiments, said antibody is an IgA, IgD, IgE, IgG, or IgM antibody. In some embodiments, said antibody is an IgG1 antibody. In some embodiments, said antibody is an IgG2 antibody. In some embodiments, said antibody is an IgG4 antibody. In some embodiments, said antibody is an antibody fragment containing an antigen-binding site. In some embodiments, said antibody is a bispecific antibody or a multispecific antibody. In some embodiments, said antibody is a monovalent antibody. In some embodiments, said antibody is a monospecific antibody. In some embodiments, said antibody is a bivalent antibody. In some embodiments, said antibody is conjugated to a cytotoxic moiety. In some embodiments, said antibody is isolated. In some embodiments, said antibody is substantially pure.

In some embodiments, said TIGIT binding agents are polyclonal antibodies. Polyclonal antibodies can be obtained by any known method. In some embodiments, polyclonal antibodies are produced by immunizing an animal (e.g., rabbit, rat, mouse, goat, donkey) with an antigen of interest (e.g., a purified peptide fragment, full-length recombinant protein, or fusion protein) via multiple subcutaneous or intraperitoneal injections. Said antigen may optionally be conjugated to a carrier such as keyhole hemocyanin (KLH) or serum albumin. Said antigen (with or without carrier protein) is diluted in sterile saline and typically combined with an adjuvant (eg complete or incomplete Freund's adjuvant) to form a stable emulsion. After a sufficient period of time, the polyclonal antibodies are isolated from the body of the immunized animal, usually from blood or ascites. These polyclonal antibodies can be purified from serum or ascites according to standard techniques in the art, including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

In some embodiments, the TIGIT binding agent is a monoclonal antibody. Monoclonal antibodies can be obtained using hybridoma methods known to the person skilled in the art. In some embodiments, using the hybridoma method, a mouse, rat, rabbit, hamster, or other suitable host animal is immunized as described above to produce antibodies that specifically bind the antigen used for immunization. In some embodiments, the lymphocytes may be immunized in vitro. In some embodiments, said antigen used for immunization may be a human protein or a fragment thereof. In some embodiments, said antigen used for immunization may be a mouse protein or a fragment thereof.

After immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. Hybridoma cells are selected using specialized media known in the art; unfused lymphocytes and myeloma cells

- 20 039540 survive the selection process. Hybridomas that produce specific monoclonal antibodies directed against a selected antigen can be identified by a variety of methods, including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, and radioimmunoassay). These hybridomas can be propagated either in culture in vitro using standard methods, or in vivo as ascitic tumors in the animal body. These monoclonal antibodies can be purified from the culture medium or ascitic fluid according to standard methods in the art, including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

In some embodiments, monoclonal antibodies can be generated using recombinant DNA techniques, as is known to those skilled in the art. Monoclonal antibody-encoding polynucleotides are isolated from mature B cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers specifically amplifying genes encoding antibody heavy and light chains and sequenced using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors that produce monoclonal antibodies when they are transfected into host cells such as E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, in other circumstances of non-protein-producing immunoglobulins.

In some other embodiments, recombinant monoclonal antibodies or fragments thereof can be isolated from phage display libraries expressing variable domains or CDRs of the desired species.

The polynucleotide(s) encoding(s) a monoclonal antibody may(may) be modified(s), for example, by using recombinant DNA technology to obtain alternative antibodies. In some embodiments, the light chain and heavy chain constant domains of, for example, a mouse monoclonal antibody can be replaced with constant regions, for example, of a human antibody to form a chimeric antibody, or to a non-immunoglobulin polypeptide to form a fusion antibody. In some embodiments, these constant regions are truncated or removed to obtain the desired monoclonal antibody fragment. Site directed mutagenesis or high density mutagenesis of the variable region(s) can be used to optimize specificity, affinity, and the like. monoclonal antibody.

In some embodiments, the TIGIT binding agent is a humanized antibody. Typically, humanized antibodies are human immunoglobulins in which amino acid residues from CDR regions are replaced with amino acid residues from CDR regions of a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that differ in the desired specificity, affinity and/or binding capacity, using methods known to the person skilled in the art. In some embodiments, some of the human immunoglobulin variable framework amino acid residues are replaced with corresponding amino acid residues in the non-human antibody. In some embodiments, the humanized antibody can be further modified by substitution of additional residues either in the framework variable region and/or within substitutional non-human residues to further refine and optimize the specificity, affinity and/or potential of the antibody. In general, a humanized antibody contains variable domain regions containing all or substantially all of the CDRs that correspond to a non-human immunoglobulin, and all or substantially all of the framework regions are of a human immunoglobulin sequence. In some embodiments, said framework regions are human immunoglobulin consensus sequence framework regions. In some embodiments, the humanized antibody may also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically a human immunoglobulin. In some embodiments, such humanized antibodies are used therapeutically because they can reduce antigenicity and the HAMA (human anti-mouse antibody) response when administered to a human subject.

In some embodiments, the TIGIT binding agent is a human antibody. Human antibodies can be directly obtained using various techniques known in the art. In some embodiments, human antibodies can be obtained from immortalized human B lymphocytes immunized in vitro, or from lymphocytes isolated from an immunized individual. In either case, cells can be obtained and isolated that produce an antibody directed against the target antigen. In some embodiments, said human antibody may be selected from a phage library that expresses human antibodies. Alternatively, to obtain antibodies and human antibody fragments in vitro from the gene repertoire of immunoglobulin variable domains of non-immunized

- 21 039540 donors, phage display technology can be used. Techniques for obtaining and using antibody phage libraries are well known in the art. Once antibodies have been identified, affinity maturation strategies known in the art, including, but not limited to, strand shuffling and site-directed mutagenesis, can be used to generate higher affinity human antibodies.

In some embodiments, human antibodies can be produced in transgenic mice that have human immunoglobulin loci. After immunization, these mice are able to produce the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

In some embodiments, said TIGIT binding agent is a bispecific antibody. Accordingly, the present invention encompasses bispecific antibodies that specifically recognize TIGIT and at least one additional target. Bispecific antibodies are capable of specifically recognizing and binding at least two different antigens or epitopes. These different epitopes may be located either on the same molecule (eg, two epitopes on TIGIT) or on different molecules (eg, one epitope is located on TIGIT and another epitope on a different protein). In some embodiments, a bispecific antibody has enhanced activity relative to an individual antibody or a combination of more than one antibody. In some embodiments, a bispecific antibody has reduced toxicity compared to an individual antibody or a combination of more than one antibody. Those skilled in the art will recognize that any therapeutic agent may have unique pharmacokinetics (PK) (eg, circulating half-life). In some embodiments, the bispecific antibody has the ability to synchronize the PK of two active binding agents, wherein the two individual binding agents have different PK profiles. In some embodiments, the bispecific antibody has the ability to focus the action of two agents in a common area (eg, the tumor and/or the tumor microenvironment). In some embodiments, the bispecific antibody has the ability to focus the action of two agents towards a common target (eg, tumor or tumor cell). In some embodiments, the bispecific antibody has the ability to target two agents to more than one biological pathway or more than one biological function. In some embodiments, the bispecific antibody has the ability to target and bring two different cells together (eg, an immune cell and a tumor cell).

In some embodiments, said bispecific antibody is a monoclonal antibody. In some embodiments, said bispecific antibody is a humanized antibody. In some embodiments, said bispecific antibody is a human antibody. In some embodiments, said bispecific antibody is an IgG1 antibody. In some embodiments, said bispecific antibody is an IgG2 antibody. In some embodiments, said bispecific antibody is an IgG4 antibody. In some embodiments, said bispecific antibody has reduced toxicity and/or reduced side effects. In some embodiments, said bispecific antibody has reduced toxicity and/or reduced side effects compared to a mixture of two individual antibodies or said antibodies as single agents. In some embodiments, said bispecific antibody has an increased therapeutic index. In some embodiments, said bispecific antibody has an increased therapeutic index compared to a mixture of said two individual antibodies or said antibodies as single agents.

In some embodiments, said antibodies can specifically recognize and bind a first antigenic target (e.g., TIGIT) as well as a second antigenic target, such as an effector molecule on an immune cell (e.g., CD2, CD3, CD28, CTLA4, PD-1, PD- L1, CD80 or CD86) or Fc receptor (eg CD64, CD32 or CD16) to focus cellular defense mechanisms on a cell expressing and/or producing the first antigenic target. In some embodiments, these antibodies can be used to direct cytotoxic agents to cells that express a particular target antigen. Said antibodies comprise an antigen binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA or TETA.

Techniques for producing bispecific antibodies are known to those skilled in the art. In some embodiments, said bispecific antibodies comprise heavy chain constant regions with amino acid modifications that constitute the interface between two heavy chains. In some embodiments, said bispecific antibodies can be generated using a ridge-to-cavity strategy. In some cases, the terms protrusions and depressions are replaced by the terms elevations and depressions. In some embodiments, said bispecific antibodies may contain hinge region variants incapable of forming disulfide bonds between heavy chains. In some embodiments, these modifications may include amino acid changes that cause changes in electrostatic interactions. In some embodiments, these modifications may include amino acid changes that result in changes in hydrophobic/hydrophilic interactions.

Bispecific antibodies can be intact antibodies or antibody fragments containing antigen-binding sites. Antibodies with more than two valences are also contemplated. For example, trispecific antibodies can be generated. Accordingly, in certain embodiments, anti-TIGIT antibodies are multispecific.

In some embodiments, the antibodies (or other polypeptides) described herein may be monospecific. In some embodiments, each of the one or more antigen-binding sites that an antibody contains is capable of binding (or binds) a homologous epitope on TIGIT.

In some embodiments, the TIGIT binding agent is an antibody fragment. Antibody fragments may have functions or abilities that are different from those of intact antibodies; for example, antibody fragments may have increased penetration into the tumor. Various techniques for obtaining antibody fragments are known, including, but not limited to, proteolytic cleavage of intact antibodies. In some embodiments, antibody fragments include an F(ab') ₂ fragment obtained by cleaving an antibody molecule with pepsin. In some embodiments, the antibody fragments include a Fab fragment obtained by reducing the disulfide bridges of F(ab') ₂ fragments. In other embodiments, antibody fragments include a Fab fragment obtained by treating an antibody molecule with papain and a reducing agent. In some embodiments, antibody fragments are generated using recombinant methods. In some embodiments, antibody fragments include Fv fragments or single chain Fv fragments (scFv). Fab, Fv, and scFv antibody fragments can be expressed and secreted in E. coli or other host cells, resulting in significant amounts of these fragments. In some embodiments, antibody fragments are isolated from antibody phage libraries as described herein. For example, methods for constructing Fab expression libraries can be used that allow rapid and efficient identification of monoclonal Fab fragments with the desired specificity for TIGIT, or derivatives, fragments, analogs, or homologues thereof. In some embodiments, the antibody fragments are linear antibody fragments. In some embodiments, antibody fragments are monospecific or bispecific. In some embodiments, said TIGIT binding agent is a scFv. Various techniques can be used to generate single chain antibodies specific for TIGIT.

In some embodiments, particularly in the case of antibody fragments, the antibody is modified to change (eg, increase or decrease) its serum half-life. This can be achieved, for example, by incorporating a recycling receptor binding epitope into the antibody fragment, by mutating an appropriate region in the antibody fragment, or by incorporating said epitope into a peptide tag, which is then fused to either end of the antibody fragment or to a region in the middle of the antibody fragment (e.g., by DNA synthesis or peptide synthesis).

Heteroconjugate antibodies are also included within the scope of the present invention. Heteroconjugated antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune cells to unwanted cells. Also provided is the possibility of obtaining heteroconjugate antibodies in vitro using methods known in the field of protein synthesis chemistry, including those involving agents for cross-linking. For example, immunotoxins can be constructed using a disulfide exchange reaction or through the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

It should be appreciated that, for the purposes of the present invention, modified antibodies may contain any type of variable region that allows antibody to target binding (ie, TIGIT). In this regard, the variable region may comprise or be derived from any type of mammalian variable region in which a humoral response and production of immunoglobulins against the desired antigen can be induced. Accordingly, the variable region modified antibodies can be derived from, for example, a human, mouse, rat, rabbit, non-human primate (eg, cynomolgus monkey, other monkeys, etc.) or rabbit. In some embodiments, both the variable and constant regions of the modified immunoglobulins are human. In other embodiments, variable regions of compatible antibodies (typically from a non-human source) can be engineered or specifically individualized to improve binding properties or reduce the immunogenicity of the molecule. To this end, variable regions suitable for use in accordance with the present invention may be humanized or otherwise altered by including borrowed amino acid sequences.

In some embodiments, both heavy and light chain variable domains are altered by at least partial replacement of one or more CDR regions and, if necessary, partial framework replacement and sequence modification and/or alteration. Although the CDR regions may be derived from an antibody of the same class, or even subclass, as the antibody from which the framework regions are derived. It is contemplated that the CDRs may be derived from another class of antibody, and often from another species. It may not be necessary to replace all CDRs with all CDRs of the donor variable region to transfer the antigen-binding capacity of one variable domain to another. It may only be necessary to transfer the residues necessary to maintain the activity of said antigen-binding site.

Regardless of the variable region changes, those skilled in the art will appreciate that the modified antibodies of the present invention include antibodies (e.g., full length antibodies or immunoreactive fragments thereof) in which at least a portion of one or more constant region domains have been deleted or otherwise altered. to provide desired biochemical characteristics, such as enhanced tumor localization or increased serum half-life, as compared to an antibody of approximately the same immunogenicity containing a native or unaltered constant region. In some embodiments, the modified antibody constant region comprises a human constant region. Constant region modifications compatible with the present invention include additions, deletions, or substitutions of one or more amino acids in one or more domains. Modified antibodies as described herein may include alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and/or the light chain constant domain (CL). In some embodiments, one or more domains are partially or completely removed from the constant regions of the modified antibodies. In some embodiments, said modified antibodies include domain deletion constructs or variants wherein the CH2 domain is completely removed (ACH2 constructs). In some embodiments, the omitted constant region domain is replaced with a short amino acid spacer (eg, 10 amino acid residues) that provides some of the molecular flexibility normally afforded by the missing constant region.

In some embodiments, modified antibodies are constructed by fusing the CH3 domain directly to the hinge region of the antibody. In other embodiments, a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains. For example, constructs can be expressed in which the CH2 domain has been removed and the remaining CH3 domain (modified or unmodified) is attached to the hinge region with a 5-20 amino acid spacer. Such a spacer may be added to keep the constant domain regulatory elements free and accessible, or to keep the hinge region flexible. However, it should be noted that amino acid spacers may, in some cases, be immunogenic and cause an undesirable immune response against said construct. Accordingly, in some embodiments, any spacer added to the construct must be relatively non-immunogenic in order to maintain the desired biological characteristics of the modified antibodies.

In some embodiments, the modified antibodies may only partially remove the constant domain or replace a few or a single amino acid. For example, a single amino acid mutation in selected regions of the CH2 domain may be sufficient to substantially reduce Fc binding. In some embodiments, a single amino acid mutation in selected regions of the CH2 domain may be sufficient to reduce Fc binding substantially and increase localization to cancer cells and/or tumor entry. Similarly, it may be desirable to simply remove a portion of one or more constant region domains that control the specific effector function (eg, C1q complement component binding) that is intended to be modulated. Such partial deletions in the constant regions can improve selected characteristics of the antibody (serum half-life) while maintaining other desirable functions associated with the subject's intact constant region domain. In addition, as mentioned above, the constant regions of the disclosed antibodies can be modified by mutation or substitution of one or more amino acids, improving the profile of the final design. It may be possible to disrupt the activity provided by a conserved binding site (eg, Fc binding) while maintaining essentially the configuration and immunogenic profile of the modified antibody. In some embodiments, one or more amino acids are added to the constant region of said modified antibodies to enhance desirable characteristics, such as decreasing or increasing effector function or providing more cytotoxin or carbohydrate attachment sites.

It is known in the art that the constant region mediates several effector functions. For example, binding of complement component C1 to the Fc region of an IgG or IgM antibody (antigen bound) activates the complement system. Complement activation is important for opsonization and lysis of cellular pathogens. Complement activation also stimulates the inflammatory response and may also be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind a cell expressing an Fc receptor (FcR). There are a number of Fc receptors specific to different classes of antibodies, including IgG (γ receptors), IgE (ε receptors), IgA (α receptors), and IgM (μ receptors). Binding of an antibody to Fc receptors on cell surfaces triggers a variety of important biological responses, including uptake and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cellular cytotoxicity, or ADCC), release of inflammatory mediators , placental transfer and control of immunoglobulin production.

In some embodiments, these modified antibodies provide altered effector functions, which in turn affect the biological profile of the administered antibody. For example, in some embodiments, deletion or inactivation (via point mutations or other means) of a constant region domain can reduce Fc receptor binding of a circulating modified antibody. In some embodiments, deletion or inactivation (by point mutations or other means) of the constant region domain may decrease Fc receptor binding of the circulating modified antibody, thereby enhancing cancer localization and/or tumor entry. In other embodiments, said constant region modifications increase the serum half-life of the antibody. In other embodiments, said constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region is modified to remove disulfide bonds or oligosaccharide moieties. Modifications to the constant region in accordance with the present invention can easily be implemented using well known biochemical or molecular engineering techniques.

In some embodiments, the TIGIT binding agent, which is an antibody, lacks one or more of the effector functions. For example, in some embodiments, said antibody does not have ADCC activity and/or does not have complement dependent cytotoxicity (CCC activity). In some embodiments, said antibody does not bind the Fc receptor and/or complement factors. In some embodiments, said antibody does not have effector function(s).

The present invention also encompasses variants and equivalents substantially homologous to recombinant, monoclonal, chimeric, humanized and human antibodies, or fragments thereof, with the antibody characteristics described herein. These variants may include, for example, conservative substitution type mutations, i.e. replacing one or more amino acids with similar amino acids.

The present invention provides methods for producing an antibody that binds TIGIT, including bispecific antibodies that specifically bind both TIGIT and a second target. In some embodiments, said method for producing an antibody that binds TIGIT involves the use of hybridoma techniques. In some embodiments, a method for making an antibody that binds human TIGIT is provided. In some embodiments, said method comprises using a polypeptide containing the mouse TIGIT extracellular domain, or a fragment thereof, as an antigen. In some embodiments, said method comprises using a polypeptide comprising a human TIGIT extracellular domain, or a fragment thereof, as an antigen. In some embodiments, said method comprises using a polypeptide comprising mouse TIGIT amino acids 29-148 as an antigen. In some embodiments, said method comprises using a polypeptide comprising human TIGIT amino acids 22-141 as an antigen. In some embodiments, said method comprises using a polypeptide comprising amino acids 29-148 of SEQ ID NO: 1 as an antigen. In some embodiments, said method comprises using a polypeptide comprising amino acids 22-141 of SEQ ID NO: 4 as an antigen. In some embodiments, said method comprises using a polypeptide comprising SEQ ID NO: 3, or a fragment thereof, as an antigen. In some embodiments, said method comprises using a polypeptide comprising SEQ ID NO: 6, or a fragment thereof, as an antigen. In some embodiments, said method for producing an antibody that binds TIGIT comprises screening a phage library. In some embodiments, said method for producing an antibody that binds TIGIT involves screening a human phage library. The present invention also provides methods for identifying an antibody that binds TIGIT. In some embodiments, said antibody is identified by FACS screening for TIGIT binding or a fragment thereof. In some embodiments, said antibody is identified by screening with ELISA ELISA for binding to TIGIT or a fragment thereof. In some embodiments, said antibody is identified by FACS screening for blocking TIGIT binding to PVR.

In some embodiments, a method for producing an anti-TIGIT antibody comprises immunizing a mammal with a polypeptide comprising amino acids 29-148 of a mouse TIGIT. In some embodiments, a method for producing an anti-TIGIT antibody comprises immunizing a mammal with a polypeptide comprising human TIGIT amino acids 22-141. In some embodiments, a method for producing an anti-TIGIT antibody comprises immunizing a mammal with a polypeptide containing a fragment (eg, a portion) of amino acids 29-148 of mouse TIGIT. In some embodiments, a method for producing an anti-TIGIT antibody comprises immunizing a mammal with a polypeptide comprising a human TIGIT amino acid fragment 22-141. In some embodiments, said method further comprises isolating antibodies or antibody-producing cells from a mammal. In some embodiments, a method for producing a monoclonal antibody that binds TIGIT comprises: (a) immunizing a mammal with a polypeptide containing a mouse TIGIT amino acid fragment 29-148; (b) isolating antibody-producing cells from the immunized mammal; and (c) fusing said antibody-producing cells with a myeloma cell line to obtain hybridoma cells. In some embodiments, a method for producing a monoclonal antibody that binds TIGIT comprises: (a) immunizing a mammal with a polypeptide comprising a human TIGIT amino acid fragment 22-141; (b) isolating antibody-producing cells from the immunized mammal; and (c) fusing said antibody-producing cells with a myeloma cell line to obtain hybridoma cells. In some embodiments, said method further comprises (d) selecting a hybridoma cell that expresses an antibody that binds TIGIT. In some embodiments, said mammal is a mouse. In some embodiments, said mammal is a rat. In some embodiments, said mammal is a rabbit. In some embodiments, said antibody is selected using a polypeptide comprising amino acids 29-148 or a fragment thereof from mouse TIGIT. In some embodiments, said antibody is selected using a polypeptide comprising amino acids 22-141 or a fragment thereof from human TIGIT. In some embodiments, said antibody binds both human TIGIT and mouse TIGIT. In some embodiments, said antibody does not bind mouse TIGIT. In some embodiments, said antibody does not bind cynomolgus TIGIT. In some embodiments, said antibody does not bind rhesus monkey TIGIT. In some embodiments, said antibody does not bind rat TIGIT. In some embodiments, said antibody binds human TIGIT and does not bind mouse TIGIT. In some embodiments, said antibody binds human TIGIT and does not bind cynomolgus TIGIT. In some embodiments, said antibody binds human TIGIT and does not bind rhesus monkey TIGIT. In some embodiments, said antibody binds human TIGIT and does not bind rat TIGIT.

In some embodiments, a method for producing an antibody that binds TIGIT comprises identifying the antibody using a membrane-bound heterodimeric molecule containing a single antigen-binding site. In some non-limiting embodiments, said antibody is identified using the methods and polypeptides described in WO 2011/100566.

In some embodiments, a method for producing an antibody that binds TIGIT comprises screening an antibody-expressing library. In some embodiments, said antibody-expressing library is a phage library. In some embodiments, said screening includes panning. In some embodiments, said antibody-expressing library is a mammalian cell library. In some embodiments, screening of said antibody-expressing library is performed using mouse TIGIT amino acids 29-148 or a fragment thereof. In some embodiments, the antibody expression library is screened using human TIGIT amino acids 22-141 or a fragment thereof. In some embodiments, said screen-identified antibody binds both human TIGIT and mouse TIGIT. In some embodiments, said screened antibody is a TIGIT antagonist.

In some embodiments, the antibody produced using the methods described herein is a TIGIT antagonist. In some embodiments, an antibody generated using the methods described herein inhibits TIGIT signaling. In some embodiments, an antibody produced using the methods described herein inhibits TIGIT phosphorylation.

In some embodiments, the antibodies described herein are isolated. In some embodiments, the antibodies described herein are substantially pure.

Binding TIGIT agents (eg antibodies) according to the present invention can be analyzed for the presence of specific binding by any method known in the art. Immunological assays that may be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore assay, FACS assay, immunofluorescence, immunocytochemistry, Western blot assay, radioimmunoassay, ELISA ELISA, immunoassay sandwich, immunoprecipitation assay, precipitation assay, diffuse gel precipitation assay, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescence immunoassay, and protein A immunoassay. Such assays are routine and well known in the art (see, for example, Ausubel et al., Editors, 1994-present, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY).

As a non-limiting example, screening for specific antibody binding to human TIGIT can be performed using an ELISA ELISA. ELISA ELISA involves obtaining an antigen (for example, TIGIT or a fragment thereof), applying the antigen to the wells of a 96-well microtiter plate, adding test antibodies conjugated to a detectable compound, such as an enzymatic substrate (for example, horseradish peroxidase or alkaline phosphatase), into the well, incubating for a period of time; and detecting the presence of an antibody bound to said antigen. In some embodiments, said test antibodies are not conjugated to a detectable compound, but instead a secondary antibody is added to the wells that recognizes said antibody (eg, an anti-Fc antibody) and is conjugated to a detectable compound. In some embodiments, instead of applying an antigen, test antibodies may be applied to the wells, an antigen (eg, TIGIT) added, and then a secondary antibody conjugated to a detectable compound. The person skilled in the art is aware of indicators that can be modified to increase the detected signal; other variants of the ELISA ELISA are also known in the art.

According to another non-limiting example, the specific binding of an antibody to TIGIT can be determined using FACS. A FACS screening assay may include generating a cDNA construct that expresses an antigen as a full-length protein (TIGIT) or a fusion protein (e.g., TIGIT-CD4TM), transfecting said construct into cells, expressing said antigen on the cell surface, mixing test antibodies with transfected cells and incubation for some period of time. Cells that bind test antibodies can be identified using a secondary antibody conjugated to the compound to be detected (eg, PE-conjugated anti-Fc antibody) and a flow cytometer. The person skilled in the art is aware of indicators that can be modified to optimize the detected signal, as well as other variants of FACS that can improve screening (for example, screening for detection of blocking antibodies).

The affinity (affinity) for the binding of an antibody or other binding agent to an antigen (eg, TIGIT), and the rate of dissociation of an antibody-antigen interaction can be determined using competitive binding assays. One example of a competitive binding assay is a radioimmunoassay involving the incubation of a labeled antigen (e.g., ³ H or ¹²⁵ I-TIGIT), or a fragment or variant thereof, with an antibody of interest in the presence of increasing amounts of unlabeled antigen, followed by detection of the antibody bound to the labeled antigen. . Antibody affinity for antigen and binding dissociation rates can be determined based on data obtained by analysis of the Scatchard plot. In some embodiments, Biacore kinetic analysis is used to determine the binding and dissociation rates of antibodies or agents that bind an antigen (eg, TIGIT). In some embodiments, the Biacore kinetic assay includes a binding and dissociation assay of antibodies to chips immobilized with an antigen (eg, TIGIT). In some embodiments, the Biacore kinetic assay comprises an antigen (eg, TIGIT) binding and dissociation assay on chips immobilized with an antibody (eg, anti-TIGIT antibody).

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises one, two, three, four, five, and/or six CDRs of an antibody 313R11, 313R12 , 313R14, or 313R19 (see Table 1). In some embodiments, said TIGIT binding agent comprises one or more of CDRs 313R11, 313R12, 313R14, or 313R19, or humanized variants thereof; two or more of the CDR 313R11 sections,

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313R12, 313R14 or 313R19 or their humanized variants; three or more of the CDRs

313R11, 313R12, 313R14 or 313R19 or their humanized variants; four or more of the plots

CDR 313R11, 313R12, 313R14 or 313R19 or their humanized variants; five or more of the CDR sections 313R11, 313R12, 313R14 or 313R19 or their humanized variants; or all six sections of CDR 313R11, 313R12, 313R14 or 313R19 or their humanized variants.

Table 1

313R11, 313R12 313R14, 313R19 Heavy chain CDR1 (HC CDR1) GSSLSSSYMS (SEQ ID NO:7) GFSLSSSYMS (SEQ ID NO: 13) NS CDR2 IIGSNGNTYYANWAKG (SEQ ID NO: 8) IIGSNGNTYYANWAKG (SEQ ID NO: 8) NA CDR3 GGYRTSGMDP (SEQ ID NO:9) GGYRTSGMDP (SEQ ID NO:9) CDR1 light chains (LC CDR1) QASQSISSYLNW (SEQ ID NO: 10) QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO:81) LCCDR2 DALKLAS (SEQ ID NO: 11) RASTLAS (SEQ ID NO: 15) LC CDR3 QQEHSVGNVDN (SEQ ID NO:12) QQEHLVAWIYN (SEQ ID NO: 16)

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: : 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9). In some embodiments, said TIGIT binding agent further comprises a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11) or RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16). In some embodiments, said TIGIT binding agent comprises a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing DALKLAS (SEQ ID NO: I) or RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16). In some embodiments, said TIGIT binding agent comprises: (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9) and (b) a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), A light chain CDR2 containing DALKLAS (SEQ ID NO: 11) or RASTLAS (SEQ ID NO: 15) and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16). In some embodiments, said TIGIT binding agent comprises: (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9) and (b) a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12 ). In some embodiments, said TIGIT binding agent comprises: (a) a heavy chain CDR1 containing GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9) and (b) a light chain CDR1 containing QASQSNIYSDLAW (SEQ ID NO: 14), a light chain CDR2 containing RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHLVAWIYN (SEQ ID NO: 16 ). In some embodiments, said TIGIT binding agent comprises: (a) a heavy chain CDR1 containing GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9) and (b) light chain CDR1 containing QASQNIYSDLAW

-28039540 (SEQ ID NO: 81), a light chain CDR2 containing RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHLVAWIYN (SEQ ID NO: 16).

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds human TIGIT, characterized in that said TIGIT binding agent comprises: (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7), GFSLSSSYMS (SEQ ID NO: 13), or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (b) a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (c) a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9) or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (d) a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14), QASQNIYSDLAW (SEQ ID NO: 81), or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (e) a light chain CDR2 containing DALKLAS (SEQ ID NO: 11), RASTLAS (SEQ ID NO: 15), or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; and (f) a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12), QQEHLVAWIYN (SEQ ID NO: 16), or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions. In some embodiments, said amino acid substitutions are conservative substitutions. In some embodiments, these substitutions are made as part of the humanization process. In some embodiments, these substitutions are made as part of the germline humanization process.

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises a heavy chain variable region characterized by at least about 80% sequence identity to SEQ ID NO: 17 , SEQ ID NO: 19 or SEQ ID NO: 32, and/or a light chain variable region differing by at least 80% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 17. According to some embodiments, said TIGIT binding agent contains a heavy variable region a chain that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 19. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region differing by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO : 32. In some embodiments, said TIGIT binding agent comprises a light chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 18. According to some variant In embodiments, said TIGIT binding agent comprises a light chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region that differs by at least about 95% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32, and/or a light chain region that differs by at least about 95% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 32, and/or a light chain variable region comprising SEQ ID NO: 18 or SEQ ID NO: 20. According to In certain embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32 and a light chain variable region comprising SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region essentially consisting of the sequence SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32, and a light chain variable region essentially consisting of the sequence SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32 and a light chain variable region a chain consisting of SEQ ID NO: 18 or SEQ ID NO: 20.

In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region comprising SEQ ID NO: 18. In some embodiments, said TIGIT binding agent

- 29 039540 agent consists of a heavy chain variable region, essentially consisting of the sequence

SEQ ID NO: 17, and a light chain variable region essentially consisting of the sequence

SEQ ID NO: 18. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 17 and a light chain variable region consisting of SEQ ID NO: 18.

In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 19 and a light chain variable region comprising SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region essentially consisting of the sequence of SEQ ID NO: 19, and a light chain variable region essentially consisting of the sequence of SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 19 and the variable a light chain region consisting of SEQ ID NO: 20.

In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 32 and a light chain variable region comprising SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region essentially consisting of the sequence of SEQ ID NO: 32, and a light chain variable region essentially consisting of the sequence of SEQ ID NO: 20. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 32 and the variable a light chain region consisting of SEQ ID NO: 20.

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises: (a) a heavy chain that shares at least 90% sequence identity with SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34 or SEQ ID NO: 56; and/or (b) a light chain that differs by at least 90% sequence identity to SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, said TIGIT binding agent comprises: (a) a heavy chain that differs by at least 95 % sequence identity of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34 or SEQ ID NO: 56; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, said TIGIT binding agent comprises a heavy chain comprising SEQ ID NO: 26, and/ or a light chain comprising SEQ ID NO: 28. In some embodiments, said TIGIT binding agent comprises a heavy chain comprising SEQ ID NO: 27 and/or a light chain comprising SEQ ID NO: 28. In some embodiments, said TIGIT binding agent the agent comprises a heavy chain comprising SEQ ID NO: 29 and/or a light chain comprising SEQ ID NO: 30. In some embodiments, said TIGIT binding agent comprises a heavy chain comprising SEQ ID NO: 34 and/or a light chain, comprising SEQ ID NO: 30. In some embodiments, said TIGIT binding agent comprises a heavy chain comprising SEQ ID NO: 56 and/or a light chain comprising SEQ ID NO: 30. In some embodiments, said TIGIT binding agent and said TIGIT binding agent comprises a heavy chain consisting essentially of the sequence of SEQ ID NO: 26 or SEQ ID NO: 27 and a light chain consisting essentially of the sequence of SEQ ID NO: 28. In some embodiments, said TIGIT binding agent comprises a heavy chain consisting essentially of the sequence of SEQ ID NO: 29, SEQ ID NO: 34, or SEQ ID NO: 56, and a light chain consisting essentially of the sequence of SEQ ID NO: 30. In some embodiments, said TIGIT binding agent comprises a heavy chain consisting of SEQ ID NO: 26 and a light chain consisting of SEQ ID NO: 28. In some embodiments, said TIGIT binding agent comprises a heavy chain consisting of SEQ ID NO: 27 and a light chain consisting of SEQ ID NO: 28. In some embodiments, said TIGIT binding agent comprises a heavy chain consisting of SEQ ID NO: 29 and a light chain consisting of SEQ ID NO: 30. In some embodiments, As an implementation, said TIGIT binding agent comprises a heavy chain consisting of SEQ ID NO: 34 and a light chain consisting of SEQ ID NO: 30. In some embodiments, said TIGIT binding agent comprises a heavy chain consisting of SEQ ID NO: 56, and a light chain consisting of SEQ ID NO: 30.

In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and the light chain variable region of the 313R11 antibody. In some embodiments, said TIGIT binding agent comprises variable regions of the 313R11 antibody, wherein said heavy chain variable region and/or light chain variable region from the 313R11 antibody has undergone affinity maturation. In some embodiments, the TIGIT binding agent comprises the heavy chain and light chain of the 313R11 antibody (with or without a leader sequence). In some embodiments, the TIGIT binding agent is a 313R11 antibody. In some embodiments, the binding

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The TIGIT agent contains a heavy chain variable region and/or a light chain variable region of the antibody 313R11, wherein said heavy chain variable region and/or light chain variable region have been humanized. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and/or the light chain variable region of the 313R11 antibody in humanized form. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313R11 antibody as part of an IgG1, IgG2, or IgG4 heavy chain.

In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of the 313R11 antibody. In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of a variant of the 313R11 antibody.

In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and the light chain variable region of the 313R12 antibody. In some embodiments, said TIGIT binding agent comprises variable regions of the 313R12 antibody, wherein said heavy chain variable region and/or light chain variable region from the 313R12 antibody has undergone affinity maturation. In some embodiments, the TIGIT binding agent comprises the heavy chain and light chain of the 313R12 antibody (with or without a leader sequence). In some embodiments, the TIGIT binding agent is a 313R12 antibody. In some embodiments, the TIGIT binding agent comprises a heavy chain variable region and/or a light chain variable region of the 313R12 antibody, wherein said heavy chain variable region and/or light chain variable region have been humanized. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and/or the light chain variable region of the 313R12 antibody in humanized form. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313R12 antibody as part of an IgG1, IgG2, or IgG4 heavy chain.

In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of the 313R12 antibody. In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of a 313R12 antibody variant.

In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and the light chain variable region of the 313R14 antibody. In some embodiments, said TIGIT binding agent comprises variable regions of the 313R14 antibody, wherein said heavy chain variable region and/or light chain variable region from the 313R14 antibody has undergone affinity maturation. In some embodiments, the TIGIT binding agent comprises the heavy chain and light chain of the 313R14 antibody (with or without a leader sequence). In some embodiments, the TIGIT binding agent is a 313R14 antibody. In some embodiments, the TIGIT binding agent comprises a heavy chain variable region and/or a light chain variable region of the 313R14 antibody, wherein said heavy chain variable region and/or light chain variable region have been humanized. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and/or the light chain variable region of the 313R14 antibody in humanized form. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313R14 antibody as part of an IgG1, IgG2, or IgG4 heavy chain.

In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of the 313R14 antibody. In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of a 313R14 antibody variant.

