Anti-TIGIT antibodies, anti-CD96 antibodies, and methods of use thereof

Abstract

The present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96) and / or TIGIT (e.g., human TIGIT) and isolated antibodies that specifically bind to TIGIT (e.g., human TIGIT). Also provided are pharmaceutical compositions comprising these multispecific molecules and antibodies, nucleic acids encoding these multispecific molecules and antibodies, expression vectors and host cells for making these multispecific molecules and antibodies, and methods of treating subjects using these multispecific molecules and antibodies.

Description

[Technical Field]

[0001] 1. Related Applications This application claims priority benefit of U.S. Provisional Application No. 63 / 201,537, filed May 4, 2021, which is incorporated herein by reference in its entirety.

[0002] 2. Sequence Listing The contents of the Sequence Listing, submitted electronically in an ASCII text file (Name: 190448_SL, Size: 293,587 bytes, Created: April 26, 2022), are incorporated herein by reference in their entirety.

[0003] 3.Technical Field The present disclosure relates to multispecific molecules that specifically bind to CD96 (eg, human CD96) and / or TIGIT (eg, human TIGIT), anti-TIGIT antibodies, and methods of using the same. [Background technology]

[0004] 4.Background technology CD96 (Cluster of Differentiation 96), also known as TACTILE (T cell activation, increased late expression), is a type I transmembrane protein in the immunoglobulin (Ig) superfamily. It contains a single Ig domain, a type I transmembrane domain, a single intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM), and a single YXXM phosphorylation motif, and is expressed on the surface of T cells and natural killer (NK) cells.

[0005] CD96 is thought to play a role in the regulation of immune cells (e.g., NK cells and T cells) and tumor metastasis. In particular, blockade of CD96 function has been shown to suppress primary tumor growth in several mouse tumor models in a CD8+ T cell-dependent manner.

[0006] Protein T cell immunoreceptor with Ig and ITIM domains (TIGIT), also known as VSIG9 or VSTM3, is a type I transmembrane protein in the immunoglobulin (Ig) superfamily. It contains a single Ig domain, a type I transmembrane domain, a single intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM), and a single immunoglobulin tail tyrosine (ITT)-like phosphorylation motif. It is expressed on activated CD4+ / CD25+ regulatory T cells (Tregs), memory CD45RO+ T cells, and natural killer (NK) cells, but not on naive T cells.

[0007] CD155 (also known as poliovirus receptor (PVR)) is highly expressed on monocytes and dendritic cells and can activate effector T cells and NK cells, as well as attenuate Treg activity through binding to its two receptors, CD226 and CD96. TIGIT has been shown to bind to CD155 and antagonize the interaction of CD155 with CD226 and CD96, thereby suppressing T cell- and NK cell-mediated immune activity.

[0008] Given the roles of human CD96 and human TIGIT in regulating immune responses, therapeutic agents designed to block CD96-ligand interactions and / or TIGIT-ligand interactions hold great promise for the treatment of diseases involving immunosuppression. Summary of the Invention

[0009] 5. Summary of the Invention The present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96) and / or TIGIT (e.g., human TIGIT) and antibodies that specifically bind to TIGIT (e.g., human TIGIT). Also provided are pharmaceutical compositions comprising these multispecific molecules and antibodies, nucleic acids encoding these multispecific molecules and antibodies, expression vectors and host cells for producing these multispecific molecules and antibodies, and methods of treating subjects using these multispecific molecules and antibodies.

[0010] In one aspect, the present disclosure provides a multispecific molecule comprising: (a) a first antigen-binding region that specifically binds to human CD96, the first antigen-binding region comprising a first VH comprising CDRs CDRH1, CDRH2, and CDRH3, and a first VL comprising CDRs CDRL1, CDRL2, and CDRL3; (i) the first VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 34, and the first VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 35; (ii) the first VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 36, and the first VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 37; or (iii) a first antigen-binding region, wherein the first VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 38, and the first VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 39; and (b) A second antigen-binding region that specifically binds to an antigen other than human CD96, the second antigen-binding region comprising a second VH comprising CDRs CDRH1, CDRH2, and CDRH3, and a second VL comprising CDRs CDRL1, CDRL2, and CDRL3.

[0011] In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the first antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 10, 11, 12, 13, 14, and 15, 16, 17, 18, 19, 20, and 21, or 22, 23, 24, 25, 26, and 27, respectively.

[0012] In certain embodiments, the second antigen-binding region specifically binds to human TIGIT. In certain embodiments, the second VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 40, and the second VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 41. In another embodiment, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the second antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively.

[0013] In another aspect, the present disclosure provides a multispecific molecule comprising: (a) a first antigen-binding region that specifically binds to an antigen other than human TIGIT, the first antigen-binding region comprising a first VH comprising CDRs CDRH1, CDRH2, and CDRH3, and a first VL comprising CDRs CDRL1, CDRL2, and CDRL3; and (b) A second antigen-binding region that specifically binds to human TIGIT, the second antigen-binding region comprising a second VH having CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 40, and a second VL having CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 41.

[0014] In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the second antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively.

[0015] In certain embodiments, the first antigen-binding region specifically binds to human CD96. In certain embodiments, the first VH comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 34, 36, or 38. In another embodiment, the amino acid sequence of the first VH consists of the amino acid sequence of SEQ ID NO: 34, 36, or 38. In another embodiment, the first VL comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 35, 37, or 39. In another embodiment, the amino acid sequence of the first VL consists of the amino acid sequence of SEQ ID NO: 35, 37, or 39.

[0016] In certain embodiments, the second VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the amino acid sequence of the second VH consists of the amino acid sequence of SEQ ID NO: 40.

[0017] In certain embodiments, the second VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the amino acid sequence of the second VL consists of the amino acid sequence of SEQ ID NO: 41.

[0018] In another aspect, the present disclosure provides a multispecific molecule comprising: (a) a first antigen-binding region that specifically binds to human CD96, wherein the antigen-binding region comprises a first VH and a first VL, wherein the first VH comprises the amino acid sequence of SEQ ID NO: 34, 36, or 38, and / or the first VL comprises the amino acid sequence of SEQ ID NO: 35, 37, or 39; and (b) a second antigen-binding region that specifically binds to an antigen other than human CD96, the second antigen-binding region comprising a second VH and a second VL.

[0019] In certain embodiments, the second antigen-binding region specifically binds to human TIGIT.

[0020] In another aspect, the present disclosure provides a multispecific molecule comprising: (a) a first antigen-binding region that specifically binds to an antigen other than human TIGIT, the first antigen-binding region comprising a first VH and a first VL; and (b) A second antigen-binding region that specifically binds to human TIGIT, wherein the antigen-binding region comprises a second VH and a second VL, and the second VH comprises the amino acid sequence of SEQ ID NO: 40 and / or the second VL comprises the amino acid sequence of SEQ ID NO: 41.

[0021] In certain embodiments, the first antigen-binding region specifically binds to human CD96. In certain embodiments, the first VH comprises the amino acid sequence of SEQ ID NO: 34, 36, or 38, and the first VL comprises the amino acid sequence of SEQ ID NO: 35, 37, or 39. In another embodiment, the amino acid sequence of the first VH consists of SEQ ID NO: 34, 36, or 38, and the amino acid sequence of the first VL consists of SEQ ID NO: 35, 37, or 39. In another embodiment, the first VH and first VL comprise the amino acid sequences of SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively. In another embodiment, the amino acid sequences of the first VH and first VL consist of the amino acid sequences of SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively. In another embodiment, the second VH comprises the amino acid sequence of SEQ ID NO: 40, and the second VL comprises the amino acid sequence of SEQ ID NO: 41. In another embodiment, the amino acid sequences of the second VH and second VL consist of the amino acid sequences of SEQ ID NOs: 40 and 41, respectively. In another embodiment, the first VH and first VL comprise the amino acid sequences of SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively, and the second VH and second VL comprise the amino acid sequences of SEQ ID NOs: 40 and 41, respectively. In another embodiment, the amino acid sequences of the first VH and first VL consist of the amino acid sequences of SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively, and the amino acid sequences of the second VH and second VL consist of the amino acid sequences of SEQ ID NOs: 40 and 41, respectively.

[0022] In certain embodiments, the first and / or second antigen-binding region comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the heavy chain constant region is an IgG1 heavy chain constant region. In other embodiments, the heavy chain constant region comprises the amino acid sequence of any one of SEQ ID NOs: 49 to 60.

[0023] In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises an N297A mutation numbered according to the EU numbering system.

[0024] In certain embodiments, the first and / or second antigen-binding region comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, and the variant heavy chain constant region binds to an FcγR with higher affinity than the wild-type heavy chain constant region binds to the FcγR. In certain embodiments, the FcγR is FcγRIIB or FcγRIIIA.

[0025] In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises S267E and L328F mutations numbered according to the EU numbering system.

[0026] In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises at least one mutation selected from the group consisting of S239D, A330L, and I332E mutations numbered according to the EU numbering system.

[0027] In certain embodiments, the first antigen-binding region comprises a first heavy chain constant region comprising an aspartic acid at amino acid position 239, an aspartic acid and a glutamic acid at amino acid positions 239 and 332, respectively, or an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively; and the second antigen-binding region comprises a second heavy chain constant region lacking aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively; Amino acid positions are numbered according to the EU numbering system.

[0028] In specific embodiments, the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 58 and 57, 59 and 57, or 60 and 57, respectively.

[0029] In certain embodiments, the first antigen-binding region comprises a first heavy chain constant region comprising aspartic acid and glutamic acid at amino acid positions 239 and 332, respectively, or aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively; and the second antigen-binding region comprises a second heavy chain constant region comprising an aspartic acid at amino acid position 239; Amino acid positions are numbered according to the EU numbering system.

[0030] In certain embodiments, the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 59 and 58, or 60 and 58, respectively.

[0031] In certain embodiments, the first heavy chain constant region comprises an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively; and the second heavy chain constant region further comprises a glutamic acid at amino acid position 332; Amino acid positions are numbered according to the EU numbering system.

[0032] In a specific embodiment, the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 60 and 59, respectively.

[0033] In certain embodiments, the first antigen-binding region comprises a first heavy chain constant region that does not contain aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively; and the second antigen-binding region comprises a second heavy chain constant region comprising an aspartic acid at amino acid position 239, an aspartic acid and a glutamic acid at amino acid positions 239 and 332, respectively, or an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively; Amino acid positions are numbered according to the EU numbering system.

[0034] In specific embodiments, the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 57 and 60, 57 and 59, or 57 and 58, respectively.

[0035] In certain embodiments, the first antigen-binding region comprises a first heavy chain constant region comprising an aspartic acid at amino acid position 239; and the second antigen-binding region comprises a second heavy chain constant region comprising aspartic acid and glutamic acid at amino acid positions 239 and 332, respectively, or aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively; Amino acid positions are numbered according to the EU numbering system.

[0036] In a specific embodiment, the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 58 and 60, or 58 and 59, respectively.

[0037] In certain embodiments, the first heavy chain constant region further comprises a glutamic acid at amino acid position 332, and the second heavy chain constant region comprises an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively; Amino acid positions are numbered according to the EU numbering system.

[0038] In a specific embodiment, the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 59 and 60, respectively.

[0039] In a specific embodiment, the first antigen-binding region comprises a first heavy chain constant region comprising a tryptophan at amino acid position 366, and the second antigen-binding region comprises a second heavy chain constant region comprising serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively; Amino acid positions are numbered according to the EU numbering system.

[0040] In certain embodiments, the first heavy chain constant region comprises SEQ ID NO: 53, 54, 55, or 56, and the second heavy chain constant region comprises SEQ ID NO: 49, 50, 51, or 52.

[0041] In certain embodiments, the first antigen-binding region comprises a first heavy chain constant region comprising serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively; and the second antigen-binding region comprises a second heavy chain constant region comprising a tryptophan at amino acid position 366; Amino acid positions are numbered according to the EU numbering system.

[0042] In certain embodiments, the first heavy chain constant region comprises SEQ ID NO: 49, 50, 51, or 52, and the second heavy chain constant region comprises SEQ ID NO: 53, 54, 55, or 56.

[0043] In certain embodiments, the first antigen-binding region comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, or 67 to 99. In certain embodiments, the first heavy chain consists of the amino acid sequence of SEQ ID NO: 1, 3, 5, or 67 to 99. In certain embodiments, the second antigen-binding region comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 7 or 100 to 110. In certain embodiments, the amino acid sequence of the second heavy chain consists of the amino acid sequence of SEQ ID NO: 7 or 100 to 110.

[0044] In certain embodiments, the multispecific molecule comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44. In certain embodiments, the first antigen-binding region comprises a first light chain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 6. In another embodiment, the first light chain consists of the amino acid sequence of SEQ ID NO: 2, 4, or 6. In certain embodiments, the second antigen-binding region comprises a second light chain comprising the amino acid sequence of SEQ ID NO: 8 or 9. In another embodiment, the amino acid sequence of the second light chain consists of the amino acid sequence of SEQ ID NO: 8 or 9.

[0045] In another aspect, the present disclosure provides a multispecific molecule comprising: (a) a first antigen-binding region that specifically binds to human CD96, the first antigen-binding region comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, or 67 to 99, and / or a first light chain comprising the amino acid sequence of SEQ ID NO: 2, 4, or 6; and (b) a second antigen-binding region that specifically binds to an antigen other than human CD96, the second antigen-binding region comprising a second heavy chain and a second light chain.

[0046] In certain embodiments, the second antigen-binding region specifically binds to human TIGIT.

[0047] In another aspect, the present disclosure provides a multispecific molecule comprising: (a) a first antigen-binding region that specifically binds to an antigen other than human TIGIT, the first antigen-binding region comprising a first heavy chain and a first light chain; and (b) A second antigen-binding region that specifically binds to human TIGIT, the second antigen-binding region comprising a second heavy chain comprising the amino acid sequence of SEQ ID NO: 7 or 100 to 110, and / or a second light chain comprising the amino acid sequence of SEQ ID NO: 8 or 9.

[0048] In certain embodiments, the first antigen-binding region specifically binds to human CD96. In certain embodiments, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 1, 3, 5, or 67-99, and / or the first light chain comprises the amino acid sequence of SEQ ID NO: 2, 4, or 6. In another embodiment, the first heavy chain comprises the amino acid sequence of SEQ ID NO: 1, 3, 5, or 67-99, and the first light chain comprises the amino acid sequence of SEQ ID NO: 2, 4, or 6. In another embodiment, the amino acid sequence of the first heavy chain consists of SEQ ID NO: 1, 3, 5, or 67-99, and the amino acid sequence of the first light chain consists of the amino acid sequence of SEQ ID NO: 2, 4, or 6.

[0049] In certain embodiments, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 7 or 100-110, and / or the second light chain comprises the amino acid sequence of SEQ ID NO: 8 or 9. In certain embodiments, the second heavy chain comprises the amino acid sequence of SEQ ID NO: 7 or 100-110, and the second light chain comprises the amino acid sequence of SEQ ID NO: 8 or 9. In another embodiment, the amino acid sequence of the second heavy chain consists of SEQ ID NO: 7, and the amino acid sequence of the second light chain consists of the amino acid sequence of SEQ ID NO: 8 or 9.

[0050] In certain embodiments, the first heavy chain and the first light chain comprise the amino acid sequences of SEQ ID NOs: 1 and 2, 3 and 4, or 5 and 6, respectively, and / or the second heavy chain and the second light chain comprise the amino acid sequences of SEQ ID NOs: 7 and 8, or 7 and 9, respectively. In certain embodiments, the first heavy chain and the first light chain comprise the amino acid sequences of SEQ ID NOs: 1 and 2, 3 and 4, or 5 and 6, respectively, or the second heavy chain and the second light chain comprise the amino acid sequences of SEQ ID NOs: 7 and 8, or 7 and 9, respectively. In another embodiment, the amino acid sequences of the first heavy chain and the first light chain consist of SEQ ID NOs: 1 and 2, 3 and 4, or 5 and 6, respectively, and the amino acid sequences of the second heavy chain and the second light chain consist of the amino acid sequences of SEQ ID NOs: 7 and 8, or 7 and 9, respectively.

[0051] In another aspect, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds human CD96 and a second antigen-binding region that specifically binds human TIGIT, wherein: (a) the first antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively; and (b) the second antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, or glutamic acid at amino acid positions 239, 330, and 332, respectively; Amino acid positions are numbered according to the EU numbering system.

[0052] In some embodiments, the first antigen-binding region comprises SEQ ID NO: 73, 84, 95, or 56, and the second antigen-binding region comprises SEQ ID NO: 103 or 49.

[0053] In another aspect, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds human CD96 and a second antigen-binding region that specifically binds human TIGIT, wherein: (a) the first antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively; and (b) the second antigen-binding region comprises aspartic acid, serine, alanine, and valine at amino acid positions 239, 366, 368, and 407, respectively; Amino acid positions are numbered according to the EU numbering system.

[0054] In certain embodiments, the first antigen-binding region comprises SEQ ID NO: 73, 84, 95, or 56, and the second antigen-binding region comprises SEQ ID NO: 104 or 50.

[0055] In another aspect, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds human CD96 and a second antigen-binding region that specifically binds human TIGIT, wherein: (a) the first antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, or glutamic acid at amino acid positions 239, 330, and 332, respectively; and (b) the second antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively; Amino acid positions are numbered according to the EU numbering system.

[0056] In certain embodiments, the first antigen-binding region comprises SEQ ID NO: 67, 78, 89, or 49, and the second antigen-binding region comprises SEQ ID NO: 7 or 56.

