isolated antibody, pharmaceutical composition, bispecific antibody, antibody-drug conjugate, isolate

专利摘要:
isolated antibodies or antigen binding portions are provided that bind to the human tigit (t-cell immunoreceptor with ig and itim domains). in some embodiments, the antibody or its antigen-binding portion has a binding affinity (kd) for human tigit of less than 5 nm. in some embodiments, the anti-tigit antibody blocks the binding of cd155 and / or cd112 to tigit.
公开号:BR112019017550A2
申请号:R112019017550
申请日:2018-02-28
公开日:2020-04-14
发明作者:Prinz Bianka；Beers Courtney；C Piasecki Julia；Peterson Scott
申请人:Seattle Genetics Inc；
IPC主号:

专利说明:

ISOLATED ANTIBODY, PHARMACEUTICAL COMPOSITION, BIESPECIFIC ANTIBODY, ANTIBODY-DRUG CONJUGATE, ISOLATED POLYNUCLEOTIDE, VECTOR, HOST CELL, METHODS OF PRODUCTION OF AN ANTIBODY AND TREATMENT OF A KIT, IN A KIT, AND A KIT, IN A KIT
BACKGROUND OF THE INVENTION [001] This application claims priority to the Patent Application
Provisional US No. 62 / 464,529, filed on February 28, 2017, and US Provisional Patent Application No. 62 / 616,779, filed on January 12, 2018, the content of each of which is incorporated by reference in this document in its entirety.
BACKGROUND OF THE INVENTION [002] TIGIT (“T-cell immunoreceptor with Ig and
ITIM ”) is an immunoreceptor that is expressed in subgroups of T cells, such as activated, memory and regulatory T cells and natural killer cells (NK). TIGIT is a member of the CD28 family of the Ig superfamily of proteins, and serves as a co-inhibitory molecule that limits the proliferation and activation of Tea cells and the function of NK cells. TIGIT mediates its immunosuppressive effect when competing with CD226 (also known as Accessory Molecule DNAX-1 or “DNAM-1”) for the same set of ligands: CD 155 (also known as poliovirus receptor or “PVR”) and CD 112 (also known as related to poliovirus 2 receptor or “PVRL2”). See, Levin et al., Eur. J. Immunol., 2011, 41: 902-915. Because the affinity of CD 155 for TIGIT is greater than its affinity for CD226, in the presence of TIGIT, CD226 signaling is inhibited, thus limiting the proliferation and activation of T cells.
[003] In melanoma patients, TIGIT expression is upregulated in specific CD8 ⁺ tumor antigen (TA) T cells and tumor infiltrating CD8 ⁺ lymphocytes (TILs). Blocking TIGIT in the
Petition 870190101574, of 10/09/2019, p. 7/108 m3 presence of cells expressing TIGIT ligand (CD 155) increased the proliferation, cytokine production and degranulation of specific CD8 ⁺ TA and TILs CD8 ⁺ cells See, Chauvin et al., J Clin Invest., 2015 , 125: 20462058. Thus, TIGIT represents a potential therapeutic target to stimulate anti-tumor T cell responses in patients, although there is still a need for better methods of blocking TIGIT and promoting antitumor responses.
BRIEF SUMMARY OF THE INVENTION [004] In one aspect, isolated antibodies or their binding portions are provided to antigens that bind to human TIGIT (T-cell immunoreceptor with Ig and ITIM domains). In some embodiments, the antibody or its antigen-binding portion has a binding affinity (Kd) for human TIGIT of less than 5 nM. In some embodiments, the antibody or its antigen-binding portion has a Kd by human TIGIT of less than 1 nM. In some embodiments, the antibody or its antigen-binding portion has a Kd by human TIGIT of less than 100 pM.
[005] In some embodiments, the antibody or its antigen-binding portion exhibits cross-reactivity with monkey cynomolgus and / or mouse TIGIT. In some embodiments, the antibody or its antigen-binding portion exhibits cross-reactivity with both the TIGIT of the cynomolgus monkey and the TIGIT of the mouse.
[006] In some embodiments, the antibody or its antigen-binding portion blocks the binding of CD 155 to TIGIT. In some embodiments, the antibody or its antigen-binding portion blocks the binding of CD 112 to TIGIT. In some embodiments, the antibody or its antigen-binding portion blocks the binding of both CD 155 and CD112 to TIGIT.
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3/123 [007] In some embodiments, the antibody or its antigen-binding portion binds to an epitope in human TIGIT that comprises amino acid positions 81 and 82. In some embodiments, the epitope comprises Phe at position 81 and / or Lys or Ser at position 82. In some embodiments, the epitope comprises Phe81 and Lys82.
[008] In some embodiments, the epitope is a discontinuous epitope.
[009] In some embodiments, the antibody or its antigen-binding portion binds to an epitope in human TIGIT that further comprises one or more of the amino acid positions 51, 52, 53, 54, 55, 73, 74, 75, 76, 77, 79, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93. In some embodiments, the epitope further comprises one or more amino acid residues selected from the group consisting of Thr51, Ala52, Gln53, Val54, Thr55, Leu73, Gly74, Trp75, His76, Ile77, Pro79, Asp83, Arg84, Val85, Ala86, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93. In some embodiments, the epitope comprises the amino acid residues Thr51, Ala52, Gln53, Val54, Thr55, Gly74, Trp75, His76, Ile77, Phe81, Lys82, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93. In some embodiments, the epitope comprises amino acid residues Ala52, Gln53, Leu73, Gly74, Trp75, Pro79, Phe81, Lys82, Asp83, Arg84, Val85 and Ala86. In some embodiments, the epitope comprises the sequence ICNADLGWHISPSFK (SEQ ID NO: 258).
[0010] In some embodiments, the antibody or its antigen-binding portion comprises one or more sequences listed in Table 3 below. In some embodiments, the antibody or its antigen-binding portion comprises one or more of:
(a) a heavy chain CDR1 comprising the sequence of any of SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 76, SEQ ID NO: 94 , SEQ ID NO: 112, SEQ ID NO: 130,
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SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 239, or SEQ ID NO: 243;
(b) a heavy chain CDR2 comprising the sequence of any of SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 42, SEQ ID NO: 60, SEQ ID NO: 78, SEQ ID NO: 96 , SEQ ID NO: 114, SEQ ID NO: 132, SEQ ID NO: 150, SEQ ID NO: 168, SEQ ID NO: 186, SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 238, or SEQ ID NO: 240;
(c) a heavy chain CDR3 comprising the sequence of any of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80, SEQ ID NO: 98 , SEQ ID NO: 116, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 170, SEQ ID NO: 188, SEQ ID NO: 206, SEQ ID NO: 223, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 244;
(d) a light chain CDR1 comprising the sequence of any of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 85, SEQ ID NO: 103 , SEQ ID NO: 121, SEQ ID NO: 139, SEQ ID NO: 157, SEQ ID NO: 175, SEQ ID NO: 193, or SEQ ID NO: 211;
(e) a light chain CDR2 comprising the sequence of any of SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 87, SEQ ID NO: 105 , SEQ ID NO: 123, SEQ ID NO: 141, SEQ ID NO: 159, SEQ ID NO: 177, SEQ ID NO: 195, or SEQ ID NO: 213; or (f) a light chain CDR3 comprising the sequence of any of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, SEQ ID NO: 89, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 143, SEQ ID NO: 161, SEQ ID NO: 179, SEQ ID NO: 197, or SEQ ID NO: 215.
[0011] In some embodiments, the antibody or its portion of
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5/123 antigen binding comprises a heavy chain of CDR1, CDR2 and CDR3 and a light chain of CDR1, CDR2 and CDR3 comprising the sequences of:
(a) SEQ ID NOs: 4, 6, 8, 13, 15 and 17, respectively; or (b) SEQ ID NOs: 22, 24, 26, 31, 33 and 35, respectively; or (c) SEQ ID NOs: 40, 42, 44, 49, 51 and 53, respectively; or (d) SEQ ID NOs: 58, 60, 62, 67, 69 and 71, respectively; or (e) SEQ ID NOs: 76, 78, 80, 85, 87 and 89, respectively; or (f) SEQ ID NOs: 94, 96, 98, 103, 105 and 107, respectively;
or (g) SEQ ID NOs: 112, 114, 116, 121, 123 and 125, respectively; or (h) SEQ ID NOs: 130, 132, 134, 139, 141 and 143, respectively; or (i) SEQ ID NOs: 148, 150, 152, 157, 159 and 161, respectively; or (j) SEQ ID NOs: 166, 168, 170, 175, 177 and 179, respectively; or (k) SEQ ID NOs: 184, 186, 188, 193, 195 and 197, respectively; or (l) SEQ ID NOs: 202, 204, 206, 211, 213 and 215, respectively; or (m) SEQ ID NOs: 221, 222, 223, 13, 15 and 17, respectively; or (n) SEQ ID NOs: 224, 225, 62, 67, 69 and 71, respectively; or (o) SEQ ID NOs: 226, 227, 228, 67, 69 and 71, respectively; or (p) SEQ ID NOs: 224, 229, 230, 67, 69 and 71, respectively;
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6/123 or (q) SEQ ID NOs: 224, 227, 230, 67, 69 and 71, respectively;
or
(r) SEQ ID We: 231, 232, 235, 103, 105 and 107, respectively; or(s) SEQ ID We: 233, 234, 236, 103, 105 and 107, respectively; or(t) SEQ ID We: 233, 234, 237, 103, 105 and 107, respectively; or(u) SEQ ID We: 166, 238, 170, 175, 177 and 179, respectively; or(v) SEQ ID We: 239, 240, 170, 175, 177 and 179, respectively; or(w) SEQ ID We: 239, 240, 241, 175, 177 and 179, respectively; or(x) SEQ ID We: 239, 240, 242, 175, 177 and 179, respectively; or(y) SEQ ID We: 243, 168, 244, 175, 177 and 179,
respectively.
[0012] In some embodiments, the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQ ID
NO: 145, SEQ ID NO: 163, SEQ ID NO: 181, SEQ ID NO: 199, SEQID
NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQID
NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQID
NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID
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NO: 257; and / or (b) a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208 .
[0013] In some embodiments, the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 245 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10; or (b) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 28; or (c) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 37 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46; or (d) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 55, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO : 248, or SEQ ID NO: 249 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 64; or
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8/123 (e) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 73 and a light chain variable region comprising an amino acid sequence that has at least 90 % sequence identity to SEQ ID NO: 82; or (f) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 91, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 100; or (g) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 109 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 118; or (h) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 136; or (i) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 145 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 154; or (j) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 163, SEQ ID NO: 253, SEQ ID
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NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 172 ; or (k) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 181 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 190; or (l) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 199 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 208.
[0014] In another aspect, antibodies or their antigen-binding portions are provided that bind to human TIGIT, wherein the antibody or its antigen-binding portion binds to an epitope in human TIGIT that comprises amino acid positions 81 and 82. In some embodiments, the epitope comprises Phe at position 81 and / or Lys or Ser at position 82. In some embodiments, the epitope comprises Phe81 and Lys82.
[0015] In some embodiments, the epitope is a discontinuous epitope.
[0016] In some embodiments, the antibody or its antigen-binding portion binds to an epitope in human TIGIT that further comprises one or more of the amino acid positions 51, 52, 53, 54, 55, 73, 74, 75, 76, 77, 79, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93. In some embodiments, the epitope further comprises one or more amino acid residues selected from the group consisting of Thr51, Ala52, Gln53, Val54, Thr55, Leu73, Gly74, Trp75, His76, Ile77, Pro79, Asp83, Arg84, Val85, Ala86, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93. In
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10/123 In some embodiments, the epitope comprises amino acid residues Thr51, Ala52, Gln53, Val54, Thr55, Gly74, Trp75, His76, Ile77, Phe81, Lys82, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93. In some embodiments, the epitope comprises amino acid residues Ala52, Gln53, Leu73, Gly74, Trp75, Pro79, Phe81, Lys82, Asp83, Arg84, Val85 and Ala86. In some embodiments, the epitope comprises the sequence ICNADLGWHISPSFK (SEQ ID NO: 258).
[0017] In yet another aspect, antibodies or their antigen binding portions are provided comprising one or more sequences disclosed in this document (for example, one or more sequences listed in Table 3 below). In some embodiments, the antibody or its antigen-binding portion comprises one or more sequences of the framework region, CDR, variable region of the heavy chain, variable region of the light chain as disclosed in this document (for example, as listed in Table 3 below) . In some embodiments, the antibody or its antigen-binding portion comprises one or more of:
(a) a heavy chain CDR1 comprising the sequence of any of SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 76, SEQ ID NO: 94 , SEQ ID NO: 112, SEQ ID NO: 130, SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 239, or SEQ ID NO: 243;
(b) a heavy chain CDR2 comprising the sequence of any of SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 42, SEQ ID NO: 60, SEQ ID NO: 78, SEQ ID NO: 96 , SEQ ID NO: 114, SEQ ID NO: 132, SEQ ID NO: 150, SEQ ID NO: 168, SEQ ID NO: 186, SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 238, or SEQ ID NO: 240;
(c) a heavy chain CDR3 comprising the sequence
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11/123 of any of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80, SEQ ID NO: 98, SEQ ID NO: 116, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 170, SEQ ID NO: 188, SEQ ID NO: 206, SEQ ID NO: 223, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO : 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 244;
(d) a light chain CDR1 comprising the sequence of any of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 85, SEQ ID NO: 103 , SEQ ID NO: 121, SEQ ID NO: 139, SEQ ID NO: 157, SEQ ID NO: 175, SEQ ID NO: 193, or SEQ ID NO: 211;
(e) a light chain CDR2 comprising the sequence of any of SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 87, SEQ ID NO: 105 , SEQ ID NO: 123, SEQ ID NO: 141, SEQ ID NO: 159, SEQ ID NO: 177, SEQ ID NO: 195, or SEQ ID NO: 213; or (f) a light chain CDR3 comprising the sequence of any of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, SEQ ID NO: 89, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 143, SEQ ID NO: 161, SEQ ID NO: 179, SEQ ID NO: 197, or SEQ ID NO: 215. [0018] In some embodiments, the antibody or its antigen-binding portion comprises a heavy chain of CDR1, CDR2 and CDR3 and a light chain of CDR1, CDR2 and CDR3 comprising the sequences of:
(a) SEQ ID NOs: 4, 6, 8, 13, 15 and 17, respectively; or (b) SEQ ID NOs: 22, 24, 26, 31, 33 and 35, respectively; or (c) SEQ ID NOs: 40, 42, 44, 49, 51 and 53, respectively; or (d) SEQ ID NOs: 58, 60, 62, 67, 69 and 71, respectively; or (e) SEQ ID NOs: 76, 78, 80, 85, 87 and 89, respectively; or (f) SEQ ID NOs: 94, 96, 98, 103, 105 and 107, respectively;
or
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(g) SEQ ID We: 112, 114, 116, 121, 123 and 125, respectively; or(h) SEQ ID We: 130, 132, 134, 139, 141 and 143, respectively; or(i) SEQ ID We: 148, 150, 152, 157, 159 and 161, respectively; or(j) SEQ ID We: 166, 168, 170, 175, 177 and 179, respectively; or(k) SEQ ID We: 184, 186, 188, 193, 195 and 197, respectively; or(D SEQ ID We: 202, 204, 206, 211, 213 and 215,
respectively; or (m) SEQ ID NOs: 221, 222, 223, 13, 15 and 17, respectively; or (n) SEQ ID NOs: 224, 225, 62, 67, 69 and 71, respectively; or (o) SEQ ID NOs: 226, 227, 228, 67, 69 and 71, respectively; or (p) SEQ ID NOs: 224, 229, 230, 67, 69 and 71, respectively; or (q) SEQ ID NOs: 224, 227, 230, 67, 69 and 71, respectively;
or
O') SEQ ID We: 231, 232, 235, 103, 105 and 107, respectively; or(s) SEQ ID We: 233, 234, 236, 103, 105 and 107, respectively; or(t) SEQ ID We: 233, 234, 237, 103, 105 and 107, respectively; or(u) SEQ ID We: 166, 238, 170, 175, 177 and 179,
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13/123 respectively; or
(v) SEQ ID We: 239, 240, 170, 175, 177 and 179, respectively; or(w) SEQ ID We: 239, 240, 241, 175, 177 and 179, respectively; or(x) SEQ ID We: 239, 240, 242, 175, 177 and 179, respectively; or(y) SEQ ID We: 243, 168, 244, 175, 177 and 179,
respectively.
[0019] In some embodiments, the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQID
NO: 145, SEQ ID NO: 163, SEQ ID NO: 181, SEQ ID NO: 199, SEQID
NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQID
NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQID
NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257; and / or (b) a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208 .
[0020] In some embodiments, the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90%
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14/123 sequence to SEQ ID NO: 1 or SEQ ID NO: 245 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10; or (b) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 28; or (c) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 37 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46; or (d) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 55, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO : 248, or SEQ ID NO: 249 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 64; or (e) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 73 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 82; or (f) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 91, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252 and a light chain variable region comprising an amino acid sequence that is at least 90%
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15/123 sequence identity to SEQ ID NO: 100; or (g) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 109 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 118; or (h) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 136; or (i) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 145 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 154; or (j) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 163, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO : 255, SEQ ID NO: 256, or SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 172; or (k) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 181 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 190; or (l) a heavy chain variable region comprising an amino acid sequence that has at least 90%
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16/123 sequence to SEQ ID NO: 199 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 208.
[0021] In some embodiments, an antibody or its antigen-binding portion as disclosed in this document exhibits synergy with an anti-PD-1 antibody or an anti-PD-Ll antibody.
[0022] In some embodiments, an antibody or its antigen-binding portion as disclosed in this document is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an entirely human antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antigen-binding fragment is a Fab, an F (ab ') 2, a scFv or a divalent scFv.
[0023] In another aspect, pharmaceutical compositions are provided comprising an isolated antibody or its antigen binding portion as described herein and a pharmaceutically acceptable carrier.
[0024] In yet another aspect, bispecific antibodies are provided comprising an isolated antibody or its antigen binding portion, as described herein.
[0025] In yet another aspect, anti-drug conjugates are provided comprising an isolated antibody or its antigen binding portion, as described in this document.
[0026] In yet another aspect, isolated polynucleotides are provided. In some embodiments, the polynucleotide comprises one or more nucleotide sequences that encode an antibody or its antigen-binding portion, as described herein. In some embodiments, the polynucleotide comprises one or more nucleotide sequences that encode a polypeptide disclosed in Table 3 below.
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In some embodiments, the polynucleotide comprises one or more nucleotide sequences that encode an antibody or its antigen-binding portion, which binds to human TIGIT, in which the isolated polynucleotide comprises:
(a) the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 56, SEQ ID NO: 74, SEQ ID NO: 92, SEQ ID NO: 110, SEQ ID NO: 128, SEQ ID NO: 146, SEQ ID NO: 164, SEQ ID NO: 182, or SEQ ID NO: 200; and / or (b) the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 29, SEQ ID NO: 47, SEQ ID NO: 65, SEQ ID NO: 83, SEQ ID NO: 101, SEQ ID NO: 119, SEQ ID NO: 137, SEQ ID NO: 155, SEQ ID NO: 173, SEQ ID NO: 191, or SEQ ID NO: 209.
[0027] In yet another aspect, vectors and host cells comprising a polynucleotide as described in this document are provided. In another aspect, methods of producing an antibody comprising culturing a host cell as described herein are provided under conditions suitable for producing the antibody.
[0028] In another aspect, kits are provided (for example, for use in a therapeutic method as described in this document). In some embodiments, the kit comprises an isolated anti-TIGIT antibody or its antigen binding portion, as described herein, or a pharmaceutical composition comprising an anti-TIGIT antibody or its antigen binding portion as described herein; and further comprises an immuno-oncological agent. In some embodiments, the immuno-oncological agent is an inhibitor of the PD-1 pathway. In some embodiments, the inhibitor of the PD-1 pathway is an anti-PD-1 antibody or an anti-PDL1 antibody. In some embodiments, the PD-1 pathway inhibitor is an antagonist or inhibitor of a T-cell co-inhibitor. In some embodiments, the immune-cancer agent is an agonist of a T-cell co-activator.
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18/123 modalities, the immuno-oncological agent is an immunostimulatory cytokine. [0029] In another aspect, methods of treating cancer in a subject are provided. In some embodiments, the method comprises administering to the subject a therapeutic amount of an isolated antibody or its antigen-binding portion as described herein, or a pharmaceutical composition as described herein, a bispecific antibody as described herein, or an anti-drug conjugate as described in this document.
[0030] In some modalities, cancer is a cancer that is enriched for expression of CD112 or CD115. In some modalities, cancer is a cancer that is enriched for T cells or natural killer cells (NK) that express TIGIT. In some embodiments, the cancer is bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer , colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma , leukemia, myeloma, or sarcoma. In certain modalities, cancer is a lymphoma or leukemia.
[0031] In some embodiments, the method further comprises administering to the subject a therapeutic amount of an immunooncological agent. In some embodiments, the immuno-cancer agent is an inhibitor of the PD-1 pathway. In some embodiments, the inhibitor of the PD-1 pathway is an anti-PD-1 antibody or an anti-PD-Ll antibody. In some embodiments, the PD-1 pathway inhibitor is an antagonist or inhibitor of a T cell co-inhibitor. In some embodiments, the immunooncological agent is an agonist of a T cell co-activator. In some embodiments, the immuno-cancer agent is an immunostimulatory cytokine.
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In some embodiments, the isolated antibody, the pharmaceutical composition, the bispecific antibody or the antibody-drug conjugate are administered simultaneously with the immuno-cancer agent. In some embodiments, the isolated antibody, the pharmaceutical composition, the bispecific antibody or the antibody-drug conjugate are administered sequentially to the immuno-cancer agent.
BRIEF DESCRIPTION OF THE FIGURES [0032] FIG. 1. Binding of 65 clones of anti-TIGIT antibodies and an irrelevant isotype control antibody with HEK 293 cells engineered to express human TIGIT (upper panel), cynomolgus monkey TIGIT (middle panel) and mouse TIGIT (lower panel).
[0033] FIG. 2. Binding of 65 clones of anti-TIGIT antibodies and an irrelevant isotype control antibody with primary human T cells (upper panel), cynomolgus monkey T cells (central panel) and mouse T cells (lower panel). For the lower panel, 35 of the 65 clones were evaluated. Of the 35 clones evaluated, 5 of the 35 did not bind to the mTIGIT-Fc protein (clones 20, 27, 55, 56 and 60), as indicated by the light green bars.
[0034] FIG. 3A-3D. (AC) Binding titration values of eight anti-TIGIT antibody clones (clones 2, 5, 13, 16, 17, 20, 25 and 54) for human TIGIT (A), mouse (B) and cynomolgus monkey (C) expressed in HEK 293 cells. Results are shown for single sample wells. (D) EC50 values of eight anti-TIGIT antibody clones (clones 2, 5, 13, 16, 17, 20, 25 and 54) for human, mouse and cynomolgus monkey TIGIT expressed in HEK 293 cells.
[0035] FIG. 4. Titration binding of clones 13 and 25 of the anti-TIGIT antibody to mouse activated splenic T cells. Results are shown for single sample wells. Clone 13 had an EC50 of 0.24 pg / ml. Clone 25 had an EC50 of 2.28 pg / ml.
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20/123 [0036] FIG. 5A-5B. Anti-TIGIT antibodies blocked the CD 155 interaction with TIGIT expressed in HEK 293 cells, for both binding of human CD 155 to HEK 293 cells expressing human TIGIT (A) and mouse CD 155 binding to HEK 293 cells expressing mouse TIGIT ( B). Results are shown for single sample wells.
[0037] FIG. 6. Anti-TIGIT antibodies blocked the interaction of human CD 112 with human TIGIT expressed in HEK 293 cells. Results are shown for single sample wells.
[0038] FIG. 7A-7B. (A) Top panel: Selected anti-TIGIT antibodies effectively blocked TIGIT-CD155 adhesion, resulting in T cell activation, as measured by a> 1.5-fold induction in luciferase activity. About 12 clones showed induction of> 1.5 times in the bioassay. Two clones did not block the TIGIT-CD155 interaction in the ForteBio assay (pink bars). The multiplicative induction was measured under no Ab control. Mean and standard deviation (SD) are from duplicate experiments; the antibodies were at 20 pg / ml. Gray bar = hlgGl isotype control. Black bar = no antibody control (defined as baseline). Bottom panel: bioassay correlation graph with TIGIT / CD155 block versus affinity with TIGIT-Fc. Activity in the bioassay correlated with affinity for the recombinant protein. (B) Dose response of 12 anti-TIGIT clones selected in the TIGIT / CD155 blocking bioassay. Clones 13 and 25, which showed strong links to all three species, showed good activity in the bioassay. Average and SD are from triplicate sample wells.
[0039] FIG. 8. Selection of anti-TIGIT antibodies synergized with anti-PD-1, resulting in the activation of T cells. Mean and SD are from wells of triplicate samples. Both clone 13 and clone 25 showed synergy with anti-PD-1 in combined bioassays.