In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and the light chain variable region of the 313R19 antibody. In some embodiments, said TIGIT binding agent comprises variable regions of the 313R19 antibody, wherein said heavy chain variable region and/or light chain variable region from the 313R19 antibody has undergone affinity maturation. In some embodiments, the TIGIT binding agent comprises the heavy chain and light chain of the 313R19 antibody (with or without a leader sequence). In some embodiments, the TIGIT binding agent is a 313R19 antibody. In some embodiments, the TIGIT binding agent comprises a heavy chain variable region and/or a light chain variable region of the 313R19 antibody, wherein said heavy chain variable region and/or light chain variable region have been humanized. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and/or the light chain variable region of the 313R19 antibody in humanized form. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313R19 antibody as part of an IgG1, IgG2, or IgG4 heavy chain.

In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of the 313R19 antibody. In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of a 313R19 antibody variant.

In some embodiments, said TIGIT binding agent comprises a heavy chain variable region encoded by a plasmid deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the terms of the Budapest Treaty dated May 27, 2015, and designated RTA-122180. In some embodiments, said TIGIT binding agent comprises a light chain variable region encoded by a plasmid deposited with ATCC, 10801 University Boulevard, Manassas, Virginia, USA under the terms of the Budapest Treaty dated May 27, 2015, and designated PTA-122181. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region encoded by a plasmid deposited with ATCC designated PTA-122180 and a light chain variable region encoded by a plasmid deposited with ATCC designated PTA122181. In some embodiments, said TIGIT binding agent comprises a heavy chain containing a heavy chain variable region encoded by a plasmid deposited with ATCC and designated PTA-122180. In some embodiments, said TIGIT binding agent comprises a light chain containing a light chain variable region encoded by a plasmid deposited with ATCC and designated PTA-122181. In some embodiments, said TIGIT binding agent comprises a heavy chain comprising a heavy chain variable region encoded by a plasmid deposited with ATCC designated PTA-122180 and a light chain comprising a light chain variable region encoded by a plasmid deposited with ATCC designated PTA- 122181.

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises one, two, three, four, five, and/or six CDRs of the 313M26 or 313M32 antibody. (see Table 2). In some embodiments, said TIGIT binding agent comprises one or more of CDRs 313M26 or 313M32; two or more of the sections CDR 313M26 or 313M32; three or more of the sections CDR 313M26 or 313M32; four or more of the sections CDR 313M26 or 313M32; five or more of the sections CDR 313M26 or 313M32; or all six sections of CDR 313M26 or 313M32.

table 2

313M26 and 313M32 Heavy chain CDR1 (HC CDR1) TSDYAWN (SEQ ID NO: 57) NS CDR2 YISYSGSTSYNPSLRS (SEQ ID NO: 58) NA CDR3 ARRQVGLGFAY (SEQ ID NO:59) Light chain CDR1 (LC) KASQDVSTAVA (SEQ ID NO:60) LCCDR2 SASYRYT (SEQ ID NO:61) LC CDR3 QQHYSTP (SEQ ID NO :62)

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), a heavy chain CDR2 containing YISYSGSTSYNPSLRS (SEQ ID NO: 58), and a heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59). In some embodiments, said TIGIT binding agent further comprises a light chain CDR1 comprising KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 comprising SASYRYT (SEQ ID NO: 61), and a light chain CDR3 comprising QQHYSTP (SEQ ID NO: 62). In some embodiments, said TIGIT binding agent comprises a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 containing SASYRYT (SEQ ID NO: 61), and a light chain CDR3 containing QQHYSTP (SEQ ID NO: 62 ). In some embodiments, said TIGIT binding agent comprises: (a) a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), a heavy chain CDR2 containing YISYSGSTSYNPSLRS (SEQ ID NO: 58), and a heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59); and (b) a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2,

- 32 039540 containing SASYRYT (SEQ ID NO: 61), and a light chain CDR3 containing QQHYSTP (SEQ ID NO: 62).

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds human TIGIT, characterized in that said TIGIT binding agent comprises: (a) a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (b) a heavy chain CDR2 containing YiSySGSTSYNPSLRS (SEQ ID NO: 58), or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (c) a heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59) or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (d) a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60) or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; (e) a light chain CDR2 containing SASYRYT (SEQ ID NO: 61) or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions; and (f) a light chain CDR3 containing QQHYSTP (SEQ ID NO: 62) or a variant thereof containing 1, 2, 3 or 4 amino acid substitutions. In some embodiments, said amino acid substitutions are conservative substitutions. In some embodiments, these substitutions are made as part of the humanization process. In some embodiments, these substitutions are made as part of the germline humanization process. In some embodiments, these substitutions are made as part of a binding optimization process.

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises a heavy chain variable region that differs by at least about 80% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 67 and/or a light chain variable region that differs by at least 80% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region that is different from at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 63. In some embodiments, said TIGIT binding agent contains the variable region of the heavy chain, distinct having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 67. In some embodiments, said binding TIGIT the agent contains a light chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 64. In some embodiments, said TIGIT binding agent comprises a light chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identity sequence SEQ ID NO: 68. In some embodiments, the uk Said TIGIT binding agent comprises a heavy chain variable region that shares at least about 95% sequence identity with SEQ ID NO: 63 or SEQ ID NO: 67 and/or a light chain variable region that shares at least about 95% sequence identity with SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 63 or SEQ ID NO: 67 and/or a light chain variable region comprising SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 63 or SEQ ID NO: 67 and a light chain variable region comprising SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region essentially consisting of the sequence SEQ ID NO: 63 or SEQ ID NO: 67, and a light chain variable region essentially consisting of the sequence of SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 63 or SEQ ID NO: 67, and a light chain variable region consisting of SEQ ID NO: 64 or SEQ ID NO: 68.

In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 63 and a light chain variable region comprising SEQ ID NO: 64. In some embodiments, said TIGIT binding agent consists of a heavy chain variable region, according to essentially consisting of the sequence of SEQ ID NO: 63, and a light chain variable region essentially consisting of the sequence of SEQ ID NO: 64. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 63, and a light chain variable region consisting of SEQ ID NO: 64.

In some embodiments, said TIGIT binding agent comprises a heavy chain variable region comprising SEQ ID NO: 67 and a light chain variable region comprising SEQ ID NO: 68. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region. a chain essentially consisting of the sequence of SEQ ID NO: 67 and a light chain variable region essentially consisting of the sequence of SEQ ID NO: 68. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region consisting of SEQ ID NO: 67 and a light chain variable region consisting of SEQ ID NO: 68.

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises: a heavy chain that shares at least 90% sequence identity with SEQ ID NO: 70, and /or a light chain that shares at least 90% sequence identity with SEQ ID NO: 72. In some embodiments, said TIGIT binding agent comprises: a heavy chain that shares at least 95% sequence identity with SEQ ID NO: 70, and/or a light chain chain having at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, said TIGIT binding agent comprises a heavy chain comprising SEQ ID NO: 70 and/or a light chain comprising SEQ ID NO: 72. According to some embodiments, said TIGIT binding agent contains a heavy chain consisting essentially of a sequence sequence of SEQ ID NO: 70, and a light chain consisting essentially of the sequence of SEQ ID NO: 72. In some embodiments, said TIGIT binding agent comprises a heavy chain consisting of SEQ ID NO: 70 and a light chain consisting of SEQ ID NO: 70. NO: 72.

In some embodiments, the present invention provides a TIGIT binding agent (e.g., an antibody) that specifically binds TIGIT, characterized in that said TIGIT binding agent comprises: a heavy chain that shares at least 90% sequence identity with SEQ ID NO: 82, and /or a light chain that shares at least 90% sequence identity with SEQ ID NO: 72. In some embodiments, said TIGIT binding agent comprises: a heavy chain that shares at least 95% sequence identity with SEQ ID NO: 82, and/or a light chain chain having at least 95% sequence identity to SEQ ID NO: 72. In some embodiments, said TIGIT binding agent comprises a heavy chain comprising SEQ ID NO: 82 and/or a light chain comprising SEQ ID NO: 72. According to some embodiments, said TIGIT binding agent contains a heavy chain consisting essentially of a sequence sequence of SEQ ID NO: 82, and a light chain consisting essentially of the sequence of SEQ ID NO: 72. In some embodiments, said TIGIT binding agent comprises a heavy chain consisting of SEQ ID NO: 82 and a light chain consisting of SEQ ID NO: 82 NO: 72.

In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and the light chain variable region of the 313M26 antibody. In some embodiments, said TIGIT binding agent comprises variable regions of the 313M26 antibody, wherein said heavy chain variable region and/or light chain variable region from the 313M26 antibody has undergone affinity maturation. In some embodiments, the TIGIT binding agent comprises the heavy chain and light chain of the 313M26 antibody (with or without a leader sequence). In some embodiments, the TIGIT binding agent is the 313M26 antibody. In some embodiments, the TIGIT binding agent comprises a heavy chain variable region and/or a light chain variable region of the 313M26 antibody, wherein said heavy chain variable region and/or light chain variable region have been humanized. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and/or the light chain variable region of the 313M26 antibody in humanized form. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313M26 antibody as part of an IgG1, IgG2, or IgG4 heavy chain.

In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of the 313M26 antibody. In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of a variant of the 313M26 antibody.

In some embodiments, the TIGIT binding agent comprises the heavy chain variable region and the light chain variable region of the 313M32 antibody. In some embodiments, said TIGIT binding agent comprises variable regions of the 313M32 antibody, wherein said heavy chain variable region and/or light chain variable region from the 313M32 antibody has undergone affinity maturation. In some embodiments, the TIGIT binding agent comprises the heavy chain and light chain of the 313M32 antibody (with or without a leader sequence). In some embodiments, the TIGIT binding agent is a 313M32 antibody. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313M32 antibody as part of the heavy chain

- 34 039540

IgG1, IgG2 or IgG4. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313M32 antibody as part of a human IgG1 heavy chain. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313M32 antibody as part of a human IgG2 heavy chain. In some embodiments, the TIGIT binding agent comprises the heavy chain variable region of the 313M32 antibody as part of a human IgG4 heavy chain. In some embodiments, a TIGIT binding agent that contains the heavy chain variable region of the 313M32 antibody as part of a human IgG4 heavy chain is referred to as a 313M33 antibody.

In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of the 313M32 antibody. In some embodiments, the TIGIT binding agent comprises, consists essentially of, or consists of a variant of the 313M32 antibody.

In some embodiments, said TIGIT binding agent comprises a heavy chain variable region encoded by a plasmid deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA, USA, under the terms of the Budapest Treaty dated August 11, 2015, and designated RTA-122346. In some embodiments, said TIGIT binding agent comprises a light chain variable region encoded by a plasmid deposited with ATCC, 10801 University Boulevard, Manassas, Virginia, USA under the terms of the Budapest Treaty dated August 11, 2015, and designated PTA-122347. In some embodiments, said TIGIT binding agent comprises a heavy chain variable region encoded by a plasmid deposited with ATCC designated PTA122346 and a light chain variable region encoded by a plasmid deposited with ATCC designated PTA-122347. In some embodiments, said TIGIT binding agent comprises a heavy chain containing a variable region encoded by a plasmid deposited with ATCC and designated PTA-122346. In some embodiments, said TIGIT binding agent comprises a light chain encoded by a plasmid deposited with ATCC and designated PTA-122347. In some embodiments, said TIGIT binding agent comprises a heavy chain containing a variable region encoded by a plasmid deposited with ATCC, designated PTA-122346, and a light chain encoded by a plasmid deposited with ATCC, designated PTA-122347.

The present invention also covers homodimeric agents/molecules and heterodimeric agents/molecules. In some embodiments, said homodimeric agents are polypeptides. In some embodiments, said heterodimeric molecules are polypeptides. Typically, a homodimeric molecule contains two identical polypeptides. Typically, a heterodimeric molecule contains at least two different polypeptides. In some embodiments, said heterodimeric molecule is capable of binding to at least two targets, such as being a bispecific agent. These targets are, for example, two different proteins on the same cell or two different proteins on two separate cells. The term shoulder can be used herein to describe the structure of a homodimeric agent, a heterodimeric agent, and/or a bispecific agent. In some embodiments, each arm contains at least one polypeptide. Typically, each arm of a heterodimeric molecule has a distinct function, such as binding two different targets. In some embodiments, one arm may contain an antigen-binding site from an antibody. In some embodiments, one arm may comprise a receptor binding portion. In some embodiments, one arm may contain a ligand. In some embodiments, one arm may contain a ligand binding region. In some embodiments, the homodimeric agent contains two identical arms. In some embodiments, the heterodimeric agent contains two different arms. In some embodiments, the bispecific agent comprises two different arms.

In some embodiments, the present invention provides a TIGIT binding agent that is a homodimeric molecule. In some embodiments, said homodimeric molecule contains two identical polypeptides. In some embodiments, the present invention provides a TIGIT binding agent that is a heterodimeric molecule. In some embodiments, said heterodimeric molecule contains at least two different polypeptides. In some embodiments, the present invention provides a TIGIT binding agent that is a heterodimeric agent. In some embodiments, the present invention provides a TIGIT binding agent that is a bispecific agent. In some embodiments, said TIGIT binding agent is a bispecific antibody.

In some embodiments, a heterodimeric agent (eg, a bispecific agent) contains a TIGIT binding agent as described herein. In some embodiments, the heterodimeric agent comprises an immune response stimulating agent or a functional fragment thereof. In some embodiments, the heterodimeric molecule has

- 35 039540 at least two functions (i) binding to TIGIT and (ii) binding to a second target. In some embodiments, the heterodimeric agent has at least two functions, (i) binding to TIGIT and (ii) non-binding function. In some embodiments, the heterodimeric molecule comprises a second immunotherapeutic agent, or a functional fragment thereof. In some embodiments, one arm of the heterodimeric molecule contains a TIGIT binding agent as described herein; and one arm of the heterodimeric molecule contains a second immunotherapeutic agent. In some embodiments, one arm of the heterodimeric agent comprises a TIGIT binding agent as described herein and one arm of the heterodimeric agent comprises an immune response stimulating agent. As used herein, the expression "immune response stimulating agent" is used in the broadest possible sense and refers to a substance that directly or indirectly stimulates the immune system by inducing activation or increasing the activity of any components of the immune system. For example, immune response stimulating agents may include cytokines as well as various antigens, including tumor antigens and antigens derived from pathogens. In some embodiments, said second immunotherapeutic agent (e.g., an immune response stimulating agent) includes, but is not limited to, a colony stimulating factor (e.g., granulocyte macrophage colony stimulating factor (GMCSF), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (GCSF), stem cell factor (SCF)), interleukin (e.g. IL-1, IL-2, IL-3, IL-7, IL-12, IL-15, IL-18), cytokine (e.g. γ-interferon), an antibody that blocks immunosuppressive functions (eg, anti-CTLA4 antibody, anti-CD28 antibody, anti-PD-1 antibody, anti-PD-L1 antibody), a Toll-like receptor (eg, TLR4, TLR7, TLR9), or a member of the B7 family (eg, CD80, CD86). In some embodiments, said immunotherapeutic agent includes, but is not limited to, an agonist antibody (eg, an anti-GITR antibody, an anti-OX40 antibody) or an agonist ligand (eg, GITRL or OX40L).

In some embodiments, said TIGIT binding agent is a heterodimeric molecule (eg, a bispecific agent) that contains a first CH3 domain and a second CH3 domain, each of which is modified to promote the formation of heteromultimers. In some embodiments, said first and second CH3 domains are modified in a ridge-to-trough pattern. In some embodiments, said first and second CH3 domains contain amino acid changes that result in altered electrostatic interactions. In some embodiments, said first and second CH3 domains contain amino acid changes that result in altered hydrophobic/hydrophilic interactions.

In some embodiments, said TIGIT binding agent is a bispecific agent that contains heavy chain constant regions selected from the group consisting of: (a) a human IgG1 first constant region, wherein the amino acids corresponding to IgG1 positions 253 and 292 (SEQ ID NO : 41), changed to glutamate or aspartate, and a second human IgG1 constant region, with the amino acids corresponding to positions 240 and 282 of IgG1 (SEQ ID NO: 41) changed to lysine; (b) a first human IgG2 constant region, with amino acids corresponding to positions 249 and 288 of IgG2 (SEQ ID NO: 42) replaced with glutamate or aspartate, and a second constant region of human IgG2, with amino acids corresponding to positions 236 and 278 of IgG2 (SEQ ID NO: 42), replaced by lysine; (c) a first human IgG3 constant region with amino acids corresponding to positions 300 and 339 of IgG3 (SEQ ID NO: 43) replaced with glutamate or aspartate, and a second human IgG3 constant region with amino acids corresponding to positions 287 and 329 of IgG3 (SEQ ID NO: 43), replaced by lysine; and (d) a first human IgG4 constant region, wherein the amino acids corresponding to positions 250 and 289 of IgG4 (SEQ ID NO: 44) are changed to glutamate or aspartate, and a second constant region of IgG4, wherein the amino acids corresponding to positions 237 and 279 of IgG4 (SEQ ID NO: 44), replaced by lysine.

In some embodiments, said TIGIT binding agent is a bispecific agent that comprises a first human IgG1 constant region with amino acid substitutions at positions corresponding to positions 253 and 292 of IgG1 (SEQ ID NO: 41), with said amino acids at positions corresponding to positions 253 and 292 IgG1 (SEQ ID NO: 41) replaced with glutamate or aspartate, and a second human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of IgG1 (SEQ ID NO: 41), with said amino acids at positions corresponding to positions 240 and 282 of IgG1 (SEQ ID NO: 41) were changed to lysine. In some embodiments, said TIGIT binding agent is a bispecific antibody that comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of IgG2 (SEQ ID NO: 42), with said amino acids at positions corresponding to positions 249 and 288 IgG2 (SEQ ID NO: 42), replaced by glutamate or aspartate, and a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of IgG2 (SEQ ID NO: 42), while

- 36 039540 these amino acids at positions corresponding to positions 236 and 278 of IgG2 (SEQ ID NO: 42) are replaced by lysine. In some embodiments, said TIGIT binding agent is a bispecific antibody that comprises a first human IgG3 constant region with amino acid substitutions at positions corresponding to positions 300 and 339 of IgG3 (SEQ ID NO: 43), with said amino acids at positions corresponding to positions 300 and 339 IgG3 (SEQ ID NO: 43) replaced with glutamate or aspartate, and a second human IgG3 constant region with amino acid substitutions at positions corresponding to positions 287 and 329 of IgG3 (SEQ ID NO: 43), with said amino acids at positions corresponding to positions 287 and 329 of IgG3 (SEQ ID NO: 43) were changed to lysine. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the first human IgG4 constant region with amino acid substitutions at positions corresponding to positions 250 and 289 of IgG4 (SEQ ID NO: 44), with said amino acids at positions corresponding to positions 250 and 289 IgG4 (SEQ ID NO: 44) replaced with glutamate or aspartate, and a second human IgG4 constant region with amino acid substitutions at positions corresponding to positions 237 and 279 of IgG4 (SEQ ID NO: 44), with said amino acids at positions corresponding to positions 237 and 279 of IgG4 (SEQ ID NO: 44) were changed to lysine.

In some embodiments, said TIGIT binding agent is a bispecific agent that comprises a first human IgG1 constant region with amino acid substitutions at positions corresponding to positions 253 and 292 of IgG1 (SEQ ID NO: 41), wherein said amino acids are replaced by glutamate, and a second a human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of IgG1 (SEQ ID NO: 41), with these amino acids replaced by lysine. In some embodiments, said TIGIT binding agent is a bispecific antibody that comprises a first human IgG1 constant region with amino acid substitutions at positions corresponding to positions 253 and 292 of IgG1 (SEQ ID NO: 41), with said amino acids replaced with aspartate, and a second a human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of IgG1 (SEQ ID NO: 41), with these amino acids replaced by lysine.

In some embodiments, said TIGIT binding agent is a bispecific agent that comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of IgG2 (SEQ ID NO: 42), wherein said amino acids are replaced with glutamate, and a second a human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of IgG2 (SEQ ID NO: 42), with these amino acids replaced by lysine. In some embodiments, said TIGIT binding agent is a bispecific antibody that comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of IgG2 (SEQ ID NO: 42), with said amino acids replaced with aspartate, and a second a human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of IgG2 (SEQ ID NO: 42), with these amino acids replaced by lysine.

In some embodiments, said TIGIT binding agent is a bispecific agent that contains the heavy chain constant region of SEQ ID NO: 45. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain constant region of SEQ ID NO: 46. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain constant region of SEQ ID NO: 47. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain constant region of SEQ ID NO: NO: 48. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the first heavy chain constant region in sequence of SEQ ID NO: 45 and the second heavy chain constant region of SEQ ID NO: 46. In some embodiments, said TIGIT binding agent is a bispecific antibody that comprises the first heavy chain constant region of SEQ ID NO: 47 and the second heavy chain constant region of SEQ ID NO: 47. chain sequence SEQ ID NO: 48.

In some embodiments, said TIGIT binding agent is a bispecific agent that contains the heavy chain constant region of SEQ ID NO: 49. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain constant region of SEQ ID NO: 50. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain constant region of SEQ ID NO: 51. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain constant region of SEQ ID NO: NO: 52. According to some implementations

said TIGIT binding agent is a bispecific antibody that comprises the first heavy chain constant region of SEQ ID NO: 49 and the second heavy chain constant region of SEQ ID NO: 50. In some embodiments, said TIGIT binding agent is a bispecific an antibody that contains the first heavy chain constant region of SEQ ID NO: 51 and the second heavy chain constant region of SEQ ID NO: 52.

In some embodiments, said TIGIT binding agent is a bispecific antibody containing a first antigen-binding site that specifically binds human TIGIT. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first antigen-binding site that specifically binds human TIGIT and a second antigen-binding site that binds a second target. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, wherein said first antigen-binding site comprises a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9). In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, wherein said first antigen-binding site comprises a heavy chain CDR1 comprising GSSLSSSYMS (SEQ ID NO: 7), a heavy chain CDR2, containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9). In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, wherein said first antigen-binding site comprises a heavy chain CDR1 comprising GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2, containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9). In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, said first antigen-binding site comprising: (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9), and (b) a second antigen-binding site, wherein said first the antigen-binding site and said second antigen-binding site contain the same (ie, identical) light chain. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, said first antigen-binding site comprising (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8) and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID N0:9), a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10) , QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11) or RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHSVGNVDN ( SEQ ID NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16) and (b) a second antigen binding site. In some embodiments, said bispecific antibody comprises a first antigen-binding site comprising a light chain CDR1 comprising QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 comprising DALKLAS (SEQ ID NO: 11) or RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16).

In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region differing by at least about 80% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32. In some embodiments, implementation, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32. In some embodiments, said bispecific antibody comprises a light chain variable region that differs by at least about 80% sequence identity to SEQ ID NO: 18, or SEQ ID NO: 20. According to some embodiments, said bispecific antibody comprises a light chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identical

- 38 039540 the sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region comprising SEQ ID NO: 17. In some embodiments, said binding agent The TIGIT agent is a bispecific antibody comprising a heavy chain first variable region comprising SEQ ID NO: 19. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a heavy chain first variable region comprising SEQ ID NO: 32. In some embodiments, said TIGIT binding agent is a heavy chain first variable region antibody comprising SEQ ID NO: 32. embodiments, said TIGIT binding agent is a bispecific antibody comprising a first light chain variable region comprising SEQ ID NO: 18. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising the first a light chain variable region comprising SEQ ID NO: 20.

In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region comprising SEQ ID NO: 17 and a first heavy chain constant region comprising SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO : 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52. In some embodiments, said TIGIT binding agent is a bispecific antibody containing the first variable region heavy chain comprising SEQ ID NO: 19 and a first heavy chain constant region comprising SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region comprising SEQ ID NO: 32 and a first heavy chain constant region, containing SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52.

In some embodiments, said TIGIT binding agent is a bispecific antibody containing a first antigen-binding site that specifically binds human TIGIT. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first antigen binding site that specifically binds human TIGIT and a second antigen binding site that binds a second target. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, said first antigen-binding site comprising a heavy chain CDR1 comprising TSDYAWN (SEQ ID NO: 57), a heavy chain CDR2, containing YISYSGSTSYNPSLRS (SEQ ID NO: 58), and a heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59). In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, wherein said first antigen-binding site comprises (a) a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), CDR2 a heavy chain containing YISYSGSTSYNPSLRS (SEQ ID NO: 58) and a heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59); and (b) a second antigen-binding site, wherein said first antigen-binding site and said second antigen-binding site contain the same (ie, identical) light chain. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising: a first antigen-binding site that specifically binds human TIGIT, said first antigen-binding site comprising: (a) a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), A heavy chain CDR2 containing YISYSGSTSYNPSLRS (SEQ ID NO: 58) and a heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59), a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 containing SASYRYT ( SEQ ID NO: 61), and a light chain CDR3 containing QQHYSTP (SEQ ID NO: 62); and (b) a second antigen binding site. In some embodiments, said bispecific antibody comprises a first antigen binding site comprising a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 containing SASYRYT (SEQ ID NO: 61), and a light chain CDR3 containing QQHYSTP (SEQ ID NO: 62).

In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region differing by at least about 80% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 67. In some embodiments, said TIGIT binding agent is is a bispecific antibody comprising a first heavy chain variable region differing by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 67. In some embodiments, said bispecific antibody comprises a light chain variable region that shares at least about 80% sequence identity with SEQ ID NO:

- 39 039540 or SEQ ID NO: 68. In some embodiments, said bispecific antibody comprises a light chain variable region that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97 % or at least about 99% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region comprising SEQ ID NO: 63. According to In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region comprising SEQ ID NO: 67. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first light chain variable region comprising SEQ ID NO: : 6 4. In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first light chain variable region comprising SEQ ID NO: 68.

In some embodiments, said TIGIT binding agent is a bispecific antibody comprising a first heavy chain variable region comprising SEQ ID NO: 63 and a first heavy chain constant region comprising SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO : 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52. In some embodiments, said TIGIT binding agent is a bispecific antibody containing the first variable region heavy chain comprising SEQ ID NO: 67 and a first heavy chain constant region comprising SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52.

In some embodiments, said TIGIT binding agent is a bispecific antibody that specifically binds human TIGIT and a second target. In some embodiments, said second target is a tumor antigen. In some embodiments, said bispecific antibody comprises a TIGIT binding agent as described herein and a second polypeptide comprising an antibody that specifically binds a tumor antigen. A bispecific antibody having binding specificity for a tumor antigen can be used to target a TIGIT binding agent to a tumor. This may be useful for inducing and/or enhancing an immune response in the vicinity of the tumor or within the tumor microenvironment. In some embodiments, a bispecific antibody can be used to induce or enhance the activity of tumor-infiltrating immune cells. In some embodiments, a bispecific antibody can be used to induce or enhance tumor infiltrating lymphocyte (TIL) activity. In some embodiments, the bispecific antibody may be used to inhibit or reduce the activity of Treg cells. In some embodiments, the bispecific antibody may be used to inhibit or reduce the activity of SPCM.

In some embodiments, said TIGIT binding agent is a bispecific antibody, characterized in that the first target is TIGIT and the second target is located on an immune response cell. In some embodiments, said second target is located on a T cell, NK cell, B cell, macrophage, dendritic cell, or myeloid cell. In some embodiments, said second target is PD-1, PD-L1, CTLA4, TIM-3, LAG-3, GITR, OX-40, GITRL, or OX-40L. In some embodiments, said second target is CD28 or 4-1BB.

In some embodiments, the bispecific antibody comprises a first arm comprising a TIGIT binding agent as described herein and a second arm comprising an antibody that specifically binds PD-1. In some embodiments, the bispecific antibody comprises a first arm comprising a TIGIT binding agent as described herein and a second arm comprising an antibody that specifically binds PD-L1. In some embodiments, the bispecific antibody comprises a first arm comprising a TIGIT binding agent as described herein and a second arm comprising an antibody that specifically binds GITR. In some embodiments, the bispecific antibody comprises a first arm containing the TIGIT binding agent described herein and a second arm containing an antibody that specifically binds OX-40. In some embodiments, the bispecific antibody comprises a first arm comprising a TIGIT binding antibody described herein and a second arm comprising an antibody specifically binding to CD40. In some embodiments, the bispecific antibody comprises a first arm comprising a TIGIT binding agent as described herein and a second arm comprising an antibody that specifically binds CTLA4. In some embodiments, the bispecific antibody comprises a first arm comprising a TIGIT binding antibody described herein and a second arm comprising an antibody specifically binding to CD28. In some embodiments, the bispecific antibody comprises a first arm comprising a TIGIT binding agent as described herein and a second arm comprising an antibody specifically binding to GITRL. In some embodiments, the bispecific antibody comprises a first arm containing the TIGIT binding agent described herein and a second arm containing an antibody that specifically binds OX-40L.

In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain variable region from the 313R11 anti-TIGIT antibody. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain variable region from the 313R12 anti-TIGIT antibody. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain variable region from the 313R14 anti-TIGIT antibody. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain variable region from an anti-TIGIT antibody 313R19 or 313R20. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the light chain variable region from an anti-TIGIT antibody 313R11 or 313R12. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains a light chain variable region from an anti-TIGIT antibody 313R14, 313R19, or 313R20.

In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain variable region from the anti-TIGIT antibody 313M26. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the light chain variable region from the 313M26 anti-TIGIT antibody.

In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the heavy chain variable region from the 313M32 anti-TIGIT antibody. In some embodiments, said TIGIT binding agent is a bispecific antibody that contains the light chain variable region from the 313M32 anti-TIGIT antibody.

In some embodiments, the bispecific agent comprises a first arm comprising a TIGIT binding agent described herein and a second arm comprising a GITRL-containing polypeptide that specifically binds GITR. In some embodiments, said second arm comprises a polypeptide comprising at least one copy of extracellular domain of GITRL. In some embodiments, the bispecific agent comprises a first arm comprising a TIGIT binding agent as described herein and a second arm comprising an OX-40L containing polypeptide that specifically binds OX-40. In some embodiments, said second arm comprises a polypeptide containing at least one copy of the extracellular domain of OX-40L. In some embodiments, the bispecific agent comprises a first arm comprising a TIGIT binding agent as described herein and a second arm comprising a CD40L containing polypeptide that specifically binds CD40. In some embodiments, said second arm comprises a polypeptide containing at least one copy of the CD40L extracellular domain. In some embodiments, the bispecific agent comprises a first arm containing the TIGIT binding agent described herein and a second arm containing a polypeptide containing a 4-1BB ligand that specifically binds 4-1BB. In some embodiments, said second arm comprises a polypeptide containing at least one copy of the extracellular domain of the 4-1BB ligand.

In some embodiments, said TIGIT binding agent is a bispecific agent that binds TIGIT to a KD of about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less; or about 0.1 nM or less. In some embodiments, said TIGIT binding agent is a bispecific agent that binds the second target with a K _D of about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less; or about 0.1 nM or less. In some embodiments, said TIGIT binding agent is a bispecific agent that binds TIGIT to a K _D of about 50 nM or less and binds the second target to a K D of about 50 nM or less. In some embodiments, said TIGIT binding agent is a bispecific agent that binds TIGIT to a KD of about 25 nM or less and binds the second target to a KD of about 25 nM or less. In some embodiments, said TIGIT binding agent is a bispecific agent that binds TIGIT to a KD of about 10 nM or less and binds the second target to a KD of about 10 nM or less.

In some embodiments, said TIGIT binding agent is a bispecific agent that binds TIGIT to a K _D of about 1 nM or less and binds the second target to a K _D of about 1 nM or less.

In some embodiments, said TIGIT binding agent is a bispecific agent that contains a single antigen-binding site with a binding affinity that is weaker than the binding affinity of the second antigen-binding site. For example, in some embodiments, said bispecific agent may bind TIGIT to a K _D in the range of about 0.1 to 1 nM, and may bind the second target to a K _D in the range of about 1 to 10 nM, or said bispecific agent may bind TIGIT with a K _D in the range of about 1 to 10 nM, and can bind the second target with a K _D in the range of about 0.1 to 1 nM. In some embodiments, said bispecific agent may bind TIGIT to a K _D in the range of about 0.1 to 1 nM, and may bind the second target to a KD in the range of about 1 to 10 nM, or said bispecific agent may bind TIGIT to a K _D in the range of about 1 to 10 nM, and can bind the second target to a KD in the range of about 0.1 to 1 nM. In some embodiments, said difference in affinity between two antigen binding sites can be about 2-fold or more, about 3-fold or more, about 5-fold or more, about 8-fold or more, about 10-fold or more, about 15 times or more, about 30 times or more, about 50 times or more, or about 100 times or more. In some embodiments, at least one amino acid residue in at least one CDR of the TIGIT antigen-binding site is replaced with a different amino acid such that the affinity of the TIGIT binding site is changed. In some embodiments, the affinity of the TIGIT binding site is increased. In some embodiments, the affinity of the TIGIT binding site is reduced. In some embodiments, at least one amino acid residue in at least one CDR of the antigen-binding site for the second target is replaced with a different amino acid such that the affinity of the second antigen-binding site is changed. In some embodiments, the affinity of the second antigen binding site is increased. In some embodiments, the affinity of the second antigen binding site is reduced. In some embodiments, the affinity of both the TIGIT binding site and the second antigen binding site is changed.

The present invention provides polypeptides, including, but not limited to, antibodies that specifically bind TIGIT. In some embodiments, the polypeptide binds human TIGIT. In some embodiments, the polypeptide binds mouse TIGIT. In some embodiments, the polypeptide binds mouse TIGIT and human TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind mouse TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind rat TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind rabbit TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind marmoset TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind dog TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind porcine TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind cynomolgus TIGIT. In some embodiments, the polypeptide binds human TIGIT and does not bind rhesus monkey TIGIT.

In some embodiments, the polypeptide comprises one, two, three, four, five, and/or six CDRs of the 313R11, 313R12, 313R14, or 313R19 antibody (see Table 1 herein). The 313R20 antibody contains the same CDRs and heavy and light chain variable domains as the 313R19 antibody, however 313R20 contains these regions and domains in IgG4 format. In some embodiments, the polypeptide comprises a CDR region(s) containing(s) up to four (ie, 0, 1, 2, 3, or 4) amino acid substitutions per CDR. In some embodiments, said heavy chain CDR region(s) are included in a heavy chain variable region. In some embodiments, said light chain CDR region(s) is/are within a light chain variable region.

In some embodiments, the present invention provides a polypeptide that specifically binds TIGIT, characterized in that said polypeptide contains an amino acid sequence that differs by at least about 80% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32 and/or an amino acid sequence that differs by at least about 80% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said polypeptide comprises an amino acid sequence that differs by at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32. In some embodiments, said polypep - 42 039540 type contains an amino acid sequence that differs from about at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 18 or SEQ ID NO: 20. According to some embodiments, said polypeptide comprises an amino acid sequence that differs by at least about 95% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32, and/or an amino acid sequence that differs by at least about 95% sequence identity SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 17 and/or an amino acid sequence comprising SEQ ID NO: 18. In some embodiments, said polypeptide comprises the sequence amino acids containing SEQ ID NO: 19, and/or an amino acid sequence containing S EQ ID NO: 20. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 32 and/or an amino acid sequence comprising SEQ ID NO: 20.

In some embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and/or SEQ ID NO: 32. As defined herein, a polypeptide may be obtained in single-stranded form or in the form of two or more associated chains. In some embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 17 and an amino acid sequence comprising SEQ ID NO: 18. In some embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 19 and an amino acid sequence comprising SEQ ID NO: 19. NO: 20. In some embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 32 and an amino acid sequence comprising SEQ ID NO: 20. In some embodiments, said polypeptide comprises an amino acid sequence consisting essentially of SEQ ID NO: 17 and an amino acid sequence consisting essentially of the sequence of SEQ ID NO: 18. In some embodiments, said polypeptide comprises an amino acid sequence consisting essentially of the sequence of SEQ ID NO: 19 and an amino acid sequence consisting essentially of sequence of SEQ ID NO: 20. In some embodiments, said polypeptide comprises an amino acid sequence consisting essentially of the sequence of SEQ ID NO: 32 and an amino acid sequence consisting essentially of the sequence of SEQ ID NO: 20. In some embodiments, said the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 17 and an amino acid sequence consisting of SEQ ID NO: 18. In some embodiments, said polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 19 and an amino acid sequence consisting of SEQ ID NO: 20. In some embodiments, said polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 32 and an amino acid sequence consisting of SEQ ID NO: 20.