[0057] In another aspect, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds human CD96 and a second antigen-binding region that specifically binds human TIGIT, wherein: (a) a first antigen-binding region comprising aspartic acid, serine, alanine, and valine at amino acid positions 239, 366, 368, and 407, respectively; (b) the second antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively; Amino acid positions are numbered according to the EU numbering system.

[0058] In certain embodiments, the first antigen-binding region comprises SEQ ID NO: 1, 3, 5, or 50, and the second antigen-binding region comprises SEQ ID NO: 7 or 56.

[0059] In another aspect, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds human CD96 and a second antigen-binding region that specifically binds human TIGIT, wherein: (a) the first antigen-binding region comprises an aspartic acid, a glutamic acid, and a tryptophan at amino acid positions 239, 332, and 366, respectively; and (b) the second antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, or glutamic acid at amino acid positions 239, 330, and 332, respectively; Amino acid positions are numbered according to the EU numbering system.

[0060] In certain embodiments, the first antigen-binding region comprises SEQ ID NO: 72, 83, 94, or 55, and the second antigen-binding region comprises SEQ ID NO: 103 or 49.

[0061] In another aspect, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds human CD96 and a second antigen-binding region that specifically binds human TIGIT, wherein: (a) the first antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, or glutamic acid at amino acid positions 239, 330, and 332, respectively; and (b) the second antigen-binding region comprises an aspartic acid, a glutamic acid, and a tryptophan at amino acid positions 239, 332, and 366, respectively; Amino acid positions are numbered according to the EU numbering system.

[0062] In certain embodiments, the first antigen-binding region comprises SEQ ID NO: 67, 78, 89, or 49, and the second antigen-binding region comprises SEQ ID NO: 102 or 55.

[0063] In another aspect, the disclosure provides an isolated antibody that specifically binds to human TIGIT, wherein the antibody comprises a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 40, and a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 41. In certain embodiments, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively. In certain embodiments, the antibody comprises the VH amino acid sequence of SEQ ID NO: 40. In another embodiment, the VL consists of the amino acid sequence of SEQ ID NO: 40. In another embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO: 41. In another embodiment, the VH amino acid sequence consists of the amino acid sequence of SEQ ID NO: 41.

[0064] In another aspect, the present disclosure provides an isolated antibody that specifically binds to human TIGIT, wherein the antibody comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 40 and 41, respectively. In certain embodiments, the VH and VL consist of the amino acid sequences of SEQ ID NOs: 40 and 41, respectively.

[0065] In some embodiments, the antibody comprises a heavy chain constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In specific embodiments, the antibody comprises an IgG1 heavy chain constant region. In other embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 57, 58, 59, or 60.

[0066] In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises an N297A mutation numbered according to the EU numbering system.

[0067] In certain embodiments, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, and the variant heavy chain constant region binds to the FcγR with higher affinity than the wild-type heavy chain constant region binds to the FcγR. In certain embodiments, the FcγR is FcγRIIB or FcγRIIIA.

[0068] In certain embodiments, the amino acid sequence of the IgG1 heavy chain constant region comprises S267E and L328F mutations numbered according to the EU numbering system.

[0069] In certain embodiments, the IgG1 heavy chain constant region comprises at least one mutation selected from the group consisting of S239D, A330L, and I332E mutations numbered according to the EU numbering system.

[0070] In certain embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence of SEQ ID NO:7.

[0071] In certain embodiments, the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 43 or 44. In certain embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 8 or 9. In another embodiment, the amino acid sequence of the light chain consists of the amino acid sequence of SEQ ID NO: 8 or 9.

[0072] In another aspect, the disclosure provides an isolated antibody that specifically binds to human TIGIT, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 107, 108, 109, or 110, and a light chain comprising the amino acid sequence of SEQ ID NO: 8 or 9. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence of SEQ ID NO: 107, 108, 109, or 110, and the amino acid sequence of the light chain consists of the amino acid sequence of SEQ ID NO: 8 or 9. In other embodiments, the heavy and light chains comprise the amino acid sequences of SEQ ID NOs: 107 and 8, 107 and 9, 108 and 8, 108 and 9, 109 and 8, 109 and 9, 110 and 8, or 110 and 9, respectively. In other embodiments, the heavy and light chain amino acid sequences consist of the amino acid sequences of SEQ ID NOs: 107 and 8, 107 and 9, 108 and 8, 108 and 9, 109 and 8, 109 and 9, 110 and 8, or 110 and 9, respectively.

[0073] In certain embodiments, the antibody is multispecific.

[0074] In certain embodiments, the multispecific molecule or isolated antibody is conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label, hi certain embodiments, the multispecific molecule or isolated antibody is conjugated to an antibody.

[0075] In another aspect, the disclosure provides an isolated polynucleotide encoding: (a) the VH, VL, heavy chain, and / or light chain of the multispecific molecules disclosed herein; (b) a first VH and a first VL of a multispecific molecule disclosed herein; (c) a second VH and a second VL of a multispecific molecule disclosed herein; (d) a first heavy chain and a first light chain of a multispecific molecule disclosed herein; or (e) A second heavy chain and a second light chain of a multispecific molecule disclosed herein.

[0076] In another aspect, the present disclosure provides isolated polynucleotides encoding the VH and / or VL, or the heavy and / or light chains, of an isolated antibody disclosed herein.

[0077] In another aspect, the present disclosure provides a vector comprising a polynucleotide disclosed herein.

[0078] In another aspect, the present disclosure provides a recombinant host cell comprising: (a) a polynucleotide disclosed herein; (b) a vector disclosed herein; (c) a first polynucleotide encoding the VH and VL of a first antigen-binding region disclosed herein, and a second polynucleotide encoding the VH and VL of a second antigen-binding region disclosed herein; (d) a first vector comprising a first polynucleotide encoding the VH and VL of a first antigen-binding region disclosed herein, and a second vector comprising a second polynucleotide encoding the VH and VL of a second antigen-binding region disclosed herein; (e) a first polynucleotide encoding the VH of a first antigen-binding region disclosed herein, a second polynucleotide encoding the VL of the first antigen-binding region disclosed herein, a third polynucleotide encoding the VH of a second antigen-binding region disclosed herein, and a fourth polynucleotide encoding the VL of the second antigen-binding region disclosed herein; (f) a first vector comprising a first polynucleotide encoding the VH of a first antigen-binding region disclosed herein, a second vector comprising a second polynucleotide encoding the VL of the first antigen-binding region disclosed herein, a third vector comprising a third polynucleotide encoding the VH of a second antigen-binding region disclosed herein, and a fourth vector comprising a fourth polynucleotide encoding the VL of the second antigen-binding region disclosed herein; (g) a first polynucleotide encoding the heavy and light chains of a first antigen-binding region disclosed herein, and a second polynucleotide encoding the heavy and light chains of a second antigen-binding region disclosed herein; (h) a first vector comprising a first polynucleotide encoding the heavy and light chains of a first antigen-binding region disclosed herein, and a second vector comprising a second polynucleotide encoding the heavy and light chains of a second antigen-binding region disclosed herein; (i) a first polynucleotide encoding the heavy chain of a first antigen-binding region disclosed herein, a second polynucleotide encoding the light chain of a first antigen-binding region disclosed herein, a third polynucleotide encoding the heavy chain of a second antigen-binding region disclosed herein, and a fourth polynucleotide encoding the VL of a second antigen-binding region disclosed herein; or (j) A first vector comprising a first polynucleotide encoding the heavy chain of a first antigen-binding region disclosed herein, a second vector comprising a second polynucleotide encoding the light chain of the first antigen-binding region disclosed herein, a third vector comprising a third polynucleotide encoding the heavy chain of a second antigen-binding region disclosed herein, and a fourth vector comprising a fourth polynucleotide encoding the light chain of the second antigen-binding region disclosed herein.

[0079] In another aspect, the present disclosure provides a recombinant host cell comprising: (a) a polynucleotide disclosed herein; (b) a vector disclosed herein; (c) a polynucleotide encoding the VH and VL of the isolated antibody disclosed herein; (d) a first vector comprising a polynucleotide encoding the VH and VL of an isolated antibody disclosed herein; (e) a first polynucleotide encoding the VH of the isolated antibody disclosed herein, and a second polynucleotide encoding the VL of the isolated antibody disclosed herein; (f) A first vector comprising a first polynucleotide encoding the VH of an isolated antibody disclosed herein, and a second vector comprising a second polynucleotide encoding the VL of an isolated antibody disclosed herein.

[0080] In another aspect, the present disclosure provides a pharmaceutical composition comprising a multispecific molecule disclosed herein, an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, or a host cell disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0081] In another aspect, the disclosure provides a method of producing a multispecific molecule or an isolated antibody, the method comprising culturing a host cell disclosed herein under appropriate conditions such that the polynucleotide is expressed and the multispecific molecule or the isolated antibody is produced.

[0082] In another aspect, the disclosure provides a method of producing a multispecific molecule, the method comprising: (a) a first polynucleotide encoding the VH and VL of a first antigen-binding region disclosed herein, and a second polynucleotide encoding the VH and VL of a second antigen-binding region disclosed herein; or (b) a first polynucleotide encoding the heavy and light chains of a first antigen-binding region disclosed herein, and a second polynucleotide encoding the heavy and light chains of a second antigen-binding region disclosed herein; This involves expressing the polynucleotide under appropriate conditions such that the polynucleotide is expressed and a multispecific molecule is produced.

[0083] In another aspect, the disclosure provides a method of producing a multispecific molecule, the method comprising: (a) a first polynucleotide encoding the VH of a first antigen-binding region disclosed herein, a second polynucleotide encoding the VL of the first antigen-binding region disclosed herein, a third polynucleotide encoding the VH of a second antigen-binding region disclosed herein, and a fourth polynucleotide encoding the VL of the second antigen-binding region disclosed herein; or (b) a first polynucleotide encoding the heavy chain of a first antigen-binding region disclosed herein, a second polynucleotide encoding the light chain of a first antigen-binding region disclosed herein, a third polynucleotide encoding the heavy chain of a second antigen-binding region disclosed herein, and a fourth polynucleotide encoding the light chain of a second antigen-binding region disclosed herein; This involves expressing the polynucleotide under appropriate conditions such that the polynucleotide is expressed and a multispecific molecule is produced.

[0084] In another aspect, the disclosure provides a method of producing a multispecific molecule, the method comprising: (a) expressing in a first cell a first polynucleotide encoding the VH and VL of a first antigen-binding region disclosed herein under conditions such that the first antigen-binding region is produced; (b) expressing in a second cell a second polynucleotide encoding the VH and VL of a second antigen-binding region disclosed herein under conditions such that the second antigen-binding region is produced; and (c) contacting the first and second antigen-binding regions produced in steps (a) and (b) under suitable conditions so that a multispecific molecule is produced.

[0085] In another aspect, the disclosure provides a method of producing a multispecific molecule, the method comprising: (a) expressing in a first cell a first polynucleotide encoding a VH of a first antigen-binding region disclosed herein and a second polynucleotide encoding a VL of the first antigen-binding region disclosed herein under conditions such that the first antigen-binding region is produced; (b) expressing in the second cell a third polynucleotide encoding the VH of the second antigen-binding region disclosed herein and a fourth polynucleotide encoding the VL of the second antigen-binding region disclosed herein under conditions such that the second antigen-binding region is produced; and (c) contacting the first and second antigen-binding regions produced in steps (a) and (b) under conditions such that a multispecific molecule is produced.

[0086] In another aspect, the disclosure provides a method of producing a multispecific molecule, the method comprising contacting a first antigen-binding region and a second antigen-binding region disclosed herein under conditions such that the multispecific molecule is produced.

[0087] In another aspect, the disclosure provides a method of producing an isolated antibody, the method comprising: (a) a polynucleotide encoding the VH and VL of an antibody disclosed herein; or (b) a polynucleotide encoding the heavy and light chains of an antibody disclosed herein, This involves expressing the polynucleotide under appropriate conditions such that the polynucleotide is expressed and the antibody is produced.

[0088] In another aspect, the disclosure provides a method of producing an isolated antibody, the method comprising: (a) a first polynucleotide encoding the VH of an antibody disclosed herein, and a second polynucleotide encoding the VL of an antibody disclosed herein; or (b) a first polynucleotide encoding the heavy chain of an antibody disclosed herein, and a second polynucleotide encoding the light chain of an antibody disclosed herein, This involves expressing the polynucleotide under appropriate conditions such that the polynucleotide is expressed and the antibody is produced.

[0089] In another aspect, the present disclosure provides a method of enhancing an immune response in a subject, the method comprising administering to the subject an effective amount of a multispecific molecule disclosed herein, an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0090] In another aspect, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject an effective amount of a multispecific molecule disclosed herein, an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0091] In certain embodiments, the multispecific molecule, isolated antibody, polynucleotide, vector, host cell, or pharmaceutical composition is administered systemically, intravenously, subcutaneously, intratumorally, or delivered to a tumor-draining lymph node.

[0092] In certain embodiments, the method further comprises administering an additional therapeutic agent to the subject. In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent. In certain embodiments, the additional therapeutic agent is a checkpoint targeting agent. In another embodiment, the checkpoint targeting agent is selected from the group consisting of an antagonistic anti-PD-1 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-CTLA-4 antibody, an antagonistic anti-TIM-3 antibody, an antagonistic anti-LAG-3 antibody, an antagonistic anti-VISTA antibody, an antagonistic anti-TIGIT antibody, an antagonistic anti-CEACAM1 antibody, an antagonistic anti-CD96 antibody, an agonist anti-GITR antibody, and an agonist anti-OX40 antibody. In another embodiment, the additional therapeutic agent is an anti-PD-1 antibody, and optionally the anti-PD-1 antibody is pembrolizumab or nivolumab. In certain embodiments, the additional therapeutic agent is an inhibitor of indoleamine-2,3-dioxygenase (IDO). In another embodiment, the inhibitor is selected from the group consisting of epacadostat, F001287, indoximod, and NLG919. In certain embodiments, the additional therapeutic agent is a vaccine. In another embodiment, the vaccine comprises a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide. In another embodiment, the heat shock protein is hsc70 and is complexed with a tumor-associated antigenic peptide. In another embodiment, the heat shock protein is gp96 protein and is complexed with a tumor-associated antigenic peptide, optionally wherein the HSPPC is derived from a tumor obtained from the subject.

[0093] In another aspect, the present disclosure provides a method of treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of a multispecific molecule disclosed herein, an isolated antibody disclosed herein, a polynucleotide disclosed herein, a vector disclosed herein, a host cell disclosed herein, or a pharmaceutical composition disclosed herein. [Brief explanation of the drawings]

[0094] 6. Brief description of the drawings [Figure 1A] 1A-1C are a series of sensorgrams showing simultaneous binding of the extracellular domains of human TIGIT and human CD96 to the multispecific molecules BA123 (FIG. 1A), BA125 (FIG. 1B), and BA127 (FIG. 1C). [Figure 1B] Same as above. [Figure 1C] Same as above.

[0095] [Figure 2A] 2A-2B are graphs showing simultaneous binding of the anti-TIGITxCD96 multispecific molecule BA127 to CHO cells engineered to express human TIGIT or human CD96, compared with the control multispecific molecules BA128, BA131, and BA133. Dual binding of cell-expressed human TIGIT and soluble His-tagged human CD96 (FIG. 2A) or cell-expressed human CD96 and soluble His-tagged human TIGIT (FIG. 2B) was detected by flow cytometry using a fluorescent dye-conjugated (Alex Fluor 488) anti-His antibody. [Figure 2B] Same as above.

[0096] [Figure 3A] Figures 3A-3I are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 3A), BA125 (Figure 3B), or BA127 (Figure 3C), or the control multispecific molecules BA129 (Figure 3D), BA130 (Figure 3E), BA131 (Figure 3F), BA133 (Figure 3G), BA134 (Figure 3H), or BA136 (Figure 3I), to CHO cells engineered to express high levels of cell surface isoform 2 of human CD96, compared to an isotype control multispecific molecule (BA128). The level of binding was assessed by median fluorescence intensity (MFI) and, in each case, plotted against the concentration of each antibody incubated with the cells, relative to the CHO cell binding of BA128. [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 3D] Same as above. [Figure 3E] Same as above. [Figure 3F] Same as above. [Figure 3G] Same as above. [Figure 3H] Same as above. [Figure 3I] Same as above.

[0097] [Figure 4A] Figures 4A-4I are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 4A), BA127 (Figure 4B), or BA125 (Figure 4C), or the control multispecific molecules BA129 (Figure 4D), BA130 (Figure 4E), BA131 (Figure 4F), BA133 (Figure 4G), BA134 (Figure 4H), or BA136 (Figure 4I), to CHO cells engineered to express high levels of cell surface isoform 1 of human CD96, compared to the isotype control multispecific molecule (BA128). The level of binding was assessed by median fluorescence intensity (MFI) and, in each case, plotted against the concentration of each antibody incubated with the cells, relative to the CHO cell binding of BA128. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 4H] Same as above. [Figure 4I] Same as above.

[0098] [Figure 5A]Figures 5A-5I are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 5A), BA125 (Figure 5B), or BA127 (Figure 5C), or the control multispecific molecules BA129 (Figure 5D), BA130 (Figure 5E), BA131 (Figure 5F), BA133 (Figure 5G), BA134 (Figure 5H), or BA136 (Figure 5I), to CHO cells engineered to express high levels of cell surface isoform 2 of cynomolgus monkey CD96, compared to the isotype control multispecific molecule (BA128). The level of binding was assessed by median fluorescence intensity (MFI) and is plotted against the concentration of each antibody incubated with the cells in each case, relative to the CHO cell binding of BA128. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 5E] Same as above. [Figure 5F] Same as above. [Figure 5G] Same as above. [Figure 5H] Same as above. [Figure 5I] Same as above.

