Antigen binding proteins

Abstract

The present disclosure relates to compositions and related methods for treating CD96 mediated diseases. The present disclosure relates to CD96 binding proteins comprising a heavy chain variable region at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 86 and a light chain variable region at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 85.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of September 25, 2020, application number: 202080080763.5, and invention name: “Antigen Binding Protein”. Technical Field

[0002] The present invention relates to antigen binding proteins and fragments thereof that specifically bind to CD96, particularly human CD96. The present invention also relates to methods for treating diseases or conditions using the antigen binding fragments, pharmaceutical compositions comprising the antigen binding fragments, and methods for manufacturing the same. Other embodiments of the present invention will become apparent from the following description. Background Art

[0003] CD96 / TACTILE ("Late expression of increased T cell activation") is a cell surface receptor in the immunoglobulin superfamily that is primarily expressed on T cells, natural killer (NK) cells, and natural killer T (NKT) cells. CD96 belongs to a family of receptors that includes CD226 and TIGIT ("T cell immunoreceptor with Ig and ITIM domains"), which are known to interact with integrin and integrin-like ligands. CD155 / NECL5 ("integrin-like protein-5") is the major ligand for all three receptors (CD96, TIGIT, and CD226). TIGIT binds to CD155 with a higher affinity (3.15 nM) than CD226 (119 nM), and CD96 binding is intermediate (37.6 nM) (Martinet L. & Smyth MJ Nat Rev Immunol. 2015 April; 15(4): 243-54). In addition to CD155, TIGIT and CD226 also bind to another ligand, CD112, with much lower affinity. Recently, a new receptor, CD112R, was discovered that also binds to CD112 (Zhu Y. et al. J Exp Med. 2016 Feb 8; 213(2): 167-76).

[0004] Among the receptors of this axis, CD226 (DNAM-1) is one of the main activating receptors of NK cells. It has been reported that CD226 enhances the cytotoxicity of NK cells to cancer cells and is essential for tumor immune surveillance (Lakshmikanth T. et al. J Clin Invest. 2009; 119(5): 1251-63; Chan CJ et al. J Immunol. 2010; 184(2): 902-11; Gilfillan S. et al. J Exp Med. 2008; 205(13): 2965-73; Iguchi-Manaka A. et al. J Exp Med. 2008; 205(13): 2959-64). In contrast, CD96 (Chan CJ et al. Nat Immunol. 2014; 15(5): 431-8) and TIGIT (Lozano E. et al. J Immunol. 2012; 188(8): 3869-75) are both known to inhibit immune responses by inhibiting NK and / or T cell function. TIGIT expression is associated with T cell exhaustion (Lozano E. et al. 2012; Kurtulus S., et al. J Clin Invest. 2015; 125(11): 4053-62) and NK cell exhaustion (Zhang Q. et al. Nat Immunol. 2018; 19(7): 723-32), and several anti-TIGIT antibodies are in clinical development.

[0005] Overall, there is much more literature and mechanistic understanding about CD226 and TIGIT relative to CD96. CD226 does not have the same classic ITAM motif as in other immune activation receptors. After ligand binding and receptor dimerization, it conducts positive signaling through a series of phosphorylation events (including PKC and Vav1 proteins). The cytoplasmic tail of TIGIT contains an ITT motif and a classic inhibitory ITIM motif. After CD155 binding, tyrosine phosphorylation of the ITT motif occurs, and immunosuppressive signaling is conducted downstream involving SHIP1. In contrast, the signaling of CD96 has not yet been elucidated. It is known that there is a potential inhibitory ITIM motif in the cytoplasmic tail of CD96, as well as a potential activation YXXM motif present in a variety of immune activation receptors (such as ICOS and CD28) (Georgiev H. et al. Front Immunol. 2018; 9: 1072).

[0006] Although CD96 was discovered more than 25 years ago (Wang PL et al. J Immunol. 1992; 148(8): 2600-8), almost nothing is known about the function of CD96, except for the fact that it is a member of the immunoglobulin family that shares the ligand CD155 with CD226 and TIGIT (Fuchs A. et al. J Immunol. 2004; 172(7): 3994-8). Subsequent publications linking CD96 to cancer have focused primarily on CD96 as a leukemic stem cell (LSC) marker. The first paper suggesting CD96 as a potential immuno-oncology target was published in 2014 by Professor Mark Smyth's laboratory; CD96 was shown to compete with CD226 for CD155 binding in NK cells, and in mice CD96 negatively regulates the production of proinflammatory cytokines, including IFNγ (γ), following LPS activation (Chan CJ et al. 2014). CD96 knockout mice as well as mice treated with anti-CD96 antibodies were less sensitive to MCA-induced sarcoma formation (Id.). In the same study, blockade of CD226 or CD155 resulted in worse outcomes, which was postulated to be due to the loss of the CD155:CD226 activation pathway (Id.).

[0007] Subsequently, further in vivo studies supporting the inhibition of CD96 for cancer treatment have been published (see, e.g., Blake SJ et al. Cancer Discov. 2016; 6(4):446-59; Brooks J. et al. 2018; 78(2):475-88; H. et al. Oncoimmunology. 2018; 7(7): e1445949). Therefore, there is a need for improved antigen binding proteins and fragments thereof targeting CD96 for treating diseases. Such compositions and related methods are provided in the present disclosure. Summary of the invention

[0008] In the first aspect, the present invention provides a CD96 binding protein. In the second aspect, the present invention also provides a nucleic acid construct encoding a CD96 binding protein. In the third aspect, the present invention provides an expression vector comprising a nucleic acid according to the second aspect. In the fourth aspect, the present invention provides a recombinant host cell comprising a nucleic acid or an expression vector as described in the preceding aspect. In the fifth aspect, the present invention also provides a method for producing a CD96 binding protein, comprising culturing a host cell as described in the preceding aspect under conditions suitable for expressing the nucleic acid sequence or vector, thereby producing a polypeptide comprising a CD96 binding protein. The sixth aspect of the present invention is a CD96 binding protein produced by the preparation method described in the preceding aspect. In the seventh aspect, the present invention also provides a pharmaceutical composition comprising a CD96 binding protein as described in any one of the preceding aspects and a pharmaceutically acceptable excipient. Another aspect of the present disclosure is a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD96 binding protein or a pharmaceutical composition as described in any one of the preceding aspects to the subject. Another aspect of the present disclosure is the method of treatment described in the preceding aspect, which also includes whether the subject expresses CD96. Another aspect of the disclosure is a CD96 binding protein or a pharmaceutical composition as described in any of the preceding aspects, for use in therapy or for treating a disease.

[0009] Provide the following:

[0010] 1. A CD96 binding protein comprising:

[0011] (a)(i) any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90 and 94 and / or CDRL1, CDRL2, CRDL3 from SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89 and 93; (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or

[0012] (b) a VH region comprising a sequence that is at least 80% identical to SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90 or 94; and / or a VL region comprising a sequence that is at least 80% identical to SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89 or 93.

[0013] 2. The CD96 binding protein of item 1, wherein the CDR of (a)(i) is:

[0014] (1) a CDRH1 selected from SEQ ID NOs: 105-125; a CDRH2 selected from SEQ ID NOs: 126-146; and / or a CDRH3 selected from SEQ ID NOs: 147-150; a CDRL1 selected from SEQ ID NOs: 97-98; a CDRL2 selected from SEQ ID NOs: 99-100; and / or a CDRL3 selected from SEQ ID NOs: 101-104.

[0015] 3. The CD96 binding protein according to any of the preceding items, wherein the binding protein comprises a CDRH3 that is 100% identical to Seq ID NO: 147, 148, 149 or 150.

[0016] 4. The CD96 binding protein of any of the preceding items, wherein the binding protein comprises: a CDRH1 that is 100% identical to SEQ ID NO: 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125; a CDRH2 that is 100% identical to SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, or 146; and / or a CDRH2 that is 100% identical to SEQ ID NO: 147, 148, 149, or 150. 100% identical CDRH3; 100% identical CDRL1 to SEQ ID NO: 97 or 98; 100% identical CDRL2 to SEQ ID NO: 99 or 100; and / or 100% identical CDRL3 to SEQ ID NO: 101, 102, 103 or 104.

[0017] 5. The CD96 binding protein according to any of the preceding items, wherein all 6 CDRs are present in the binding protein.

[0018] 6. A CD96 binding protein comprising the following 6 CDRs:

[0019] CDRH1 of SEQ ID NO: 115;

[0020] CDRH2 of SEQ ID NO: 145; and

[0021] CDRH3 of SEQ ID NO: 147; and

[0022] CDRL1 of SEQ ID NO: 97;

[0023] CDRL2 of SEQ ID NO: 99; and

[0024] CDRL3 of SEQ ID NO:101.

[0025] 7. The CD96 binding protein according to item 6, wherein the binding protein comprises:

[0026] A VH region that is 75% identical to SEQ ID NO: 86; and / or

[0027] A VL region that is 75% identical to SEQ ID NO:85.

[0028] 8. The CD96 binding protein according to any one of the preceding items, wherein the binding protein comprises:

[0029] a VH region that is 100% identical to SEQ ID NO: 86; and / or

[0030] A VL region that is 100% identical to SEQ ID NO:85.

[0031] 9. The CD96 binding protein according to any of the preceding items, wherein the binding protein comprises a synthetic polypeptide, a humanized sequence or a chimeric sequence.

[0032] 10. The CD96 binding protein according to any of the preceding items, wherein the protein binds to CD96 with a binding affinity (KD) equal to or better than that of CD155 or with a binding affinity (KD) that can prevent or replace the binding of CD155 to CD96.

[0033] 11. The CD96 binding protein according to any of the preceding items, which binds to CD96 with a binding affinity (KD) of at least about 30 nM.

[0034] 12. The CD96 binding protein according to any one of the preceding items, which is an antibody and which comprises an antibody Fc region, which can bind to an Fcγ receptor and / or can promote IFNγ release.

[0035] 13. The CD96 binding protein according to item 12, wherein the Fc region is wild-type IgG1 Fc or a functional variant thereof.

[0036] 14. A CD96 binding protein which is an antibody, and wherein the binding protein comprises: a heavy chain that is 100% identical to the amino acid sequence of SEQ ID NO: 165; and / or a light chain that is 100% identical to the amino acid sequence of SEQ ID NO: 166.

[0037] 15. A CD96 binding protein which is an antibody, and wherein the binding protein comprises: a heavy chain that is 100% identical to the amino acid sequence of SEQ ID NO: 170; and / or a light chain that is 100% identical to the amino acid sequence of SEQ ID NO: 169.

[0038] 16. A CD96 binding protein which is an antibody, and wherein the binding protein comprises: a heavy chain that is 100% identical to the amino acid sequence of SEQ ID NO: 174; and / or a light chain that is 100% identical to the amino acid sequence of SEQ ID NO: 173.

[0039] 17. A nucleic acid sequence encoding a CD96 binding protein as defined in any one of the preceding items.

[0040] 18. A nucleic acid sequence according to claim 17, wherein the sequence comprises SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 167, 172 or 176 encoding a heavy chain; and / or SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 168, 171 or 175 encoding a light chain.

[0041] 19. An expression vector comprising a nucleic acid sequence as defined according to item 17 or 18.

[0042] 20. An expression vector according to item 19, which is selected from the following: (i) a vector encoding the nucleic acid sequence of the heavy chain of SEQ ID NO 167 and the light chain of SEQ ID NO 168, (ii) a vector encoding the nucleic acid sequence of the heavy chain of SEQ ID NO 172 and the light chain of SEQ ID NO 171, and (iii) a vector encoding the nucleic acid sequence of the heavy chain of SEQ ID NO 176 and the light chain of SEQ ID NO 175.

[0043] 21. A recombinant host cell comprising a nucleic acid sequence as defined in item 17 or 18 or an expression vector as defined in item 19 or 20.

[0044] 22. A method for producing a C96 binding protein, the method comprising culturing a host cell as defined in item 21 under conditions suitable for expressing the nucleic acid sequence or vector, thereby producing a polypeptide comprising the CD96 binding protein.

[0045] 23. A CD96 binding protein produced by the method according to item 22.

[0046] 24. A cell line engineered to express a CD96 binding protein according to any one of items 1 to 16.

[0047] 25. A pharmaceutical composition comprising a CD96 binding protein as defined in any one of items 1 to 16 and a pharmaceutically acceptable excipient.

[0048] 26. A pharmaceutical composition according to item 25, comprising a CD96 binding protein as defined in any one of items 6, 8 or 14-16 and a pharmaceutically acceptable excipient.

[0049] 27. A method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD96 binding protein as defined in any one of items 1 to 16 or a pharmaceutical composition as defined in any one of items 25-26.

[0050] 28. A method for treating a disease in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a CD96 binding protein as defined in any one of items 6, 8 or 14-16.

[0051] 29. The method according to item 27 or 28, further comprising determining whether the subject expresses CD96.

[0052] 30. A CD96 binding protein as defined in any one of items 1 to 16, or a pharmaceutical composition as defined in any one of items 25-26, for use in therapy.

[0053] 31. A CD96 binding protein as defined in any one of items 1 to 16 or a pharmaceutical composition as defined in any one of items 25-26 for use in treating a disease in a subject.