[0040] FIG. 9A-9H. (A-D) Titration connection (A-C) and values of
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EC50 (D) for binding to human TIGIT (A), mouse (B) and monkey cynomolgus (C) expressed in HEK 293 cells for fully human anti-TIGIT clone 13 (cl3 hlgGl ”) and IgGl chimeras of the clone 13 from mouse (“cl3 mlgGl”) and mouse IgG2a (“cl3 mIgG2a”). Mean and SD are from duplicate sample wells. (EF) Antibodies cl3 hlgGl, cl3 mlgGl, and cl3 mIgG2a blocked the interaction of CD155 with TIGIT expressed in HEK 293 cells, for both binding of human CD155 to HEK 293 cells expressing human TIGIT (E) and mouse CD 155 binding to cells HEK 293 expressing mouse TIGIT (F). The results are for single sample wells. (G) Cl3 hlgGl, cl3 mlgGl and cl3 m! GG2a antibodies blocked the interaction of human CD112 with human TIGIT expressed in HEK 293 cells. The results are for single sample wells. (H) Dose response of clones of parental and chimeric anti-TIGIT antibodies cl313 hlgGl, cl3 mlgGl and cl3 m! GG2a in TIGIT / CD155 blocking bioassay. Average and SD are from triplicate sample wells. [0041] FIG. 10A-10K. Anti-TIGIT antibodies that can adhere to the activation of Fcgamma receptors mediated antitumor efficacy in a mouse CT26 syngenic tumor model. (A) Average tumor volume of the group. (BK) Tumor volume of the individual animal for groups 1 to 10. PR = Partial response (the tumor volume is 50% or less of its volume on day 1 for three consecutive measurements and equal to or greater than 13.5 mm ³ for one or more of these three measurements). CR = Complete Response (tumor volume is less than 13.5 mm ³ for three consecutive measurements).
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction [0042] As described in this document, antibodies with high affinity for human TIGIT (T-cell immunoreceptor with the Ig and ITIM domains) have been identified, and also have cross-reactivity with one or both mouse TIGIT and monkey cynomolgus TIGIT , what
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22/123 inhibit the interaction between TIGIT and CD 155. These antibodies also exhibit synergy with anti-PD-1 antibodies. Thus, the anti-TIGIT antibodies described in this document can be used in a number of therapeutic applications, such as for the treatment of various cancers, either as a single agent or in combination with another therapeutic agent such as anti-PD-1 agents or anti-PD-Ll agents.
[0043] Therefore, in one aspect, the present invention provides compositions, kits and methods of treatment comprising an antibody or antigen-binding portion of an antibody, which binds to human TIGIT.
II. Definitions [0044] Unless otherwise defined, the technical and scientific terms used in this document have the same meaning as commonly understood by a person skilled in the art. See, for example, Lackie, Dictionary of Cell and Molecular Biology, Elsevier (2007 4th ed.); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described in this document can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain frequently used terms in this document and are not intended to limit the scope of this disclosure.
[0045] As used in this document, the singular forms one (a), and the (a) include the referring plural forms, unless the content clearly indicates otherwise. Thus, for example, reference to an antibody optionally includes a combination of two or more of such molecules, and the like.
[0046] The term about, as used in this document, refers to the usual margin of error for the respective value readily known by the person skilled in this technical field.
Petition 870190101574, of 10/09/2019, p. 28/108 / 123 [0047] As used in this document, the term TIGIT refers to T cell immunoreceptor with Ig and ITIM domains. The protein encoded by the TIGIT gene is a member of the CD28 family of the Ig superfamily of proteins. TIGIT is expressed in several classes of T cells and in natural killer cells (NK) and mediates its immunosuppressive effect competing with CD226 for the CD155 and CD112 ligands. See, Levin et al., Eur. J. Immunol., 2011, 41: 902-915. TIGIT is also referred to in the art as WUCAM (Cell Adhesion Molecule at the University of Washington) and VSTM3 (HUGO designation). See Levin et al., Eur J Immunol, 2011, 41: 902-915. Therefore, the reference to TIGIT throughout this application also includes a reference to WUCAM and / or VSTM3, unless otherwise stated or apparent in a context. The sequences of human TIGIT proteins and nucleotides are established in, for example, Genbank Accession No. NM173799 (SEQ ID NO: 217) and NP776160 (SEQ ID NO: 218), respectively.
[0048] The term cancer refers to a disease characterized by uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether they are characterized as malignant, benign, soft or hard tissues, and cancers of all stages and degrees, including pre- and post-metastatic cancers. Examples of different types of cancer include, but are not limited to, digestive and gastrointestinal cancers such as gastric cancer (eg stomach cancer), colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer , bile duct cancer, cancer of the small intestine and cancer of the esophagus; breast cancer; lung cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer, ovarian cancer; kidney cancer; cancer of the central nervous system; skin cancer (eg, melanoma); lymphoma; gliomas; choriocarcinomas; cancers of
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24/123 head and neck; osteogenic sarcomas; and blood cancers. As used herein, a tumor comprises one or more cancer cells.
[0049] The term antibody refers to a polypeptide encoded by an immunoglobulin gene or its functional fragments that specifically bind and recognize an antigen (e.g., human TIGIT), a particular cell surface marker or any desired target. Typically, the "variable region" contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in binding specificity and affinity. See, Fundamental Immunology 7th Edition, Paul, ed., Wolters Kluwer Health / Lippincott Williams & Wilkins (2013). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes like IgG, IgM, IgA, IgD and IgE respectively.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having a light chain (about 25 kD) and a heavy chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (Vl) and variable heavy chain (Vh) refer to these light and heavy chains respectively.
[0051] An "isotype" is a class of antibodies defined by the constant region of the heavy chain. Immunoglobulin genes include genes from the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region. Light chains are classified as kappa or lambda. Heavy chains are
Petition 870190101574, of 10/09/2019, p. 30/108 / 123 classified as gamma, mu, alpha, delta, or epsilon, which in turn define the IgG, IgM, IgA, IgD and IgE isotype classes respectively.
[0052] As used in this document, complementarity determining region (CDR) refers to the three hypervariable regions in each chain that interrupt the four framework regions established by the light and heavy chain variable regions. CDRs are primarily responsible for binding to an antigen epitope. The CDRs of each chain are typically referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus, and are usually also identified by the chain on which the specific CDR is located. Thus, a Vh CDR3 is located in the variable domain of the antibody heavy chain in which it is found, whereas a V1 CDR1 is CDR1 in the variable domain of the antibody light chain in which it is found.
[0053] The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is, the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs in three-dimensional space.
[0054] The amino acid sequences of CDR and framework regions can be determined using several definitions well known in the art, for example, Kabat, Chothia, international database ImMunoGeneTics (IMGT) and AbM (see, for example, Johnson and Wu, Nucleic Acids Res. 2000 Jan 1; 28 (1): 214-218 and Johnson et al., Nucleic Acids Res., 29: 205-206 (2001); Chothia and Lesk, (1987) J. Mol. Biol. 196 : 901-917; Chothia et al. (1989) Nature 342, 877-883; Chothia et al. (1992) J. Biol. 227, 799-817; Al-Lazikani et al., J. Mol.Biol 1997, 273 (4)). Unless otherwise stated, CDRs are determined according to Kabat. Definitions of antigen combination sites are also described below: Ruiz et al. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc Nucleic
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Acids Res. Jan 1; 29 (1): 207-9 (2001); MacCallum et al., J. Mol. Biol., 262: 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al., Methods Enzymol., 203: 121-153, (1991); Pedersen et al., Immunomethods, 1, 126, (1992); and Rees et al., In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996).
[0055] The terms "antigen binding portion" or "antigen binding fragment" are used interchangeably in this document and refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (for example, example, TIGIT). It has been shown that the antigen-binding function of an antibody can be performed by fragments of an antibody in its entirety. Examples of antigen-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains), an F (ab ') 2 fragment (a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region), single chain Fv (scFv), complementarity determining regions (CDR), VL (light chain variable region), VH (heavy chain variable region), disulfide linked Fvs ( dsFv) and any combination thereof or any other functional portion of an immunoglobulin peptide capable of binding the target antigen (see, for example, Fundamental Immunology, supra). As appreciated by a person skilled in the art, several antibody fragments can be obtained by a variety of methods, for example, digesting an intact antibody with an enzyme, such as pepsin; or synthesis again. Antibody fragments are often synthesized again chemically or using recombinant DNA methodology. Thus, the term antibody, as used in this document, includes fragments of antibodies produced either by modifying whole antibodies, or those newly synthesized
Petition 870190101574, of 10/09/2019, p. 32/108 / 123 using recombinant DNA methodologies (eg, single-stranded Fv) and those identified using phage display libraries and yeast-based antibody libraries (see, for example, McCafferty et al., (1990) Nature 348: 552; Y. Xu et al., PEDS, 2013, 26: 663-670; WO 2009/036379; WO 2010/105256; and WO 2012/009568). The term antibody also includes divalent or bispecific molecules, diabodies, tribodies and tetrabodies. Bivalent and bispecific molecules are described, for example, in Kostelny et al. (1992) J. Immunol. 148: 1547, Pack and Pluckthun (1992) Biochemistry 31: 1579, Hollinger et al. (1993), PVÁS. USA 90: 6444, Gruber et al. (1994) J Immunol. 152: 5368, Zhu et al. (1997) Protein Sci. 6: 781, Hu et al. (1996) Cancer Res. 56: 3055, Adams et al. (1993) Cancer Res. 53: 4026, and McCartney, et al. (1995) Protein Eng. 8: 301.
[0056] A monoclonal antibody refers to a clonal preparation of antibodies with a unique binding specificity and affinity for a given epitope on an antigen. A "polyclonal antibody" refers to a preparation of antibodies that are raised against a single antigen, but with different binding and affinity specificities.
[0057] An "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See, for example, Morrison et al., PNAS USA, 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44: 65-92 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Padlan, Molec. Immun. , 28: 489-498 (1991); Padlan, Molec. Immun., 31 (3): 169-217 (1994).
[0058] As used in this document, the term chimeric antibody refers to an antibody molecule in which (a) the constant region, or its portion, is altered, replaced or exchanged so that the site
Petition 870190101574, of 10/09/2019, p. 33/108 / 123 antigen binding (variable region, CDR or its portion) is linked to a constant region of a different or altered class, effector function and / or species, or an entirely different molecule that gives new properties to the chimeric antibody (for example, an enzyme, toxin, hormone, growth factor, drug, etc.); or (b) the variable region, or its portion, is altered, replaced or exchanged for a variable region that has a different or altered antigen specificity (for example, CDR and framework regions of different species).
[0059] The term epitope refers to the area or region of an antigen to which an antibody specifically binds, that is, an area or region in physical contact with the antibody, and may include some amino acids or portions of some amino acids, for example example, 5 or 6, or more, for example, 20 or more amino acids, or portions of those amino acids. In some cases, the epitope includes non-protein components, for example, from a carbohydrate, nucleic acid or lipid. In some cases, the epitope is a three-dimensional portion. So, for example, when the target is a protein, the epitope can be comprised of consecutive amino acids, or amino acids from different parts of the protein that are brought close by the fold of proteins (for example, a discontinuous epitope). The same goes for other types of target molecules that form three-dimensional structures.
[0060] The phrase "specifically binds" refers to a molecule (for example, antibody or antibody fragment) that binds to a target with greater affinity, avidity, more readily and / or longer duration to that target in a sample than to a non-target compound. In some embodiments, an antibody or its antigen-binding portion that specifically binds to a target is an antibody or its antigen-binding portion that binds to the target with at least 2 times greater affinity than non-target compounds, for example at least 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 25 times, 50 times or 100 times
Petition 870190101574, of 10/09/2019, p. 34/108 / 123 more affinity. For example, an antibody that specifically binds to TIGIT will typically bind to TIGIT with an affinity at least 2 times greater than with a target other than TIGIT. A person ordinarily skilled in the art understands by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target.
[0061] The term "binding affinity" is used in this document as a measure of the strength of a non-covalent interaction between two molecules, for example, an antibody or its fragment, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).
[0062] The binding affinity between two molecules, for example, an antibody or its fragment, and an antigen, through a monovalent interaction, can be quantified by determining the dissociation constant (Kd). In turn, Kd can be determined by measuring the kinetics of complex formation and dissociation using, for example, the Surface Plasmonic Resonance (SPR) (Biacore ™) method. The rate constants corresponding to the association and dissociation of a monovalent complex are referred to as association rate constants k _a (or k _on ) and dissociation rate constant kd (or k _O ff), respectively. Kd is related to k _a and kd through the equation Kd = kd! K _a . The value of the dissociation constant can be determined directly by well-known methods, and can be calculated even for complex mixtures by methods such as those, for example, established in Caceci et al. (1984, Byte 9: 340-362). For example, Kd can be established using a nitrocellulose double filter binding filter assay such as that disclosed by Wong and Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 54285432). Other standard assays to assess ligand binding capacity
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30/123 such as antibodies to target antigens are known in the art, including, for example, ELISAs, Western blots, RIAs and flow cytometry analysis, and other assays exemplified elsewhere in this document. The binding kinetics and binding affinity of the antibody can also be assessed by standard assays known in the art or as described in the Examples section below, such as Plasmonic Surface Resonance (SPR), for example using a Biacore ™ system; kinetic exclusion assays, such as KinExA®; and BioLayer interferometry (for example, using the ForteBio® Octet platform). In some embodiments, the binding affinity is determined using a BioLayer interferometry assay. See, for example, Wilson et al., Biochemistry and Molecular Biology Education, 38: 400-407 (2010); Dysinger et al, J. Immunol. Methods, 379: SO41 (2012); and Estep et al., Mabs, 2013, 5: 270-278.
[0063] The term cross-reacts, as used here, refers to the ability of an antibody to bind to an antigen other than the antigen against which the antibody was raised. In some embodiments, cross-reactivity refers to the ability of an antibody to bind to an antigen of another species other than the antigen against which the antibody was raised. As a non-limiting example, an anti-TIGIT antibody as described herein that is created against a human TIGIT antigen may exhibit cross-reactivity with TIGIT of a different species (for example, mouse or monkey).
[0064] The terms polypeptide, peptide and protein are used interchangeably in this to refer to a polymer of amino acid residues. The terms apply to amino acid polymers, in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used in this document, the terms encompass
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31/123 amino acid chains of any length, including full length proteins, where the amino acid residues are linked by covalent peptide bonds.
[0065] The term amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that work in a similar way to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are subsequently modified, for example, hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is, an α carbon that is linked to a hydrogen, a carboxyl group, an amine group and an R group, for example, homoserine, norleucine, methionine sulfoxide, methyl methionine sulfonium. Such analogs have modified R groups (for example, norleukin) or modified peptide backbones, but maintain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that works in a similar way to a naturally occurring amino acid.
[0066] Amino acids can be referred to in this document by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPACIUB Biochemical Nomenclature Commission. Nudeotids, likewise, can be referred to by their commonly accepted one-letter codes.
[0067] As used herein, the terms nucleic acid and polynucleotide interchangeably refer to nucleotide strands of any length and include DNA and RNA. Nudeotids can be deoxyribonucleotides, ribonucleotides, modified nudeotides or
Petition 870190101574, of 10/09/2019, p. 37/108 m3 bases, and / or its analogs or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated in this document include single and double stranded DNA, single and double stranded RNA and hybrid molecules having mixtures of DNA and single and double stranded RNA.
[0068] The term isolated, as used in reference to a nucleic acid or protein (for example, antibody), denotes that the nucleic acid or protein is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state. It can be in a dry or aqueous solution. Purity and homogeneity are usually determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from the open reading frames that flank the gene and encode proteins other than the protein encoded by the gene of interest. The term purified denotes that a nucleic acid or protein essentially forms a band in an electrophoretic gel. In particular, it means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
[0069] The term "immune-cancer agent" refers to an agent that enhances, stimulates or upregulates an immune response against cancer in an individual (for example, stimulating an immune response to inhibit tumor growth). In some embodiments, an immunoocnological agent is a small molecule, antibody, peptide, protein, circular peptide, peptidomimetic, polynucleotide, inhibitor RNA, aptamer,
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33/123 drug compound or other compound. In some embodiments, an immuno-cancer agent is an antagonist or inhibitor of the PD-1 or PD-
1.
[0070] The terms "subject", "patient", "individual" and the like are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, mice, goats, pigs and others mammal species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but it usually refers to an individual under medical supervision. A patient can be an individual seeking treatment, monitoring, adjustment or modification of an existing therapeutic regime, etc.
[0071] The terms "therapy", "treatment" and "improvement" refer to any reduction in the severity of symptoms. In the case of cancer treatment, treatment may refer to the reduction, for example, of tumor size, number of cancer cells, growth rate, metastatic activity, cell death of non-cancer cells, etc. As used in this document, the terms "treat" and "prevent" are not intended to be absolute terms. Treatment and prevention can refer to any delay in onset, improvement in symptoms, improvement in patient survival, increased time or rate of survival, etc. Treatment and prevention can be complete (with no remaining detectable symptoms) or partial, so that symptoms are less frequent or severe than in a patient without the treatment described in this document. The effect of treatment can be compared to an individual or group of individuals who have not received treatment, or to the same patient before treatment or at a different time during treatment. In some aspects, the severity of the disease is reduced by at least 10%, when compared, for example, to the individual prior to administration or to a control individual who is not being treated. In some ways, the severity of the disease is reduced by at least 25%,
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50%, 75%, 80% or 90%, or in some cases, no longer detectable using standard diagnostic techniques.
[0072] As used herein, a "therapeutic amount" or "therapeutically effective amount" of an agent (for example, an antibody as described in this document) is an amount of the agent that prevents, relieves, lessens or reduces the severity of symptoms of a disease (for example, cancer) in a subject. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease in the therapeutic effect of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75% , 80%, 90% or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease in “times”. For example, a therapeutically effective amount can have at least 1.2 times, 1.5 times, 2 times, 5 times, or more effect on a control.
[0073] The terms "administer", "administered" or "administering" refer to methods of administering agents, compounds or compositions to the desired site of biological action. These methods include, but are not limited to, topical administration, parenteral administration, intravenous administration, intradermal administration, intramuscular administration, colon administration, rectal administration or intraperitoneal administration. Administration techniques that are optionally used with the agents and methods described in this document include, for example, as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, ed. current; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, PA.
III. Antibodies to TIGIT [0074] In one aspect, antibodies and antigen-binding portions of antibodies that bind to human TIGIT (T-cell immunoreceptor with Ig and ITIM domains) are provided. As described in this document, in
Petition 870190101574, of 10/09/2019, p. 40/108 / 123 In some embodiments, the anti-TIGIT antibody inhibits the interaction between TIGIT and one or both of the CD155 and CD112 ligands. In some embodiments, the anti-TIGIT antibody inhibits the interaction between TIGIT and CD 155 in a functional bioassay, allowing CD155-CD226 signaling to occur. In some embodiments, the anti-TIGIT antibody exhibits synergy with an anti-PD-1 agent (for example, an anti-PD-1 antibody) or an anti-PD-Ll agent (for example, an anti-PD-Ll antibody ).
Characteristics of Anti-TIGIT Antibodies [0075] In some embodiments, an anti-TIGIT antibody binds to the human TIGIT protein (SEQ ID NO: 218) or its high affinity portion. In some embodiments, the antibody has a binding affinity (Kd) for human TIGIT of less than 5 nM, less than 1 nM, less than 500 pM, less than 250 pM, less than 150 pM, less than 100 pM, less than 50 pM, less than 40 pM, less than 30 pM, less than 20 pM or less than about 10 pM. In some embodiments, the antibody has a binding affinity (Kd) for human TIGIT of less than 50 pM. In some embodiments, the antibody has a Kd by human TIGIT in the range of about 1 pM to about 5 nM, for example, about 1 pM to about 1 nM, about 1 pM to about 500 pM, about 5 pM to about 250 pM, or about 10 pM to about 100 pM.
[0076] In some embodiments, in addition to binding to human TIGIT with high affinity, the anti-TIGIT antibody exhibits cross-reactivity with monkey cynomolgus (“cyno”) TIGIT (for example, a cyno TIGIT protein with the sequence of SEQ ID NO: 219) and / or mouse TIGIT (for example, a mouse TIGIT protein with the sequence of SEQ ID NO: 220). In some embodiments, the anti-TIGIT antibody binds to mouse TIGIT (for example, a mouse TIGIT with the sequence of SEQ ID NO: 220) with a binding affinity (Kd) of 100 nM or less. In some embodiments, the anti-TIGIT antibody binds to the
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Human TIGIT with a Kd of 5 nM or less and cross-reacts with mouse TIGIT with a Kd of 100 nM or less. In some embodiments, an anti-TIGIT antibody that binds to a human TIGIT also cross-reacts with both monkey cynomolgus and mouse TIGIT.
[0077] In some embodiments, the cross-reactivity of the antibody is determined by detecting the specific binding of the anti-TIGIT antibody to TIGIT that is expressed in a cell (for example, a cell line that expresses human TIGIT, cyno TIGIT or TIGIT de mouse or a primary cell that endogenously express TIGIT (e.g., primary T cells that endogenously express human TIGIT, cyno TIGIT or mouse TIGIT). In some embodiments, antibody binding and antibody cross-reactivity is determined by detecting specific binding of the anti-TIGIT antibody to purified or recombinant TIGIT (for example, purified or recombinant human TIGIT, purified or recombinant cyan TIGIT or TIGIT de purified or recombinant mouse) or a chimeric protein comprising TIGIT (e.g., a Fe fusion protein comprising human TIGIT, cyano TIGIT or mouse TIGIT, or a His-tagged protein comprising human TIGIT, cyano TIGIT or mouse TIGIT) .
[0078] Methods for analyzing binding affinity, binding kinetics and cross-reactivity are known in the art. See, for example, Ernst et al., Determination of Equilibrium Dissociation Constants, Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009). These methods include, but are not limited to, solid phase binding assays (eg ELISA assay), immunoprecipitation, surface plasmon resonance (SPR, eg Biacore ™ (GE Healthcare, Piscataway, NJ)), kinetic exclusion (for example, KinExA ®), flow cytometry, fluorescence activated cell separator (FACS), BioLayer interferometry
Petition 870190101574, of 10/09/2019, p. 42/108 / 123 (e.g., Octet ™ (FortéBio, Inc., Menlo Park, CA)) and Western blot analysis. SPR techniques are reviewed, for example, in Hahnfeld et al. Determination of Kinetic Data Using SPR Biosensors, Molecular Diagnosis of Infectious Diseases (2004). In a typical SPR experiment, an interactant (target or marker agent) is immobilized on an SPR-active, on a gold-plated glass slide in a flow cell, and a sample containing the other interactant is introduced to flow through the surface. When light of a given wavelength is reflected on the surface, changes in the optical reflectivity of gold indicate the bond and the kinetics of the bond. In some embodiments, kinetic exclusion assays are used to determine affinity. This technique is described, for example, in Darling et al., Assay and Drug Development Technologies Vol. 2, number 6 647-657 (2004). In some embodiments, BioLayer interferometry assays are used to determine affinity. This technique is described, for example, in Wilson et al., Biochemistry and Molecular Biology Education, 38: 400-407 (2010); Dysinger et al., J. Immunol. Methods, 379: 30-41 (2012).
[0079] In some embodiments, the anti-TIGIT antibodies and their antigen-binding portions of the present disclosure inhibit the interaction between TIGIT and the CD 155 ligand. In some embodiments, the anti-TIGIT antibodies and their antigen-binding portions inhibit the interaction between TIGIT and the CD 112 ligand. In some embodiments, antiTIGIT antibodies and their antigen binding portions inhibit the interaction between TIGIT and both CD155 and CD112 ligands.
[0080] In some embodiments, the ability of an antiTIGIT antibody to inhibit interactions between TIGIT and CD 155 and / or CD112 is assessed by measuring whether the physical interactions between TIGIT and CD 155 or CD112 decrease in a binding assay. In some embodiments, the bond test is a competitive bond test. The test can be performed on several
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38/123 formats, such as, but not limited to an ELISA assay, flow cytometry, a surface plasmon resonance (SPR) assay (eg, Biacore ™) or BioLayer interferometry (eg, ForteBio Octet ™). See, for example, Duff et al., Biochem J., 2009, 419: 577-584; Dysinger et al., J. Immunol. Methods, 379: 30-41 (2012); and Estep et al., Mabs, 2013, 5: 270-278.
[0081] In some embodiments, the anti-TIGIT antibody inhibits the interaction between TIGIT and CD 155 in a functional bioassay, such as a functional cellular assay in which the inhibition of the TIGIT / CD155 interaction is assessed by measuring the activation of the CD155-CD226 signaling in the cell (for example, via activation of a downstream reporter). An exemplary non-limiting functional cell assay is described in the Examples section below. In this exemplary functional assay, luciferase expression requires TCR adhesion and a CD155-CD226 costimulatory signal. A first cell (also known as an "effector T cell") expresses a complex of TCR, TIGIT and CD226 on the cell surface and contains a luciferase gene. A second cell (also known as an “artificial antigen presenting cell”) expresses an activator of TCR and CD 155. Co-culture of cells in the absence of anti-TIGIT antibody results in a TIGITCD155 interaction that inhibits co-stimulation of the effector cell for CD155-CD226, preventing expression of luciferase by the effector cell. In the presence of an anti-TIGIT antibody that inhibits the interaction between TIGIT and CD 155, CD 155 and CD226 are able to interact and produce a co-stimulatory signal that drives the expression of luciferase in the first cell. Such functional cell assays are described in the art, for example, Cong et al., Genetic Engineering and Biotechnology News, 2015, 35 (10): 16-17, and are also commercially available (for example, TIGIT / CD155 Blockade Bioassay Kit, Promega Corp., Madison, WI). In some embodiments, an anti-TIGIT antibody that inhibits the interaction between TIGIT and CD 155 increases the level or
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39/123 amount of activation of the CD155-CD226 signaling (for example, as measured in a cell assay such as the TIGIT / CD155 Blockade Bioassay Kit) by at least 10%, at least 20%, at least 30%, at least 40% , at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to the level or amount of CD155-CD226 signaling in the absence of the anti-TIGIT antibody. In some embodiments, an anti-TIGIT antibody that inhibits the interaction between TIGIT and CD 155 increases the level or amount of activation of the CD155-CD226 signaling (for example, as measured in a cell assay such as the TIGIT / CD155 Blockade Bioassay Kit) at least about 1.2 times, at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times or more, compared to the level or amount of CD155-CD226 signaling in the absence of the antibody anti-TIGIT.