[0099] [Figure 6A] Figures 6A-6I are a series of graphs showing blockade of human CD155-Fc binding to CHO cells engineered to express high levels of cell surface isoform 2 of human CD96 by the anti-TIGITxCD96 multispecific molecules BA123 (Figure 6A), BA125 (Figure 6B), or BA127 (Figure 6C), or the control multispecific molecules BA129 (Figure 6D), BA130 (Figure 6E), BA131 (Figure 6F), BA133 (Figure 6G), BA134 (Figure 6H), or BA136 (Figure 6I). The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response against the concentration of each antibody incubated with the cells, in each case compared to blockade by the isotype control multispecific molecule (BA128). [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 6E] Same as above. [Figure 6F] Same as above. [Figure 6G] Same as above. [Figure 6H] Same as above. [Figure 6I] Same as above.

[0100] [Figure 7A] Figures 7A-7I are a series of graphs showing blockade of human CD155-Fc binding to CHO cells engineered to express high levels of cell surface isoform 2 of cynomolgus monkey CD96 by the anti-TIGITxCD96 multispecific molecules BA123 (Figure 7A), BA125 (Figure 7B), or BA127 (Figure 7C), or the control multispecific molecules BA129 (Figure 7D), BA130 (Figure 7E), BA131 (Figure 7F), BA133 (Figure 7G), BA134 (Figure 7H), or BA136 (Figure 7I). The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response against the concentration of each multispecific molecule incubated with the cells, in each case compared to blockade by the isotype control multispecific molecule (BA128). [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] Same as above. [Figure 7H] Same as above. [Figure 7I] Same as above.

[0101] [Figure 8A]Figures 8A-8I are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 8A), BA125 (Figure 8B), or BA127 (Figure 8C), or the control multispecific molecules BA129 (Figure 8D), BA130 (Figure 8E), BA131 (Figure 8F), BA133 (Figure 8G), BA134 (Figure 8H), or BA136 (Figure 8I), to CHO cells engineered to express high levels of cell surface human TIGIT. The level of binding was assessed by median fluorescence intensity (MFI) and, in each case, plotted against the concentration of each antibody incubated with the cells, relative to CHO cell binding of the isotype control multispecific molecule (BA128). [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above. [Figure 8E] Same as above. [Figure 8F] Same as above. [Figure 8G] Same as above. [Figure 8H] Same as above. [Figure 8I] Same as above.

[0102] [Figure 9A] Figures 9A-9I are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 9A), BA125 (Figure 9B), or BA127 (Figure 9C), or the control multispecific molecules BA129 (Figure 9D), BA130 (Figure 9E), BA131 (Figure 9F), BA133 (Figure 9G), BA134 (Figure 9H), or BA136 (Figure 9I), to CHO cells engineered to express high levels of cell surface cynomolgus monkey TIGIT. The level of binding was assessed by median fluorescence intensity (MFI), in each case plotted against the concentration of each antibody incubated with the cells, relative to the binding of the isotype control multispecific molecule (BA128). [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 9G] Same as above. [Figure 9H] Same as above. [Figure 9I] Same as above.

[0103] [Figure 10A] Figures 10A-10I are a series of graphs showing blockade of human CD155-Fc binding to CHO cells engineered to express high levels of cell surface human TIGIT by the anti-TIGITxCD96 multispecific molecules BA123 (Figure 10A), BA125 (Figure 10B), or BA127 (Figure 10C), or the control multispecific molecules BA129 (Figure 10D), BA134 (Figure 10E), BA131 (Figure 10F), BA133 (Figure 10G), BA130 (Figure 10H), or BA136 (Figure 10I). The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response in each case against the concentration of each antibody incubated with the cells, compared to blockade by the isotype control multispecific molecule (BA128). [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 10F] Same as above. [Figure 10G] Same as above. [Figure 10H] Same as above. [Figure 10I] Same as above.

[0104] [Figure 11A]Figures 11A-11I are a series of graphs showing blockade of human CD155-Fc binding to CHO cells engineered to express high levels of cell surface cynomolgus monkey TIGIT by the anti-TIGITxCD96 multispecific molecules BA123 (Figure 11A), BA125 (Figure 11B), or BA127 (Figure 11C), or the control multispecific molecules BA129 (Figure 11D), BA130 (Figure 11E), BA131 (Figure 11F), BA133 (Figure 11G), BA134 (Figure 11H), or BA136 (Figure 11I). The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response in each case against the concentration of each antibody incubated with the cells, compared to blockade by the isotype control multispecific molecule (BA128). [Figure 11B] Same as above. [Figure 11C] Same as above. [Figure 11D] Same as above. [Figure 11E] Same as above. [Figure 11F] Same as above. [Figure 11G] Same as above. [Figure 11H] Same as above. [Figure 11I] Same as above.

[0105] [Figure 12A] Figures 12A-12I are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 12A), BA125 (Figure 12B), or BA127 (Figure 12C), or the control multispecific molecules BA129 (Figure 12D), BA130 (Figure 12E), BA131 (Figure 12F), BA133 (Figure 12G), BA134 (Figure 12H), or BA136 (Figure 12I), to CHO cells engineered to coexpress high levels of cell surface human TIGIT and human CD96 isoform 2. The level of binding was assessed by median fluorescence intensity (MFI) and in each case plotted against the concentration of each antibody incubated with the cells, relative to the binding of the isotype control multispecific molecule (BA128). [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 12F] Same as above. [Figure 12G] Same as above. [Figure 12H] Same as above. [Figure 12I] Same as above.

[0106] [Figure 13A] Figures 13A-13I are a series of graphs showing blockade of human CD155-Fc binding to CHO cells engineered to co-express high levels of cell surface human TIGIT and human CD96 isoform 2 by the anti-TIGITxCD96 multispecific molecules BA123 (Figure 13A), BA125 (Figure 13B), or BA127 (Figure 13C), or the control multispecific molecules BA129 (Figure 13D), BA130 (Figure 13E), BA131 (Figure 13F), BA133 (Figure 13G), BA134 (Figure 13H), or BA136 (Figure 13I). The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response in each case against the concentration of each antibody incubated with the cells, compared to blockade by the isotype control multispecific molecule (BA128). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 13H] Same as above. [Figure 13I] Same as above.

[0107] [Figure 14A]Figures 14A-14F are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 14A), BA125 (Figure 14B), or BA127 (Figure 14C), or the control multispecific molecules BA129 (Figure 14D), BA130 (Figure 14E), and BA131 (Figure 14F), to CHO cells engineered to express high levels of the cell surface variant V / V of human FcγRIIIa. The level of binding was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 14E] Same as above. [Figure 14F] Same as above.

[0108] [Figure 15A] Figures 15A-15F are a series of graphs showing binding of the anti-TIGITxCD96 multispecific molecules BA123 (Figure 15A), BA125 (Figure 15B), or BA127 (Figure 15C), or the control multispecific molecules BA129 (Figure 15D), BA130 (Figure 15E), and BA131 (Figure 15F), to CHO cells engineered to express high levels of the cell surface variant F / F of human FcγRIIIa. The level of binding was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 15E] Same as above. [Figure 15F] Same as above.

[0109] [Figure 16A]Figures 16A-16C are a series of graphs showing the binding of anti-CD96 antibodies BA143 (Figure 16A), BA144 (Figure 16B), and BA145 (Figure 16C) to CHO cells engineered to express high levels of cell surface isoform 2 of human CD96, compared to an isotype control antibody (BA146). The level of binding was assessed by median fluorescence intensity (MFI) and, in each case, plotted against the concentration of each antibody incubated with the cells, relative to the CHO cell binding of BA146. [Figure 16B] Same as above. [Figure 16C] Same as above.

[0110] [Figure 17A] Figures 17A-17C are a series of graphs showing the binding of anti-CD96 antibodies BA143 (Figure 17A), BA144 (Figure 17B), and BA145 (Figure 17C) to CHO cells engineered to express high levels of cell surface isoform 2 of cynomolgus CD96, compared to an isotype control antibody (BA146). The level of binding was assessed by median fluorescence intensity (MFI) and, in each case, plotted against the concentration of each antibody incubated with the cells, relative to the CHO cell binding of BA146. [Figure 17B] Same as above. [Figure 17C] Same as above.

[0111] [Figure 18A] Figures 18A-18C are a series of graphs showing blockade of human CD155-Fc binding to CHO cells engineered to express high levels of human CD96 cell surface isoform 2 by anti-CD96 antibodies BA143 (Figure 18A), BA144 (Figure 18B), and BA145 (Figure 18C). The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response against the concentration of each antibody incubated with the cells, in each case compared to blockade by an isotype control antibody (BA146). [Figure 18B] Same as above. [Figure 18C] Same as above.

[0112] [Figure 19A] Figures 19A-19C are a series of graphs showing blockade of human CD155-Fc binding to CHO cells engineered to express high levels of cell surface isoform 2 of cynomolgus monkey CD96 by anti-CD96 antibodies BA143 (Figure 19A), BA144 (Figure 19B), and BA145 (Figure 19C). The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response against the concentration of each antibody incubated with the cells, in each case compared to blockade by an isotype control antibody (BA146). [Figure 19B] Same as above. [Figure 19C] Same as above.

[0113] [Figure 20] Figure 20 is a graph showing binding of the anti-TIGIT IgG1 antibody BA148 to CHO cells engineered to express high levels of cell surface human TIGIT, compared to an isotype control antibody (BA149). The level of binding was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells, in each case relative to the CHO cell binding of BA149.

[0114] [Figure 21] 21 is a graph showing binding of anti-TIGIT antibody BA148 to CHO cells engineered to express high levels of cell surface cynomolgus monkey TIGIT compared to an isotype control antibody (BA149). The level of binding was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells in each case relative to the CHO cell binding of BA149.

[0115] [Figure 22]Figure 22 is a graph showing blocking of human CD155-Fc binding to CHO cells engineered to express high levels of human TIGIT by anti-TIGIT antibody BA148. The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response versus the concentration of each antibody incubated with the cells, relative to blockade by an isotype control antibody (BA149).

[0116] [Figure 23] 23 is a graph showing blockade of human CD155-Fc binding to CHO cells engineered to express high levels of cynomolgus monkey TIGIT by anti-TIGIT antibody BA148. The level of CD155-Fc binding was assessed by median fluorescence intensity (MFI) and plotted as percent maximal response versus the concentration of each antibody incubated with the cells, relative to blockade by an isotype control antibody (BA149).

[0117] [Figure 24A] Figures 24A-24D are a series of graphs showing binding of anti-TIGIT antibody BA148 and anti-CD96 antibodies BA143, BA144, and BA145 to CHO cells engineered to express high levels of human FcγRIIIa variant V / V on their surface. The level of binding of BA143 (Figure 24A), BA144 (Figure 24B), BA145 (Figure 24C), and BA148 (Figure 24D) was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above.

[0118] [Figure 25A]Figures 25A-25D are a series of graphs showing binding of the anti-TIGIT IgG1 antibody BA148 and the anti-CD96 IgG1 WT antibodies BA143, BA144, and BA145 to CHO cells engineered to express high cell surface levels of the human FcγRIIIa variant F / F. The level of binding of BA143 (Figure 25A), BA144 (Figure 25B), BA145 (Figure 25C), and BA148 (Figure 25D) was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above.

[0119] [Figure 26A] Figures 26A-26F are a series of graphs showing the ability of BA127, BA143, BA148, or BA128 to bind to activated human T cells in three different donors. Binding to CD4+ T cells (Figures 26A, 26B, and 26C) and CD8+ T cells (Figures 26D, 26E, and 26F) was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells. [Figure 26B] Same as above. [Figure 26C] Same as above. [Figure 26D] Same as above. [Figure 26E] Same as above. [Figure 26F] Same as above.

[0120] [Figure 27A] Figures 27A-27F are a series of graphs showing the ability of BA127 or BA128 to bind to activated human T cells in three different donors. Binding to CD4+ T cells (Figures 27A, 27B, and 27C) and CD8+ T cells (Figures 27D, 27E, and 27F) was assessed by median fluorescence intensity (MFI) and plotted against the concentration of each antibody incubated with the cells. [Figure 27B]Same as above. [Figure 27C] Same as above. [Figure 27D] Same as above. [Figure 27E] Same as above. [Figure 27F] Same as above.

[0121] [Figure 28A] Figures 28A-C are a series of graphs showing the ability of BA123, BA125, BA127, BA128, BA129, BA130, and BA131 multispecific molecules to promote IL-2 secretion by SEA-stimulated PBMCs from a single donor across a range of multispecific molecule concentrations. Each panel represents an independent experiment using the same donor. [Figure 28B] Same as above. [Figure 28C] Same as above.

[0122] [Figure 29] FIG. 29 is a graph showing the ability of the BA127 and BA128 multispecific molecules and the BA143 and BA148 antibodies to promote IL-2 secretion by SEA-stimulated PBMC from a single donor across a range of multispecific molecule concentrations.

[0123] [Figure 30A] Figures 30A-30F are a series of graphs showing the ability of BA127 and BA128 to enhance IL-2 secretion by SEA-stimulated PBMC from six different donors across a wide range of antibody concentrations. [Figure 30B] Same as above. [Figure 30C] Same as above. [Figure 30D] Same as above. [Figure 30E] Same as above. [Figure 30F] Same as above.

[0124] [Figure 31A]Figures 31A-31B are graphs showing the ability of BA125, BA127, BA128, and BA133 (Figure 31A), and BA127, BA143, BA146, anti-TIGIT monospecific reference antibody 1, and anti-TIGIT monospecific reference antibody 2 (Figure 31B) to block the binding of TIGIT expressed on Jurkat cells to CD155 expressed on CHO cells. Figure 31C is a graph showing the ability of BA127, BA131, BA128, and anti-TIGIT reference antibody 3 to block the binding of TIGIT expressed on Jurkat cells to CD155 expressed on CHO cells. Blockade is expressed as the fold change in NFAT-luciferase signal across a range of antibody concentrations. [Figure 31B] Same as above. [Figure 31C] Same as above.

[0125] [Figure 32A] Figures 32A-B are graphs showing the ability of the anti-TIGITxCD96 multispecific molecule BA127, a reference anti-TIGIT antibody, and an isotype control antibody to induce IL-2 cytokine secretion in primary healthy donor human PBMCs stimulated with suboptimal concentrations of SEA superantigen from two donors over a wide range of concentrations. [Figure 32B] Same as above.

[0126] [Figure 33A] Figures 33A-33E are a series of graphs showing tumor volume over time in a mouse colorectal cancer model, in which mice were administered a bispecific isotype control (Figure 33A), an anti-TIGIT mouse surrogate monospecific antibody (Figure 33B), an anti-CD96 mouse surrogate monospecific antibody (Figure 33C), both anti-TIGIT and anti-CD96 mouse surrogate monospecific antibodies (Figure 33D), or an anti-TIGITxCD96 mouse surrogate multispecific molecule (Figure 33E). [Figure 33B] Same as above. [Figure 33C] Same as above. [Figure 33D] Same as above. [Figure 33E] Same as above.

[0127] [Figure 34A] Figure 34A is a graph showing mean tumor volume over time in a mouse colorectal cancer model, where mice were administered both anti-TIGIT and anti-CD96 murine surrogate monospecific antibodies, anti-TIGITxCD96 murine surrogate multispecific molecules, Fc-silenced anti-TIGITxCD96 murine surrogate multispecific molecules, or an isotype control. Figures 34B-34E are a series of graphs showing individual tumor volumes over time for each individual mouse administered both anti-TIGIT and anti-CD96 murine surrogate monospecific antibodies (Figure 34C), anti-TIGITxCD96 murine surrogate multispecific molecules (Figure 34D), Fc-silenced anti-TIGITxCD96 murine surrogate multispecific molecules (Figure 34E), or an isotype control (Figure 34B). [Figure 34B] Same as above. [Figure 34C] Same as above. [Figure 34D] Same as above. [Figure 34E] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0128] 7. MODE FOR CARRYING OUT THE INVENTION The present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CDCD96) and / or TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and isolated anti-TIGIT antibodies. Also provided are pharmaceutical compositions comprising these multispecific molecules and antibodies, nucleic acids encoding these multispecific molecules and antibodies, expression vectors and host cells for making these multispecific molecules and antibodies, and methods of treating subjects using these multispecific molecules and antibodies. The multispecific molecules and antibodies disclosed herein are particularly useful for increasing immune cell activation and are therefore useful in treating cancer in a subject or treating or preventing an infectious disease in a subject.

[0129] 7.1 Definition As used herein, the term "CD96" refers to cluster of differentiation 96, also known as TACTILE (T cell activation, increased late expression), which in humans is encoded by the CD96 gene. As used herein, the term "human CD96" refers to the CD96 protein encoded by the wild-type human CD96 gene (e.g., GenBank™ Accession No. NM_005816.5), fragments, or variants thereof. Exemplary extracellular portions of human CD96 are provided herein as SEQ ID NOS: 61-65. Exemplary extracellular portions of cynomolgus monkey CD96 are provided herein as SEQ ID NOS: 66, 111, and 112.

[0130] As used herein, the term "TIGIT" refers to a T cell immunoreceptor with an Ig domain and an ITIM domain (also known as VSIG9 or VSTM3) encoded by the TIGIT gene in humans. As used herein, the term "human TIGIT" refers to the TIGIT protein encoded by the wild-type human TIGIT gene (e.g., GenBank™ Accession No. NM_173799.3) or the extracellular domain of such a protein. Exemplary amino acid sequences of the extracellular domains of mature human TIGIT protein and cynomolgus monkey TIGIT protein are provided as SEQ ID NOs: 113 and 114, respectively.