[0054] 32. The CD96 binding protein for use according to item 31, wherein the patient is a human subject.

[0055] 33. The CD96 binding protein for use according to items 31-32 for treating solid tumors.

[0056] 34. A CD96 binding protein for use according to items 31-32, wherein the protein is an antibody according to items 14-16 and the solid tumor is selected from ovarian cancer, lung cancer (e.g., NSCLC), gastric cancer, bladder cancer, colorectal cancer, liver cancer (e.g., HCC), renal cancer (e.g., RCC) and head and neck squamous cell carcinoma (HNSCC).

[0057] 35. Use of a CD96 binding protein as defined in any one of items 1 to 16 or a pharmaceutical composition as defined in any one of items 25-26 in the manufacture of a medicament for treating a disease.

[0058] 36. A pharmaceutical composition according to any one of items 25-26, further comprising a PD-1 antagonist and / or an anti-TIGIT antibody.

[0059] 37. A pharmaceutical composition according to item 36, wherein the PD-1 antagonist is an antibody comprising a heavy chain sequence of SEQ ID NO: 160 and a light chain sequence of SEQ ID NO: 162.

[0060] 38. The method according to item 27 or 28, further comprising administering a PD-1 antagonist and / or an anti-TIGIT antibody.

[0061] 39. A method according to claim 38, wherein the disease is cancer and wherein the CD96 binding protein and the PD-1 antagonist and / or anti-TIGIT antibody are administered to a patient, such as a human patient, simultaneously or sequentially in any order.

[0062] 40. The method of item 39, wherein the cancer is a solid tumor and wherein the PD1 antagonist is an antibody comprising the heavy chain sequence of SEQ ID NO: 160 and the light chain sequence of SEQ ID NO: 162.

[0063] 41. A method according to claim 40, wherein the solid tumor is selected from ovarian cancer, lung cancer (e.g., NSCLC), gastric cancer, bladder cancer, colorectal cancer, liver cancer (e.g., HCC), renal cancer (e.g., RCC), and head and neck squamous cell carcinoma (HNSCC).

[0064] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, presently preferred embodiments are shown in the accompanying drawings. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 CD96 binding protein and human CD3 + T cells (A), and the ability of CD96 binding protein to prevent CD155 binding to CHO cells expressing human CD96 (B).

[0066] Figure 2 Shown are solution equilibrium titration (SET) data for binding of CD96 binding proteins to human (a), cynomolgus monkey (b), or mouse (c) CD96.

[0067] Figure 3 Shown is the binding of CD96 binding proteins to HEK cells transiently transfected with human or cynomolgus CD96 isoforms.

[0068] Figure 4 The expression of CD96 binding protein (relative to isotype control) in primary human T cells (total CD3 + T cells relative to CD4 + or CD8 + subgroups).

[0069] Figure 5 Binding of CD96 binding protein to activated primary cynomolgus monkey T cells is shown.

[0070] Figure 6 Imaging cytometry was used to show the expression of CD8 + CD96 internalization in T cells using imaging cytometry; CD8 + Representative cell images of T cells (grey = CD8 staining, white = CD96 binding protein-PE staining)

[0071] Figure 7 It is shown that CD96 binding protein, when pre-complexed with membrane CD96 in primary human T cells, prevents CD155:Fc from binding to the T cells.

[0072] Figure 8 Shown is the displacement of CD155:Fc by CD96 binding protein pre-bound to human T cells.

[0073] Fig. 9Human Fcγ reporter assay data are shown, indicating that binding of CD96 binding protein to CD96 on primary human T cells does not induce cross-linking and / or signaling through Fcγ receptors.

[0074] Fig.10 Shown in CD4 + and CD8 + Human ADCC target cell killing assay data in T cells; in the presence of CD96 binding protein, in CD4 + or CD8 + No evidence of increased cell death was observed in T cells.

[0075] Fig.11 Shown in CD4 + Human CDC target cell killing assay in T cells; no evidence of induced complement-dependent cytotoxicity was observed from CD96 binding protein.

[0076] Fig.12 Shown are EC50 data for IFNγ release by CD96 binding proteins (expressed from CHO or HEK cells) in a mixed PMBC-MLR assay.

[0077] Fig.13 Shown in the presence or absence of CD4 + (A) or CD8 + (B) Activity of CD96 binding protein (expressed from HEK cells) on IFNγ production in the mixed PBMC-MLR assay in the context of T cells.

[0078] Fig.14 Shown are the effects of CD96 binding protein on IFNγ and granzyme B secretion in a mixed PBMC-MLR assay.

[0079] Fig.15 Shown is the expression of IFNγ in different cell populations on day 3 in a mixed PBMC-MLR assay in the presence of CD96 binding protein and controls. + The frequency of cells.

[0080] Fig.16 Shown is the increase in CD4 + 、CD8 + CD96 in NK cell populations + The frequency of cells.

[0081] Fig.17 Shown is the increase in CD4 + 、CD8 +and the expression levels of CD96 in NK cell populations.

[0082] Fig.18 FAC characterization is shown, demonstrating the role of CD96 binding protein in the mixed PBMC-MLR assay for CD226 + Single positive relative to CD226 + CD96 + Effect of the ratio of double-positive NK cells.

[0083] Fig.19 FAC characterization is shown, demonstrating the effect of CD96 binding protein on IFNγ in the total NK cell population in a mixed PBMC-MLR assay + G JZ + Effect of double positive cells.

[0084] Fig. 20 Shown is the inhibitory effect of plate-bound CD155-Fc on IFNγ production in a human PBMC assay.

[0085] Fig.21 The activity of CD96 binding protein alone or in combination with anti-PD1 or anti-TIGIT antibodies in a renal cancer TIL functional assay is shown.

[0086] Fig. 22 Data from bioluminescent imaging studies of CD96 binding protein in the NK cell-dependent B16F10 melanoma lung colonization model are shown, showing representative bioluminescent images acquired approximately 15 minutes after injection of B16F10 RFluc melanoma cells.

[0087] Fig.23 Shown are lung bioluminescent signals at day 14 after B16F10 cell injection in mice without depletion; with CD4, CD8 or NK cell depletion.

[0088] Fig.24 In vivo bioluminescent lung signals at days 14 and 20 are shown, demonstrating the effect of CD96 binding protein (relative to control) on lung metastasis in different groups.

[0089] Fig.25 The CD4+ / -treated cells treated with CD96 binding protein at day 20 (end of study) are shown. + / CD8 + Images of depleted mouse lungs relative to controls.

[0090] Fig.26 Shown is IFNγ production in CD155-coated PBMC assays in the presence of anti-TIGIT mAb, in the presence of CD96 binding protein or isotype control as assessed by MSD.

[0091] Fig. 27 Shown is TNFα production in CD155-coated PBMC assay in the presence of anti-TIGIT mAb, in the presence of CD96 binding protein or isotype control as assessed by MSD.

[0092] Fig.28 Shown is IFNγ production in a CD155-coated PBMC assay in the presence or absence of anti-TIGIT mAb for 3 days, in the presence of CD96 binding protein or isotype control as assessed by MSD. DETAILED DESCRIPTION

[0093] The present disclosure provides CD96 binding proteins, nucleic acids encoding the proteins, and related subject matter.

[0094] As used herein and in the claims, singular forms of nouns include plural references unless the context clearly dictates otherwise.Thus, for example, reference to a "peptide chain" refers to one or more peptide chains and includes equivalents thereof known to those skilled in the art.

[0095] As used herein and in the claims, the term "comprising" encompasses "including" or "consisting of", e.g., a composition "comprising X" may consist solely of X, or may include additional things, e.g., X+Y.

[0096] The term "consisting essentially of limits the scope of a feature to the specified materials or steps, and those materials or steps that do not materially affect the basic characteristics of the claimed feature.

[0097] The term "consisting of does not include the presence of any additional components.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used to implement or test the method of the present disclosure, exemplary compositions and methods are described herein. Any aspect and embodiment of the present disclosure described herein can also be combined. For example, the subject matter of any dependent or independent claim disclosed herein can be combined in multiples (for example, based on the independent claim to which they belong, one or more references of each dependent claim can be combined into a single claim).

[0099] The ranges provided herein include all values ​​within the particular range described as well as values ​​around the endpoints of the particular range.The figures and tables of the present disclosure also depict ranges and discrete values ​​that can constitute elements of any method disclosed herein.

[0100] The concentrations described herein are determined at ambient temperature and pressure. For example, this may be at room temperature or at a temperature and pressure within a particular portion of a process stream. Preferably, the concentrations are determined at standard conditions of 25°C and 1 bar pressure.

[0101] The term "about" means values ​​that are within two standard deviations of the mean for any particular measurement.

[0102] "Affinity" is the strength with which one molecule binds to another. The binding affinity of an antigen binding protein to its target can be measured by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetic methods (e.g., BIACORE TM analysis) to determine.

[0103] As used herein, the term "antigen" refers to a macromolecular structure that is selectively recognized by an antigen binding protein. Antigens include, but are not limited to, proteins (with or without polysaccharides) or protein compositions comprising one or more T cell epitopes. As contemplated herein, the target binding domain of an antigen binding protein may recognize the sugar side chains of a glycoprotein rather than a specific amino acid sequence of a macromolecule. Therefore, a sugar moiety or a sulfated sugar moiety is used as an antigen.

[0104] As used herein, the term "antigen binding protein" refers to isolated proteins, antibodies, antibody fragments (e.g., Fab, etc.) and other antibody-derived protein constructs, such as those comprising a domain (e.g., domain antibodies, etc.) capable of binding to CD96. Such alternative antibody formats include triabodies, tetrabodies, minibodies and minibodies. Alternative scaffolds are also included, in which one or more CDRs of any molecule according to the present disclosure can be arranged on a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, SpA scaffold, LDL receptor class A domain, avimer (see, e.g., U.S. Patent Application Publication Nos. 2005 / 0053973, 2005 / 0089932, 2005 / 0164301) or EGF domain. ABPs also include antigen-binding fragments of such antibodies or other molecules. In addition, when paired with an appropriate light chain, the ABP may comprise a VH region (e.g., scFV, diabody, tribody or tetrabody, Tandabs, etc.) of the invention that forms a full-length antibody, (Fab')2 fragment, Fab fragment, bispecific or biparatopic molecule, or its equivalent. The ABP may comprise an antibody that is IgG1, IgG2, IgG3 or IgG4; IgM; IgA, IgE or IgD or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. The ABP may also be a chimeric antibody of the type described in WO86 / 01533, comprising an antigen binding region and a non-immunoglobulin region. The antigen-binding protein of the present disclosure may be provided as a lyophilized powder containing an antibody and an excipient, which excipient may be reconstituted with a pharmaceutically acceptable carrier (e.g., sterile water). The reconstituted pharmaceutical composition may then be administered subcutaneously or intravenously (e.g., using further dilution). The antigen binding proteins of the present disclosure can also be provided as liquid preparations containing antibodies, excipients and pharmaceutically acceptable carriers. The liquid pharmaceutical composition can then be administered subcutaneously or intravenously (e.g., using further dilution). The terms "ABP", "antigen binding protein" and "binding protein" are used interchangeably herein. As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no restriction on the maximum number of amino acids that can contain a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to as, for example, peptides, oligopeptides and oligomers in the art, and longer chains, which are commonly referred to as proteins in the art, and there are many types of them. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc.The polypeptides include natural peptides, recombinant peptides, synthetic peptides or a combination thereof.

[0105] As used herein, the term "antibody variant" means an antibody that is different from a parent antibody by at least one amino acid modification (e.g., by having different amino acid side chains), post-translational modification or other modification in at least one heavy chain, light chain or combination thereof that results in a structural change (e.g., different amino acid side chains, different post-translational modification or other modification) relative to the parent antibody. The structural change can be determined directly by a variety of methods well known in the art (e.g., LC-MS, direct sequencing), or indirectly by methods such as isoelectric focusing. Such methods are well known to those of ordinary skill in the art.

[0106] As used herein, the term "epitope" refers to the portion of an antigen that contacts a specific binding domain. An epitope can be linear or conformational / discontinuous. A conformational or discontinuous epitope comprises amino acid residues separated by other sequences, i.e., in a discontinuous sequence of the primary sequence of the antigen. Although the residues may be from different regions of the peptide chain, they are very close in the three-dimensional structure of the antigen. In the case of a multimeric antigen, a conformational or discontinuous epitope may include residues from different peptide chains. The specific residues contained in the epitope may be determined by a computer modeling program or by a three-dimensional structure obtained by methods known in the art (e.g., X-ray crystallography). As considered herein, the term epitope includes post-translational modifications to polypeptides that can be recognized by antigen binding proteins or domains (e.g., sugar moieties of glycosylated proteins).

[0107] As used herein, the term "isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment (e.g., a host cell).

[0108] CD96 (cluster of differentiation 96), also known as TACTILE, is a receptor expressed on T cells and NK cells that has sequence similarity to CD226 (DNAM01) and is a type 1 transmembrane glycoprotein belonging to the immunoglobulin superfamily.