In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO : 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 55, and/or SEQ ID NO: 56. In some embodiments, implementation of the specified polypeptide contains an amino acid sequence containing SEQ ID NO: 21, and an amino acid sequence containing SEQ ID NO: 23. According to some embodiments, the implementation of the specified polypeptide contains an amino acid sequence containing SEQ ID NO: 22, and an amino acid sequence containing SEQ ID NO: : 23. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 24 and an amino acid sequence comprising SEQ ID NO: 25. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 33 and an amino acid sequence comprising SEQ ID NO: 25. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 55 and an amino acid sequence comprising SEQ ID NO: 25. In some embodiments, said polypeptide comprises said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 26 and an amino acid sequence comprising SEQ ID NO: 28. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 27 and an amino acid sequence comprising SEQ ID NO: 28. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 29 and an amino acid sequence comprising SEQ ID NO: 30. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 34 and an amino acid sequence , containing SEQ ID NO: 30. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 56 and an amino acid sequence comprising SEQ ID NO: 30.

- 43 039540

In some embodiments, the TIGIT binding agent comprises a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18,

SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID

NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 56.

In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 21 and an amino acid sequence consisting of SEQ ID NO: 23. In some embodiments, the polypeptide contains an amino acid sequence consisting of SEQ ID NO: 22 and an amino acid sequence, consisting of SEQ ID NO: 23. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 24 and an amino acid sequence consisting of SEQ ID NO: 25. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 25. NO: 33 and an amino acid sequence consisting of SEQ ID NO: 25. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 55 and an amino acid sequence consisting of SEQ ID NO: 25. In some embodiments, the polypeptide contains a sequence of amino acids consisting of SEQ ID NO: 26 and an amino acid sequence consisting of SEQ ID NO: 28. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 27 and an amino acid sequence consisting of SEQ ID NO: 28. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 29 and an amino acid sequence of SEQ ID NO: 30. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 34 and an amino acid sequence of consisting of SEQ ID NO: 30. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 56 and an amino acid sequence consisting of SEQ ID NO: 30.

In some embodiments, the polypeptide comprises one, two, three, four, five, and/or six CDRs of the 313M26 or 313M32 antibody (see Table 2 herein). In some embodiments, the polypeptide comprises CDR regions containing up to four (ie, 0, 1, 2, 3, or 4) amino acid substitutions per CDR. In some embodiments, said heavy chain CDR region(s) are included in the heavy chain variable region. In some embodiments, said light chain CDR region(s) are included in the light chain variable region.

In some embodiments, the present invention provides a polypeptide that specifically binds TIGIT, characterized in that said polypeptide contains an amino acid sequence that differs by at least about 80% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 67, and/or the sequence amino acids that differ by at least about 80% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said polypeptide comprises an amino acid sequence that differs by at least about 85%, at least about 90%, at least at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 67. In some embodiments, said polypeptide contains an amino acid sequence that differs by at least about 85%, by me having at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, said polypeptide comprises an amino acid sequence, having at least about 95% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 167 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 64 or SEQ ID NO: 68. In some embodiments, implementation of the specified polypeptide contains an amino acid sequence containing SEQ ID NO: 63, and/or an amino acid sequence containing SEQ ID NO: 64. According to some embodiments, the specified polypeptide contains an amino acid sequence containing SEQ ID NO: 67, and/or an amino acid sequence, containing SEQ ID NO: 68.

In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 82, and/or SEQ ID NO: 83. As defined herein, a polypeptide can be produced in single chain form or as two or more associated chains. In some embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 63 and an amino acid sequence comprising SEQ ID NO: 64. In some embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 67 and an amino acid sequence comprising SEQ ID NO: 67 NO: 68. In some embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 69 and an amino acid sequence comprising SEQ ID NO: 71. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 70 and an amino acid sequence comprising SEQ ID NO: 72. In some embodiments, said polypeptide comprises an amino acid sequence comprising SEQ ID NO: 82 and an amino acid sequence comprising SEQ ID NO: 72.

In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 63 and an amino acid sequence of SEQ ID NO: 64. In some embodiments, the polypeptide comprises an amino acid sequence of SEQ ID NO: 67 and an amino acid sequence of consisting of SEQ ID NO: 68. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 69 and an amino acid sequence consisting of SEQ ID NO: 71. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 71. NO: 70 and an amino acid sequence consisting of SEQ ID NO: 72. In some embodiments, the polypeptide comprises an amino acid sequence consisting of SEQ ID NO: 82 and an amino acid sequence consisting of SEQ ID NO: 72.

Many proteins, including antibodies, contain a signal sequence that directs the transport of these proteins to different locations. Typically, signal sequences (also referred to as signal peptides or leader sequences) are located at the N terminus of the resulting polypeptides. They target the polypeptide to the endoplasmic reticulum and proteins are sorted to their destination, for example, to the interior of the organelle, to the inner membrane, to the outer membrane of the cell, or to the extracellular space by secretion. Most of the signal sequences are cleaved from the protein by a signal peptidase after transport of the proteins to the endoplasmic reticulum. Cleavage of a signal sequence from a polypeptide usually occurs at a specific site in the amino acid sequence and depends on the amino acid residues within the signal sequence. Although there is typically one specific cleavage site, the signal peptidase may recognize and/or use more than one cleavage site, resulting in inhomogeneous N-terminus of the polypeptide. For example, the use of different cleavage sites within a signal sequence may result in the expression of a polypeptide with different N-terminal amino acids. Accordingly, in some embodiments, the polypeptides as described herein may contain a mixture of polypeptides with different N-termini. In some embodiments, the length of said N-termini differs by 1, 2, 3, 4, or 5 amino acids. In some embodiments, said polypeptide is substantially homogeneous, i. polypeptides contain the same N-terminus. In some embodiments, the polypeptide signal sequence contains one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) amino acid substitutions and/or deletions compared to natural or original signal sequence. In some embodiments, the polypeptide signal sequence contains amino acid substitutions and/or deletions that allow a single cleavage site to dominate, resulting in a substantially homogeneous single N-terminal polypeptide. In some embodiments, the signal sequence of the polypeptide affects the level of expression of the polypeptide, such as increasing or decreasing expression.

In some embodiments, an antibody competes for specific binding to TIGIT with a TIGIT binding agent described herein.

In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising: (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9) and (b) a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11) or RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16).

In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising: (a) a heavy chain variable region comprising SEQ ID NO: 17, SEQ ID NO: 19, or SeQ ID NO: 32, and (b) a heavy chain variable region light chain comprising SEQ ID NO: 18 or SEQ ID NO: 20. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain variable region comprising SEQ ID NO: 17 and a light chain variable region , containing SEQ ID NO: 18. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent containing a heavy chain variable region containing SEQ ID NO: 19 and a light chain variable region containing SEQ ID NO: 20. In some embodiments, the antibody competes for specific binding with TIGIT

- 45 039540 with a TIGIT binding agent containing a heavy chain variable region containing SEQ ID

NO: 32, and a light chain variable region containing SEQ ID NO: 20.

In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain comprising SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, or SEQ ID NO: 56 , and a light chain containing SEQ ID NO: 28 or SEQ ID NO: 30. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent containing a heavy chain containing SEQ ID NO: 26 and a light chain containing SEQ ID NO: 28. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain containing SEQ ID NO: 27 and a light chain containing SEQ ID NO: 28. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain comprising SEQ ID NO: 29 and a light chain comprising SEQ ID NO: 30. In some embodiments, ant an antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain containing SEQ ID NO: 34 and a light chain containing SEQ ID NO: 30. In some embodiments, an antibody competes for specific binding to TIGIT with a TIGIT binding agent, containing a heavy chain containing SEQ ID NO: 56 and a light chain containing SEQ ID NO: 30.

In some embodiments, the antibody competes with the 313R11 antibody for specific binding to TIGIT. In some embodiments, the antibody competes with the 313R12 antibody for specific binding to TIGIT. In some embodiments, the antibody competes with the 313R14 antibody for specific binding to human TIGIT. In some embodiments, the antibody competes with the 313R19 antibody for specific binding to human TIGIT. In some embodiments, the antibody competes with the 313R20 antibody for specific binding to human TIGIT.

In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, wherein said reference antibody is a 313R11 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, wherein said reference antibody is a 313R12 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, wherein said reference antibody is a 313R14 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, wherein said reference antibody is a 313R19 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, wherein said reference antibody is a 313R20 antibody.

In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the TIGIT binding agent of the present invention. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313R11 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313R12 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313R14 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313R19 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313R20 antibody.

In another embodiment, the antibody binds an epitope on TIGIT overlapping with an epitope on TIGIT that binds a TIGIT binding agent of the present invention. In some embodiments, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313R11 antibody. In another embodiment, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313R12 antibody. In some embodiments, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313R14 antibody. In some embodiments, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313R19 antibody. In some embodiments, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313R20 antibody.

In some embodiments, an antibody competes for specific binding to TIGIT with a TIGIT binding agent described herein. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising: (a) a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), a heavy chain CDR2 containing YISYSGSTSYNPSLRS (SEQ ID NO: 58), and A heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59) and (b) a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 containing SASYRYT (SEQ ID NO: 61) and a light chain CDR3, containing

- 46 039540

QQHYSTP (SEQ ID NO: 62).

In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising (a) a heavy chain variable region comprising SEQ ID NO: 63 or SEQ ID NO: 67 and (b) a light chain variable region comprising SEQ ID NO: : 64 or SEQ ID NO: 68. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain variable region comprising SEQ ID NO: 63 and a light chain variable region comprising SEQ ID NO: 64 In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain variable region comprising SEQ ID NO: 167 and a light chain variable region comprising SEQ ID NO: 68. In some embodiments, the antibody competes for specific binding to TIGIT with a TIGIT binding agent containing a heavy chain containing SEQ ID NO: 70 and a light chain comprising SEQ ID NO: 72. In some embodiments, an antibody competes for specific binding to TIGIT with a TIGIT binding agent comprising a heavy chain comprising SEQ ID NO: 82 and a light chain comprising SEQ ID NO: 72 .

In some embodiments, the antibody competes with the 313M26 antibody for specific binding to TIGIT. In some embodiments, the antibody competes with the 313M32 antibody for specific binding to TIGIT. In some embodiments, the antibody competes with the 313M32 antibody for specific binding to human TIGIT. In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, said reference antibody being a 313M26 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, wherein said reference antibody is a 313M32 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to TIGIT, said reference antibody being a 313M33 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to human TIGIT, said reference antibody being a 313M32 antibody. In some embodiments, the antibody competes with a reference antibody for specific binding to human TIGIT, said reference antibody being a 313M33 antibody.

In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the TIGIT binding agent described herein. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313M26 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313M32 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on TIGIT as the 313M33 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on human TIGIT as the 313M32 antibody. In some embodiments, the antibody binds the same epitope or substantially the same epitope on human TIGIT as the 313M33 antibody.

In another embodiment, the antibody binds an epitope on TIGIT overlapping with an epitope on TIGIT that binds the TIGIT binding agent described herein. In some embodiments, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313M26 antibody. In another embodiment, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313M32 antibody. In another embodiment, said antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the 313M33 antibody. In another embodiment, said antibody binds an epitope on TIGIT that overlaps with an epitope on human TIGIT that binds the 313M32 antibody. In another embodiment, said antibody binds an epitope on TIGIT that overlaps with an epitope on human TIGIT that binds the 313M33 antibody.

In some embodiments, the antibody competes for binding to an epitope containing the amino acids included in the sequence of SEQ ID NO: 79 with a TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing the amino acids included in the sequence of SEQ ID NO: 80 with a TIGIT binding agent described herein. In some embodiments, an antibody competes for binding to an epitope containing the amino acids included in SEQ ID NO: 79 and SEQ ID NO: 80 with a TIGIT binding agent described herein. In some embodiments, an antibody competes for binding to an epitope containing amino acids Q62 and I109 of SEQ ID NO: 4 with a TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing the amino acids Q62 and

- 47 039540

T119 of SEQ ID NO: 4, with the TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing amino acids Q64 and I109 of SEQ ID NO: 4 with a TIGIT binding agent described herein. In some embodiments, an antibody competes for binding to an epitope containing the Q64 and T119 amino acids of SEQ ID NO: 4 with a TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing the amino acids Q62, Q64, and I109 of SEQ ID NO: 4 with the TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing amino acids Q62, Q64 and T119 of SEQ ID NO: 4 with a TIGIT binding agent described herein. In some embodiments, an antibody competes for binding to an epitope containing amino acids Q62, I109, and T119 of SEQ ID NO: 4 with a TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing amino acids Q64, I109 and T119 of SEQ ID NO: 4 with a TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing amino acids Q62, Q64, I109, and T119 of SEQ ID NO: 4 with the TIGIT binding agent described herein. In some embodiments, the antibody competes for binding to an epitope containing at least one amino acid selected from the group consisting of: N58, E60, Q62, Q64, L65, F107, I109, H111, T117, T119, G120, and R121 of the SEQ sequence ID NO: 4, with the TIGIT binding agent described herein.

In some embodiments, said TIGIT binding agent (eg, antibody) described herein binds TIGIT and modulates TIGIT activity. In some embodiments, said TIGIT binding agent is a TIGIT antagonist and reduces TIGIT activity. In some embodiments, said TIGIT binding agent inhibits TIGIT activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%. , at least about 90% or about 100%. In some embodiments, the TIGIT binding agent that inhibits TIGIT activity is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that inhibits human TIGIT activity is a humanized version of a 313R11 antibody, 313R12 antibody, 313R14 antibody, 313R19 antibody, or 313R20 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT activity is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that inhibits human TIGIT activity is a humanized version of a 313M26 antibody (eg, a 313M32 antibody). In some embodiments, the TIGIT binding agent that inhibits TIGIT activity is the 313M32 antibody.

In some embodiments, the TIGIT binding agents described herein bind TIGIT and inhibit or reduce TIGIT signaling. In some embodiments, said TIGIT binding agent (e.g., antibody) inhibits TIGIT signaling by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least at least about 75%, at least about 90%, or about 100%. In some embodiments, said TIGIT binding agent inhibits mouse TIGIT signaling. In some embodiments, said TIGIT binding agent inhibits human TIGIT signaling. In some embodiments, said TIGIT binding agent inhibits human and mouse TIGIT signaling. In some embodiments, the TIGIT binding agent that inhibits TIGIT signaling is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT signaling is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT signaling is the 313M32 antibody.

Phosphorylation of the cytoplasmic tail of TIGIT occurs after interaction with its PVR counterreceptor. TIGIT phosphorylation represents the start of a cascade that includes downstream events affecting other known signaling pathways. Accordingly, an assessment of TIGIT phosphorylation can provide information on TIGIT activity and TIGIT signaling.

Phosphorylation assays are known to those skilled in the art and are widely used to monitor protein activation and/or pathway activation. These assays can be used to monitor the effect of various treatment options on target protein and/or target pathway activation. For example, an in vitro phosphorylation assay can be used to assess the effect of an antagonist

- 48 039540

TIGIT to PVR-induced TIGIT activation.

In some embodiments, said TIGIT binding agent (eg, antibody) inhibits binding of TIGIT to the receptor. In some embodiments, said TIGIT binding agent inhibits TIGIT binding to PVR. In some embodiments, said TIGIT binding agent inhibits TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4. In some embodiments, said inhibition of binding of a TIGIT binding agent to PVR is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%. % or at least about 95%. In some embodiments, said inhibition of the binding of a TIGIT binding agent to PVR-L2, PVR-L3, and/or PVR-L4 is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In some embodiments, the TIGIT binding agent that inhibits TIGIT binding to PVR is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4 is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT binding to PVR is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT binding to PVR is the 313M32 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4 is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that inhibits TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4 is the 313M32 antibody.

In some embodiments, said TIGIT binding agent (eg, antibody) blocks TIGIT from binding to the receptor. In some embodiments, said TIGIT binding agent blocks TIGIT from binding to the PVR. In some embodiments, said blocking of binding to a PVR TIGIT binding agent is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%. % or at least about 95%. In some embodiments, said TIGIT binding agent blocks TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4. In some embodiments, the binding of the TIGIT binding agent to PVR-L2, PVR-L3, and/or PVR-L4 is blocked by at least about 10%, at least about 25%, at least about 50%, at least at least about 75%, at least about 90%, or at least about 95%. In some embodiments, the TIGIT binding agent that blocks TIGIT binding to PVR is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that blocks TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4 is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that blocks TIGIT binding to PVR is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that blocks TIGIT binding to PVR is the 313M32 antibody. In some embodiments, the TIGIT binding agent that blocks TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4 is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that blocks TIGIT binding to PVR-L2, PVR-L3, and/or PVR-L4 is the 313M32 antibody.

Binding assays are known to those skilled in the art and are described herein. Binding assays can be used to monitor the effect of a test agent on the interaction between a target protein and a binding partner of said protein (eg, a receptor or ligand). For example, an in vitro binding assay can be used to evaluate whether a TIGIT antagonist blocks the interaction of TIGIT with PVR.

In some embodiments, the TIGIT binding agents described herein have one or more of the following effects: inhibit tumor cell proliferation; inhibit tumor growth; reduce tumor tumorigenicity; death of tumor cells, enhance or additionally stimulate the immune response, enhance or additionally stimulate the antitumor response, increase the cytolytic activity of immune cells, increase the killing of tumor cells, increase the killing of tumor cells by immune cells, induce cell differentiation in tumors, differentiation of tumorigenic cells

- 49 039540 current into non-tumorogenic cells, induce expression of differentiation markers in tumor cells, prevent tumor cell metastasis, decrease tumor cell survival, increase cell contact dependent growth inhibition, increase tumor cell apoptosis, reduce epithelial-mesenchymal transition (EMT), or reduce the survival of tumor cells. In some embodiments, said agents have one or more of the following effects: inhibit viral infection, inhibit chronic viral infection, reduce viral load, induce cell death of virus-infected cells, or reduce the number or percentage of virus-infected cells.

In some embodiments, the TIGIT binding agents described herein inhibit tumor growth. In some embodiments, said TIGIT binding agents inhibit tumor growth in vivo (eg, in a mouse model and/or in a human cancer patient). In some embodiments, tumor growth is inhibited at least about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 50-fold, about 100-fold, or about 1000-fold compared to an untreated tumor.

In some embodiments, the TIGIT binding agents described herein reduce the tumorigenicity of a tumor. In some embodiments, said TIGIT binding agents reduce tumor tumorigenicity in an animal model, such as a mouse model. In some embodiments, said mouse model is a xenograft mouse model. In some embodiments, the TIGIT binding agent does not bind mouse TIGIT and is ineffective in a mouse model. In some embodiments, a surrogate TIGIT binding agent that binds mouse TIGIT is used in a mouse model. In some embodiments, said TIGIT binding agents reduce the tumorigenicity of a tumor containing cancer stem cells in an animal model, such as a mouse model. In some embodiments, the number or frequency of cancer stem cells in a tumor is reduced by at least about 2-fold, about 3-fold, about 5-fold, about 10-fold, about 50-fold, about 100-fold, or about 1000-fold. In some embodiments, the reduction in the number or frequency of cancer stem cells is determined by serial dilution analysis using an animal model. Additional examples and guidance on the use of serial dilution assays to determine the reduction in the number or frequency of cancer stem cells in a tumor can be found, for example, in international publication WO 2008/042236; US Patent Publication No. 2008/0064049; and US Patent Publication No. 2008/0178305.

In some embodiments, the agents (eg, polypeptides and/or antibodies) described herein bind TIGIT and modulate an immune response. In some embodiments, the TIGIT binding agent described herein activates and/or enhances an immune response. In some embodiments, the TIGIT binding agent enhances cellular immunity, stimulates cellular immunity, or enhances cellular immunity. In some embodiments, the TIGIT binding agent enhances innate cellular immunity, stimulates innate cellular immunity, or enhances innate cellular immunity. In some embodiments, the TIGIT binding agent enhances adaptive cellular immunity, stimulates adaptive cellular immunity, or enhances adaptive cellular immunity. In some embodiments, the TIGIT binding agent increases T cell activity, stimulates T cell activity, or enhances T cell activity. In some embodiments, the TIGIT binding agent increases cytolytic T-cell (CTL) activity, stimulates cytolytic T-cell (CTL) activity, or enhances cytolytic T-cell (CTL) activity. In some embodiments, the TIGIT binding agent increases the activity, stimulates the activity, or enhances the activity of NK cells. In some embodiments, the TIGIT binding agent increases the activity, stimulates the activity, or enhances the activity of lymphokine-activated killer (LAK) cells. In some embodiments, the TIGIT binding agent increases the activity, stimulates the activity, or enhances the activity of tumor infiltrating lymphocytes (TIF). In some embodiments, the TIGIT binding agent inhibits or reduces the activity of Treg cells. In some embodiments, the TIGIT binding agent inhibits or reduces the activity of SCMP. In some embodiments, the TIGIT binding agent increases tumor cell killing, promotes tumor cell killing, or enhances tumor cell killing. In some embodiments, the TIGIT binding agent increases the inhibition, stimulates the inhibition, or enhances the inhibition of tumor growth.

In some embodiments, the agent described herein is a human TIGIT antagonist. In some embodiments, said agent is a TIGIT antagonist and activates and/or enhances an immune response. According to some

- 50 039540 variants of implementation of the specified agent is a TIGIT antagonist and activates and/or increases the activity of NK cells. In some embodiments, said agent increases said activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90% or about 100%. In some embodiments, said agent is a TIGIT antagonist and activates T cells and/or increases T cell activity (eg, T cell cytolytic activity). In some embodiments, said agent increases said activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90% or about 100%. In some embodiments, said agent is a TIGIT antagonist and induces and/or enhances a Th1-type immune response. In general, a Th1-type immune response includes the production of interferon-gamma (IFN-γ), IL-2, and tumor necrosis factor-beta (TNF-β). In contrast to the Th1-type immune response, the Th2-type immune response usually includes the production of IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13. In some embodiments, said agent is a TIGIT antagonist and induces and/or increases the production of cytokines or lymphokines. In some embodiments, said induction and/or increase in cytokine or lymphokine production may be an indirect effect.

In some embodiments, the TIGIT binding agent described herein increases NK cell activation. In some embodiments, the TIGIT binding agent increases T cell activation. In some embodiments, said activation of NK cells and/or T cells by a TIGIT binding agent results in an increase in the level of activation of NK cells and/or T cells by at least about 10%, at least about 25%, at least at least about 50%, at least about 75%, at least about 90%, or at least about 95%. In some embodiments, the TIGIT binding agent that increases NK cell activation is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that increases NK cell activation is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that increases NK cell activation is the 313M32 antibody.

In some embodiments, said TIGIT binding agent (eg, antibody) is an antagonist of regulatory T cell (Treg) activity. In some embodiments, the TIGIT binding agent described herein inhibits or reduces the activity of Treg cells. In some embodiments, inhibition of Treg cell activity by the TIGIT binding agent results in inhibition of Treg cell suppressor activity by at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the TIGIT binding agent that inhibits Treg activity is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that inhibits Treg activity is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that inhibits Treg activity is the 313M32 antibody.

In some embodiments, said TIGIT binding agent (eg, antibody) is a myeloid-derived suppressor cell (MSC) antagonist. In some embodiments, said TIGIT binding agent inhibits the activity of SCMP. In some embodiments, said TIGIT binding agent inhibits SCMP activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%. , at least about 90% or about 100%. In some embodiments, the TIGIT binding agent that inhibits SCMP activity is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that inhibits SCMP activity is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that inhibits SCMP activity is the 313M32 antibody.

In some embodiments, said TIGIT binding agent (eg, antibody) enhances natural killer (NK) cell activity. In some embodiments, said TIGIT binding agent increases NK cell activity by at least about

- 51 039540 positively by 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%. In some embodiments, the TIGIT binding agent that enhances NK cell activity is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that enhances NK cell activity is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that enhances NK cell activity is the 313M32 antibody.

In some embodiments, said TIGIT binding agent (eg, antibody) increases the activity of tumor infiltrating lymphocytes (TILs). In some embodiments, said TIGIT binding agent increases TIL activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%. , at least about 90% or about 100%. In some embodiments, the TIGIT binding agent that increases the activity of TIL cells is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that increases the activity of TIL cells is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that increases the activity of TIL cells is the 313M32 antibody.

In some embodiments, said TIGIT binding agent (eg, antibody) increases or enhances the activity of lymphokine-activated killer (LAK) cells. In some embodiments, said TIGIT binding agent increases LAK activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%. , at least about 90% or about 100%. In some embodiments, the TIGIT binding agent that increases the activity of LAK cells is a 313R11 antibody, a 313R12 antibody, a 313R14 antibody, a 313R19 antibody, or a 313R20 antibody. In some embodiments, the TIGIT binding agent that increases the activity of LAK cells is a 313M26 antibody or a 313M32 antibody. In some embodiments, the TIGIT binding agent that increases the activity of LAK cells is the 313M32 antibody.

In vivo and in vitro assays to determine whether a TIGIT binding agent (or candidate binding agent) modulates an immune response are known in the art or under development. In some embodiments, a functional assay can be used that detects T cell activation. In some embodiments, a functional assay can be used that detects T cell proliferation. In some embodiments, a functional assay can be used that detects NK activity. In some embodiments, a functional assay can be used that detects CTL activity. In some embodiments, a functional assay may be used that detects Treg activity. In some embodiments, a functional assay can be used that detects SPCM activity. In some embodiments, a functional assay can be used that detects the production of cytokines or lymphokines, or cells that produce cytokines or lymphokines. In some embodiments, an ELISpot assay is used to measure the frequency of antigen-specific T cells. In some embodiments, the ELISpot assay is used to measure cytokine release/production and/or to measure the number of cytokine-producing cells. In some embodiments, cytokine assays are used to identify a Th1-type response. In some embodiments, cytokine assays are used to identify a Th2-type response. In some embodiments, cytokine assays are used to identify a Th17-type response. In some embodiments, the FACS assay is used to measure activation markers on immune cells, including, but not limited to, T cells, B cells, NK cells, macrophages, and/or myeloid cells.

In some embodiments, the TIGIT binding agents described herein have a circulating half-life in mice, rats, cynomolgus monkeys, or humans of at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, or at least about 2 weeks. In some embodiments, said TIGIT binding agent is an IgG (e.g., IgG1, IgG2, or IgG4) antibody that has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 2 hours, at least about 5 hours. , at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, or at least about 2 weeks. Methods for increasing (or decreasing) the half-life of agents such as polypeptides and antibodies are known in the art. For example, known methods for increasing the circulating half-life of IgG antibodies include introducing mutations in the Fc region that increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn) at pH 6.0. Known methods for increasing the circulating half-life of antibody fragments lacking an Fc region include techniques such as pegylation.

In some embodiments described herein, TIGIT binding agents are polypeptides. In some embodiments, said polypeptides are recombinant polypeptides, naturally occurring polypeptides, or synthetic polypeptides containing an antibody or fragment thereof that bind TIGIT. It is known in the art that certain amino acid sequences of the present invention may vary without significant effect on protein structure or function. Accordingly, the present invention further includes polypeptide variants that exhibit significant activity, or include regions of an antibody or fragment thereof that binds TIGIT. In some embodiments, amino acid sequence variants of TIGIT-binding polypeptides include deletions, insertions, inversions, repeats, and/or other types of substitutions.

These polypeptides, their analogs and variants can be further modified to include additional chemical moieties not normally included in the polypeptide. Said derivatized fragments may improve or otherwise modulate the solubility, biological half-life and/or absorption of the polypeptide. Said fragments may also reduce or eliminate unwanted side effects of said polypeptides and variants thereof. An overview of the chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 22 ^st Edition, 2012, Pharmaceutical Press, London.

In some embodiments, the polypeptides described herein are isolated. In some embodiments, the polypeptides described herein are substantially isolated.

The polypeptides described herein can be obtained by any suitable method known in the art. Such methods range from direct protein synthesis methods to designing a DNA sequence encoding polypeptide sequences and expressing said sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the specified sequence can be subjected to mutagenesis using site-directed mutagenesis to obtain its functional analogues.

In some embodiments, a DNA sequence encoding a polypeptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the choice of codons preferred by the host cell in which the recombinant polypeptide of interest is to be produced. Standard methods can be used to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. In addition, a DNA oligomer containing a nucleotide sequence encoding a particular isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain sticky 5' or 3' ends for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or other method), polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operably linked to an expression control sequence suitable for protein expression in the desired host. Proper assembly can be confirmed by nucleotide sequencing, enzyme restriction mapping, and/or expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high levels of expression of a transfected gene in a host, said gene must be operably linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

In some embodiments, recombinant expression vectors are used to amplify and express DNA encoding anti-human TIGIT antibodies or fragments thereof. For example, recombinant expression vectors may be replicable DNA constructs containing synthetic or cDNA-derived DNA fragments encoding the polypeptide chain of a TIGIT binding agent, such as an anti-TIGIT antibody or fragment thereof, operably linked to appropriate transcriptional and/or translational regulatory

- 53 039540 elements derived from the genes of mammals, microorganisms, viruses or insects. A transcription unit typically contains a combination of the following sequences: (1) a genetic element or elements that play a regulatory role in gene expression, such as transcriptional promoters or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) suitable transcription and translation initiation and termination sequences. Regulatory elements may include an operator sequence to control transcription. Additionally, the ability to replicate in the host, usually provided by the origin of replication, and a gene for selection to facilitate recognition of transformants can be included. Regions of DNA are functionally linked if they are functionally related to each other. For example, signal peptide DNA (secretory leader) is operably linked to polypeptide DNA if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of said sequence; or a ribosome binding site is operably linked to a coding sequence if it is located in such a way as to allow translation. In some embodiments, building blocks for use in yeast expression systems include a leader sequence that allows extracellular secretion of the translated protein by the host cell. In other embodiments, in situations where the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. Then the specified residue may optionally be cleaved from the expressed recombinant protein to obtain the final product.

The choice of expression control sequence and expression vector depends on the choice of the host. A wide range of expression host/vector combinations can be used. Suitable expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Suitable expression vectors for bacterial hosts include known bacterial plasmids such as those from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and plasmids for a wider range of hosts such as M13 and other filamentous single stranded DNA phages.

Binding TIGIT agents (eg, polypeptides or antibodies) according to the present invention can be expressed from one or more vectors. For example, in some embodiments, one heavy chain polypeptide expresses one vector, a second heavy chain polypeptide expresses a second vector, and a light chain polypeptide expresses a third vector. In some embodiments, the first heavy chain polypeptide and the light chain polypeptide express one vector, and the second heavy chain polypeptide expresses a second vector. In some embodiments, the two heavy chain polypeptides are expressed by one vector and the light chain polypeptide is expressed by a second vector. In some embodiments, three polypeptides are expressed from a single vector. Accordingly, in some embodiments, the first heavy chain polypeptide, the second heavy chain polypeptide, and the light chain polypeptide express a single vector.

Suitable host cells for expression of a TIGIT-binding polypeptide or antibody (or TIGIT protein for use as an antigen) include prokaryotic, yeast, insect, or higher eukaryotic cells under the control of suitable promoters. Prokaryotes include Gram-negative or Gram-positive organisms such as E. coli or Bacillus. Higher eukaryotic cells include stable cell lines derived from mammalian cells as described below. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian host cells, as well as methods for producing proteins, including producing antibodies, are well known in the art.

Various mammalian cell culture systems can be used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells may be desirable because said proteins are usually properly folded, suitably modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (derived from monkey kidney), L-929 (derived from mouse fibroblast), C127 (derived from mouse mammary tumor), 3T3 (derived from fibroblast mice), CHO (derived from Chinese hamster ovary), HeLa (derived from human cervical cancer), BHK (derived from hamster kidney fibroblasts), HEK-293 (derived from human embryonic kidney), and variants thereof. Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, an appropriate promoter and enhancer linked to the genes for putative expression, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' non-translated sequences. such as essential ribosome binding sites, poly-adenylation site, donor and acceptor splicing sites, and transcription termination sequences.

Expression of recombinant proteins in insect cell culture systems (eg, baculovirus) is also a reliable way to obtain properly folded biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art.

Accordingly, the present invention provides cells containing the TIGIT binding agents described herein. In some embodiments, said cells produce the TIGIT binding agents described herein. In some embodiments, said cells produce an antibody. In some embodiments, said cells produce an antibody that binds mouse TIGIT. In some embodiments, said cells produce an antibody that binds human TIGIT. In some embodiments, said cells produce an antibody that binds mouse TIGIT and human TIGIT. In some embodiments, said cells produce the 313R11 antibody. In some embodiments, said cells produce the 313R12 antibody. In some embodiments, said cells produce the 313R14 antibody. In some embodiments, said cells produce the 313R19 antibody. In some embodiments, said cells produce the 313R20 antibody. In some embodiments, said cells produce the 313M26 antibody. In some embodiments, said cells produce the 313M32 antibody. In some embodiments, said cells produce the 313M33 antibody. In some embodiments, said cells produce a bispecific antibody that binds TIGIT. In some embodiments, said cells produce a bispecific antibody that binds TIGIT and a second target. In some embodiments, said cell is a hybridoma cell. In some embodiments, said cell is a mammalian cell. In some embodiments, said cell is a prokaryotic cell. In some embodiments, said cell is a eukaryotic cell.

Proteins produced by the transformed host may be purified according to any suitable method. Standard methods include chromatography (eg, ion exchange, affinity, and size exclusion chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione S transferase can be attached to the protein to allow for easy purification by passing through a suitable affinity column. Affinity chromatography used to purify immunoglobulins may include protein A, protein G, and protein L chromatography. Isolated proteins can be physically characterized using techniques such as proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high performance liquid chromatography (HPLC) and X-ray crystallography. The purity of isolated proteins can be determined using techniques known to those skilled in the art, including, but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).

In some embodiments, supernatants from expression systems that secrete recombinant protein into culture media can first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, said concentrate may be applied to a suitable purification matrix. In some embodiments, an anion exchange resin may be used, such as a matrix or substrate with pendent diethylaminoethyl (DEAE) groups. The matrices may be acrylamide, agarose, dextran, cellulose, or other types commonly used for protein purification. In some embodiments, a cation exchange step may be carried out. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. In some embodiments, hydroxyapatite media can be used, including, but not limited to, ceramic hydroxyapatite (CHT). In some embodiments, one or more reverse phase HPLC steps using hydrophobic RP-HPLC media, such as methyl pendant or other aliphatic groups on silica gel, can be used to further purify the recombinant protein (eg, TIGIT binding agent). Some or all of the above purification steps, in various combinations, can be used to obtain a homogeneous recombinant protein.

In some embodiments, heterodimeric proteins, such as bispecific antibodies, are purified according to any of the methods described herein. In some embodiments, anti-TIGIT bispecific antibodies are isolated and/or purified with

- 55 039540 using at least one chromatographic step. In some embodiments, said at least one chromatographic step includes affinity chromatography. In some embodiments, said at least one chromatographic step further comprises anion exchange chromatography. In some embodiments, the isolated and/or purified antibody product contains at least 90% heterodimeric antibody. In some embodiments, the isolated and/or purified antibody product contains at least 95%, 96%, 97%, 98%, or 99% heterodimeric antibody. In some embodiments, the isolated and/or purified antibody product contains approximately 100% heterodimeric antibody.

In some embodiments, a polypeptide produced in a bacterial culture can be isolated, for example, by initial extraction from the cell pellet followed by one or more steps of concentration, salting out, water ion exchange, or size exclusion chromatography. HPLC may be used for the final purification steps. Microbial cells used to express the recombinant protein may be disrupted using any convenient method, including freeze-thaw cycle, sonication, mechanical disruption, or the use of cell-lysing agents.

In some embodiments, said TIGIT binding agent is a polypeptide that is not an antibody or does not contain an immunoglobulin Fc region. Various methods for identifying and producing non-antibody polypeptides that bind protein targets with high affinity are known in the art. In some embodiments, phage display or mammalian display technology may be used to generate and/or identify a TIGIT binding polypeptide. In some embodiments, said polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, lipocalin, fibronectin domain, ankyrin repeat consensus domain, and thioredoxin. Various methods for identifying and obtaining non-antibody polypeptides that bind a protein target with high affinity are known in the art. In some embodiments, phage display technology may be used to generate and/or identify a binding polypeptide. In some embodiments, a binding polypeptide display technique in mammalian cells may be used to obtain and/or identify.

It may be desirable to further modify the polypeptide to increase (or decrease) the serum half-life. This can be achieved, for example, by incorporating a recycling receptor binding epitope into the polypeptide by mutating an appropriate region in the polypeptide, or by incorporating said epitope into a peptide tag, which is then fused to either end of the polypeptide or to a region in the middle of the polypeptide (e.g., by DNA synthesis or peptide synthesis).