[0131] As used herein, a "multispecific molecule" is a molecule that contains two or more antigen-binding regions that specifically bind to different antigens.

[0132] As used herein, "antigen-binding region" refers to the portion of a multispecific molecule or antibody that contains the amino acid residues that confer its specificity for an antigen to the multispecific molecule or antigen. Examples of antigen-binding regions include antibody complementarity-determining regions (CDRs), heavy chain variable regions, light chain variable regions, heavy chains, light chains, and any fragments thereof. Antigen-binding regions can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans.

[0133] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFvs), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (e.g., including anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies, or classes (e.g., human IgG1 or IgG4) or subclasses thereof. In certain embodiments, antibodies are humanized monoclonal antibodies. In another specific embodiment, antibodies are human monoclonal antibodies.

[0134] A "multispecific antibody" is an antibody that specifically binds to two or more different antigens, or to two or more different regions of the same antigen (e.g., a bispecific antibody). Multispecific antibodies include bispecific antibodies that contain two different antigen-binding sites (excluding the Fc region). Multispecific antibodies can include, for example, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing two heavy chain and two light chain molecules, antibody light chain monomers, heteroconjugate antibodies, linked single-chain antibodies or linked single-chain Fvs (scFv), camelized antibodies, affibodies, linked Fab fragments, F(ab')2 fragments, chemically linked Fvs, and disulfide-linked Fvs (sdFv). Multispecific antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, the multispecific antibodies described herein are IgG antibodies, or classes (e.g., human IgG1, IgG2, or IgG4) or subclasses thereof.

[0135] As used herein, the term "CDR" or "complementarity-determining region" refers to the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. These particular regions are described, for example, by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), by Chothia et al., J. Mol. Biol. 196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996), all of which are incorporated herein by reference in their entireties, where the definitions include overlapping or subsets of amino acid residues when compared against each other. CDRH1, CDRH2, and CDRH3 represent the heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent the light chain CDRs.

[0136] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically the amino-terminal approximately 110-120 or 110-125 amino acids in a mature heavy chain and approximately 90-115 amino acids in a mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen. The variability in sequence is concentrated in those regions called complementarity-determining regions (CDRs), while the more highly conserved regions in a variable region are called framework regions (FRs). While not wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of an antibody with an antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0137] As used herein, the terms "VH" and "VL" refer to antibody heavy and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0138] As used herein, the term "constant region" is a term commonly used in the art. A constant region is an antibody portion, e.g., the carboxyl-terminal portion of the light and / or heavy chain, that is not directly involved in binding the antibody to an antigen but may exhibit various effector functions, such as interaction with Fc receptors (e.g., Fc gamma receptors).

[0139] As used herein, the term "heavy chain," when used in reference to an antibody, can refer to any of the different types, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4, respectively.

[0140] As used herein, the term "light chain," when used in reference to an antibody, can refer to any of the different types, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain.

[0141] As used herein, the terms "specifically bind," "specifically recognize," "immunospecifically bind," and "immunospecifically recognize" are similar terms in the context of antibodies and refer to a molecule that binds to an antigen (e.g., an epitope or immune complex) as such binding is understood by one of skill in the art. For example, a molecule that specifically binds to an antigen may generally bind to other peptides or polypeptides with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, Idaho), or other assays known in the art. In certain embodiments, a molecule that specifically binds to an antigen has a K that is lower than the K that would be expected if the molecule were to nonspecifically bind to another antigen. A At least 2 logs (e.g., 10 times), 2.5 logs, 3 logs, 4 logs or more than A binds to the antigen.

[0142] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0143] As used herein, the terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures as described herein. Methods of "treatment" employ administration of an antibody to a subject having or prone to having a disease or disorder to prevent, cure, delay, reduce the severity of, or ameliorate a disease or disorder or one or more symptoms of a recurrent disease or disorder, or to extend the subject's survival beyond that expected in the absence of such treatment.

[0144] As used herein, the term "effective amount" in reference to the administration of a therapy to a subject refers to the amount of therapy that achieves the desired prophylactic or therapeutic effect.

[0145] As used herein, the term "subject" includes any human or non-human animal. In certain embodiments, the subject is a human or non-human mammal. In certain embodiments, the subject is a human.

[0146] As used herein with respect to an antibody or polynucleotide, the term "isolated" refers to an antibody or polynucleotide that is separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids, or carbohydrates, etc.) that are present in the natural source of the antibody or polynucleotide. All examples of "isolated antibodies" described herein are additionally contemplated as antibodies that may, but need not be isolated. All examples of "isolated polynucleotides" described herein are additionally contemplated as polynucleotides that may, but need not be isolated. All examples of "antibodies" described herein are additionally contemplated as antibodies that may, but need not be isolated. All examples of "polynucleotides" described herein are additionally contemplated as polynucleotides that may, but need not be isolated.

[0147] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87:2264-2268, as modified in Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., to score=100 and wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, for example, for score=50 and word length=3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search that detects distant relationships between molecules (ibid.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, e.g., the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).Another specific, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0148] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0149] 7.2 Multispecific Molecules that Bind CD96 and / or TIGIT and Anti-TIGIT Antibodies In one aspect, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and / or TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT). For example, the multispecific molecules provided herein can comprise a first antigen-binding region that binds to CD96 and a second antigen-binding region that binds to an antigen other than CD96. The multispecific molecules provided herein can also comprise a first antigen-binding region that binds to an antigen other than TIGIT and a second antigen-binding region that binds TIGIT. Also provided herein are multispecific molecules comprising a first antigen-binding region that binds to CD96 and a second antigen-binding region that binds TIGIT. The amino acid sequences of exemplary anti-CD96 and anti-TIGIT antigen-binding regions are shown in Table 1 and Table 2, respectively.

[0150] In another aspect, the present disclosure provides antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT). The amino acid sequences of exemplary antibodies are shown in Table 2.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

[0151] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH that comprises one, two, or all three of the CDRs of the VH shown in Table 1. In certain embodiments, the first antigen-binding region comprises a CDRH1 of the VH shown in Table 1. In certain embodiments, the first antigen-binding region comprises a CDRH2 of the VH shown in Table 1. In certain embodiments, the first antigen-binding region comprises a CDRH3 of the VH shown in Table 1.

[0152] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VL that comprises one, two, or all three of the CDRs of a VL disclosed in Table 1. In certain embodiments, the first antigen-binding region comprises CDRL1 of the VL shown in Table 1. In certain embodiments, the first antigen-binding region comprises CDRL2 of the VL shown in Table 1. In certain embodiments, the first antigen-binding region comprises CDRL3 of the VL shown in Table 1.

[0153] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VH that comprises one, two, or all three CDRs of the VH shown in Table 2. In certain embodiments, the second antigen-binding region comprises CDRH1 of the VH shown in Table 2. In certain embodiments, the second antigen-binding region comprises CDRH2 of the VH shown in Table 2. In certain embodiments, the second antigen-binding region comprises CDRH3 of the VH shown in Table 2.

[0154] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VL that comprises one, two, or all three of the CDRs of the VL shown in Table 2. In certain embodiments, the second antigen-binding region comprises CDRH1 of the VL shown in Table 2. In certain embodiments, the second antigen-binding region comprises CDRH2 of the VL shown in Table 2. In certain embodiments, the second antigen-binding region comprises CDRH3 of the VL shown in Table 2.

[0155] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a VH comprising one, two, or all three of the CDRs of the VH shown in Table 1, and the second antigen-binding region comprises a VH comprising one, two, or all three of the CDRs of the VH shown in Table 2. In certain embodiments, the first antigen-binding region comprises a CDRH1 of the VH shown in Table 1, and the second antigen-binding region comprises a CDRH1 of the VH shown in Table 2. In certain embodiments, the first antigen-binding region comprises a CDRH2 of the VH shown in Table 1, and the second antigen-binding region comprises a CDRH2 of the VH shown in Table 2. In certain embodiments, the first antigen-binding region comprises a CDRH3 of a VH shown in Table 1, and the second antigen-binding region comprises a CDRH3 of a VH shown in Table 2.

[0156] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a VL comprising one, two, or all three of the CDRs of a VL shown in Table 1, and the second antigen-binding region comprises a VL comprising one, two, or all three of the CDRs of a VL shown in Table 2. In certain embodiments, the first antigen-binding region comprises a CDRH1 of a VL shown in Table 1, and the second antigen-binding region comprises a CDRH1 of a VL shown in Table 2. In certain embodiments, the first antigen-binding region comprises a CDRH2 of a VL shown in Table 1, and the second antigen-binding region comprises a CDRH2 of a VL shown in Table 2. In certain embodiments, the first antigen-binding region comprises a CDRH3 of a VL shown in Table 1, and the second antigen-binding region comprises a CDRH3 of a VL shown in Table 2.

[0157] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the antibody comprises a VH domain comprising one, two, or all three of the CDRs of the VH domain shown in Table 2. In certain embodiments, the antibody comprises a CDRH1 of the VH domain shown in Table 2. In certain embodiments, the antibody comprises a CDRH2 of the VH domain shown in Table 2. In certain embodiments, the antibody comprises a CDRH3 of the VH domain shown in Table 2.

[0158] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the antibody comprises a VL domain comprising one, two, or all three of the CDRs of the VL domain disclosed in Table 2. In certain embodiments, the antibody comprises CDRL1 of the VL domain shown in Table 2. In certain embodiments, the antibody comprises CDRL2 of the VL domain shown in Table 2. In certain embodiments, the antibody comprises CDRL3 of the VL domain shown in Table 2.

[0159] The individual CDRs of the multispecific molecules or antibodies disclosed herein can be determined according to any CDR numbering scheme known in the art.

[0160] In certain embodiments, one or more of the CDRs of the multispecific molecules or antibodies disclosed herein can be determined according to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteins of immunological interest (1991), each of which is incorporated herein by reference in its entirety.

[0161] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 1 herein, as determined by the Kabat numbering scheme.

[0162] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 2 herein, as determined by the Kabat numbering scheme.

[0163] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 1 herein, and the second antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 2 herein, as determined by the Kabat numbering scheme.

[0164] In certain embodiments, the present disclosure provides antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and comprise the CDRs of the antibodies disclosed in Table 2 herein, as determined by the Kabat numbering scheme.

[0165] In certain embodiments, one or more of the CDRs of the multispecific molecules or antibodies disclosed herein may be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226, all of which are incorporated by reference in their entirety).

[0166] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 1 herein, as determined by the Chothia numbering system.

[0167] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 2 herein, as determined by the Chothia numbering system.

[0168] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 1 herein, and the second antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 2 herein, as determined by the Chothia numbering system.

[0169] In certain embodiments, the present disclosure provides antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and comprise the CDRs of the antibodies disclosed in Table 2 herein, as determined by the Chothia numbering system.

[0170] In certain embodiments, one or more of the CDRs of the multispecific molecules or antibodies disclosed herein can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745, incorporated herein by reference in its entirety. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), incorporated herein by reference in its entirety.

[0171] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 1 herein, as determined by the MacCallum numbering system.

[0172] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 2 herein, as determined by the MacCallum numbering system.

[0173] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 1 herein, and the second antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 2 herein, as determined by the MacCallum numbering system.

[0174] In certain embodiments, the present disclosure provides antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and comprise the CDRs of the antibodies disclosed in Table 2 herein, as determined by the MacCallum numbering scheme.

[0175] In certain embodiments, the CDRs of the multispecific molecules or antibodies disclosed herein can be determined according to the IMGT numbering system described in Lefranc MP, (1999) The Immunologist 7:132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212, each of which is incorporated herein by reference in its entirety; and Lefranc MP et al., (2009) Nucleic Acids Res 37:D1006-D1012.

[0176] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 1 herein, as determined by the IMGT numbering system.

[0177] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 2 herein, as determined by the IMGT numbering system.

[0178] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 1 herein, and the second antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 2 herein, as determined by the IMGT numbering system.

[0179] In certain embodiments, the present disclosure provides antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and comprise the CDRs of the antibodies disclosed in Table 2 herein, as determined by the IMGT numbering scheme.

[0180] In certain embodiments, the CDRs of the multispecific molecules or antibodies disclosed herein can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between the Kabat CDRs and the Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0181] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 1 herein, as determined by the AbM numbering scheme.

[0182] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 2 herein, as determined by the AbM numbering scheme.

[0183] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 1 herein, and the second antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 2 herein, as determined by the AbM numbering scheme.

[0184] In certain embodiments, the present disclosure provides antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and comprise the CDRs of the antibodies disclosed in Table 2 herein, as determined by the AbM numbering scheme.

[0185] In certain embodiments, the CDRs of the antibodies disclosed herein can be determined according to the AHo numbering system as described in Honegger and Pluckthun, A., J. Mol. Biol. 309:657-670 (2001), which is incorporated herein by reference in its entirety.

[0186] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 1 herein, as determined by the AHo numbering system.

[0187] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises the CDRs of the antigen-binding regions disclosed in Table 2 herein, as determined by the AHo numbering system.

[0188] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 1 herein, and the second antigen-binding region comprises the CDRs of the antigen-binding region disclosed in Table 2 herein, as determined by the AHo numbering system.

[0189] In certain embodiments, the present disclosure provides antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and comprise the CDRs of the antibodies disclosed in Table 2 herein, as determined by the AHo numbering system.

[0190] In certain embodiments, the individual CDRs of the multispecific molecules or antibodies disclosed herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable regions predicted to contact the epitope region of CD96 or TIGIT.

[0191] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions of the VH set forth in SEQ ID NO: 34, 36, or 38, and a VL comprising the amino acid sequences of the CDRL1, CDRL2, and CDRL3 regions of the VL set forth in SEQ ID NO: 35, 37, or 39, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable regions that are predicted to contact the epitope region of CD96 (e.g., human CD96 or cynomolgus CD96).

[0192] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VH comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions of the VH set forth in SEQ ID NO: 40, and a VL comprising the amino acid sequences of the CDRL1, CDRL2, and CDRL3 regions of the VL set forth in SEQ ID NO: 41, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable regions that are predicted to contact the epitope region of TIGIT (e.g., human TIGIT or cynomolgus TIGIT).

[0193] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a VH comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions of the VH set forth in SEQ ID NO: 34, 36, or 38, and a VL comprising the amino acid sequences of the CDRL1, CDRL2, and CDRL3 regions of the VL set forth in SEQ ID NO: 35, 37, or 39; and the second antigen-binding region comprises a VH comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions of the VL set forth in SEQ ID NO: 40. and a VL comprising the amino acid sequences of the CDRH1, CDRH2, and CDRH3 regions of the VL set forth in SEQ ID NO: 41, wherein each CDR is independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of the multispecific molecule, wherein the structural analysis identifies residues in the variable regions that are predicted to contact the epitope regions of CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), respectively.

[0194] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, 16, 17, and 18, or 22, 23, and 24, respectively.

[0195] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, 19, 20, and 21, or 25, 26, and 27, respectively.

[0196] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 10, 11, 12, 13, 14, and 15, 16, 17, 18, 19, 20, and 21, or 22, 23, 24, 25, 26, and 27, respectively.

[0197] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 34, 36, or 38. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 34, 36, or 38. In certain embodiments, the amino acid sequence of the VH consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, and 38.

[0198] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 35, 37, or 39. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 35, 37, or 39. In certain embodiments, the amino acid sequence of the VL consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 35, 37, or 39.

[0199] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 35, 37, or 39. and a VL comprising an amino acid sequence that is 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a VH ...L comprising an amino acid sequence that is 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a VL comprising an amino acid sequence that is 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a VL comprising an amino acid sequence that is 86, 87, 88, 89, In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 37. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 36, and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 37.In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a VH comprising the amino acid sequence of SEQ ID NO: 38 and a VL comprising the amino acid sequence of SEQ ID NO: 39. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 38, and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 39.

[0200] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises the VH and VL amino acid sequences set forth in SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively. In certain embodiments, the VH and VL amino acid sequences consist of amino acid sequences selected from the group consisting of SEQ ID NOs: 34 and 35, 36 and 37, and 38 and 39, respectively.

[0201] In certain embodiments, the present disclosure provides multispecific molecules that cross-compete for binding to CD96 (e.g., human CD96 or cynomolgus CD96) with a multispecific molecule comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively.

[0202] In certain embodiments, the present disclosure provides multispecific molecules that bind to the same or overlapping epitope of CD96 (e.g., an epitope of human CD96 or an epitope of cynomolgus monkey CD96) as the multispecific molecules described herein, e.g., multispecific molecules comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively.

[0203] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the second antigen-binding region comprises a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

[0204] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the second antigen-binding region comprises a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.

[0205] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the second antigen-binding region comprises a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively.

[0206] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 40. In a specific embodiment, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO:40.

[0207] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 41. In a specific embodiment, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO:41.

[0208] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the second antigen-binding region has at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 80%) similarity to the amino acid sequence set forth in SEQ ID NO: 40. and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a VH comprising the amino acid sequence of SEQ ID NO: 40, and a VL comprising the amino acid sequence of SEQ ID NO: 41. In a specific embodiment, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO:40, and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO:41.

[0209] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT, and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises the VH and VL amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively. In certain embodiments, the VH and VL amino acid sequences consist of the amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively.

[0210] In certain embodiments, the present disclosure provides multispecific molecules that cross-compete with a multispecific molecule comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively, for binding to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT).

[0211] In certain embodiments, the present disclosure provides multispecific molecules that bind to the same or overlapping epitope of TIGIT (e.g., an epitope of human TIGIT or an epitope of cynomolgus monkey TIGIT) as the multispecific molecules described herein, e.g., multispecific molecules comprising the VH and VL amino acid sequences shown in SEQ ID NOs: 40 and 41, respectively.