[0109] Human CD96 has 3 subtypes: two membranes (V1; V2) and a soluble form. The longer V1 isoform is a 585 amino acid protein with a MW of 65,634Da. The shorter isoform V2 (569aa) has a MW of 63,888Da. V2 differs from V1 in that the Ig fold of the second domain has a short deletion. It is reported that the first domain of CD96 contains the epitope required for CD155 binding, while the second domain regulates the amplitude / intensity of binding. CD96V2 binds much more strongly to CD155 than CD96V1, and is also the main expression form except for acute myeloid leukemia AML (cells). The soluble form of CD96 (sCD96) is almost unknown. It has been reported that sCD96 (1-3ng / ml) is detected in the blood of healthy donors, and higher levels are detected in hepatitis B virus (HBV) HBV / cirrhosis patients.

[0110] Based on mRNA analysis of normal human tissues, the highest expression of CD96 was observed in hematopoietic cells (probes for V1 and V2 detecting CD96). CD96 expression was also observed in tissues containing a large number of lymphocytes, such as the spleen, lungs, thyroid gland, and small intestine. In hematopoietic cells, CD96 is most enriched in T cells and NK cells, and is expressed at a lower level in some B cells. Like CD96, TIGIT and CD226 are most enriched in hematopoietic cells and are expressed in T cells and NK cells. CD226 is also expressed in conventional DC cells. The ligand CD155 shows a wide range of expression patterns in normal human tissues, and is also expressed in DC cells and macrophages (antigen presenting cells). CD155 is not expressed in T cells or NK cells.

[0111] The binding affinity (KD) of the antigen binding protein-target antigen interaction can be 1 mM or less, 100 nM or less, 10 nM or less, 2 nM or less, or 1 nM or less. Alternatively, the KD can be 5 to 10 nM; or 1 to 2 nM. The KD can be 1 pM to 500 pM; or 500 pM to 1 nM. For example, certain useful such variants have a binding affinity (KD) of at least about 40 nM or at least about 35 nM or at least about 30 nM, such as about 10 pM to about 30 nM.

[0112] The binding affinity of an antigen binding protein is determined by the association constant (Ka) and the dissociation constant (Kd) (KD = Kd / Ka). Binding affinity can be measured by BIACORE TMBinding affinity can be measured by FORTEBIO, for example, by capturing the test antibody on a protein A coated sensor surface and allowing the target antigen to flow through the surface. Alternatively, binding affinity can be measured by FORTEBIO, for example, by capturing the test antibody receptor on a protein A coated pin and allowing the target antigen to flow through the surface. Alternatively, binding affinity (KD) can be measured by using MSD-SET analysis (MSD solution equilibrium titration), for example, by titrating the test antibody onto a standard binding MSD plate and using MSD SECTOR IMAGER to detect. MSD-SET determines the solution phase, equilibrium affinity of the antibody. This known method relies on free antigen when detecting equilibrium in a series of titrated antibody concentrations.

[0113] Kd can be 1x10-3 Ms-1 or less, 1x10-4 Ms-1 or less, or 1x10-5 Ms-1 or less. Kd can be 1x10-5 Ms-1 to 1x10-4 Ms-1; or 1x10-4 Ms-1 to 1x10-3 Ms-1. A slow Kd can result in slow dissociation of the antigen binding protein-target antigen complex and increased neutralization of the target antigen.

[0114] As used herein, the term "specific antigen binding activity" means antigen binding activity as measured, for example, by surface plasmon resonance (SPR). CD96 specific binding activity can be measured by SPR using BIACORE TM The activity is determined instrumentally, e.g. in binding mode. It is the binding activity divided by the total protein content in the sample.

[0115] As used herein, the terms "VH" and "VL" refer to the heavy chain variable region and the light chain variable region, respectively, of an antigen binding protein.

[0116] "CDR" is defined as the complementary determining region amino acid sequence of an antigen binding protein. These are the hypervariable regions of the heavy and light chains of immunoglobulins. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Therefore, as used herein, "CDR" refers to all three heavy chain CDRs, all three light chain CDRs, all heavy chain and light chain CDRs, or at least one CDR, wherein at least one CDR is CDRH3. The framework region follows each of these CDR regions. Acceptable heavy chain variable regions and light chain variable region framework 1, framework 2, and framework 3 regions are easily identified by those of ordinary skill in the art. Acceptable heavy chain constant regions (including hinge regions) and light chain constant regions are also easily identified by those of ordinary skill in the art. Acceptable antibody isotypes are also easily identified by those of ordinary skill in the art.

[0117] Throughout this specification, the amino acid residues in the variable domain sequences and the full-length antibody sequences are numbered according to the Kabat numbering convention. Similarly, the terms "CDR", "CDRL1", "CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" used in this specification follow the Kabat numbering convention.

[0118] It will be apparent to those skilled in the art that there are alternative numbering conventions for amino acid residues in variable domain sequences and full-length antibody sequences. There are also alternative numbering conventions for CDR sequences, such as those set forth according to the Chothia numbering convention. The structure and protein folding of antibodies may mean that other residues are considered part of the CDR sequence, and the skilled artisan understands this.

[0119] Other numbering conventions for CDR sequences available to the skilled person include the "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlapping region can be determined using at least two of the Kabat, Chothia, AbM and contact methods to provide a "minimum binding unit". The minimum binding unit can be a sub-portion of a CDR.

[0120] Table 1 below shows one definition of each CDR or binding unit using each numbering convention. The variable domain amino acid sequences are numbered using the Kabat numbering scheme in Table 1. It should be noted that some CDR definitions may differ depending on the individual publication used.

[0121] Table 1

[0122]

[0123] In one embodiment of the present disclosure is a CD96 binding protein comprising: (a) (i) any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90 and 94 and / or CDRL1, CDRL2, CRDL3 from SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89 and 93; (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or (b) a VH region comprising a CDR variant of SEQ ID NO: 90, or 94; and / or a VL region comprising a sequence that is at least 80% identical (or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical) to a sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90 or 94; and / or a VL region comprising a sequence that is at least 80% identical to a sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89 or 93.

[0124] In another embodiment, the CD96 binding protein comprises: (a) (i) any one or combination of the following CDRs: CDRH1, CDRH2, CDRH3 from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90 and 94 and / or CDRL1, CDRL2, CRDL3 from SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89 and 93; (ii) a CDR variant of (i), wherein the variant has 1, 2 or 3 amino acid modifications; or (b) a VH region comprising SEQ ID NO: NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90 or 94; and / or a VL region comprising a sequence that is at least 80% identical (or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical) to the sequence of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89 or 93.

[0125] Another embodiment of the present disclosure is the CD96 binding protein described in the preceding aspects, which comprises a CDRH1 selected from SEQ ID NOs: 105-125; a CDRH2 selected from SEQ ID NOs: 126-146; and / or a CDRH3 selected from SEQ ID NOs: 147-150; a CDRL1 selected from SEQ ID NOs: 97-98; a CDRL2 selected from SEQ ID NOs: 99-100; and / or a CDRL3 selected from SEQ ID NOs: 101-104.

[0126] Another embodiment of the disclosure is the CD96 binding protein of any of the preceding aspects, wherein the binding protein comprises a CDRH3 that is 100% identical to SEQ ID NO: 147, 148, 149 or 150.

[0127] Another embodiment of the disclosure is a CD96 binding protein of any of the preceding aspects, comprising a CDRH1 that is 100% identical to SEQ ID NO: 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125; a CDRH2 that is 100% identical to SEQ ID NO: 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, or 146; and / or a CDRH2 that is 100% identical to SEQ ID NO: 147, 148, 149, or 150. 100% identical CDRH3; 100% identical CDRL1 to SEQ ID NO:97 or 98; 100% identical CDRL2 to SEQ ID NO:99 or 100; and / or 100% identical CDRL3 to SEQ ID NO:101, 102, 103 or 104.

[0128] Another embodiment of the disclosure is the CD96 binding protein of any of the preceding aspects, wherein all six CDRs are present in the binding protein.

[0129] Another embodiment of the present disclosure is the CD96 binding protein of any of the preceding aspects, comprising a CDRH1 of SEQ ID NO: 115; a CDRH2 of SEQ ID NO: 145; and a CDRH3 of SEQ ID NO: 147; and / or a CDRL1 of SEQ ID NO: 97; a CDRL2 of SEQ ID NO: 99; and a CDRL3 of SEQ ID NO: 101.

[0130] Another embodiment of the disclosure is the CD96 binding protein of any of the preceding aspects, wherein the binding protein comprises: a VH region that is 75% identical to SEQ ID NO: 86; and / or a VL region that is 75% identical to SEQ ID NO: 85 (or at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to these sequences). Another aspect of the disclosure is the CD96 binding protein of any of the preceding aspects, wherein the binding protein comprises: a VH region that is 100% identical to SEQ ID NO: 86; and / or a VL region that is 100% identical to SEQ ID NO: 85.

[0131] In one embodiment of the disclosure, the invention provides a CD96 binding protein comprising any CDR as described herein (alone or in the combination described), and further comprising an Fc region that can bind to an Fcγ receptor and / or can promote IFNγ release. Such an Fc region can be a wild-type IgG1 Fc.

[0132] In one embodiment of the disclosure, the invention provides a CD96 binding protein comprising any VH region as described herein (alone or in the combination described) and further comprising an Fc region that can bind to an Fcγ receptor and / or can promote IFNγ release. Such an Fc region can be a wild-type IgG1 Fc.

[0133] In one embodiment of the disclosure, the invention provides a CD96 binding protein comprising any VL region as described herein (alone or in the combination described) and further comprising an Fc region that can bind to an Fcγ receptor and / or can promote IFNγ release. Such an Fc region can be a wild-type IgG1 Fc.

[0134] Another embodiment of the present disclosure is a CD96 binding protein as described in any of the preceding aspects, which is an antibody, wherein the binding protein comprises: a (complete) heavy chain that is 75% identical (or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical) to SEQ ID NO: 165; and / or a (complete) light chain that is 75% identical (or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical) to SEQ ID NO: 166.

[0135] Another aspect of the disclosure is the CD96 binding protein of any of the preceding aspects, wherein the binding protein comprises: a VH region that is 100% identical to SEQ ID NO:86; and / or a VL region that is 100% identical to SEQ ID NO:85.

[0136] Another aspect of the present disclosure is the CD96 binding protein of any of the preceding aspects, which is an antibody and wherein the binding protein comprises: a (complete) heavy chain that is 100% identical to the amino acid sequence of SEQ ID NO: 165; and / or a (complete) light chain that is 100% identical to the amino acid sequence of SEQ ID NO: 166 (CDRs are underlined in SEQ ID NOs: 165 and 166); or is an antibody that binds to CD96 and wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID NO: 167 and / or the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 168.

[0137] Another aspect of the present disclosure is the CD96 binding protein of any of the preceding aspects, which is an antibody, and wherein the binding protein comprises: a (complete) heavy chain that is 100% identical to the amino acid sequence of SEQ ID NO: 170; and / or a (complete) light chain that is 100% identical to the amino acid sequence of SEQ ID NO: 169 (CDRs are underlined), or is an antibody that binds to CD96 and wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID NO: 172 and / or the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 171.

[0138] Another aspect of the present disclosure is the CD96 binding protein of any of the preceding aspects, which is an antibody and wherein the binding protein comprises: a (complete) heavy chain that is 100% identical to the amino acid sequence of SEQ ID NO: 174; and / or a (complete) light chain that is 100% identical to the amino acid sequence of SEQ ID NO: 173 (CDRs are underlined), or is an antibody that binds to CD96 and wherein the heavy chain is encoded by the nucleic acid sequence of SEQ ID NO: 176 and / or the light chain is encoded by the nucleic acid sequence of SEQ ID NO: 175.

[0139] The invention includes binding proteins that are 100% identical to any of the amino acid sequences described herein as well as proteins that are variants of the amino acid sequences described herein (e.g., sequences that are at least 75% identical or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to the sequences herein).

[0140] The invention also includes nucleic acids encoding binding proteins of the invention, including nucleic acids that are 100% identical to any nucleic acid sequence described herein, as well as nucleic acids that are variants of the sequences described herein (e.g., sequences that are at least 75% identical or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to the sequences herein).

[0141] The CDRs and variable regions having the amino acid sequences provided in this application have binding affinity for CD96 that is equal to or better than the binding affinity for CD155 and / or are variable regions that can prevent or replace the binding of CD155 to CD96 (examples of binding assays that can be used to determine this are set forth in Examples 1 and 2 herein). Useful variants of the sequences described herein are variants in which the variable regions have binding affinity for CD96 that is equal to or better than CD155 or can prevent or replace the binding of CD155 to CD96 (examples that can be used to determine this are set forth in Examples 1 and 2 herein). For example, when binding affinity is determined, for example, by MSD-SET assay as described in detail herein, certain useful such variants have a binding affinity (KD) of at least about 40 nM or at least about 35 nM or at least about 30 nM, such as from about 10 pM to about 30 nM.

[0142] Useful CD96 binding proteins (e.g., antibodies) according to the present invention may comprise such variable regions and an Fc region as described herein, wherein the Fc region can bind to an Fcγ receptor and / or can promote IFNγ release. Such an Fc region according to the present invention is a wild-type IgG1 Fc or a functional variant thereof (e.g., an Fc-ineffective variant, which region can bind to an Fcγ receptor and / or can promote IFNγ release). Thus, any CDR and / or variable region of the present invention can be combined with an Fc region of the present invention, such as a wild-type IgG1 Fc or a functional variant thereof.