[0082] In some embodiments, an anti-TIGIT antibody that binds to human TIGIT (and optionally exhibits cross-reactivity with monkey cynomolgus and / or mouse TIGIT and / or optionally inhibits the interaction between TIGIT and CD 155 and / or CD 112) exhibits synergy with an anti-PD-1 agent. (for example, an anti-PD-1 antibody). In some embodiments, the anti-TIGIT antibody magnifies the effect of the anti-PD-1 agent (for example, anti-PD-1 antibody) at least about 1.2 times, at least about 1.5 times, at least at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times or more.
[0083] In some embodiments, the anti-TIGIT antibody exhibits synergy with an anti-PD-1 agent (for example, an anti-PD-1 antibody) in
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40/123 a functional bioassay, such as a functional cell assay in which the inhibition of TIGIT signaling and inhibition of PD-1 signaling is assessed by measuring the activation of signaling in an effector cell. An exemplary non-limiting functional cell assay is described in the Examples section below. In this exemplary functional assay, a first cell (also known as an "effector T cell") expresses a complex of TCR, TIGIT, CD226 and PD-1 on the cell surface and contains a luciferase gene. A second cell (also known as an “artificial antigen presenting cell”) expresses an activator of TCR, CD155 and PD-L1. The expression of the luciferase gene by the effector cell is activated by one or both of the (1) blockade of the TIGIT-CD155 interaction, thus allowing the CD155-CD226 interaction and subsequent co-stimulation of luciferase expression by the effector cell or (2) blockade of PD-1 / PD-L1 interaction, thus alleviating the inhibition of luciferase expression by the effector cell. The level of luciferase expression in the absence or presence of anti-TIGIT antibodies and anti-PD-1 agents or anti-PD-Ll agents can be measured and quantified to determine whether an anti-TIGIT antibody exhibits synergy with the anti-PD agent -1 or the antiPD-L1 agent. Such functional cell assays are described in the art (for example, Cong et al., Genetic Engineering and Biotechnology News, 2015, 35 (10): 16-17), and are also commercially available (for example, TIGIT / PD-1 Blockade Bioassay Kit, Promega Corp., Madison, WI).
[0084] In some embodiments, the effectiveness of an anti-TIGIT antibody, as well as whether the anti-TIGIT antibody inhibits synergistically with an anti-PD-1 agent (for example, an anti-PD-1 antibody) or an antiPD-Ll agent (for example, an anti-PD-Ll antibody), can be measured using an in vivo model, for example, an in vivo tumor model. For example, the effectiveness of an anti-TIGIT antibody as described herein, or the effectiveness of an anti-TIGIT antibody as described herein when administered in combination with an anti-PD-1 agent or an anti-PD-Ll agent can be
Petition 870190101574, of 10/09/2019, p. 46/108 / 123 evaluated using a syngeneic mouse tumor model. Suitable syngeneic tumor models are described in the art. See, for example, Rios-Doria et al., Neoplasia, 2015, 17: 661-670; and Moynihan et al., Nature Medicine, 2016, doi: 10.1038 / nm.4200. In some embodiments, an anti-TIGIT antibody reduces the size of a tumor or the total number of tumors in an in vivo model by at least 10%, at least 20%, at least 30%, at least 40%, at least 50 %, at least 60%, at least 70%, at least 80%, at least 90% or more compared to a control or reference value (for example, compared to the size of the tumor or total number of tumors in an untreated control).
[0085] In some embodiments, an anti-TIGIT antibody recognizes a human TIGIT epitope comprising one or both of the amino acid positions 81 and 82, numbered with reference to SEQ ID NO: 218. In some embodiments, an anti-TIGIT antibody recognizes an epitope comprising Phe at position 81. In some embodiments, an antiTIGIT antibody recognizes an epitope comprising Lys or Ser at position 82. In some embodiments, an anti-TIGIT antibody recognizes an epitope which comprises Phe at position 81 and Lys at position 82. In some embodiments, an anti-TIGIT antibody recognizes an epitope comprising Phe at position 81 and Ser at position 82.
[0086] In some embodiments, an anti-TIGIT antibody recognizes a linear epitope comprising one or both amino acid positions 81 and 82 (e.g., a discontinuous epitope comprising Phe at position 81 and Lys or Ser at position 82). In some embodiments, an anti-TIGIT antibody recognizes a discontinuous epitope that comprises one or both positions of amino acids 81 and 82 (e.g., a discontinuous epitope that comprises Phe at position 81 and Lys or Ser at position 82).
[0087] In some embodiments, an anti-TIGIT antibody binds to an epitope in human TIGIT that further comprises one or more (eg
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42/123 example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more) of amino acid positions 51, 52, 53, 54, 55, 73 , 74, 75, 76, 77, 79, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93. In some embodiments, an anti-TIGIT antibody binds to an epitope in human TIGIT that further comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more) of the following: Thr in position 51, Wing in position 52, Glu or Gin in position 53, Go in position 54, Thr in position 55, Leu in position 73, Gly in position 74, Trp in position 75, His in position 76, Vai or Ile in position 77, Ser or Pro in position 79, Asp in position 83, Arg in position 84, Vai in position 85, Vai or Ala in position 86, Pro in position 87, Gly in position 88, Pro in position 89, Ser or Gly in position 90, Leu in position 91, Gly at position 92, or Leu at position 93. In some embodiments, an anti-TIGIT antibody binds to an epitope in human TIGIT that further comprises one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more) of the amino acid residues Thr51, Ala52, Gln53, Val54, Thr55, Leu73, Gly74, Trp75, His76, Ile77, Pro79, Asp83, Arg84, Val85, A186, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93.
[0088] In some embodiments, an anti-TIGIT antibody recognizes an epitope comprising Phe at position 81 and Lys or Ser at position 82, and further comprises Thr at position 51, Ala at position 52, Glu or Gin at position 53, Vai at position 54 and / or Thr at position 55. In some embodiments, an anti-TIGIT antibody recognizes an epitope comprising Phe at position 81 and Lys or Ser at position 82, and further comprising Gly at position 74, Trp at position 75, His at position 76 and / or Vai or Ile at position 77. In some embodiments, an anti-TIGIT antibody recognizes an epitope comprising Phe at position 81 and Lys or Ser at position 82, and further comprising Pro at position 87, Gly at position 88, Pro in position 89, Ser or Gly in position 90, Leu in position 91, Gly in position 92 and / or Leu in position 93. In some embodiments, an anti-TIGIT antibody
Petition 870190101574, of 10/09/2019, p. 48/108 / 123 recognizes an epitope comprising the amino acid residues Thr51, Ala52, Gln53, Val54, Thr55, Gly74, Trp75, His76, Ile77, Phe81, Lys82, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93.
[0089] In some embodiments, an anti-TIGIT antibody recognizes an epitope comprising Phe at position 81 and Lys or Ser at position 82, and further comprises Ala at position 52 and / or Glu or Gin at position 53. In some embodiments, an anti-TIGIT antibody recognizes an epitope comprising Phe at position 81 and Lys or Ser at position 82, and further comprises Leu at position 73, Gly at position 74 and / or Trp at position 75. In some embodiments, an anti- TIGIT recognizes an epitope comprising Phe at position 81 and Lys or Ser at position 82, and further comprises Asp at position 83, Arg at position 84, Vai at position 85 and / or Vai or Ala at position 86. In some embodiments, a antiTIGIT antibody recognizes an epitope comprising amino acid residues Ala52, Gln53, Leu73, Gly74, Trp75, Pro79, Phe81, Lys82, Asp83, Arg84, Val85 and Ala86.
[0090] In some embodiments, an anti-TIGIT antibody recognizes a human TIGIT epitope comprising the sequence ICNADLGWHISPSFK (SEQ ID NO: 258), which corresponds to residues 6882 of human TIGIT (SEQ ID NO: 218). In some embodiments, an anti-TIGIT antibody recognizes a human TIGIT epitope consisting of the sequence ICNADLGWHISPSFK (SEQ ID NO: 258).
Anti-TIGIT Antibody Sequences [0091] In some embodiments, an anti-TIGIT antibody that binds to human TIGIT and that optionally exhibits cross-reactivity with monkey cynomolgus and / or mouse TIGIT comprises a light chain sequence, or a a portion of it, and / or a heavy chain sequence, or a portion of it, derived from any of the following antibodies described in this document: Clone 2, Clone 2C, Clone 3, Clone 5, Clone 13,
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Clone 13A, Clone 13B, Clone 13C, Clone 13D, Clone 14, Clone 16, Clone 16C, Clone 16D, Clone 16E, Clone 18, Clone 21, Clone 22, Clone 25, Clone 25A, Clone 25B, Clone 25D, Clone 25D , Clone 25E, Clone 27 or Clone 54. The amino acid sequences of the CDR, the light chain variable domain (VL) and the heavy chain variable domain (VH) of the anti-TIGIT Clone 2, Clone 2C, Clone 3 antibodies, Clone 5, Clone 13, Clone 13A, Clone 13B, Clone 13C, Clone 13D, Clone 14, Clone 16, Clone 16C, Clone 16D, Clone 16E, Clone 18, Clone 21, Clone 22, Clone 25, Clone 25B, Clone 25B , Clone 25C, Clone 25D, Clone 25E, Clone 27, and Clone 54 are set out in Table 3 below.
[0092] In some embodiments, an anti-TIGIT antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92% , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) with SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID
NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQ ID NO: 145, SEQID
NO: 163, SEQ ID NO: 181, SEQ ID NO: 199, SEQ ID NO: 245, SEQID
NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQID
NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQID
NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257. In some embodiments, an anti-TIGIT antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID
NO: 109, SEQ ID
NO: 181, SEQ ID
NO: 247, SEQ ID
NO: 251, SEQ ID
NO: 127, SEQ ID
NO: 199, SEQ ID
NO: 248, SEQ ID
NO: 252, SEQ ID
NO: 145, SEQ ID
NO: 245, SEQ ID
NO: 249, SEQ ID
NO: 253, SEQ ID
NO: 163, SEQ ID
NO: 246, SEQ ID
NO: 250, SEQ ID
NO: 254, SEQ ID
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NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257. In some embodiments, a VH sequence having at least 90% sequence identity to a reference sequence (for example, SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73 SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQ ID NO: 145, SEQ ID NO: 163, SEQ ID
NO: 181, SEQ ID NO: 199, SEQ ID NO: 245, SEQ ID NO: 246, SEQID
NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQID
NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQID
NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257) contain one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (for example, conservative substitutions), inserts or deletions relative to the reference sequence but retain the ability to bind to human TIGIT and, optionally, retain the ability to block the binding of CD 155 and / or CD112 to TIGIT.
[0093] In some embodiments, an anti-TIGIT antibody comprises a light chain variable region (VL) comprising an amino acid sequence that has at least 90% sequence identity (for example, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208. In some embodiments, an anti-TIGIT antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208. In some embodiments, a VL sequence with at least 90% sequence identity with a reference sequence (for example, SEQ ID NO: 10,
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SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208) contain one, two, three, four, five, six, seven, eight, nine, ten or more substitutions (for example, conservative substitutions), inserts or deletions related to the reference sequence, but retain the ability to bind to human TIGIT and, optionally, retain the ability to block binding of CD 155 and / or CD112 to TIGIT.
[0094] In some embodiments, an anti-TIGIT antibody comprises a heavy chain variable region comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1, SEQ ID NO: 19 , SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQ ID NO: 145, SEQ ID NO: 163, SEQ ID NO: 181, SEQ ID NO: 199, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO : 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257, and further comprises a light chain variable region that comprises an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, p at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208. In some embodiments, an anti-TIGIT antibody comprises a heavy chain variable region that comprises the sequence of
Petition 870190101574, of 10/09/2019, p. 52/108 / 123 amino acids of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQ ID NO: 145, SEQ ID NO: 163, SEQ ID NO: 181, SEQ ID NO: 199, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO : 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256 , or SEQ ID NO: 257 and further comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190 or SEQ ID NO: 208.
[0095] In some embodiments, an anti-TIGIT antibody comprises:
(a) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 1 or SEQ ID NO: 245 and a VL comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least minus 99% sequence identity) to SEQ ID NO: 10;
(b) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 19 and a VL comprising an amino acid sequence with at least 90% sequence identity
Petition 870190101574, of 10/09/2019, p. 53/108 / 123 (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 28;
(c) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 37 and a VL comprising an amino acid sequence with at least 90% sequence identity (by example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity sequence) to SEQ ID NO: 46;
(d) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to any of SEQ ID NO: 55, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, or SEQ ID NO: 249 and a VL comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 64;
(e) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 73 and a VL comprising an amino acid sequence with at least 90% sequence identity
Petition 870190101574, of 10/09/2019, p. 54/108 / 123 (for example, at least 91%, minus 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least minus 99% sequence identity) to SEQ ID NO: 82;
(f) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to any of SEQ ID NO: 91, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO : 252 and a VL comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 100;
(g) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 109 and a VL comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity sequence) to SEQ ID NO: 118;
(h) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 127 and a VL comprising an amino acid sequence with at least 90% sequence identity
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50/123 (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 136;
(i) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 145 and a VL comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity ) to SEQ ID NO: 154;
(j) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to any of SEQ ID NO: 163, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257 and a VL comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93% at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 172;
(k) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 181 and a VL comprising an amino acid sequence with at least 90% sequence identity
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51/123 (for example, at least 91%, minus 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 % sequence identity) to SEQ ID NO: 190; or (1) a VH comprising an amino acid sequence with at least 90% sequence identity (for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to SEQ ID NO: 199 and a VL comprising an amino acid sequence with at least 90% sequence identity ( for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity sequence) to SEQ ID NO: 208.
[0096] In some embodiments, an anti-TIGIT antibody comprises:
(a) a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL comprising the amino acid sequence of SEQ ID NO: 10;
(b) a VH comprising the amino acid sequence of
SEQ ID NO: 19 and a VL comprising the amino acid sequence
SEQ ID NO: 28;
(c) a VH comprising the amino acid sequence of
SEQ ID NO: 37 and a VL comprising the amino acid sequence
SEQ ID NO: 46;
(d) a VH comprising the amino acid sequence of
SEQ ID NO: 55 and a VL comprising the amino acid sequence
SEQ ID NO: 64;
(e) a VH comprising the amino acid sequence of
SEQ ID NO: 73 and a VL comprising the amino acid sequence
SEQ ID NO: 82;
Petition 870190101574, of 10/09/2019, p. 57/108 m3 (f) a VH comprising the amino acid sequence of SEQ ID NO: 91 and a VL comprising the amino acid sequence of SEQ ID NO: 100;
(g) a VH comprising the amino acid sequence of SEQ ID NO: 109 and a VL comprising the amino acid sequence of SEQ ID NO: 118;
(h) a VH comprising the amino acid sequence of SEQ ID NO: 127 and a VL comprising the amino acid sequence of SEQ ID NO: 136;
(i) a VH comprising the amino acid sequence of SEQ ID NO: 145 and a VL comprising the amino acid sequence of SEQ ID NO: 154;
(j) a VH comprising the amino acid sequence of SEQ ID NO: 163 and a VL comprising the amino acid sequence of SEQ ID NO: 172;
(k) a VH comprising the amino acid sequence of SEQ ID NO: 181 and a VL comprising the amino acid sequence of SEQ ID NO: 190;
(l) a VH comprising the amino acid sequence of SEQ ID NO: 199 and a VL comprising the amino acid sequence of SEQ ID NO: 208; or (m) a VH comprising the amino acid sequence of SEQ ID NO: 245 and a VL comprising the amino acid sequence of SEQ ID NO: 10; or (n) a VH comprising the amino acid sequence of SEQ ID NO: 246 and a VL comprising the amino acid sequence of SEQ ID NO: 64; or (o) a VH comprising the amino acid sequence of SEQ ID NO: 247 and a VL comprising the amino acid sequence of SEQ ID NO: 247
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SEQ ID NO: 64; or (p) a VH comprising the amino acid sequence of SEQ ID NO: 248 and a VL comprising the amino acid sequence of SEQ ID NO: 64;
(q) a VH comprising the amino acid sequence of SEQ ID NO: 249 and a VL comprising the amino acid sequence of SEQ ID NO: 64; or (r) a VH comprising the amino acid sequence of SEQ ID NO: 250 and a VL comprising the amino acid sequence of SEQ ID NO: 100; or (s) a VH comprising the amino acid sequence of SEQ ID NO: 251 and a VL comprising the amino acid sequence of SEQ ID NO: 100; or (t) a VH comprising the amino acid sequence of SEQ ID NO: 252 and a VL comprising the amino acid sequence of SEQ ID NO: 100; or (u) a VH comprising the amino acid sequence of SEQ ID NO: 253 and a VL comprising the amino acid sequence of SEQ ID NO: 172; or (v) a VH comprising the amino acid sequence of SEQ ID NO: 254 and a VL comprising the amino acid sequence of SEQ ID NO: 172; or (w) a VH comprising the amino acid sequence of SEQ ID NO: 255 and a VL comprising the amino acid sequence of SEQ ID NO: 172; or (x) a VH comprising the amino acid sequence of SEQ ID NO: 256 and a VL comprising the amino acid sequence of SEQ ID NO: 172; or (y) a VH comprising the amino acid sequence of
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SEQ ID NO: 257 and a VL comprising the amino acid sequence of SEQ ID NO: 172.
[0097] In some embodiments, an anti-TIGIT antibody comprises one or more (for example, one, two, three, four, five or more) of: a heavy chain CDR1 sequence comprising the amino acid sequence of any one SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 76, SEQ ID NO: 94, SEQ ID NO: 112, SEQ ID NO: 130, SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 231, SEQ ID NO : 233, SEQ ID NO: 239, or SEQ ID NO: 243;
a heavy chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 42, SEQ ID NO: 60, SEQ ID NO: 78, SEQ ID NO : 96, SEQ ID NO: 114, SEQ ID NO: 132, SEQ ID NO: 150, SEQ ID NO: 168, SEQ ID NO: 186, SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 225 , SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 238, or SEQ ID NO: 240;
a heavy chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80, SEQ ID NO : 98, SEQ ID NO: 116, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 170, SEQ ID NO: 188, SEQ ID NO: 206, SEQ ID NO: 223, SEQ ID NO: 228 , SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 244;
a light chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 85, SEQ ID NO : 103, SEQ ID NO: 121, SEQ ID NO: 139, SEQ ID NO: 157, SEQ ID NO: 175, SEQ ID NO: 193, or SEQ ID NO: 211;
Petition 870190101574, of 10/09/2019, p. 60/108 / 123 a light chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 87, SEQ ID NO: 105, SEQ ID NO: 123, SEQ ID NO: 141, SEQ ID NO: 159, SEQ ID NO: 177, SEQ ID NO: 195, or SEQ ID NO: 213; and / or a light chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, SEQ ID NO: 89, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 143, SEQ ID NO: 161, SEQ ID NO: 179, SEQ ID NO: 197, or SEQ ID NO: 215.
[0098] In some embodiments, an anti-TIGIT antibody comprises a heavy chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 76, SEQ ID NO: 94, SEQ ID NO: 112, SEQ ID NO: 130, SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 239, or SEQ ID NO: 243; a CDR2 sequence of heavy chain comprising the amino acid sequence of any of SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 42, SEQ ID NO: 60, SEQ ID NO: 78, SEQ ID NO: 96, SEQ ID NO: 114, SEQ ID NO: 132, SEQ ID NO: 150, SEQ ID NO: 168, SEQ ID NO: 186, SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 238, or SEQ ID NO: 240; and a heavy chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80, SEQ ID NO : 98, SEQ ID NO: 116, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 170, SEQ ID NO: 188, SEQ ID NO: 206, SEQ ID NO: 223, SEQ ID NO: 228 , SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID
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NO: 237, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 244.
[0099] In some embodiments, an anti-TIGIT antibody comprises a light chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 85, SEQ ID NO: 103, SEQ ID NO: 121, SEQ ID NO: 139, SEQ ID NO: 157, SEQ ID NO: 175, SEQ ID NO: 193, or SEQ ID NO : 211; a light chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 87, SEQ ID NO : 105, SEQ ID NO: 123, SEQ ID NO: 141, SEQ ID NO: 159, SEQ ID NO: 177, SEQ ID NO: 195, or SEQ ID NO: 213; and a light chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, SEQ ID NO: 89, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 143, SEQ ID NO: 161, SEQ ID NO: 179, SEQ ID NO: 197, or SEQ ID NO: 215.
[00100] In some embodiments, an anti-TIGIT antibody comprises:
(i) a heavy chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 76, SEQ ID NO: 94, SEQ ID NO: 112, SEQ ID NO: 130, SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 239, or SEQ ID NO: 243; and (ii) a heavy chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 42, SEQ ID NO: 60, SEQ ID NO: 78 , SEQ ID NO: 96, SEQ ID NO: 114, SEQ ID NO: 132, SEQ ID NO: 150, SEQ ID NO: 168, SEQ ID NO: 186, SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID
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NO: 227, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID
NO: 238, or SEQ ID NO: 240; and (iii) a heavy chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80 , SEQ ID
NO: 98, SEQ ID NO: 116, SEQ ID NO: 134, SEQ ID NO: 152, SEQID
NO: 170, SEQ ID NO: 188, SEQ ID NO: 206, SEQ ID NO: 223, SEQID
NO: 228, SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID NO: 236, SEQID
NO: 237, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 244; and (iv) a light chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 85 , SEQ ID NO: 103, SEQ ID NO: 121, SEQ ID NO: 139, SEQ ID NO: 157, SEQ ID NO: 175, SEQ ID NO: 193, or SEQ ID NO: 211; and (v) a light chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 87 , SEQ ID NO: 105, SEQ ID NO: 123, SEQ ID NO: 141, SEQ ID NO: 159, SEQ ID NO: 177, SEQ ID NO: 195, or SEQ ID NO: 213; and (vi) a light chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, SEQ ID NO: 89 , SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 143, SEQ ID NO: 161, SEQ ID NO: 179, SEQ ID NO: 197, or SEQ ID NO: 215.
[00101] In some embodiments, an anti-TIGIT antibody comprises: (i) a heavy chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 221; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 222; (iii) a sequence of
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Heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 223; (iv) a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 13; (v) a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 15; and (vi) a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 17.
[00102] In some embodiments, an anti-TIGIT antibody comprises: (i) a heavy chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 58, SEQ ID NO: 224, or SEQ ID NO: 226; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 60, SEQ ID NO: 225, SEQ ID NO: 227 or SEQ ID NO: 229; (iii) a heavy chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 62, SEQ ID NO: 228 or SEQ ID NO: 230; (iv) a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 67; (v) a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 69; and (vi) a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 71.
[00103] In some embodiments, an anti-TIGIT antibody comprises: (i) a heavy chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 94, SEQ ID NO: 231, or SEQ ID NO: 233; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 96, SEQ ID NO: 232 or SEQ ID NO: 234; (iii) a heavy chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 98, SEQ ID NO: 235, SEQ ID NO: 236 or SEQ ID NO: 237; (iv) a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 103; (v) a sequence of CDR2
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59/123 light chain comprising the amino acid sequence of SEQ ID NO: 105; and (vi) a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 107.
[00104] In some embodiments, an anti-TIGIT antibody comprises: (i) a heavy chain CDR1 sequence comprising the amino acid sequence of any of SEQ ID NO: 166, SEQ ID NO: 239, or SEQ ID NO: 243; (ii) a heavy chain CDR2 sequence comprising the amino acid sequence of any of SEQ ID NO: 168, SEQ ID NO: 238 or SEQ ID NO: 240; (iii) a heavy chain CDR3 sequence comprising the amino acid sequence of any of SEQ ID NO: 170, SEQ ID NO: 241, SEQ ID NO: 242 or SEQ ID NO: 244; (iv) a light chain CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 175; (v) a light chain CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 177; and (vi) a light chain CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 179.
[00105] In some embodiments, an anti-TIGIT antibody comprises a heavy chain CDR1-3 and a light chain CDR1-3 that comprises the amino acid sequences of:
(a) SEQ ID NOs: 4, 6, 8, 13, 15 and 17, respectively;
(b) SEQ ID NOs: 22, 24, 26, 31, 33 and 35, respectively;
(c) SEQ ID NOs: 40, 42, 44, 49, 51 and 53, respectively;
(d) SEQ ID NOs: 58, 60, 62, 67, 69 and 71, respectively;
(e) SEQ ID NOs: 76, 78, 80, 85, 87 and 89, respectively;
(f) SEQ ID NOs: 94, 96, 98, 103, 105 and 107, respectively;
(g) SEQ ID NOs: 112, 114, 116, 121, 123 and 125, respectively;
(h) SEQ ID NOs: 130, 132, 134, 139, 141 and 143, respectively;
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(i) SEQ ID We: 148, 150, 152, 157, 159 and 161, respectively;(j) SEQ ID We: 166, 168, 170, 175, 177 and 179, respectively;(k) SEQ ID We: 184, 186, 188, 193, 195 and 197, respectively;(D SEQ ID We: 202, 204, 206, 211, 213 and 215,
respectively; or (m) SEQ ID NOs: 221, 222, 223, 13, 15 and 17, respectively; or (n) SEQ ID NOs: 224, 225, 62, 67, 69 and 71, respectively; or (o) SEQ ID NOs: 226, 227, 228, 67, 69 and 71, respectively; or (p) SEQ ID NOs: 224, 229, 230, 67, 69 and 71, respectively; or (q) SEQ ID NOs: 224, 227, 230, 67, 69 and 71, respectively;
or
O') SEQ ID We: 231, 232, 235, 103, 105 and 107, respectively; or(s) SEQ ID We: 233, 234, 236, 103, 105 and 107, respectively; or(t) SEQ ID We: 233, 234, 237, 103, 105 and 107, respectively; or(u) SEQ ID We: 166, 238, 170, 175, 177 and 179, respectively; or(v) SEQ ID We: 239, 240, 170, 175, 177 and 179, respectively; or(w) SEQ ID We: 239, 240, 241, 175, 177 and 179,
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61/123 respectively; or (x) SEQ ID NOs: 239, 240, 242, 175, 177 and 179, respectively; or (y) SEQ ID NOs: 243, 168, 244, 175, 177 and 179, respectively [00106] In some embodiments, the antibody further includes a framework, such as a human immunoglobulin framework. For example, in some embodiments, an antibody comprises a CDR as described in this document, and further comprises a human acceptor framework, for example, a human immunoglobulin framework or a human consensus framework. Human immunoglobulin frameworks can be part of the human antibody, or a non-human antibody can be humanized by replacing one or more endogenous frameworks by human framework region (s). Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using the best fit method (see, for example, Sims et al., J. Immunol. 151: 2296 (1993 )); framework regions derived from the consensus human antibody sequence of a specific subgroup of variable regions of light or heavy chain (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); and Presta et al., J. Immunol., 151: 2623 (1993)); mature human framework regions (somatically mutated) or germline framework regions (see, for example, Almagro and Fransson, Front. Biosci. 13: 16191633 (2008)); and framework regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)). Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences. For example, lineage DNA sequences
Petition 870190101574, of 10/09/2019, p. 67/108 m3 germline for genes from variable regions of human heavy and light chains can be found in the germline variable gene sequence database “VBASE2” for human and mouse sequences.