[0212] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding molecule comprises a first VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, 16, 17, and 18, or 22, 23, and 24, respectively, and the second antigen-binding region comprises a second VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

[0213] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding molecule comprises a first VL comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, 19, 20, and 21, or 25, 26, and 27, respectively, and the second antigen-binding region comprises a second VL comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.

[0214] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding molecule comprises a first VH comprising CDRH1, CDRH2, and CDRH3 regions, and a first VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions are and the second antigen-binding region comprises a second VH comprising CDRH1, CDRH2, and CDRH3 regions, and a second VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively.

[0215] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region shares at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 100%) of the amino acid sequence set forth in SEQ ID NO: 34, 36, or 38. and the second antigen-binding region comprises a first VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:40, and the second antigen-binding region comprises a second VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:40. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a first VH comprising the amino acid sequence set forth in SEQ ID NO: 34, 36, or 38, and the second antigen-binding region comprises a second VH comprising the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the amino acid sequence of the first VH consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, and 38, and the amino acid sequence of the second VH consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0216] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region shares at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 100%) of the amino acid sequence set forth in SEQ ID NO: 35, 37, or 39. and the second antigen-binding region comprises a first VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:41, and the second antigen-binding region comprises a second VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:41. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a first VL comprising the amino acid sequence set forth in SEQ ID NO: 35, 37, or 39, and the second antigen-binding region comprises a second VL comprising the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the amino acid sequence of the first VL consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, and 39, and the amino acid sequence of the second VL consists of the amino acid sequence set forth in SEQ ID NO: 41.

[0217] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a first VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 35, 37, or 39. and a first VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 40, and a second antigen-binding region comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 40. , 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:41, and a second VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO:41. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a first VH comprising the amino acid sequence of SEQ ID NO: 34, 36, or 38, and a first VL comprising the amino acid sequence of SEQ ID NO: 35, 37, or 39, and the second antigen-binding region comprises a second VH comprising the amino acid sequence of SEQ ID NO: 40, and a second VL comprising the amino acid sequence of SEQ ID NO: 41.In a specific embodiment, the amino acid sequence of the first VH consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 36, and 38, the amino acid sequence of the first VL consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, and 39, the amino acid sequence of the second VH consists of the amino acid sequence set forth in SEQ ID NO: 40, and the amino acid sequence of the second VL consists of the amino acid sequence set forth in SEQ ID NO: 41.

[0218] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a first VH and a first VL comprising the amino acid sequences set forth in SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively, and the second antigen-binding region comprises a second VH and a second VL comprising the amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively. In certain embodiments, the amino acid sequences of the first VH and the first VL consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 35, 36 and 37, and 38 and 39, respectively, and the amino acid sequences of the second VH and the second VL consist of the amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively.

[0219] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that cross-competes with a multispecific molecule comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively, for binding to CD96 (e.g., human CD96 or cynomolgus CD96), and a second antigen-binding region that cross-competes with a multispecific molecule comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively, for binding to TIGIT (e.g., human TIGIT or cynomolgus TIGIT).

[0220] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that binds to the same or overlapping epitope of CD96 (e.g., an epitope of human CD96 or an epitope of cynomolgus monkey CD96) as a multispecific molecule described herein, e.g., a multispecific molecule comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively, and a second antigen-binding region that binds to the same or overlapping epitope of TIGIT (e.g., an epitope of human TIGIT or an epitope of cynomolgus monkey TIGIT) as a multispecific molecule described herein, e.g., a multispecific molecule comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively.

[0221] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the isolated antibody comprises a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.

[0222] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the isolated antibody comprises a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.

[0223] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the isolated antibody comprises a VH comprising CDRH1, CDRH2, and CDRH3 regions, and a VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise the amino acid sequences set forth in SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively.

[0224] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 40.

[0225] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), comprising a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), comprising a VL comprising the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 41.

[0226] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), comprising a VH comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 40, and a VL comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), comprising a VH comprising the amino acid sequence of SEQ ID NO: 40 and a VL comprising the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the amino acid sequence of the VH consists of the amino acid sequence set forth in SEQ ID NO: 40, and the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 41.

[0227] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), comprising the VH and VL amino acid sequences set forth, respectively, in SEQ ID NOs: 40 and 41. In certain embodiments, the VH and VL amino acid sequences consist of the amino acid sequences set forth, respectively, in SEQ ID NOs: 40 and 41.

[0228] In certain embodiments, the present disclosure provides isolated antibodies that cross-compete for binding to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) with an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 40 and 41, respectively.

[0229] In certain embodiments, the present disclosure provides an isolated antibody that binds to the same or overlapping epitope of TIGIT (e.g., an epitope of human TIGIT or an epitope of cynomolgus monkey TIGIT) as an antibody described herein, for example, an antibody comprising the VH and VL amino acid sequences shown in SEQ ID NOs: 40 and 41, respectively.

[0230] In certain embodiments, the epitopes of a multispecific molecule or antibody may be determined by, for example, NMR spectroscopy, surface plasmon resonance (BIAcore®), X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled to mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen NE (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251:6300-6303, all of which are incorporated herein by reference in their entireties). Polyspecific molecule:antigen or antibody:antigen crystals may be examined using well-known X-ray diffraction techniques and refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; e.g., Meth Enzymol (1985) volumes 114 & 115, eds. Wyckoff HW et al.; U.S. Patent Application No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323, all of which are incorporated herein by reference in their entireties. Mutagenesis mapping studies can be accomplished using any method known to those of skill in the art.For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, e.g., Champe M et al., (1995), supra, and Cunningham BC & Wells JA (1989), supra. In certain embodiments, the epitope of a multispecific molecule or antibody is determined using alanine scanning mutagenesis. Multispecific molecules or antibodies that recognize and bind to the same or overlapping epitopes of CD96 (e.g., human CD96 or cynomolgus CD96) and / or TIGIT (e.g., human TIGIT or cynomolgus TIGIT) can also be identified using routine techniques, such as immunoassays, by demonstrating, for example, the ability of one multispecific molecule or antibody to block the binding of another multispecific molecule or antibody to a target antigen, i.e., by competitive binding assays. Competitive binding assays can also be used to determine whether two multispecific molecules or antibodies have similar binding specificities for an epitope. Competitive binding can be determined in an assay in which the immunoglobulin inhibits specific binding of a reference multispecific molecule or antibody to a common antigen under test, such as CD96 (e.g., human CD96 or cynomolgus CD96) and / or TIGIT (e.g., human TIGIT or cynomolgus TIGIT).Many types of competitive binding assays are known, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct label assay, solid-phase direct label sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid-phase direct biotin-avidin EIA (see Cheung RC et al., (1986) J Immunol 137:3614-9); al., (1990) Virology 176:546-52); and direct labeling RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entireties. Typically, such assays involve the use of purified antigen (e.g., CD96, such as human CD96 or cynomolgus CD96, or TIGIT, such as human TIGIT or cynomolgus TIGIT) bound to a solid surface or cells bearing either of these: an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when a competing multispecific molecule or antibody is present in excess, it will inhibit specific binding of a reference multispecific molecule or antibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. Competitive binding assays can be designed in a number of different formats, using either labeled antigen or labeled multispecific molecules or antibodies.In a typical version of this assay, antigen is immobilized on a 96-well plate, and the ability of unlabeled polyspecific molecules or antibodies to block the binding of labeled antibodies to the antigen is then measured using radioactive or enzyme labels. For further details, see, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M et al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, ed. Harlow E & Lane D editors (supra), pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0231] In certain embodiments, the multispecific molecule inhibits binding of human CD96 to human CD155 (also known as the poliovirus receptor (PVR)). In certain embodiments, binding of human CD96 to human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of human CD96 to human CD155 in the absence of the multispecific molecule.

[0232] In certain embodiments, the multispecific molecule inhibits the binding of a soluble fragment of human CD96 to a soluble fragment of human CD 155. In certain embodiments, binding of the soluble fragment of human CD96 to the soluble fragment of human CD 155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of the soluble fragment of human CD96 to the soluble fragment of human CD 155 in the absence of the multispecific molecule.

[0233] In certain embodiments, the antibody inhibits binding of CD96-expressing cells to a soluble fragment of human CD 155. In certain embodiments, binding of CD96-expressing cells to a soluble fragment of human CD 155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of CD96-expressing cells to a soluble fragment of human CD 155 in the absence of the multispecific molecule.

[0234] In certain embodiments, the multispecific molecule inhibits binding of CD96-expressing cells to cells expressing human CD155. In certain embodiments, binding of CD96-expressing cells to CD155-expressing cells is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of CD96-expressing cells to CD155-expressing cells in the absence of the multispecific molecule.

[0235] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, or 67-99. In certain embodiments, the amino acid sequence of the heavy chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 67-99.

[0236] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6. In certain embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6.

[0237] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a heavy chain and a light chain, wherein the heavy chain and light chain comprise the amino acid sequences of SEQ ID NOs: 1 and 2, 3 and 4, or 5 and 6, respectively. In certain embodiments, the amino acid sequences of the heavy chain and light chain consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, 3 and 4, and 5 and 6, respectively.

[0238] In certain embodiments, the multispecific molecule inhibits binding of human TIGIT to human CD155 (also known as the poliovirus receptor (PVR)). In certain embodiments, binding of human TIGIT to human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of human TIGIT to human CD155 in the absence of the multispecific molecule.

[0239] In certain embodiments, the multispecific molecule inhibits the binding of a soluble fragment of human TIGIT to a soluble fragment of human CD 155. In certain embodiments, binding of the soluble fragment of human TIGIT to the soluble fragment of human CD 155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of the soluble fragment of human TIGIT to the soluble fragment of human CD 155 in the absence of the multispecific molecule.

[0240] In certain embodiments, the multispecific molecule inhibits binding of TIGIT-expressing cells to a soluble fragment of human CD155. In certain embodiments, binding of TIGIT-expressing cells to a soluble fragment of human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of TIGIT-expressing cells to a soluble fragment of human CD155 in the absence of the multispecific molecule.

[0241] In certain embodiments, the multispecific molecule inhibits binding of TIGIT-expressing cells to cells expressing human CD 155. In certain embodiments, binding of TIGIT-expressing cells to CD155-expressing cells is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of TIGIT-expressing cells to CD155-expressing cells in the absence of the multispecific molecule.

[0242] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 7 or 100 to 110. In certain embodiments, the amino acid sequence of the heavy chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 100 to 110.

[0243] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 8 or 9. In certain embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.

[0244] In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a heavy chain and a light chain, wherein the heavy chain and the light chain comprise the amino acid sequences of SEQ ID NOs: 7 and 8, or 7 and 9, respectively. In certain embodiments, the amino acid sequences of the heavy chain and the light chain consist of amino acid sequences selected from the group consisting of SEQ ID NOs: 7 and 8, and 7 and 9, respectively.

[0245] In certain embodiments, the multispecific molecule inhibits the binding of human CD96 and human TIGIT to human CD155 (also known as the poliovirus receptor (PVR)). In certain embodiments, the binding of human CD96 and human TIGIT to human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to the binding of human CD96 and human TIGIT to human CD155 in the absence of the multispecific molecule.

[0246] In certain embodiments, the multispecific molecule inhibits the binding of soluble fragments of human CD96 and human TIGIT to soluble fragments of human CD 155. In certain embodiments, the binding of soluble molecules of human CD96 and soluble fragments of human TIGIT to soluble fragments of human CD 155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to the binding of soluble molecules of human CD96 and soluble fragments of human TIGIT to soluble fragments of human CD 155 in the absence of the multispecific molecule.

[0247] In certain embodiments, the multispecific molecule inhibits binding of cells expressing CD96, TIGIT, or CD96 and TIGIT to a soluble fragment of human CD155. In certain embodiments, binding of cells expressing CD96, TIGIT, or CD96 and TIGIT to a soluble fragment of human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of cells expressing CD96, TIGIT, or CD96 and TIGIT to a soluble fragment of human CD155 in the absence of the multispecific molecule.

[0248] In certain embodiments, the multispecific molecule inhibits binding of cells expressing CD96, TIGIT, or CD96 and TIGIT to cells expressing human CD 155. In certain embodiments, binding of cells expressing CD96, TIGIT, or CD96 and TIGIT to CD155-expressing cells is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the multispecific molecule compared to binding of cells expressing CD96, TIGIT, or CD96 and TIGIT to CD155-expressing cells in the absence of the multispecific molecule.

[0249] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a first heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, or 67-99, and the second antigen-binding region comprises a second heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 7 or 100-110. In certain embodiments, the amino acid sequence of the first heavy chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 67-99, and the amino acid sequence of the second heavy chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 100-110.

[0250] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a first light chain comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6, and the second antigen-binding region comprises a second light chain comprising the amino acid sequence set forth in SEQ ID NO: 8 or 9. In certain embodiments, the amino acid sequence of the first light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, and 6, and the amino acid sequence of the second light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.

[0251] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a first heavy chain and a first light chain, wherein the first heavy chain and the first light chain comprise the amino acid sequences of SEQ ID NOs: 1 and 2, 3 and 4, or 5 and 6, respectively, and the second antigen-binding region comprises a second heavy chain and a second light chain, wherein the second heavy chain and the second light chain comprise the amino acid sequences of SEQ ID NOs: 7 and 8, or 7 and 9, respectively. In certain embodiments, the amino acid sequences of the first heavy chain and the first light chain consist of amino acid sequences selected from the group consisting of SEQ ID NOs: 1 and 2, 3 and 4, and 5 and 6, respectively, and the amino acid sequences of the second heavy chain and the second light chain consist of amino acid sequences selected from the group consisting of SEQ ID NOs: 7 and 8, and 7 and 9, respectively.

[0252] In certain embodiments, the antibody inhibits binding of human TIGIT to human CD155 (also known as poliovirus receptor (PVR)). In certain embodiments, binding of human TIGIT to human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody compared to binding of human TIGIT to human CD155 in the absence of the antibody.

[0253] In certain embodiments, the antibody inhibits the binding of a soluble fragment of human TIGIT to a soluble fragment of human CD155. In certain embodiments, the binding of the soluble fragment of human TIGIT to the soluble fragment of human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody compared to the binding of the soluble fragment of human TIGIT to the soluble fragment of human CD155 in the absence of the antibody.

[0254] In certain embodiments, the antibody inhibits binding of TIGIT-expressing cells to a soluble fragment of human CD155. In certain embodiments, binding of TIGIT-expressing cells to a soluble fragment of human CD155 is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody compared to binding of TIGIT-expressing cells to a soluble fragment of human CD155 in the absence of the antibody.

[0255] In certain embodiments, the antibody inhibits binding of TIGIT-expressing cells to cells expressing human CD155. In certain embodiments, binding of TIGIT-expressing cells to CD155-expressing cells is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% in the presence of the antibody compared to binding of TIGIT-expressing cells to CD155-expressing cells in the absence of the antibody.

[0256] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 107, 108, 109, or 110. In certain embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 107, 108, 109, or 110.

[0257] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 8 or 9. In certain embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9.

[0258] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), comprising a heavy chain and a light chain, wherein the heavy chain and light chain comprise the amino acid sequences of SEQ ID NOs: 107 and 8, 107 and 9, 108 and 8, 108 and 9, 109 and 8, 109 and 9, 110 and 8, or 110 and 9, respectively.

[0259] In certain embodiments, the amino acid sequences of the heavy and light chains consist of the amino acid sequences selected from the group consisting of SEQ ID NOs: 107 and 8, 107 and 9, 108 and 8, 108 and 9, 109 and 8, 109 and 9, 110 and 8, or 110 and 9, respectively.

[0260] In certain embodiments, the multispecific molecules or antibodies disclosed herein are conjugated to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In certain embodiments, a cytotoxic agent is capable of inducing death or destruction of a cell that comes into contact with it. In certain embodiments, a cytostatic agent is capable of preventing or substantially reducing the proliferation and / or inhibiting the activity or function of a cell that comes into contact with it. In certain embodiments, the cytotoxic or cytostatic agent is a chemotherapeutic agent. In certain embodiments, the radionuclide is an isotope 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 Re. In certain embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0261] Any immunoglobulin (Ig) constant region can be used in the multispecific molecules or antibodies disclosed herein. In specific embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0262] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 49 to 60. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96, wherein the first antigen-binding region comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42.

[0263] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the second antigen-binding region comprises a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 49 to 60. In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 43 or 44.

[0264] In certain embodiments, a multispecific molecule comprises a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 50, and the second antigen-binding region comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 56. In certain embodiments, the present disclosure provides a multispecific molecule comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42, and the second antigen-binding region comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 43 or 44.

[0265] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 57, 58, 59, or 60. In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 43 or 44.

[0266] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1)) and / or the hinge region (residues 216-230, numbered according to the EU numbering system) of the multispecific molecules or antibodies described herein to alter one or more functional properties of the multispecific molecules or antibodies, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0267] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of a multispecific molecule or antibody described herein such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region can be altered to, for example, facilitate association of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the multispecific molecule or antibody.

[0268] In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into an IgG constant region, or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc fragment), to alter (e.g., decrease or increase) the half-life of the multispecific molecule or antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated by reference in their entirety. In certain embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region, or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc fragment), to decrease the half-life of the multispecific molecule or antibody in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitutions, insertions, or deletions) are introduced into the IgG constant region, or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc fragment), to increase the half-life of the multispecific molecule or antibody in vivo. In certain embodiments, the multispecific molecule or antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU numbering system. In certain embodiments, the IgG1 constant region of a multispecific molecule or antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU numbering system. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety.Mutant IgGs of this type are referred to as "YTE variants" and have been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same multispecific molecule or antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In certain embodiments, the multispecific molecule or antibody comprises an IgG constant region comprising one, two, three, or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU numbering system.