[0143] Another embodiment of the disclosure is the CD96 binding protein of any of the preceding aspects, wherein the binding protein comprises a synthetic polypeptide, a humanized sequence, or a chimeric sequence.

[0144] As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0145] The term "domain" refers to a folded protein structure that retains its tertiary structure independent of the rest of the protein. Typically, domains are responsible for discrete functional properties of a protein and, in many cases, can be added, removed, or transferred to other proteins without loss of function of the rest of the protein and / or the domain.

[0146] The term "nucleic acid" or "polynucleotide" refers to a polymer of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and single-stranded or double-stranded forms thereof. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly specified. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0147] In one embodiment of the present disclosure is a nucleic acid sequence encoding the CD96 binding protein of any of the preceding embodiments.

[0148] An additional embodiment of the disclosure is a nucleic acid sequence as described in the preceding embodiments, wherein the sequence comprises SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, or 96 encoding a heavy chain; and / or SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, or 95 encoding a light chain. Another embodiment of the disclosure is an expression vector comprising the nucleic acid of any of the preceding embodiments.

[0149] The compositions described herein can be produced by many conventional techniques. For example, the compositions can be expressed and purified in a recombinant expression system. In one embodiment, the compositions are produced by a method of culturing host cells under conditions suitable for expression of the CD96 binding protein described in any of the preceding embodiments, wherein the compositions are expressed and optionally purified and optionally formulated in a pharmaceutical composition.

[0150] Many different expression systems and purification schemes can be used to produce compositions. Typically, host cells are transformed with recombinant expression vectors encoding antibodies. A wide range of host cells can be used, including eukaryotic cell lines (e.g., CHO, Perc6, HEK293, HeLa, NS0) of mammalian origin. Suitable host cells include mammalian cells, such as CHO (e.g., CHOK1 and CHO-DG44).

[0151] The host cell may be an isolated host cell. The host cell is not typically part of a multicellular organism (e.g., a plant or an animal). The host cell may be a non-human host cell.

[0152] Appropriate cloning and expression vectors and cloning methods for use with eukaryotic or mammalian cellular hosts are known in the art.

[0153] Cells can be cultured under conditions that promote antibody expression. For example, a production bioreactor is used to culture cells. The volume of the production bioreactor can be: (i) about 20,000 liters, about 10,000 liters; about 5,000 liters; about 2,000 liters; about 1,000 liters; or about 500 liters; (ii) 500 to 20,000 liters; or 500 to 10,000 liters; 500 to 5,000 liters; 1,000 to 10,000 liters, or 2,000 to 10,000 liters. For example, cells can be cultured in a production bioreactor at a pH of about 6.75 to pH 7.00. Alternatively, cells can be cultured in a production bioreactor for about 12 to about 18 days. Alternatively, cells can be cultured in a production bioreactor at a pH of about 6.75 to pH 7.00 for about 12 to about 18 days. This culturing step can help control the level of deamidated antibody variants, eg, reduce the level of deamidated antibody variants.

[0154] The composition can be recovered and purified by conventional protein purification procedures. For example, the composition can be harvested directly from the culture medium. Harvesting cell cultures can be clarified, for example by centrifugation and / or depth filtration. Purification is performed after the recovery of the composition to ensure sufficient purity.

[0155] Another embodiment of the present invention is a recombinant host cell comprising a nucleic acid sequence or expression vector as described in any of the preceding embodiments. Another embodiment of the present disclosure is a method for producing a CD96 binding protein, comprising culturing a host cell as described in the preceding embodiments under conditions suitable for expressing the nucleic acid sequence or vector, thereby producing a polypeptide comprising a CD96 binding protein. Another embodiment of the present disclosure is a CD96 binding protein produced by the production method described in the preceding embodiments. Another embodiment of the present disclosure is in a cell line engineered to express the CD96 binding protein as described in any of the preceding embodiments.

[0156] "Percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is an "identity" value, expressed as a percentage, calculated by the BLASTN algorithm when the subject nucleic acid sequence and the query nucleic acid sequence have 100% query coverage after performing paired BLASTN comparisons. This paired BLASTN comparison between a query nucleic acid sequence and a subject nucleic acid sequence is performed using the default settings of the BLASTN algorithm available on the website of the National Center for Biotechnology Institute, with filters for low complexity regions turned off. Importantly, the query nucleic acid sequence can be described by a nucleic acid sequence identified in one or more claims herein.

[0157] "Percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is an "identity" value, expressed as a percentage, calculated by the BLASTN algorithm when the subject nucleic acid sequence and the query nucleic acid sequence have 100% query coverage after performing paired BLASTN comparisons. This paired BLASTN comparison between a query nucleic acid sequence and a subject nucleic acid sequence is performed using the default settings of the BLASTN algorithm available on the website of the National Center for Biotechnology Institute, with filters for low complexity regions turned off. Importantly, the query nucleic acid sequence can be described by a nucleic acid sequence identified in one or more claims herein.

[0158] The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer amino acid or nucleotide changes compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the subject sequence. Such changes include at least one amino acid deletion, substitution (including conservative and non-conservative substitutions) or insertion, and wherein the changes may occur at the amino terminal or carboxyl terminal position of the query sequence or anywhere between those terminal positions, interspersed individually in the amino acids or nucleotides in the query sequence or in one or more contiguous groups within the query sequence.

[0159] As used herein, "sequence identity" is the degree of relatedness between two or more amino acid sequences or two or more nucleic acid sequences, as determined by comparing the sequences. Comparison of sequences and determination of sequence identity can be accomplished using a mathematical algorithm; those skilled in the art will be aware of computer programs that can be used to align two sequences and determine the percent identity between them. Those skilled in the art will appreciate that different algorithms may produce slightly different results.

[0160] As used herein, the term "specific binding" and grammatical variants thereof with respect to antibodies mean antibodies or antibody fragments that recognize and bind to specific antigens, but do not substantially recognize or bind to other molecules in the sample. For example, an antibody that specifically binds to an antigen from one species may also bind to an antigen from one or more species. However, this cross-species reactivity itself does not change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen may also bind to different allele forms of the antigen. However, this cross-reaction itself does not change the specific classification of the antibody. In some cases, the term "specific binding (specific binding or specifically binding)" can be used to refer to the interaction of an antibody, protein or peptide with a second chemical substance, to mean that the interaction depends on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical substance; for example, antibodies generally recognize and bind to specific protein structures, not proteins. If the antibody is specific to epitope "A", then in a reaction containing labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A bound to the antibody.

[0161] As used herein, the term "pharmaceutical composition" means a composition suitable for administration to a patient.

[0162] The pharmaceutical compositions described herein may comprise a purified preparation of a CD96 binding protein described herein.

[0163] For example, a pharmaceutical preparation may include a purified preparation of a CD96 binding agent described herein in combination with a pharmaceutically acceptable carrier.

[0164] Typically, such pharmaceutical compositions contain a pharmaceutically acceptable carrier as known and required by accepted pharmaceutical practice. Examples of such carriers include sterile carriers, such as saline, Ringers solution or dextrose solution, optionally buffered to a pH in the range of 5 to 8 with a suitable buffer.

[0165] The pharmaceutical composition can be administered by injection or infusion (e.g., intravenously, intraperitoneally, intradermally, subcutaneously, intramuscularly or intraportally). Such compositions are suitably free of visible particulate matter. The pharmaceutical composition can comprise 1 mg to 10 g of antigen-binding protein, e.g., 5 mg to 1 g of antigen-binding protein. Alternatively, the composition can comprise 5 mg to 500 mg of antigen-binding protein, e.g., 5 mg to 50 mg.

[0166] The method for preparing such pharmaceutical compositions is well known to those skilled in the art. The pharmaceutical composition can include 1 mg to 10 g of antigen-binding proteins in a unit dosage form, optionally together with instructions for use. According to methods well known to those skilled in the art or obvious, the pharmaceutical composition can be lyophilized (freeze-dried) to reconstruct before administration. When the antibody has an IgG1 isotype, a copper chelator (e.g., citrate (e.g., sodium citrate) or EDTA or histidine) can be added to the pharmaceutical composition to reduce the copper-mediated degradation degree of the isotype antibody. The pharmaceutical composition can also include a solubilizing agent (e.g., arginine), a surfactant / anti-aggregating agent (e.g., polysorbate 80) and an inert gas (e.g., nitrogen) to replace the oxygen in the headspace of the vial.

[0167] In one embodiment of the present disclosure is a pharmaceutical composition comprising a CD96 binding protein as described in any one of the preceding embodiments and a pharmaceutically acceptable excipient.Another embodiment of the present disclosure is a pharmaceutical composition comprising a therapeutically effective amount of a CD96 binding protein as described in any one of the preceding embodiments.

[0168] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by reducing tumor growth rate, reducing tumor volume, reducing the number of tumor cells, reducing the number of metastases, increasing life expectancy, or improving various physiological symptoms associated with cancer conditions. "Anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the present invention to prevent tumorigenesis in the first place.

[0169] As used herein, the terms "cancer", "neoplasm" and "tumor" are used interchangeably and, in the singular or plural, refer to cells that have undergone malignant transformation that renders them pathological to the host organism. Illustrative examples of cells that can be targeted by the compositions and methods contemplated in particular embodiments include, but are not limited to, the following cancers: synovial sarcoma, non-small cell lung cancer (NSCLC), myxoid round cell liposarcoma (MRCLS) and multiple myeloma (MM). Primary cancer cells can be easily distinguished from non-cancerous cells by mature techniques, particularly histological examinations. As used herein, the definition of cancer cells includes not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells and in vitro cultures and cell lines derived from cancer cells. When referring to cancer types that typically manifest as solid tumors, "clinically detectable" tumors are tumors that are detectable based on tumor masses; for example, by procedures such as computed tomography (CT) scans, magnetic resonance imaging (MRI), X-rays, ultrasound or palpation on physical examinations, and / or tumors that are detectable due to the expression of one or more cancer-specific antigens in samples that can be obtained from patients. The tumor can be a hematopoietic (or blood or hematological or blood-related) cancer, such as a cancer derived from blood cells or immune cells, which can be referred to as a "liquid tumor". Specific examples of clinical conditions based on blood tumors include leukemias, such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphocytic leukemia; plasma cell malignancies, such as multiple myeloma, MGUS, and Waldenstrom's macroglobulinemia; lymphomas, such as non-Hodgkin's lymphoma, Hodgkin's lymphoma; and the like.

[0170] Cancer can be any cancer in which there is an abnormal number of mother cells or unwanted cell proliferation or is diagnosed as a blood cancer, including lymphoid and myeloid malignancies. Myeloid malignancies include but are not limited to acute myeloid (or myeloid or myeloid or myeloblastic) leukemia (undifferentiated or differentiated), acute promyelocytic (or promyelocytic (promyelocytic or promyelogenous or promyeloblastic)) leukemia, acute myelomonocytic (or myelomonocytic) leukemia, acute monocytic (or monocytic) leukemia, erythroleukemia and megakaryoblastic (or megakaryoblastic) leukemia. These leukemias can be collectively referred to as acute myeloid (or myeloid or myeloid) leukemia (AML). Myeloid malignancies also include myeloproliferative diseases (MPD), which include but are not limited to chronic myeloid (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis) and polycythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts transformed (RAEBT); and myelofibrosis (MFS) with or without unexplained myeloid metaplasia.

[0171] Hematopoietic cancers also include lymphoid malignancies, which can affect lymph nodes, spleen, bone marrow, peripheral blood and / or extranodal sites. Lymphomas include B-cell malignancies, including but not limited to B-cell non-Hodgkin lymphoma (B-NHL). B-NHL can be indolent (or low-grade), intermediate (or aggressive) or high-grade (very aggressive). Indolent B-cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL), including lymph node MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemic lesions, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or 3B) lymphoma, and primary mediastinal lymphoma (PML). High-grade B-NHLs include Burkitt lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV-associated (or AIDS-associated) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocytic (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman's disease. NHL may also include T-cell non-Hodgkin's lymphoma (T-NHL), which includes, but is not limited to, T-cell non-Hodgkin's lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disease (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sezary syndrome.

[0172] Hematopoietic cancer also includes Hodgkin lymphoma (or disease), including classical Hodgkin lymphoma, nodular sclerosing Hodgkin lymphoma, mixed cell type Hodgkin lymphoma, lymphocyte-based (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma and lymphocyte depletion type Hodgkin lymphoma. Hematopoietic cancer also includes plasma cell disease or cancer, such as including multiple myeloma (MM) of smoldering MM, monoclonal gammopathy (MGUS) of unknown significance (or unknown or unclear), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's macroglobulinemia, plasma cell leukemia and primary amyloidosis (AL). Hematopoietic cancer can also include other cancers of other hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. Tissues comprising hematopoietic cells, referred to herein as "hematopoietic cell tissue," include bone marrow; peripheral blood; thymus; and peripheral lymphoid tissues, such as spleen, lymph nodes, mucosa-associated lymphoid tissue (e.g., gut-associated lymphoid tissue), tonsils, Peyer's patches, and appendix, as well as lymphoid tissues associated with other mucosa, such as the bronchial lining.