Heteroconjugate molecules are also within the scope of the present invention. Heteroconjugate molecules consist of two covalently linked polypeptides. Such molecules have been proposed, for example, to target immune cells to unwanted cells such as tumor cells. It is also contemplated that heteroconjugate molecules can be prepared in vitro using known methods of protein synthesis chemistry, including crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or through the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

In some embodiments, TIGIT binding agents may be used in any of a number of conjugated forms (ie, immunoconjugate or radioconjugate) or in unconjugated form. In some embodiments, these agents may be used in unconjugated form to engage the subject's natural defense mechanisms, including complement dependent cytotoxicity (CCC) and antibody dependent cellular cytotoxicity (ADCC) to eliminate malignant or cancerous cells.

In some embodiments, said TIGIT binding agent is conjugated to a cytotoxic agent. In some embodiments, said TIGIT binding agent is an antibody conjugated to an ADC (antibody drug conjugate) cytotoxic agent. In some embodiments, said cytotoxic agent is a chemotherapeutic agent, including but not limited to methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, pyrrolobenzodiazepines (PBDs), or other intercalating agents. In some embodiments, said cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria toxin A chain, non-binding diphtheria toxin active fragments, exotoxin A chain, exotoxin A chain, A ricin, abrin A chain, modessin A chain, α-sarcin, Aleurites fordii proteins, dianthine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S),

- 56 039540 Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecins. In some embodiments, said cytotoxic agent is a radioisotope for producing a radioconjugate or radioconjugated antibody. Various radionuclides are available for making radioconjugated antibodies, including but not limited to ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹ⁿ In, ¹³¹ In, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re and ²¹² Bi. Conjugates of the antibody with one or more small molecule toxins, such as calicheamicins, maytansinoids, trichotenes, and CC1065, and derivatives of these toxins having toxin activity, may also be used. Antibody-cytotoxic agent conjugates can be prepared using various bifunctional protein-binding agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bisazido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene -2,6-diisocyanate) and bi-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

III. Polynucleotides.

In some embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode an agent described herein. The term polynucleotides that encode a polypeptide encompasses a polynucleotide that includes only the coding sequences for said polypeptide, as well as a polynucleotide that includes additional coding and/or non-coding sequences. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA and synthetic DNA; they may be double-stranded or single-stranded; single stranded DNA can be a coding strand or a non-coding (antisense) strand.

In some embodiments, said polynucleotide comprises a polynucleotide (e.g., a nucleotide sequence) encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO : 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 , SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 55, and SEQ ID NO: 56.

In some embodiments, the polynucleotide comprises a polynucleotide whose nucleotide sequence is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and according to some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 55, and SEQ ID NO: 56. Also provided is a polynucleotide that contains a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SE Q ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 55 and SEQ ID NO: 56. In some embodiments, said hybridization takes place under conditions of high stringency. High stringency conditions are known to those skilled in the art and may include, but are not limited to, (1) using low ionic strength and high wash temperature, e.g. 15 mM sodium chloride/1.5 mM sodium citrate (1x SSC) with 0.1 % sodium dodecyl sulfate at 50°C; (2) using in the hybridization a denaturing agent such as formamide, for example 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 in 5x SSC (0.75 M NaCl, 75 mM sodium citrate) at 42°C; or (3) using 50% formamide, 5x SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS and 10% dextran sulfate at 42°C, followed by a wash with 0.2x SSC containing 50% formamide at 55°C followed by a high stringency wash including 0.1x SSC containing EDTA at 55°C .

In some embodiments, said polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO : 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO: 83.

In some embodiments, the polynucleotide comprises a polynucleotide whose nucleotide sequence is at least about 80% identical, at least

- 57 039540 is about 85% identical, at least about 90% identical, at least about 95% identical, and, in some embodiments, at least about 96%, 97, 98, or 99% identical to the polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 82, and SEQ ID NO: 83. Also provided is a polynucleotide that contains a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO: 83.

In some embodiments, said polynucleotide comprises a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO : 76 and SEQ ID NO: 84. In some embodiments, the polynucleotide comprises a polynucleotide whose nucleotide sequence is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95 % identical and, in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 73 , SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 84. Also provided is a polynucleotide that contains a polynucleotide that hybridizes to a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 84. In some embodiments, these hybridization techniques are performed under conditions high rigidity as described above.

In some embodiments, the polynucleotide comprises a mature polypeptide coding sequence fused within the same reading frame to a polynucleotide that aids in, for example, the expression and secretion of the polypeptide from the host cell (e.g., a leader sequence that functions as a secretory sequence to direct transport of the polypeptide out of the cell). ). Said leader sequence-containing polypeptide is a precursor protein and may contain a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide. These polynucleotides may also encode a precursor protein, which is a mature protein containing additional 5'-terminal amino acid residues. The mature protein containing the prosequence is the precursor protein and is the inactive form of the protein. After cleavage of the prosequence, the active mature protein remains.

In some embodiments, the polynucleotide comprises a mature polypeptide coding sequence fused within the same reading frame to a marker sequence that, for example, purifies the encoded polypeptide. For example, the marker sequence may be a hexa-histidine tag delivered by the pQE-9 vector to allow purification of the mature polypeptide fused to said marker if a bacterial host is used, or the marker sequence may be a hemagglutinin (HA) tag derived from influenza virus hemagglutinin protein, if used in a mammalian host (eg, COS-7 cells). In some embodiments, the marker sequence is a FLAG tag, a peptide with the sequence DYKDDDDK (SEQ ID NO: 40), which can be used in combination with other affinity tags.

The present invention also relates to variants of the polynucleotides described herein, characterized in that said variant encodes, for example, fragments, analogs and/or derivatives.

In some embodiments, the present invention provides a polynucleotide comprising a polynucleotide whose nucleotide sequence is at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical and, in some embodiments, at least about 96%, 97%, 98%, or 99% identical to a polynucleotide encoding a polypeptide containing a TIGIT binding agent described in the document.

In this document, when referring to a polynucleotide, the nucleotide sequence of which is at least, for example, 95% identical to the reference nucleotide sequence, it is assumed that the nucleotide sequence of the specified polynucleotide is identical to the specified reference sequence, except that the specified polynucleotide sequence may include up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide whose nucleotide sequence is at least 95% identical to the reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence can be deleted or replaced with another nucleotide, or a set of nucleotides, up to 5% of the total number of nucleotides in the reference sequence , maybe

- 58 039540 be built into the indicated reference sequence. Said reference sequence mutations may occur at the 5' or 3' end positions of the reference sequence of nucleotides, or anywhere between these terminal positions, either individually distributed between nucleotides of the reference sequence, or as one or more contiguous groups within the reference sequence.

Polynucleotide variants may contain changes in coding regions, non-coding regions, or both. In some embodiments, a variant polynucleotide contains changes that result in silent substitutions, additions, or deletions, but does not change the properties or activities of the encoded polypeptide. In some embodiments, the polynucleotide variant contains silent substitutions that do not alter the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Variant polynucleotides can be made for various purposes, such as optimizing codon expression in a particular host (ie, changing codons in human mRNA to codons preferred by a bacterial host such as E. coli). In some embodiments, the polynucleotide variant contains at least one silent mutation in a non-coding or coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate or change the expression (or expression levels) of the encoded polypeptide. In some embodiments, a variant polynucleotide is obtained to increase expression of the encoded polypeptide. In some embodiments, a variant polynucleotide is produced to reduce expression of the encoded polypeptide. In some embodiments, the polynucleotide variant provides increased expression of the encoded polypeptide compared to the original polynucleotide sequence. In some embodiments, the polynucleotide variant provides reduced expression of the encoded polypeptide compared to the original polynucleotide sequence.

In some embodiments, at least one polynucleotide variant is prepared (without altering the amino acid sequence of the encoded polypeptide) to increase production of the heterodimeric molecule. In some embodiments, at least one polynucleotide variant is obtained (without altering the amino acid sequence of the encoded polypeptide) to increase production of the bispecific antibody.

In some embodiments, said polynucleotides are isolated. In some embodiments, said polynucleotides are substantially pure.

Also provided are vectors and cells containing the polynucleotides described herein. In some embodiments, the expression vector contains a polynucleotide molecule. In some embodiments, the host cell contains an expression vector containing said polynucleotide molecule. In some embodiments, the host cell comprises a polynucleotide molecule.

IV. Methods of application and pharmaceutical compositions.

The TIGIT binding agents of the present invention are suitable for a variety of uses including, but not limited to, therapeutic methods such as cancer treatment. In some embodiments, these therapeutic methods include cancer immunotherapy. In some embodiments, the TIGIT binding agent is suitable for activating an immune response, stimulating an immune response, increasing and/or enhancing an immune response, inhibiting tumor growth, shrinking a tumor, increasing tumor cell apoptosis, and/or decreasing tumorigenicity. In some embodiments, the TIGIT binding agent of the present invention is also suitable for immunotherapy directed against pathogens such as viruses. In some embodiments, the TIGIT binding agent is suitable for activating an immune response, stimulating an immune response, increasing and/or enhancing an immune response, inhibiting viral infection, reducing viral infection, increasing apoptosis of virus-infected cells, and/or increasing the killing of virus-infected cells. These methods of application may be carried out in vitro, ex vivo or in vivo methods.

The present invention provides methods for activating an immune response in a subject using a TIGIT binding agent described herein. In some embodiments, the present invention provides methods for stimulating an immune response in a subject using a TIGIT binding agent described herein. In some embodiments, the present invention provides methods for enhancing an immune response in a subject using a TIGIT binding agent described herein. In some embodiments, the present invention provides methods for enhancing an immune response in a subject using a TIGIT binding agent described herein. In some embodiments, said activation of an immune response, stimulation of an immune response, enhancement and/or enhancement of an immune response includes enhancement of cellular immunity. According to some embodiments, said activation of the immune response, stimulation of the immune response, enhancement and/or enhancement of the immune response comprises an increase in T-cell activity. In some embodiments, said activation of an immune response, stimulation of an immune response, enhancement and/or enhancement of an immune response comprises an increase in CTL activity. In some embodiments, said activation of the immune response, stimulation of the immune response, enhancement and/or enhancement of the immune response comprises an increase in the activity of NK cells. In some embodiments, said activation of an immune response, stimulation of an immune response, enhancement and/or enhancement of an immune response includes an increase in T cell activity and an increase in NK cell activity. In some embodiments, said activation of an immune response, stimulation of an immune response, enhancement and/or enhancement of an immune response includes an increase in CTL activity and an increase in NK cell activity. In some embodiments, said activation of an immune response, stimulation of an immune response, enhancement and/or enhancement of an immune response comprises inhibition or reduction of suppressive activity of Treg cells. In some embodiments, said activation of the immune response, stimulation of the immune response, increase and/or enhancement of the immune response includes inhibition or reduction of the suppressor activity of SCMP cells. In some embodiments, said immune response is the result of antigenic stimulation. In some embodiments, said antigenic challenge is a tumor cell. In some embodiments, said antigenic challenge is cancer. In some embodiments, said antigenic challenge is a pathogen. In some embodiments, said antigenic challenge is a virus. In some embodiments, said antigenic challenge is a virus-infected cell. In some embodiments of any of the methods described herein, the TIGIT binding agent is an anti-TIGIT antibody. In some embodiments of any of the methods described herein, the TIGIT binding agent is the 313M32 antibody.

In some embodiments, a method for enhancing an immune response in a subject comprises administering to said subject a therapeutically effective amount of a TIGIT binding agent described herein, said agent being an antibody that specifically binds the extracellular domain of TIGIT. In some embodiments, a method for enhancing an immune response in a subject comprises administering to said subject a therapeutically effective amount of a TIGIT binding agent described herein, said agent being an antibody that specifically binds to the extracellular domain of human TIGIT. In some embodiments, said method for enhancing an immune response in a subject comprises administering to said subject a therapeutically effective amount of a 313M32 antibody.

In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament to activate an immune response, stimulate an immune response, increase and/or enhance an immune response. In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a cellular immunity enhancing medicament. In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament to increase T cell activity. In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament to increase CTL activity. In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament to increase NK activity. In some embodiments, the present invention provides the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament to inhibit or reduce the suppressive activity of Treg cells. In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament to inhibit or reduce the suppressive activity of SCMP cells.

The present invention also provides methods for inhibiting and/or reducing TIGIT signaling in a cell, comprising contacting said cell with an effective amount of a TIGIT binding agent described herein. In some embodiments, said method of inhibiting and/or reducing TIGIT signaling in a cell comprises contacting said cell with an effective amount of the 313M32 antibody. In some embodiments, the present invention provides the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament to inhibit

- 60 039540 and/or reduction of TIGIT signaling in the cell. In some embodiments, said cell is a T cell. In some embodiments, said cell is an activated T cell. In some embodiments, said cell is an NK cell. In some embodiments, said cell is a Treg. In some embodiments, said cell is a SCMP. In some embodiments, said method is an in vivo method, characterized in that said step of bringing said cell into contact with said agent comprises administering a therapeutically effective amount of a TIGIT binding agent to said subject. In some embodiments, said method is an in vitro or ex vivo method.

The present invention also provides methods for inhibiting tumor growth using a TIGIT binding agent described herein. In some embodiments, said method for inhibiting tumor growth comprises the use of the 313M32 antibody. In some embodiments, said method for inhibiting tumor growth comprises contacting a mixture of cells with a TIGIT binding agent in vitro. For example, immortalized cells or cancer cells mixed with immune cells (eg, T cells or NK cells) are cultured in a medium to which a test agent that binds TIGIT is added. In some embodiments, tumor cells are isolated from a patient sample, such as, for example, a tissue biopsy, a pleural effusion, or a blood sample mixed with immune cells (e.g., T cells and/or NK cells), and cultured in a medium in which is added by the test agent that binds TIGIT. In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament for inhibiting tumor or tumor cell growth. In some embodiments, the TIGIT binding agent increases the activity, stimulates the activity, and/or enhances the activity of immune cells. In some embodiments, the TIGIT binding agent inhibits the growth of tumor cells.

In some embodiments, said method for inhibiting tumor growth comprises contacting said tumor or tumor cells with a TIGIT binding agent as described herein in vivo. In some embodiments, contacting a tumor or tumor cell with a TIGIT binding agent is performed in an animal model. For example, the test agent may be administered to mice bearing tumors. In some embodiments, the TIGIT binding agent increases the activity, stimulates the activity, and/or enhances the activity of immune cells in mice. In some embodiments, the TIGIT binding agent inhibits tumor growth. In some embodiments, the TIGIT binding agent promotes tumor regression. In some embodiments, the TIGIT binding agent is administered to the animal simultaneously with or shortly after tumor cell administration to prevent tumor growth (prevention model). In some embodiments, the TIGIT binding agent is administered as a therapeutic agent after the tumors have reached a certain size or become mature (therapeutic model).

In some embodiments, said method for inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a TIGIT binding agent described herein. In some embodiments, said subject is a human. In some embodiments, said subject has a tumor or said subject had a tumor that has been at least partially removed. In some embodiments, said method for inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a 313M32 antibody.

Furthermore, the present invention provides a method for inhibiting tumor growth in a subject, comprising administering to said subject a therapeutically effective amount of a TIGIT binding agent described herein. In some embodiments, the present invention provides for the use of a TIGIT binding agent described herein in the preparation or formulation of a medicament for inhibiting tumor or tumor cell growth. In some embodiments, said tumor contains cancer stem cells. In some embodiments, the incidence of cancer stem cells in a tumor is reduced by administering said agent. In some embodiments, a method of reducing the incidence of cancer stem cells in a tumor in a subject is provided, comprising administering to said subject a therapeutically effective amount of a TIGIT binding agent.

In addition, the present invention provides a method for reducing the tumorigenicity of a tumor in a subject, comprising administering to said subject a therapeutically effective amount of a TIGIT binding agent described herein. In some embodiments, said tumor contains cancer stem cells. According to some embodiments of the

- 61 039540 tumor morogenicity is reduced by reducing the frequency of occurrence of cancer stem cells in the tumor. In some embodiments, these methods include the use of TIGIT binding agents described herein. In some embodiments, the incidence of cancer stem cells in a tumor is reduced by the administration of a TIGIT binding agent described herein. In some embodiments, a method for reducing the tumorigenicity of a tumor in a subject comprises administering to said subject a therapeutically effective amount of a 313M32 antibody.

In some embodiments, said tumor is a solid tumor. In some embodiments, said tumor is a tumor selected from the group consisting of: colon and rectal tumors, pancreatic tumors, lung tumors, ovarian tumors, liver tumors, breast tumors, kidney tumors, prostate tumors, neuroendocrine tumors, gastrointestinal tumors, melanomas, cervical tumors, bladder tumors, glioblastomas, and head and neck tumors. In some embodiments, said tumor is a colon and rectal tumor. In some embodiments, said tumor is an ovarian tumor. In some embodiments, said tumor is a lung tumor. In some embodiments, said tumor is a pancreatic tumor. In some embodiments, said tumor is a melanoma tumor.

The present invention provides methods for treating cancer comprising administering to a subject a therapeutically effective amount of a TIGIT binding agent described herein (eg, a subject in need of treatment). In some embodiments, the present invention provides the use of a TIGIT binding agent described herein in the manufacture or preparation of a medicament for the treatment of cancer. In some embodiments, the TIGIT binding agent binds human TIGIT and inhibits or reduces cancer growth. In some embodiments, said subject is a human. In some embodiments, said subject has a cancerous tumor. In some embodiments, the tumor in said subject has been at least partially resected. In some embodiments, a method of treating cancer in a subject comprises administering to said subject a therapeutically effective amount of a 313M32 antibody.

In some embodiments, said cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer. tract, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, glioblastoma, and head and neck cancer. In some embodiments, said cancer is pancreatic cancer. In some embodiments, said cancer is ovarian cancer. In some embodiments, said cancer is colorectal cancer. In some embodiments, said cancer is breast cancer. In some embodiments, said cancer is prostate cancer. In some embodiments, said cancer is lung cancer. In some embodiments, said cancer is melanoma.

In some embodiments, said cancer is blood cancer. In some embodiments, said blood cancer is leukemia. In other embodiments, said blood cancer is lymphoma. In a particular embodiment, said cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin's lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).

In some embodiments of the methods described herein, the method further comprises the step of determining the expression level of PD-L1 in said tumor or cancer. In some embodiments, PD-L1 expression level determination is performed prior to treatment, with a TIGIT binding agent described herein. In some embodiments, if the tumor or cancer is overexpressing PD-L1, the subject is administered a TIGIT binding agent. In some embodiments, the method includes: (i) obtaining a sample of a cancer or tumor from a subject; (ii) measuring the expression level of PD-L1 in said sample; and (iii) administering an effective amount of a TIGIT binding agent to said subject if said tumor or cancer is overexpressed by PD-L1. In some embodiments, said sample is a biopsy sample. In some embodiments, said sample comprises tumor cells, tumor-infiltrating immune cells, stromal cells, and any combination thereof.

- 62 039540 In some embodiments, said sample is a formalin-fixed paraffin-embedded (FFPE) sample. In some embodiments, said sample is an archival, fresh, or frozen tissue sample. In some embodiments, the level of PD-L1 expression in said sample is compared to a predetermined level of PD-L1 expression. In some embodiments, said predetermined expression level of PD-L1 expression is the level of PD-L1 expression in a reference tumor sample, a reference normal tissue sample, a set of reference tumor samples, or a set of reference normal tissue samples. In some embodiments, the expression level of PD-L1 is determined using immunohistochemical (IHC) analysis. In some embodiments, the expression level of PD-L1 is determined using an assay that includes an H-index score. In some embodiments, the expression level of PDL1 is determined using an antibody that specifically binds PD-L1. In some embodiments, PD-L1 is detected on tumor cells. In some embodiments, PD-L1 is detected on tumor-infiltrating immune cells.

Combination therapy with two or more therapeutic agents often uses agents that work through different mechanisms of action, although this is not necessary. Combination therapy using agents with different mechanisms of action may provide additive or synergistic effects. Combination therapy may allow the use of a lower dose of each agent than monotherapy, thus reducing toxic side effects and/or increasing the therapeutic index of said agent(s). Combination therapy may reduce the chance of developing resistant cancer cells. In some embodiments, the combination therapy includes a therapeutic agent that affects (eg enhances or activates said response) an immune response and a therapeutic agent that affects (eg inhibits or kills) tumor/cancer cells.

In some embodiments, the combination of an agent described herein and at least one additional therapeutic agent provides additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of said agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a reduction in toxicity and/or side effects of said agent. In some embodiments, the combination therapy results in a reduction in toxicity and/or side effects of the additional therapeutic agent(s).

In some embodiments, in addition to administering a TIGIT binding agent described herein, the method or treatment further comprises administering at least one additional therapeutic agent. The additional therapeutic agent may be administered prior to administration, simultaneously with administration, or after administration of said agent. In some embodiments, said at least one additional therapeutic agent includes 1, 2, 3 or more additional therapeutic agents.

Therapeutic agents that may be administered in combination with the agents described herein include chemotherapeutic agents. Accordingly, in some embodiments, said method or treatment comprises administering an agent of the present invention in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with an agent may occur prior to administration, simultaneously with administration, or after administration of the chemotherapeutic agents. Combined administration may include co-administration, either in the same pharmaceutical formulation or using separate formulations, or sequential administration in any order, however, as a rule, within a certain period of time, so that all active agents can exercise their biological activity simultaneously. . The preparation and dosing regimens of such chemotherapeutic agents may be carried out in accordance with the manufacturer's instructions or may be determined empirically by the practitioner. The formulation and dosing regimens for such chemotherapy are also described in The Chemotherapy Source Book, 4th ^Edition , 2008, MC Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.

Suitable classes of therapeutic agents include, for example, antitubulin agents, auristatins, DNA minor groove binding agents, inhibitors of DNA replication, alkylating agents (for example, platinum complexes such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemosensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. . In some embodiments, said second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.

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Chemotherapeutic agents suitable for use in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; mustargens such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, autramycin, azaserine, bleomycins, sactinomycin, calicheamicin, carabicin, carminomycin, carsinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, epirubicin, esorubicin, idarubicin, marsellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxyfluridine, enocytabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; adrenal depressants such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcin; diaziquon; elfornitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); taxoids such as paclitaxel (TAXOL) and docetaxel (TAXOTHER); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbin; novantron; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamycins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above agents. Chemotherapeutic agents also include anti-hormonal agents that act as regulators or inhibitors of the action of hormones on tumors, such as antiestrogen, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapriston, and toremifene (FARESTON ); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above agents. In some embodiments, said additional therapeutic agent is cisplatin. In some embodiments, said additional therapeutic agent is carboplatin.

In some embodiments, said chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents that interfere with the action of the topoisomerase enzyme (eg, topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. In some embodiments, said additional therapeutic agent is irinotecan.

In some embodiments, said chemotherapeutic agent is an antimetabolite. An antimetabolite is a chemical substance with a structure similar to that of a metabolite required for normal biochemical reactions, but different enough to interfere with one or more of the normal functions of cells, such as cell division. Antimetabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, sodium methotrexate, raltitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, and pharmaceutically acceptable salts, acids or derivatives of any of the above. In some embodiments, said additional therapeutic agent is gemcitabine.

In some embodiments, said chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, said agent is

- 64 039540 san. In some embodiments, said agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In some embodiments, said agent is paclitaxel (TAXOL), docetaxel (TAXOTHER), albumin-bound paclitaxel (ABRAXAN), DHA-paclitaxel, or PG-paclitaxel. In certain other alternative embodiments, said antimitotic agent comprises a vinca alkaloid such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, said antimitotic agent is an Eg5 kinesin inhibitor or a mitotic kinase inhibitor such as Aurora A or Plk1. In some embodiments, said additional therapeutic agent is paclitaxel. In some embodiments, said additional therapeutic agent is albumin-bound paclitaxel (ABRAXAN).

In some embodiments, the additional therapeutic agent comprises an agent such as a small molecule. For example, treatment may involve the combined administration of an agent of the present invention and a small molecule that acts as an inhibitor of tumor-associated antigens, including, but not limited to, rEGFR, HER2 (ErbB2), and/or VEGF. In some embodiments, an agent of the present invention is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefinitib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatinib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RENENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, the additional therapeutic agent comprises an mTOR inhibitor.

In some embodiments, said additional therapeutic agent is an agent that inhibits the cancer stem cell pathway. In some embodiments, said additional therapeutic agent is a Notch pathway inhibitor. In some embodiments, said additional therapeutic agent is a Wnt pathway inhibitor.

In some embodiments, said additional therapeutic agent is a BMP pathway inhibitor. In some embodiments, said additional therapeutic agent is a Hippo pathway inhibitor. In some embodiments, said additional therapeutic agent is an inhibitor of the RSPO/LGR pathway. In some embodiments, said additional therapeutic agent is an inhibitor of the mTOR/AKR pathway.

In some embodiments, the additional therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment may involve the combined administration of an agent of the present invention and antibodies against tumor-associated antigens, including, but not limited to, antibodies that bind rEGF, HER2/ErbB2, and/or VEGF. In some embodiments, said additional therapeutic agent is an antibody specific for a tumor stem cell marker. In some embodiments, said additional therapeutic agent is an antibody that binds a component of the Notch pathway. In some embodiments, said additional therapeutic agent is an antibody that binds a component of the Wnt pathway. In some embodiments, said additional therapeutic agent is an antibody that inhibits the cancer stem cell pathway. In some embodiments, said additional therapeutic agent is a Notch pathway inhibitor. In some embodiments, said additional therapeutic agent is a Wnt pathway inhibitor. In some embodiments, said additional therapeutic agent is a BMP pathway inhibitor. In some embodiments, said additional therapeutic agent is an antibody that inhibits β-catenin-mediated signaling. In some embodiments, said additional therapeutic agent is an antibody that is an angiogenesis inhibitor (eg, an anti-VEGF or VEGF receptor antibody). In some embodiments, said additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

In some embodiments, in addition to administering a TIGIT binding agent described herein, said method or treatment further comprises administering at least one additional immunotherapeutic agent. In some embodiments, said additional immunotherapeutic agent is an immune response stimulating agent. In some embodiments, said immunotherapeutic agent (e.g., an immune response stimulating agent) includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimuli).

- 65 039540 stimulating factor (GM-CSF), macrophage colony-stimulating factor (MCSF), granulocyte colony-stimulating factor (GCSF), stem cell factor (SCF)), interleukin (e.g. IL1, IL-2, IL-3, IL-7, IL -12, IL-15, IL-18), an antibody that blocks immunosuppressive functions (e.g., anti-CTLA4 antibody, anti-CD28 antibody, anti-CD3 antibody, anti-PD1 antibody, anti-PD-L1 antibody), antibody that enhances the functions of the immune cells (eg, anti-GITR antibody or anti-OX-40 antibody), Toll-like receptor (eg, TLR4, TLR7, TLR9), soluble ligand (eg, GITRL or OX-40L), or member of the B7 family (eg, CD80, CD86 ). An additional immunotherapeutic agent (eg, an immune response stimulating agent) may be administered prior to administration, simultaneously with administration, or after administration of the TIGIT binding agent. Also provided are pharmaceutical compositions comprising a TIGIT binding agent and an additional immunotherapeutic agent (eg, agent(s) that stimulates an immune response). In some embodiments, said immunotherapeutic agent comprises 1, 2, 3 or more immunotherapeutic agents. In some embodiments, said immune response stimulating agent comprises 1, 2, 3 or more immune response stimulating agents.

In some embodiments, said additional therapeutic agent is an antibody that is an immune checkpoint inhibitor. In some embodiments, said immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-CD28 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, an anti-GITR antibody, or an anti-OX40 antibody. In some embodiments, said immune checkpoint inhibitor is an anti-4-1BB antibody. In some embodiments, said additional therapeutic agent is an anti-PD-1 antibody selected from the groups consisting of: nivolumab (OPDIVO), pembrolizumab (KEYTRUDA), or pidilizumab. In some embodiments, said additional therapeutic agent is an anti-PD-1 antibody selected from the groups consisting of: MEDI0680, REGN2810, BGB-A317, and PDR001. In some embodiments, said additional therapeutic agent is an anti-PD-L1 antibody selected from the group consisting of: BMS935559 (MDX-1105), atezolizumab (MPDL3280A), durvalumab (MEDI4736), or avelumab (MSB0010718C). In some embodiments, said additional therapeutic agent is an anti-CTLA-4 antibody selected from the group consisting of: ipilimumab (EPBOI) or tremelimumab. In some embodiments, said additional therapeutic agent is an anti-LAG-3 antibody selected from the group consisting of: BMS-986016 and LAG525. In some embodiments, said additional therapeutic agent is an anti-OX-40 antibody selected from the group consisting of: MEDI6469, MEDI0562, and MOXR0916. In some embodiments, said additional therapeutic agent is an anti-4-1BB antibody selected from the group consisting of: PF-05082566.

In addition, treatment with a TIGIT binding agent described herein may include combination treatment with other biological molecules, e.g., one or more cytokines (e.g., lymphokines, interleukins, interferons, tumor necrosis factors, and/or growth factors), or may be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by the attending physician.

In some embodiments, said TIGIT binding agent may be administered in combination with a biological molecule selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), bone morphogenetic proteins (BMP), brain-derived neurotrophic factor (BDNF), EGF, erythropoietin (EPO), fibroblast growth factor (FGF), glial cell neurotrophic factor (GDNF), GCSF, GMCSF, GDF9, hepatocyte growth factor (HGF), hepatoma growth factor (HDGF), IGF, migration stimulating factor, myostatin (GDF -8), nerve cell growth factor (NGF), neurotrophins, TRF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, PFR (placental growth factor), IFN-γ, IL-1, IL-2 , IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15 and IL-18. In some embodiments, said TIGIT binding agent may be administered in combination with a biological molecule selected from the group consisting of: macrophage colony stimulating factor (MCSF) and stem cell factor (SCF).

In some embodiments, treatment with a TIGIT binding agent described herein may be accompanied by surgical removal of tumors, removal of cancer cells, or any other surgical treatment deemed necessary by the attending physician.

In some embodiments, the treatment comprises administering a TIGIT binding agent of the present invention in combination with radiation therapy. Treatment with an agent can be carried out before, simultaneously with, or after radiation therapy. Dosing regimens for such radiation therapy may be determined by the medical practitioner.

In some embodiments, treatment comprises administering a TIGIT binding agent of the present invention in combination with antiviral therapy. Treatment with an agent may occur before, concurrently with, or after antiviral therapy. The choice of antiviral drug for use in combination therapy depends on the virus with which the subject is infected.

Combined administration may include co-administration in the same pharmaceutical composition, or the use of separate compositions, or sequential administration in any order, however, as a rule, within a certain period of time, so that all active agents can exercise their biological activity simultaneously.

It should be understood that the combination of a TIGIT binding agent described herein and at least one additional therapeutic agent may be administered in any order or simultaneously. In some embodiments, said agent is administered to patients previously treated with a second therapeutic agent. In some other embodiments, said TIGIT binding agent and the second therapeutic agent are administered substantially simultaneously or concurrently. For example, the subject may receive the agent while undergoing treatment with a second therapeutic agent (eg, undergoing chemotherapy). In some embodiments, the TIGIT binding agent is administered within a 1 year period from treatment with the second therapeutic agent. In some other alternative embodiments, the TIGIT binding agent is administered within a period of 10, 8, 6, 4, or 2 months from any treatment with the second therapeutic agent. In some other embodiments, the TIGIT binding agent is administered within a period of 4, 3, 2, or 1 weeks from any treatment with the second therapeutic agent. In some embodiments, the agent is administered within a period of 5, 4, 3, 2, or 1 days from any treatment with the second therapeutic agent. It should also be borne in mind that these two (or more) agents or treatments can be administered to the specified subject/carried out to the specified subject within a few hours or minutes (ie, essentially simultaneously).

The appropriate dosage of the TIGIT binding agent of the present invention for the treatment of a disease depends on the type of disease being treated, the severity and course of the disease, the response of the disease, whether the agent is being administered for a therapeutic or prophylactic purpose, prior therapy, the patient's clinical history, and the like. . is entirely at the discretion of the attending physician. Said TIGIT binding agent may be administered once or during a course of treatment lasting from several days to several months, or until a cure or alleviation of the disease state (eg reduction in tumor size) is achieved. Optimal dosing regimens can be calculated based on measurements of drug accumulation in the patient's body and vary depending on the relative potency of the individual agent. The administering physician can determine optimal dosages, dosing techniques, and frequency of repetition. In some embodiments, the dosage is 0.01 μg to 100 mg/kg body weight, 0.1 μg to 100 mg/kg body weight, 1 μg to 100 mg/kg body weight, 1 mg to 100 mg/kg kg body weight, 1 mg to 80 mg/kg body weight, 10 mg to 100 mg/kg body weight, 10 mg to 75 mg/kg body weight, or 10 mg to 50 mg/kg body weight. In some embodiments, the dosage of said agent is from about 0.1 mg to about 20 mg/kg of body weight. In some embodiments, the dosage of said agent is about 0.5 mg/kg of body weight. In some embodiments, the dosage of said agent is about 1 mg/kg of body weight. In some embodiments, the dosage of said agent is about 1.5 mg/kg body weight. In some embodiments, the dosage of said agent is about 2 mg/kg body weight. In some embodiments, the dosage of said agent is about 2.5 mg/kg body weight. In some embodiments, the dosage of said agent is about 5 mg/kg body weight. In some embodiments, the dosage of said agent is about 7.5 mg/kg body weight. In some embodiments, the dosage of said agent is about 10 mg/kg body weight. In some embodiments, the dosage of said agent is about 12.5 mg/kg body weight. In some embodiments, the dosage of said agent is about 15 mg/kg body weight. In some embodiments, the dose may be administered once or more daily, weekly, monthly, or yearly. In some embodiments, said agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks.

In some embodiments, a first higher loading dose of the TIGIT binding agent may be administered, followed by one or more lower doses. In some embodiments, the frequency of administration may also vary. In some embodiments, the dosing regimen may include administering a first dose followed by additional doses (or maintenance doses) once a week, once every two weeks, once every three weeks, or once every month. For example, the dosing regimen may include the administration of a first loading dose followed by a weekly maintenance dose of, for example, half of said first dose; or the dosing regimen may include the introduction of the first loading dose, followed by the introduction of maintenance doses of, for example, half of the specified first dose, every other week; or the dosing regimen may include the introduction of the first three doses within 3 weeks, followed by the introduction of maintenance doses, for example, the introduction of the same amount every second week.

As is known to those skilled in the art, administration of any therapeutic agent may result in side effects and/or toxicity. In some cases, these side effects and/or toxicity are so severe that it precludes the introduction of a particular agent in a therapeutically effective dose. In some cases, drug therapy must be stopped and other agents tried. However, many agents of the same therapeutic class often exhibit similar side effects and/or toxicity, meaning that the patient is forced to either discontinue therapy or, if possible, suffer from unpleasant side effects associated with the therapeutic agent.

In some embodiments, the dosing regimen may be limited to a specific number of administrations or cycles. In some embodiments, said agent is administered over 3, 4, 5, 6, 7, 8 or more cycles. For example, said agent is administered every 2 weeks for 6 cycles, administered every 3 weeks for 6 cycles, administered every 2 weeks for 4 cycles, administered every 3 weeks for 4 cycles, and the like. Dosing regimens can be determined and then modified by those skilled in the art.

The present invention provides methods for administering the TIGIT binding agents described herein to a subject, including using an intermittent dosing strategy to administer one or more agents that can reduce side effects and/or toxicity associated with administration of the agent, a chemotherapeutic agent, and the like. In some embodiments, a method of treating cancer in a human subject comprises administering to said subject a therapeutically effective dose of an agent in combination with a therapeutically effective dose of a chemotherapeutic agent, one or both of said agents being administered in accordance with an intermittent dosing strategy. In some embodiments, said intermittent dosing strategy comprises administering to said subject a first dose of an agent and administering subsequent doses of said agent approximately every 2 weeks in a single dose. In some embodiments, said intermittent dosing strategy comprises administering to said subject a first dose of an agent and administering subsequent doses of said agent approximately every 3 weeks in a single dose. In some embodiments, said intermittent dosing strategy comprises administering to said subject a first dose of an agent and administering subsequent doses of said agent approximately every 4 weeks once. In some embodiments, said agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.