[0269] In certain embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1), numbered according to the EU numbering system) and / or the hinge region (residues 216-230, numbered according to the EU numbering system) of the multispecific molecules or antibodies described herein, e.g., to increase or decrease the affinity of the multispecific molecule or antibody for an Fc receptor (e.g., an activating Fc receptor) on the surface of an effector cell. Mutations in the Fc region of multispecific molecules or antibodies that decrease or increase the affinity of the multispecific molecule or antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptors of multispecific molecules or antibodies that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, all of which are incorporated by reference herein in their entirety.

[0270] In certain embodiments, the multispecific molecule or antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγRIIB with higher affinity than the wild-type heavy chain constant region binds to FcγRIIB. In certain embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, e.g., a variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more substitutions selected from the group consisting of S267E and L328F, P238D and L328E, P238D and E233D, G237D, H268D, P271G, and A330R, a set of amino acid mutations selected from the group consisting of P238D, E233D, G237D, H268D, P271G, and A330R, G236D and S267E, S239D and S267E, V262E, S267E, and L328F, and V264E, S267E, and L328F, according to the EU numbering system. In certain embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0271] In a further embodiment, one, two, or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function of the multispecific molecule or antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be substituted with a different amino acid residue such that the multispecific molecule or antibody has altered affinity for an effector ligand but retains the antigen-binding ability of the parent multispecific molecule or antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, each of which is incorporated by reference in its entirety. In certain embodiments, deletion or inactivation (through point mutation or other means) of the constant region domain may reduce Fc receptor binding of circulating multispecific molecules or antibodies, thereby increasing tumor localization. See, e.g., U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In certain embodiments, one or more amino acid substitutions may be introduced into the Fc region of a multispecific molecule or antibody described herein to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604, which is incorporated herein by reference in its entirety).In various embodiments, one or more of the following mutations in the constant region of the multispecific molecules or antibodies described herein, numbered according to the EU numbering system, may be made: N297A substitution, N297Q substitution, L234A substitution, L234F substitution, L235A substitution, L235F substitution, L235V substitution, L237A substitution, S239D substitution, E233P substitution, L234V substitution, L235A substitution, C236 deletion, P238A substitution, S239D substitution, F243L substitution, D265A substitution, S267E substitution, L328F substitution, R292P substitution, Y300L substitution, A327Q substitution, P329A substitution, A330L substitution, I332E substitution, or P396L substitution.

[0272] In certain embodiments, a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of a multispecific molecule or antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235A, L237A, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of a multispecific molecule or antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of a multispecific molecule or antibody described herein. In certain embodiments, a mutation selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of a multispecific molecule or antibody described herein. In certain embodiments, a mutation selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of a multispecific molecule or antibody described herein. In certain embodiments, a mutation selected from the group consisting of S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of a multispecific molecule or antibody described herein.

[0273] In certain embodiments, the multispecific molecules or antibodies described herein comprise an IgG1 constant region with an N297Q or N297A amino acid substitution, numbered according to the EU numbering system. In certain embodiments, the multispecific molecules or antibodies described herein comprise an IgG1 constant region with a mutation selected from the group consisting of D265A, P329A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the multispecific molecules or antibodies described herein comprise an IgG1 constant region with an mutation selected from the group consisting of L234A, L235A, and combinations thereof, numbered according to the EU numbering system. In another embodiment, the multispecific molecules or antibodies described herein comprise an IgG1 constant region with an mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof, numbered according to the EU numbering system. In certain embodiments, the amino acid residues in the constant regions of the multispecific molecules or antibodies described herein at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain, numbered according to the EU numbering system, are not L, L, and D, respectively. This approach is described in detail in International Publication WO 14 / 108483, which is incorporated herein by reference in its entirety. In certain embodiments, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain, numbered according to the EU numbering system, are F, E, and A, or A, A, and A, respectively.

[0274] In certain embodiments, one or more amino acids selected from amino acid residues 329, 331, and 322 in the constant region of a multispecific molecule or antibody described herein, numbered according to the EU numbering system, can be substituted with a different amino acid residue such that the multispecific molecule or antibody has altered C1q binding and / or reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In certain embodiments, one or more amino acid residues within amino acid positions 231-238 in the N-terminal region of the CH2 domain of a multispecific molecule or antibody described herein, numbered according to the EU numbering system, are altered, thereby altering the ability of the multispecific molecule or antibody to fix complement. This approach is further described in International Publication WO 94 / 29351, which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region of the multispecific molecules or antibodies described herein is modified to increase the ability of the multispecific molecule or antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to modify the Fc region of the multispecific molecules or antibodies to increase the ability of the multispecific molecules or antibodies to mediate antibody-dependent cellular cytotoxicity (ADCC), and / or to modify the Fc region of the multispecific molecules or antibodies to increase the ability of the multispecific molecules or antibodies to mediate antibody-dependent cellular cytotoxicity (ADCC), and / or to modify the Fc region of the multispecific molecules or antibodies to increase the ability of the multispecific molecules or antibodies to mediate antibody-dependent cellular cytotoxicity (ADCC), and / or to modify the Fc region of the multispecific molecules or antibodies to increase the ability of the multispecific molecules or antibodies to mediate antibody-dependent cellular cytotoxicity (ADCC), and / or to modify the Fc region of the multispecific molecules or antibodies to mediate antibody-dependent ... Mutating (e.g., introducing amino acid substitutions) one or more amino acids at positions 96, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 increases the affinity of the antibody for an Fcγ receptor.This approach is further described in International Publication No. WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0275] In certain embodiments, the multispecific molecules or antibodies described herein comprise a modified IgG1 constant region, where the modification increases the ability of the multispecific molecule or antibody to mediate antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, 0.1, 1, or 10 μg / mL of the multispecific molecule or antibody is capable of inducing cell death of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of CD96-expressing and / or TIGIT-expressing cells within 1, 2, or 3 hours, as assessed by methods described herein and / or known to those of skill in the art. In certain embodiments, the modified IgG1 constant region comprises S239D and I332E substitutions, numbered according to the EU numbering system. In certain embodiments, the modified IgG1 constant region comprises S239D, A330L, and I332E substitutions, numbered according to the EU numbering system. In certain embodiments, the modified IgG1 constant region comprises the following substitutions, numbered according to the EU numbering system: L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the antibody is capable of inducing cell death in effector T cells and Tregs, wherein the percentage of Tregs undergoing cell death is at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold higher than the percentage of effector T cells undergoing cell death.

[0276] In certain embodiments, the multispecific molecules described herein comprise a first and a second heavy chain constant region, wherein the first heavy chain constant region and the second heavy chain constant region comprise different amino acid substitutions.

[0277] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising an S239D mutation, and the second antigen-binding region comprises a heavy chain constant region that does not comprise the S239D, A330L, and I332E mutations.

[0278] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that does not comprise the S239D, A330L, and I332E mutations.

[0279] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that does not comprise the S239D, A330L, and I332E mutations.

[0280] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising an S239D mutation, and the second antigen-binding region comprises a heavy chain constant region comprising an S239D and an I332E mutation.

[0281] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising an S239D mutation, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations.

[0282] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising an S239D mutation.

[0283] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations.

[0284] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising an S239D mutation.

[0285] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations.

[0286] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that does not contain the S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that comprises the S239D mutation.

[0287] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that does not contain the S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that comprises the S239D and I332E mutations.

[0288] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that does not include the S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that includes the S239D, A330L, and I332E mutations.

[0289] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising an S239D mutation, and the second antigen-binding region comprises a heavy chain constant region that does not comprise the S239D, A330L, and I332E mutations.

[0290] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that does not comprise S239D, A330L, and I332E mutations.

[0291] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that does not comprise the S239D, A330L, and I332E mutations.

[0292] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising an S239D mutation, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations.

[0293] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising an S239D mutation, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations.

[0294] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising the S239D mutation.

[0295] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations.

[0296] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising an S239D mutation.

[0297] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and I332E mutations.

[0298] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that does not contain the S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that comprises the S239D mutation.

[0299] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that does not contain the S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that comprises the S239D and I332E mutations.

[0300] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that does not include the S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that includes the S239D, A330L, and I332E mutations.

[0301] In certain embodiments, multispecific molecules that bind to CD96 and / or TIGIT comprise "knob-into-hole" mutations, wherein the multispecific molecules comprise a T366W mutation in the "knob chain" and a T366S, L368A, Y407V mutation in the "hole chain", and optionally an additional interchain disulfide bridge between the CH3 domains, e.g., by introducing a Y349C mutation in the "knob chain" and an E356C or S354C mutation in the "hole chain", a R409D, K370E mutation in the "knob chain" and a D399K, E357K mutation in the "hole chain", a T366W mutation in the "knob chain" and a T366S, L368A, Y407V mutation in the "hole chain", and T366S, L368A, Y407V mutations in the "knob strand" and D399K, E357K mutations in the "hole strand"; Y349C, T366W mutations in one of the strands and E356C, T366S, L368A, Y407V mutations in the corresponding strand; Y349C, T366W mutations in one strand and S354C, T and Y349C, T366W in one chain and S354C, T366S, L368A, Y407V in the corresponding chain (numbering according to the EU numbering system).

[0302] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising a T366W mutation, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations.

[0303] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation.

[0304] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96), and a second antigen-binding region that specifically binds to TIGIT (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising a T366W mutation, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations.

[0305] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation.

[0306] In certain embodiments, the multispecific molecules described herein comprise first and second heavy chain constant regions, wherein the first heavy chain constant region and the second heavy chain constant region comprise knob-into-hole substitutions and further comprise additional amino acid substitutions that differ in the first antigen-binding region and the second antigen-binding region.

[0307] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not S239D, A330L, and I332E mutations.

[0308] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not S239D, A330L, and I332E mutations.

[0309] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not the S239D, A330L, and I332E mutations.

[0310] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations.

[0311] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations.

[0312] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations.

[0313] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations.

[0314] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations.

[0315] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations.

[0316] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, but not S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations.

[0317] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations but not S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations.

[0318] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations but not S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations.

[0319] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, but not S239D, A330L, and I332E mutations.

[0320] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, but not S239D, A330L, and I332E mutations.

[0321] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, but not S239D, A330L, and I332E mutations.

[0322] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations.

[0323] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations.

[0324] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations.

[0325] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations.

[0326] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations.

[0327] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations.

[0328] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that comprises a T366W mutation but does not include S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that comprises S239D, T366S, L368A, and Y407V mutations.

[0329] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that comprises a T366W mutation but does not include S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that comprises S239D, I332E, T366S, L368A, and Y407V mutations.

[0330] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to an antigen other than CD96 (e.g., TIGIT, e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region that comprises a T366W mutation but does not include S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region that comprises S239D, A330L, I332E, T366S, L368A, and Y407V mutations.

[0331] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not S239D, A330L, and I332E mutations.

[0332] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not S239D, A330L, and I332E mutations.

[0333] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not the S239D, A330L, and I332E mutations.

[0334] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations.

[0335] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations.

[0336] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations.

[0337] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations.

[0338] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations.

[0339] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations.

[0340] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations but not S239D, A330L, and I332E, and the second antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations.

[0341] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations but not S239D, A330L, and I332E, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations.

[0342] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations but not S239D, A330L, and I332E, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations.

[0343] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, but not S239D, A330L, and I332E mutations.

[0344] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, but not S239D, A330L, and I332E mutations.

[0345] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising T366S, L368A, and Y407V mutations, but not S239D, A330L, and I332E mutations.

[0346] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations.

[0347] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations.

[0348] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations.

[0349] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations.

[0350] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations.

[0351] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, and T366W mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations.

[0352] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus monkey CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not including S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, T366S, L368A, and Y407V mutations.

[0353] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not including S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, I332E, T366S, L368A, and Y407V mutations.

[0354] In certain embodiments, the present disclosure provides multispecific molecules comprising a first antigen-binding region that specifically binds to an antigen other than TIGIT (e.g., CD96, e.g., human CD96 or cynomolgus CD96) and a second antigen-binding region that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), wherein the first antigen-binding region comprises a heavy chain constant region comprising a T366W mutation but not S239D, A330L, and I332E mutations, and the second antigen-binding region comprises a heavy chain constant region comprising S239D, A330L, I332E, T366S, L368A, and Y407V mutations.

[0355] In certain embodiments, the multispecific molecules or antibodies described herein comprise the constant region of an IgG4 antibody, in which the serine at amino acid residue 228 of the heavy chain, numbered according to the EU numbering system, is substituted with a proline.

[0356] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96), wherein the multispecific molecules comprise a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 49-60.

[0357] In certain embodiments, any of the constant region mutations or modifications described herein may be introduced into one or both of the heavy chain constant regions of the multispecific molecules or antibodies described herein that have two heavy chain constant regions.

[0358] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and function as antagonists (e.g., decrease or inhibit CD96 activity).

[0359] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and reduce or inhibit CD96 (e.g., human CD96 or cynomolgus CD96) activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relative to CD96 (e.g., human CD96 or cynomolgus CD96) activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by methods described herein and / or methods known to one of skill in the art. In certain embodiments, the present disclosure provides a method for specifically binding to CD96 (e.g., human CD96 or cynomolgus CD96) that enhances CD96 (e.g., human CD96 or cynomolgus CD96) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or greater than CD96 (e.g., human CD96 or cynomolgus CD96) activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96)), as assessed by methods described herein and / or methods known to one of skill in the art. fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more. Non-limiting examples of CD96 (e.g., human CD96 or cynomolgus CD96) activity can include CD96 (e.g., human CD96 or cynomolgus CD96) signaling, CD96 (e.g., human CD96 or cynomolgus CD96) binding to its ligand (e.g., CD155) or a fragment and / or fusion protein thereof, activation of T cells (e.g., T cells expressing human CD96), activation of natural killer (NK) cells, reduction or inhibition of Tregs, increased cytokine (e.g., IL-2) production, and increased activity of CD155 (e.g., human CD155).In certain embodiments, increased CD96 (eg, human CD96 or cynomolgus CD96) activity is assessed as described in the Examples.

[0360] In certain embodiments, the present disclosure provides a method for the detection of CD96 (e.g., human CD96 or cynomolgus CD96) that specifically binds to its ligand (e.g., CD155) or fragment and / or fusion protein, as compared to CD96 (e.g., human CD96 or cynomolgus CD96) binding to this ligand without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by methods described herein and / or methods known to one of skill in the art. In one embodiment, multispecific molecules are provided that reduce or inhibit CD96 or cynomolgus CD96) activity by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In certain embodiments, the present disclosure provides antibodies that specifically bind CD96 (e.g., human CD96 or cynomolgus CD96) and enhance CD96 (e.g., human CD96 or cynomolgus CD96) binding to its ligand (e.g., CD155 (e.g., human or cynomolgus CD155) or a fragment and / or fusion protein thereof) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, or greater than ... Multispecific molecules are provided that increase the antibody binding by 20-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0361] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and activate T cells (e.g., T cells expressing human CD96). In certain embodiments, the T cells are memory T cells. In certain embodiments, the T cells are primary CD3-expressing T cells. In certain embodiments, the T cells are CD96-expressing Jurkat cells. In certain embodiments, the multispecific molecules disclosed herein increase the activity of nuclear factor of activated T cells (NFAT) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to NFAT activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase NFAT activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to NFAT activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)). In certain embodiments, the multispecific molecules increase NFAT activity in the presence of a ligand for CD96 (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells (e.g., monocytes or dendritic cells) expressing a ligand for CD96.

[0362] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and increase cytokine production (e.g., IL-2) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relative to cytokine production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and increase cytokine production (e.g., IL-2) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more relative to cytokine production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the multispecific molecules increase cytokine production (e.g., IL-2) in the presence of a ligand for CD96 (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells (e.g., monocytes or dendritic cells) expressing a ligand for CD96. In certain embodiments, the multispecific molecules increase IL-2 production compared to IL-2 production without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)).

[0363] In certain embodiments, the present disclosure provides a method for the treatment of staphylococcal enterotoxin-1 (PD-1)-associated IFNγ and / or IL-2 production by a multispecific antibody that specifically binds to CD96 (e.g., human CD96 or cynomolgus CD96), alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), compared to IFNγ and / or IL-2 production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by a method described herein or a method known to one of skill in the art. In one embodiment, multispecific molecules are provided that increase IFNγ and / or IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to SEQ ID NO: 1A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0364] In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A (SEA) in the presence of a multispecific molecule described herein that specifically binds CD96 (e.g., human CD96 or cynomolgus monkey CD96) show a significant increase in the number of cells expressing the multispecific molecule without any multispecific molecule or an unrelated multispecific molecule (e.g., CD96 (e.g., human CD96 or cynomolgus monkey CD96) as assessed by the methods described herein or by methods known to one of skill in the art). In some embodiments, the multispecific molecule (multispecific molecule that does not specifically bind to monkey CD96) increases IFNγ and / or IL-2 production by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to IFNγ and / or IL-2 production from PBMCs stimulated with SEA alone.

[0365] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and increase or promote memory recall of memory T cells. In certain embodiments, the memory T cells are CD8 effector memory T cells. In certain embodiments, the memory T cells are CD4 effector memory T cells. In certain embodiments, the multispecific molecules increase the number of memory T cells that proliferate when memory T cells are contacted with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to the number of memory T cells that proliferate when memory T cells are contacted with their cognate antigen in the absence of any multispecific molecule or in the presence of an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecule increases cytokine (e.g., IFNγ, TNFα) production by memory T cells upon contact with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to cytokine production by memory T cells upon contact with their cognate antigen in the absence of any multispecific molecule or in the presence of an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by methods described herein or known to one of skill in the art.