[0173] In one embodiment, the cancer that can be treated by the binding protein of the present invention can include solid tumors (e.g., recurrent, metastatic or advanced solid tumors). Examples of such solid tumors include ovarian cancer, lung cancer (e.g., NSCLC), gastric cancer, bladder cancer, colorectal cancer, liver cancer (e.g., HCC), renal cancer (e.g., RCC) and head and neck squamous cell carcinoma (HNSCC).

[0174] As used herein, the term "treatment" means treatment and / or prevention. A therapeutic effect is obtained by suppressing, alleviating or eradicating the disease state.

[0175] As used herein, "therapeutically effective amount" or "effective amount" means an amount that provides a therapeutic or preventive benefit or elicits a biological or medical response in a tissue, system, or subject that a researcher, veterinarian, physician, or other clinician is seeking. Therapeutically effective amounts and treatment regimens are generally determined empirically and may depend on factors such as the age, weight, and health of the patient and the disease or condition to be treated. These factors are within the purview of the attending physician.

[0176] As used herein, the term "treatment" and its grammatical variants mean therapeutic therapy. With respect to a particular condition, treatment means: (1) a condition that improves or prevents one or more biological manifestations of a condition, (2) interfering with (a) one or more points of the biological cascade that causes or is responsible for the condition or (b) one or more biological manifestations of a condition, (3) alleviating one or more symptoms, effects or side effects associated with the condition or its treatment, (4) slowing the progression of a condition or one or more biological manifestations of a condition, and / or (5) curing the condition or one or more biological manifestations of the condition by eliminating one or more biological manifestations of the condition or reducing them to an undetectable level (considered to be a state of remission of the manifestation, during which no additional treatment is required) over a period of time. Those skilled in the art will understand that the duration of remission of a particular disease or condition is considered. Preventive therapy is also contemplated. Those skilled in the art will understand that "prevention" is not an absolute term. In medicine, "prevention" is understood to refer to the prophylactic administration of a drug to significantly reduce the likelihood or severity of a condition or its biological manifestation, or to delay the onset of such a condition or its biological manifestation. Preventive therapy is appropriate, for example, when the subject is considered to be at high risk for developing cancer, such as when the subject has a strong family history of cancer or when the subject has been exposed to a carcinogen.

[0177] The terms "individual", "subject" and "patient" are used interchangeably herein. In one embodiment, the subject is a mammal, such as a primate, such as a marmoset or monkey, or a human. In another embodiment, the subject is a human.

[0178] The dosage of the antigen-binding protein administered to the subject is generally 1 μg / kg to 150 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.5 mg / kg to 50 mg / kg, 1 to 25 mg / kg, about 0.3 mg / kg to about 3 mg / kg or 1 to 10 mg / kg of the subject's body weight. For example, the dosage can be 10 mg / kg, 30 mg / kg or 60 mg / kg. The dosage can also be 10 mg / kg to 110 mg / mg, 15 mg / kg to 25 mg / kg or 15 mg / kg to 100 mg / kg. The antigen-binding protein can be administered, for example, parenterally, subcutaneously, intravenously or intramuscularly. The dosage can also be administered based on each subject, for example, about 20 mg / subject to about 750 mg / subject, about 75 mg / subject to about 750 mg / subject, about 20 mg / subject to about 200 mg / subject. The dosage can be any discrete sub-range with these dosage ranges. For example, the dose may also be administered subcutaneously on a per-subject basis, such as about 100 mg / subject (e.g., once every 4 weeks), or 300 mg / subject (or other doses may be administered subcutaneously, provided that approximately the same or comparable bioavailability as intravenous administration is achieved - e.g., 3 doses of 100 mg / subject to achieve a total dose of 300 mg / subject administered subcutaneously).

[0179] Any type of range provided herein includes all values ​​within the particular range stated and values ​​about the endpoints of the particular range.

[0180] If desired, an effective daily dose of an antibody or antigen binding protein of the disclosure (e.g., as a pharmaceutical composition) may be administered in two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage form.

[0181] The dosage can be administered by slow continuous infusion over a period of 2 to 24 hours (eg, 2 to 12 hours or 2 to 6 hours). Such administration can result in reduced side effects.

[0182] As required, the administration of dosage can be repeated once or more times, for example, 3 times a day, once a day, once every 2 days, once a week, once every 14 days, once a month, once every 3 months, once every 4 months, once every 6 months or once every 12 months. Antigen-binding proteins can be administered by maintenance therapy, for example, once a week for 6 months or longer. Antigen-binding proteins can be administered by intermittent therapy, for example, 3 to 6 months in the cycle, then 3 to 6 months without administration, then again administering antigen-binding proteins for 3 to 6 months, etc.

[0183] For example, the dose may be administered subcutaneously once every 14 or 28 days in the form of multiple doses on each day of administration. In one embodiment, the dose of the composition is 100 mg once every 4 weeks (28 days).

[0184] The antigen binding proteins can be administered to a subject in a manner that targets the treatment to a specific site.

[0185] The CD96 binding protein in the method of the present disclosure can be combined or co-administered with one or more other therapeutic agents (e.g., antibodies, small molecule inhibitors) or used in combination with cell therapy. As used herein, the term "co-administered" means the simultaneous administration or separate sequential administration of a CD96 binding protein as described herein and one or more other active agents (including chemotherapy and radiotherapy) known to be useful for treating cancer in any manner. As used herein, the term one or more other active agents include any compounds or therapeutic agents known or showing favorable properties when administered to a patient in need of cancer treatment. Preferably, if the administration is not simultaneous, the compounds are administered at end times close to each other. In addition, it is not important whether the compounds are administered in the same dosage form, for example, one compound can be administered by injection and another compound can be administered orally.

[0186] Generally, in the cancer treatment of the present invention, any antitumor agent active to the susceptible tumor to be treated can be co-administered. Examples of such drugs can be found in VT Devita, TS Lawrence and SA Rosenberg (Editors) Cancer Principles and Practice of Oncology, 10th edition (September 5, 2014), Lippincott Williams & Wilkins Publishers. Those of ordinary skill in the art can discern which drug combinations are useful based on the specific characteristics of the drug and the cancer involved. Typical antitumor agents that can be used in the present invention include, but are not limited to, anti-microtubule agents or anti-mitotic agents; platinum coordination complexes; alkylating agents; antibiotics; topoisomerase I inhibitors; topoisomerase II inhibitors; antimetabolites; hormones and hormone analogs; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutics; pro-apoptotic agents; cell cycle signaling inhibitors; proteasome inhibitors; heat shock protein inhibitors; cancer metabolism inhibitors; and cancer gene therapy agents.

[0187] An example of one or more additional active ingredients for combination or co-administration with the CD96 binding proteins disclosed herein is an anti-tumor agent. Examples of anti-tumor agents include, but are not limited to, chemotherapeutic agents; immunomodulators; immunomodulators; and immunostimulatory adjuvants.

[0188] The currently disclosed CD96 binding proteins can also be used in combination with anti-TIGIT antibodies. Such a combination can further enhance CD155 / CD226 activation. Combinations with anti-TIGIT antibodies can be used to treat solid tumors, such as kidney tumors, such as renal cell carcinoma (RCC). An example of such an anti-TIGIT antibody is tiragolumab.

[0189] Anti-microtubule agents or anti-mitotic agents are cycle-specific agents that are active against tumor cell microtubules during the M phase or mitosis of the cell cycle. Examples of anti-microtubule agents include, but are not limited to, diterpenes and vinca alkaloids.

[0190] Platinum coordination complexes are non-periodic specific anticancer agents that interact with DNA. Platinum complexes enter tumor cells, hydrate, and form intra- and inter-strand crosslinks with DNA, causing adverse biological effects on tumors. Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin.

[0191] Alkylating agents are non-periodic anticancer specific agents and strong electrophilic agents. Typically, alkylating agents form covalent bonds with DNA through nucleophilic moieties (e.g., phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups) of DNA molecules by alkylation. Such alkylation destroys nucleic acid functions that lead to cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards, such as cyclophosphamide, melphalan, and chlorambucil; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine; and triazenes, such as dacarbazine.

[0192] Antibiotic antitumor agents are non-cycle specific agents that can bind to or intercalate with DNA. This action disrupts the normal function of nucleic acids, leading to cell death. Examples of antibiotic antitumor agents include, but are not limited to, actinomycins, such as dactinomycin; anthracyclines, such as daunorubicin and doxorubicin; and bleomycin.

[0193] Topoisomerase I inhibitors include, but are not limited to, camptothecin. The cytotoxic activity of camptothecin is believed to be related to its topoisomerase I inhibitory activity.

[0194] Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins. Epipodophyllotoxins are cycle-specific antitumor agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and G2 phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA, causing DNA strand breaks. Strand breaks accumulate and cell death ensues. Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide.

[0195] Antimetabolite tumor agents are cycle-specific antitumor agents that act in the S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis to limit DNA synthesis. Therefore, the S phase does not proceed and cell death ensues. Examples of antimetabolite antitumor agents include, but are not limited to, fluorouracil, methotrexate, cytarabine, mercaptopurine, thioguanine, and gemcitabine.

[0196] Hormones and hormone analogs are useful compounds for treating cancer where there is a relationship between the hormone and the growth and / or lack of growth of the cancer. Examples of hormones and hormone analogs that can be used for cancer treatment include, but are not limited to, adrenocortical steroids, such as prednisone and prednisolone; aminoglutethimide and other aromatase inhibitors, such as anastrozole, letrozole, vorozole, and exemestane; progestins, such as megestrol acetate; estrogens, androgens, and antiandrogens, such as flutamide, nilutamide, bicalutamide, cyproterone acetate, and 5α-reductase inhibitors (e.g., finasteride and dutasteride); antiestrogens, such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxifene, and selective estrogen receptor modulators (SERMS); gonadotropin-releasing hormone (GnRH) and its analogs, which stimulate the release of luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH), LHRH agonists and antagonists (e.g., gonadotropin acetate and leuprolide).

[0197] Signal transduction pathway inhibitors are those inhibitors that block or inhibit the chemical processes that cause changes in cells. As used herein, the change is cell proliferation or differentiation. Signal transduction inhibitors that can be used for the present invention include but are not limited to inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2 / SH3 domain blockers, serine / threonine kinases, phosphatidylinositol-3 kinases, inositol signaling and Ras oncogenes.

[0198] Several protein tyrosine kinases catalyze the phosphorylation of specific tyrosyl residues in various proteins involved in regulating cell growth. Such protein tyrosine kinases can be broadly classified as either receptor or non-receptor kinases.

[0199] Receptor tyrosine kinases are transmembrane proteins with an extracellular ligand binding domain, a transmembrane domain, and a tyrosine kinase domain. Receptor tyrosine kinases are involved in regulating cell growth and are commonly referred to as growth factor receptors. Inappropriate or uncontrolled activation of many of these kinases, such as by overexpression or mutation (i.e., abnormal kinase growth factor receptor activity), has been shown to lead to uncontrolled cell growth. Therefore, the abnormal activity of such kinases is associated with malignant tissue growth. Therefore, inhibitors of such kinases can provide cancer treatment methods. Growth factor receptors include, for example, epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), erbB2, erbB4, vascular endothelial growth factor receptor (VEGFR), tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains (TIE-2), insulin growth factor-I (IGFI) receptor, macrophage colony stimulating factor (Cfms), BTK, ckit, cmet, fibroblast growth factor (FGF) receptor, Trk receptor (TrkA, TrkB and TrkC), ephrin (eph) receptor and RET proto-oncogene. Several growth receptor inhibitors are under development, including ligand antagonists, antibodies, tyrosine kinase inhibitors and antisense oligonucleotides. Growth factor receptors and agents that inhibit growth factor receptor function are described, for example, in Kath JC, Exp. Opin. Ther. Patents, 10(6): 803-818 (2000); Shawver LK et al., Drug Discov. Today, 2(2): 50-63 (1997); and Lofts, FJ and Gullick WJ, "Growth factor receptors as targets." in New Molecular Targets for Cancer Chemotherapy, Kerr DJ and Workman P. (eds.), (June 27, 1994), CRC Press. Non-limiting examples of growth factor receptor inhibitors include pazopanib and sorafenib.

[0200] Tyrosine kinases that are not growth factor receptor kinases are referred to as non-receptor tyrosine kinases. Non-receptor tyrosine kinases that can be used in the present invention are targets or potential targets of anticancer drugs, including cSrc, Lck, Fyn, Yes, Jak, cAbl, FAK (focal adhesion kinase), Brutons tyrosine kinase and Bcr-Abl. Such non-receptor kinases and agents that inhibit the function of non-receptor tyrosine kinases are described in Sinha S. and Corey SJ, J. Hematother. Stem Cell Res., 8 (5): 465-480 (2004) and Bolen, JB, Brugge, JS, Annu. Rev. Immunol., 15: 371-404 (1997).