The present invention provides compositions containing TIGIT binding agents described herein. The present invention also provides pharmaceutical compositions containing the TIGIT binding agents described herein and a pharmaceutically acceptable carrier. In some embodiments, said pharmaceutical compositions are suitable for use in immunotherapy. In some embodiments, these compositions are suitable for use in tumor growth inhibition. In some embodiments, said pharmaceutical compositions are suitable for use in inhibiting tumor growth in a subject (eg, a human patient). In some embodiments, said compositions are suitable for use in the treatment of cancer. In some embodiments, said pharmaceutical compositions are suitable for use in the treatment of cancer in a subject (eg, a human patient).

Formulations for storage and use are prepared by combining a purified antibody or agent of the present invention with a pharmaceutically acceptable vehicle (eg, carrier or excipient). Those skilled in the art generally consider pharmaceutically acceptable carriers, excipients and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

Suitable pharmaceutically acceptable bases include, but are not limited to, non-toxic buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives such as ammonium octadecyldimethylbenzyl chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; low molecular weight polypeptides (eg, less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as

- 68 039540 sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as protein complexes with Zn; and nonionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22nd ^Edition ,

2012, Pharmaceutical Press, London.).

Pharmaceutical compositions according to the present invention may be administered by any of a number of methods, both topical and systemic. The introduction can be carried out locally through the use of epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; through the lungs by inhalation or insufflation of powders or aerosols, including through a nebulizer, intratracheally and intranasally; orally; or parenterally, including intravenously, intra-arterially, intratumorally (into tumors), subcutaneously, intraperitoneally, intramuscularly (eg, by injection or infusion), or intracranially (eg, intrathecally or intraventricularly).

The specified therapeutic composition can be represented by a single dosage form. Such formulations include tablets, pills, capsules, powders, granules, aqueous or non-aqueous solutions or suspensions, or suppositories. In solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier. Standard tabletting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dibasic calcium phosphate or gums, and diluents (eg, water). These ingredients can be used to form a solid precomposition containing a homogeneous mixture with a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. Said solid precomposition is then subdivided into dosage units of the type described above. For tablets, pills, etc. The formulation or composition may be coated or otherwise formulated to provide a dosage form that provides a sustained release benefit. For example, a tablet or pill may contain an internal composition coated with an external component. In addition, these two components can be separated by an enteric layer, which serves to protect against disintegration and allows the internal component to pass intact through the stomach or delay its release. Various materials can be used for such enteric layers or coatings, such materials include a range of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

The TIGIT binding agents described herein may also be microencapsulated. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or as macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22 ^st Edition, 2012, Pharmaceutical Press, London.

In some embodiments, the pharmaceutical compositions comprise an agent of the present invention in combination with liposomes. Methods for preparing liposomes are known to those skilled in the art. For example, some liposomes can be prepared by reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters with a given pore size to obtain liposomes of the desired diameter.

In some embodiments, sustained release formulations containing TIGIT binding agents as described herein can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the agent, characterized in that said matrices are in the form of molded articles (eg, films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7-ethyl H-glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid/glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of a lactic acid/glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

V. Selected implementation options.

Embodiment 1 provides an isolated antibody specifically binding to the extracellular domain of TIGIT that comprises: (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7) or GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG ( SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP (SEQ ID NO: 9), and/or (b) a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11) or RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID

- 69 039540

NO: 12) or QQEHLVAWIYN (SEQ ID NO: 16).

In Embodiment 2, the antibody of Embodiment 1 comprises: (a) a heavy chain CDR1 containing GSSLSSSYMS (SEQ ID NO: 7), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP ( SEQ ID NO: 9), and/or (b) a light chain CDR1 containing QASQSISSYLNW (SEQ ID NO: 10), a light chain CDR2 containing DALKLAS (SEQ ID NO: 11), and a light chain CDR3 containing QQEHSVGNVDN (SEQ ID NO: 12).

In Embodiment 3, the antibody of Embodiment 1 comprises: (a) a heavy chain CDR1 containing GFSLSSSYMS (SEQ ID NO: 13), a heavy chain CDR2 containing IIGSNGNTYYANWAKG (SEQ ID NO: 8), and a heavy chain CDR3 containing GGYRTSGMDP ( SEQ ID NO: 9); and/or (b) a light chain CDR1 containing QASQNIYSDLAW (SEQ ID NO: 81), a light chain CDR2 containing RASTLAS (SEQ ID NO: 15), and a light chain CDR3 containing QQEHLVAWIYN (SEQ ID NO: 16).

In Embodiment 4, an isolated antibody that specifically binds TIGIT comprises: (a) a heavy chain variable region that shares at least 90% sequence identity with SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32; and/or (b) a light chain variable region that shares at least 90% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20.

In Embodiment 5, an antibody of any one of Embodiments 1-4 comprises: (a) a heavy chain variable region differing by at least 95% sequence identity to SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 32; and/or (b) a light chain variable region that shares at least 95% sequence identity with SEQ ID NO: 18 or SEQ ID NO: 20.

In Embodiment 6, the antibody of Embodiment 5 comprises: (a) a heavy chain variable region comprising SEQ ID NO: 17; and (b) a light chain variable region comprising SEQ ID NO: 18.

In Embodiment 7, the antibody of Embodiment 5 comprises: (a) a heavy chain variable region comprising SEQ ID NO: 19; and (b) a light chain variable region comprising SEQ ID NO: 20.

In Embodiment 8, the antibody of Embodiment 5 comprises: (a) a heavy chain variable region comprising SEQ ID NO: 32; and (b) a light chain variable region comprising SEQ ID NO: 20.

In Embodiment 9, the antibody of any one of Embodiments 1-8 is a monoclonal antibody.

In Embodiment 10, the antibody of any one of Embodiments 1-9 is a humanized antibody.

In Embodiment 11, the antibody of any one of Embodiments 1-9 is a human antibody.

In Embodiment 12, the antibody of any one of Embodiments 1-10 is a recombinant antibody or a chimeric antibody.

In Embodiment 13, the antibody of any one of Embodiments 1-12 is a bispecific antibody.

In Embodiment 14, the antibody of any one of Embodiments 1-13 is an antibody fragment containing an antigen-binding site.

In Embodiment 15, the antibody of any one of Embodiments 1-13 is an IgG antibody.

In Embodiment 16, the antibody of Embodiment 15 is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody.

In Embodiment 17, the antibody comprises: (a) a heavy chain amino acid sequence selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, and SEQ ID NO: 56; and (b) a light chain amino acid sequence selected from the group consisting of: SEQ ID NO: 28 and SEQ ID NO: 30.

In Embodiment 18, the antibody of Embodiment 17 comprises the heavy chain amino acid sequence as set forth in SEQ ID NO: 26 and the light chain amino acid sequence as set forth in SEQ ID NO: 28.

In Embodiment 19, the antibody of Embodiment 17 comprises the heavy chain amino acid sequence as set forth in SEQ ID NO: 27 and the light chain amino acid sequence as set forth in SEQ ID NO: 28.

In Embodiment 20, the antibody of Embodiment 17 comprises the heavy chain amino acid sequence as set forth in SEQ ID NO: 29 and the light chain amino acid sequence as set forth in SEQ ID NO: 30.

In Embodiment 21, the antibody of Embodiment 17 comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 34 and the light chain amino acid sequence set forth in SEQ ID NO: 30.

In Embodiment 22, the antibody of Embodiment 17 comprises the heavy chain amino acid sequence as set forth in SEQ ID NO: 56 and the light chain amino acid sequence as set forth in SEQ ID NO: 30.

In embodiment 23, the antibody comprises a heavy chain variable region and a light chain variable region from an antibody selected from the group consisting of: 313R11, 313R12, 313R14, 313R19, and 313R20.

In Embodiment 24, the antibody is selected from the group consisting of: 313R11, 313R12, 313R14, 313R19, and 313R20.

In Embodiment 25, the antibody comprises a heavy chain variable region encoded by a plasmid deposited with ATCC as PTA-122180.

In Embodiment 26, the antibody contains a light chain variable region encoded by a plasmid deposited with ATCC as PTA-122181.

In Embodiment 27, the antibody comprises a polypeptide containing a heavy chain variable region encoded by a plasmid deposited with ATCC under PTA-121180.

In Embodiment 28, the antibody comprises a polypeptide containing a light chain variable region encoded by a plasmid deposited with ATCC under PTA-122181.

In Embodiment 29, the antibody comprises a heavy chain variable region encoded by a plasmid deposited with ATCC number PTA-122180 and a light chain variable region encoded by a plasmid deposited with ATCC number PTA-122181.

According to embodiment 30, the antibody comprises a polypeptide encoded by a plasmid deposited with ATCC number PTA-122180 and a plasmid deposited with ATCC number PTA122181.

In Embodiment 31, the isolated antibody competes with the antibody of any of Embodiments 1-30 for specific binding to TIGIT.

In Embodiment 32, the isolated antibody binds the same epitope on TIGIT as the antibody of any of Embodiments 1-30.

In Embodiment 33, the isolated antibody binds an epitope on TIGIT that overlaps with an epitope on TIGIT that binds the antibody of any one of Embodiments 1-30.

In Embodiment 34, the antibody of any one of Embodiments 1-33 inhibits TIGIT binding to the poliovirus receptor (PVR).

In Embodiment 35, the antibody of any one of Embodiments 1-33 inhibits or blocks the interaction between TIGIT and PVR.

In Embodiment 36, the antibody of any one of Embodiments 1-33 inhibits TIGIT signaling.

In Embodiment 37, the antibody of any one of Embodiments 1-33 is a TIGIT-mediated signaling antagonist.

In Embodiment 38, the antibody of any one of Embodiments 1-33 inhibits TIGIT activation.

In Embodiment 39, the antibody of any one of Embodiments 1-33 inhibits TIGIT phosphorylation.

In Embodiment 40, an antibody according to any one of Embodiments 1-33 reduces TIGIT expression on the cell surface.

In Embodiment 41, the antibody of any one of Embodiments 1-33 induces and/or enhances an immune response.

Embodiment 42 provides an antibody according to Embodiment 41, characterized in that said immune response is directed to a tumor or tumor cell.

Embodiment 43 provides an antibody according to Embodiment 41, characterized in that said immune response is directed to a virus or virus-infected cell.

In Embodiment 44, an antibody according to any one of Embodiments 1-33 enhances cellular immunity.

In Embodiment 45, an antibody according to any one of Embodiments 1-33 enhances T cell activity.

In Embodiment 46, an antibody according to any one of Embodiments 1-33 enhances cytolytic T-cell (CTL) activity.

In Embodiment 47, an antibody according to any one of Embodiments 1-33 enhances natural killer (NK) cell activity.

In Embodiment 48, an antibody according to any one of Embodiments 1-33 that increases IL-2 production and/or the number of IL-2 producing cells.

In Embodiment 49, the antibody of any one of Embodiments 1-33 increases IFN-γ production and/or the number of IFN-γ producing cells.

In Embodiment 50, the antibody of any one of Embodiments 1-33 enhances a Th1-type immune response.

In Embodiment 51, the antibody of any one of Embodiments 1-33 reduces IL-4 production and/or the number of IL-4 producing cells.

In Embodiment 52, the antibody of any one of Embodiments 1-33 reduces IL-10 and/or the number of IL-10 producing cells.

In Embodiment 53, an antibody according to any one of Embodiments 1-33 reduces a Th2-type immune response.

In Embodiment 54, the antibody of any one of Embodiments 1-33 inhibits and/or reduces the suppressor activity of regulatory T cells (Treg).

In Embodiment 55, the antibody of any one of Embodiments 1-33 inhibits and/or reduces the suppressor activity of myeloid-derived suppressor cells (MSC cells).

In Embodiment 56, the antibody of any one of Embodiments 1-55 inhibits tumor growth.

In Embodiment 57, the heterodimeric agent comprises an antibody according to any one of Embodiments 1-33.

According to embodiment 58, the bispecific agent comprises a) a first arm that specifically binds TIGIT, and b) a second arm, said first arm comprising an antibody according to any one of embodiments 1-33.

Embodiment 59 provides a bispecific agent according to Embodiment 58, characterized in that said second arm contains an antigen-binding site from an antibody.

Embodiment 60 provides a bispecific agent according to Embodiment 58, wherein said second arm specifically binds PD-1, PD-L1, CTLA4, TIM-3, LAG-3, OX-40, or GITR.

Embodiment 61 provides a bispecific agent according to Embodiment 58, characterized in that said second arm specifically binds a tumor antigen.

Embodiment 62 provides a bispecific agent according to Embodiment 58, wherein said second arm comprises an immune response stimulating agent.

Embodiment 63 provides a bispecific agent according to Embodiment 62, wherein said immune response stimulating agent is selected from the group consisting of: granulocyte macrophage colony stimulating factor (GMCSF), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor ( GCSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), ligand 4-1BB, GiTRL, OX-40L, anti-CD3, anti-CTLA4, anti-PD-1, anti-PD-L1, anti-GITR, anti-OX-40, anti-LAG-3, and anti-TIM- 3.

Embodiment 64 provides a bispecific agent according to any one of Embodiments 58-63, wherein said first arm contains a first CH3 domain and said second arm contains a second CH3 domain, each modified to promote heterodimer formation.

Embodiment 65 provides a bispecific agent according to Embodiment 64, characterized in that said first and second CH3 domains are modified based on electrostatic effects.

Embodiment 66 provides a bispecific agent according to any one of Embodiments 58-63, wherein said first arm comprises a first human IgG1 constant region with amino acid substitutions at positions corresponding to positions 253 and 292 of SEQ ID NO: 41, wherein said amino acids are replaced to glutamate or aspartate, and said second arm contains a second human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of SEQ ID NO: 41, with said amino acids replaced with lysine.

Embodiment 67 provides a bispecific agent according to any one of Embodiments 58-63, wherein said first arm comprises a first human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of SEQ ID NO: 41, wherein said amino acids are replaced to lysine, and said second arm contains a second human IgG1 constant region with amino acid substitutions at positions corresponding to positions 253 and 292 of SEQ ID NO: 41, said amino acids being replaced by glutamate or aspartate.

Embodiment 68 provides a bispecific agent according to any one of Embodiments 58-63, wherein said first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:42, wherein in this case, said amino acids are replaced with glutamate or aspartate, and said second arm contains a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO: 42, with said amino acids replaced with lysine.

Embodiment 69 provides a bispecific agent according to any one of Embodiments 58-63, wherein said first arm comprises a first human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO: 42, wherein said amino acids are replaced to lysine, and said second arm contains a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO: 42, said amino acids being replaced by glutamate or aspartate.

Embodiment 70 provides a bispecific agent according to Embodiment 64, characterized in that said first and second CH3 domains are modified in a ridge-in-trough pattern.

In Embodiment 71, the bispecific agent according to any one of Embodiments 58-70 inhibits TIGIT binding to PVR.

According to embodiment 72, the bispecific agent according to any one of embodiments 58-70 inhibits or blocks the interaction between TIGIT and PVR.

In Embodiment 73, the bispecific agent of any one of Embodiments 58-70 inhibits TIGIT signaling.

In Embodiment 74, the bispecific agent of any one of Embodiments 58-70 is a TIGIT-mediated signaling antagonist.

According to embodiment 75, the bispecific agent according to any one of embodiments 58-70 inhibits TIGIT activation.

According to embodiment 76, the bispecific agent according to any one of embodiments 58-70 inhibits TIGIT phosphorylation.

In Embodiment 77, the bispecific agent of any one of Embodiments 58-70 reduces TIGIT expression on the cell surface.

In Embodiment 78, the bispecific agent of any one of Embodiments 58-70 induces and/or enhances an immune response.

Embodiment 79 provides a bispecific agent according to Embodiment 78, characterized in that said immune response is directed to a tumor or tumor cell.

Embodiment 80 provides a bispecific agent according to Embodiment 78, characterized in that said immune response is directed to a virus or a virus-infected cell.

In Embodiment 81, the bispecific agent according to any one of Embodiments 58-70 enhances cellular immunity.

In Embodiment 82, a bispecific agent according to any one of Embodiments 58-70 increases T cell activity.

In Embodiment 83, the bispecific agent according to any one of Embodiments 58-70 increases CTL activity.

In Embodiment 84, the bispecific agent according to any one of Embodiments 58-70 increases NK cell activity.

In Embodiment 85, the bispecific agent of any one of Embodiments 58-70 increases IL-2 production and/or the number of IL-2 producing cells.

According to embodiment 86, the bispecific agent according to any of embodiments 58-70 increases IFN-γ production and/or the number of IFN-γ producing cells.

In Embodiment 87, the bispecific agent of any one of Embodiments 58-70 enhances a Th1-type immune response.

In Embodiment 88, the bispecific agent of any one of Embodiments 58-70 reduces IL-4 production and/or the number of IL-4 producing cells.

Embodiment 89 provides a bispecific agent according to any one of Embodiments 58-70 that reduces IL-10 and/or the number of IL-10 producing cells.

In Embodiment 90, the bispecific agent of any one of Embodiments 58-70 reduces a Th2-type immune response.

In Embodiment 91, the bispecific agent of any one of Embodiments 58-70 inhibits and/or reduces the suppressor activity of Treg cells.

In Embodiment 92, the bispecific agent of any one of Embodiments 58-70 inhibits and/or reduces the suppressor activity of SCMP cells.

- 73 039540

In Embodiment 93, the bispecific agent of any one of Embodiments 58-92 inhibits tumor growth.

According to embodiment 94, the polypeptide contains a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 55, and SEQ ID NO: 56.

According to embodiment 95, the cell contains or produces an antibody, bispecific agent, or polypeptide according to any one of embodiments 1-94.

In Embodiment 96, the composition comprises an antibody, bispecific agent, or polypeptide according to any one of Embodiments 1-94.

In Embodiment 97, the pharmaceutical composition comprises an antibody, bispecific agent, or polypeptide according to any one of Embodiments 1-94, and a pharmaceutically acceptable carrier.

According to embodiment 98, the isolated polynucleotide molecule contains a polynucleotide that encodes an antibody, a bispecific agent, or a polypeptide according to any one of embodiments 1-94.

In Embodiment 99, the vector contains a polynucleotide according to Embodiment 98.

In embodiment 100, the isolated cell contains a polynucleotide according to embodiment 98 or a vector according to embodiment 99.

According to embodiment 101, a method of inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of an antibody, bispecific agent, or polypeptide according to any one of embodiments 1-94.

Embodiment 102 provides a method for inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of an antibody according to any one of Embodiments 1-33.

Embodiment 103 provides the method of Embodiment 101 or Embodiment 102, wherein said immune response is directed against a tumor or cancer.

Embodiment 104 provides a method for inhibiting tumor cell growth, characterized in that said method comprises contacting said tumor cells with an effective amount of an antibody, bispecific agent, or polypeptide according to any one of Embodiments 1-94.

Embodiment 105 provides a method for inhibiting tumor growth in a subject, the method comprising administering to said subject a therapeutically effective amount of an antibody, bispecific agent, or polypeptide according to any one of Embodiments 1-94.

Embodiment 106 provides a method for inhibiting tumor growth in a subject, the method comprising administering to said subject a therapeutically effective amount of an antibody according to any one of Embodiments 1-33.

Embodiment 107 provides a method according to any one of Embodiments 103-106, wherein said tumor or tumor cell is selected from the group consisting of a colon and rectal tumor, an ovarian tumor, a pancreatic tumor, a lung tumor, a liver tumor, a tumor breast, kidney tumors, prostate tumors, gastrointestinal tumors, melanomas, cervical tumors, bladder tumors, glioblastomas and head and neck tumors.

Embodiment 108 provides a method of treating cancer in a subject, characterized in that said method comprises administering to said subject a therapeutically effective amount of an antibody, bispecific agent, or polypeptide according to any one of Embodiments 1-94.

Embodiment 109 provides a method of treating cancer in a subject, characterized in that said method comprises administering to said subject a therapeutically effective amount of an antibody according to any one of Embodiments 1-33.

Embodiment 110 provides the method of Embodiment 108 or Embodiment 109, wherein said cancer is selected from the group consisting of colon cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer.

Embodiment 111 provides a method according to any one of Embodiments 101-110, which further comprises administering at least one additional therapeutic agent.

Embodiment 112 provides the method of Embodiment 111, wherein said additional therapeutic agent is a chemotherapeutic agent.

Embodiment 113 provides the method of Embodiment 111, wherein said additional therapeutic agent is an antibody.

Embodiment 114 provides the method of Embodiment 111, wherein said additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-LAG-3 antibody, or an anti-TIM-3 antibody.

Embodiment 115 provides the method of Embodiment 111, wherein said additional therapeutic agent is an immune response stimulating agent.

Embodiment 116 provides a method according to Embodiment 115, wherein said immune response stimulating agent is selected from the group consisting of: GM-CSF, MCSF, GCSF, IL-2, IL-3, IL-12, IL-15 , B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, GITRL, OX-40 ligand, anti-CD3 antibody, anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-GITR antibody , antibodies against OX-40, antibodies against LAG-3 and antibodies against TIM-3.

Embodiment 117 provides the method of Embodiment 111, wherein said additional therapeutic agent is an inhibitor of the Notch pathway, the Wnt pathway, or the RSPO/LGR pathway.

According to embodiment 118, the method according to any one of embodiments 101-103 or 105-117, wherein said subject is a human.

According to embodiment 119, the method according to any one of embodiments 101-103 or 105-118, wherein said subject has had a tumor removed or a cancer has been removed.

According to embodiment 120, the method of any one of embodiments 101-119, wherein said tumor or cancer expresses PD-L1.

In Embodiment 121, the method of any one of Embodiments 101-120, further comprising the step of determining the expression level of PD-L1 in said tumor or cancer.

Embodiment 122 provides the method of Embodiment 121, wherein the determination of the expression level of PD-L1 is performed prior to treatment or contact with an antibody.

Embodiment 123 provides a method according to Embodiment 121 or Embodiment 122, characterized in that, if said tumor or cancer is overexpressed by PD-L1, said antibody, said bispecific agent, or polypeptide: (a) administers the specified subject; or (b) brought into contact with said tumor or tumor cell.

Embodiment 124 provides a plasmid deposited with ATCC and assigned the number PTA-122180.

Embodiment 125 provides a plasmid deposited with ATCC and assigned the number PTA-122181.

Embodiment 126 provides an isolated antibody specifically binding to the extracellular domain of human TIGIT that comprises: (a) a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), a heavy chain CDR2 containing YISYSGSTSYNPSLRS (SEQ ID NO: 58), and A heavy chain CDR3 containing ARRQVGLGFAY (SEQ ID NO: 59) and/or (b) a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 containing SASYRYT (SEQ ID NO: 61) and a CDR3 light chain containing QQHYSTP (SEQ ID NO: 62).

Embodiment 127 provides an isolated antibody specifically binding to human TIGIT that comprises: (a) a heavy chain variable region that shares at least 90% sequence identity with SEQ ID NO: 63 or SEQ ID NO: 67; and/or (b) a light chain variable region that shares at least 90% sequence identity with SEQ ID NO: 64 or SEQ ID NO: 68.

In Embodiment 128, the antibody of Embodiment 126 or Embodiment 127 comprises: (a) a heavy chain variable region differing by at least 95% sequence identity to SEQ ID NO: 63 or SEQ ID NO: 67; and/or (b) a light chain variable region that shares at least 95% sequence identity with SEQ ID NO: 64 or SEQ ID NO: 68.

In Embodiment 129, the antibody of Embodiment 128 comprises a heavy chain variable region comprising SEQ ID NO: 63 and a light chain variable region comprising SEQ ID NO: 64.

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In embodiment 130, the antibody of embodiment 128 comprises a heavy chain variable region comprising SEQ ID NO: 67 and a light chain variable region comprising SEQ ID NO: 68.

In Embodiment 131, the antibody of any one of Embodiments 126-130 is a monoclonal antibody.

In Embodiment 132, the antibody of any one of Embodiments 126-128, 130, or 131 is a humanized antibody.

In embodiment 133, the antibody of embodiment 126 is a human antibody.

In Embodiment 134, the antibody of any one of Embodiments 126-133 is a recombinant antibody or a chimeric antibody.

In Embodiment 135, the antibody of any one of Embodiments 126-134 is a bispecific antibody.

In Embodiment 136, the antibody of any one of Embodiments 126-135 is an antibody fragment containing an antigen binding site.

In Embodiment 137, the antibody of any one of Embodiments 126-136 is an IgG antibody.

In Embodiment 138, the antibody of Embodiment 137 is an IgG1 antibody, an IgG2 antibody, or an IgG4 antibody.

Embodiment 139 provides an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 70 and the light chain amino acid sequence set forth in SEQ ID NO: 72.

Embodiment 140 provides an antibody comprising the same heavy chain variable region and light chain variable region amino acid sequences as the 313M32 antibody.

Embodiment 141 provides an antibody comprising a heavy chain variable region encoded by a plasmid deposited with ATCC under PTA-122346.

Embodiment 142 provides an antibody comprising a light chain variable region encoded by a plasmid deposited with ATCC under PTA-122347.

Embodiment 143 provides an antibody containing a light chain encoded by a plasmid deposited with ATCC under PTA-122347.

Embodiment 144 provides an antibody comprising a heavy chain variable region encoded by a plasmid deposited with ATCC number PTA-122346 and a light chain variable region encoded by a plasmid deposited with ATCC number PTA-122347.

Embodiment 145 provides an antibody comprising a heavy chain variable region encoded by a plasmid deposited with ATCC number PTA-122346 and a light chain encoded by a plasmid deposited with ATCC number PTA-122347.

In Embodiment 146, the antibody of any of Embodiments 126-145 does not bind: (a) mouse TIGIT; (b) rat TIGIT; (c) TIGIT of the cynomolgus monkey; and/or (d) TIGIT rhesus monkey.

Embodiment 147 provides an isolated antibody that competes with an antibody according to any of embodiments 126-146 for specific binding to TIGIT.

Embodiment 148 provides an isolated antibody that binds the same epitope on TIGIT as an antibody of any of Embodiments 126-146.

Embodiment 149 provides an isolated antibody that binds an epitope on TIGIT overlapping with an epitope on TIGIT that binds an antibody according to any of embodiments 126-146.

Embodiment 150 provides an isolated antibody specifically binding to human TIGIT, characterized in that said antibody binds an epitope comprising: (a) amino acids constituting the sequence of SEQ ID NO: 79; (b) amino acids included in the sequence of SEQ ID NO: 80; (c) amino acids included in the sequences of SEQ ID NO: 79 and SEQ ID NO: 80; (d) amino acids Q62 and I109 of SEQ ID NO: 4; (e) amino acids Q62 and T119 of SEQ ID NO: 4; (f) amino acids Q64 and I109 of SEQ ID NO: 4; (g) amino acids Q64 and T119 of SEQ ID NO: 4; (h) amino acids Q62, Q64 and I109 of SEQ ID NO: 4; (i) amino acids Q62, Q64 and T119 of SEQ ID NO: 4; (j) amino acids Q62, I109 and T119 of SEQ ID NO: 4; (k) amino acids Q64, I109 and T119 of SEQ ID NO: 4; or (1) amino acids Q62, Q64, I109 and T119 of SEQ ID NO: 4.

Embodiment 151 provides an antibody according to embodiment 150, characterized in that said antibody binds an epitope containing the amino acids included in SEQ ID NO: 79 and SEQ ID NO: 80.

Embodiment 152 provides an antibody according to embodiment 150, characterized in that said antibody binds an epitope containing at least something

- 76 039540 one of the amino acids N58, E60, Q62, Q64, L65, F107, I109, H111, T117, T119 and G120 sequences

SEQ ID NO: 4.

Embodiment 153 provides an antibody according to any of the embodiments

150-152, characterized in that said epitope is a conformational epitope.

In Embodiment 154, the antibody of any one of Embodiments 126-153 inhibits TIGIT binding to the poliovirus receptor (PVR).

In Embodiment 155, the antibody of any one of Embodiments 126-153 inhibits or blocks the interaction between TIGIT and PVR.

In Embodiment 156, the antibody of any one of Embodiments 126-153 inhibits TIGIT signaling.

In Embodiment 157, the antibody of any one of Embodiments 126-153 is a TIGIT-mediated signaling antagonist.

In Embodiment 158, the antibody of any one of Embodiments 126-153 inhibits TIGIT activation.

In Embodiment 159, the antibody of any one of Embodiments 126-153 inhibits TIGIT phosphorylation.

In Embodiment 160, an antibody according to any one of Embodiments 126-153 reduces TIGIT expression on the cell surface.

In Embodiment 161, the antibody of any one of Embodiments 126-153 induces and/or enhances an immune response.

Embodiment 162 provides an antibody of Embodiment 161, characterized in that said immune response is directed to a tumor or tumor cell.

In Embodiment 163, an antibody according to any one of Embodiments 126-153 enhances cellular immunity.

In Embodiment 164, an antibody according to any one of Embodiments 126-153 enhances T cell activity.

In Embodiment 165, an antibody according to any one of Embodiments 126-153 enhances cytolytic T-cell (CTL) activity.

In Embodiment 166, an antibody according to any one of Embodiments 126-153 enhances natural killer (NK) cell activity.

According to embodiment 167, an antibody according to any one of embodiments 126-153 increases IL-2 production and/or the number of IL-2 producing cells.

According to embodiment 168, an antibody according to any of embodiments 126-153 increases IFN-γ production and/or the number of IFN-γ producing cells.

In Embodiment 169, an antibody according to any one of Embodiments 126-153 enhances a Th1-type immune response.

According to embodiment 170, an antibody according to any one of embodiments 126-153 reduces IL-4 production and/or the number of IL-4 producing cells.

In Embodiment 171, the antibody of any one of Embodiments 126-153 reduces IL-10 production and/or the number of IL-10 producing cells.

In Embodiment 172, an antibody according to any one of Embodiments 126-153 reduces a Th2-type immune response.

In Embodiment 173, the antibody of any one of Embodiments 126-153 inhibits and/or reduces the suppressor activity of regulatory T cells (Treg).

In Embodiment 174, the antibody of any one of Embodiments 126-153 inhibits and/or reduces the suppressor activity of myeloid-derived suppressor cells (MSC cells).

In Embodiment 175, the antibody of any one of Embodiments 126-174 inhibits tumor growth.

Embodiment 176 provides a heterodimeric agent that comprises an antibody according to any of embodiments 126-146.

Embodiment 177 provides a bispecific agent comprising: a) a first arm that specifically binds TIGIT, and b) a second arm, said first arm comprising an antibody according to any of embodiments 126-146.

Embodiment 178 provides a bispecific agent according to Embodiment 177, characterized in that said second arm contains an antigen-binding site from an antibody.

Embodiment 179 provides the bispecific agent of claim 177, wherein said second arm specifically binds PD-1, PD-L1, CTLA-4, TIM-3, LAG-3, OX-40, or GITR.

Embodiment 180 provides the bispecific agent of claim 177, wherein said second arm specifically binds a tumor antigen.

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Embodiment 181 provides a bispecific agent according to Embodiment 177, characterized in that said second arm contains an immunotherapeutic agent.

According to embodiment 182, a bispecific agent is provided according to embodiment 181, characterized in that said immunotherapeutic agent is selected from the group consisting of: granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (MCSF), granulocyte colony-stimulating factor (GCSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (Cd86), ligand 4-1BB, GITRL, OX-40L, anti-Cd3, anti-CTLA4, anti-PD-1, anti-PD-L1, anti-GITR, anti-OX40, anti-4-1BB, anti-LAG-3, and anti-TIM- 3.

Embodiment 183 provides a bispecific agent according to any one of Embodiments 177-182, wherein said first arm contains a first CH3 domain and said second arm contains a second CH3 domain, each modified to promote heterodimer formation.

Embodiment 184 provides a bispecific agent according to Embodiment 183, wherein said first and second CH3 domains are modified based on electrostatic effects.

Embodiment 185 provides a bispecific agent according to any one of Embodiments 177-182, characterized in that said first arm comprises a first human IgG1 constant region with amino acid substitutions at positions corresponding to positions 253 and 292 of SEQ ID NO: 41, wherein said amino acids are replaced with glutamate or aspartate, and said second arm contains a second human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of SEQ ID NO: 41, with said amino acids replaced with lysine.

Embodiment 186 provides a bispecific agent according to any one of Embodiments 177-182, characterized in that said first arm comprises a first human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of SEQ ID NO: 41, wherein said amino acids are changed to lysine, and said second arm contains a second human IgG1 constant region with amino acid substitutions at positions corresponding to positions 253 and 292 of SEQ ID NO: 41, said amino acids being changed to glutamate or aspartate.

Embodiment 187 provides a bispecific agent according to Embodiment 183, characterized in that said first and second CH3 domains are modified in a ridge-to-trough pattern.

In Embodiment 188, the bispecific agent of any one of Embodiments 177-187 inhibits TIGIT binding to PVR.

In Embodiment 189, the bispecific agent according to any one of Embodiments 177-187 inhibits or blocks the interaction between TIGIT and PVR.

According to embodiment 190, the bispecific agent according to any one of embodiments 177-187 inhibits TIGIT signaling.

In Embodiment 191, the bispecific agent of any one of Embodiments 177-187 is a TIGIT-mediated signaling antagonist.

According to embodiment 192, the bispecific agent according to any one of embodiments 177-187 inhibits the activation of TIGIT.

According to embodiment 193, the bispecific agent according to any one of embodiments 177-187 inhibits TIGIT phosphorylation.

In Embodiment 194, a bispecific agent according to any one of Embodiments 177-187 reduces expression of TIGIT on the cell surface.

In Embodiment 195, the bispecific agent of any one of Embodiments 177-187 induces and/or enhances an immune response.

Embodiment 196 provides a bispecific agent according to Embodiment 195, characterized in that said immune response is directed to a tumor or tumor cell.

In Embodiment 197, a bispecific agent according to any one of Embodiments 177-187 enhances cellular immunity.

In Embodiment 198, a bispecific agent according to any one of Embodiments 177-187 increases T cell activity.

In Embodiment 199, a bispecific agent according to any one of Embodiments 177-187 increases CTL activity.

In Embodiment 200, the bispecific agent of any one of Embodiments 177-187 increases NK cell activity.

In Embodiment 201, the bispecific agent according to any one of embodiments 177-187 increases IL-2 production and/or the number of IL-2 producing cells.

According to embodiment 202, the bispecific agent according to any one of embodiments 177-187 increases IFN-γ production and/or the number of IFN-γ producing cells.

In Embodiment 203, the bispecific agent of any of Embodiments 177-187 enhances a Th1-type immune response.

According to embodiment 204, the bispecific agent according to any one of embodiments 177-187 reduces IL-4 production and/or the number of IL-4 producing cells.

In Embodiment 205, the bispecific agent of any one of Embodiments 177-187 reduces IL-10 production and/or the number of IL-10 producing cells.

In Embodiment 206, the bispecific agent of any one of Embodiments 177-187 reduces a Th2-type immune response.

In Embodiment 207, the bispecific agent of any one of Embodiments 177-187 inhibits and/or reduces the suppressor activity of Treg cells.

In Embodiment 208, the bispecific agent of any one of Embodiments 177-187 inhibits and/or reduces the suppressor activity of SCMP cells.

In Embodiment 209, the bispecific agent of any one of Embodiments 177-208 inhibits tumor growth.

Embodiment 210 provides a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO: 63, SEQ ID NO: 64, SeQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO : 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 82 and SEQ ID NO: 83.

Embodiment 211 provides a cell containing or producing an antibody, bispecific agent, or polypeptide according to any of embodiments 126-210.

Embodiment 212 provides a composition comprising an antibody, bispecific agent, or polypeptide according to any of embodiments 126-210.

Embodiment 213 provides a pharmaceutical composition comprising an antibody, bispecific agent, or polypeptide according to any one of Embodiments 126-210, and a pharmaceutically acceptable carrier.

Embodiment 214 provides an isolated polynucleotide molecule comprising a polynucleotide that encodes an antibody, a bispecific agent, or a polypeptide according to any of embodiments 126-210.

Embodiment 215 provides an isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 84.

Embodiment 216 provides a vector containing a polynucleotide according to Embodiment 214 or Embodiment 215.

Embodiment 217 provides an isolated cell containing the polynucleotide of Embodiment 214 or Embodiment 215.

Embodiment 218 provides an isolated cell containing the vector of Embodiment 216.

Embodiment 219 provides a method for inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of an antibody, bispecific agent, or polypeptide according to any of embodiments 126-210.

According to embodiment 220, a method of inducing an immune response, activating an immune response, stimulating an immune response, enhancing, enhancing, or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of an antibody according to any one of embodiments 126-153.

Embodiment 221 provides the method of Embodiment 219 or Embodiment 220, wherein said immune response is directed against a tumor or cancer.