[00107] In some embodiments, an anti-TIGIT antibody comprises one or more heavy chain framework regions (FR1, FR2, FR3 and / or FR4) comprising an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 39, SEQ ID NO: 41,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID
NO: 61, SEQ ID NO: 63, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 111, SEQ ID
NO: 117, SEQ ID
NO: 135, SEQ ID
NO: 153, SEQ ID
NO: 171, SEQ ID
NO: 129, SEQ ID
NO: 147, SEQ ID
NO: 165, SEQ ID
NO: 183, SEQ ID
NO: 113, SEQ ID
NO: 131, SEQ ID
NO: 149, SEQ ID
NO: 167, SEQ ID
NO: 185, SEQ ID
NO: 115, SEQ ID
NO: 133, SEQ ID
NO: 151, SEQ ID
NO: 169, SEQ ID
NO: 187, SEQ ID
NO: 189, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, or SEQ ID NO: 207.
[00108] In some embodiments, an anti-TIGIT antibody comprises one or more light chain framework regions (FR1, FR2, FR3 and / or FR4) comprising an amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 14 , SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO : 88, SEQ ID NO: 90, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124 , SEQ ID NO: 126, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID
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NO: 144, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQID
NO: 162, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQID
NO: 180, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 196, SEQID
NO: 198, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214 or SEQID
NO: 216.
[00109] In some embodiments, the anti-TIGIT antibodies of the present disclosure do not compete for binding with the antibodies described in US 2009/0258013, US 2016/0176963, US 2016/0376365 or WO 2016/028656. In some embodiments, the anti-TIGIT antibodies of the present disclosure do not bind to the same epitope as the antibodies described in US 2009/0258013, US 2016/0176963, US 2016/0376365 or WO 2016/028656.
Preparation of Antibodies [00110] To prepare an antibody that binds to TIGIT, many procedures known in the art can be used. See, for example, Kohler & Milstein, Nature 256: 495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., Pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd ed 1986..
[00111] The genes encoding the heavy and light chains of an antibody of interest can be cloned from a cell, for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce an antibody recombinant monoclonal. Libraries of genes that encode heavy and light chains of monoclonal antibodies can also be made from hybridoma cells or plasma. In addition, the phage display or yeast display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to antigens
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64/123 selected (see, for example, McCafferty et al., Nature 348: 552-554 (1990); Marks et al., Biotechnology 10: 779-783 (1992), Lou et al. (2010) PEDS 23: 311; and Chao et al., Nature Protocols, 1: 755-768 (2006)). Alternatively, antibodies and antibody sequences can be isolated and / or identified using a yeast-based antibody delivery system, as described in, for example, Xu et al., Protein Eng Des Sei, 2013, 26: 663- 670; WO 2009/036379; WO 2010/105256; and WO 2012/009568. The random combinations of gene products of heavy and light chains generate a large pool of antibodies with different antigenic specificity (see, e.g., Kuby, Immunology (3rd ed. 1997)). Techniques for producing single chain antibodies or recombinant antibodies (US Patent 4,946,778, US Patent No. 4,816,567) can also be adapted to produce antibodies. Antibodies can also be taken bispecific, that is, capable of recognizing two different antigens (see, for example, WO 93/08829, Traunecker et al., EMBO J. 10: 3655-3659 (1991); and Suresh et al. , Methods in Enzymology 121: 210 (1986)). The antibodies can also be heteroconjugated, for example, two covalently joined antibodies, or antibodies covalently linked to immunotoxins (see, for example, US Patent No. 4,676,980, WO 91/00360; and WO 92/200373).
[00112] Antibodies can be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell, such as a hybridoma, or a CHO cell. Many of these systems are widely available from commercial suppliers. In embodiments in which an antibody comprises both Vh and Vl regions, the Vh and Vl regions can be expressed using a single vector, for example, in a dicistronic expression unit, or be under the control of different promoters. In other modalities, the region of
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Vh and Vl can be expressed using separate vectors. A Vh or Vl region, as described in this document, can optionally comprise a methionine at the N-terminus.
[00113] Methods for humanizing or primatizing non-human antibodies are also known in the art. In general, the humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often called import residues, which are normally taken from a variable import domain. Humanization can essentially be carried out following the method of Winter et al. (See, for example, Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992)), by replacing rodent CDR or CDR sequences with the corresponding sequences of an antibody human. Accordingly, such humanized antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies. Transgenic mice, or other organisms, such as other mammals, can be used to express human or humanized antibodies (see, for example, US Patent No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633. 425; 5,661,016, Marks et al., Bio / Technology 10: 779-783 (1992), Lonberg et al., Nature 368: 856-859 (1994), Morrison, Nature 368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14: 845-51 (1996). Neuberger, Nature Biotechnology 14: 826 (1996) and Lonberg & Huszar, Intern. Rev. Immunol. 13: 65-93 (1995)).
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66/123 [00114] As an alternative to humanization, human antibodies can be generated. As a non-limiting example, transgenic animals (for example, mice) can be produced that are capable, after immunization, of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been reported that the homozygous deletion of the antibody heavy chain binding region (JH) gene in mutant chimeric and germline mice results in complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene matrix in such germline mutant mice will result in the production of human antibodies upon exposure to antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. Know. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immun., 7:33 (1993); and US Patent Nos. 5,591,669, 5,589,369 and 5,545,807.
[00115] In some embodiments, antibody fragments (such as Fab, Fab ', F (ab') 2, scFv or diabody) are generated. Several techniques have been developed for the production of antibody fragments. Traditionally, these fragments are derived from proteolytic digestion of intact antibodies (see, for example, Morimoto et al., J. Biochem. Biophys. Meth., 24: 107-117 (1992); and Brennan et al., Science, 229 : 81 (1985)). However, these fragments can now be produced directly using recombinant host cells. For example, antibody fragments can also be isolated from antibody phage libraries. Alternatively, Fab'-SH fragments can be recovered directly from E. coli cells and chemically joined to form F (ab ') 2 fragments (see, for example, Carter et al., BioTechnology, 10: 163-167 (1992)). According to another approach, F (ab ') 2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be evident for
Petition 870190101574, of 10/09/2019, p. 72/108 / 123 those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv (scFv) fragment. See, for example, PCT Publication No. WO 93/16185; and US Patent Nos. 5,571,894 and 5,587,458. The antibody fragment can also be a linear antibody as described, for example, in US Patent No. 5,641,870.
[00116] In some embodiments, the antibody or antibody fragment can be conjugated to another molecule, for example, polyethylene glycol (pegylation) or serum albumin, to provide an extended half-life in vivo. Examples of pegylation of antibody fragments are provided in Knight et al. Platelets 15: 409, 2004 (for abciximab); Pedley et al., Br. J. Cancer 70: 1126, 1994 (for an anti-CEA antibody); Chapman et al., Nature Biotech. 17: 780, 1999; and Humphreys, et al., Protein Eng. Des. 20: 227, 2007).
[00117] In some embodiments, multispecific antibodies are provided that comprise an anti-TIGIT antibody or antigen binding fragment, as described in this document, for example, a bispecific antibody. Multispecific antibodies are antibodies that have binding specificities for at least two different sites. In some embodiments, the multispecific antibody has a binding specificity for TIGIT (e.g., human TIGIT) and has a binding specificity for at least one other antigen. Methods for producing multispecific antibodies include, but are not limited to, recombinant coexpression of two heavy and light chain pairs in a host cell (see, for example, Zuo et al., Protein Eng Des Sei, 2000, 13: 361 367); “knobs-into-holes” interaction (see, for example, Ridgway et al., Protein Eng Des Sei, 1996, 9: 617-721); diabody body (see, for example, Hollinger et al., PNAS (USA), 1993, 90: 6444-6448); and intramolecular trimerization (see, for example, Alvarez-Cienfuegos et al., Scientific Reports, 2016, doi: /10.1038/srep28643); See also Spiess et al., Molecular
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Immunology, 2015, 67 (2), Part A: 95-106.
[00118] In some embodiments, antibody-drug conjugates are provided that comprise an anti-TIGIT antibody or antigen binding fragment as described herein. In anticancer drug conjugates, a monoclonal antibody having a binding specificity for an antigen (eg, TIGIT) is covalently linked to a cytotoxic drug. Methods for preparing antibody-drug conjugates are described, for example, in Chudasama et al., Nature Chemistry, 2016, 8: 114-119; WO 2013/068874; and US 8,535,678.
Nucleic Acids, Vectors and Host Cells [00119] In some embodiments, anti-TIGIT antibodies, as described in this document, are prepared using recombinant methods. Accordingly, in some respects, the invention provides isolated nucleic acids comprising a nucleic acid sequence that encodes any of the anti-TIGIT antibodies as described herein (for example, any one or more of the CDRs described herein); vectors that comprise these nucleic acids; and host cells into which the nucleic acids are introduced which are used to replicate the nucleic acids encoding the antibody and / or to express the antibodies. In some embodiments, the host cell is eukaryotic, for example, a Chinese hamster ovary (CHO) cell; or a human cell.
[00120] In some embodiments, a polynucleotide (for example, an isolated polynucleotide) comprises a nucleotide sequence that encodes an antibody or its antigen-binding portion as described in this document (for example, as described in the Section above entitled Anti Antibody Sequences) -TIGIT). In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes one or more amino acid sequences (for example, CDR, heavy chain, light chain and / or framework regions) disclosed in Table 3 below. In
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69/123 In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes an amino acid sequence with at least 85% sequence identity (for example, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to a sequence (for example, a sequence CDR, heavy chain, light chain or framework region) disclosed in Table 3 below.
[00121] In some embodiments, a polynucleotide (e.g., an isolated polynucleotide) comprises a nucleotide sequence that encodes a variable region of the heavy chain as described in this document. In some embodiments, a polynucleotide comprises a nucleotide sequence that encodes a variable region of the heavy chain that comprises an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO : 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQ ID NO: 145, SEQ ID NO: 163, SEQ ID NO: 181 , SEQ ID NO: 199, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 56, SEQ ID NO: 74, SEQ ID NO: 92, SEQ ID NO: : 110, SEQ ID NO: 128, SEQ ID NO: 146, SEQ ID NO: 164, SEQ ID NO: 182, or SEQ ID NO: 200.
[00122] In some embodiments, a polynucleotide (e.g., an isolated polynucleotide) comprises a nucleotide sequence that encodes a light chain variable region as described in this document. In some embodiments, a polynucleotide comprises a sequence
Petition 870190101574, of 10/09/2019, p. 75/108 / 123 nucleotide encoding a light chain variable region comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136,
SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208. In some embodiments, a polynucleotide comprises the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 29, SEQ ID NO: 47, SEQ ID
NO: 65, SEQ ID NO: 83, SEQ ID NO: 101, SEQ ID NO: 119, SEQ ID NO: 137,
SEQ ID NO: 155, SEQ ID NO: 173, SEQ ID NO: 191, or SEQ ID NO: 209.
[00123] In some embodiments, the polynucleotide comprises a nucleotide sequence that encodes a variable region of heavy chain and a variable region of light chain, as described in this document. In some embodiments, a polynucleotide comprises a nucleotide sequence that encodes a variable region of heavy chain comprising an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID
NO: 127,
NO: 199,
NO: 248,
NO: 252,
SEQ ID
SEQ ID
SEQ ID
SEQ ID
NO: 145,
NO: 245,
NO: 249,
NO: 253,
SEQ ID
SEQ ID
SEQ ID
SEQ ID
NO: 163,
NO: 246,
NO: 250,
NO: 254,
SEQ ID
SEQ ID
SEQ ID
SEQ ID
NO: 181,
NO: 247,
NO: 251,
NO: 255,
SEQ ID
SEQ ID
SEQ ID
SEQ ID
NO: 256, or SEQ ID NO: 257 and encoding a variable region of the light chain that comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 56, SEQ ID NO: 74, SEQ ID NO: 92, SEQ ID NO: : 110, SEQ ID
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NO: 128, SEQ ID NO: 146, SEQ ID NO: 164, SEQ ID NO: 182, or SEQ ID NO: 200, and further comprises the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 29, SEQ ID NO: 47, SEQ ID NO: 65, SEQ ID NO: 83, SEQ ID NO: 101, SEQ ID NO: 119, SEQ ID NO: 137, SEQ ID NO: 155, SEQ ID NO: 173, SEQ ID NO: 191 or SEQ ID NO: 209.
[00124] In an additional aspect, methods are provided to produce an anti-TIGIT antibody as described in this document. In some embodiments, the method includes culturing a host cell as described herein (for example, a host cell expressing a polynucleotide or vector as described herein) under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).
[00125] Suitable vectors containing polynucleotides that encode antibodies of the present disclosure, or fragments thereof, include cloning vectors and expression vectors. Although the selected cloning vector may vary according to the host cell being used, useful cloning vectors generally have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and / or may transport genes for a marker that can be used to select clones that contain the vector. Examples include bacteriophage plasmids and viruses, for example, pUC18, pUC19, Bluescript (for example, pBS SK +) and their derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNA and carrier vectors, such as PS A3 and pAT28. Cloning vectors are available from commercial suppliers, such as BioRad, Stratagene and Invitrogen.
[00126] Expression vectors are generally replicable polynucleotide constructs that contain a nucleic acid of the present disclosure. The expression vector can be replicable in host cells
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72/123 as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenovirus, adeno-associated viruses, retrovirus, and any other vector.
IV. Therapeutic Methods Using Anti-TIGIT Antibodies [00127] In another aspect, methods of treating or preventing cancer in a subject are provided. In some embodiments, the method comprises administering to the subject a therapeutic amount of an anti-TIGIT antibody or antigen-binding fragment as described herein or a pharmaceutical composition comprising an anti-TIGIT antibody or antigen-binding fragment as described herein . In some modalities, the subject is a human, for example, an adult or a human child.
[00128] In some modalities, the cancer is a cancer or a cancer cell that is enriched for expression of CD 112 and / or CD 155. In some modalities, cancers enriched with CD 112 and / or CD 155 are identified by immunoassay -histochemistry of tumor samples using antibodies specific for CD 112 or CD 155. In some embodiments, the expression of CD112 or CD 155 is enriched or increased in tumor cells or leukocytes infiltrating tumors. In some embodiments, cancer is identified based on the assessment of CD 112 and / or CD 155 mRNA levels in tumor samples (for example, by methods known in the art, such as quantitative RT-PCR). In some embodiments, measurements of CD112 or CD155 soluble in blood samples obtained from cancer patients can be used to identify a cancer that is enriched for expression of CD112 and / or CD155. In some embodiments, the method involves obtaining a sample from an individual (for example, a tumor sample or a blood sample), measuring the level of CD112 and / or CD155 in the subject's sample and
Petition 870190101574, of 10/09/2019, p. 78/108 / 123 comparing the CD 112 and / or CD 155 level in the subject sample with a control value (for example, a sample from a healthy control subject or a determined CD 112 and / or CD 155 expression level for a population of healthy controls). In some embodiments, the method comprises determining that the level of CD 112 and / or CD 155 in the subject sample is greater than a control value and subsequently administering an anti-TIGIT antibody to the individual as described herein.
[00129] In some modalities, cancer is a cancer or a cancer cell that is enriched for T cells or natural killer cells (NK) that express TIGIT. In some embodiments, cancers enriched by TIGIT are identified by immunohistochemical evaluation of tumor samples using antibodies specific to TIGIT. In some embodiments, an antibody that is specific for T cells or NK cells (for example, anti-CD3, anti-CD4, anti-CD8, anti-CD25 or anti-CD56) is used to determine a subset or subsets of infiltrating cells tumor that express TIGIT. In some modalities, cancer is identified based on the assessment of TIGIT mRNA levels in tumor samples. In some embodiments, measurements of soluble TIGIT can be used in blood samples obtained from cancer patients (optionally in combination with an antibody that is specific for T cells or NK cells) to identify a cancer enriched for T cells or NK cells that express TIGIT . In some embodiments, the method involves obtaining a sample from a subject (for example, a tumor sample or a blood sample), measuring the level of TIGIT in the subject's sample, optionally detecting the presence of T cells or NK cells (for example, using an antibody that is specific for T cells or NK cells such as anti-CD3, anti-CD4, anti-CD8, anti-CD25 or anti CD56) and comparing the level of TIGIT in the subject's sample to a value of control (for example, a sample from a healthy control individual or a
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74/123 TIGIT expression level determined for a population of healthy controls). In some embodiments, the method comprises determining that the level of TIGIT in the subject's sample is greater than a control value and subsequently administering to the individual an anti-TIGIT antibody as described in this document.
[00130] In some modalities, cancer is bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer , colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer (for example, melanoma ), neoplasm of the central nervous system, lymphoma, leukemia, myeloma, or sarcoma. In some modalities, cancer is stomach cancer. In some modalities, cancer is lung cancer. In some modalities, cancer is skin cancer (for example, melanoma). In some modalities, cancer is metastatic cancer. In some embodiments, cancer is lymphoma or leukemia, including but not limited to acute myeloid, chronic myeloid, acute lymphoid or chronic lymphoid leukemia, diffuse large B cell lymphoma, follicular lymphoma, mantle cell lymphoma, small lymphoid lymphoma , primary large B cell mediastinal lymphoma, splenic marginal B cell lymphoma or extranodal marginal B cell lymphoma.
[00131] In some embodiments, the method further comprises administering to the subject a therapeutic amount of an immunooncological agent. In some embodiments, the immuno-oncological agent is an agent (for example, an antibody, small molecule or peptide) that antagonizes or inhibits a component of an immune pathway, such as the PD-1 pathway, the CTLA- 4, the Lag3 pathway, or the TIM-3 pathway. In some modalities, the immuno-oncological agent is an agonist of a co-activator of
Petition 870190101574, of 10/09/2019, p. 80/108 / 123 T cells (ie, a protein agonist that stimulates T cell activation) targeting the OX-40 pathway, the 4-1BB pathway (CD 137), the CD27 pathway, the CD27 pathway ICOS, or the GITR pathway.
[00132] In some embodiments, the immuno-oncological agent is an inhibitor of the PD-1 pathway. In some embodiments, the PD-1 pathway inhibitor is an anti-PD-1 antibody or anti-PD-Ll antibody, such as but not limited to pembrolizumab, nivolumab, durvalumab, pidilizumab or atezolizumab. Inhibitors of the PD-1 pathway are described in the art. See, for example, Dolan et al., Cancer Control, 2014, 21: 231-237; Luke et al., Oncotarget, 2014, 6: 3479-3492; US 2016/0222113; US 2016/0272708; US 2016/0272712; and US 2016/0319019.
[00133] In some embodiments, the immuno-oncological agent is an agonist of a T cell co-activator. In some embodiments, the immuno-oncological agent is an agonist of CD28, CD28H, CD3, 4-1BB (CD137), ICOS, 0X40, GITR, CD27 or CD40. In some modalities, the immuno-oncological agent is an immunostimulatory cytokine. In some embodiments, the immunostimulatory cytokine is a granulocyte and macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 1 ( IL-1), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15) or gamma interferon (IFN-γ).
[00134] In some embodiments, treatment with an anti-TIGIT antibody as described in this document is combined with one or more other cancer treatments, such as surgery, radiation or chemotherapy. In some embodiments, the chemotherapeutic agent is an alkylating agent (for example, cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mecloretamine, uramustine, thiotepa, nitrosoureas or temozolomide), an anthracycline (for example, doxorubicin, adriamycin,
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76/123 daunorubicin, epirubicin or mitoxantrone), a cytoskeleton disruptor (eg, paclitaxel or docetaxel), histone deacetylase inhibitor (eg, vorinostat or romidepsin), a topoisomerase inhibitor (eg, irinotecan, topotano, ansacretin, topotano, etoposide, or teniposide), a kinase inhibitor (for example, bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, or vismodegibe), a nucleoside analog or precursor analog (for example, azacitidine, azathioprine, capecitabine, fluorine, aitarabina hydroxyurea, mercaptopurine, methotrexate or thioguanine), a peptide antibiotic (eg actinomycin or bleomycin), a platinum-based agent (eg cisplatin, oxaliplatin or carboplatin) or a plant alkaloid (eg vincristine, vimblastine , vinorelbine, vindesine, podophyllotoxin, paclitaxel or docetaxel).
[00135] In some embodiments, the anti-TIGIT antibody (and optionally an immuno-cancer agent or other therapeutic treatment) is administered in a therapeutically effective amount or dose. A daily dose in the range of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg can be used at about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg, can be used. The dosages, however, can be varied according to several factors, including the chosen route of administration, the formulation of the composition, the patient's response, the severity of the condition, the subject's weight and the judgment of the prescribing physician. The dosage can be increased or decreased over time, as required by an individual patient. In certain cases, a patient initially receives a low dose, which is then increased to an effective dose tolerable to the patient. The determination of an effective amount is well within the ability of those skilled in the art.
[00136] The route of administration of an anti-TIGIT antibody or
Petition 870190101574, of 10/09/2019, p. 82/108 / 123 pharmaceutical composition comprising an anti-TIGIT antibody (and optionally an immuno-oncological agent or other therapeutic treatment) can be oral, intraperitoneal, transdermal, subcutaneous, intravenous, intramuscular, inhalation, topical, intralesional, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or optical, or any other methods known in the art. In some embodiments, the anti-TIGIT antibody (and optionally an immuno-oncological agent) is administered orally, intravenously or intraperitoneally.
[00137] Co-administered therapeutic agents (for example, anti-TIGIT antibody and an immuno-cancer agent or other therapeutic treatment) can be administered together or separately, simultaneously or at different times. When administered, therapeutic agents independently can be administered once, twice, three, four times a day or more or less frequently, as needed. In some embodiments, the therapeutic agents administered are administered once daily. In some embodiments, the therapeutic agents administered are administered at the same time one more time, for example, as a mixture. In some embodiments, one or more of the therapeutic agents is administered in a sustained release formulation.
[00138] In some embodiments, the anti-TIGIT antibody and other therapeutic treatment (for example, an immuno-cancer agent) are administered concurrently. In some embodiments, the antiTIGIT antibody and other therapeutic treatment (for example, an immunooncological agent) are administered sequentially. For example, in some embodiments, an anti-TIGIT antibody is administered first, for example, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 days or more before administering an immunological agent. In some modalities, an immuno-cancer agent is administered first,
Petition 870190101574, of 10/09/2019, p. 83/108 for example, for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 days or more before administering an antiTIGIT antibody.
[00139] In some embodiments, the anti-TIGIT antibody (and optionally the immuno-oncological agent) is administered to the subject over an extended period of time, for example, for at least 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350 days or more.
V. Compositions and Kits [00140] In another aspect, compositions and kits are provided that comprise an anti-TIGIT antibody for use in the treatment or prevention of cancer in a subject.
Pharmaceutical Compositions [00141] In some embodiments, pharmaceutical compositions are provided that comprise an anti-TIGIT antibody for use in administering to a subject with a cancer. In some embodiments, the anti-TIGIT antibody is as described in Section III above, for example, an anti-TIGIT antibody having a binding affinity, activity, cross-reactivity, epitope recognition and / or one or more CDR, VH sequences and / or VL as disclosed in Section III above.
[00142] In some embodiments, an anti-TIGIT antibody and an immuno-cancer agent (for example, an inhibitor of the PD-1 pathway as described in this document) are formulated in pharmaceutical compositions, together or separately, as described in this document . In some embodiments, the immuno-oncological agent is an inhibitor of the PD-1 pathway or an inhibitor of the CTLA-4 pathway. In some embodiments, the immunooncological agent is an agonist of a T cell co-activator. In some embodiments, the inhibitor of the PD-1 pathway is an anti-PD-1 antibody or anti-PD-Ll antibody, such as but not limited to pembrolizumab, nivolumab, durvalumab, pidilizumab or atezolizumab.
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79/123 [00143] Guidance for the preparation of formulations for use in the present invention are found, for example, in Remington: The Science and Practice of Pharmacy, 21 ed, 2006, supra, "Martindale:. The Complete Drug Reference, Sweetman, 2005, London: Pharmaceutical Press; Niazi, Handbook of Pharmaceutical Manufacturing Formulations, 2004, CRC Press; and Gibson, Pharmaceutical Preformulation and Formulation: A Practical Guide from Candidate Drug Selection to Commercial Dosage Form, 2001, Interpharm Press, which are hereby incorporated by reference. The pharmaceutical compositions described in this document can be manufactured in a manner that is known to those skilled in the art, that is, by means of conventional mixing, dissolving, granulating, drug-making, emulsifying, encapsulating, trapping or lyophilizing processes. The following methods and excipients are exemplary only and are in no way limiting.