[0201] SH2 / SH3 domain blockers are agents that disrupt SH2 or SH3 domain binding in a variety of enzymes or adaptor proteins, including PI3-K p85 subunit, Src family kinases, adaptor molecules (Shc, Crk, Nck, Grb2) and Ras-GAP. The SH2 / SH3 domain as a target for anticancer drugs is discussed in Smithgall TE, J. Pharmacol. Toxicol. Methods, 34(3): 125-32 (1995).

[0202] Inhibitors of serine / threonine kinases include, but are not limited to, MAP kinase cascade blockers, including blockers of Raf kinase (rafk), mitogen- or extracellular-regulated kinase (MEK), and extracellular-regulated kinase (ERK); protein kinase C family member blockers, including PKC blockers (α, β, γ, ε, μ, λ, ι, ζ); IkB kinase (IKKa, IKKb); PKB family kinases; AKT kinase family members; TGFβ receptor kinase; and mammalian target of rapamycin (mTOR) inhibitors, including, but not limited to, rapamycin (FK506) and rapalogs, RAD001, or everolimus CCI-779 or temsirolimus, AP23573, AZD8055, WYE-354, WYE-600, WYE-687 and Pp121. Examples of serine / threonine kinase inhibitors include, but are not limited to, trametinib, dabrafenib and the Akt inhibitor afuresertib and N-{(1S)-2-amino-1-[(3,4-difluorophenyl)methyl]ethyl}-5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)-2-furancarboxamide.

[0203] Inhibitors of members of the phosphatidylinositol 3-kinase family, including blockers of PI3-kinase, ATM, DNA-PK and Ku, can also be used in the present invention. Such kinases are discussed in Abraham RT, Curr. Opin. Immunol., 8(3): 412-418 (1996); Canman CE and Lim DS, Oncogene, 17(25): 3301-3308 (1998); Jackson SP, Int. J. Biochem. Cell Biol., 29(7): 935-938 (1997); and Zhong H. et al., Cancer Res., 60(6): 1541-1545 (2000).

[0204] Also useful in the present invention are inositol signaling inhibitors, such as phospholipase C blockers and inositol analogs. Such signaling inhibitors are described in Powis G. and Kozikowski A., "Inhibitors of Myo-Inositol Signaling." in New Molecular Targets for Cancer Chemotherapy, Kerr DJ and Workman P. (eds.), (June 27, 1994), CRC Press.

[0205] Another class of signal transduction pathway inhibitors is inhibitors of Ras oncogenes. Such inhibitors include inhibitors of farnesyl transferase, geranyl-geranyl transferase and CAAX proteases as well as antisense oligonucleotides, ribozymes and other immunotherapies. Such inhibitors have been shown to block ras activation in cells containing wild-type mutant ras, thereby acting as antiproliferative agents. Ras oncogene inhibition is discussed in Scharovsky OG et al., J. Biomed. Sci., 7(4): 292-298 (2000); Ashby M. N., Curr. Opin. Lipidol., 9(2): 99-102 (1998); and Bennett CF and Cowsert LM, Biochem. Biophys. Acta., 1489(1): 19-30 (1999).

[0206] Antagonists of receptor kinase ligand binding can also be used as signal transduction inhibitors. Such signal transduction pathway inhibitors include humanized antibodies or other antagonists that use the extracellular ligand binding domain of receptor tyrosine kinases. Examples of antibodies or other antagonists of receptor kinase ligand binding include, but are not limited to, cetuximab Trastuzumab Trastuzumab Pertuzumab ErbB inhibitors, including lapatinib, erlotinib, and gefitinib; and 2C3 VEGFR2-specific antibody (see Brekken RA et al., Cancer Res., 60(18):5117-5124 (2000)).

[0207] Non-receptor kinase angiogenesis inhibitors can also be used in the present invention. Inhibitors of angiogenesis-related VEGFR and TIE2 are discussed above with respect to signal transduction inhibitors (both receptors are receptor tyrosine kinases). Angiogenesis is often associated with erbB2 / EGFR signaling, because inhibitors of erbB2 and EGFR have been shown to inhibit angiogenesis, primarily VEGF expression. Therefore, non-receptor tyrosine kinase inhibitors can be used in combination with the EGFR / erbB2 inhibitors of the present invention. For example, anti-VEGF antibodies, which do not recognize VEGFR (receptor tyrosine kinase), but bind to ligands; small molecule inhibitors of integrins (αβ3), which inhibit angiogenesis; endostatin and angiostatin (non-RTK) can also prove useful in combination with the disclosed compounds. (See Bruns CJ et al., Cancer Res., 60(11):2926-2935 (2000); Schreiber AB et al., Science, 232(4755):1250-1253 (1986); Yen L. et al., Oncogene, 19(31):3460-3469 (2000)).

[0208] Reagents used in immunotherapy regimens can also be used in combination with the present invention. There are many immunotherapy strategies for generating immune responses against erbB2 or EGFR. These strategies are generally in the field of tumor vaccination. By inhibiting the erbB2 / EGFR signaling pathway using a combination of small molecule inhibitors, the efficacy of the immunotherapy approach can be greatly enhanced. Discussions of immunotherapy / tumor vaccine approaches are found in Reilly RT et al., Cancer Res., 60(13):3569-3576 (2000); and Chen Y. et al., Cancer Res., 58(9):1965-1971 (1998).

[0209] Reagents for pro-apoptotic regimens (e.g., Bcl-2 antisense oligonucleotides) can also be used in the combination of the present invention. Members of the Bcl-2 protein family block apoptosis. Therefore, upregulation of Bcl-2 is associated with chemoresistance. Studies have shown that epidermal growth factor (EGF) stimulates anti-apoptotic members of the Bcl-2 family (i.e., Mcl-1). Therefore, strategies aimed at downregulating Bcl-2 expression in tumors have shown clinical benefits. Such pro-apoptotic strategies using Bcl-2 antisense oligonucleotide strategies are discussed in Waters JS et al., J. Clin. Oncol., 18(9): 1812-1823 (2000); and Kitada S. et al., Antisense Res. Dev., 4(2): 71-79 (1994).

[0210] Cell cycle signaling inhibitors inhibit molecules involved in cell cycle control. The protein kinase family known as cyclin-dependent kinases (CDKs) and their interaction with the protein family known as cyclins control the progression of the eukaryotic cell cycle. The coordinated activation and inactivation of different cyclin / CDK complexes are necessary for the normal progression of the cell cycle. Several cell cycle signaling inhibitors are under development. For example, examples of cyclin-dependent kinases (including CDK2, CDK4 and CDK6) and their inhibitors are described in, for example, Rosania GR and Chang YT, Exp. Opin. Ther. Patents, 10 (2): 215-230 (2000). In addition, p21WAF1 / CIP1 is described as an effective universal inhibitor of cyclin-dependent kinases (Cdks) (Ball KL, Prog. Cell Cycle Res., 3: 125-134 (1997)). Compounds known to induce p21WAF1 / CIP1 expression participate in inhibiting cell proliferation and have tumor suppressor activity (Richon VM et al., Proc. Natl. Acad. Sci. USA, 97 (18): 10014-10019 (2000)), and are included as cell cycle signal inhibitors. Histone deacetylase (HDAC) inhibitors participate in the transcriptional activation of p21WAF1 / CIP1 (Vigushin DM and Coombes RC, Anticancer Drugs, 13 (1): 1-13 (2002)), and are suitable cell cycle signaling inhibitors for combined use herein. Examples of such HDAC inhibitors include, but are not limited to, vorinostat, romidepsin, panobinostat, valproic acid and mocetinostat.

[0211] Proteasome inhibitors are drugs that block the action of the proteasome (a cellular complex that breaks down proteins, such as the p53 protein). Several proteasome inhibitors are on the market or are being studied for the treatment of cancer. Suitable proteasome inhibitors for use in combination herein include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-yl, salinosporamide A, and carfilzomib.

[0212] 70 kilodalton heat shock proteins (Hsp70s) and 90 kilodalton heat shock proteins (Hsp90s) are a family of heat shock proteins that are commonly expressed. Hsp70 and Hsp90 are overexpressed in certain cancer types. Several Hsp70 and Hsp90 inhibitors are being studied for the treatment of cancer. Examples of Hsp70 and Hsp90 inhibitors for use in combination herein include, but are not limited to, tanspiramycin and radicicol.

[0213] Many tumor cells display a metabolism that is significantly different from that of normal tissues. For example, the rate of glycolysis (the metabolic process that converts glucose into pyruvate) is increased, and the resulting pyruvate is reduced to lactate instead of being further oxidized in the mitochondria via the tricarboxylic acid (TCA) cycle. This effect is common even under aerobic conditions and is known as the Warburg Effect.

[0214] Lactate dehydrogenase A (LDH-A) is an isoform of lactate dehydrogenase expressed in muscle cells, which plays a key role in tumor cell metabolism by reducing pyruvate to lactate and then exporting it to the extracellular space. The enzyme has been shown to be upregulated in many tumor types. The changes in glucose metabolism described in the Warburg effect are essential for the growth and proliferation of cancer cells, and knocking out LDH-A using RNA-i has been shown to lead to a reduction in cell proliferation and tumor growth in xenograft models (Tennant DA et al., Nat. Rev. Cancer, 10(4): 267-277 (2010); Fantin VR et al., Cancer Cell, 9(6): 425-434 (2006)).

[0215] High levels of fatty acid synthase (FAS) have been found in cancer precursor lesions. Pharmacological inhibition of FAS affects the expression of key oncogenes involved in cancer development and maintenance. Alli PM et al., Oncogene, 24(1):39-46 (2005).

[0216] Cancer metabolism inhibitors, including LDH-A inhibitors and fatty acid biosynthesis inhibitors (or FAS inhibitors), are suitable for use in combination herein.

[0217] Cancer gene therapy involves the selective transfer of recombinant DNA / RNA using viral or non-viral gene delivery vectors to alter cancer calls for therapeutic purposes. Examples of cancer gene therapy include, but are not limited to, suicide and oncolytic gene therapy and adoptive T cell therapy.

[0218] As used herein, "immunomodulator" refers to any substance that affects the immune system, including monoclonal antibodies. The CD96 binding protein of the present invention can be considered an immunomodulator. Immunomodulators can be used as anti-tumor agents for the treatment of cancer. For example, immunomodulators include, but are not limited to, antibodies or other antagonists of CTLA-4, such as ipilimumab. Antibodies or other antagonists of PD-1, such as dotalimumab, nivolumab Pembrolizumab ) and cimiprilimab Other immunomodulators include, but are not limited to, antibodies or other antagonists of PD-L1, OX-40, LAG3, TIM-3, 41BB, and GITR.

[0219] As used herein, "PD-1 antagonist" means any compound or biological molecule that blocks the binding of PD-L1 expressed on cancer cells to PD-1 expressed on immune cells (T cells, B cells or NKT cells), and preferably also blocks the binding of PD-L2 expressed on cancer cells to PD-1 expressed by immune cells. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL1, B7H1, B7-4, CD274 and B7-H for PD-L1; PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2. The human PD-1 amino acid sequence can be found in NCBI locus number: NP_005009. The human PD-L1 and PD-L2 amino acid sequences can be found in NCBI locus numbers: NP_054862 and NP_079515, respectively.

[0220] PD-1 antagonists that can be used in any aspect of the present invention include monoclonal antibodies (mAbs) or antigen-binding fragments thereof that specifically bind to PD-1 or PD-L1, and preferably specifically bind to human PD-1 or human PD-L1. mAbs can be human antibodies, humanized antibodies, or chimeric antibodies, and can include human constant regions. In some embodiments, the human constant region is selected from IgG1, IgG2, IgG3, and IgG4 constant regions, and in preferred embodiments, the human constant region is IgG1 or IgG4 constant region. In some embodiments, the antigen-binding fragment is selected from Fab, Fab'-SH, F(ab')2, scFv, and Fv fragments.

[0221] Examples of mAbs that bind to human PD-1 and can be used in various aspects and embodiments of the present invention are described in U.S. Pat. No. 8,552,154; U.S. Pat. No. 8,354,509; U.S. Pat. No. 8,168,757; U.S. Pat. No. 8,008,449; U.S. Pat. No. 7,521,051; U.S. Pat. No. 7,488,802;

[0222] Described in WO2004072286; WO2004056875; and WO2004004771.

[0223] Other PD-1 antagonists that can be used in any aspect and embodiment of the present invention include immunoadhesins that specifically bind to PD-1, and preferably specifically bind to human PD-1, such as fusion proteins containing an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin molecule). Examples of immunoadhesin molecules that specifically bind to PD-1 are described in WO2010027827 and WO2011066342. Specific fusion proteins used as PD-1 antagonists in the methods of treatment, medicaments, and uses of the present invention include AMP-224 (also known as B7-DCIg), which is a PD-L2-FC fusion protein and binds to human PD-1.

[0224] Nivolumab is a A commercially available humanized monoclonal anti-PD-1 antibody. Nivolumab is suitable for the treatment of some unresectable or metastatic melanomas. Nivolumab binds to and blocks the activation of the Ig superfamily transmembrane protein PD-1 through its ligands PD-L1 and PD-L2, leading to T cell activation and cell-mediated immune responses against tumor cells or pathogens. Activated PD-1 negatively regulates T cell activation and effector function by inhibiting P13k / Akt pathway activation. Other names for nivolumab include: BMS-936558, MDX-1106, and ONO-4538. The amino acid sequence of nivolumab and methods of use and preparation are disclosed in U.S. Patent No. US 8,008,449.