Embodiment 222 provides a method for inhibiting tumor cell growth, characterized in that said method comprises contacting said tumor cells with an effective amount of an antibody, bispecific agent, or polypeptide according to any of embodiments 126-210.

Embodiment 223 provides a method for inhibiting tumor growth in a subject, the method comprising administering to said subject a therapeutically effective amount of an antibody, bispecific agent, or polypeptide according to any of embodiments 126-210.

Embodiment 224 provides a method for inhibiting tumor growth in a subject, the method comprising administering to said subject a therapeutically effective amount of an antibody according to any one of Embodiments 126-153.

Embodiment 225 provides a method according to any of the embodiments

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221-224, characterized in that said tumor or tumor cell is selected from the group consisting of colon and rectum tumor, ovarian tumor, pancreatic tumor, lung tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, tumor gastrointestinal tract, melanomas, cervical tumors, bladder tumors, glioblastomas, and head and neck tumors.

Embodiment 226 provides a method of treating cancer in a subject, characterized in that said method comprises administering to said subject a therapeutically effective amount of an antibody, bispecific agent, or polypeptide according to any one of embodiments 126-210.

Embodiment 227 provides a method of treating cancer in a subject, characterized in that said method comprises administering to said subject a therapeutically effective amount of an antibody according to any of embodiments 126-153.

Embodiment 228 provides the method of Embodiment 226 or Embodiment 227, wherein said cancer is selected from the group consisting of colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer.

In Embodiment 229, the method of any of Embodiments 219-228 further comprises administering at least one additional therapeutic agent.

Embodiment 230 provides the method of Embodiment 229, wherein said additional therapeutic agent is a chemotherapeutic agent.

Embodiment 231 provides the method of Embodiment 229, wherein said additional therapeutic agent is an antibody.

Embodiment 232 provides the method of Embodiment 229, wherein said additional therapeutic agent is an immunotherapeutic agent.

Embodiment 233 provides a method according to Embodiment 232, wherein said immunotherapeutic agent is selected from the group consisting of: GM-CSF, MCSF, GCSF, IL-2, IL-3, IL-12, IL-15, B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, GITRL, OX-40 ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-GITR antibody, antibody anti-OX-40, anti-4-1BB, anti-LAG-3, and anti-TIM-3.

Embodiment 234 provides the method of Embodiment 229, wherein said additional therapeutic agent is an inhibitor of the Notch pathway, the Wnt pathway, or the RSPO/LGR pathway.

Embodiment 235 provides a method according to any of embodiments 219-221 or 223-234, wherein said subject has had a tumor or cancer removed.

Embodiment 236 provides a method according to any of embodiments 221-235, wherein said tumor or cancer expresses PD-L1.

Embodiment 237 provides a method according to any one of Embodiments 221-236, further comprising the step of determining the expression level of PD-L1 in said tumor or cancer.

Embodiment 238 provides the method of Embodiment 237, wherein the determination of the level of PD-L1 expression is performed prior to treatment or contact with an antibody.

Embodiment 239 provides a method according to Embodiment 236 or Embodiment 237, wherein if said tumor or cancer is overexpressed by PD-L1, said antibody, said bispecific agent, or polypeptide: (a) administers the specified subject; or (b) contact with said tumor or tumor cell.

Embodiment 240 provides a plasmid deposited with ATCC and assigned the number PTA-122346.

Embodiment 241 provides a plasmid deposited with ATCC and assigned the number PTA-122347.

Embodiment 242 provides an antibody comprising the heavy chain amino acid sequence set forth in SEQ ID NO: 82 and the light chain amino acid sequence set forth in SEQ ID NO: 72.

VI. Screening.

The present invention provides screening methods for identifying TIGIT binding agents that modulate an immune response. In some embodiments, the present invention provides methods for screening candidate agents, including but not limited to

- 80 039540 listed, polypeptides, antibodies, peptides, peptidomimetics, small molecules, compounds or other drugs that modulate the immune response.

In some embodiments, a screening method for determining a candidate TIGIT binding agent that modulates an immune response comprises determining whether said agent has an effect on immune cells. In some embodiments, a screening method for determining a candidate TIGIT binding agent that modulates an immune response includes determining whether said agent is capable of increasing immune cell activity. In some embodiments, a screening method for determining a candidate TIGIT binding agent that modulates an immune response includes determining if said agent is capable of increasing the activity of cytolytic cells such as CTL and/or NK cells. In some embodiments, a screening method for determining a candidate TIGIT binding agent that modulates an immune response includes determining if said agent is capable of reducing the activity of immunosuppressive cells such as Treg or SCMP cells.

VII. Kits containing the agents described herein.

The present invention provides kits that contain the TIGIT binding agents described herein and can be used to implement the methods described herein. In some embodiments, the kit contains at least one purified TIGIT binding agent in one or more containers. In some embodiments, said kits contain all components necessary and/or sufficient to perform a detection assay, including all controls, assay instructions, and all necessary analysis and reporting software. One of skill in the art will appreciate that the disclosed TIGIT binding agents of the present invention can be readily included in kits in one of the well known formats well known in the art.

Additionally, kits are provided that contain a TIGIT binding agent, as well as at least one additional therapeutic agent. In some embodiments, said second (or more) therapeutic agent is a chemotherapeutic agent. In some embodiments, said second (or more) therapeutic agent is an antibody.

Embodiments of the present invention may be further defined by reference to the following non-limiting examples, which detail the preparation of certain antibodies of the present invention and methods of using the antibodies of the present invention. Those skilled in the art will appreciate that many modifications to both materials and methods can be made without departing from the scope of the present invention.

Examples

Example 1 Obtaining antibodies against TIGIT.

Anti-TIGIT antibodies were generated using the extracellular domain of mouse TIGIT to immunize rabbits. Using flow cytometry, the supernatants were screened for antibody binding to mouse TIGIT and human TIGIT, and 9 antibody clones were selected for further characterization (FIG. 1). Supernatants from these antibodies were screened for the ability to specifically block the interaction of mouse TIGIT with the corresponding counter-receptor, mouse PVR. Four antibodies were shown to block the TIGIT/PVR interaction. Importantly, two of these antibodies were able to bind human TIGIT (313RM and 313Rb2).

The 313Rb1 heavy chain and light chain variable regions were cloned into mouse IgG1 format for chimeric 313R11 antibody and mouse IgG2a format for chimeric 313R12 antibody. The sequences from CDRs 313R11 and 313R12 are: (i) heavy chain CDR1 GSSLSSSYMS (SEQ ID NO: 7), (ii) heavy chain CDR2 IIGSNGNTYYANWAKG (SEQ ID NO: 8), (in) heavy chain CDR3 GGYRTSGMDP (SEQ ID NO: : 9), (iv) QASQSISSYLNW light chain CDR1 (SEQ ID NO: 10), (v) DALKLAS light chain CDR2 (SEQ ID NO: 11), and (vi) QQEHSVGNVDN light chain CDR3 (SEQ ID NO: 12). The heavy chain variable region sequence of 313R11 and 313R12 is represented by SEQ ID NO: 17, and the light chain variable region sequence of 313R11 and 313R12 is represented by SEQ ID NO: 18. The 313R11 heavy chain sequence is represented by SEQ ID NO: 21 or SEQ ID NO: 26 (without signal sequence). The 313R12 heavy chain sequence is represented by SEQ ID NO: 22 or SEQ ID NO: 27 (no signal sequence). The light chain sequence of 313R11 and 313R12 is represented by SEQ ID NO: 23 or SEQ ID NO: 28 (no signal sequence).

The 313Rb2 heavy chain and light chain variable regions were cloned into human IgG1 format to generate the 313R14 antibody. The CDR regions were modified to increase the affinity of human TIGIT, and the frameworks were optimized and/or humanized to produce several variants, including 313R19. The sequences from CDRs 313R14 and 313R19 are: (i) CDR1

- 81 039540 heavy chain GFSLSSSYMS (SEQ ID NO: 13), (ii) heavy chain CDR2 IIGSNGNTYYANWAKG (SEQ ID NO: 8), (iii) heavy chain CDR3 GGYRTSGMDP (SEQ ID NO: 9), (iv) light chain CDR1 QASQSNIYSDLAW (SEQ ID NO: 14) or QASQNIYSDLAW (SEQ ID NO: 81), (v) RASTLAS light chain CDR2 (SEQ ID NO: 15) and (vi) QQEHLVAWIYN light chain CDR3 (SEQ ID NO: 16). The 313R14 heavy chain variable region sequence is represented by SEQ ID NO: 19 and the 313R14 light chain variable region sequence is represented by SEQ ID NO: 20. The 313R19 heavy chain variable region sequence is represented by SEQ ID NO: 32 and the 313R19 light chain variable region sequence is represented by SEQ ID NO: 20. The 313R14 heavy chain sequence is represented by SEQ ID NO: 24 or SEQ ID NO: 29 (no signal sequence). The 313R19 heavy chain sequence is represented by SEQ ID NO: 33 or SEQ ID NO: 34 (no signal sequence).

The light chain sequence of 313R14 and 313R19 is represented by SEQ ID NO: 25 or SEQ ID NO: 30 (no signal sequence).

A plasmid encoding a polypeptide containing the heavy chain variable region of the 313R19 antibody has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, USA, under the terms of the Budapest Treaty dated May 27, 2015, and is designated PTA-122180 . A plasmid encoding a polypeptide containing the light chain variable region of the 313R19 antibody has been deposited with the ATCC database, 10801 University Boulevard, Manassas, Virginia, USA under the terms of the Budapest Treaty dated May 27, 2015, and is designated PTA-122181.

Example 2 TIGIT signaling and TIGIT phosphorylation.

As described herein, after the interaction of TIGIT with its counter-receptor PVR, TIGIT is phosphorylated along the cytoplasmic tail. TIGIT phosphorylation represents the start of a cascade that includes downstream events affecting other known signaling pathways. Accordingly, assessment of TIGIT phosphorylation as well as the phosphorylation status of downstream proteins can provide information on TIGIT function and TIGIT antagonist effect.

The Jurkat CD4+ human T cell line lacks human or mouse TIGIT expression as assessed by real-time PCR and FACS (data not shown). To obtain a TIGIT-expressing cell line, Jurkat cells were infected with a lentiviral construct expressing mouse TIGIT (mTIGIT) labeled with FLAG and green fluorescent protein (GFP). The E.G7-OVA mouse lymphoma cell line lacks human or mouse PVR expression as assessed by real-time PCR and/or FACS. E.G7-OVA cells constitutively synthesize and secrete ovalbumin (OVA), and C57BL/6 mice immunized with E.G7-OVA cells generate H- ^2Kb -restricted cytotoxic lymphocytes specific for the OVA 258276 peptide. The PVR cell line E.G7-OVA cells were infected with a lentiviral construct expressing mouse PVR (mPVR) and GFP. GFP positive cells from each infected cell line were sorted into 96-well plates using the FACSAria II instrument (BD Biosciences) and the isolated single cells were expanded into clones. GFP positivity and mTIGIT or mPVR expression were confirmed in these single cell derived clones by FACS analysis and real time PCR (data not shown).

To evaluate TIGIT phosphorylation in response to PVR, cell lines were incubated in serum-free media for 2 h at 37°C. Jurkat-mTIGIT cells were mixed with stock E.G7OVA cells or E.G7-OVA-mPVR cells at a cell ratio of 5:1 and incubated for 5 min at 37°C in the presence of a tyrosine phosphatase inhibitor (10 mM sodium orthovanadate, New England Biolabs). Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody-coated magnetic beads that captured FLAG-labeled mTIGIT proteins. Immunoprecipitates were assessed using Western blot analysis. The presence of phosphorylated TIGIT was assessed using an antiphosphotyrosine antibody (Cell Signaling Technology). The presence of total TIGIT was assessed using an anti-FLAG antibody (Cell Signaling Technology).

In T cells, TIGIT was strongly phosphorylated in response to mPVR expressing tumor cells compared to phosphorylation in response to non-PVR expressing cells (FIG. 2A).

To evaluate other signaling pathways potentially affected by TIGIT, cell lines were incubated in serum-free media for 2 h at 37°C. Jurkat-mTIGIT cells were combined with stock E.G7-OVA cells or E.G7-OVA-mPVR cells at a cell ratio of 5:1 and incubated at 37°C for 0, 5, 15, 45, or 60 min. Cell lysates were prepared and evaluated by Western blot analysis. The levels of phosphorylated SHP1 (containing SH2 tyrosine phosphatase 1) and Erk1/2 were determined using an anti-PhosPho-SHPI antibody (Tyr564) and an anti-phospho-Erk1/2 antibody (Thr202/Tyr204), respectively (both antibodies from Cell Signaling Technology). The level of total Erk1/2 was assessed using an antibody against Erk1/2 (Cell Signaling Technology). SHP1 is an important negative regulator of cytokine-mediated signaling pathways, including the JAK/STAT pathway, while Erk1/2 are protein kinases involved in the Ras-Raf-MEK-ERK signaling cascade.

As shown in FIG. 2B, specific activation of SHP1 and Erk1/2 occurred upon incubation of TIGIT-expressing T cells with mPVR-expressing tumor cells. Based on phosphorylation, activation was observed as early as 5 min and was observed to start to decrease after 45 min in the case of Erk1/2 and after 60 min in the case of SHP1. SHP1 and Erk1/2 activation did not occur in response to mPVR in native Jurkat T cells not expressing mTIGIT (data not shown).

To investigate whether TIGIT antagonists inhibit TIGIT activity, a TIGIT phosphorylation assay was performed in the presence of anti-TIGIT antibodies. As described above, JurkatmTIGIT cells were mixed with stock E.G7-OVA cells or E.G7-OVA-mPVR cells at a cell ratio of 5:1 and incubated for 5 min at 37°C in the presence of 10 mM sodium orthovanadate and 20 µg/ml control antibody (polyclonal mouse IgG; Sigma), anti-TIGIT 313R11, anti-TIGIT 313R12, or anti-TIGIT 313Rb1. Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody-coated magnetic beads that captured FLAG-labeled mTIGIT proteins. Immunoprecipitates were assessed using Western blot analysis. The presence of phosphorylated TIGIT was assessed using an antiphosphotyrosine antibody. The presence of total TIGIT was assessed using an anti-FLAG antibody.

As previously shown, potent mTIGIT phosphorylation occurred when mTIGIT-expressing Jurkat T cells were combined with mPVR-expressing tumor cells. Phosphorylation of mTIGIT in response to interaction with mPVR was reduced and/or suppressed when cells were incubated in the presence of anti-TIGIT antibodies. This decrease in TIGIT phosphorylation was most pronounced for the anti-TIGIT antibody 313R12 (FIG. 3).

Additional experiments were performed to investigate whether TIGIT antagonists inhibit TIGIT signaling. Similar to the studies described herein, Jurkat-mTIGIT cells were mixed with stock E.G7-OVA cells or E.G7-OVA-mPVR cells at a cell ratio of 5:1 and incubated for 15 min at 37°C in the presence of amplification concentrations (0-40 µg/ml) of anti-TIGIT 313R11 antibody, anti-TIGIT 313R12 antibody, or anti-TIGIT 313Rb1 antibody. Cell lysates were prepared and evaluated by Western blot analysis. Phosphorylated SHP1 (SH2-containing tyrosine phosphatase 1) and phosphorylated Erk1/2 were detected using an anti-PhosPho-SHPI antibody (Tyr562) and an anti-phosphoErk1/2 antibody (Thr202/Tyr204), respectively. The level of total SHP1 was assessed using an antibody against SHP1. The level of total Erk1/2 was assessed using an antibody against Erk1/2.

Phosphorylation of Erk1/2 in response to the mTIGIT-mPVR interaction inhibited anti-TIGIT antibodies in a clear dose-dependent manner (FIG. 4). Phosphorylation of SHP1 in response to the mTIGIT-mPVR interaction dose-dependently inhibited the anti-TIGIT antibody 313Rb1 and 313#11. In the experiment shown, the 313R12 antibody was shown to have only a weak effect on SHP1 phosphorylation.

These results strongly suggest that anti-TIGIT antibodies inhibit TIGIT function and affect downstream signaling pathways.

Example 3 TIGIT mediated inhibition of cytokine production.

The T-cell hybridoma ^B3Z86 /90.14 (B3Z) stably expresses the OVA-specific H-2K α T cell receptor (TCR). B3Z cells were infected with a lentiviral construct expressing mouse TIGIT (mTIGIT) and an mTIGIT clone (B3Z-mTIGIT) was generated from a single cell. The E.G7OVA cells of the mouse lymphoma cell line stably express OVA and can activate the OVA-specific TCR on B3Z cells, resulting in IL-2 secretion. To determine the effect of mTIGIT/mPVR interactions on antigen-specific IL-2 secretion, stock B3Z cells or B3Z-mTIGIT cells were incubated with stock E.G7-OVA cells or E.G7-OVA-mPVR cells (described above, see Example 2) in a 2:1 ratio in 12-well plates. After 24 hours, the concentrations of IL-2 in cell-free culture supernatants were measured using ELISA ELISA (R&D Systems). Data are expressed as the ratio of IL-2 secretion by parental B3Z cells or B3ZmTIGIT T cells in response to mPVR expressing tumor cells and IL-2 secretion in response to mPVR negative tumor cells.

For the original B3Z T cells, IL-2 secretion was similar regardless of mPVR expression on OVA-presenting tumor cells (ie, a ratio of approximately 1.0). Conversely, IL-2 secretion by B3Z-mTIGIT T cells was lower in the presence of mPVR-expressing tumor cells than in the presence of mPVR-negative tumor cells (i.e. ratio < 1.0) (FIG. 5A). When B3Z-mTIGIT T cells were pretreated with 20 μg/ml anti-TIGIT 313R11 or 313R12 antibody prior to co-culture with E.G7-OVA cells, inhibition of IL-2 production no longer occurred (FIG. 5B).

These results suggest that mTIGIT activation by mPVR inhibits T cell activation in response to antigen, as evidenced by a decrease in IL-2 production. In addition, blockade of TIGIT by a TIGIT antagonist inhibits TIGIT activation and appears to reduce TIGIT-associated inhibition of T-cell activity, as indicated by the pronounced production

IL-2. These data support the theory that TIGIT inhibition can disinhibit T cell suppression and enhance the immune response.

Example 4 TIGIT mediated inhibition of NK cell cytotoxicity.

Human natural killer (NK) cells NK-92 were infected with a lentiviral construct expressing FLAG-labeled mTIGIT and GFP, and a clonal cell line (NK92-mTIGIT) was obtained as described above. Human B cell line 721.221 cells were infected with a lentiviral construct expressing mPVR and GFP and a clonal cell line (721.221mPVR) was obtained. The absence of human or mouse PVR expression by the original 721.221 cells was confirmed by FACS and real-time PCR (data not shown). The ability of parental NK92 cells or NK-92-mTIGIT cells to lyse parental 721.221 tumor cells or 721.221mPVR cells was tested in a standard 4 hour calcein release assay. NK-92 or NK-92-mTIGIT cells were seeded in 96-well V-bottom plates. Target 721.221 or 721-221-mPVR cells were labeled with 10 μM acetoxymethyl-calcein (calcein AM) (Life Technologies) for 1 h at 37°C and then combined with NK-92 cells with an effector:target cell ratio of 25: 1 or 12:1. After a 4-hour incubation at 37° C., cell-free supernatants were collected and calcein release was quantified on a fluorometer with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. In a subsequent experiment, NK-92 and NK-92-mTIGIT cells were incubated in the presence of 20 μg/ml anti-TIGIT 313R11 antibody, 313R12 antibody, or control antibody for X minutes. These cells are then used in a calcein release assay as described above.

Percent specific cell lysis is defined as follows: % lysis=100χ(ERSR)/(MR-SR) where ER, SR and MR correspond to experimental, spontaneous and maximum release of calcein, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (ie, in the absence of effector cells); maximum release is determined by lysis of target cells with an equal volume of 10% SDS. Data are presented as the ratio of the cytotoxicity of NK-92 cells (baseline or NK-92-mTIGIT) against target 721.221-mPVR cells to the cytotoxicity of NK-92 cells (baseline or NK-92-mTIGIT) against baseline (negative). mPVR) target cells 721.221.

As shown in FIG. 6A, cytotoxicity of parental NK-92 cells against mPVR positive and mPVR negative target 721.221 cells was similar (i.e., the ratio was approximately 1.0). In contrast, the cytotoxicity of NK-92-mTIGIT cells against 721.221-mPVR cells was lower than that against mPVR-negative parental 721.221 cells (i.e., the ratio was < 1.0). When the ratio of effector cells to target cells was 25:1 or 12:1, there were no differences in the results. When NK-92 or NK-92-mTIGIT cells were treated with anti-TIGIT 313R11 antibody or 313R12 antibody prior to use in the cytotoxicity assay, inhibition of cell-mediated cytotoxicity was reduced and/or suppressed (FIG. 6B).

These results suggest that mTIGIT activation by mPVR inhibits NK cell-mediated cytotoxicity. Importantly, these results indicate that a TIGIT antagonist can interfere with the PVR-TIGIT interaction and lead to the restoration or disinhibition of the cytotoxic potential of NK cells.

Example 5 In vivo inhibition of tumor growth by an anti-TIGIT antibody.

CT26.WT mouse colon tumor cells were implanted subcutaneously (30,000 cells/mouse) in the posterior lateral region of 6-8 week old Balb/c mice. The tumors were allowed to grow for 10 days to reach a volume of approximately 50 mm ³ . Mice were treated with 0.25 mg/mouse anti-TIGIT 313R11 antibody (mIgG1 antibody), anti-TIGIT 313R12 antibody (mIgG2a antibody), mIgG1 control antibody, or mIgG2a control antibody (n=10 per control antibody group and 20 per experimental antibody group). ). Mice were dosed by intraperitoneal injection twice a week for three weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper on days 10, 15, 18, 22, 25 and 29.

As shown in FIG. 7A, the 313R12 anti-TIGIT antibody inhibited tumor growth by almost 75% compared to the isotype matched control antibody. In 9 mice, tumors regressed to undetectable levels by day 29. The anti-TIGIT 313R11 antibody only inhibited tumor growth by approximately 15% compared to the isotype matched control antibody. Antibodies 313R11 and 313R12 differ only in IgG isotype, since 313R12 is an IgG2a antibody and 313R11 is an IgG1 antibody. These results suggest that the antibody isotype may have a significant effect on the therapeutic efficacy of anti-TIGIT antibodies. Mouse IgG2 antibodies (equivalent to human IgG1 antibodies) are known to have increased

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ADCC activity compared to mouse IgG1 antibodies (equivalent to human IgG2) and this biological characteristic may play a role in the pronounced antitumor effect of the antibody

313R12.

This experiment was repeated with an anti-TIGIT 313R12 antibody and a mIgG2a control antibody. As described above, CT26.WT cells were implanted subcutaneously (30,000 cells/mouse) in the posterior lateral region of 6-8 week old Balb/c mice. The tumors were allowed to grow for 7 days to reach a volume of approximately 55 mm ³ . Mice were treated with 0.25 mg/mouse of anti-TIGIT 313R12 antibody or mIgG2a control antibody (n=10 per group). Mice were dosed by intraperitoneal injection twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper on days 7, 14, 17, 21, 25, 28 and 31.

Similar to the observations in the previous study, the anti-TIGIT 313R12 antibody inhibited the growth of CT26.WT tumors; tumor growth inhibition was observed in all 10 mice (FIG. 7B). In addition, 9 out of 10 mice treated with 313R12 antibody regressed tumors with a decrease in initial size, with 8 tumors regressing to undetectable levels after 2 weeks of treatment. In the present study, three tumors regressed in the control group treated with the IgG2 antibody, however, it is known that tumor regression can occur in untreated immunocompetent mice, in particular with a small starting number of tumor cells. However, complete inhibition of growth and/or regression of tumors in the 313R12 antibody-treated group demonstrated that anti-TIGIT antibody treatment has a significant therapeutic effect, which may be due to modulation and/or enhancement of the subject's immune response.

Successful cancer immunotherapy involves building long-term antitumor immunity. To study the development of long-term immunity with fresh CT26.WT tumor cells, mice from the studies described herein were stimulated in which CT26.WT tumors regressed to undetectable levels after treatment with an anti-TIGIT antibody. Eleven mice that showed complete tumor regression were injected with an increased number of CT26.WT cells (60,000 cells/mouse) on day 132 (102 days after the last dose of anti-TIGIT 313R12 antibody). As a control, 10 previously unstimulated mice were used, which were injected with CT26.WT cells in the same amount. Mice were not treated; tumor growth was monitored and tumor volumes were measured with an electronic caliper.

In the control group, all 10 mice developed large tumors by day 23 and were sacrificed. Conversely, 10 mice that had previously rejected CT26.WT tumors did not grow tumors. The remaining mice showed slight tumor growth 14 days after injection, however, the tumor completely regressed by 21 days. These 11 mice were then restimulated with 150,000 CT26.WT cells (5 times the number of cells relative to the first dose). In this case, 10 previously unstimulated mice were also used as controls, which were injected with CT26.WT cells in the same amount (150,000 cells/mouse). As in the previous experiment, all 10 mice in the control group developed large tumors and were sacrificed on day 22. Six mice did not develop tumors, and another 2 mice developed small tumors that completely regressed by day 22. An additional 3 mice had small tumors that appeared to have regressed or stabilized. These results are summarized in table. 3 and are presented as the percentage of tumor-free mice in each group.

Table 3

First stimulation of 30,000 cells 1st re-stimulation 60,000 cells 2nd restimulation 150,000 cells Control mice 10% (1/10) 0% (0/10) 0% (0/10) Mice treated with 313R12 antibody ¹ 55% (11/20) 100% (11/11) 72% (8/11)

¹ Mice treated with 313R12 antibody only after the first tumor challenge.

These results indicate that anti-TIGIT treatment can provide a strong and effective antitumor response. It is important to note that this anti-tumor response appears to result in long-term immunity and protection against tumor cells.

Example 6 Effector activity of anti-TIGIT IgG2 antibody.

Glycosylation of the IgG Fc region has been shown to be essential for optimal expression of biological activity mediated by FcyRI, FcyII, FcyRIII and complement component C1q (ie ADCC and CDC). For example, the heavy chain glycosylation site of a mouse IgG2a antibody

- 85 039540 is located at position 314 of asparagine residue of SEQ ID NO: 22. Replacing said asparagine residue with an alanine residue results in deglycosylation of the antibody and a decrease in ADCC activity. A deglycosylated variant of the anti-TIGIT antibody 313R12 containing an alanine residue at position 314 was obtained and named 313R13.

The activity of the anti-TIGIT 313R13 antibody was compared with that of the anti-TIGIT 313R12 antibody to investigate the effect of deglycosylation on antitumor activity. CT26.WT cells were implanted (30,000 cells/mouse) in the posterior lateral region of 6-8 week old Balb/c mice. The tumors were allowed to grow for 10 days to reach a volume of approximately 50 mm ³ . Mice were treated with 0.25 mg/mouse anti-TIGIT 313R12 antibody, anti-TIGIT 313R13 antibody, or mIgG2a control antibody (n=10 per control antibody group and 20 per experimental antibody group). Mice were dosed by intraperitoneal injection twice a week for three weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper on days 7, 10, 14, 17 and 21.

As shown in FIG. 8, the 313R12 anti-TIGIT antibody showed significant tumor growth inhibition compared to the isotype matched control. Conversely, the deglycosylated anti-TIGIT 313R13 antibody did not show significant antitumor activity.

Since the deglycosylated antibody is expected to have no or limited ADCC activity, these results suggest that ADCC may play an important role in the pronounced antitumor activity of the 313R12 anti-TIGIT antibody.

Example 7 IFN-γ, IL-2, IL-4 and IL-10 ELISpot assays.

ELISpot is a highly sensitive immunoassay for the detection of cytokine-secreting cells. Briefly, the ELISpot assay employs a capture antibody specific for the desired cytokine, previously coated on microplate wells. Stimulated cells are dispensed into wells and immobilized antibody placed in close proximity to any cytokine-secreting cell binds the secreted cytokine. The standard washing and incubation steps are then carried out with suitable detection reagents. For example, a combination of a biotinylated detection antibody followed by the addition of alkaline phosphatase conjugated streptavidin and a colored substrate solution is widely used. In the sites of localization of cytokines, a colored precipitate is formed, which looks like a spot (spot), while each individual spot corresponds to an individual cytokine-secreting cell. Slot counting can be done using an automated reading system or manually using a microscope. In some embodiments, each well is imaged using an automated reading system and the total slot count, slot area, or total optical density (TOD) is used for quantitation.

IFN-γ secreting cells were detected using the mouse IFN-γ ELISpot kit (MabTech). Cells were isolated from spleens of mice bearing CT26.WT tumors treated with anti-TIGIT 313R12 antibody (n=6) or isotype control antibody (n=6) (see Example 5). Splenocytes (5 x 105/well) from each mouse in each experimental group were distributed into a 96-well plate coated with an antibody specific for mouse IFN-γ. These cells were cultured in the presence or absence of the tumor-specific ^CD8 + T cell peptide AH-1 and incubated for 48 hours. murine leukemia provirus endogenous to the CT26.WT cell line. IFN-γ secreting cells were detected according to the manufacturer's instructions. Images were captured using a Bioreader 6000-Fz (BioSys) instrument and the number of slots, slot area, and total optical density were determined. Results are presented as total optical density; data are expressed as mean ± SEM

As shown in FIG. 9A, IFN-γ secreting cells were significantly increased in anti-TIGIT 313R12 treated mice compared to control antibody treated mice. It is important to note that the number of IFN-γ secreting cells was increased only in the presence of the AN-1 peptide. These results show that the 313R12 anti-TIGIT antibody activated tumor-specific CD8+ T cells. Said activation may be direct or indirect, ie. by inhibition of suppressor cells.

IL-2 secreting cells were detected using the mouse IL-2 ELISpot kit (MabTech). Cells were isolated from spleens of tumor-bearing mice treated with anti-TIGIT 313R12 antibody (n=6) or isotype matched control antibody (n=6). Splenocytes (5 x 105/well) from each mouse in each experimental group were dispensed into a 96-well plate coated with an antibody specific for mouse IL-2. These cells were incubated for 48 hours. IL-2 secreting cells were detected according to the manufacturer's instructions. Image capture was performed using a Bioreader 6000-F-z (BioSys) instrument and the number of slots, slot area and total optical density were determined. The results are presented as a general

- 86 039540 optical density; data are expressed as mean ± S.E.M.

As shown in FIG. 9B, IL-2 secreting cells were not significantly reduced in anti-TIGIT 313R12 treated mice compared to control antibody treated mice. The decrease in the number of secreting IL-2 cells was equivalent in the presence or absence of the AN-1 peptide.

IL-4 secreting cells were detected using a mouse IL-4 ELIspot kit (MabTech). Cells were isolated from spleens of tumor-bearing mice treated with anti-TIGIT 313R12 antibody (n=6) or isotype matched control antibody (n=6). Splenocytes (5 x 105/well) from each mouse in each experimental group were distributed into a 96-well plate coated with an antibody specific for mouse IL-4. These cells were incubated for 48 hours. IL-4 secreting cells were detected according to the manufacturer's instructions. Image capture was performed using a Bioreader 6000-F-z (BioSys) instrument and the number of slots, slot area and total optical density were determined. Results are presented as total optical density; data are expressed as mean ± S.E.M.

As shown in FIG. 9C, IL-4 secreting cells were significantly reduced in anti-TIGIT 313R12 treated mice compared to control antibody treated mice.

IL-10 secreting cells were detected using the mouse IL-10 ELIspot kit (MabTech). Cells were isolated from spleens of tumor-bearing mice treated with anti-TIGIT 313R12 antibody (n=6) or isotype matched control antibody (n=6). Splenocytes (5 x 105/well) from each mouse in each experimental group were distributed into a 96-well plate coated with an antibody specific for mouse IL-10. These cells were incubated for 48 hours. IL-10 secreting cells were detected according to the manufacturer's instructions. Image capture was performed using a Bioreader 6000-F-z (BioSys) instrument and the number of slots, slot area and total optical density were determined. Results are presented as total optical density; data are expressed as mean ± S.E.M.

As shown in FIG. 9D, IL-10 secreting cells were significantly reduced in mice treated with anti-TIGIT 313R12 antibody compared to mice treated with control antibody.

IFN-γ is typically produced by NK cells, Th1 CD4+ T cells, CD8+ T cells, antigen presenting cells, and B cells. Research results suggest that IFN-γ plays a role in tumor immunity and may be a regulator of antitumor activity mediated by other cytokines, in particular IL-12 and IL-2. Accordingly, treatment with an anti-TIGIT antibody that results in an increase in IFN-γ should enhance antitumor immunity.

IL-4 is produced by CD4+ Th2 cells and induces the differentiation of naive CD4+ T cells into Th2 cells. In addition, IL-4 inhibits the formation of Th1 cells, inhibits the production of Th1 cytokines such as TNF-α and IL-12, and inhibits macrophage activation. IL-10 is usually produced by Treg cells and helper T cells. Initially, IL-10 was identified as a Th2 cytokine that modulates the growth and/or differentiation of innate immune cells and suppresses the activation and effector functions of T cells, in particular cytotoxic T cells. More recently, IL-10 has been shown to have some immunostimulatory effects and, accordingly, is considered to have pleiotropic functions. The significant reduction in IL-4 and IL-10 producing cells in anti-TIGIT treated mice in the present study suggests that TIGIT targeting may result in inhibition of Th2 responses and may inhibit Treg cells and/or Treg function.

Example 8 Cell Cytotoxicity Assays

For T-cell cytotoxicity assays, spleen cells from CT26.WT tumor bearing mice were harvested as described above (Example 5). Cells were seeded in 96-well V-bottom plates in RPMI 1640 culture medium (Gibco/Life Technologies, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L- glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco). Target CT26.WT cells were labeled with 10 μM calcein AM (Life Technologies) for 1 h at 37° C. and then combined with splenocytes in an effector:target (E:T) ratio of 50:1. After incubation for 4 hours at 37° C., cell-free supernatants were collected and calcein release was quantified on a fluorometer with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Percent specific cell lysis was defined as % lysis=100χ(ER-SR)/(MR-SR), where ER, SR, and MR correspond to experimental, spontaneous, and maximum calcein release, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (ie, in the absence of effector cells); maximum release is determined by lysis of target cells with an equal volume of 10% SDS.

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As shown in FIG. 10, CD8+ cytotoxic T cells from mice bearing CT26.WT tumors when these mice were treated with anti-TIGIT 313R12 antibody showed an increased ability to kill target CT26.WT cells compared to cells from mice treated with a control antibody.

These results suggest that an anti-TIGIT antibody can increase antigen-specific cytotoxic T-cell activity and, accordingly, enhance the antitumor immune response. This increase in cytotoxic T cell activity may be due to the direct or indirect effect of the anti-TIGIT antibody, i.e. inhibition of the effect of suppressor cells.

Example 9 Regulatory T cell (Treg) assay.

Regulatory T cells (Treg) play a critical role in maintaining homeostasis and preventing autoimmune responses. Treg cells are a small subgroup of T cells, most of which are CD4+ cells and express CD25 (IL-2 receptor α-chain) and other Treg-associated molecular markers. Foxp3, a transcription factor, has been identified as a factor in the development and function of Treg cells. Foxp3 has been considered a specific marker for identifying and identifying Treg cells and separating Treg cells from other T cell subpopulations. However, the specificity of Foxp3 for Treg cells in human cells has been disputed. Another subpopulation of cells besides CD4+ Treg cells is CD8+ Treg cells, and Foxp3 may not be as important for their development and function as it is for CD4+ Treg cells.

The functionality of Treg cells in anti-TIGIT treated mice was assessed by determining the effect of Treg cells on proliferation of naive CD4+ or CD8+ T cells. Raw T cells were purified from the spleens of untreated mice using an enrichment column for mouse CD3+ T cells (R&D Systems). These purified T cells were labeled with 5 μM violet tracking dye (VTD; Life Technologies). 2x105 VTD-labeled T cells were incubated with anti-CD3 and anti-CD28 antibody coated beads to stimulate cell proliferation. Treg cells were isolated from spleens of mice bearing CT26.WT tumors (see Example 5) treated with anti-TIGIT 313R12 antibody (n=6) or isotype control antibody (n=5) using a mouse Treg isolation kit (Miltenyi Biotec ). To determine the effect of Treg cells on T cell proliferation, stimulated VTD-labeled T cells (effectors) were co-cultured with isolated Treg cells from the spleens of mice treated with anti-TIGIT 313R12 antibody and mice treated with control antibody (effector:Treg ratio = 1: 0.5). On day 4, cells were washed and incubated with anti-mouse CD4 or anti-mouse CD8 antibodies. Cells were assessed by FACS analysis using the FACSCanto II instrument and FACSDiva v6.1.3 software (BD Biosciences). VTD signals decrease 2-fold with labeled cell division, so the assay gate was set between the maximum signal obtained in said assay without Treg cells and the minimum signal obtained without stimulation with anti-CD3/CD28 antibodies. The percentage of cells in the indicated area (VTD underexpression) on CD4+ T cells and CD8+ T cells was used to calculate the proliferation of CD4+ and CD8+ T cells. The percentage of suppression was calculated as

[maximum signal - (signal from sample/maximum signal)]x100.