[00144] In some embodiments, an anti-TIGIT antibody (and optionally an immuno-oncological agent) is prepared for administration in a sustained release, controlled release, prolonged release, scheduled release or delayed release formulation, for example, in matrices permeable to solid hydrophobic polymers containing the therapeutic agent. Various types of sustained release materials have been established and are well known to those skilled in the art. Current extended release formulations include film-coated tablets, multiparticulate or pellet systems, matrix technologies using hydrophilic or lipophilic materials, and wax-based tablets with pore-forming excipients (see, for example, Huang, et al. Drug Dev Pharm. 29:79 (2003); Peamchob et al. Drug Dev. Ind. Pharm. 29: 925 (2003); Maggi et al. Eur. J. Pharm. Biofarma. 55:99 (2003); Khanvilkar , et al., Drug Dev. Ind. Pharm. 228: 601 (2002); and Schmidt et al., Int. J. Pharm. 216: 9 (2001)). Sustained-release management systems can,
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80/123 depending on their preparation, release the compounds over hours or days, for example, over 4, 6, 8, 10, 12, 16, 20, 24 hours or more. Typically, sustained release formulations can be prepared using naturally occurring or synthetic polymers, for example, vinylpyrrolidone polymer, such as polyvinylpyrrolidone (PVP); hydrophilic carboxyvinyl polymers; hydrophobic and / or hydrophilic hydrocolloids, such as methylcellulose, ethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose; and carboxypolymethylene.
[00145] For oral administration, an anti-TIGIT antibody (and optionally an immuno-oncological agent) can be readily formulated in combination with pharmaceutically acceptable carriers that are well known in the art. These carriers allow the compounds to be formulated as tablets, pills, pills, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablet or pill cores. Suitable excipients include, for example, fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
[00146] The anti-TIGIT antibody (and optionally the immunooncological agent) can be formulated for parenteral administration by injection,
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81/123 for example, by bolus injection or continuous infusion. For injection, the compound or compounds may be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable oils or the like, glycerides of synthetic aliphatic acids, esters of higher aliphatic acids or propylene glycol; and, if desired, with conventional additives, such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. In some embodiments, the compounds can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or saline buffer. Injection formulations can be presented in unit dosage form, for example, in ampoules or in multiple dose containers, with an added preservative. The compositions can take these forms, as suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulating agents, such as suspending, stabilizing and / or dispersing agents.
[00147] The anti-TIGIT antibody (and optionally the immunooncological agent) can be administered systematically by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. For topical administration, the agents are formulated in ointment, creams, ointments, powders and gels. In one embodiment, the transdermal delivery agent can be DMSO. Transdermal delivery systems may include, for example, adhesives. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Exemplary transdermal delivery formulations include those described in US Patent No. 6,589,549; 6,544,548; 6,517,864; 6,512,010; 6,465,006; 6,379,696; 6,312,717 and 6,310,177, each of which are here
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82/123 incorporated by reference.
[00148] In some embodiments, a pharmaceutical composition comprises an acceptable carrier and / or excipients. A pharmaceutically acceptable carrier includes any solvents, dispersion media or coatings that are physiologically compatible and that, preferably, do not interfere or otherwise inhibit the activity of the therapeutic agent. In some embodiments, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent (s). Physiologically acceptable compounds may include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and / or buffers. Other pharmaceutically acceptable carriers and their formulations are well known and generally described in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005. Various pharmaceutically acceptable excipients are well known in the art and can be found for example in Handbook of Pharmaceutical Excipients (5th ed., Ed. Rowe et al. Pharmaceutical Press, Washington, DC).
[00149] Dosages and desired drug concentration of the pharmaceutical compositions of the disclosure may vary depending on the particular intended use. Determination of the appropriate dosage or route of administration is well within the skill of a person skilled in the art. Suitable dosages are also described in Section IV above.
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Kits [00150] In some modalities, kits are provided for use in treating a subject with cancer. In some modalities, the kit comprises:
an anti-TIGIT antibody; and an immuno-oncological agent.
[00151] In some embodiments, the anti-TIGIT antibody is as described in Section III above, for example, an anti-TIGIT antibody having a binding affinity, activity, cross-reactivity, epitope recognition and / or one or more sequences of CDR, VH and / or VL as disclosed in Section III above. In some embodiments, the immuno-oncological agent is an inhibitor of the PD-1 pathway or an inhibitor of the CTLA-4 pathway. In some embodiments, the immuno-cancer agent is an agonist of a T cell co-activator. In some embodiments, the inhibitor of the PD-1 pathway is an anti-PD-1 antibody or an anti-PD-Ll antibody. In some modalities, the immuno-oncological agent is pembrolizumab, nivolumab, durvalumab, pidilizumab or atezolizumab.
[00152] In some embodiments, the kits may also comprise instructional materials containing directions (that is, protocols) for practicing the methods of this invention (for example, instructions for using the kit to treat cancer). While instructional materials typically comprise printed or written materials, they are not limited to such. Any means capable of storing such instructions and communicating them to an end user is covered by this disclosure. Such media include, but are not limited to, electronic storage media (for example, magnetic disks, tapes, cartridges, chips), optical media (for example, CD-ROM) and the like. Such means may include addresses of internet sites that provide such instructional materials.
SAW. Examples
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84/123 [00153] The following examples are offered to illustrate, but not limit, the claimed invention.
Example 1: Generation of anti-TIGIT antibodies [00154] Fully human anti-TIGIT monoclonal antibodies were generated using a yeast base antibody presentation system (see, for example, Xu et al, “Addressing polyspecificity of antibodies selected from an in vitro yeast presentation system: a FACS-based, high-throughput selection and analytical tool ”, PEDS, 2013, 26: 663-670; WO 2009/036379; WO 2010/105256; and WO 2012/009568). Eight unexposed human libraries of synthetic yeasts, each with a diversity of ~ 10 ^9, were examined. For the first two selection cycles, a magnetic ball screening technique was performed using the Miltenyi MACS system, as described previously (see, for example, Siegel et al, “High efficiency recovery and epitope-specific sorting of an scFv yeast display library, ”J Immunol Methods, 2004, 286: 141-153). Briefly, yeast cells (~ 10 ¹⁰ cells / library) were incubated with 5 ml of biotinylated 10 nM Fe fusion antigen for 30 minutes at 30 ° C in wash buffer (phosphate buffered saline (PBS) / serum albumin) 0.1% bovine (BSA)). After washing once with 40 ml ice cold wash buffer, the cell pellet was resuspended in 20 ml of wash buffer and microspheres were added Streptavidin (500 μΐ) to the yeast and incubated for 15 minutes at 4 C. Then , the yeasts were pelleted, resuspended in 20 ml of wash buffer and loaded onto a Miltenyi LS column. After the 20 ml was transferred, the column was washed 3 times with 3 ml of wash buffer. The column was then removed from the magnetic field and the yeast was eluted with 5 ml of growth medium and then grown overnight. The following selection cycles were performed using flow cytometry. Approximately 2x10 ⁷ yeast was pelleted, washed three times with washing buffer and
Petition 870190101574, of 10/09/2019, p. 90/108 / 123 incubated at 30 ° C with 10 nM Fc fusion antigen and decreasing concentrations of biotinylated monomeric antigen (100 to 1 nM) under equilibrium conditions, 10 nM biotinylated Fc fusion antigens or 100 nM monomeric antigens from different species in order to obtain species cross-reactivity, or with a poly specificity depletion reagent (PSR) to remove non-specific antibodies from the selection. For PSR depletion, the libraries were incubated with a 1:10 dilution of biotinylated PSR reagent, as described previously (see, for example, Xu et al, supra). The yeast was then washed twice with washing buffer and stained with LC-FITC (diluted 1: 100) and with secondary reagents SA-633 (diluted 1: 500) or EA-PE (extravidin-R-PE, diluted 1 : 50) for 15 minutes at 4 ° C. After washing twice with wash buffer, the cell pellets were resuspended in 0.3 ml of wash buffer and transferred to separation tubes with filter cap. The classification was performed using a FACS ARIA classifier (BD Biosciences) and the classification doors were determined to select antibodies with desired characteristics. Selection cycles were repeated until a population with all the desired characteristics was obtained. After the final classification cycle, yeasts were plated and individual colonies were selected for characterization.
[00155] Antigens included recombinant human dimeric TIGIT-Fc (Acro Biosystems TIT-H5254), human monomeric TIGIT (Bell Biological 10917-H08H), mouse dimeric TIGIT-Fc (R&D Systems, 7267-TG) and mouse monomeric TIGIT (Bell Biologies 50939-M08H).
[00156] Campaign not exposed: 744 clones were sequenced, producing 345 unique clones (single CDRH3). 18 VH germline strains were represented in the clones.
[00157] Discontinuous diversification campaign for light chains: The
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86/123 heavy chain plasmids (VH) from a cluster of enriched ligands from the sixth cycle of unexposed discovery selections were extracted from yeast by crushing and capturing, propagated and subsequently purified from E. Coli, and then transformed into a library of light chains with a diversity of 10 ⁷ .
[00158] The selections were made essentially under the same conditions as for the unexposed discovery. Briefly, a cycle of enrichment of magnetic spheres was followed by three cycles of selections by flow cytometry. In the enrichment cycle for magnetic spheres, the biotinylated Fc fusion antigen of 10 nM was used. The first cycle in flow cytometry consisted of a positive selection cycle using 100 nM biotinylated monovalent antigen. This was followed by a second cycle, which consisted of a negative selection cycle for PSR depletion. The last (third) cycle consisted of a positive selection cycle, in which the monovalent antigen was titrated to 100 nM, 10 nM, 1 nM. For all libraries, yeasts from the 1 nM classification of this third cycle were plated and individual colonies were harvested and characterized. In total, 728 clones were sequenced, producing 350 unique HC / LC combinations (93 unique CDRH3s).
[00159] A total of 695 unique clones were identified among the unexposed and discontinuous light chain mixing campaigns.
Example 2: Characterization of anti-TIGIT Antibodies [00160] Sixty-five (65) clones were selected for production and further evaluation representing 12 VH of germline and 9 VL of germline.
Production and purification of antibodies [00161] Yeast clones were cultured to saturation and then induced for 48 h at 30 ° C with agitation. After induction, yeast cells were pelleted and supernatants were collected for
Petition 870190101574, of 10/09/2019, p. 92/108 / 123 purification. The IgGs were purified using a Protein A column and eluted with acetic acid, pH 2.0. Fab fragments were generated by papain digestion and purified using KappaSelect (GE Healthcare LifeSciences).
Binding of anti-TIGIT antibodies to recombinant human and mouse proteins [00162] ForteBio's affinity measurements were performed on an Octet RED384 generally as previously described (see, for example, Estep et al., “High throughput solution-based measurement of antibodyantigen affinity and epitope binning, ”, Mabs, 2013, 5: 270-278). Briefly, ForteBio's affinity measurements were performed by loading IgGs in line to the AHQ sensors. The sensors were balanced off line in assay buffer for 30 minutes and then monitored in line for 60 seconds to establish the baseline. Sensors with loaded IgGs were exposed to 100 nM antigen (dimeric Fe fusion antigen or monomeric antigen) for 3 minutes, and then transferred to assay buffer for 3 minutes for dissociated measurement. All binding and dissociation kinetics were analyzed using the 1: 1 binding model.
[00163] Of the 65 IgG clones, 43 had an affinity for the TIGIT monomer of <100 nM. Of the 65 IgG clones, 34 cross-reacted with mouse TIGIT-Fc. The binding affinity for selected clones is shown in Table 1 below.
Characterization of epitope / ligand competition assay [00164] The characterization of epitope / ligand block was performed using a standard sandwich cross-assay in the ForteBio Octet RED384 system. The anti-control target IgG was loaded on the AHQ sensors and the Fe-binding sites not occupied on the sensor were blocked with an irrelevant human IgG1 antibody. The sensors were
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88/123 then exposed to the 100 nM target antigen followed by a second ligand or anti-target antibody (human CD155-Fc (Sino Biological, 10109-H02H)). Additional binding by the second antibody or ligand after combining the antigen indicates an unoccupied (non-competing) epitope, while no binding indicates epitope blocking (competitor or ligand blocker).
[00165] Four characterizing antibodies (not mutually exclusive) were used for the evaluation of the bin group and five overlapping characterizing profiles were identified. 63 of the 65 antiTIGIT antibodies competed with the ligand for binding to hTIGIT-Fc. Characterizing profiles and binder competition results for selected clones are shown in Table 1 below.
Table 1. Epitope characterization data, ligand competition and affinity data for selected anti-TIGIT clones
Clone Bin group code CD Competition 155 IgG Kd of human TIGIT-Fc (M) Kd of human TIGIT (M) IgG monomer Mouse TIGIT-Fc (M) IgG Kd 2 1,2,3,4 Yes 9.56E-10 1.01E-08 2.03E-09 3 1,2,3,4 Yes 2.77E-09 7.36E-08 5.64E-09 5 1,2,3,4 Yes 9.85E-10 1.41E-08 3.25E-09 13 1,2,3 Yes 5.43E-10 2.56E-09 1.16E-10 14 1,2,3 Yes 2.01E-09 5.87E-08 2.43E-09 16 1,2,3 Yes 6.90E-10 2.06E-09 1.05E-08 18 1,2,3 Yes 2.39E-09 5.08E-08 8.82E-09 21 1,2,3 Yes 5.85E-10 2.18E-09 N.B. 22 1,2,3 Yes 7.90E-10 1.38E-08 1.05E-08 25 1,2,3 Yes 6.20E-10 6.18E-10 1.10E-09 27 1,2,3 Yes 5.58E-10 2.32E-09 N.B. 54 1,2,3 Yes 6.89E-10 3.49E-09 N.B.
Comments:
NB = Non-Binding under the conditions of this test [00166] The bin group code and CD155 competition data were generated in the ForteBio Octet RED384 system using a standard sandwich cross-block assay as described in Example 2.
[00167] KD affinity data was generated in the ForteBio Octet RED384 system as described in Example 2.
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Binding of anti-TIGIT antibodies to overexpressed human, mouse and cynomolgus monkey TIGIT in HEK 293 cells [00168] HEK 293 cells were engineered to stably express high levels of human TIGIT, mouse or monkey cynomolgus by lentiviral transduction. Approximately 100,000 parental HEK 293 cells (TIGIT negative) or HEK 293 cells that overexpress in human, mouse or cynomolgus monkey were stained with 100 nM of each anti-TIGIT antibody for 5 minutes at room temperature. The cells were then washed twice with wash buffer and incubated with anti-human IgG conjugated to PE for 15 minutes on ice. The cells were then washed twice with wash buffer and analyzed by flow cytometry on a FACS Canto II instrument (BD Biosciences). The Fold over background (FOB) calculation was done as the median fluorescence intensity (MFI) of the anti-TIGIT clone bound to target-positive cells divided by the MFI of the anti-TIGIT clone bound to target-negative cells.
[00169] As shown in Figure 1, all 65 antibodies showed specific binding to the 293-hTIGIT line (FOB> 10, as indicated by the horizontal black line in the graph). 53 clones specifically linked the 293-cyTIGIT line while 31 clones specifically linked the 293-mTIGIT line.
Polyspecificity Reagent (PSR) Assay [00170] Assessment of binding to a polyspecificity reagent was conducted to determine specificity for TIGIT as previously described (see, for example, Xu et al, supra). Briefly, the biotinylated PSR reagent diluted 1:10 of the stock was incubated with yeast-presenting IgG for 20 minutes on ice. The cells were washed and marked with EA-PE (extravidin-R-PE) and read on a FACS analyzer. The polyspecific binding score on a scale of 0 to 1 is correlated with IgG
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90/123 control with low, medium and high nonspecific binding with a score of 0 indicating no binding and a score of 1 indicating a very high nonspecific binding.
[00171] Sixty-two (62) of the 65 clones were classified as non-polyspecific ligands with a PSR score <0.10. Three clones scored as low polyspecific ligands (PSR score 0.100.33).
Hydrophobic interaction chromatography assay [00172] Hydrophobic interaction chromatography (HIC) was performed as previously described (Estep et al., Supra). Briefly, 5pg IgG samples were mixed with a mobile phase A solution (1.8 M ammonium sulfate and 0.1 M sodium phosphate at pH 6.6) to obtain a final concentration of 1 M sulfate of ammonia from fence before analysis. A Sepax Proteomix HIC butyl-NP5 column was used with a linear gradient of mobile phase A and mobile phase solution B (0.1 M sodium phosphate, pH 6.5) for 20 minutes at a flow interval of 1 mL / minute with 280 nM UV absorbance monitoring.
[00173] Increased antibody retention in hydrophobic columns has been correlated with increased hydrophobicity and a propensity for poor expression, aggregation or precipitation during purification. Five of the 65 clones had high HIC retention times> 11.5 minutes, 10 clones had an average HIC retention time of 10.5 - 11.5 minutes and the rest of the clones had low HIC retention times.
Example 3: Binding of anti-TIGIT antibodies to human, mouse and cynomolgus monkey TIGIT endogenously expressed in primary cells [00174] Sixty-five (65) antibodies that have been shown to be specific for recombinant human TIGIT protein and human TIGIT expressed in HEK cells 293 were assessed for their ability to bind to
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Endogenous TIGIT in primary human peripheral blood T cells. The antibodies were also evaluated for cross-reactivity with cynomolgus TIGIT in peripheral blood T cells and 35 of the 65 clones were evaluated for cross-reactivity with mouse TIGIT in activated splenic T cells.
[00175] Human T pan cells were negatively isolated from the leukapheresis product to 99% purity. 100,000 cells were stained at 4 ° C for 30 minutes with 20 pg / ml of each anti-TIGIT antibody. Anti-TIGIT antibodies were detected with polyclonal goat anti-human IgG conjugated to PE (Jackson ImmunoResearch 109-116-098). The samples were analyzed using a CytoFLEX flow cytometer. The percentage of TIGIT + of the lymphocyte population synchronized with FSC / SSC for each antibody was determined using only anti-human IgG staining to determine the threshold for positivity.
[00176] Cynomolgus white blood cells were isolated from whole blood by lysis of red blood cells (eBioscience 00-4300). 200,000 cells were stained at 4 ° C for 30 minutes with 20 pg / ml of each anti-TIGIT antibody. Anti-TIGIT antibodies were detected with polyclonal goat anti-human IgG adsorbed against monkey immunoglobulins conjugated with AlexaFluor647 (SouthemBiotech 2049-31) and T cells were identified by counter staining with FITC-conjugated SP34 anti-CD3 clone ( BD Pharmingen 556611). The samples were analyzed using a CytoFLEX flow cytometer. The percentage of TIGIT + of the CD3 + population was determined for each antibody using only anti-human IgG-PE staining to determine the threshold for positivity.
[00177] BALB / c mouse T cells were isolated from the spleens by negative selection (Stem Cell Technologies 19851A) to> 99% purity. The cells were activated for 24 hours with the anti-CD3 clone 145-2C11 attached to the plate (BioLegend 100302) to upregulate TIGIT.
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200,000 activated cells were stained at 4 ° C for 30 minutes with 20pg / ml of each anti-TIGIT antibody (35 of 65 clones tested). Anti-TIGIT antibodies were detected with polyclonal goat anti-human IgG conjugated to PE (Jackson ImmunoResearch 109-116-098). The samples were analyzed using a FACSCalibur flow cytometer. The median fluorescence intensity of the lymphocyte population synchronized by FSC / SSC for each antibody was determined.
[00178] Figure 2 shows the binding of 65 clones of antiTIGIT antibodies and an isotype control antibody irrelevant to primary human T cells, monkey cynomolgus and mouse. Both clones 13 and 25 showed strong binding to all three T cell species.
Titratable binding of anti-TIGIT antibodies to the cell surface expressed in TIGIT [00179] HEK 293 cells were engineered to stably express high levels of human TIGIT, mouse or monkey cynomolgus by lentiviral transduction. 200,000 293-TIGIT cells were stained at 4 ° C for 30 minutes with a 10-point triple titer (30 to 0.002 pg / ml) of each anti-TIGIT antibody. AntiTIGIT antibodies were detected with polyclonal goat anti-human IgG conjugated to PE (Jackson ImmunoResearch 109-116-098). The samples were analyzed using a CytoFLEX flow cytometer. The median fluorescence intensity of the lymphocyte population synchronized by FSC / SSC for each antibody was determined. Nonlinear regression of data transformed into Log (X) was used to generate EC50 values in GraphPad Prism 6. None of the anti-TIGIT antibodies showed binding to parental HEK 293 cells (TIGIT-) (data not shown). Figure 3A-C shows the binding titration and Figure 3D shows the binding EC50 of eight antiTIGIT antibody clones (clone 2, clone 5, clone 13, clone 16, clone 17, clone 20, clone 25, and clone 54 ) human, monkey cynomolgus and mouse TIGIT
Petition 870190101574, of 10/09/2019, p. 98/108 / 123 expressed in HEK 293 cells.
[00180] C57BL / 6 mouse T cells were isolated from the spleens by negative selection (Stem Cell Technologies 19851A) to> 99% purity. The cells were activated for 24 hours with the anti-CD3 clone 145-2C11 attached to the plate (BioLegend 100302) to upregulate TIGIT. 200,000 cells were stained at 4 ° C for 30 minutes with an 8-point triplicate (30 to 0.014 pg / ml) titration of each anti-TIGIT antibody. Anti-TIGIT antibodies were detected with polyclonal goat anti-human IgG conjugated to PE (Jackson ImmunoResearch 109-116-098). The samples were analyzed using a FACSCalibur flow cytometer. The median fluorescence intensity of the lymphocyte population synchronized by FSC / SSC for each antibody was determined. Nonlinear regression of data transformed into Log (X) was used to generate EC50 values in GraphPad Prism 6. Figure 4 shows the binding and EC50 titration of binding of anti-TIGIT clones 13 and 25 to mouse activated splenic T cells .
Example 4: Anti-TIGIT antibodies block the binding of CD155 and CD112 ligands to TIGIT expressed on the cell surface [00181] HEK 293 cells were engineered to stably express high levels of human or mouse TIGIT by lentiviral transduction. hCD155-Fc (Biological Bell 10109-H02H), hCD112-Fc (Biological Bell 10005-H02H) and mCD155-Fc (Biological Bell 50259-M03H) were conjugated to AlexaFluor647 (ThermoFisher A30009). 200,000 293-hTIGIT or 293-mTIGIT cells were co-incubated with 1 pg / ml CD155-Fc-AlexaFluor647 or 5 pg / ml CD112-Fc-AlexaFluor647 and a 12-point double titration (10 to 0.005 pg / ml) of each anti-TIGIT antibody or an isotype control antibody. The samples were analyzed using a CytoFLEX flow cytometer. The median fluorescence intensity of the lymphocyte population synchronized by FSC / SSC was determined for
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94/123 each antibody. The percentage block was calculated in relation to the MFI of the control without antibody. Non-linear regression of the data transformed into Log (X) was performed using GraphPad Prism 6.
[00182] As shown in Figure 5A-B, six clones of anti-TIGIT antibodies (clone 2, clone 5, clone 13, clone 17, clone 25 and clone 55) and five of the six clones (clone 2, clone 5 , clone 13, clone 17 and clone 25) significantly blocked the interaction of CD 155 with TIGIT expressed in HEK 293 cells for both human TIGIT / human CD155 and for mouse TIGIT / mouse CD155. Clone 55 specifically binds to human TIGIT but did not compete with hCD155-Fc for binding to hTIGIT-Fc in the ForteBio Octet ligand competition assay. Similarly, clone 55 did not effectively block the interaction of hCD155 with the 293-hTIGIT cell line. Clone 2, clone 5, clone 13, clone 17 and clone 25 were also able to disrupt the binding of human CD112 to human TIGIT. As observed for CD 155, clone 55 was much less effective in blocking the CD112-TIGIT interaction. See Figure 6.
Example 5: In vitro activity of anti-TIGIT antibodies in a TIGIT / CD155 blocking bioassay [00183] The activity of anti-TIGIT antibodies can be functionally characterized using a TIGIT / CD155 blocking bioassay (for example, TIGIT / CD155 Blockade Bioassay Kit, Promega Corp., Madison, WI), in which the expression of a reporter gene is induced or enhanced when an antibody blocks the TIGIT / CD155 interaction. The TIGIT / CD155 blocking bioassay comprises two types of cells: an effector cell expressing TIGIT, CD226 and a TCR complex on the cell surface and containing a luciferase reporter gene; and an artificial antigen presenting cell that expresses CD 155 and a TCR activator on the cell surface. In this bioassay, luciferase expression requires TCR adhesion plus a costimulatory signal. The CD155-TIGIT interaction
Petition 870190101574, of 10/09/2019, p. 100/108 / 123 has greater affinity than the CD155-CD226 interaction, resulting in liquid inhibitory signaling and without expression of luciferase. Blocking the CD155-TIGIT interaction allows for CD155-CD226 costimulation, stimulating luciferase expression.
[00184] Jurkat effector cells that express both TIGIT and CD226 were co-cultured with CHO-K1 artificial antigen presenting cells (aAPCs) that express a TCR and CD155 activator. Jurkat effector cells contain a luciferase reporter gene driven by the IL-2 promoter. In the absence of anti-TIGIT blocking antibodies, CD155-TIGIT adhesion leads to T cell co-inhibition and the absence of IL-2 promoter activity. After the addition of anti-TIGIT antibodies, the CD155-TIGIT interaction is interrupted, allowing CD 155 to associate with CD226 to send a co-stimulatory signal and boost luciferase expression.