[0366] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and activate NK cells. In certain embodiments, the NK cells are isolated. In certain embodiments, the NK cells are in a mixed culture of PBMCs. In certain embodiments, the multispecific molecules disclosed herein increase the level of CD107a expression in NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the level of CD107a expression in NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase the level of CD107a expression in NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to the level of CD107a expression in NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by methods described herein or known to one of skill in the art.In certain embodiments, the multispecific molecules disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96)), as assessed by methods described herein or known to one of skill in the art.

[0367] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to TIGIT (eg, human TIGIT or cynomolgus TIGIT) and function as antagonists (eg, decrease or inhibit TIGIT activity).

[0368] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and reduce or inhibit TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by methods described herein and / or methods known to one of skill in the art. In certain embodiments, the present disclosure provides a method for specifically binding to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT)), as assessed by the methods described herein and / or methods known to one of skill in the art. In some embodiments, multispecific molecules are provided that reduce or inhibit TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more compared to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) activity. Non-limiting examples of TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity may include TIGIT (e.g., human TIGIT or cynomolgus TIGIT) signaling, TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to its ligand (e.g., CD155) or a fragment and / or fusion protein thereof, activation of T cells (e.g., T cells expressing human TIGIT), activation of natural killer (NK) cells, reduction or inhibition of Tregs, increased cytokine (e.g., IL-2) production, and increased activity of CD155 (e.g., human CD155).In certain embodiments, an increase in TIGIT (eg, human TIGIT or cynomolgus TIGIT) activity is assessed as described in the Examples.

[0369] In certain embodiments, the present disclosure provides a method for the treatment of TIGIT (e.g., human TIGIT or cynomolgus TIGIT) that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and enhances TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to its ligand (e.g., CD155) or a fragment and / or fusion protein thereof by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more, as assessed by a method described herein or a method known to one of skill in the art, compared to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to this ligand without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and reduce or inhibit TIGIT (e.g., human TIGIT) binding to its ligand (e.g., C) by 90%, 95%, 98%, or 99%, as compared to TIGIT (e.g., human TIGIT) binding to this ligand without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. Provided are multispecific molecules that increase TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to CD155 (e.g., human or cynomolgus CD155) or fragments and / or fusion proteins thereof) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0370] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and activate T cells (e.g., T cells expressing human TIGIT). In certain embodiments, the T cells are memory T cells. In certain embodiments, the T cells are primary CD3-expressing T cells. In certain embodiments, the T cells are TIGIT-expressing Jurkat cells. In certain embodiments, the multispecific molecules disclosed herein increase the activity of nuclear factor of activated T cells (NFAT) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the NFAT activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase the activity of NFAT by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, as assessed by methods described herein or known to one of skill in the art, relative to the NFAT activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain embodiments, the multispecific molecule increases NFAT activity in the presence of a ligand of TIGIT (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells expressing a ligand of TIGIT (e.g., monocytes or dendritic cells).

[0371] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increase cytokine production (e.g., IL-2) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to cytokine production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increase cytokine production (IL-2) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to cytokine production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the multispecific molecules increase cytokine production (e.g., IL-2) in the presence of a ligand for TIGIT (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells (e.g., monocytes or dendritic cells) expressing a ligand for TIGIT. In certain embodiments, the multispecific molecules increase IL-2 production relative to IL-2 production without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).

[0372] In certain embodiments, the present disclosure provides a method for the treatment of staphylococcal enterotoxin-1 (STI)-associated inflammatory bowel disease (IEHD) by a multispecific antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT), alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), compared to IFNγ and / or IL-2 production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. The present invention provides multispecific molecules that increase IFNγ and / or IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to SEQ ID NO: 1A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0373] In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA) in the presence of a multispecific molecule described herein that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) are stimulated with SEA without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). IFNγ and / or IL-2 production was increased by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein or known to one of skill in the art compared to IFNγ and / or IL-2 production from stimulated PBMCs alone.

[0374] In certain embodiments, the present disclosure provides molecules that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increase or promote memory recall of memory T cells. In certain embodiments, the memory T cells are CD8 effector memory T cells. In certain embodiments, the memory T cells are CD4 effector memory T cells. In certain embodiments, the multispecific molecules increase the number of proliferating memory T cells when the memory T cells are contacted with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to the number of proliferating memory T cells when the memory T cells are contacted with their cognate antigen in the absence of any multispecific molecule or in the presence of an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecule increases cytokine (e.g., IFNγ, TNFα) production by memory T cells when they are contacted with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein or known to one of skill in the art, compared to cytokine production by memory T cells when they are contacted with their cognate antigen in the absence of any multispecific molecule or in the presence of an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).

[0375] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and activate NK cells. In certain embodiments, the NK cells are isolated. In certain embodiments, the NK cells are in a mixed culture of PBMCs. In certain embodiments, the multispecific molecules disclosed herein increase the expression level of CD107a in NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the expression level of CD107a in NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase the expression level of CD107a in NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to the expression level of CD107a in NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by methods described herein or known to one of skill in the art.In certain embodiments, the multispecific molecules disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by methods described herein or known to one of skill in the art.

[0376] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and function as antagonists (e.g., reduce or inhibit CD96 and TIGIT activity).

[0377] In certain embodiments, the present disclosure provides a method for the production of CD96 (e.g., human CD96 or cynomolgus monkey CD96) that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) or TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. Multispecific molecules are provided that reduce or inhibit CD96 (e.g., human CD96 or cynomolgus CD96) and TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to human CD96 or cynomolgus CD96) and TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity. In certain embodiments, the present disclosure provides a method for the production of CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) that specifically binds to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96) or TIGIT (e.g., human TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. Multispecific molecules are provided that reduce or inhibit CD96 (e.g., human CD96 or cynomolgus CD96) and TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more relative to T (e.g., human TIGIT or cynomolgus TIGIT) activity.Non-limiting examples of CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) activities may include CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) signaling, CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) binding to its ligand (e.g., CD155) or a fragment and / or fusion protein thereof, activation of T cells (e.g., T cells expressing human CD96 and / or TIGIT), activation of natural killer (NK) cells, reduction or inhibition of Tregs, increased cytokine (e.g., IL-2) production, and increased activity of CD155 (e.g., human CD155). In certain embodiments, increases in CD96 (eg, human CD96 or cynomolgus CD96) and TIGIT (eg, human TIGIT or cynomolgus TIGIT) activity are assessed as described in the Examples.

[0378] In certain embodiments, the present disclosure provides a method for the detection of CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) by immunohistochemistry, in which the target polypeptide is specifically bound to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and the target polypeptide is specifically bound to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) to its ligand without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96 or cynomolgus monkey CD96) or TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. and a fusion protein thereof. The present invention provides multispecific molecules that reduce or inhibit binding of CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) to its ligand (e.g., CD155) or fragment and / or fusion protein thereof by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) binding.In certain embodiments, the present disclosure provides antibodies that specifically bind CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) to their ligands without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind CD96 (e.g., human CD96 or cynomolgus monkey CD96) or TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. and TIGIT) to its ligand (e.g., CD155 (e.g., human or cynomolgus CD155) or fragments and / or fusion proteins thereof) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0379] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) and activate T cells (e.g., T cells expressing human CD96 and / or human TIGIT). In certain embodiments, the T cells are memory T cells. In certain embodiments, the T cells are primary CD3-expressing T cells. In certain embodiments, the T cells are CD96-expressing and / or TIGIT-expressing Jurkat cells. In certain embodiments, the multispecific molecules disclosed herein increase the activity of nuclear factor of activated T cells (NFAT) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to NFAT activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase the activity of NFAT by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to the NFAT activity without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecule increases NFAT activity in the presence of a ligand for CD96 and / or TIGIT (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells expressing a ligand for CD96 and / or TIGIT (e.g., monocytes or dendritic cells).

[0380] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increase cytokine production (e.g., IL-2) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relative to cytokine production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the present disclosure provides antibodies that specifically bind to CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) without any multispecific molecule or with an unrelated multispecific molecule (e.g., CD96 (e.g., human CD96 or cynomolgus monkey CD96) or TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), as assessed by a method described herein or a method known to one of skill in the art. Provided are multispecific molecules that increase cytokine production (e.g., IL-2) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to cytokine production with a multispecific molecule that does not specifically bind to cynomolgus monkey TIGIT. In certain embodiments, the multispecific molecule increases cytokine production (e.g., IL-2) in the presence of a ligand for CD96 and / or TIGIT (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells (e.g., monocytes or dendritic cells) expressing a ligand for CD96 and / or TIGIT.In certain embodiments, the multispecific molecules increase the production of IL-2 compared to IL-2 production without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).

[0381] In certain embodiments, the present disclosure provides a method for treating staphylococcal enterotoxin-dependent leukemia (LEL)-associated leukemia (LEK ...), which specifically binds CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), compared to IFNγ and / or IL-2 production without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind CD96 (e.g., human CD96 or cynomolgus monkey CD96) or TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In one embodiment, multispecific molecules are provided that increase IFNγ and / or IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to SEQ ID NO: 1A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0382] In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcal enterotoxin A (SEA) in the presence of a multispecific molecule described herein that specifically binds CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) show a significant increase in the number of cells stimulated with Staphylococcus enterotoxin A (SEA) in the presence of a multispecific molecule described herein that specifically binds CD96 (e.g., human CD96 or cynomolgus monkey CD96) and TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT), as assessed by methods described herein or known to one of skill in the art, without any multispecific molecule or in the presence of an unrelated multispecific molecule (e.g., CD96 (e.g., human CD96 or cynomolgus monkey TIGIT)). In some embodiments, the IFNγ and / or IL-2 production is increased by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to IFNγ and / or IL-2 production from PBMCs stimulated with SEA alone (e.g., a polyspecific molecule that does not specifically bind to human TIGIT or cynomolgus TIGIT) or SEA (e.g., a polyspecific molecule that does not specifically bind to human TIGIT or cynomolgus TIGIT).

[0383] In certain embodiments, the present disclosure provides molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increase or promote memory recall of memory T cells. In certain embodiments, the memory T cells are CD8 effector memory T cells. In certain embodiments, the memory T cells are CD4 effector memory T cells. In certain embodiments, the multispecific molecules increase the number of proliferating memory T cells when contacted with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold compared to the number of proliferating memory T cells when contacted with their cognate antigen in the absence of any multispecific molecule or in the presence of an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecule is selected from those that exhibit a potent inhibitory effect on memory T cells when they contact their cognate antigen in the absence of any multispecific molecule or in the presence of an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. and increasing cytokine (e.g., IFNγ, TNFα) production from memory T cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold relative to cytokine production from memory T cells upon contact with their cognate antigen.

[0384] In certain embodiments, the present disclosure provides multispecific molecules that specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) and TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and activate NK cells. In certain embodiments, the NK cells are isolated. In certain embodiments, the NK cells are in a mixed culture of PBMCs. In certain embodiments, the multispecific molecules disclosed herein increase the expression level of CD107a in NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the expression level of CD107a in NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., an antibody that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein increase the expression level of CD107a in NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to the expression level of CD107a in NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by methods described herein or known to one of skill in the art.In certain embodiments, the multispecific molecules disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein or known to one of skill in the art. In certain embodiments, the multispecific molecules disclosed herein are capable of inhibiting cytokine production from NK cells without any multispecific molecule or with an unrelated multispecific molecule (e.g., a multispecific molecule that does not specifically bind to CD96 (e.g., human CD96 or cynomolgus CD96) or TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein or by methods known to one of skill in the art. The present invention provides an anti-inflammatory drug that increases cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells.

[0385] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to TIGIT (eg, human TIGIT or cynomolgus TIGIT) and function as antagonists (eg, reduce or inhibit TIGIT activity).

[0386] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and reduce or inhibit TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein and / or methods known to those skilled in the art, compared to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain embodiments, the present disclosure provides a method for specifically binding to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by the methods described herein and / or methods known to those skilled in the art. In some embodiments, the present invention provides isolated antibodies that reduce or inhibit TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more compared to TIGIT (e.g., human TIGIT or cynomolgus monkey TIGIT) activity. Non-limiting examples of TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity may include TIGIT (e.g., human TIGIT or cynomolgus TIGIT) signaling, TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to its ligand (e.g., CD155) or a fragment and / or fusion protein thereof, activation of T cells (e.g., T cells expressing human TIGIT), activation of natural killer (NK) cells, reduction or inhibition of Tregs, increased cytokine (e.g., IL-2) production, and increased activity of CD155 (e.g., human CD155).In certain embodiments, an increase in TIGIT (eg, human TIGIT or cynomolgus TIGIT) activity is assessed as described in the Examples.

[0387] In certain embodiments, the present disclosure provides an antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and enhances TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to its ligand (e.g., CD155 or a fragment and / or fusion protein thereof) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more compared to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to this ligand without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and reduce or inhibit TIGIT (e.g., human TIGIT) binding to its ligand (e.g., CD15) by 90%, 95%, 98%, or 99%, as compared to TIGIT (e.g., human TIGIT) binding to this ligand without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. and (c) increasing TIGIT (e.g., human TIGIT or cynomolgus TIGIT) binding to a target protein (e.g., human or cynomolgus CD155) or a fragment and / or fusion protein thereof) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0388] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and activates a T cell (e.g., a T cell expressing human TIGIT). In certain embodiments, the T cell is a memory T cell. In certain embodiments, the T cell is a primary CD3-expressing T cell. In certain embodiments, the T cell is a TIGIT-expressing Jurkat cell. In certain embodiments, the antibodies disclosed herein increase nuclear factor of activated T cells (NFAT) activity by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to NFAT activity without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the antibodies disclosed herein increase NFAT activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, as assessed by methods described herein or known to one of skill in the art, compared to NFAT activity without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain embodiments, the antibody increases NFAT activity in the presence of a ligand of TIGIT (e.g., CD155) or a fragment and / or fusion protein thereof, and / or a cell (e.g., monocyte or dendritic cell) expressing a ligand of TIGIT.

[0389] In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increase cytokine production (e.g., IL-2) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to cytokine production without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the present disclosure provides isolated antibodies that specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increase cytokine production (IL-2) by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to cytokine production without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the antibody increases cytokine production (e.g., IL-2) in the presence of a TIGIT ligand (e.g., CD155) or a fragment and / or fusion protein thereof, and / or cells (e.g., monocytes or dendritic cells) expressing the TIGIT ligand. In certain embodiments, the antibody increases IL-2 production compared to IL-2 production without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).

[0390] In certain embodiments, the present disclosure provides a method for the treatment of staphylococcal enterotoxin-dependent inflammatory bowel disease (IEDH) by an antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and, alone or in combination with an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), reduces IFNγ and / or IL-2 production compared to IFNγ and / or IL-2 production without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. Provided are isolated antibodies that increase IFNγ and / or IL-2 production in human peripheral blood mononuclear cells (PBMCs) in response to SEQ ID NO: 1A (SEA) stimulation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

[0391] In certain embodiments, human peripheral blood mononuclear cells (PBMCs) stimulated with Staphylococcus enterotoxin A (SEA) in the presence of an antibody described herein that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) are compared with PBMCs stimulated with SEA alone without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). IFNγ and / or IL-2 production is increased by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold as assessed by methods described herein or known to one of skill in the art compared to IFNγ and / or IL-2 production from PBMCs.

[0392] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and increases or promotes memory recall of memory T cells. In certain embodiments, the memory T cells are CD8 effector memory T cells. In certain embodiments, the memory T cells are CD4 effector memory T cells. In certain embodiments, the antibodies increase the number of proliferating memory T cells when the memory T cells are contacted with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by a method described herein or a method known to one of skill in the art, compared to the number of proliferating memory T cells when the memory T cells are contacted with their cognate antigen in the absence of any antibody or in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)). In certain embodiments, the antibody increases cytokine (e.g., IFNγ, TNFα) production by memory T cells when they are contacted with their cognate antigen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by a method described herein or a method known to one of skill in the art, compared to cytokine production by memory T cells when they are contacted with their cognate antigen in the absence of any antibody or in the presence of an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)).

[0393] In certain embodiments, the present disclosure provides an isolated antibody that specifically binds to TIGIT (e.g., human TIGIT or cynomolgus TIGIT) and activates NK cells. In certain embodiments, the NK cells are isolated. In certain embodiments, the NK cells are in a mixed culture of PBMCs. In certain embodiments, the antibodies disclosed herein increase the expression level of CD107a in NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to the expression level of CD107a in NK cells without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the antibodies disclosed herein increase the expression level of CD107a in NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more, compared to the expression level of CD107a in NK cells without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art.In certain embodiments, the antibodies disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art. In certain embodiments, the antibodies disclosed herein increase cytokine production (e.g., IFNγ and / or TNFα) from NK cells by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more compared to cytokine production (e.g., IFNγ and / or TNFα) from NK cells without any antibody or with an unrelated antibody (e.g., an antibody that does not specifically bind to TIGIT (e.g., human TIGIT or cynomolgus TIGIT)), as assessed by a method described herein or a method known to one of skill in the art.