As shown in FIG. 11, treatment with anti-TIGIT antibody 313R12 did not have any observable effect on the suppressor function of spleen-derived Treg cells against CD4+ T cell proliferation. Conversely, treatment with anti-TIGIT 313R12 reduced the suppressor function of spleen-derived Treg cells on CD8+ T cell proliferation by approximately 30%.

These results suggest that treatment with an anti-TIGIT antibody may result in a reduction and/or suppression of Treg function, ie. disinhibition of the immune response. Decreased Treg function or suppression of Treg may enhance the overall antitumor immune response.

Example 10 Immune cell characterization of CT26.WT tumor bearing mice treated with anti-TIGIT antibody 313R12.

To better characterize the immune cells of CT26.WT tumor bearing mice treated with anti-TIGIT 313R12 antibody, splenocytes isolated from individual mice were analyzed by FACS. CT26.WT cells (20,000 cells/mouse) were subcutaneously injected into the posterior lateral region of 6 Balb/c mice aged 6-8 weeks. The tumors were allowed to grow for 10 days until reaching a size of approximately 50 mm ³ . Mice were treated with 0.25 mg/mouse of anti-TIGIT 313R12 antibody or isotype matched control antibody (n=10-20 per group). Mice were dosed by intraperitoneal injection twice a week. Tumor growth was monitored and tumor volumes were measured with an electronic caliper. Six mice from each treatment group were used for analysis. On day 32 after cell injection (1 day after the last dose of antibody), splenocytes were isolated. Splenocytes (1x106) were incubated for 10 min with recombinant Fc protein to block non-specific binding, and then incubated with fluorochrome-conjugated antibodies in 100 µl of FACS staining buffer (HBSS with 2% heat-inactivated calf serum) for 20 min on ice. Unbound antibodies were removed by washing, and dead cells were labeled with fixed dye to detect viability. Cells were fixed in 2% paraformaldehyde for 20 min at room temperature and analyzed using the FACSCanto II instrument and FACSDiva v6.1.3 software (BD Biosciences).

Total T cells were identified using anti-mouse CD3e, CD4+ T cells using anti-mouse CD4, and CD8+ T cells using anti-mouse CD8. Central memory cells were identified using anti-mouse/human CD44 antibody, and effector memory cells were identified using anti-human CD62L antibody. FACS staining was performed as described above using anti-mouse CD8b (clone 53-5.8, BioLegend), anti-mouse CD4 (clone GK1.5, BioLegend), anti-mouse CD62L (clone MEL-14, BioLegend) and anti- Mouse CD44 (clone 1M7, BioLegend). Cells were analyzed for expression of CD44 ^hlgh CD62L ^hlgh corresponding to central memory cells and expression of CD44 ^high CD62Ll ^ow corresponding to effector memory cells (gated by CD8+ T cells).

FACS analysis showed that the proportion of the T cell population of total living splenocytes in mice bearing CT26.WT tumors treated with the anti-TIGIT 313R12 antibody was increased compared to the T cell population in mice treated with the control antibody (Fig. 12A ). The percentage of CD4+ T cells (of total living cells) as well as the percentage of CD8+ T cells (of total living cells) increased in mice treated with 313R12 antibody compared to mice treated with control antibody (Fig. 12B and 12C). In addition, the percentage of central memory cells in the CD4+ T cell population increased (FIG. 12D). However, the percentage of central memory cells in the CD8+ T cell population did not increase (FIG. 12E).

These data showed that treatment with anti-TIGIT antibody increased the percentage of both CD4+ and CD8+ T cell populations. These results are consistent with enhanced immune response.

To evaluate the effect of anti-TIGIT antibody on target expression, TIGIT expression on Treg cells was analyzed by flow cytometry. As described herein, Treg cells are suppressor cells; CD4+ Treg cells are known to express the transcription factor Foxp3. As described above, splenocytes from mice carrying CT26.WT tumors treated with anti-TIGIT 313R12 antibody or control antibody were isolated. Splenocytes (1x106) were incubated for 10 min with recombinant Fc protein to block non-specific binding, and then incubated with anti-TIGIT 313R12 antibody and anti-mouse CD4 antibody in 100 µl of FACS staining buffer (HBSS with 2% heat-inactivated calf serum) for 20 mines on ice. After washing with FACS staining buffer, cells were labeled with fixable dye to detect viable cells, fixed, and permeabilized overnight. After permeabilization, cells were washed twice with 1X permeabilization buffer. Cells were stained with anti-mouse Foxp3 antibody for 30 min on ice, washed and fixed with 2% formaldehyde for 20 min at room temperature. Flow cytometry was performed using the FACSCanto II instrument and data analysis was performed using FACSDiva v6.1.3 software (BD Biosciences).

As shown in FIG. 13A, the percentage of TIGIT-expressing splenocytes of total splenocytes was reduced in CT26.WT tumor-bearing mice treated with anti-TIGIT 313R12 antibody compared to splenocytes from tumor-bearing mice treated with a control antibody. The percentage of Foxp3+CD4+ cells (corresponding to Treg cells) of total CD4+ cells was approximately 22-23% in both groups of mice and did not appear to be affected by anti-TIGIT antibody treatment (FIG. 13B). However, the percentage of TIGIT-positive Treg cells (of total Treg cells) was dramatically reduced in the splenocyte population of CT26.WT tumor-bearing mice treated with anti-TIGIT 313R12 antibody compared to the percentage of tumor-bearing mouse splenocytes treated with control antibody (Fig. 13C). Conversely, the percentage of TIGIT-negative Treg cells was not significantly increased in the splenocyte population of CT26.WT tumor-bearing mice treated with the anti-TIGIT 313R12 antibody compared to the splenocyte population of tumor-bearing mice treated with control antibody (Fig. 13D).

These results suggest that the 313R12 anti-TIGIT antibody conferred depletion on TIGIT positive cells, in particular Treg cells. However, this reduction in the number of TIGIT positive cells as assessed by FACS may be due to internalization of TIGIT proteins and/or downregulation of TIGIT expression on the cell surface rather than actual cell depletion.

Myeloid-derived suppressor cells (MSCM cells) are a heterogeneous family of myeloid cells and have been shown to be potent suppressors of both adaptive and innate immunity. SCMP cells suppress proliferation and activation of CD4+

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T cells and CD8+ T cells and facilitate the formation of Treg cells. SCMP cells are believed to contribute to cancer progression by inhibiting the antitumor immune response, stimulating angiogenesis, and creating a pre-metastatic environment. SCMP cells are characterized by the expression on the cell surface of the myeloid differentiation antigens Gr1 and CD11b. Expression of Gr1 is mainly restricted to cells of the granulocytic lineage, and CD11b is a cell surface integrin found on the surface of macrophages, granulocytes, NK cells and T cells. In mice, SKMP cells can be subdivided into two subpopulations, granulocytic SKMP cells (G-SKMP) and monocytic SKMP cells (M-SKMP), based in part on these markers. HSCMP cells typically have multilobed nuclei and a CD11b+Gr1 ^{hlgh phenotype} , while MSCMP cells have a monocytic morphology and a CD11b+Gr1 ^low phenotype. Both populations of SCMP cells have been shown to suppress the T cell response through several mechanisms, including increased production of arginase, inducible nitric oxide synthase (iNOS), nitric oxide, and reactive oxygen species. Accordingly, SCMP cells contribute to the immunosuppressive tumor microenvironment and may limit the effects of the antitumor immune response.

Myeloid cells, in particular CMPS cells of CT26.WT tumor-bearing mice treated with anti-TIGIT 313R12 antibody or control antibody, were analyzed by FACS. Splenocytes (1x106) were incubated for 10 min with recombinant Fc protein to block non-specific binding, and then incubated with fluorochrome-conjugated antibodies in 100 µl of FACS staining buffer (HBSS with 2% heat-inactivated calf serum) for 20 min on ice. These cells were then incubated with anti-mouse CD11b and anti-mouse Gr1. Unbound antibodies were removed by washing and cells were labeled with fixed dye to detect viability. Cells were fixed in 2% paraformaldehyde for 20 min at room temperature and analyzed using the FACSCanto II instrument and FACSDiva v6.1.3 software (BD Biosciences).

As shown in FIG. 14A, the percentage of CD11b+ myeloid cells (of total living cells) from the spleens of CT26.WT tumor bearing mice treated with anti-TIGIT 313R12 antibody was reduced by approximately 25% compared to the percentage of CD11b+ myeloid cells from tumor bearing mice treated with control antibody. The percentage of CD11b+Gr1+ SCMP cells (of total CD11b+ myeloid cells) in the spleens of tumor-bearing mice treated with anti-TIGIT 313R12 antibody was reduced by approximately 45% compared to the percentage of CD11b+Gr1+ SCMP cells of tumor-bearing mice treated with control antibody (FIG. 14B). In the CMPS population, the percentage of CMCM cells (identified as CD11b+Gr1 ^high ) from the spleens of CT26.WT tumor-bearing mice treated with anti-TIGIT 313R12 antibody decreased by approximately 66%, but the percentage of CMCM cells (identified as CD11b+Gr1 ^low ) from The spleens of mice bearing CT26.WT tumors treated with anti-TIGIT antibody 313R12 increased by approximately 30% (FIGS. 14C and 14D, respectively).

These results suggest that anti-TIGIT treatment may enhance the antitumor response by reducing the suppressor activity of specific immune cells, ie. SCMP cells.

Example 11 In vivo inhibition of tumor growth by anti-TIGIT antibody.

Renca is a Balb/c derived renal adenocarcinoma cell line from ATCC. Renca cells (5x105 cells/mouse) were implanted subcutaneously in the posterior lateral region of 6-8 week old Balb/c mice. The tumors were allowed to grow for 7 days until reaching a volume of approximately 43 mm ³ . Mice were treated with 0.25 mg/mouse of anti-TIGIT 313R12 antibody (mIgG2a antibody) or control mIgG2a antibody (n=10 per group). Mice were dosed by intraperitoneal injection twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper on days 7, 10, 14, 17, 21 and 24.

As shown in FIG. 15, anti-TIGIT antibody 313R12 significantly inhibited tumor growth compared to isotype matched control antibody. All control mice were sacrificed by day 28 due to the size of the tumors. Conversely, 8 out of 10 313R12 treated mice had tumor regression by day 28, and 5 of these mice had no detectable tumors. Five of the original ten mice were kept; detectable tumors were absent for at least 100 days after injection.

These results indicate the effectiveness of the anti-tumor activity of the antibody against TIGIT 313R12 in several different types of tumors.

Example 12 In vivo inhibition of tumor growth by anti-TIGIT antibody. Dose study.

Since anti-TIGIT antibody has been shown to effectively inhibit tumor growth, a dose range study was performed. CT26.WT mouse colon tumor cells were implanted subcutaneously (30,000 cells/mouse) in Balb/c mice; the tumors were allowed to grow until they reached an average size of approximately 35 mm ³ . Mice were treated with 30, 15, 10, 5, 3, 1, or 0.5 mg/kg of anti-TIGIT 313R12 antibody or control antibody (n=10 per group). Mice were dosed by intraperitoneal injection once a week for a total of 3 doses. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points.

In FIG. 16 shows the average tumor volumes for each experimental group. Treatment with anti-TIGIT antibody 313R12 expression inhibited the growth of CT26.WT tumors at all doses, including a minimum level of 0.5 mg/kg. As shown in Table. 4, at day 20, tumors regressed in at least 40% of mice at all dose levels. In all treated groups, individual mice tumors regressed to an undetectable level. A dose-response curve was not observed in the present experiment, which may be due to the size of the tumors at the start of treatment.

Table 4

Dose, mg/kg Number of mice with tumor regression Number of mice with undetected tumors Control antibody 1/10 0/10 0.5 8/10 3/10 one 6/10 1/10 3 8/10 4/10 5 7/10 3/10 ten 5/10 3/10 fifteen 4/10 2/10 thirty 7/10 4/10

These results demonstrate the activity of the anti-TIGIT 313R12 antibody as an immunotherapeutic agent. In general, these results were unexpected in that a small amount of anti-TIGIT antibody was required to provide a significant antitumor effect.

Example 13 In vivo inhibition of tumor growth by anti-TIGIT antibody and anti-PD-L1 antibody.

CT26.WT mouse colon tumor cells were implanted subcutaneously (30,000 cells/mouse) in Balb/c mice; on the first day of treatment (day 10 after implantation), the average size of the tumors was approximately 105 mm ³ . Mice were treated with 0.25 mg/mouse of anti-TIGIT 313R12 antibody, anti-PD-L1 antibody, combination of 313R12 antibody and anti-PD-L1 antibody, or control antibody (n=10-20 per group). Mice were injected with antibodies twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper.

As shown in FIG. 17B, treatment with anti-TIGIT antibody 313R12 expression inhibited the growth of CT26.WT tumors in a significant percentage of mice. It should be noted that treatment with the anti-TIGIT 313R12 antibody can not only inhibit tumor growth in most mice, but also induce regression of individual tumors, often to undetectable levels. Anti-PD-L1 antibody treatment was significantly less successful in inhibiting tumor growth when used as the sole agent (FIG. 17C). Treatment with the combination of anti-TIGIT 313R12 antibody and anti-PD-L1 antibody inhibited tumor growth to a greater extent than either agent alone (FIG. 17D). The average tumor volume is shown in Fig. 17E; the survival percentage of mice in each group is shown in FIG. 17F.

One way to evaluate the presence and/or functionality of a population of antitumor memory cells is to re-stimulate previously treated mice with fresh tumor cells. Mice (from the studies described above) previously treated with anti-TIGIT 313R12 antibody, anti-mPD-L1 antibody, or a combination of 313R12 antibody and anti-mPD-L1 antibody were used in the restimulation study. Mice that had complete tumor regression and had no detectable tumors for at least 128 days after the first tumor injection were restimulated with CT26.WT tumor cells (30,000 cells). Mice that were subjected to re-tumor stimulation received the last dose of treatment 100 days prior to re-stimulation. As a control group, unstimulated Balb/c mice (n=10) were used, which were injected with CT26.WT tumor cells (30,000 cells). Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points. Data are expressed as mean ± S.E.M.

The mean volume of CT26.WT tumors in previously unstimulated mice increased steadily up to day 28 and was approximately 1750 mm ³ . In a previous experiment, only two mice previously treated with anti-PD-L1 antibody had complete tumor regression, however, these two mice showed complete immunity to re-tumor challenge. Four mice previously treated with anti-TIGIT antibody had complete tumor regression; none of these mice showed tumor growth after re-stimulation; they demonstrated complete immunity to repeated tumor stimulation. In addition, 7 mice previously treated with the combination of 313R12 antibody and anti-PD-L1 antibody had complete tumor regression; these mice showed complete immunity to tumor stimulation.

- 91 039540

Mice treated with anti-TIGIT antibody, alone or in combination with anti-PD-L1 antibody, appear to be well protected when restimulated with CT26.WT tumor cells. These results suggest the presence of immunogenic memory after treatment with an anti-TIGIT antibody, either alone or in combination with a checkpoint inhibitor.

Example 14 In vivo inhibition of tumor growth by anti-TIGIT antibody and anti-PD-1 antibody.

CT26.WT mouse colon tumor cells were implanted subcutaneously (30,000 cells/mouse) in Balb/c mice; on the first day of treatment (7 days after implantation) the average size of the tumors was approximately 62 mm ³ . Mice (n=15) were treated with 12.5 mg of anti-TIGIT 313R12 antibody, anti-PD-1 antibody, combination of 313R12 antibody and anti-PD-1 antibody, or control antibody (n=15 per group). Mice were injected with these antibodies by intraperitoneal injection twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with an electronic caliper.

As shown in FIG. 18B, treatment with anti-TIGIT antibody 313R12 significantly inhibited tumor growth. As can be seen from the examples above, treatment with 313R12 can not only inhibit tumor growth, but also induce regression of some tumors. Treatment with the combination of anti-TIGIT 313R12 antibody and anti-PD-1 antibody inhibited tumor growth to a greater extent than treatment with either agent alone (FIG. 18D). The mean tumor volume in the four groups at day 24 is shown in FIG. 18E.

Mice previously treated with anti-TIGIT 313R12, anti-mPD-1, or a combination of 313R12 and anti-mPD-L1 were used in the restimulation study. Mice that had complete tumor regression and were free of detectable tumors for at least 100 days after the first tumor injection were restimulated with CT26.WT tumor cells (60,000 cells). As a control group, unstimulated Balb/c mice (n=10) were used, which were injected with CT26.WT tumor cells (60,000 cells). Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points. Data are expressed as mean ± S.E.M.

The mean volume of CT26.WT tumors in previously unstimulated mice increased steadily; on day 21, the mean tumor volume was approximately 1250 mm ³ . In a previous experiment, 13 mice were obtained that had complete regression of tumors previously treated with a combination of 313R12 antibody and anti-PD-1 antibody; these mice showed complete immunity to tumor stimulation.

These results further support the idea that the anti-TIGIT 313R12 antibody is a very potent immunotherapeutic agent, even when administered as a single agent. In addition, the effectiveness of the anti-TIGIT antibody can be further enhanced by combination with other immunotherapeutic agents.

Example 15 Preparation of anti-TIGIT monoclonal antibodies.

Antibodies were generated against amino acids 22-141 of recombinant human TIGIT (R&D Systems) and/or amino acids 22-138 of recombinant human TIGIT (Sino Biological Inc.). Mice (n=3) were immunized with TIGIT using standard techniques. The sera of individual mice were screened for anti-human TIGIT antibodies approximately 70 days after the first immunization using FACS analysis. The animal with the highest antibody titer was selected for the last additional antigen challenge, after which spleen cells were isolated to obtain a hybridoma. SP2/0 cells were used as fusion partners with mouse spleen cells. Hybridoma cells were seeded at 1 cell per well in 96-well plates and the supernatants were screened against human TIGIT by FACS analysis.

For FACS screening for anti-TIGIT antibodies, a chimeric fusion protein was constructed to express the extracellular domain of human TIGIT (FLAG-hTIGITCD4TM-GFP) on the cell surface and transfected into HEK-293T cells. After 48 h, the transfected cells were suspended in ice-cold PBS containing 2% PBS and heparin and incubated on ice in the presence of 50 µl of hybridoma supernatants for 30 min. A second incubation was performed with 100 μl of PE-conjugated anti-human secondary antibody Fc to detect antibody-bound cells. Cells were incubated with anti-FLAG antibody (Sigma-Aldrich) as positive control and anti-FE antibody as negative control. These cells were analyzed on a FACSCalibur instrument (BD Biosciences) and processed using FlowJo software.

Several human TIGIT-binding hybridomas were identified and the 313M26 antibody was selected. The amino acid sequences of the heavy chain variable region and the light chain variable region of 313M26 are represented by SEQ ID NO: 63 and SEQ ID NO: 64, respectively. The 313M26 heavy chain variable region and light chain variable region nucleotide sequences are represented by SEQ ID NO: 65 and SEQ ID NO: 66, respectively.

The 313M26 antibody was humanized using standard techniques known to those skilled in the art.

- 92 039540 of the art. The humanized version of 313M26 is referred to herein as 313M32 and is an IgG1 antibody. The humanized 313M32 heavy chain variable region was formatted onto an IgG4 backbone; its combination with the 313M32 light chain is called 313M33. The amino acid sequences of the heavy chain variable region and the light chain variable region of 313M32 (and 313M33) are represented by SEQ ID NO: 67 and SEQ ID NO: 68, respectively. The nucleotide sequences of the heavy chain variable region and the light chain variable region of 313M32 are represented by SEQ ID NO: 73 and SEQ ID NO: 74, respectively. The 313M26/313M32 heavy chain and light chain CDR regions are listed in Table 1. 2 herein (SEQ ID NOs: 57-62). The amino acid sequence of the heavy chain 313M32 with the predicted signal sequence is represented by the sequence of SEQ ID NO: 69; and without the signal sequence - the sequence of SEQ ID NO: 70; the 313M32 light chain amino acid sequence with the predicted signal sequence is represented by SEQ ID NO: 71, and without the signal sequence by SEQ ID NO: 72. The 313M32 heavy chain and light chain nucleotide sequences are represented by SEQ ID NO: 75 and SEQ ID NO: 76 , respectively. The 313M33 heavy chain amino acid sequence with the predicted signal sequence is represented by SEQ ID NO: 83, and without the signal sequence by SEQ ID NO: 82; the 313M33 light chain amino acid sequence with the predicted signal sequence is represented by SEQ ID NO: 71, and without the signal sequence by SEQ ID NO: 72. The 313M33 heavy chain and light chain nucleotide sequences are represented by SEQ ID NO: 84 and SEQ ID NO: 76 , respectively.

The plasmid encoding the heavy chain variable region of the 313M32 antibody has been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Virginia, USA under the terms of the Budapest Treaty dated August 11, 2015, and is designated PTA-122346. The plasmid encoding the light chain of the 313M32 antibody was deposited with the ATCC database, 10801 University Boulevard, Manassas, Virginia, USA, under the terms of the Budapest Treaty dated August 11, 2015, and is designated PTA-122347.

The humanized anti-TIGIT antibody 313M32 was screened for binding to TIGIT proteins from several different species. 313M32 was determined to potently bind human TIGIT and not bind TIGIT of any other species tested, including mice, rats, guinea pigs, rabbits, marmosets, pigs, dogs, rhesus monkeys, and cynomolgus monkeys. In this it differs from another anti-TIGIT antibody produced by OncoMed Pharmaceuticals, 313R19, which binds TIGIT of all the indicated species except guinea pig. The 313R19 antibody is a humanized version of the rabbit anti-TIGIT antibody (313R12), which was originally identified as binding both mouse and human TIGIT. In table. 5 summarizes information on anti-TIGIT antibodies and binding to TIGIT in mice, humans, cynomolgus monkeys, and cynomolgus monkeys.

Example 16 FACS analysis of blocking human TIGIT binding to PVR anti-TIGIT antibody.

Human TIGIT cell surface protein was prepared by ligating human TIGIT amino acids 22-141 into the CD4 transmembrane domain and GFP C-terminal protein tag using standard recombinant DNA techniques (hTIGIT-CD4TM-GFP). PVR-Fc constructs were generated using standard recombinant DNA techniques. Specifically, the human PVR extracellular domain was ligated in-frame into the rabbit Fc region, and the recombinant hPVR-rbFc protein was expressed in CHO cells. The fusion proteins were purified from the cell culture medium using protein A chromatography.

HEK-293T cells were transiently transfected with the hTIGIT-CD4TM-GFP construct. After 16 h, the transfected cells were suspended in ice-cold HBSS containing 2% PBS and heparin and incubated on ice with 0.5 μg/ml hPVR-rbFc fusion protein in the presence of anti-TIGIT antibodies 313R19, 313M26, or 313M32 for 60 min. The 313R19 antibody is a humanized version of the rabbit anti-TIGIT antibody. 313R19 binds mouse and human TIGIT. These antibodies were tested at concentrations of 10, 2 and 0.4 μg/ml. As a control, cells were incubated without antibody or without hPVR-rbFc. Second incubation with 100 µl PE-conjugated secondary antibody

- 93 039540 against rabbit Fc was performed to detect cells that bind the hPVR-rbFc fusion protein. These cells were analyzed on the FACSCanto instrument (BD Biosciences); data were processed using FlowJo software.

As shown in FIG. 19A, in the absence of any anti-TIGIT antibody, hPVR-rbFc strongly bound to hTIGIT expressed on the surface of HEK-293T cells. All three anti-TIGIT antibodies blocked the binding of hPVR-rbFc to hTIGIT at a maximum concentration of 10 μg/ml. However, at lower concentrations of 2 and 0.4 μg/ml antibody, the 313R19 antibody did not block the binding of hPVR-rbFc to TIGIT. Conversely, the anti-hTIGIT antibody 313M32 expression blocked the binding of hPVR-rbFc to hTIGIT at 2 μg/ml, and the level of blockade was higher than that of the parent mouse antibody 313M26. These results suggest that the affinity of the humanized 313M32 antibody for hTIGIT was higher than that of the original 313M26 antibody and the 313R19 antibody. An additional experiment was performed using 313R19 antibody and 313M32 antibody at concentrations of 10, 5, 2.5, and 1.25 μg/ml (FIG. 19B). The results of these two studies suggest that the affinity of the 313M32 antibody for hTIGIT is approximately 5-fold higher than that of the 313R19 antibody. Additionally, preliminary results from the Biacore assay indicated that the affinity of 313R19 was 2 nM and the affinity of 313M32 was 0.4 nM.

Example 17 TIGIT signaling and TIGIT phosphorylation.

Phosphorylation of TIGIT along the cytoplasmic tail occurs after interaction with its PVR counterreceptor. TIGIT phosphorylation represents the start of a cascade that includes downstream events affecting other known signaling pathways. Accordingly, an assessment of TIGIT phosphorylation can provide information on the function of TIGIT and the effect of a TIGIT antagonist.

The Jurkat human CD4+ T cell line lacks human TIGIT expression as assessed by real-time PCR and FACS (data not shown). To obtain a TIGIT-expressing cell line, Jurkat cells were infected with a lentiviral construct expressing human TIGIT (hTIGIT) labeled with FLAG and green fluorescent protein (GFP). GFP positive cells were sorted into 96-well plates using a FACSAria II cell sorter (BD Biosciences), single cells were expanded into clones, and a clonal cell line (Jurkat-hTIGIT) was selected.

Human B-cell lymphoma cells 721.221 lack PVR expression as assessed by real-time PCR and FACS (data not shown). To obtain a PVR-expressing cell line, 721.221 cells were infected with a lentiviral construct expressing human PVR (hPVR) and GFP. GFP positive cells were sorted into 96-well plates using a FACSAria II cell sorter (BD Biosciences), single cells were expanded into clones, and a clonal cell line (721.221-hPVR) was selected.

To evaluate TIGIT phosphorylation in response to PVR, Jurkat-hTIGIT cells were incubated in serum-free media for 2 h at 37°C. These cells were then pre-treated with 20 μg/ml of anti-TIGIT 313R19, 313M26 or 313M32 antibody or control antibody for 20 minutes at room temperature. Jurkat-hTIGIT cells were mixed with stock 721.221 cells or 721.221-mPVR cells at a cell ratio of 5:1 and incubated for 5 min at 37° C. in the presence of a tyrosine phosphatase inhibitor (10 mM sodium orthovanadate, New England Biolabs). Cell lysates were prepared and immunoprecipitated with anti-FLAG antibody-coated magnetic beads that captured FLAG-labeled hTIGIT proteins. Immunoprecipitates were separated on 4-12% bis-Tris gels (Novex/Life Technologies) and transferred to nitrocellulose membranes using the iBlot 2 apparatus (Life Technologies). The membranes were incubated with an anti-tyrosine antibody conjugated to horseradish peroxidase and phosphorylated with TIGIT (pTIGIT), visualized using standard detection reagents. The presence of total TIGIT was assessed using an anti-FLAG antibody (Cell Signaling Technology).

In response to hPVR-expressing hTIGIT tumor cells, pronounced phosphorylation was observed in Jurkat cells. In the presence of anti-TIGIT antibodies, in particular the 313M32 antibody, this phosphorylation was inhibited (FIG. 20).

Example 18 Epitope Mapping.

Epitope mapping of anti-hTIGIT antibodies.

To identify the 313M32 antibody binding epitope, analysis was performed with a number of human TIGIT variants with specific amino acid substitutions. These variants were based on amino acid sequence differences between human TIGIT and cynomolgus TIGIT, SEQ ID NO: 4 and SEQ ID NO: 77, respectively. The amino acid sequence of rhesus monkey TIGIT (SEQ ID NO: 78) is identical to cynomolgus TIGIT (FIG. 21). In amino acid variants, amino acid residues present in the cynomolgus TIGIT are inserted at the appropriate positions in the human TIGIT. The resulting hTIGIT variants contain substitutions at positions (1) E36K and I41V; (2) T51M, (3) Q62H and Q64H, (4) D72E, (5) S78Y and S80A, (6) V100M, (7) I109T and T119R; and (8) G135S. Each hTIGIT variant received

- 94 039540 using the hTIGIT (AK 22-141)-CD4TM-GFP construct described in Example 15.

TIGIT variants were assessed by FACS with anti-hTIGIT antibodies. HEK-293T cells were transiently transfected with hTIGIT-CD4TM-GFP, mTIGIT-CD4TM-GFP, or one of the hTIGIT-CD4TM-GFP constructs. After 16 hours, the transfected cells were suspended in ice-cold HBSS containing 2% FBS and incubated on ice with 1 μg/ml of anti-hTIGIT antibodies 313R12, 313M32, or 313M34 for 30 min. 313M34 is an anti-hTIGIT antibody that was derived from the amino acid sequences of the 10A7 antibody described in US Publication 2009/0258013. Antibody 10A7 is an anti-mouse TIGIT antibody that binds both mouse TIGIT and human TIGIT. A second incubation with 50 μl of PE-conjugated anti-Fc secondary antibody was performed to detect cells that bind antibodies. Cells were analyzed on a FACSCanto instrument (BD Biosciences) and data was processed using FlowJo software.

As shown in FIG. 22, the 313M32 antibody did not bind to the TIGIT 3 variant with substitutions at amino acid residue positions 62 and 64, and the TIGIT 7 variant with substitutions at amino acid residue positions 109 and 119. Strong binding of this antibody to hTIGIT and TIGIT variants 1, 2, 4, 5, 6, and 8. Amino acid residues 62, 64, 109, and 119 are highlighted in black in the crystal structure image of PVR-associated TIGIT (FIG. 23). The human TIGIT structure is represented as a sphere and the PVR structure as a ribbon. The amino acid positions indicate that the 313M32 antibody binds hTIGIT in a region that would block binding and/or interfere with PVR binding. Of note, there is also no binding of the 313R12 antibody to the TIGIT 7 variant with substitutions at amino acid residue positions 109 and 119, suggesting that 313R12 binds an overlapping epitope with the epitope that binds 313M32. Interestingly, the binding of the 313M34 antibody to TIGIT is not affected by the amino acid substitutions in the TIGIT 3 variant or variant 7, but is affected by the amino acid residue change at position 100 of the TIGIT 6 variant. This result indicates that the 313M34 antibody does not bind the same epitope as the 313M32 antibody. . The relative positions of the critical binding residues for antibodies 313M32 and 313M34 on TIGIT are marked in the image in FIG. 23.

Competitive studies were performed to confirm that antibodies 313M26 (the original mouse anti-hTIGIT antibody for 313M32) and 313R19 (a humanized rabbit anti-hTIGIT antibody derived from 313R12) bind a common epitope. Since the Fc parts of antibodies 313M26 and 313R19 are different, i.e. are mouse Fc and human Fc, respectively, binding of one antibody (313R19) in the presence or absence of a second antibody (313M26) can be determined. Briefly, HEK-293T cells were transiently transfected with an expression vector encoding human TIGIT-CD4TM-GFP. Cells were incubated for 1 hour with 0.5 µg of 313M19 antibody and excess of 313M26 antibody (50 to 0.78 µg/mL, 2-fold dilutions). Cells were washed and incubated with a fluorescently labeled secondary antibody specific for human Fc and analyzed by flow cytometry.

As shown in FIG. 24A, 313M26 competed with 313R19 for binding to hTIGIT, and an excess of 313M26 effectively blocked 313R19 binding to hTIGIT. These data show that 313M26/313M32 and 313R12/313R19 bind to overlapping epitopes and are consistent with the epitope analysis presented in FIG. 23. In parallel experiments, the ability of 313R19 or 313M32 to compete with 313M34 for binding to TIGIT was investigated. As shown in FIG. 24B, competition with 313M34 for binding to hTIGIT was not observed for either the 313R19 antibody or the 313M32 antibody. These results show that 313R19 and 313M32 bind to a different epitope from the epitope that binds 313M34. These results are also consistent with the epitope analysis presented in FIG. 23.

Example 19 Cytokine production.

Recruitment of TIGIT by PVR (CD155) expressed on tumor cells or antigen presenting cells has been shown to down-regulate T-cell effector functions and inhibit the antitumor immune response (see, e.g., Joller et al., 2014, Immunity, 40: 569-581). The ability of anti-TIGIT antibodies 313R19 and 313M32 to block PVR-mediated inhibition of cytokine secretion by T cells was investigated. Activated human T cell blasts were obtained by culturing primary human T cells for 10 days with phytohemagglutinin (PHA, 2 μg/ml) and IL-2 (4 ng/ml). The blasts were kept in PHA/IL-2-free media for 24 h, resuspended at a density of 5x105 cells/mL, and treated with control antibody (polyclonal human IgG, 25 µg/mL), anti-TIGIT antibody 313R19 or 313M32 at a concentration of 10 or 25 μg/ml for 15 min at room temperature. Cells were then plated in wells that were coated overnight at 4°C with anti-CD3 antibody (Ebioscience), human PVR-Fc, or anti-CD3 and hPVR-Fc (all concentrations were 10 μg/m). After 48 hours, cell-free culture supernatants were collected and ELISA was performed for the cytokines IFN-γ and IL-2 (R&D Systems). The results are expressed as the level of cytokine production by cells activated with anti-CD3 antibody alone, with the level of anti-CD3 cytokine antibody taken as 1.0.

As seen in FIG. 25, hPVR-Fc does not induce the production of either IFN-γ or IL-2 in activated

- 95 039540 cells. However, hPVR-Fc markedly inhibits cytokine production induced by anti-CD3 recruitment. This inhibition is abolished by anti-TIGIT antibodies 313R19 and 313M32, and at a higher dose of 25 μg/ml, the production of IFN-γ or IL-2 is actually increased compared to the amount produced by the anti-CD3 antibody. Activated T cells express both TIGIT and CD226, both of which can interact with PVR, however the TIGIT/PVR interaction is inhibitory and the CD226/PVR interaction is activating. Without being bound by a particular theory, it is assumed that when activated T cells come into contact with PVR, the TIGIT/PVR interaction wins because TIGIT binds PVR with greater force than CD226. In addition, TIGIT levels appear to be higher on activated T cells than CD226 levels, so the overall outcome of the interaction is inhibitory. For example, in FIG. 25 compares an anti-CD3 antibody alone with a combination of an anti-CD3 antibody and PVR. Blocking TIGIT with 313R19 or 313M32 abolishes TIGIT-mediated inhibition, but CD226/PVR interaction theoretically still occurs; thus, the interaction is activating. Accordingly, as seen in FIG. 25, when TIGIT is blocked and T cells are stimulated with the combination of anti-CD3 antibody and PVR, IFN-γ levels are actually higher than when stimulated with anti-CD3 antibody alone.

These results demonstrate that blockade of human TIGIT with an anti-TIGIT antibody can resolve TIGIT/PVR interactions and restore cytokine production favorable for an antitumor response.

Example 20 Antibody Dependent Cell Mediated Cytotoxicity Assay.

Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to the killing of an antibody-coated target cell by a cytotoxic effector cell by a non-phagocytic process, typically characterized by the release of cytotoxic granules or the expression of cell death-inducing molecules. Effector cells mediating ADCC include natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils, and dendritic cells. The ability of anti-TIGIT antibodies 313R19 and 313M32 to mediate NK-cell ADCC was investigated. ADCC was tested in a standard 4-hour calcein release assay using primary human NK cells as effectors and parental Jurkat or JurkathTIGIT cells as targets. Human NK cells were isolated directly from fresh peripheral blood buffy coat samples (Stanford Blood Center, Palo Alto, CA) by 30 min incubation with a RosetteSep cocktail (Stem Cell Technologies) prior to Ficoll density gradient centrifugation (Ficoll-Hypaque, Sigma). Target Jurkat or Jurkat-hTIGIT parent cells were labeled with 10 μM calcein AM (Life Technologies) for 1 h at 37° C. and then treated with control polyclonal IgG antibody, anti-TIGIT 313R19, or anti-TIGIT 313M32 (all at 10 μg/ ml) for 15 minutes at room temperature. After incubation, Jurkat or Jurkat-hTIGIT cells were combined with NK cells at a ratio of effector:target cells of 20:1 in 96-well V-bottom plates. After a 4-hour incubation at 37° C., cell-free supernatants were collected and calcein release was quantified on a fluorometer with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The percentage of specific cell lysis was determined as follows:

% lysis=100χ(ER-SR)/(MR-SR), where ER, SR and MR correspond to experimental release, spontaneous release and maximum release of calcein, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (ie, in the absence of effector cells); maximum release is determined by lysis of target cells with an equal volume of 10% SDS. For both cell lines, Jurkat and Jurkat-hTIGIT, the percentage of ADCC is defined as the percentage of specific lysis in the presence of experimental antibody (313R19 or 313M32) minus the percentage of specific lysis in the presence of control antibody.