[00185] aAPCs were plated in 96-well plates and allowed to adhere overnight. The following day, 20 pg / ml of each antiTIGIT antibody or an isotype control antibody and Jurkat effector cells were added to the plate. After a 6 hour incubation at 37 ° C, the cells were lysed and a luciferase substrate was added. Luciferase activity was quantified in a plate reader. Luciferase activity was calculated as an unfolding of the signal in the control without antibody.
[00186] As shown in Figures 7A-7B, 12 clones of anti-TIGIT antibodies demonstrated functional block in this bioassay.
Example 6: In vitro activity of anti-TIGIT antibodies in a TIGIT / PD-1 combination bioassay [00187] The synergistic activity of anti-TIGIT antibodies in combination with anti-PD-1 agents (for example, anti-PD- 1) can be functionally characterized using a TIGIT / PD-1 combination bioassay, in which the expression of a reporter gene is enhanced
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96/123 when antibodies block the TIGIT / CD155 interaction and the PD1 / PD-L1 interaction. The bioassay comprises two types of cells: an effector cell expressing TIGIT, CD226, PD-1 and a TCR complex on the cell surface and containing a luciferase reporter gene; and an artificial antigen presenting cell that expresses CD155, PD-L1 and a TCR activator on the cell surface. In this bioassay, luciferase expression requires TCR adhesion plus a costimulatory signal. The CD155-TIGIT interaction has greater affinity than the CD155-CD226 interaction, resulting in liquid inhibitory signaling and without expression of luciferase. In addition, binding of PD-L1 to PD-1 inhibits luciferase expression. Blocking the CD155-TIGIT interaction and the PD-1 / PD-L1 interaction alleviates inhibition and allows for the co-stimulation of CD155-CD226, boosting luciferase expression.
[00188] Jurkat effector cells that express PD-1, TIGIT and CD226 were co-cultured with CHOK1 artificial antigen presenting cells (aAPCs) that express a TCR activator, PD-L1 and CD 155. Jurkat effector cells contain a gene luciferase reporter directed by the IL-2 promoter. In the absence of anti-TIGIT blocking antibodies, the coupling of PD-L1-PD-1 and CD155-TIGIT leads to co-inhibition of T cells and the absence of IL-2 promoter activity. After the addition of anti-PD-1 and anti-TIGIT antibodies, the PD-L1-PD-1 interaction is blocked, releasing a coinibitory signal, and the CD155-TIGIT interaction is stopped, allowing CD 155 to associate with CD226 to send a co-stimulatory signal and boost the production of luciferase.
[00189] aAPCs were plated in 96-well plates and allowed to adhere overnight. The following day, Jurkat effector cells and a 2.5-fold titre of 10 points (100 to 0.03 pg / ml) of each anti-TIGIT antibody alone, or anti-PD-1 antibody (EH12 clone) were added to the plate. 2H7, BioLegend, San Diego, CA), or each anti-TIGIT antibody + anti-PD-1 antibody (1: 1 ratio). After a 6-hour incubation at 37 ° C, the
Petition 870190101574, of 10/09/2019, p. 102/108 / 123 cells were lysed and luciferase substrate was added. Luciferase activity was quantified in a plate reader. Luciferase activity was calculated as an unfolding of the signal in the control without antibody. As shown in Figure 8, neither anti-TIGIT nor anti-PD-1 alone led to dramatic Jurkat activation, however, the combination of both anti-TIGIT 13 and clone 25 with anti-PD-1 produced an strong activation.
Example 7: In vivo activity of anti-TIGIT antibodies in a CT26 syngenic tumor model in BALB / c mice [00190] Based on the affinity for murine TIGIT, the antiTIGIT 13 clone was chosen for evaluation in a murine syngene tumor model. Chimeras of IgGl and IgG2a were generated from mice of the parental fully human antiTIGIT clone 13 for in vivo experiments in order to address the question of whether the Fe isotype has an effect on the in vivo efficacy of antagonistic TIGIT antibodies. In vitro, the chimeric antibodies showed activity similar to the parental antibody hlgGl with respect to (1) binding to human, mouse and cynomolgus monkey TIGIT, (2) binding block of ligand CD 155 and CD 112 to TIGIT expressed on cell surface and (3) activity in the CD155-TIGIT blocking bioassay. See Figure 9A-9H.
[00191] BALB / c mice at 8 weeks of age with an average body weight of 19g were obtained from Charles River Laboratories. The mice were implanted subcutaneously with 300,000 CT26 colon cancer cells on the right lateral flank. Tumors were allowed to progress until the mean tumor volume of the group was 72 mm ³ (range 48-88 mm ³ ) on the seventh day after tumor inoculation. The animals were allocated to 10 n = IO treatment groups by matching pairs in such a way that the mean tumor volume of the group was similar in all treatment groups. The tumor length and width were measured and the tumor volume was calculated using the formula
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Volume (mm ³ ) = 0.5 * Length * Width ² , where length is the longest dimension. The 13 mlgGl anti-TIGIT, 13 mIgG2a antiTIGIT and RMP1-14 anti-PD-1 clones (BioXCell) were diluted to the appropriate concentration for dosing in sterile PBS. Sterile PBS was used as vehicle control. TIGIT antibodies were dosed at 5 or 20 mg / kg via intraperitoneal injection twice a week for 3 weeks (6 doses in total). The anti-PD-1 antibody was dosed at 5 mg / kg via intraperitoneal injection twice a week for 2 weeks (4 doses in total). Dosage started on the day of allocation (day 1 of the study). The measurements of tumor volume and body weight were collected twice a week until the mice reached a limit tumor volume of 2000 mm ³ . None of the animals exhibited loss of body weight in relation to the pre-dose weights, indicating exceptional tolerability of all test agents.
[00192] As shown in Figure 10A, anti-mPD-1 alone had no effect on tumor progression. The mlgGl anti-TIGIT chimera of clone 13 (“13-1”), which does not bind efficiently to activating Fcgamma receptors, did not mediate any antitumor activity, either as a single agent or in combination with anti-PD-1. In contrast, the mIgG2a chimera of clone 13 (“13-2”), which is capable of binding to activating Fcgamma receptors, delayed tumor progression (86.5% (5 mg / kg) or 74.4% (20 mg / kg) of tumor growth inhibition on day 18). Three out of ten animals in the 5 mg / kg single agent 13-2 group had complete tumor regressions that were stable until the end of the study (study day 46). In the single agent group 13-2 of 20 mg / kg, two of the ten animals showed partial tumor regressions (defined as tumor volume <50% of the initial volume by three consecutive measurements). Figure 10A shows that the addition of antiPD-1 to the chimera of clone 13 mIgG2a (13-2) did not increase efficacy over 13-2 alone (day 18 tumor growth inhibition) by 53.8% (5 mg / kg anti-TIGIT + 5 mg / kg anti-PD-1) vs 86.5% (5 mg / kg anti-TIGIT
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99/123 alone) and 89.6% (20 mg / kg anti-TIGIT + 5 mg / kg anti-PD-1) vs 74.4% (20 mg / kg anti-TIGIT alone). Similar numbers of complete and partial responders were observed in the combination groups. See, for example, Figures 10B-10K.
Example 8: Antibody Optimization and Optimized Antibody Characterization [00193] Antibody clones 2, 13, 16 and 25 from the production of the primary discovery were selected for further affinity maturation. The optimization of antibodies was carried out by introducing diversities in the variable region of the heavy chain. Two optimization cycles were applied to the strains above. The first cycle was composed by a diversification approach of CDRH1 and CDRH2, while in the second cycle a CDRH3 mutagenesis approach was applied.
[00194] CDRH1 and CDRH2 approach: The CDRH3 of a single antibody was recombined in a library previously prepared with variants of CDRH1 and CDRH2 of a variety of Ix10 ⁸ . The selections were then made with one MACS cycle and four FACS cycles, as described for the unexposed discovery.
[00195] In the first FACS cycle, the libraries were separated for 1 nM monomeric TIGIT binding. The second FACS cycle was a PSR depletion cycle to reduce poly-specificity. The last two cycles were positive selection cycles using parental IgG or Fab for high affinity pressure. Fab / IgG pressure was performed as follows: the antigen was incubated with parental IgG or Fab 10 times and then incubated with the yeast libraries. Enriched selections for IgGs with better affinities than parental IgG or Fab. The cross-reactivity of species was verified in the last two FACS cycles.
[00196] Mutagenesis of CDRH3: Libraries were generated with diversification of CDRH3 through randomization of positions in CDRH3.
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The selections were made with a MACS cycle and three FACS cycles, as previously described. Negative PSR selections, species cross-reactivity, affinity pressure and classification were performed to obtain a population with the desired characteristics.
Kp MSD-SET measurements [00197] Balance affinity measurements were generally performed as previously described (Estep et al., Supra). Briefly, equilibrium titrations of the solution (SET) were performed in PBS + 0.1% IgG-Free BSA (PBSF) with biotinylated human TIGIT-His monomer kept constant at 50 pM and incubated with 3 to 5 times dilutions in series of antibody from 5 nM. Antibodies (20 nM in PBS) were coated on standard binding MSD-ECL plates overnight at 4 ° C or room temperature for 30 minutes. The plates were then blocked by 1% BSA for 30 minutes with agitation at 700 rpm, followed by three washes with wash buffer (PBSF + 0.05% Tween 20). SET samples were applied and incubated on the plates for 150 seconds with shaking at 700 rpm followed by a wash. The antigen captured on a plate was detected with 250 ng / mL of sulfotag-labeled streptavidin in PBSF by incubation on the plate for 3 minutes. The plates were washed three times with wash buffer and then read on the MSD Sector Imager 2400 instrument using lx Read Buffer T with surfactant. The percentage of free antigen was plotted as a function of the antibody titrated in Prism and adjusted to a quadratic equation to extract Kd. To improve performance, liquid handling robots were used in MSD-SET experiments, including preparation of SET samples.
[00198] The binding of the optimized antibodies to His-tagged human TIGIT, TIGIT-Fc cyno and mouse TIGIT-Fc was measured using the ForteBio system as described above. Optimized antibodies have also been tested for ligand block in a
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101/123 CD15 ligand competition assay and for binding to parental HEK cell lines, human TIGIT HEK, cyno TIGIT HEK, and mouse TIGIT HEK, as described above.
[00199] Affinity data and cell binding data for affinity-optimized antibodies are shown in Table 2 below.
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Table 2. Affinity and Cell Binding Data for Affinity-Optimized Antibodies
clone index Ki> IgG from human monovalent TIGITIlis (M) (ForteBio) IgG's KoTIGIT-Fc (M) avid for cyno (ForteBio) IgG's KoTIGIT-Fc (M) avid Murino (ForteBio) Ki) IgG from human (M) TIGIT-His (MSD) Cellular connectionHuman TIGIT and IIEK cell (FOBFold OverBackground) Cellular connectionCyno TIGIT and IIEK cell (FOB Fold OverBackground) Mouse TIGIT cell link and IIEK cell (FOB Fold Over Background) 2 8.18E-09 1.34E-09 1.76E-09 AT 158 162 73 2C 5.18E-1O 9.84E-10 3.92E-10 1.60E-11 193 224 100 13 2.63E-09 1.04E-09 3.41E-10 AT 212 224 119 13A 6.27E-10 1.12E-09 3.70E-10 2.50E-11 206 240 115 13B 6.10E-10 1.05E-09 3.3OE-1O 5.30E-12 201 235 102 13C 5.63E-10 1.07E-09 3.29E-10 8.60E-12 194 281 116 13D 5.71E-10 1.16E-09 3.64E-10 5.00E-12 190 245 116 16 2.52E-09 4.67E-09 9.07E-09 AT 192 27 19 16C 9.11E-10 4.25E-09 8.01E-10 6.30E-12 208 157 99 16D 5.96E-10 1.15E-09 2.63E-09 1.30E-11 199 241 63 16E 7.78E-10 1.36E-09 3.70E-09 1.10E-11 195 186 56 25 1.27E-09 1.50E-09 9.67E-10 AT 205 247 117 25A 1.10E-09 1.64E-09 8.23E-10 1.80E-11 207 238 119 25B 1.16E-09 1.40E-09 7.19E-10 2.20E-11 222 291 129 25C 6.97E-10 1.24E-09 4.94E-10 5.60E-12 216 286 124 25D 8.46E-10 1.18E-09 5.8OE-1O 2.70E-11 225 272 137 25E 8.51E-10 1.18E-09 5.66E-10 1.30E-11 204 252 116
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Example 9: Epitope mapping [00200] The epitopes of two of the monoclonal antibodies disclosed in this document, Clone 13 and Clone, were characterized by peptide arrangement.
25. To reconstruct epitopes of the target molecule, a library of peptide-based epitope mimetics was synthesized using solid phase Fmoc synthesis. An amino-functionalized polypropylene support was obtained by grafting with a patented formulation of hydrophilic polymer, followed by reaction with t-butyloxycarbonylhexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with hydroxybenzotriazole (HOBt) and subsequent cleavage of acidic cleavage groups (TFA). Standard Fmoc peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by modified JANUS liquid handling stations (Perkin Elmer).
[00201] The synthesis of structural mimetics was performed using the patented Scaffolds Chemically Bound Peptides (CLIPS) technology (Pepscan). CLIPS technology allows you to structure peptides into single loops, double loops, triple loops, leaf folds, helix folds and their combinations. CLIPS models are coupled to cysteine residues. The multiple cysteine side chains on the peptides are coupled to one or two CLIPS models. For example, a 0.5 mM solution of P2-CLIPS (2,6-bis (bromomethyl) pyridine) is dissolved in ammonia bicarbonate (20 mM, pH 7.8) / acetonitrile (1: 3 (v / v)). This solution is added to the peptide arrangements. The CLIPS model will bind to side chains of two cysteines as if present in the solid phase bound peptides of the peptide arrays (455-well plate with 3 μΐ wells). The peptide arrangements are gently agitated in the solution for 30 to 60 minutes while completely covered in solution. Finally, the peptide arrangements are washed extensively with excess H2O and sonicated in disruption buffer containing 1% SDS / 0.1% beta-mercaptoethanol in PBS (pH
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7.2) at 70 ° C for 30 minutes, followed by sonication in H2O for another 45 minutes. The CLIPS T3 transporter peptides were produced in a similar manner but with three cysteines.
[00202] Different sets of peptides were synthesized according to the following models. Set 1 comprised a set of linear peptides having a length of 15 amino acids derived from the human TIGIT target sequence with a displacement of a residue. Set 2 comprised a set of linear peptides from Set 1, but with residues at positions 10 and 11 replaced by Ala. When a native Ala occurred in any position, he was replaced by Gly. Set 3 comprised a set of linear peptides from Set 1, which contained Cys residues. In this set, the native Cys were replaced by Cysacetamidomethyl ("Cys-acm"). Set 4 comprised a set of linear peptides having a length of 17 amino acids derived from the human TIGIT target sequence with a displacement of a residue. In positions 1 and 17 were Cys residues, used to create mimetic bonds through CLIPS mP2. Native Cys have been replaced by Cysacm. Set 6 comprised a set of linear peptides having a length of 22 amino acids derived from the human TIGIT target sequence with a displacement of a residue. Residues at positions lie 12 were replaced by the PG standard, while residues Cys were placed at positions 1 and 22 to create a mP2 restricted mimetic. Native Cys residues were replaced by Cys-acm. Set 7 contained a set of linear peptides having a length of 27 amino acids. At positions 1-11 and 17-27 were 11mer peptide sequences derived from the target sequence and joined via the GGSGG linker. The combinations were made based on information from UniProt on disulfide bonding for human TIGIT. Set 8 comprised a set of combinatorial peptides having a
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105/123 length of 33 amino acids. Positions 2-16 and 18-32 were the 15-mer peptides derived from the human TIGIT target sequence. In positions 1, 17 and 33, there were the Cys residues used to create discontinuous mimetics using T3 CLIPS.
[00203] The binding of the antibody to each of the synthesized peptides was tested in a pepscan-based ELISA. The peptide arrays were incubated with primary antibody solution (overnight at 4 ° C). After washing, the peptide arrays were incubated with a 1/1000 dilution of a goat anti-human HRP conjugate (Southern Biotech) for one hour at 25 ° C. After washing, the 2,2'-azino-di-3-ethylbenzothiazoline peroxidase substrate (ABTS) and 20 μ / ml of 3% H2O2 were added. After one hour, color development was measured. Color development was quantified with a charge-coupled device (CCD) - camera and an image processing system. The values obtained from the CCD camera range from 0 to 3000 mAU, similar to a standard 96 well ELISA plate reader.
[00204] To verify the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel. These were screened with commercial antibodies 3C9 and 57.9 (Posthumus et al., J. Virol., 1990, 64: 3304-3309).
[00205] For clone 13, when tested under conditions of high restriction, clone 13 binds weakly to the discontinuous mimetic epitope. The antibody was also tested under conditions of moderate restriction and detectable binding of the antibody was observed. The highest signal intensities were recorded with discontinuous epitope mimetics containing the central stretches 68lCNADLGWHISPSFK ₈₂ , 42lLQCHLSSTTAQV ₅₄ , 108CIYHTYPDGTYTGRI122 · A weaker additional binding was observed with peptides containing the peptide fragment 80SFKDRVG linear and simple was
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106/123 generally lower and was only observed for standards 6 ₈ ICNADLGWHISPSFK ₈₂ , 1O ₈ CIYHTYPDGTYTGRI122 and ₈₀ SFKDRVAPGPG ₉ 0.
[00206] For clone 25, when tested under conditions of high restriction, clone 25 was detectably bound to peptides from all clusters. The strongest link was observed with discontinuous epitope mimetics. Although binding to peptides containing residues in stretch 6 ₈ ICNADLGWHISPSFK ₈ 2 was also observed in other sets, binding to peptide stretch 50TTAQVTQ56 was only observed in combination with 68lCNADLGWHISPSFK ₈ 2. An additional weaker binding was observed with peptides containing stretch of peptide ₈₀ SFKDRVAPGPGLGL ₉ 3.
[00207] Based on these epitope mapping results for Clone 13 and Clone 25, refined mapping of the Clone 13 and Clone 25 epitopes was performed using the methods described above using the following sets of peptides. Set 1 comprised a library of single residue epitope mutants based on the sequence CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC. ADHIQRY waste has been subjected to replacement. Headings 1, 17, 19, 30 and 33 have not been replaced. The native Cys residues were replaced by Cys-acm (denoted “2”). Set 2 comprised a mobile mutant double wing library derived from the sequence
CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC. Headings 1, 17 and 33 have not been replaced. The native Cys residues were replaced by Cysacm. Set 3 comprised a library of single residue epitope mutants based on the sequence CKDRVAPGPGLGLTLQCI2NADLGWHISPSFKC. ADHIQRY residues were used for the replacement. Positions 1, 2, 17, 19, 30 and 33 have not been replaced. Assembly 4 comprised a double wing library
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CKDRVAPGPGLGLTLQCI2NADLGWHISPSFKC. Headings 1, 17 and 33 have not been replaced.
[00208] Clone 13 was tested with four series of discontinuous epitope mutants derived from CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC peptides and
CKDRVAPGPGLGLTLQCI2NADLGWHISPSFKC under conditions of high and moderate restriction. Data analysis indicated that, in all cases, substitutions of residues 8iFKs2 with single residues or double Ala prevented Clonel3 from binding. Unique mutations of other residues within the discontinuous epitope mimetic had no drastic binding effects. On the contrary, mutants of the double-winged epitope exhibited a more pronounced effect on binding when compared with the series of single-residue mutants for the corresponding discontinuous mimetics. It was also found that the substitutions of residues by 51AQVT55 double wing in CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC significantly impacted Clonel3 binding. Signal intensities recorded for Clone 13 with epitope mimetics derived from the CKDRVAPGPGLGLTLQCI2NADLGWHISPSFKC sequence were lower than those recorded with
CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC. It was also found that, in addition to 8iFKs2, 74GWHI77 double-wing substitutions remarkably reduce the binding of Clone 13. In addition, double-wing mutations within the 87PGPGLGL93 stretch weakened the binding somewhat.
[00209] Clone 25 was tested on four series of peptide-derived epitope mutants derived from peptides
CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC and
CKDRVAPGPGLGLTLQCI2NADLGWHISPSFKC under conditions of high and moderate restriction. The analysis of data collected from sets
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108/123 individual mutant epitopes indicated that single or double substitutions of siFKs2 residues dramatically affected binding. Substitutions for single wastes from other wastes within CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC and
CKDRVAPGPGLGLTLQCI2NADLGWHISPSFKC did not cause a noticeable decrease in signal strengths. A series of double mutant Ala mobile exhibited more pronounced effects on the binding of Clone 25 to the mimic. In addition to 8iFKs2, double-wing substitutions of residues 52AQ53 and P79 also moderately affected antibody binding to epitope mimetic
CILQ2HLSSTTAQVTQCI2NADLGWHISPSFKC. Analysis of the binding of Clone 25 to two mutant Ala series derived from CKDRVAPGPGLGLTLQCI2NADLGWHISPSFKC again confirmed the importance of 8iFKs2, but also indicated that double Ala substitutions of residues 73LGW75 and 82KDRVA86 moderately affected the binding.
[00210] In summary, for the monoclonal antibodies Clone 13 and Clone 25 it was found that residues 8iFKs2 were essential for the binding of both antibodies to the TIGIT epitope mimetic. For Clone 13, residues 51TAQVT55, 74GWHI77 and 87PGPGLGL93 were also found to contribute to binding. For Clone 25, it was also found that residues 52AQ53,73LGW75, P79 and 82KDRVA86 contribute to the binding.