[0225] Pembrolizumab is available as A commercially available humanized monoclonal anti-PD-1 antibody. Pembrolizumab is indicated for the treatment of some unresectable or metastatic melanomas. The amino acid sequence of pembrolizumab and methods of use are disclosed in U.S. Pat. No. 8,168,757.

[0226] In one embodiment, the PD-1 antagonist comprises any one or combination of the following CDRs:

[0227] CDRH1: SYDMS (SEQ ID NO: 151)

[0228] CDRH2: TISGGGSYTYYQDSVKG (SEQ ID NO: 152)

[0229] CDRH3:PYYAMDY (SEQ ID NO: 153)

[0230] CDRL1: KASQDVGTAVA (SEQ ID NO: 154)

[0231] CDRL2: WASTLHT (SEQ ID NO: 155)

[0232] CDRL3: QHYSSYPWT (SEQ ID NO: 156)

[0233] In one embodiment, the PD-1 antagonist comprises a heavy chain variable region CDR1 ("CDRH1") comprising an amino acid sequence having one or two amino acid variations relative to the amino acid sequence set forth in SEQ ID NO: 151 ("CDR variant").

[0234] In one embodiment, the PD-1 antagonist comprises a heavy chain variable region CDR2 ("CDRH2") comprising an amino acid sequence having five or fewer (e.g., four or fewer, three or fewer, two or fewer, or one) amino acid variations relative to the amino acid sequence set forth in SEQ ID NO: 152 ("CDR variant"). In another embodiment, CDRH2 comprises an amino acid sequence having one or two amino acid variations relative to the amino acid sequence set forth in SEQ ID NO: 152.

[0235] In one embodiment, the PD-1 antagonist comprises a heavy chain variable region CDR3 ("CDRH3") comprising an amino acid sequence having one or two amino acid variations relative to the amino acid sequence set forth in SEQ ID NO: 153 ("CDR variant").

[0236] In one embodiment, the PD-1 antagonist comprises a light chain variable region CDR1 ("CDRL1") comprising an amino acid sequence having three or fewer (e.g., one or two) amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 154 ("CDR variant").

[0237] In one embodiment, the PD-1 antagonist comprises a light chain variable region CDR2 ("CDRL2") comprising an amino acid sequence having one or two amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 155 ("CDR variant").

[0238] In one embodiment, the PD-1 antagonist comprises a light chain variable region CDR3 ("CDRL3") comprising an amino acid sequence having three or fewer (e.g., one or two) amino acid variations relative to the amino acid sequence set forth in SEQ ID NO: 156 ("CDR variant"). In a specific embodiment, CDRL3 comprises an amino acid sequence having one amino acid variation relative to the amino acid sequence set forth in SEQ ID NO: 156. In another embodiment, the variant CDRL3 comprises the amino acid sequence set forth in SEQ ID NO: 157.

[0239] In one embodiment, the PD-1 antagonist comprises CDRH1, which comprises an amino acid sequence having at most one amino acid variation relative to the amino acid sequence shown in SEQ ID NO: 151; CDRH2, which comprises an amino acid sequence having at most five amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 152; CDRH3, which comprises an amino acid sequence having at most one amino acid variation relative to the amino acid sequence shown in SEQ ID NO: 153; CDRL1, which comprises an amino acid sequence having at most three amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 154; CDRL2, which comprises an amino acid sequence having at most one amino acid variation relative to the amino acid sequence shown in SEQ ID NO: 155; and / or CDRL3, which comprises an amino acid sequence having at most three amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 156.

[0240] In one embodiment of the present invention, the PD-1 antagonist comprises CDRH1 (SEQ ID NO: 151), CDRH2 (SEQ ID NO: 151) and CDRH3 (SEQ ID NO: 153) in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 158. In some embodiments, the anti-PD-1 antibody of the present invention comprises a heavy chain variable region having at least 90% sequence identity with SEQ ID NO: 158. Suitably, the PD-1 antagonist of the present invention may comprise a heavy chain variable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 158.

[0241] PD-1 antagonist heavy chain (V H )Variable region:

[0242] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYAMDYWGQGTTVTVSS (SEQ ID NO: 158)

[0243] In one embodiment, the PD-1 antagonist comprises a heavy chain variable region (“V H ”), which comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 158. In one embodiment, V H Comprising an amino acid sequence having at least one amino acid variation relative to the amino acid sequence shown in SEQ ID NO: 158 (e.g., having 1 to 5, e.g., 1 to 3, in particular up to 2 amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 158).

[0244] In one embodiment of the present invention, the PD-1 antagonist comprises CDRL1 (SEQ ID NO: 154), CDRL2 (SEQ ID NO: 155) and CDRL3 (SEQ ID NO: 156) in the light chain variable region having the amino acid sequence shown in SEQ ID NO: 159. In one embodiment, the PD-1 antagonist of the present invention comprises the heavy chain variable region of SEQ ID NO: 158 and the light chain variable region of SEQ ID NO: 159.

[0245] In some embodiments, the PD-1 antagonist of the present invention comprises a light chain variable region having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 159. Suitably, the PD-1 antagonist of the present invention may comprise a light chain variable region having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 159.

[0246] PD-1 antagonist light chain (V L )Variable region:

[0247] DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTLHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTKLEIK(SEQ ID NO: 159)

[0248] In one embodiment, the PD-1 antagonist comprises a light chain variable region (“V L ”), which comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 159. In one embodiment, V L An amino acid sequence comprising at least one amino acid variation relative to the amino acid sequence shown in SEQ ID NO: 159 (e.g., 1 to 5, e.g., 1 to 3, in particular up to 2 amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 159).

[0249] In one embodiment, the PD-1 antagonist comprises V H , which comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 158; and V L The PD-1 antagonist comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 159. In one embodiment, the PD-1 antagonist comprises a V sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 158. H and / or a V sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 159 L .

[0250] In one embodiment, the PD-1 antagonist comprises a V having the amino acid sequence shown in SEQ ID NO: 158. H and V having the amino acid sequence shown in SEQ ID NO: 159 L .

[0251] In one embodiment, the PD-1 antagonist is a monoclonal antibody comprising a heavy chain (HC) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:160.

[0252] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSTISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYYAMDYWGQGTTV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP CPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:160)

[0253] In one embodiment, the HC comprises an amino acid sequence having at least one amino acid variation (e.g., 1 to 10, such as 1 to 7, in particular up to 6 amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 160). In another embodiment, the HC comprises one, two, three, four, five, six or seven amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 160.

[0254] In one embodiment, the HC chain comprises a variation at position 380 and / or 385 of SEQ ID NO: 160. The asparagine residues at these positions may be modified, for example, by deamidation (converting an asparagine (N) residue to an aspartic acid (D) residue). Thus, in one embodiment, the HC comprises the amino acid sequence of SEQ ID NO: 162 (N380D), SEQ ID NO: 163 (N385D), or SEQ ID NO: 164 (N380D and N385D).

[0255] In one embodiment, the PD-1 antagonist is a monoclonal antibody comprising a light chain (LC) amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO:161.

[0256] DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWASTLHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:161)

[0257] In one embodiment, LC comprises an amino acid sequence having at least one amino acid variation (e.g., 1 to 10, such as 1 to 5, in particular up to 3 amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 161). In another embodiment, LC comprises one, two or three amino acid variations relative to the amino acid sequence shown in SEQ ID NO: 161.

[0258] In one embodiment, the PD-1 antagonist comprises a HC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 160; and a LC comprising an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 161. Thus, the antibody is an antibody having a heavy chain that is at least about 90% identical to the heavy chain amino acid sequence of SEQ ID NO: 160 and / or having a light chain that is at least about 90% identical to the light chain amino acid sequence of SEQ ID NO: 161.

[0259] In one embodiment, the PD-1 antagonist comprises a heavy chain amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:160 and / or a light chain amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:161.

[0260] In one embodiment, the PD-1 antagonist comprises the heavy chain sequence of SEQ ID NO: 160 and the light chain sequence of SEQ ID NO: 161. In one embodiment, the antibody is dotalizumab comprising the heavy chain sequence of SEQ ID NO: 160 and the light chain sequence of SEQ ID NO:162.

[0261] Anti-PD-L1 antibodies and methods for their preparation are known in the art. Such antibodies against PD-L1 can be polyclonal or monoclonal, and / or recombinant, and / or humanized. PD-L1 antibodies are being developed as immunomodulators for the treatment of cancer.

[0262] Exemplary PD-L1 antibodies are disclosed in U.S. Pat. No. 9,212,224; U.S. Pat. No. 8,779,108; U.S. Pat. No. 8,552,154; U.S. Pat. No. 8,383,796; U.S. Pat. No. 8,217,149; U.S. Patent Publication No. 20110280877; WO2013079174; and WO2013019906. Additional exemplary antibodies to PD-L1 (also known as CD274 or B7-H1) and methods of use are disclosed in U.S. Pat. No. 8,168,179; U.S. Pat. No. 7,943,743; U.S. Pat. No. 7,595,048; WO2014055897; WO2013019906; and WO2010077634. Specific anti-human PD-L1 monoclonal antibodies used as PD-1 antagonists in the treatment methods, drugs and uses of the present invention include MPDL3280A, BMS-936559, MEDI4736, and MSB0010718C.

[0263] Atezolizumab is available as A commercially available fully humanized monoclonal anti-PD-L1 antibody. Atezolizumab is indicated for the treatment of some locally advanced or metastatic urothelial carcinomas. Atezolizumab blocks the interaction of PD-L1 with PD-1 and CD80. Other exemplary PD-L1 antibodies include avelumab Durvalumab

[0264] CD134, also known as OX40, is a member of the TNFR receptor superfamily and, unlike CD28, is not constitutively expressed on resting naive T cells. OX40 is a secondary co-stimulatory molecule expressed 24 to 72 hours after activation; its ligand OX40L is also not expressed on resting antigen-presenting cells, but after their activation. The expression of OX40 depends on the full activation of T cells; in the absence of CD28, the expression of OX40 is delayed and the level is reduced fourfold. OX-40 antibodies, OX-40 fusion proteins and methods of using them are disclosed in U.S. Patent Nos.: US 7,504,101; US ​​7,758,852; US 7,858,765; US 7,550,140; US 7,960,515; WO2012027328; WO2013028231.

[0265] Additional examples of one or more additional active ingredients (anti-tumor agents) for use in combination or co-administration with the presently disclosed CD96 binding proteins are antibodies or other antagonists of CD20, retinoids, or other kinase inhibitors. Examples of such antibodies or antagonists include, but are not limited to, rituximab ( and ), Ofatumumab and bexarotene

[0266] Additional examples of one or more additional active ingredients (anti-tumor agents) for use or co-administration in combination with the presently disclosed CD96 binding proteins are Toll-like receptor 4 (TLR4) antagonists, including but not limited to aminoalkylaminoglucosidoside phosphates (AGP).

[0267] AGP is known to be used as vaccine adjuvant and immunostimulant, for stimulating cytokine production, activated macrophage, promoting innate immune response and increasing antibody production in immune animals. AGP is a synthetic ligand of TLR4. AGP and its immunomodulatory effect by TLR4 are disclosed in patent publications such as WO 2006016997, WO 2001090129 and / or U.S. Patent number 6,113,918, and have been reported in the literature. Other AGP derivatives are disclosed in U.S. Patent number 7,129,219, U.S. Patent number 6,911,434 and U.S. Patent number 6,525,028. Some AGP serves as the agonist of TLR4, and others are considered to TLR4 antagonists.

[0268] A further non-limiting example of one or more additional active ingredients (anti-tumor agents) for use or co-administration in combination with the presently disclosed CD96 binding proteins is an antibody to ICOS.

[0269] The CDRs of a murine antibody to human ICOS with agonist activity are shown in PCT / EP2012 / 055735 (WO2012131004). Antibodies to ICOS are also disclosed in WO 2008137915, WO 2010056804, EP 1374902, EP1374901 and EP1125585.

[0270] Another example of one or more additional active ingredients (anti-tumor agents) for use or co-administration in combination with the presently disclosed CD96 binding proteins is a poly ADP ribose polymerase (PARP) inhibitor. Non-limiting examples of such inhibitors include niraparib, olaparib, rucaparib and talazoparib.

[0271] Additional non-limiting examples of one or more additional active ingredients (anti-tumor agents) used or co-administered in combination with the presently disclosed CD96 binding proteins are STING modulating compounds, CD39 inhibitors, and A2a and A2a adenosine antagonists.