As shown in FIG. 26, the percentage of ADCC increased significantly in the presence of anti-TIGIT antibodies 313R19 and 313M32 when TIGIT was expressed by target cells. The ability of anti-TIGIT antibodies to induce ADCC may indicate several mechanisms by which anti-TIGIT antibodies enhance the immune response to tumor cells.

Example 21 In vivo inhibition of tumor growth in humanized mice with an anti-TIGIT antibody.

A humanized mouse model was used to investigate the efficacy of anti-TIGIT antibody treatment of a human tumor. Humanized mice were obtained from Jackson Laboratories. These mice are obtained by injecting human hematopoietic stem cells (CD34+ cells) into irradiated NSG mice. After 15 weeks, the presence of mature human lymphocytes is confirmed by flow cytometry. Each mouse was injected subcutaneously with patient-derived melanoma tumor cells (OMP-M9, 75,000 cells/mouse). The tumors were allowed to grow for 19 days until an average volume of approximately 50 mm ³ was reached. carrier mice

- 96,039,540 tumors were randomized into groups (n=8 mice per group). Tumor-bearing mice were treated with either a control antibody, an anti-TIGIT 313R19 antibody, or an anti-TIGIT antibody.

313M32. Mice were dosed at 1 or 5 mg/kg every 5 days. Tumor growth was monitored and tumor volumes were measured with an electronic caliper at predetermined time points.

As shown in FIG. 27, tumor growth was inhibited in mice treated with 313R19 antibody and 313M32 antibody compared to controls. These results indicate that TIGIT targeting effectively enhances the antitumor immune response of human lymphocytes and contributes to the inhibition of human tumor growth in vivo. In this experiment, the combination of anti-TIGIT antibody 313R19 or 313M32 and anti-hPD-1 antibody did not inhibit tumor growth to any greater extent than anti-TIGIT antibody when used as the only agents (data not shown).

Antibodies 313M32 and 313R19 have been found to inhibit tumor growth in the context of human lymphocytes, and have similar activity in vivo. These results demonstrate that humanized mouse models carrying patient-derived xenografts can be used to investigate the anti-TIGIT antibody 313M32 (which binds only human TIGIT) in parallel with preclinical studies using anti-TIGIT antibodies 313R12 and 313R19 and mouse tumor models.

Example 22 Anti-TIGIT Antibody Pharmacokinetics.

As described herein, the 313R12 anti-TIGIT antibody binds preferentially to mouse TIGIT and is a mouse surrogate molecule used in most preclinical efficacy studies. PK antibodies against TIGIT 313R12 were determined in C57BL-6J mice at doses of 0.1, 1 and 10 mg/kg. Blood samples were taken over 21 days. The 313R12 antibody showed non-linear 2-compartment PK characteristics at the dose levels studied. This concentration-dependent clearance is believed to be a typical feature of monoclonal antibodies, for which target-mediated clearance may be an important component of the overall elimination mechanism when the target is abundant and readily available to the drug. At a dose of 10 mg/kg, the concentration-time profile of 313R12 approached linear over 21 days, suggesting saturation of the available target molecules. Estimation of the final half-life in the case of non-linear PK data is not possible.

PK antibodies against TIGIT 313R19 were determined in Balb/c mice. One dose of 10 mg/kg was administered to each mouse intravenously or intraperitoneally. Blood samples were taken over 21 days. The 313R19 antibody showed linear two-compartment PK characteristics with typical IgG clearance. The estimated terminal phase half-life in mice at a dose of 10 mg/kg was 7.6 days.

PK antibodies against TIGIT 313R19 were also determined in the cynomolgus monkey. Each animal received 4 doses of 10, 30 and 100 mg/kg twice a week. Each dosing group included 4 males; the study was terminated after the fourth dose without a recovery period. The 313R19 antibody in the cynomolgus monkey showed linear two-compartment PK characteristics with typical IgG clearance. The estimated terminal half-life in the cynomolgus monkey was 15.3 days.

The pharmacokinetics of anti-TIGIT antibody 313M32 was determined in Balb/c mice. Each mouse was administered a single dose of 1 or 10 mg/kg intravenously or 10 mg/kg intraperitoneally. Blood samples were taken over 21 days. The 313M32 antibody in mice showed linear two-compartment FC characteristics consistent with the fact that there was no detectable binding of 313M32 to mouse TIGIT. Clearance was slow and tentatively demonstrated typical IgG FA behavior. The estimated terminal half-life in mice was 11.7 days.

It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes may be made by one of skill in the art in light of such examples and embodiments, and such modifications or changes are included within the scope and spirit of this inventions.

All publications, patents, patent applications, internet sites, and accession numbers/database sequences, including both polynucleotide and polypeptide sequences referenced herein, are incorporated herein by reference herein in their entirety and for any purpose in to the same extent as if each individual publication, patent, patent application, internet site or accession number/database sequence were specifically individually listed for inclusion by reference.

The sequences disclosed herein are as follows:

- 97 039540

Mouse TIGIT amino acid sequence (SEQ ID NO: 1)

MHGWLLLWVQGLIQAAFLATAIGATAGTIDTKRNISAEEGGSVILQCHFSSDTAEVTQV DWKQQDQLLAIYSVDLGWHVASVFSDRWPGPSLGLTFQSLTMNDTGEYFCTYHTYPGGI YKGRIFLKVQE S S DDRNGLAQFQTAPLGGTMAAVLGLICLMVTGVTVLARKDKSIRMHSI ESGLGRTEAEPQEWNLRSLSSPGSPVQTQTAPAGPCGEQAEDDYADPQEYFNVLSYRSLE SFIAVSKTG

Mouse TIGIT amino acid sequence without predicted signal sequence (SEQ ID NO:2)

TIDTKRNISAEEGGSVILQCHFSSDTAEVTQVDWKQQDQLLAIYSVDLGWHVASVFSDRV VPGPSLGLTFQSLTMNDTGEYFCTYHTYPGGIYKGRIFLKVQESSDDRNGLAQFQTAPLG GTMAAVLGLIC LMVTGVTVLARKDKSIRMHSIE S GL GRTEAE PQEWNLRS L S S P GS PVQT QTAPAG P C GE QAE D DYAD P QE YFNVL SYRSLESFIAVSKTG

Mouse TIGIT extracellular domain amino acid sequence (SEQ ID NO:3)

TIDTKRNISAEEGGSVILQCHFSSDTAEVTQVDWKQQDQLLAIYSVDLGWHVASVFSDRV VPGPSLGLTFQSLTMNDTGEYFCTYHTYPGGIYKGRIFLKVQESSDDRNGLAQFQTAPLG

Human TIGIT amino acid sequence (SEQ ID NO:4)

MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWE QQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTG RIFLEVGNLESSVAEHGARFQIPLLGAMAATLWICTAVIVWALTRKKKALRIHSVEGDLR RKSAGQEEWSPSAPSPPGSCVAGLEAPLSTG

Human TIGIT amino acid sequence without predicted signal sequence (SEQ ID NO:5)

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSF KDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP LLGAMAATLWICTAVIVWALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCV QAEAPAGLCGEQRGCSEDCAELLHDYFNVLSYRS

Human TIGIT extracellular domain amino acid sequence (SEQ ID NO: 6)

MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSF KDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP

313R.il/313R12 heavy chain CDR1 (SEQ ID NO:7)

GSSLSSSYMS

- 98 039540

313R11/313R12/313R14/313R19 heavy chain CDR2 (SEQ ID NO:8)

IIGSNGNTYYANWAKG

313R11/313R12/313R14/313R19 heavy chain CDR3 (SEQ ID NO:9)

GGYRTSGMDP

313R11/313R12 Light chain CDR1 (SEQ ID NO:10)

QASQSISSYLNW

313R11/313R12 Light chain CDR2 (SEQIDNO:11)

DALKLAS

313R11/313R12 light chain CDR3 (SEQ ID NO:12)

QQEHSVGNVDN

313R14/313R19 Heavy chain CDR1 (SEQ ID NO:13)

GFSLSSSYMS

313R14/313R19 CDR1 light chain option! (SEQ ID NO:14)

QASQSNIYSDLAW

313R14/313R19 light chain CDR2 (SEQ ID NO:15)

RAST LAS

313R14/313R19 light chain CDR3 (SEQ ID NO:16)

QQEHLVAWIYN

313R11/313R12 Heavy chain variable region amino acid sequence (SEQ ID

NO: 17)

QVQLESEGGLFKPTDTLTLTCTVSGSSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYAN

WAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSS

313R11/313R12 Light chain variable region amino acid sequence (SEQ ID

NO: 18)

- 99 039540

DIWTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPS

RFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVVVKR

313R14 Heavy chain variable region amino acid sequence (SEQ ID NO:19)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPPGKGLEWIGIIGSNGNTYYA

NWAKGRVTISKT S T TVE LKLS SVTAADTAVYYCARGGYRT S GMDPWGQGT LVTVS S

313R14/313R19/313R20 Light chain variable region amino acid sequence (SEQ ID NO:20)

DIQMTQSPSSLSASVGDRVTITCQASQNIYSDLAWYQQKPGKAPKLLIYRASTLASGVPS

RFSGSGSGTDFTLTISSLQPEDFATYYCQQEHLVAWIYNVFGQGTKVEIKR

313R11 Heavy chain amino acid sequence (mlgGl) with underlined signal sequence (SEQ ID NO:21)

MKHLWFFLLLVAAPRWVLSQVQLESEGGLFKPTDTLTLTCTVSGSSLSSSYMSWVRQAPG

KGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSG

MDPWGPGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSL

SSGVHTFPAVLESDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKP

CICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQT

QPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVY

TIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSK

LNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

313R12 Heavy chain amino acid sequence (mIgG2) with underlined signal sequence (SEQ ID NO:22)

MKHLWFFLLLVAAPRWVLSQVQLESEGGLFKPTDTLTLTCTVSGSSLSSSYMSWVRQAPG

KGLEWIGIIGSNGNTYYANWAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSG

MDPWGPGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSL

SSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP

CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVE

VHTAQTQTHREDYNSTLRWSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSV RAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGS

- 100 039540

YFMYSKLRVEKKNWVERNS YSCSWHEGLHNHHTTTKSFSRTPGK

313R11/313R12 Light chain amino acid sequence with underlined signal sequence (SEQ ID NO:23)

MKHLWFFLLLVAAPRWVLSDIVMTQTPASVEVAVGGTVTIKCQAS QSIS SYLNWYQQKPG

QPPKLLIYDALKLASGVPSRFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVF GGGTEVWKRTDAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNG VLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

313R14 Heavy chain amino acid sequence (hlgGl) with underlined signal sequence (SEQ ID NO:24)

MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPP

GKGLEWIGIIGSNGNT YYANWAKGRVTISKTSTTVELKLSS VTAAD TAVYYCARGGYRT S

GMDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

313R14/313R19/313R20 Light chain amino acid sequence with underlined signal sequence (SEQ ID NO:25)

MVLQTQVFISLLLWISGAYGDIQMTQS PS S L SASVGDRVTITCQAS QNIYS DLAWYQQKP GKAPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQEHLVAWIYNV FGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

313R11 Heavy chain amino acid sequence (mlgGl) without signal sequence (SEQ ID NO:26)

QVQLESEGGLFKPTDTLTLTCTVSGSSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYAN WAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSAKTTP PSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLESDLYTLS

- 101 039540

SSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPK DVLTITLTPKVTCVWDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPI MHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCM ITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVL HEGLHNHHTEKSLSHSPGK

313R12 Heavy chain amino acid sequence (m!gG2) without signal sequence (SEQ ID NO:27)

QVQLESEGGLFKPTDTLTLTCTVSGSSLSSSYMSWVRQAPGKGLEWIGIIGSNGNTYYAN WAKGRFTISKTSTTVELKITSPTTEDTATYFCARGGYRTSGMDPWGPGTLVTVSSAKTTA PSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLS SSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFI FPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRV

VSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQ VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS YSCSWHEGLHNHHTTTKSFSRTPGK

313R11/313R12 Light chain amino acid sequence without signal sequence (SEQ ID NO:28)

DIWTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYDALKLASGVPS RFSGSGSGTEYTLTISGVESADAATYYCQQEHSVGNVDNVFGGGTEVWKRTDAAPTVSI FPPSSEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSS

T LT L TKDEYERHNS YT CEATHKT S T S PIVKS FNRNE C

313R14 Heavy chain amino acid sequence (hlgGl) without signal sequence (SEQ ID NO:29)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPPGKGLEWIGIIGSNGNTYYA NWAKGRVTISKT S T TVE LKLS SVTAADTAVYYCARGGYRT S GMDPWGQGT LVTVS SAS T K GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR

- 102 039540

VFS CS VMHEALHNHYTQKSLSLSPGK

313R14/313R19/313R20 Light chain amino acid sequence without predicted signal sequence (SEQ ID ΝΌ:30)

DIQMTQSPSSLSASVGDRVTITCQASQNIYSDLAWYQQKPGKAPKLLIYRASTLASGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQEHLVAWIYNVFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSKDSPVEKHKSS

Peptide AH-1 (SEQ ID NO:31)

SPSYVYHQF

313R19/313R20 Heavy chain variable region amino acid sequence (SEQ ID NO: 32)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPPGKGLEWIGIIGSNGNTYYA NWAKGRVTISKSSNQVSLKLSSVTAADTAVYYCARGGYRTSGMDPWGQGTLVTVSS

313R19 Heavy chain amino acid sequence (IgGl) with underlined signal sequence (SEQ ID NO:33)

MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPP GKGLEWIGIIGSNGNTYYANWAKGRVTISKSSNQVSLKLSSVTAADTAVYYCARGGYRTS GMDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

313R19 Heavy chain amino acid sequence (IgGl) without signal sequence (SEQ ID NO:34)

- 103 039540

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPPGKGLEWIGIIGSNGNTYYA NWAKGRVTISKSSNQVSLKLSSVTAADTAVYYCARGGYRTSGMDPWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

Linker (SEQ ID NO:35)

ESGGGGVT

Linker (SEQ ID NO :3 6)

LESGGGGVT

Linker (SEQ ID NO :3 7)

GRAQVT

Linker (SEQ ID NO :3 8)

WRAQVT

Linker (SEQ ID NO :3 9)

ARGRAQVT

FLAG tag (SEQ ID NO :40)

DYKDDDDK

Human IgGl heavy chain constant region (SEQ ID NO:41)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

- 104 039540

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG2 heavy chain constant region (SEQ ID NO:42)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR WSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG3 heavy chain constant region (SEQ ID NO:43)

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSC DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHE ALHNRFTQKSLSLSPGK

Human IgG4 heavy chain constant region (SEQ ID NO:44)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK

Human IgGl Fc Region (version 13A) (SEQ ID NO:45)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVPGTCSHALSH

- 105 039540

Human IgGl Fc Region (version 13B) (SEQ ID NO:46)

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPVVMKLDS DGSFFLYSELTVDKSRWQQALK

Human IgGl Fc Region (version 13A) (SEQ ID NO:47)

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgGl Fc Region (version 13B) (SEQ ID NO:48)

EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSELTVCSVKW

Human IgG2 Fc Region (version 13A) (SEQ ID NO:49)

CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP

Human IgG2 Fc Region (version 13B) (SEQ ID NO: 50)

CVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS

Human IgG2 Fc Region (version 13A) (SEQ ID NO:51)

TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE

- 106 039540

VQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG2 Fc Region (version 13A) (SEQ ID NO:52)

TKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENALNYK TTPPMLKSDGSFFLYTGNHEQVKLS

Human IgG2 Fc Region (version 13B) (SEQ ID NO: 53)

TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPE

VQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Human IgG2 Fc Region (version 13B) (SEQ ID NO: 54)

TKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVWDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRWSVLTWHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSELTVDKHLS

313R20 Heavy chain amino acid sequence (IgG4) with underlined signal sequence (SEQ ID NO:55)

MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPP GKGLEWIGIIGSNGNTYYANWAKGRVTISKSSNQVSLKLSSVTAADTAVYYCARGGYRTS GMDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPRE

- 107 039540

PQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF

LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

313R20 Heavy chain (IgG4) amino acid sequence without signal sequence (SEQ ID NO:56)

QVQLQESGPGLVKPSETLSLTCTVSGFSLSSSYMSWIRQPPGKGLEWIGIIGSNGNTYYA

NWAKGRVTISKSSNQVSLKLSSVTAADTAVYYCARGGYRTSGMDPWGQGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWS VLTVLHQDWLNGKEYKCKVSNKGLPS SIEKTISKAKGQPRE PQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK

313M26/313M32 Heavy chain CDR1 (SEQ ID NO: 57)

TSDYAWN

313M26/313M32 heavy chain CDR2 (SEQ ID NO: 58)

YISYSGSTSYNPSLRS

313M26/313M32 heavy chain CDR3 (SEQ ID NO: 59)

ARRQVGLGFAY

313M26/313M32 light chain CDR1 (SEQ ID NO:60)

KASQDVSTAVA

313M26/313M32 light chain CDR2 (SEQ ID NO:61)

SASYRYT

313M26/313M32 light chain CDR3 (SEQ ID NO:62)

QQHYSTP

313M26 Heavy chain variable region amino acid sequence (SEQ ID NO:63)

- 108 039540

DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWVRQFPGNKLEWMGYISYSGSTSY

NPSLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRQVGLGFAYWGQGTLVTVSS

313M26 Light chain variable region amino acid sequence (SEQ ID NO:64)

DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPD

RFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFG

313M26 Heavy chain variable region nucleotide sequence (SEQ ID NO:65)

GATGTGCAGCTTCAGGAGTCAGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTC

ACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGGTCCGGCAG

TTTCCAGGAAAACAAACTGGAGTGGATGGGCTACATAAGCTACAGTGGTAGCACTAGCTAC

AAC C CAT C T C T CAGAAG T C GAAT CTCTATCACTC GAGACACAT C CAAGAAC CAG T T C T T C

CTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGGAGACAG

GTCGGGCTGGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCAGCTCA

313M26 Light chain variable region nucleotide sequence (SEQ ID NO:66)

GACATTGTGATGACCAGTCTCACAAATTCATGTCCACATCAGTTGGAGACAGGGTCAGC

ATCACCTGCAAGGCCAGTCAGGATGTGAGTACTGCTGTAGCCTGGTATCAACAGAAACCA

GGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGAT

CGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCAGCAGTGTGTCAGGCT

GAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGTGGACGTTCGGT

313M32 Heavy chain variable region amino acid sequence (SEQ ID NO:67)

QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQPPGKGLEWIGYISYSGSTSY

NPSLRSRVTISRDTSKNQFFLKLSSVTAADTAVYYCARRQVGLGFAYWGQGTLVTVSS

313M32 Light chain variable region amino acid sequence (SEQ ID NO:68)

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPS

RFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPWTFG

313M32 Heavy chain (IgGl) amino acid sequence with predicted signal sequence underlined (SEQ ID NO:69)

- 109 039540

MDWTWRILFLVAAATGAHSQVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQP PGKGLEWIGYISYSGSTSYNPSLRSRVTISRDTSKNQFFLKLSSVTAADTAVYYCARRQV GLGFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

313M32 Heavy chain amino acid sequence (IgGl) without signal sequence (SEQ ID NO:70)

QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQPPGKGLEWIGYISYSGSTSY NPSLRSRVTISRDTSKNQFFLKLSSVTAADTAVYYCARRQVGLGFAYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK

313M32 Light chain amino acid sequence with underlined predicted signal sequence (SEQ ID NO:71)

MVLQTQVFISLLLWISGAYGDIQMT Q S P S S L SASVG DRVTIT CKAS QDVS TAVAWYQQK P GKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPWTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSNTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

313M32 Light chain amino acid sequence without signal sequence (SEQ ID NO:72)

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYRYTGVPS RFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPWTFGQGTKVEIKRTVAAPSVFIFPP

- 110 039540

SDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSNTLT

L S KADYEKHKVYACEVTHQGL S S PVT KS FNRGE C

313M32 Heavy chain variable region nucleotide sequence (SEQ ID NO:73)

CAGGTCCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCTGAGACCCTGTCCCTC

ACCTGCGCTGTCTCTGGTTACTCCATCACCTCCGATTATGCCTGGAACTGGATTCGGCAG

CCCCCAGGGAAGGGGCTGGAGTGGATTGGGTACATAAGCTACTCTGGTAGCACTAGCTAC

AAC CCATCTCTCCGGT CAC GG G T CACAATAT CAC G G GACACAT C CAAGAAC CAG T T C T T C

CTGAAGCTGTCCTCTGTGACCGCCGCTGACACCGCCGTGTGTATTACTGTGCAAGGAGACAG

GTCGGGCTGGGGTTTGCTTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCA

313M32 Light chain variable region nucleotide sequence (SEQ ID NO:74)

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACC

ATCACTTGCAAGGCTTCTCAGGATGTGTCTACTGCTGTTGCCTGGTATCAGCAGAAACCA

GGGAAAGCCCCTAAGCTCCTGATCTACTCTGCATCCTATCGGTACACTGGGGTCCCATCA

AGGTTCTCCGGATCTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCT

GAAGATAT T GCAACATAT TAC T GT CAGCAACAT TAT T C TAC T C S T T GGACAT T C GG C

313M32 Heavy chain (IgGl) nucleotide sequence (SEQ ID NO:75)

ATGGACTGGACCTGGAGGATACTCTTTCTCGTGGCTGCAGCCACAGGAGCCCACTCCCAG

GTCCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCTGAGACCCTGTCCCTCACC

TGCGCTGTCTCTGGTTACTCCATCACCTCCGATTATGCCTGGAACTGGATTCGGCAGCCC

CCAGGGAAGGGGCTGGAGTGGATTGGGTACATAAGCTACTCTGGTAGCACTAGCTACAAC

CCATCTCTCCGGTCACGGGTCACAATATCACGGGACACATCCAAGAACCAGTTCTTCCTG

AAGCTGTCCTCTGTGACCGCCGCTGACACCGCCGTGTGTATTACTGTGCAAGGAGACAGGTC

GGGCTGGGGTTTGCTTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCAGCCAGCACA

AAGGGCCCTCCGTGTTCCCTCTGGCCCCTTCCTCCAAGTCCACCTCCGGCGGCACCGCC

GCTCTGGGCTGCCTGGTGAAGGACTACTTCCCTGAGCCTGTGACCGTGTCCTGGAACTCT

GGCGCCCCTGACCTCTGGCGTGCACACCTTCCCAGCCGTGCTGCAGTCCTCCGGCCTGTAC

TCCCTGTCCTCCGTGGTGACCGTGCCTTCCTCCTCCCTGGGCACCCCAGACCTACATCTGC

AACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGC

- 111 039540

GACAAGACCCACACCTGCCCTCCCTGCCCTGCCCCTGAGCTGCTGGGCGGACCTTCCGTG TTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCCCGGACCCCTGAGGTGACC TGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAGGTGAAGTTCAATTGGTACGTGGAC GGCGTGGAGGTGCACAACGCTAAGACCAAGCCAAGGGAGGAGCAGTACAACTCCACCTAC CGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAG TGCAAGGTCTCCAACAAGGCCCTGCCCGCTCCCATCGAGAAAACCATCTCCAAGGCCAAG GGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCACCCAGCCGGGAGGAGATGACCAAG AACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCTTCCGATATCGCCGTGGAG TGGGAGTCTAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCC GACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGC AACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGC CTGTCTCTGTCTCCTGGCAAGTGA

313M32 Light chain nucleotide sequence (SEQ ID NO:76)

ATGGTGCTCCAGACCCAGGTCTTCATTTCCCTGCTGCTCTGGATCAGCGGAGCCTACGGG GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACC ATCACTTGCAAGGCTTCTCAGGATGTGTCTACTGCTGTTGCCTGGTATCAGCAGAAACCA GGGAAAGCCCCTAAGCTCCTGATCTACTCTGCATCCTATCGGTACACTGGGGTCCCATCA AGGTTCTCCGGATCTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCT GAAGATAT T GCAACATAT TAC T GT CAGCAACAT TAT T TAC T C C C T T GGACAT T C GG C CAA GGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCTCCA TCTGATGAGCAGCTCAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT CCCAGAGAGGCCAAAGTCCAGTGGAAGGTGGATAACGCCCTCCAATCCGGCAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAACACCCTGACA C T GAGCAAAGCAGAC TAC GAGAAACACAAAG TCTATGCCTGC GAAG T CAC C CAT CAG G G C

CTGTCTTCCCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGCTAA

TIGIT cynomolgus monkey (SEQ ID NO :77)

MRWCLFLIWAQGLRQAPLASGMMTGTIETTGNISAKKGGSVILQCHLSSTMAQVTQWWE QHDHSLLAIRNAELGWHIYPAFKDRVAPGPGLGLTLQSLTMNDTGEYFCTYHTYPDGTYR

- 112 039540

GRIFLEVLESSVAEHSARFQIPLLGAMAMMLWICIAVIVWVLARKKKSLRIHSVESGL QRKSTGQEEQIPSAPSPPGSCVQAEAAPAGLCGEQQGDDCAELHDYFNVLSYRSLGSCSF FTETG

Rhesus monkey TIGIT (SEQ ID NO:78)

MRWCLFLIWAQGLRQAPLASGMMTGTIETTGNISAKKGGSVILQCHLSSTMAQVTQVNWE QHDHSLLAIRNAELGWHIYPAFKDRVAPGPGLGLTLQSLTMNDTGEYFCTYHTYPDGTYR

Amino acids 55-70 human TIGIT (SEQ ID NO:79)

T QVNWEQQD QL LAICN

Human TIGIT amino acids 105-122 (SEQ ID NO:80)

EYFCIYHTYPDGTYTGRI

313R14/313R19 Light chain CDR1 (SEQ ID NO:81)

QASQNIYSDLAW

313M33 Heavy chain amino acid sequence (IgG4) without signal sequence (SEQ ID NO:82)

QVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQPPGKGLEWIGYISYSGSTSY NPSLRSRVTISRDTSKNQFFLKLSSVTAADTAVYYCARRQVGLGFAYWGQGTLVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFL FPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK

- 113 039540

313M33 Heavy chain amino acid sequence (IgG4) with signal sequence (SEQ ID ΝΌ:83)

MDWTWRILFLVAAATGAHSQVQLQESGPGLVKPSETLSLTCAVSGYSITSDYAWNWIRQP

PGKGLEWIGYISYSGSTSYNPSLRSRVTISRDTSKNQFFLKLSSVTAADTAVYYCARRQV

GLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS

GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG

PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE

VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP

REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

313M33 Heavy chain (IgG4) nucleotide sequence (SEQ ID NO: 84)

ATGGACTGGACCTGGAGGATACTCTTTCTCGTGGCTGCAGCCACAGGAGCCCACTCCCAG

GTCCAGCTGCAGGAGTCTGGCCCAGGACTGGTGAAGCCTTCTGAGACCCTGTCCCTCACC

TGCGCTGTCTCTGGTTACTCCATCACCTCCGATTATGCCTGGAACTGGATTCGGCAGCCC

CCAGGGAAGGGGCTGGAGTGGATTGGGTACATAAGCTACTCTGGTAGCACTAGCTACAAC

CCATCTCTCCGGTCACGGGTCACAATATCACGGGACACATCCAAGAACCAGTTCTTCCTG

AAGCTGTCCTCTGTGACCGCCGCTGACACCGCCGTGTGTATTACTGTGCAAGGAGACAGGTC

GGGCTGGGGTTTGCTTACTGGGGCCAAGGAACCCTGGTCACCGTCAGCTCAGCCAGCACA

AAGGGCCCATCCGTCTTCCCCCTGGCACCCTGCTCCCGGAGCACCTCCGAGAGCACAGCC

GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCCGTTACCGTGTCTTGGAACTCC

GGCGCACTGACCAGCGGCGTGCACACCTTCCCTGCTGTCCTCCAATCCTCTGGACTCTAC

TCCCTCTCCTCCGTGGTGACAGTGCCCTCCAGCAGCCTGGGCACTAAGACCCTACACCTGC

AACGTCGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGA

CCCCCATGCCCACCTTGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTC

CCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACTTGCGTGGTG

GTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTATGTGGATGGCGTGGAG

GTTCATAATGCCAAGACAAAGCCTCGGGAGGAGCAGTTCAACAGCACCTACCGGGTGGTC

AGCGTCCTCACCGTCCTGCACCAAGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTC

TCCAACAAAGGGCTCCCATCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGGGAGCCACAGGTGTACACCCTGCCCCCATCCCAAGAGGAGATGACCAAGAACCAAGTG

TCCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGTGGAGTGGGAGAGC

AATGGGCAGCCTGAGAACAACTACAAGACCACTCCTCCCGTGCTGGACTCCGACGGCTCC TTCTTCCTCTACTCCCGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGCAATGTCTTC TCCTGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTG TCTCTGGGCAAATGA

CLAIM

Claims (35)

CLAIM 1. An isolated antibody specifically binding to the extracellular domain of TIGIT, which contains: a heavy chain CDR1 containing TSDYAWN (SEQ ID NO: 57), a heavy chain CDR2 containing YISYSGSTSYNPSLRS (SEQ ID NO: 58), and a heavy chain CDR3 containing ARRQVGLGFAY ( SEQ ID NO: 59), and a light chain CDR1 containing KASQDVSTAVA (SEQ ID NO: 60), a light chain CDR2 containing SASYRYT (SEQ ID NO: 61), and a light chain CDR3 containing QQHYSTP (SEQ ID NO: 62) . 2. An antibody according to claim 1, which contains a heavy chain variable region at least 95% identical to the sequence of SEQ ID NO: 67, and a light chain variable region at least 95% identical to the sequence of SEQ ID NO: 68. 3. An antibody according to claim 1, which contains a heavy chain variable region at least 95% identical to the sequence of SEQ ID NO: 63, and a light chain variable region at least 95% identical to the sequence of SEQ ID NO: 64. 4. An antibody according to any one of claims 1 to 3, which is a monoclonal antibody. 5. An antibody according to claim 1, which contains the amino acid sequence of the heavy chain SEQ ID NO: - 114 039540 70 and the light chain amino acid sequence of SEQ ID NO: 72. 6. An antibody containing a heavy chain variable region encoded by a plasmid deposited with the American Type Culture Collection (ATCC) under the number PTA-122346, and a light chain variable region encoded by a plasmid deposited with the ATCC under the number PTA-122347, and the specified antibody is specifically binds the extracellular domain of TIGIT. 7. Antibody according to any one of claims 1-6, which binds human TIGIT but does not bind: (a) mouse TIGIT; (b) rat TIGIT; (c) TIGIT of the cynomolgus monkey; and/or (d) TIGIT rhesus monkey. 8. An antibody according to any one of claims 1 to 7, which: (a) inhibits TIGIT binding to the poliovirus receptor (PVR); (b) inhibits or blocks the interaction of TIGIT and PVR; (c) inhibits TIGIT signaling; (d) inhibits TIGIT activation; (e) inhibits TIGIT phosphorylation; and/or (f) reduces TIGIT expression on the cell surface. 9. An antibody according to any one of claims 1 to 8 which induces and/or enhances an immune response. 10. An antibody according to claim 9, characterized in that said immune response is directed to a tumor or tumor cell. 11. An antibody according to any one of claims 1-7, which: (a) enhances cellular immunity; (b) increases T cell activity; (c) increases cytolytic T-cell (CTL) activity; (d) increases natural killer (NK) cell activity; (e) increases the production of IL-2 and/or the number of producing IL-2 cells; (f) increases the production of IFN-γ and/or the number of producing IFN-γ cells; (g) enhances the Th1-type immune response; (h) reduces the production of IL-4 and/or the number of producing IL-4 cells; (i) reduces the production of IL-10 and/or the number of producing IL-10 cells; (j) reduces the Th2-type immune response; (k) inhibits and/or reduces the suppressor activity of regulatory T cells (Treg); and/or (l) inhibits and/or reduces the suppressor activity of myeloid-derived suppressor cells (MSCs). 12. An antibody according to any one of claims 1 to 3, which is a humanized antibody. 13. An antibody according to any one of claims 1 to 3, which is a human antibody. 14. An antibody according to any one of claims 1 to 3, which is a chimeric antibody. 15. An antibody according to any one of claims 1 to 3, which is an IgG1 antibody. 16. An antibody according to any one of claims 1 to 3, which is an IgG2 antibody. 17. An antibody according to any one of claims 1 to 3, which is an IgG4 antibody. 18. A heterodimeric agent containing an antibody according to any one of claims 1-11. 19. Bispecific agent containing: a) the first arm that specifically binds TIGIT; and b) a second arm, said first arm comprising an antibody according to any one of claims 1-11. 20. A bispecific agent according to claim 19, characterized in that said second arm contains an antigen-binding site from an antibody. 21. A pharmaceutical composition for inducing, stimulating, enhancing or prolonging an immune response in a subject, comprising an antibody according to any one of claims 1 to 11, a heterodimeric agent according to claim 18 or a bispecific agent according to claim 19 or 20, and a pharmaceutically acceptable carrier. 22. An isolated polynucleotide molecule containing a polynucleotide that encodes an antibody according to any one of claims 1-11. 23. An isolated polynucleotide encoding a polypeptide specifically binding to the TIGIT extracellular domain, comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76. 24. A vector containing a polynucleotide according to claim 22 or 23. 25. A cell containing a polynucleotide according to claim 22 or 23 or a vector according to claim 24. 26. A method of inducing, stimulating, enhancing or prolonging an immune response in a subject, comprising administering a therapeutically effective amount of an antibody according to any one of claims 1 to 11, a heterodimeric agent according to claim 18, or a bispecific agent according to claim 19 or 20. 27. A method for inhibiting tumor growth in a subject, characterized in that said includes administering to said subject a therapeutically effective amount of an antibody according to any one of claims 1 to 11, a heterodimeric agent according to claim 18, or a bispecific agent according to claim 19 or 20. 28. The method according to claim 27, characterized in that said tumor is selected from the group consisting of a tumor of the colon and rectum, an ovarian tumor, a pancreatic tumor, a lung tumor, a liver tumor, a breast tumor, a kidney tumor, a prostate tumor, gastrointestinal tumors, melanomas, cervical tumors, bladder tumors, glioblastomas, and head and neck tumors. 29. A method of treating cancer in a subject, characterized in that said method comprises administering to said subject a therapeutically effective amount of an antibody according to any one of claims 1 to 11, a heterodimeric agent according to claim 18, or a bispecific agent according to claim 19 or 20. 30. The method according to claim 29, characterized in that said cancer is selected from the group consisting of colon and rectal cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, and head and neck cancer. 31. The method of any one of claims 26-30, which further comprises administering at least one additional therapeutic agent to the subject, wherein said additional therapeutic agent is an immunotherapeutic agent selected from the group consisting of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony stimulating factor (MCSF), granulocyte colony stimulating factor (GCSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 ( CD80), B7-2 (CD86), 4-1BB ligand, GITRL, OX-40 ligand (OX-40L), anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA4 antibodies, anti-LAG-3 antibodies, antibodies anti-CD3, anti-GITR antibodies, anti-OX-40 antibodies and anti-TIM-3 antibodies. 32. Plasmid deposited with ATCC with assigned number PTA-122346. 33. Plasmid deposited with ATCC with assigned number PTA-122347. 34. A pharmaceutical composition for inhibiting tumor growth in a subject, comprising an antibody according to any one of claims 1 to 11, a heterodimeric agent according to claim 18 or a bispecific agent according to claim 19 or 20, and a pharmaceutically acceptable carrier. 35. A pharmaceutical composition for the treatment of cancer in a subject, comprising an antibody according to any one of claims 1 to 11, a heterodimeric agent according to claim 18 or a bispecific agent according to claim 19 or 20, and a pharmaceutically acceptable carrier.