Table 3. Listing of Informal Strings
Name SEQ ID NO Sequence VH protein fromClone 2 1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARGQYYYGSSSRGYYYMDVWGQGTTVTVSS VH DNA from Clone 2 2 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACCACTACATGGACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTACTAGAAACAAAGCTAACAGTTACACCACAGAATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTATCTGCAAATGAACAGCCTGAAAACCGAGGACACGGCGGTGTACTACTGCGCCAGAGGCCAGTACTACTACGGCAGCAGCAGCAGAGGTTACTACTACATGGACGTATGGGGCCAGGGAACAACCGTCACCGTCTCCTCA
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FR1 VH of Clone 2 3 EVQLVESGGGLVQPGGSLRLSCAASG CDR1 VH doClone 2 4 FTFSDHYMD FR2 VH of Clone 2 5 WVRQAPGKGLEWVG CDR2 VH doClone 2 6 RTRNKANSYTTEYAASVKG FR3 VH of Clone 2 7 RFTISRDDSKNSLYLQMNSLKTEDTAVYYC CDR3 VH doClone 2 8 ARGQYYYGSSSRGYYYMDV FR4 VH of Clone 2 9 WGQGTTVTVSS VL protein ofClones 2 and 2C 10 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAVPSPLTFGGGTKVEIK VL DNA from Clone 2 11 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTT AGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGG CTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGC ATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCA CTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGTCCCCAGTCCTCTCACTTTTGGCGGAGG GACCAAGGTTGAGATCAAA FR1 VL of Clone 2 12 EIVLTQSPGTLSLSPGERATLSC CDR1 VL dosClones 2 and 2C 13 RASQSVSSSYLA FR2 VL of Clone 2 14 WYQQKPGQAPRLLIY CDR2 VL dosClones 2 and 2C 15 GASSRAT FR3 VL of Clone 2 16 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC CDR3 VL dosClones 2 and 2C 17 QQAVPSPLT FR4 VL of Clone 2 18 FGGGTKVEIK VH protein fromClone 3 19 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARGQYYYGSSSRGYYYMDVWGQGTTVTVSS VH DNA from Clone 3 20 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTG GAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTC AGTGACCACTACATGGACTGGGTCCGCCAGGCTCCAGGGAAGG GGCTGGAGTGGGTTGGCCGTACTAGAAACAAAGCTAACAGTTA CACCACAGAATACGCCGCGTCTGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAGAACTCACTGTATCTGCAAATGAACA GCCTGAAAACCGAGGACACGGCGGTGTACTACTGCGCCAGAGG CCAGTACTACTACGGCAGCAGCAGCAGAGGTTACTACTACATG GACGTATGGGGCCAGGGA FR1 VH of Clone 3 21 EVQLVESGGGLVQPGGSLRLSCAASG CDR1 VH doClone 3 22 FTFSDHYMD FR2 VH of Clone 3 23 WVRQAPGKGLEWVG CDR2 VH doClone 3 24 RTRNKANSYTTEYAASVKG FR3 VH from Clone 3 25 RFTISRDDSKNSLYLQMNSLKTEDTAVYYC CDR3 VH doClone 3 26 ARGQYYYGSSSRGYYYMDV
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FR4 VH of Clone 3 27 WGQGTTVTVSS VL proteinClone 3 28 EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQVGPPLTFGGGTKVEIK VL DNA from Clone 3 29 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTT AGGAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGG CTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGC ATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCA CTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTAT TACTGTCAGCAGGTCGGACCCCCCCTCACTTTTGGCGGAGGGAC CAAGGTTGAGATCAAA FR1 VL of Clone 3 30 EIVLTQSPGTLSLSPGERATLSC CDR1 VL doClone 3 31 RASQSVRSSYLA FR2 VL of Clone 3 32 WYQQKPGQAPRLLIY CDR2 VL ofClone 3 33 GASSRAT FR3 VL of Clone 3 34 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC CDR3 VL doClone 3 35 QQVGPPLT FR4 VL of Clone 3 36 FGGGTKVEIK VH protein fromClone 5 37 EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGPRYQDRAGMDVWGQGTTVTVSS VH DNA from Clone 5 38 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCACCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGGGCCCCAGATACCAAGACAGGGCAGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCTCCTCA FR1 VH of Clone 5 39 EVQLLESGGGLVQPGGSLRLSCAASG CDR1 VH doClone 5 40 FTFSTYAMS FR2 VH of Clone 5 41 WVRQAPGKGLEWVS CDR2 VH doClone 5 42 AISGSGGSTYYADSVKG FR3 VH of Clone 5 43 RFTISRDNSKNTLYLQMNSLRAEDTAVYYC CDR3 VH doClone 5 44 AKGPRYQDRAGMDV FR4 VH of Clone 5 45 WGQGTTVTVSS VL proteinClone 5 46 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLATPYTFGGGTKVEIK VL DNA of Clone 5 47 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCACATCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAAAGCCTCGCCACTCCTTACACTTTTGGCGGAGGGACC AAGGTTGAGATCAAA FR1 VL of Clone 5 48 DIQMTQSPSSLSASVGDRVTITC
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CDR1 VL doClone 5 49 RASQSISSYLN FR2 VL of Clone 5 50 WYQQKPGKAPKLLIY CDR2 VL ofClone 5 51 AASSLQS FR3 VL of Clone 5 52 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR3 VL doClone 5 53 QQSLATPYT FR4 VL of Clone 5 54 FGGGTKVEIK VH protein fromClone 13 55 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGSIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSEVGAILGYVWFDPWGQGTLVTVSS VH DNA from Clone 13 56 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGCATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCCAGAGGCCCTTCTGAAGTAGGAGCAATACTCGGATATGTATGGTTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA FR1 VH of Clone 13 57 QVQLVQSGAEVKKPGSSVKVSCKASG CDR1 VH doClone 13 58 GTFSSYAIS FR2 VH of Clone 13 59 WVRQAPGQGLEWMG CDR2 VH doClone 13 60 SIIPIFGTANYAQKFQG FR3 VH of Clone 13 61 RVTITADESTSTAYMELSSLRSEDTAVYYC CDR3 VH dosClones 13 and 13A 62 ARGPSEVGAILGYVWFDP FR4 VH of Clone 13 63 WGQGTLVTVSS VL Protein from Clones 13, 13 A, 13B, 13C, and 13D 64 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARRIPITFGGGTKVEIK VL DNA of Clone 13 65 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAGGCAAGACGAATCCCTATCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA FR1 VL of Clone 13 66 DIVMTQSPLSLPVTPGEPASISC CDR1 VL of Clones 13, 13 A, 13B, 13C, and 13D 67 RSSQSLLHSNGYNYLD FR2 VL of Clone 13 68 WYLQKPGQSPQLLIY CDR2 VL of Clones 13, 13 A, 13B, 13C, and 13D 69 LGSNRAS FR3 VL of Clone 13 70 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC CDR3 VL of Clones 13, 13 A, 13B, 13C, and 13D 71 MQARRIPIT
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FR4 VL of Clone 13 72 FGGGTKVEIK VH protein fromClone 14 73 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGSIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSEVGAILGYVWFDPWGQGTLVTVSS DNAVHdo Clone 14 74 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGCATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCCAGAGGCCCTTCTGAAGTAGGAGCAATACTCGGATATGTATGGTTCGACCCATGGGGACAGGGTACATTGGTCACCGTCTCCTCA FR1 VH of Clone 14 75 QVQLVQSGAEVKKPGSSVKVSCKASG CDR1 VH doClone 14 76 GTFSSYAIS FR2 VH of Clone 14 77 WVRQAPGQGLEWMG CDR2 VH doClone 14 78 SIIPIFGTANYAQKFQG FR3 VH of Clone 14 79 RVTITADESTSTAYMELSSLRSEDTAVYYC CDR3 VH doClone 14 80 ARGPSEVGAILGYVWFDP FR4 VH of Clone 14 81 WGQGTLVTVSS VL Protein Clone 14 82 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQAKRLPLTFGGGTKVEIK VL DNA of Clone 14 83 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGT TGGGGTTTATTACTGCATGCAGGCAAAACGACTCCCTCTCACTT TTGGCGGAGGGACCAAGGTTGAGATCAAA FR1 VL of Clone 14 84 DIVMTQSPLSLPVTPGEPASISC CDR1 VL doClone 14 85 RSSQSLLHSNGYNYLD FR2 VL of Clone 14 86 WYLQKPGQSPQLLIY CDR2 VL ofClone 14 87 LGSNRAS FR3 VL of Clone 14 88 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC CDR3 VL doClone 14 89 MQAKRLPLT FR4 VL of Clone 14 90 FGGGTKVEIK VH protein fromClone 16 91 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTASYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARQSTWHKLYGTDVWGQGTTVTVSS
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DNAVH of Clone 16 92 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTG GGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTC AGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAG GGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCA AGCTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGG ACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAG ATCTGAGGACACGGCGGTGTACTACTGCGCAAGACAGAGCACCTGGCACAAATTGTACGGAACGGACGTATGGGGCCAGGGAACAA CTGTCACCGTCTCCTCA FR1 VH of Clone 16 93 QVQLVQSGAEVKKPGSSVKVSCKASG CDR1 VH doClone 16 94 GTFSSYAIS FR2 VH of Clone 16 95 WVRQAPGQGLEWMG CDR2 VH doClone 16 96 GIIPIFGTASYAQKFQG FR3 VH of Clone 16 97 RVTITADESTSTAYMELSSLRSEDTAVYYC CDR3 VH doClone 16 98 ARQSTWHKLYGTDV FR4 VH of Clone 16 99 WGQGTTVTVSS VL Protein from Clones 16, 16C, 16D, and 16E 100 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDSLPPTFGGGTKVEIK VL DNA of Clone 16 101 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAGCAGGGAGACAGTCTCCCTCCTACTTTTGGCGGAGGGACC AAGGTTGAGATCAAA FR1 VL of Clone 16 102 DIQMTQSPSSVSASVGDRVTITC CDR1 VL of Clones 16, 16C, 16D, and 16E 103 RASQGISSWLA FR2 VL of Clone 16 104 WYQQKPGKAPKLLIY CDR2 VL of Clones 16, 16C, 16D, and 16E 105 AASSLQS FR3 VL of Clone 16 106 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR3 VL of Clones 16, 16C, 16D, and 16E 107 QQGDSLPPT FR4 VL of Clone 16 108 FGGGTKVEIK VH protein fromClone 18 109 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMSWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARVRYGYADGMDVWGQGTTVTVSS
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DNAVH of Clone 18 110 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGTCATGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCCAGAGTGAGGTACGGATACGCAGACGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCTCCTCA FR1 VH of Clone 18 111 QVQLVQSGAEVKKPGASVKVSCKASG CDR1 VH doClone 18 112 YTFTSYYMS FR2 VH of Clone 18 113 WVRQAPGQGLEWMG CDR2 VH doClone 18 114 IINPSGGSTSYAQKFQG FR3 VH of Clone 18 115 RVTMTRDTSTSTV YMELS SLRSEDTAV YYC CDR3 VH doClone 18 116 ARVRYGYADGMDV FR4 VH of Clone 18 117 WGQGTTVTVSS VL proteinClone 18 118 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYHEPFTFGGGTKVEIK VL DNA of Clone 18 119 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCACATCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAAGTATACCACCTCCCTTTCACTTTTGGCGGAGGGACC AAGGTTGAGATCAAA FR1 VL of Clone 18 120 DIQMTQSPSSESASVGDRVTITC CDR1 VL doClone 18 121 RASQSISSYEN FR2 VL of Clone 18 122 WYQQKPGKAPKEEIY CDR2 VL ofClone 18 123 GASSEQS FR3 VL of Clone 18 124 GVPSRFSGSGSGTDFTETISSEQPEDFATYYC CDR3 VL doClone 18 125 QQVYHEPFT FR4 VL of Clone 18 126 FGGGTKVEIK VH protein fromClone 21 127 QEQEQESGPGEVKPSETESETCTVSGGSISSSSYYWGWIRQPPGKGEEWIGSIYYSGSTYYNPSEKSRVTISVDTSKNQFSEKESSVTAADTAVYYCARDPEYQDAPFDYWGQGTEVTVSS VH DNA from Clone 21 128 CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTT CGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATC AGCAGTAGTAGTTACTACTGGGGCTGGATCCGCCAGCCGCTGAGGGGGTCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCCG TAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTG ACCGCCGCAGACACGGCGGTGTACTACTGCGCCAGAGATCCTTT GTACCAAGACGGCCCTG
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FR1 VH21 of Clone 129 QLQLQESGPGLVKPSETLSLTCTVSG CDR1Clone 21 VH of 130 GSISSSSYYWG FR2 VH21 of Clone 131 WIRQPPGKGLEWIG CDR2Clone 21 VH of 132 SIYYSGSTYYNPSLKS FR3 VH21 of Clone 133 RVTISVDTSKNQFSLKLSSVTAADTAVYYC CDR3Clone 21 VH of 134 ARDPLYQDAPFDY FR4 VH21 of Clone 135 WGQGTLVTVSS ProteinClone 21 SAW of 136 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRANFPTFGGGTKVEIK VL DNA of Clone 21 137 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGAGAGCCAACTTCCCTACTTTTGGCGGAGGGACCAA GGTTGAGATCAAA FR1 VL21 of Clone 138 EIVLTQSPATLSLSPGERATLSC CDR1Clone 21 VL of 139 RASQSVSSYLA FR2 VL 21 of Clone 140 WYQQKPGQAPRLLIY CDR2Clone 21 VL of 141 DASNRAT FR3 VL21 of Clone 142 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC CDR3Clone 21 VL of 143 QQRANFPT FR4 VL21 of Clone 144 FGGGTKVEIK ProteinClone 22 VH of 145 QVQLQESGPGLVKPSETLSLTCAVSGYSISSGYYWAWIRQPPGKGLEWIGSIYHSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARQGYYYGSSGSVDFDLWGRGTLVTVSS VH DNA from Clone 22 146 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTT CGGAGACCCTGTCCCTCACCTGCGCTGTCTCTGGTTACTCCATC AGCAGTGGTTACTACTGGGCTTGGATCCGGCAGCCCGGGGGGGGGGGGGGGGGGGGCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAG ACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGTTCTGTGACC GCCGCAGACACGGCGGTGTACTACTGCGCCAGGCAGGGATACT ACTACGGCAGGGGGGGGAGGTACCTTGGTCACCGTCTCCTCA FR1 VH22 of Clone 147 QVQLQESGPGLVKPSETLSLTCAVSG CDR1Clone 22 VH of 148 YSISSGYYWA FR2 VH22 of Clone 149 WIRQPPGKGLEWIG CDR2Clone 22 VH of 150 SIYHSGSTYYNPSLKS FR3 VH22 of Clone 151 RVTISVDTSKNQFSLKLSSVTAADTAVYYC
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CDR3 VH doClone 22 152 ARQGYYYGSSGSVDFDL FR4 VH of Clone 22 153 WGRGTLVTVSS VL proteinClone 22 154 DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPK lliyaasnlqsgvpsrfsgsgsgtdftltisslqpedfatyycqqan SFPPWTFGGGTKVEIK VL DNA of Clone 22 155 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGGCAAATAGTCTCCCTCCTTGGACTTTTGGCGGAGGG ACCAAGGTTGAGATCAAA FR1 VL of Clone 22 156 DIQMTQSPSSVSASVGDRVTITC CDR1 VL doClone 22 157 RASQGISSWFA FR2 VL of Clone 22 158 wyqqkpgkapkeeiy CDR2 VL ofClone 22 159 aasneqs FR3 VL of Clone 22 160 gvpsrfsgsgsgtdftetisseqpedfatyyc CDR3 VL doClone 22 161 qqanseppwt FR4 VL of Clone 22 162 fgggtkveik VH protein fromClone 25 163 qvqevqsgaevkkpgasvkvsckasgytftsyaiswvrqapgqg eewmgwisayngntnyaqkeqgrvtmttdtststaymeersers ddtavyycardessfwsgdvegafdiwgqgtmvtvss VH DNA from Clone 25 164 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGCCATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCGGTGTACTACTGCGCAAGGGATTTGTCTAGCTTCTGGAGCGGAGACGTGTTAGGAGCCTTCGACATATGGGGTCAGGGTACAATGGTCACCGTCTCCTCA FR1 VH of Clone 25 165 QVQFVQSGAEVKKPGASVKVSCKASG CDR1 VH dosClones 25 and 25A 166 ytftsyais FR2 VH of Clone 25 167 wvrqapgqgeewmg Clones CDR225 and 25E 168 wisayngntnyaqkeqg FR3 VH of Clone 25 169 rvtmttdtststaymeersersddtavyyc VH CDR3 of Clones 25, 25A, and 25B 170 ardessfwsgdvegafdi FR4 VH of Clone 25 171 WGQGTMVTVSS VL Protein from Clones 25, 25A, 25B, 25C, 25D, and 25E 172 diqmtqspssesasvgdrvtitcrasqsissyenwyqqkpgkapke eiyaasseqsgvpsrfsgsgsgtdftetisseqpedfatyycqqsvpp rtfgggtkveik
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VL DNA of Clone 25 173 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCACATCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAGCAAAGCGTCCCCCCCAGGACTTTTGGCGGAGGGACCAA GGTTGAGATCAAA FR1 VL of Clone 25 174 DIQMTQSPSSLSASVGDRVTITC CDR1 VL of Clones 25, 25A, 25B, 25C, 25D, and 25E 175 RASQSISSYLN FR2 VL of Clone 25 176 WYQQKPGKAPKLLIY CDR2 VL of Clones 25, 25A, 25B, 25C, 25D, and 25E 177 AASSLQS FR3 VL of Clone 25 178 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC CDR3 VL of Clones 25, 25A, 25B, 25C, 25D, and 25E 179 QQSVPPRT FR4 VL of Clone 25 180 FGGGTKVEIK VH protein fromClone 27 181 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLSSFWSGDVLGAFDIWGQGTMVTVSS VH DNA from Clone 27 182 CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGCCATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCGGTGTACTACTGCGCAAGGGATTTGTCTAGCTTCTGGAGCGGAGACGTGTTAGGAGCCTTCGACATATGGGGTCAGGGTACAATGGTCACCGTCTCCTCA FR1 VH of Clone 27 183 QVQLVQSGAEVKKPGASVKVSCKASG CDR1 VH doClone 27 184 YTFTSYAIS FR2 VH of Clone 27 185 WVRQAPGQGLEWMG CDR2 VH doClone 27 186 WISAYNGNTNYAQKLQG FR3 VH of Clone 27 187 RVTMTTDTSTSTAYMELRSLRSDDTAVYYC CDR3 VH doClone 27 188 ARDLSSFWSGDVLGAFDI FR4 VH of Clone 27 189 WGQGTMVTVSS VL proteinClone 27 190 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQHAN HITFGGGTKVEIK
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VL DNA of Clone 27 191 GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCCACTGGTATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGAGTTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCACGCCAATCACATCACTTTTGGCGGAGGGACCAA GGTTGAGATCAAA FR1 VL of Clone 27 192 EIVMTQSPATLSVSPGERATLSC CDR1 VL doClone 27 193 RASQSVSSNLA FR2 VL of Clone 27 194 WYQQKPGQAPRLLIY CDR2 VL ofClone 27 195 GASTRAT FR3 VL of Clone 27 196 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC CDR3 VL doClone 27 197 QQHANHIT FR4 VL of Clone 27 198 FGGGTKVEIK VH protein fromClone 54 199 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARASDSYGVGLYYGMDVWGQGTTVTVSS VH DNA from Clone 54 200 CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAATAATCAACCCTAGTGGTGGTAGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCGGTGTACTACTGCGCTAGGGCATCTGACTCCTACGGAGTGGGCCTCTACTACGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCTCCTCA FR1 VH of Clone 54 201 QVQLVQSGAEVKKPGASVKVSCKASG CDR1 VH doClone 54 202 YTFTSYYMH FR2 VH of Clone 54 203 WVRQAPGQGLEWMG CDR2 VH doClone 54 204 IINPSGGSTSYAQKFQG FR3 VH of Clone 54 205 RVTMTRDTSTSTV YMELS SLRSEDTAV YYC CDR3 VH doClone 54 206 ARASDSYGVGLYYGMDV FR4 VH of Clone 54 207 WGQGTTVTVSS VL proteinClone 54 208 EIVLTQSPGTLSLSPGERATLSCRASQSVRSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYYVSPLTFGGGTKVEIK VL DNA of Clone 54 209 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCC AGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTT AGGAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGG CTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGC ATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCA CTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTAT TACTGTCAGCAGTACTACGTCAGTCCTCTCACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA FR1 VL of Clone 54 210 EIVLTQSPGTLSLSPGERATLSC
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CDR1Clone 54 VL of 211 RASQSVRSSYLA FR2 VL 54 of Clone 212 WYQQKPGQAPRLLIY CDR2Clone 54 VL of 213 GASSRAT FR3 VL 54 of Clone 214 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC CDR3Clone 54 VL of 215 QQYYVSPLT FR4 VL 54 of Clone 216 FGGGTKVEIK
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Human TIGIT cDNA: sequence (GenBank Record No. NM_173799.3)
CGTCCTATCTGCAGTCGGCTACTTTCAGTGGCAGAAGAGGCCAC
ATCTGCTTCCTGTAGGCCCTCTGGGCAGAAGCATGCGCTGGTGT
CTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGC
CTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATT
TCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTC
CTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAG
GACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACAT
CTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGG
GCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTA
CTTCTGCATCTATCACACCTACCCTGATGGGACGTACACTGGGA
GAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGG
TGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGC
TGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACT
AGAAAGAAGAAAGCCCTCAGAATCCATTCTGTGGAAGGTGACC
TCAGGAGAAAATCAGCTGGACAGGAGGAATGGAGCCCCAGTGC
TCCCTCACCCCCAGGAAGCTGTGTCCAGGCAGAAGCTGCACCTG
CTGGGCTCTGTGGAGAGCAGCGGGGAGAGGACTGTGCCGAGCT
GCATGACTACTTCAATGTCCTGAGTTACAGAAGCCTGGGTAACT
GCAGCTTCTTCACAGAGACTGGTTAGCAACCAGAGGCATCTTCT
GGAAGATACACTTTTGTCTTTGCTATTATAGATGAATATATAAG
CAGCTGTACTCTCCATCAGTGCTGCGTGTGTGTGTGTGTGTGTA
TGTGTGTGTGTGTTCAGTTGAGTGAATAAATGTCATCCTCTTCTC
CATCTTCATTTCCTTGGCCTTTTCGTTCTATTCCATTTTGCATTAT
GGCAGGCCTAGGGTGAGTAACGTGGATCTTGATCATAAATGCA
AAATTAAAAAATATCTTGACCTGGTTTTAAATCTGGCAGTTTGA
GCAGATCCTATGTCTCTGAGAGACACATTCCTCATAATGGCCAG
CATTTTGGGCTACAAGGTTTTGTGGTTGATGATGAGGATGGCAT
GACTGCAGAGCCATCCTCATCTCATTTTTTCACGTCATTTTCAGT
AACTTTCACTCATTCAAAGGCAGGTTATAAGTAAGTCCTGGTAG
CAGCCTCTATGGGGAGATTTGAGAGTGACTAAATCTTGGTATCT
GCCCTCAAGAACTTACAGTTAAATGGGGAGACAATGTTGTCAT
GAAAAGGTATTATAGTAAGGAGAGAAGGAGACATACACAGGC
CTTCAGGAAGAGACGACAGTTTGGGGTGAGGTAGTTGGCATAG
GCTTATCTGTGATGAAGTGGCCTGGGAGCACCAAGGGGATGTT
GAGGCTAGTCTGGGAGGAGCAGGAGTTTTGTCTAGGGAACTTG
TAGGAAATTCTTGGAGCTGAAAGTCCCACAAAGAAGGCCCTGG
CACCAAGGGAGTCAGCAAACTTCAGATTTTATTCTCTGGGCAGG
CATTTCAAGTTTCCTTTTGCTGTGACATACTCATCCATTAGACAG
CCTGATACAGGCCTGTAGCCTCTTCCGGCCGTGTGTGCTGGGGA
AGCCCCAGGAAACGCACATGCCCACACAGGGAGCCAAGTCGTA
GCATTTGGGCCTTGATCTACCTTTTCTGCATCAATACACTCTTGA
GCCTTTGAAAAAAGAACGTTTCCCACTAAAAAGAAAATGTGGA
TTTTTAAAATAGGGACTCTTCCTAGGGGAAAAAGGGGGGCTGG
GAGTGATAGAGGGTTTAAAAAATAAACACCTTCAAACTAACTT
CTTCGAACCCTTTTATTCACTCCCTGACGACTTTGTGCTGGGGTT
GGGGTAACTGAACCGCTTATTTCTGTTTAATTGCATTCAGGCTG
GATCTTAGAAGACTTTTATCCTTCCACCATCTCTCTCAGAGGAA
TGAGCGGGGAGGTTGGATTTACTGGTGACTGATTTTCTTTCATG
GGCCAAGGAACTGAAAGAGAATGTGAAGCAAGGTTGTGTCTTG
CGCATGGTTAAAAATAAAGCATTGTCCTGCTTCCTAAGACTTAG
ACTGGGGTTGACAATTGTTTTAGCAACAAGACAATTCAACTATT
TCTCCTAGGATTTTTATTATTATTATTTTTTCACTTTTCTACCAAA
TGGGTTACATAGGAAGAATGAACTGAAATCTGTCCAGAGCTCC
AAGTCCTTTGGAAGAAAGATTAGATGAACGTAAAAATGTTGTT
GTTTGCTGTGGCAGTTTACAGCATTTTTCTTGCAAAATTAGTGC
AAATCTGTTGGAAATAGAACACAATTCACAAATTGGAAGTGAA
CTAAAATGTAATGACGAAAAGGGAGTAGTGTTTTGATTTGGAG
GAGGTGTATATTCGGCAGAGGTTGGACTGAGAGTTGGGTGTTAT
TTAACATAATTATGGTAATTGGGAAACATTTATAAACACTATTG
GGATGGTGATAAAATACAAAAGGGCCTATAGATGTTAGAAATG
GGTCAGGTTACTGAAATGGGATTCAATTTGAAAAAAATTTTTTT
AAATAGAACTCACTGAACTAGATTCTCCTCTGAGAACCAGAGA
AGACCATTTCATAGTTGGATTCCTGGAGACATGCGCTATCCACC
ACGTAGCCACTTTCCACATGTGGCCATCAACCACTTAAGATGGG
GTTAGTTTAAATCAAGATGTGCTGTTATAATTGGTATAAGCATA
TATGAATAATAAGAATACTATTTCAGTAGTTTTGGTATATTGTG
10/9/2019, p. 127/176
AAATCACACTAGATTCTGGAGATTTAA
121/123
Human TIGIT protein (GenBank Registration No.NP_776160.2) 218 MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPEEGAMAATEVVICTAVIVVVAETRKKKAERIHSVEGDERRKSAGQEEWSPSAPSPPGSCVQAEAAPAGECGEQRGEDCAEEHDYFNV ESYRSEGNCSFFTETG TIGIT protein fromcynomolgus monkey 219 MAFEVAPPMQFVYEEKTECVFNMVFAKPGFSETVFSHRESFTVESAVGYFRWQKRPHEEPVSPEGRSMRWCEFEIWAQGERQAPEASGMMTGTIETTGNISAKKGGSVIEQCHESSTMAQVTQVNWEQHDHSEEAIRNAEEGWHIYPAFKDRVAPGPGEGETEQSETMNDTGEYFCTYHTYPDGTYRGRIFEEVEESSVAEHSARFQIPEEGAMAMMEVVICIAVIVVVVEARKKKSERIHSVESGEQRKSTGQEEQIPSAPSPPGSCVQAEAAPAGECGEQQGDDCAEEHDYFNVESYRSEGSCSFFTETG Mouse TIGIT protein 220 MHGWEEEVWVQGEIQAAFEATGATAGTIDTKRNISAEEGGSVIEQCHFSSDTAEVTQVDWKQQDQEEAIYSVDEGWHVASVFSDRVVPGPSEGETFQSETMNDTGEYFCTYHTYPGGIYKGRIFEKVQESSVAQFQTAPEGGTMAAVEGEICEMVTGVTVEARKKSIRMHSIESGEGRTE AEPQEWNERSESSPGSPVQTQTAPAGPCGEQAEDDYADPQEYFNV ESYRSEESFIAVSKTG CDR1 VH doClone 2C 221 FTFTDYYMD CDR2 VH doClone 2C 222 RTRNKVNSYYTEYAASVKG CDR3 VH doClone 2C 223 ARGQYYYGSDRRGYYYMDV VH CDR1 of Clones 13A, 13C, and 13D 224 GTFESSAIS CDR2 VH doClone 13A 225 SIIPYFGTANYAQKFQG CDR1 VH doClone 13B 226 GTFSAWAIS CDR2 VH dosClones 13B and 13D 227 SIIPYFGKANYAQKFQG CDR3 VH doClone 13B 228 ARGPSEVSGIEGYVWFDP CDR2 VH doClone 13C 229 SIIPEFGKANYAQKFQG CDR3 VH dosClones 13C and 13D 230 ARGPSEVKGIEGYVWFDP CDR1 VH doClone 16C 231 GTFREYAIS CDR2 VH doClone 16C 232 GIHPIFGTARYAQKFQG CDR1 VH dosClones 16D and 16E 233 GTFSDYPIS VH CDR2 of Clones 16B, 16D and 16E 234 GIIPIVGGANYAQKFQG CDR3 VH doClone 16C 235 TRQSTWHKEYGTDV CDR3 VH do16D Clone 236 TRQSTWHKEFGTDV CDR3 VH doClone 16E 237 ARQSTWHKVYGTDV CDR2 VH doClone 25A 238 WISAYNGNTKYAQKEQG VH CDR1 of Clones 25B, 25C, and 25D 239 YTFTSYPIG
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VH CDR2 of Clones 25B, 25C, and 25D 240 WISSYNGNTNYAQKLQG CDR3 VH doClone 25C 241 ARG AS SFWSGD VLG AFDI CDR3 VH doClone 25D 242 ARDLKSFWSGDVLGAFDI CDR1 VH doClone 25E 243 YTFTSYAIA CDR3 VH doClone 25E 244 ARSGSSFWSGDVLGAFDI VH of Clone 2C 245 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGLEWVGRTRNKVNSYYTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARGQYYYGSDRRGYYYMDVWGQGTTVTVSS VH of Clone 13A 246 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEWMGSLIPYFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSEVGAILGYVWFDPWGQGTLVTVSS VH of Clone 13B 247 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSAWAISWVRQAPGQGLEWMGSIIPYFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSEVSGILGYVWFDPWGQGTLVTVSS ClH 13C VH 248 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEWMGSIIPLFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSEVKGILGYVWFDPWGQGTLVTVSS VH of Clone 13D 249 QVQLVQSGAEVKKPGSSVKVSCKASGGTFLSSAISWVRQAPGQGLEWMGSIIPYFGKANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGPSEVKGILGYVWFDPWGQGTLVTVSS ClH 16C VH 250 QVQLVQSGAEVKKPGSSVKVSCKASGGTFREYAISWVRQAPGQGLEWMGGIHPIFGTARYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRQSTWHKLYGTDVWGQGTTVTVSS VH of Clone 16D 251 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYPISWVRQAPGQGLEWMGGIIPIVGGANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCTRQSTWHKLFGTDVWGQGTTVTVSS ClH 16E VH 252 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSDYPISWVRQAPGQGLEWMGGIIPIVGGANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARQSTWHKVYGTDVWGQGTTVTVSS VH of Clone 25A 253 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAISWVRQAPGQGLEWMGWISAYNGNTKYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLSSFWSGDVLGAFDIWGQGTMVTVSS ClH 25B VH 254 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYPIGWVRQAPGQGLEWMGWISSYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLSSFWSGDVLGAFDIWGQGTMVTVSS ClH 25C VH 255 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYPIGWVRQAPGQGLEWMGWISSYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGASSFWSGDVLGAFDIWGQGTMVTVSS VH of Clone 25D 256 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYPIGWVRQAPGQGLEWMGWISSYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLKSFWSGDVLGAFDIWGQGTMVTVSS VH of Clone 25E 257 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAIAWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSGSSFWSGDVLGAFDIWGQGTMVTVSS HTIGIT 68-82 epitope 258 ICNADLGWHISPSFK
[00211] Although the invention set out above has been described in some detail by way of illustration and example for the sake of clarity of understanding, a person skilled in the art will appreciate that many modifications and variations of this invention can be made without departing from the
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123/123 its spirit and scope. The specific modalities described in this document are offered by way of example only and are not intended to be limiting in any way. It is intended that the specification and the examples are considered as examples only, with the true scope and spirit of the invention being indicated by the following claims. [00212] All publications, patents, patent applications and / or other documents cited in this document are incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, patent application and / or another document was individually indicated to be incorporated as a reference for all purposes.