[0394] 7.3 Pharmaceutical Compositions Provided herein are compositions comprising anti-CD96 (e.g., human CD96 or cynomolgus CD96) and / or anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) multispecific molecules or isolated anti-TIGIT antibodies disclosed herein, having a desired degree of purity, in a physiologically acceptable carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium 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 (less than about 10 residues) polypeptides, serum albumin, gelatin or proteins such as immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0395] In certain embodiments, the pharmaceutical composition comprises an anti-CD96 (e.g., human CD96 or cynomolgus CD96) and / or anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) multispecific molecule or isolated anti-TIGIT antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of an anti-CD96 (e.g., human CD96 or cynomolgus CD96) and / or anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) multispecific molecule or isolated anti-TIGIT antibody disclosed herein, and optionally one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In certain embodiments, the multispecific molecule or antibody is the only active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein increase or promote CD96 (e.g., human CD96 or cynomolgus CD96) and / or TIGIT (e.g., human TIGIT or cynomolgus TIGIT) activity and may be useful in treating conditions such as cancer or infectious diseases. In a specific embodiment, the present invention relates to a pharmaceutical composition of the present invention comprising an anti-CD96 (e.g., human CD96 or cynomolgus CD96) and / or anti-TIGIT (e.g., human TIGIT or cynomolgus TIGIT) multispecific molecule or isolated anti-TIGIT antibody of the present invention for use as a medicament. In another embodiment, the present invention relates to a pharmaceutical composition of the present invention for use in a method for the treatment of cancer or an infectious disease.

[0396] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous solvents, non-aqueous solvents, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous solvents include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous parenteral solvents include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral preparations packaged in multidose containers, including phenol or cresol, mercuric, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium disulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestrants or chelating agents include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible solvents, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0397] Pharmaceutical compositions can be formulated for any r...

Claims

1. A multispecific molecule comprising: (a) a first antigen-binding region that specifically binds to human CD96, the first antigen-binding region comprising a first VH comprising CDRs CDRH1, CDRH2, and CDRH3, and a first VL comprising CDRs CDRL1, CDRL2, and CDRL3; (i) the first VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 38, and the first VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 39; (ii) the first VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 34, and the first VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 35; or (iii) a first antigen-binding region, wherein the first VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 36, and the first VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 37; and (b) a second antigen-binding region that specifically binds to an antigen other than human CD96, said second antigen-binding region comprising a second VH comprising CDRs CDRH1, CDRH2, and CDRH3, and a second VL comprising CDRs CDRL1, CDRL2, and CDRL3.

2. (a) the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the first antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 10, 11, 12, 13, 14, and 15, 16, 17, 18, 19, 20, and 21, or 22, 23, 24, 25, 26, and 27, respectively; (b) the first VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 34, 36, or 38; and / or the first VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 35, 37, or 39; (c) the first VH amino acid sequence comprises the amino acid sequence of SEQ ID NO: 34, 36, or 38; and / or the first VL amino acid sequence comprises the amino acid sequence of SEQ ID NO: 35, 37, or 39; or (d) the first VH and the first VL comprise the amino acid sequences of SEQ ID NOs: 34 and 35, 36 and 37, or 38 and 39, respectively.

3. The multispecific molecule of claim 1 or 2, wherein the second antigen-binding region specifically binds to human TIGIT, and optionally the second VH comprises CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 40, and the second VL comprises CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO:

41.

4. (a) the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of the second antigen-binding region comprise the amino acid sequences of SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively; (b) the second VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40; and / or the second VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41; (c) the amino acid sequence of the second VH comprises the amino acid sequence of SEQ ID NO: 40; and / or the amino acid sequence of the second VL comprises the amino acid sequence of SEQ ID NO: 41; or (d) the second VH and the second VL comprise the amino acid sequences of SEQ ID NOs: 40 and 41, respectively.

5. (a) the first and / or second antigen-binding region comprises a heavy chain constant region, the heavy chain constant region comprising: (i) human IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ , and IgA ２ Selected from the group consisting of: (ii) IgG １ A heavy chain constant region; (iii) comprises any one of the amino acid sequences of SEQ ID NOs: 49-60; (iv) an IgG containing the N297A mutation numbered according to the EU numbering system; １ A heavy chain constant region; (v) comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a higher affinity than the wild-type heavy chain constant region binds to the FcγR, optionally wherein the FcγR is FcγRIIB or FcγRIIIA; (vi) an IgG comprising the S267E and L328F mutations numbered according to the EU numbering system; １ a heavy chain constant region; and / or (vii) an IgG comprising at least one mutation selected from the group consisting of S239D, A330L, and I332E mutations numbered according to the EU numbering system; １ a heavy chain constant region; and / or (b) the first and / or second antigen-binding region comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44.

6. (a) the first antigen-binding region comprises a first heavy chain constant region comprising an aspartic acid at amino acid position 239, an aspartic acid and a glutamic acid at amino acid positions 239 and 332, respectively, or an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively, and the second antigen-binding region comprises a second heavy chain constant region that does not comprise an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively; (b) the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 58 and 57, 59 and 57, or 60 and 57, respectively; (c) the first antigen-binding region comprises a first heavy chain constant region comprising aspartic acid and glutamic acid at amino acid positions 239 and 332, respectively, or aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively, and the second antigen-binding region comprises a second heavy chain constant region comprising an aspartic acid at amino acid position 239; (d) the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 59 and 58, or 60 and 58, respectively; (e) the first heavy chain constant region comprises an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively, and the second heavy chain constant region further comprises a glutamic acid at amino acid position 332; (f) the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 60 and 59, respectively; (g) the first antigen-binding region comprises a first heavy chain constant region that does not contain aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively, and the second antigen-binding region comprises a second heavy chain constant region that comprises an aspartic acid at amino acid position 239, an aspartic acid and a glutamic acid at amino acid positions 239 and 332, respectively, or an aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively; (h) the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 57 and 60, 57 and 59, or 57 and 58, respectively; (i) the first antigen-binding region comprises a first heavy chain constant region comprising an aspartic acid at amino acid position 239, and the second antigen-binding region comprises a second heavy chain constant region comprising an aspartic acid and a glutamic acid at amino acid positions 239 and 332, respectively, or an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively; (j) the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 58 and 60, or 58 and 59, respectively; (k) the first heavy chain constant region comprises an aspartic acid at amino acid position 239 and a glutamic acid at amino acid position 332, and the second heavy chain constant region comprises an aspartic acid, a leucine, and a glutamic acid at amino acid positions 239, 330, and 332, respectively; (l) the first heavy chain constant region and the second heavy chain constant region comprise SEQ ID NOs: 59 and 60, respectively; (m) the first antigen-binding region comprises a first heavy chain constant region comprising a tryptophan at amino acid position 366, and the second antigen-binding region comprises a second heavy chain constant region comprising a serine, an alanine, and a valine at amino acid positions 366, 368, and 407, respectively; (n) the first heavy chain constant region comprises SEQ ID NO: 53, 54, 55, or 56, and the second heavy chain constant region comprises SEQ ID NO: 49, 50, 51, or 52; (o) the first antigen-binding region comprises a first heavy chain constant region comprising serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, and the second antigen-binding region comprises a second heavy chain constant region comprising tryptophan at amino acid position 366; or (p) the first heavy chain constant region comprises SEQ ID NO: 49, 50, 51, or 52, and the second heavy chain constant region comprises SEQ ID NO: 53, 54, 55, or 56; The multispecific molecule of claim 5 , wherein the amino acid positions are numbered according to the EU numbering system.

7. (a) the first antigen-binding region comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 1, 3, 5, or 67-99; and / or the first light chain comprises the amino acid sequence of SEQ ID NO: 2, 4, or 6; (b) the second antigen-binding region comprises a second heavy chain comprising the amino acid sequence of SEQ ID NO: 7 or 100-110; and / or the second antigen-binding region comprises a second light chain comprising the amino acid sequence of SEQ ID NO: 8 or 9; (c) the first heavy chain and the first light chain comprise the amino acid sequences of SEQ ID NOs: 1 and 2, 3 and 4, or 5 and 6, respectively, and / or the second heavy chain and the second light chain comprise the amino acid sequences of SEQ ID NOs: 7 and 8, or 7 and 9, respectively; or (d) the first heavy chain and the first light chain comprise the amino acid sequences of SEQ ID NOs: 5 and 6, respectively, and the second heavy chain and the second light chain comprise the amino acid sequences of SEQ ID NOs: 7 and 9, respectively.

8. 1. A multispecific molecule comprising a first antigen-binding region that specifically binds to human CD96 and a second antigen-binding region that specifically binds to human TIGIT: (a) the first antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively, and the second antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, or glutamic acid at amino acid positions 239, 330, and 332, respectively; (b) the first antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively, and the second antigen-binding region comprises aspartic acid, serine, alanine, and valine at amino acid positions 239, 366, 368, and 407, respectively; (c) the first antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively, and the second antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively; (d) the first antigen-binding region comprises aspartic acid, serine, alanine, and valine at amino acid positions 239, 366, 368, and 407, respectively, and the second antigen-binding region comprises aspartic acid, leucine, glutamic acid, and tryptophan at amino acid positions 239, 330, 332, and 366, respectively; (e) the first antigen-binding region comprises aspartic acid, glutamic acid, and tryptophan at amino acid positions 239, 332, and 366, respectively, and the second antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, or glutamic acid at amino acid positions 239, 330, and 332, respectively; (f) the first antigen-binding region comprises serine, alanine, and valine at amino acid positions 366, 368, and 407, respectively, but does not comprise aspartic acid, leucine, and glutamic acid at amino acid positions 239, 330, and 332, respectively, and the second antigen-binding region comprises aspartic acid, glutamic acid, and tryptophan at amino acid positions 239, 332, and 366, respectively; A multispecific molecule, wherein said amino acid positions are numbered according to the EU numbering system.

9. An isolated antibody that specifically binds to human TIGIT, the antibody comprising a VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 40, and a VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO:

41.

10. (a) the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of SEQ ID NOs: 28, 29, 30, 31, 32, and 33, respectively; (b) the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 40; and / or the VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 41; and / or (c) the VH and VL comprise the amino acid sequences of SEQ ID NOs: 40 and 41, respectively.

11. (a) the antibody comprises a heavy chain constant region, the heavy chain constant region comprising: (i) human IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ , and IgA ２ Selected from the group consisting of: (ii) IgG １ A heavy chain constant region; (iii) comprises the amino acid sequence of SEQ ID NO: 57, 58, 59, or 60; (iv) an IgG containing the N297A mutation numbered according to the EU numbering system; １ A heavy chain constant region; (v) a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a higher affinity than the wild-type heavy chain constant region binds to the FcγR, optionally wherein the FcγR is FcγRIIB or FcγRIIIA; (vi) an IgG comprising the S267E and L328F mutations numbered according to the EU numbering system; １ a heavy chain constant region; and / or (vii) an IgG comprising at least one mutation selected from the group consisting of S239D, A330L, and I332E mutations numbered according to the EU numbering system; １ a heavy chain constant region; and / or (b) the antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 43 or 44.

12. (a) the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 or 100-110; and / or a light chain comprising the amino acid sequence of SEQ ID NO: 8 or 9; (b) the antibody comprises heavy and light chains comprising the amino acid sequences of SEQ ID NOs: 7 and 9, 7 and 8, 107 and 8, 107 and 9, 108 and 8, 108 and 9, 109 and 8, 109 and 9, 110 and 8, or 110 and 9, respectively; or (c) The isolated antibody of any one of claims 9 to 11, wherein the antibody comprises a heavy chain and a light chain comprising the amino acid sequences of SEQ ID NOs: 7 and 9, respectively.

13. 13. The multispecific molecule or isolated antibody of any one of claims 1 to 12, wherein the multispecific molecule or isolated antibody is conjugated to an antibody, a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label.

14. 1. An isolated polynucleotide comprising: (a) the VH, VL, heavy chain and / or light chain of the multispecific molecule of any one of claims 1 to 8 or the isolated antibody of any one of claims 9 to 12, (b) the first VH and the first VL of the multispecific molecule of any one of claims 1 to 8; (c) the second VH and the second VL of the multispecific molecule of any one of claims 1 to 8, (d) the first heavy chain and the first light chain of the multispecific molecule of any one of claims 1 to 8, (e) an isolated polynucleotide encoding the second heavy chain and the second light chain of the multispecific molecule of any one of claims 1 to 8.

15. A vector comprising the polynucleotide of claim 14.

16. 1. A recombinant host cell comprising: (a) the polynucleotide of claim 14, (b) the vector according to claim 15 ; (c) a first polynucleotide encoding the VH and VL of a first antigen-binding region according to any one of claims 1 to 8, and a second polynucleotide encoding the VH and VL of a second antigen-binding region according to any one of claims 1 to 8; (d) a first vector comprising a first polynucleotide encoding the VH and VL of a first antigen-binding region according to any one of claims 1 to 8, and a second vector comprising a second polynucleotide encoding the VH and VL of a second antigen-binding region according to any one of claims 1 to 8; (e) a first polynucleotide encoding the VH of the first antigen-binding region according to any one of claims 1 to 8, a second polynucleotide encoding the VL of the first antigen-binding region according to any one of claims 1 to 8, a third polynucleotide encoding the VH of the second antigen-binding region according to any one of claims 1 to 8, and a fourth polynucleotide encoding the VL of the second antigen-binding region according to any one of claims 1 to 8; (f) a first vector comprising a first polynucleotide encoding the VH of the first antigen-binding region according to any one of claims 1 to 8, a second vector comprising a second polynucleotide encoding the VL of the first antigen-binding region according to any one of claims 1 to 8, a third vector comprising a third polynucleotide encoding the VH of the second antigen-binding region according to any one of claims 1 to 8, and a fourth vector comprising a fourth polynucleotide encoding the VL of the second antigen-binding region according to any one of claims 1 to 8; (g) a first polynucleotide encoding the heavy and light chains of a first antigen-binding region according to any one of claims 1 to 8, and a second polynucleotide encoding the heavy and light chains of a second antigen-binding region according to any one of claims 1 to 8; (h) a first vector comprising a first polynucleotide encoding the heavy and light chains of a first antigen-binding region according to any one of claims 1 to 8, and a second vector comprising a second polynucleotide encoding the heavy and light chains of a second antigen-binding region according to any one of claims 1 to 8; (i) a first polynucleotide encoding a heavy chain of a first antigen-binding region according to any one of claims 1 to 8, a second polynucleotide encoding a light chain of the first antigen-binding region according to any one of claims 1 to 8, a third polynucleotide encoding a heavy chain of a second antigen-binding region according to any one of claims 1 to 8, and a fourth polynucleotide encoding a light chain of the second antigen-binding region according to any one of claims 1 to 8; (j) a first vector comprising a first polynucleotide encoding the heavy chain of the first antigen-binding region according to any one of claims 1 to 8, a second vector comprising a second polynucleotide encoding the light chain of the first antigen-binding region according to any one of claims 1 to 8, a third vector comprising a third polynucleotide encoding the heavy chain of the second antigen-binding region according to any one of claims 1 to 8, and a fourth vector comprising a fourth polynucleotide encoding the light chain of the second antigen-binding region according to any one of claims 1 to 8; or (k) a polynucleotide encoding the VH and VL, or the heavy and light chains, of the isolated antibody according to any one of claims 9 to 12; (l) a vector comprising a polynucleotide encoding the VH and VL, or the heavy and light chains, of the isolated antibody according to any one of claims 9 to 12; (m) a first polynucleotide encoding the VH or heavy chain of the isolated antibody of any one of claims 9 to 12, and a second polynucleotide encoding the VL or light chain of the isolated antibody of any one of claims 9 to 12; or (n) A recombinant host cell comprising a first vector comprising a first polynucleotide encoding the VH or heavy chain of the isolated antibody of any one of claims 9 to 12, and a second vector comprising a second polynucleotide encoding the VL or light chain of the isolated antibody of any one of claims 9 to 12.

17. A pharmaceutical composition comprising a multispecific molecule according to any one of claims 1 to 8, an isolated antibody according to any one of claims 9 to 12, a polynucleotide according to claim 14, a vector according to claim 15, or a host cell according to claim 16, and a pharma- ceutical acceptable carrier or excipient.

18. 20. A method for producing a multispecific molecule or an isolated antibody, the method comprising culturing a host cell described in claim 16 under suitable conditions such that a polynucleotide is expressed and the multispecific molecule or isolated antibody is produced.

19. (i) a method of enhancing an immune response in a subject; (ii) a method of treating cancer in a subject; or (iii) Methods of Treating an Infectious Disease in a Subject 18. The multispecific molecule of any one of claims 1 to 8, the isolated antibody of any one of claims 9 to 12, the polynucleotide of claim 14, the vector of claim 15, the host cell of claim 16, or the pharmaceutical composition of claim 17 for use in a method comprising administering to the subject an effective amount of the multispecific molecule, the isolated antibody, the polynucleotide, the vector, the host cell, or the pharmaceutical composition.

20. (a) the multispecific molecule, isolated antibody, polynucleotide, vector, host cell, or pharmaceutical composition is administered systemically, intravenously, subcutaneously, or intratumorally, or delivered to a tumor-draining lymph node; (b) the method further comprises administering to the subject an additional therapeutic agent, (i) is a chemotherapeutic agent; (ii) optionally a checkpoint targeting agent selected from the group consisting of an antagonist anti-PD-1 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, an antagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-VISTA antibody, an antagonist anti-TIGIT antibody, an antagonist anti-CEACAM1 antibody, an antagonist anti-CD96 antibody, an agonist anti-GITR antibody, and an agonist anti-OX40 antibody; (iii) optionally an inhibitor of indoleamine-2,3-dioxygenase (IDO) selected from the group consisting of epacadostat, F001287, indoximod, and NLG919; or (iv) optionally a vaccine comprising a heat shock protein peptide complex (HSPPC) comprising a heat shock protein complexed with an antigenic peptide; 20. The multispecific molecule, the isolated antibody, the polynucleotide, the vector, the host cell, or the pharmaceutical composition of claim 19.

Images