权利要求:
Claims (69)
[1]
1. Isolated antibody, or its antigen-binding portion, that binds to human TIGIT (T-cell immunoreceptor with Ig and ITIM domains), characterized by the fact that the antibody or its antigen-binding portion has a binding affinity (KD) for human TIGIT less than 5 nM.
[2]
An isolated antibody according to claim 1, characterized by the fact that the antibody or its antigen-binding portion has a KD for human TIGIT of less than 1 nM.
[3]
An isolated antibody according to claim 1, characterized by the fact that the antibody or its antigen binding portion has a KD for human TIGIT below 100 pM.
[4]
An isolated antibody according to any one of claims 1 to 3, characterized in that the antibody or its antigen-binding portion is cross-reactive with monkey cynomolgus TIGIT and / or mouse TIGIT.
[5]
An isolated antibody according to claim 4, characterized by the fact that the antibody or its antigen-binding portion is cross-reactive with monkey cynomolgus TIGIT and mouse TIGIT.
[6]
An isolated antibody according to any one of claims 1 to 5, characterized in that the antibody or its antigen-binding portion blocks the binding of CD 155 to TIGIT.
[7]
An isolated antibody according to any one of claims 1 to 5, characterized in that the antibody or its antigen-binding portion blocks the binding of CD112 to TIGIT.
[8]
An isolated antibody according to any one of claims 1 to 5, characterized in that the antibody or its antigen-binding portion blocks the binding of CD 155 and CD112 to TIGIT.
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2/19
[9]
An isolated antibody according to any one of claims 1 to 8, characterized in that the antibody or its antigen-binding portion binds to an epitope in human TIGIT that comprises one or both of the amino acid positions 81 and 82.
[10]
An isolated antibody according to claim 9, characterized by the fact that the epitope comprises Phe at position 81.
[11]
An isolated antibody according to claim 8, characterized by the fact that the epitope comprises Lys or Ser at position 82.
[12]
Isolated antibody according to any one of claims 9 to 11, characterized in that the epitope comprises Phe at position 81 and Lys or Ser at position 82.
[13]
An isolated antibody according to claim 12, characterized by the fact that the epitope comprises Phe81 and Lys82.
[14]
An isolated antibody according to any one of claims 9 to 13, characterized in that the epitope is a discontinuous epitope.
[15]
An isolated antibody according to any one of claims 9 to 14, characterized in that the antibody or its antigen-binding portion binds to an epitope in human TIGIT that further comprises one or more of the amino acid positions 51, 52 , 53, 54, 55, 73, 74, 75, 76, 77, 79, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93.
[16]
16. Isolated antibody according to claim 15, characterized by the fact that the epitope further comprises one or more amino acid residues selected from the group consisting of Thr51, Ala52, Gln53, Val54, Thr55, Leu73, Gly74, Trp75, His76, Ile77 , Pro79, Asp83, Arg84, Val85, Ala86, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93.
[17]
17. Isolated antibody according to claim 16, characterized by the fact that the epitope comprises the residues of
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3/19 amino acids Thr51, Ala52, Gln53, Val54, Thr55, Gly74, Trp75, His76, Ile77, Phe81, Lys82, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93.
[18]
18. Isolated antibody according to claim 16, characterized in that the epitope comprises amino acid residues Ala52, Gln53, Leu73, Gly74, Trp75, Pro79, Phe81, Lys82, Asp83, Arg84, Val85 and Ala86.
[19]
19. Isolated antibody according to any of claims 9 to 18, characterized in that the epitope comprises the sequence ICNADLGWHISPSFK.
[20]
20. Isolated antibody according to any one of claims 1 to 19, characterized in that the antibody or its antigen-binding portion comprises one or more of:
(a) a heavy chain CDR1 comprising the sequence of any of SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 76, SEQ ID NO: 94 , SEQ ID NO: 112, SEQ ID NO: 130, SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 239, or SEQ ID NO: 243;
(b) a heavy chain CDR2 comprising the sequence of any of SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 42, SEQ ID NO: 60, SEQ ID NO: 78, SEQ ID NO: 96 , SEQ ID NO: 114, SEQ ID NO: 132, SEQ ID NO: 150, SEQ ID NO: 168, SEQ ID NO: 186, SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 238, or SEQ ID NO: 240;
(c) a heavy chain CDR3 comprising the sequence of any of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80, SEQ ID NO: 98 , SEQ ID NO: 116, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 170, SEQ ID NO: 188, SEQ ID NO: 206, SEQ ID NO: 223, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID
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4/19
NO: 236, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 244;
(d) a light chain CDR1 comprising the sequence of any of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 85, SEQ ID NO: 103 , SEQ ID NO: 121, SEQ ID NO: 139, SEQ ID NO: 157, SEQ ID NO: 175, SEQ ID NO: 193, or SEQ ID NO: 211;
(e) a light chain CDR2 comprising the sequence of any of SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 87, SEQ ID NO: 105 , SEQ ID NO: 123, SEQ ID NO: 141, SEQ ID NO: 159, SEQ ID NO: 177, SEQ ID NO: 195, or SEQ ID NO: 213; or (f) a light chain CDR3 comprising the sequence of any of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, SEQ ID NO: 89, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 143, SEQ ID NO: 161, SEQ ID NO: 179, SEQ ID NO: 197, or SEQ ID NO: 215.
[21]
21. An isolated antibody according to claim 20, characterized in that the antibody or its antigen binding portion comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR and CDR3 comprising the sequences of:
(a) SEQ ID NOs: 4, 6, 8, 13, 15 and 17, respectively; or (b) SEQ ID NOs: 22, 24, 26, 31, 33 and 35, respectively; or (c) SEQ ID NOs: 40, 42, 44, 49, 51 and 53, respectively; or (d) SEQ ID NOs: 58, 60, 62, 67, 69 and 71, respectively; or (e) SEQ ID NOs: 76, 78, 80, 85, 87 and 89, respectively; or (f) SEQ ID NOs: 94, 96, 98, 103, 105 and 107, respectively; or (g) SEQ ID NOs: 112, 114, 116, 121, 123 and 125, respectively; or (h) SEQ ID NOs: 130, 132, 134, 139, 141 and 143, respectively; or
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5/19 (i) SEQ ID NOs: 148, 150, 152, 157, 159 and 161, respectively; or (j) SEQ ID NOs: 166, 168, 170, 175, 177 and 179, respectively; or (k) SEQ ID NOs: 184, 186, 188, 193, 195 and 197, respectively; or (l) SEQ ID NOs: 202, 204, 206, 211, 213 and 215, respectively; or (m) SEQ ID NOs: 221, 222, 223, 13, 15 and 17, respectively; or (n) SEQ ID NOs: 224, 225, 62, 67, 69 and 71, respectively; or (o) SEQ ID NOs: 226, 227, 228, 67, 69 and 71, respectively; or (p) SEQ ID NOs: 224, 229, 230, 67, 69 and 71, respectively; or (q) SEQ ID NOs: 224, 227, 230, 67, 69 and 71, respectively; or (r) SEQ ID NOs: 231, 232, 235, 103, 105 and 107, respectively; or (s) SEQ ID NOs: 233, 234, 236, 103, 105 and 107, respectively; or (t) SEQ ID NOs: 233, 234, 237, 103, 105 and 105, respectively; or (u) SEQ ID NOs: 166, 238, 170, 175, 177 and 179, respectively; or (v) SEQ ID NOs: 239, 240, 170, 175, 177 and 179, respectively; or (w) SEQ ID NOs: 239, 240, 241, 175, 177 and179,
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6/19 respectively; or (x) SEQ ID NOs: 239, 240, 242, 175, 177 and 179, respectively; or (y) SEQ ID NOs: 243, 168, 244, 175, 177 and 179, respectively.
[22]
22. Isolated antibody according to any one of claims 1 to 21, characterized in that the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQID
NO: 145, SEQ ID NO: 163, SEQ ID NO: 181, SEQ ID NO: 199, SEQID
NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQID
NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQID
NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257; and / or (b) a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208 .
[23]
23. Isolated antibody according to claim 22, characterized in that the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 245 and a light chain variable region comprising an amino acid sequence that has fur
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7/19 minus 90% sequence identity to SEQ ID NO: 10; or (b) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 28; or (c) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 37 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46; or (d) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 55, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO : 248, or SEQ ID NO: 249 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 64; or (e) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 73 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 82; or (f) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 91, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 100; or (g) a heavy chain variable region comprising a
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8/19 amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 109 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 118 ; or (h) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 136; or (i) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 145 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 154; or (j) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 163, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO : 255, SEQ ID NO: 256, or SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 172; or (k) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 181 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 190; or (l) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 199 and a light chain variable region comprising an amino acid sequence that has at least 90%
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9/19 sequence identity to SEQ ID NO: 208.
[24]
24. Isolated antibody according to any one of claims 1 to 23, characterized in that the antibody or its antigen-binding portion is synergistic with an anti-PD1 antibody or an anti-PD-Ll antibody.
[25]
25. Isolated antibody, or its antigen-binding portion, that binds to human TIGIT, characterized by the fact that the antibody or its antigen-binding portion binds to an epitope in human TIGIT that comprises the positions of amino acids 81 and 82.
[26]
26. Isolated antibody according to claim 25, characterized by the fact that the epitope comprises Phe at position 81 and / or Lys or Ser at position 82.
[27]
27. Isolated antibody according to claim 26, characterized in that the epitope comprises Phe81 and Lys82.
[28]
28. Isolated antibody according to any one of claims 25 to 27, characterized in that the epitope is a discontinuous epitope.
[29]
29. An isolated antibody according to any one of claims 25 to 28, characterized in that the antibody or its antigen-binding portion binds to an epitope in human TIGIT that further comprises one or more of the amino acid positions 51, 52 , 53, 54, 55, 73, 74, 75, 76, 77, 79, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93.
[30]
30. Isolated antibody according to claim 29, characterized in that the epitope further comprises one or more amino acid residues selected from the group consisting of Thr51, Ala52, Gln53, Val54, Thr55, Leu73, Gly74, Trp75, His76, Ile77 , Pro79, Asp83, Arg84, Val85, Ala86, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93.
[31]
31. The isolated antibody according to claim 30, characterized by the fact that the epitope comprises residues of
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10/19 amino acids Thr51, Ala52, Gln53, Val54, Thr55, Gly74, Trp75, His76, Ile77, Phe81, Lys82, Pro87, Gly88, Pro89, Gly90, Leu91, Gly92 and Leu93.
[32]
32. Isolated antibody according to claim 30, characterized in that the epitope comprises amino acid residues Ala52, Gln53, Leu73, Gly74, Trp75, Pro79, Phe81, Lys82, Asp83, Arg84, Val85 and Ala86.
[33]
33. Isolated antibody according to any of claims 25 to 32, characterized in that the epitope comprises the sequence ICNADLGWHISPSFK.
[34]
34. An isolated antibody according to any one of claims 25 to 32, characterized in that the antibody or its antigen-binding portion comprises one or more of:
(a) a heavy chain CDR1 comprising the sequence of any of SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 76, SEQ ID NO: 94 , SEQ ID NO: 112, SEQ ID NO: 130, SEQ ID NO: 148, SEQ ID NO: 166, SEQ ID NO: 184, SEQ ID NO: 202, SEQ ID NO: 221, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 239, or SEQ ID NO: 243;
(b) a heavy chain CDR2 comprising the sequence of any of SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 42, SEQ ID NO: 60, SEQ ID NO: 78, SEQ ID NO: 96 , SEQ ID NO: 114, SEQ ID NO: 132, SEQ ID NO: 150, SEQ ID NO: 168, SEQ ID NO: 186, SEQ ID NO: 204, SEQ ID NO: 222, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 238, or SEQ ID NO: 240;
(c) a heavy chain CDR3 comprising the sequence of any of SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 44, SEQ ID NO: 62, SEQ ID NO: 80, SEQ ID NO: 98 , SEQ ID NO: 116, SEQ ID NO: 134, SEQ ID NO: 152, SEQ ID NO: 170, SEQ ID NO: 188, SEQ ID NO: 206, SEQ ID NO: 223, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 235, SEQ ID
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11/19
NO: 236, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 242, or SEQ ID NO: 244;
(d) a light chain CDR1 comprising the sequence of any of SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 85, SEQ ID NO: 103 , SEQ ID NO: 121, SEQ ID NO: 139, SEQ ID NO: 157, SEQ ID NO: 175, SEQ ID NO: 193, or SEQ ID NO: 211;
(e) a light chain CDR2 comprising the sequence of any of SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 87, SEQ ID NO: 105 , SEQ ID NO: 123, SEQ ID NO: 141, SEQ ID NO: 159, SEQ ID NO: 177, SEQ ID NO: 195, or SEQ ID NO: 213; or (f) a light chain CDR3 comprising the sequence of any of SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 53, SEQ ID NO: 71, SEQ ID NO: 89, SEQ ID NO: 107, SEQ ID NO: 125, SEQ ID NO: 143, SEQ ID NO: 161, SEQ ID NO: 179, SEQ ID NO: 197, or SEQ ID NO: 215.
[35]
35. An isolated antibody according to claim 34, characterized in that the antibody or its antigen-binding portion comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR and CDR3 comprising the sequences of:
(a) SEQ ID NOs: 4, 6, 8, 13, 15 and 17, respectively; or (b) SEQ ID NOs: 22, 24, 26, 31, 33 and 35, respectively; or (c) SEQ ID NOs: 40, 42, 44, 49, 51 and 53, respectively; or (d) SEQ ID NOs: 58, 60, 62, 67, 69 and 71, respectively; or (e) SEQ ID NOs: 76, 78, 80, 85, 87 and 89, respectively; or (f) SEQ ID NOs: 94, 96, 98, 103, 105 and 107, respectively; or (g) SEQ ID NOs: 112, 114, 116, 121, 123 and 125, respectively; or (h) SEQ ID NOs: 130, 132, 134, 139, 141 and 143, respectively; or
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12/19 (i) SEQ ID NOs: 148, 150, 152, 157, 159 and 161, respectively; or (j) SEQ ID NOs: 166, 168, 170, 175, 177 and 179, respectively; or (k) SEQ ID NOs: 184, 186, 188, 193, 195 and 197, respectively; or (l) SEQ ID NOs: 202, 204, 206, 211, 213 and 215, respectively; or (m) SEQ ID NOs: 221, 222, 223, 13, 15 and 17, respectively; or (n) SEQ ID NOs: 224, 225, 62, 67, 69 and 71, respectively; or (o) SEQ ID NOs: 226, 227, 228, 67, 69 and 71, respectively; or (p) SEQ ID NOs: 224, 229, 230, 67, 69 and 71, respectively; or (q) SEQ ID NOs: 224, 227, 230, 67, 69 and 71, respectively; or (r) SEQ ID NOs: 231, 232, 235, 103, 105 and 107, respectively; or (s) SEQ ID NOs: 233, 234, 236, 103, 105 and 107, respectively; or (t) SEQ ID NOs: 233, 234, 237, 103, 105 and 105, respectively; or (u) SEQ ID NOs: 166, 238, 170, 175, 177 and 179, respectively; or (v) SEQ ID NOs: 239, 240, 170, 175, 177 and 179, respectively; or (w) SEQ ID NOs: 239, 240, 241, 175, 177 and179,
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13/19 respectively; or (x) SEQ ID NOs: 239, 240, 242, 175, 177 and 179, respectively; or (y) SEQ ID NOs: 243, 168, 244, 175, 177 and 179, respectively.
[36]
36. An isolated antibody according to claim 34, characterized in that the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 37, SEQ ID NO: 55, SEQ ID NO: 73, SEQ ID NO: 91, SEQ ID NO: 109, SEQ ID NO: 127, SEQID
NO: 145, SEQ ID NO: 163, SEQ ID NO: 181, SEQ ID NO: 199, SEQID
NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQID
NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQID
NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 257; and / or (b) a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 10, SEQ ID NO: 28, SEQ ID NO: 46, SEQ ID NO: 64, SEQ ID NO: 82, SEQ ID NO: 100, SEQ ID NO: 118, SEQ ID NO: 136, SEQ ID NO: 154, SEQ ID NO: 172, SEQ ID NO: 190, or SEQ ID NO: 208 .
[37]
37. An isolated antibody according to claim 36, characterized in that the antibody or its antigen-binding portion comprises:
(a) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 245 and a light chain variable region comprising an amino acid sequence that has fur
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14/19 minus 90% sequence identity to SEQ ID NO: 10; or (b) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 19 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 28; or (c) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 37 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 46; or (d) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 55, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO : 248, or SEQ ID NO: 249 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 64; or (e) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 73 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 82; or (f) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 91, SEQ ID NO: 250, SEQ ID NO: 251, or SEQ ID NO: 252 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 100; or (g) a heavy chain variable region comprising a
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15/19 amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 109 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 118 ; or (h) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 127 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 136; or (i) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 145 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 154; or (j) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to any of SEQ ID NO: 163, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO : 255, SEQ ID NO: 256, or SEQ ID NO: 257 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 172; or (k) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 181 and a light chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 190; or (l) a heavy chain variable region comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 199 and a light chain variable region comprising an amino acid sequence that has at least 90%
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16/19 sequence identity to SEQ ID NO: 208.
[38]
38. Isolated antibody according to any one of claims 1 to 37, characterized in that the antibody is a monoclonal antibody.
[39]
39. Isolated antibody according to any one of claims 1 to 38, characterized in that the antibody is a humanized antibody.
[40]
40. Isolated antibody according to any one of claims 1 to 38, characterized in that the antibody is a fully human antibody.
[41]
41. Isolated antibody according to any one of claims 1 to 38, characterized in that the antibody is a chimeric antibody.
[42]
42. An isolated antibody according to any of claims 1 to 38, characterized in that the antigen binding fragment is a Fab, an F (ab ') 2, a scFv, or a divalent scFv.
[43]
43. Pharmaceutical composition, characterized in that it comprises the isolated antibody as defined in any one of claims 1 to 42, and a pharmaceutically acceptable carrier.
[44]
44. Bispecific antibody, characterized in that it comprises the antibody as defined in any one of claims 1 to 42.
[45]
45. Antibody-drug conjugate, characterized in that it comprises the antibody as defined in any one of claims 1 to 42.
[46]
46. Isolated polynucleotide, characterized in that it comprises a nucleotide sequence that encodes the antibody as defined in any one of claims 1 to 42.
[47]
47. Isolated polynucleotide, characterized by the fact that
Petition 870190101572, of 10/09/2019, p. 146/176
17/19 comprises a nucleotide sequence encoding an antibody, or its antigen-binding portion, which binds to human TIGIT, in which the isolated polynucleotide comprises:
(a) the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 38, SEQ ID NO: 56, SEQ ID NO: 74, SEQ ID NO: 92, SEQ ID NO: 110, SEQ ID NO: 128, SEQ ID NO: 146, SEQ ID NO: 164, SEQ ID NO: 182, or SEQ ID NO: 200; and / or (b) the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 29, SEQ ID NO: 47, SEQ ID NO: 65, SEQ ID NO: 83, SEQ ID NO: 101, SEQ ID NO: 119, SEQ ID NO: 137, SEQ ID NO: 155, SEQ ID NO: 173, SEQ ID NO: 191, or SEQ ID NO: 209.
[48]
48. Vector, characterized by the fact that it comprises the polynucleotide as defined in claim 46 or 47.
[49]
49. Host cell, characterized by the fact that it comprises the polynucleotide as defined in claim 46 or 47, or the vector as defined in claim 48.
[50]
50. Method of producing an antibody, characterized in that it comprises culturing the host cell as defined in claim 49, under conditions suitable for the production of the antibody.
[51]
51. Kit, characterized by the fact that it comprises:
the isolated antibody as defined in any one of claims 1 to 42, the pharmaceutical composition as defined in claim 43; and an immuno-oncological agent.
[52]
52. Kit according to claim 51, characterized by the fact that the immuno-oncological agent is an inhibitor of the PD-1 pathway.
[53]
53. Kit according to claim 52, characterized in that the inhibitor of the PD-1 pathway is an anti-PD1 antibody or an anti-PD-Ll antibody.
Petition 870190101572, of 10/09/2019, p. 147/176
18/19
[54]
54. Kit according to claim 51, characterized by the fact that the immuno-oncological agent is an antagonist or inhibitor of a co-inhibitor of T cells.
[55]
55. Kit according to claim 51, characterized in that the immuno-oncological agent is an agonist of a T cell co-activator.
[56]
56. Kit according to claim 51, characterized by the fact that the immuno-oncological agent is an immunostimulatory cytokine.
[57]
57. Method of treating cancer in a subject, the method being characterized by the fact that it comprises administering to the subject a therapeutic amount of the isolated antibody as defined in any one of claims 1 to 42, of the pharmaceutical composition as defined in claim 43, the bispecific antibody as defined in claim 44, or the antibody-drug conjugate as defined in claim 45.
[58]
58. Method according to claim 57, characterized by the fact that cancer is a cancer that is enriched for the expression of CD112 or CD155.
[59]
59. Method according to claim 57, characterized by the fact that cancer is a cancer that is enriched for T cells or natural killer cells (NK) that express TIGIT.
[60]
60. Method according to any of claims 57 to 59, characterized in that the cancer is bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, cancer of testicle, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, lymphoma, leukemia, myeloma, or
Petition 870190101572, of 10/09/2019, p. 148/176
19/19 sarcoma.
[61]
61. Method according to claim 60, characterized by the fact that the cancer is a lymphoma or a leukemia.
[62]
62. The method of any one of claims 57 to 61, characterized in that it further comprises administering to the subject a therapeutic amount of an immuno-oncological agent.
[63]
63. The method of claim 62, characterized in that the immuno-oncological agent is an inhibitor of the PD-1 pathway.
[64]
64. The method of claim 63, characterized in that the inhibitor of the PD-1 pathway is an anti-PD1 antibody or an anti-PD-Ll antibody.
[65]
65. The method of claim 62, characterized in that the immuno-cancer agent is an antagonist or inhibitor of a T cell co-inhibitor.
[66]
66. The method of claim 62, characterized by the fact that the immuno-oncological agent is an agonist of a T cell co-activator.
[67]
67. Method according to claim 62, characterized by the fact that the immuno-oncological agent is an immunostimulatory cytokine.
[68]
68. Method according to any one of claims 62 to 67, characterized in that the isolated antibody, the pharmaceutical composition, the bispecific antibody, or the antibody-drug conjugate is administered simultaneously with the immuno-oncological agent.
[69]
69. Method according to any one of claims 62 to 67, characterized in that the isolated antibody, the pharmaceutical composition, the bispecific antibody, or the antibody-drug conjugate is administered sequentially to the immuno-oncological agent.

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WO2018160704A1|2018-09-07|

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US20200040082A1|2020-02-06|

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法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762464529P| true| 2017-02-28|2017-02-28|

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US201862616779P| true| 2018-01-12|2018-01-12|

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PCT/US2018/020239|WO2018160704A1|2017-02-28|2018-02-28|Anti-tigit antibodies|

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