[0272] The selected anti-tumor agents that can be used in combination with the CD96 binding protein or a pharmaceutically acceptable salt thereof include, but are not limited to, abarelix, abemaciclib, abiraterone, afatinib, aflibercept, aldoxorubicin, alectinib, alemtuzumab, arsenic trioxide, asparaginase, axitinib, AZD-9291, belinostat, bendamustine, bevacizumab, blinatumomab, bosutinib, brentuximab, cabazitaxel, cabozantinib, capecitabine, ceritinib, clofarabine, cobimetinib, crizotinib, daratumumab, dasatinib, degarelix, denosumab, dinutuximab, docetaxel, elotuzumab, entinostat, enzalutamide, epirubicin, eribulin, filgrastim, flumatinib, fulvestrant, furoquine ab, gemtuzumab ozogamicin, ibrutinib, ibrutinib, idelalisib, imatinib, irinotecan, ixabepilone, ixazomib, lenalidomide, lenvatinib, folinic acid, dichloromethyl diethylamide, necituzumab, nelarabine, netupitant, nilotinib, obinutuzumab, olaparib, omacetaxine, osimertinib, oxaliplatin, paclitaxel, palbociclib, palonosetron, panitumumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, plerixafor, pomalidomide, ponatinib, pralatrexate, quizartinib, radium 223, ramucirumab, regorafenib, rolapitant, ricapapab, sipuleucel-T, sonidegi, sunitinib, talimogene laherparepvec, tipiracil, topotecan, trabectinib, trifluridine, triptorelin, uridine, vandetanib, veliparib, vemurafenib, venetoclax, vincristine, vismodegib, and zoledronic acid.

[0273] Treatment may be therapeutic, prophylactic or preventative. A subject is a subject in need thereof. Those in need of treatment may include individuals already suffering from a particular medical disease, in addition to those who may develop the disease in the future.

[0274] Thus, the methods, antigen binding proteins and compositions of the disclosure described herein can be used for preventive or prophylactic treatments (if indicated). In this case, the methods, antigen binding proteins and compositions of the disclosure can be used to prevent or delay the onset of one or more aspects or symptoms of the disease. The subject can be asymptomatic. The subject can have a genetic predisposition to the disease. A preventive effective amount of antigen binding proteins is administered to such an individual. A preventive effective amount is an amount that prevents or delays the onset of one or more aspects or symptoms of the disease described herein.

[0275] The methods, antigen binding proteins and compositions of the present disclosure need not affect a complete cure, or eradicate every symptom or manifestation of a disease to constitute a viable therapeutic treatment. As is recognized in the art, drugs used as therapeutic agents in therapeutic methods can reduce the severity of a given disease state, but do not need to eliminate every manifestation of the disease to be considered a useful therapeutic agent. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of the disease to constitute a viable prophylactic. It is sufficient to simply reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect) or reduce the likelihood of the disease occurring (e.g., by delaying the onset of the disease) or worsening in a subject.

[0276] Another aspect of the present disclosure is a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD96 binding protein or pharmaceutical composition as described in any one of the aforementioned aspects. Another aspect of the present disclosure is the method of treatment described in the aforementioned aspects, which further comprises whether the subject expresses CD96.

[0277] Another aspect of the present disclosure is a CD96 binding protein or a pharmaceutical composition as described in any of the preceding aspects, for use in therapy or for treating a disease.

[0278] Another aspect of the present disclosure is the use of the CD96 binding protein or pharmaceutical composition as claimed in any one of the preceding claims in the manufacture of a medicament for treating a disease.

[0279] Another aspect of the present disclosure is a pharmaceutical composition comprising a therapeutically effective amount of the CD96 binding protein as described in any one of the preceding aspects.

[0280] One embodiment of the present disclosure is a method for treating a disease in a subject (e.g., a human subject) in need thereof, comprising administering to the subject a therapeutically effective amount of a CD96 binding protein as described in any one of the preceding embodiments or a pharmaceutical composition as described in any one of the preceding embodiments.

[0281] One embodiment of the present disclosure is a method for treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a CD96 binding or pharmaceutical composition as described in any of the preceding embodiments to the subject. Another embodiment of the present disclosure is a method for treating a disease in a subject in need thereof, further comprising determining whether the subject expresses CD96. Another embodiment of the present disclosure is a CD96 binding protein or drug as described in any of the preceding embodiments, which is used for treatment. Another embodiment of the present disclosure is a CD96 binding protein or pharmaceutical composition as described in any of the preceding embodiments, which is used for treatment of a disease. Another embodiment is the use of a CD96 binding protein or pharmaceutical composition as described in any of the preceding embodiments in the manufacture of a medicament for treating a disease. Another embodiment of the present disclosure is a method for treating a disease or treatment, comprising administering to the subject a therapeutically effective amount of a CD96 binding or pharmaceutical composition as described in any of the preceding embodiments to the subject, wherein the disease to be treated is cancer. In another aspect, the cancer is a solid tumor (e.g., a recurrent, metastatic, or advanced solid tumor). The cancer may be: liver cancer (e.g. HCC), ovarian cancer, lung cancer such as non-small cell lung cancer (NSCLC), kidney cancer (e.g. RCC), colon cancer, gastric cancer, bladder cancer, head and neck squamous cell carcinoma (HNSCC), or it may be leukemia and / or any B-cell malignancy.

[0282] Example

[0283] Example 1

[0284] Generation of CD96 binding protein

[0285] Binding protein generation

[0286] Fully human antibodies specific for human CD96 were isolated from a naive eukaryotic library using biotinylated recombinant human and cynomolgus CD96 in combination with cell sorting and FACS-based selection techniques. The heavy chain output from the naive eukaryotic library selection was shuffled with a light chain library and further selections were performed to identify the best light chain pairings.

[0287] Affinity maturation was performed to improve antibody affinity / potency. This involved integrating the CDRH3 of antibody 42Y073-86F08-1 (GAGYYGDKDPMDV of SEQ ID NO: 2) into a pre-made library with CDRH1 or CDRH2 diversity. This diverse library was further selected and lead molecules were identified and sequenced. The amino acid and nucleic acid sequences of the variable light and heavy chains of the lead molecules are shown in SEQ ID NOs: 1-96. The amino acid sequences of the CDR regions are shown in SEQ ID NOs: 97-150. Fc selection

[0288] During the process of selecting lead molecules, binding proteins were evaluated as human IgG1 WT molecules or Fc-invalid human IgG1 molecules. In order to prepare Fc-invalid molecules, amino acid residues at positions 234, 235 and 237 (particularly L234 and L235, or L235 and G237) were mutated to alanine, thereby generating hIgG1'LALA' (L234A / L235A) and hIgG1'LAGA' (L235A / G237A) Fc-invalid human IgG1 molecules. These binding proteins exhibited neutralization of CD96 binding and CD155 binding. Three CD96 binding proteins 42Y073-86F08-66 (86F08-66), 42Y073-86F08-16 (86F08-16) and 42Y073-86F04-23 (86F04-23) were selected for further evaluation. Binding protein 42Y073-86F08-66 is also known as GSK6097608 (monoclonal antibody). The primary T cell binding and CD155 neutralization activities of these three CD96 binding proteins were evaluated using different IgG variants (human IgG1, IgG2, IgG4PE or IgG1 Fc null). Figure 1 As shown, changes in the Fc did not affect binding to cells expressing CD96 or neutralization of CD155 binding to cells expressing CD96.

[0289] Example 2

[0290] Characterization of CD96-binding proteins

[0291] Binds to CHO cells expressing human or cynomolgus CD96

[0292] CHO cells expressing human CD96v2 or cynomolgus CD96v2 were prepared in PBS. Cynomolgus CD96v2 cells were stained with 1 μM purple proliferation dye (VPD450). Cells were diluted in preheated culture medium (RPMI containing 10% fetal bovine serum) and incubated for another 10 minutes at 37°C, then centrifuged at 400g for 5 minutes and resuspended in flow buffer (PBS+0.5% BSA+2mM EDTA) to 1.5x 106 / ml. Dyed CHO-cyCD96 and unstained CHO-huCD96 cells were combined and added to the wells of a 384-well assay plate at 4°C for 1h with the dilution of the test anti-CD96 binding protein. Cells were further incubated with anti-IgG APC conjugate antibodies, and the amount of surface-bound antibodies on each of CHO-cyCD96 and CHO-huCD96 cells was determined by flow cytometry. The median fluorescence intensity (MFI) values ​​were fitted using a 4-parameter logistic model to calculate the EC50 for binding. The data are summarized in Table 2.

[0293] Neutralization of human CD155 binding to CHO cells expressing human CD96v2

[0294] CD96 binding protein was prepared in PBS in a U-shaped bottom 96-well plate. CHO cells expressing human CD96v2 were added and incubated at room temperature for 30 minutes. After washing the cells 3 times by centrifugation, human CD155-Fc-AF647 conjugate was added for 30 minutes at room temperature. After washing three times, the cells were fixed and the CD155-Fc-AF647 staining level was analyzed by flow cytometry. The median fluorescence intensity (MFI) value was fitted using a 4-parameter logistic model to calculate the IC50 value. The data are summarized in Table 2.

[0295] Binding of CD96 binding protein to primary human T cells.

[0296] Human PBMCs were isolated from leukoreduction filters using density gradient centrifugation with Histopaque-1077. CD3 T cells were then isolated using a total T cell isolation kit (Miltenyi) according to the manufacturer's instructions. + T cells. Serial dilutions of the test CD96 binding protein were incubated with human T cells at 4°C for 1 h. After washing 3 times in buffer, anti-human IgG-APC conjugate was added for 1 h at 4°C. After washing three times, the cells were fixed and APC staining was analyzed by flow cytometry. The median fluorescence intensity (MFI) values ​​were fitted using a 4-parameter logistic model to calculate the EC50 of binding. The data are summarized in Table 2.

[0297] Table 2. Binding data and neutralization of human CD155 binding to hCD96v2 expressing CHO cells by CD96 binding proteins

[0298]

[0299]

[0300] 42Y073-86F08-66 was selected for further characterization

[0301] To help select lead molecules, the biophysical properties of anti-CD96 binding proteins were evaluated. Immunogenicity, deamidation, glycosylation, oxidation, aspartate isomerization were predicted by computer analysis, and aggregation, fragmentation, and chemical degradation properties were evaluated under the following conditions: 50mM sodium phosphate pH 7.5 and 50mM sodium acetate pH 5.0 at 10mg / ml or 1mg / ml, as well as unstressed and stressed (heat stress at 40°C for 2 weeks) conditions. Anti-CD96 binding proteins were expressed in a HEK transient system and purified using protein A affinity and size exclusion chromatography. The anti-CD96 binding proteins were scored as follows:

[0302] Table 3 - Sample preparation risks:

[0303]

[0304] Table 4 - Aggregation and fragmentation risk by aSEC (analytical size exclusion chromatography):

[0305]

[0306] Table 5 - Aggregation risk by DLS (Dynamic Light Scattering):

[0307]

[0308]

[0309] *Rh = hydrodynamic radius

[0310] Antigen binding by BIAcore:

[0311] Antigen binding changes between unstressed and stressed samples within ±10% are considered to be within an acceptable range (low risk). Molecules outside this range may indicate potential risk due to loss of binding activity.

[0312] The data are summarized in Tables 6 and 7. In Table 6, the positions in the sequence where the predicted risks were identified (Kabat numbering). In Table 7, the observed risks and the conditions under which they were observed use the following abbreviations: Agg = aggregation; Increased mass % = increased heterogeneity; Loss of binding = observed loss of binding greater than the assay limit; Increased binding = increased binding (indicating a possible tendency to aggregate); HMwS = high molecular weight species; LMwS = low molecular weight species; Rh = hydrodynamic radius (where a larger radius indicates possible aggregation); PBS = phosphate buffered saline; U = unstressed; S = stressed; A = 50 mM sodium acetate pH 5.0; and P = 50 mM sodium phosphate pH 7.5.

[0313] 42Y073-86F08-66, 42Y073-86F08-16, and 42Y073-86F04-23 showed minimal development liability and minimal computer risk. However, 42Y073-86F08-66 was selected for development and further characterization because the following properties were observed:

[0314] The percentage of acceptable levels of total high molecular weight species (HMwS; <5%), low molecular weight species (LMwS; <2.5%), and monomers (95%) as determined by analytical size exclusion chromatography (aSEC);

[0315] Expected values ​​for size distribution (>98% in SEC peak 2) and hydrodynamic radius (4.5-7 nm) as determined by dynamic light scattering (DLS);

[0316] Minimal unexpected changes in chemical heterogeneity between unstressed and stressed as determined by capillary isoelectric focusing (cIEF); and

[0317] • For antigen binding, <10% change was determined by antigen binding assay.

[0318] Table 6. Computer biophysical analysis results of CD96 binding proteins

[0319]

[0320]

[0321]

[0322] Table 7. Results of further biophysical analysis of CD96 binding proteins

[0323]

[0324]

[0325] Epitope binning of CD96 binding proteins

[0326] Epitope binding competition assays were performed to determine the CD96 epitopes bound to the anti-CD96 binding proteins presented herein. In short, HuCD96-His was incubated at room temperature for 1 h with the first CD96 binding protein, and then the binding of the second binding protein captured on the protein A sensor was measured by BLI. If the binding of the second CD96 binding protein was observed, the two binding proteins were considered to be non-competitive and were assigned to different epitope bins. If the binding of the second CD96 binding protein was not seen, the two binding proteins were considered to be competitive and were assigned to the same epitope bin. The same binding protein as the first and second binding proteins was used, and a self-bin control was included for each CD96 binding protein.

[0327] Except for 42Y073-2B04-46, competition between all CD96 binding proteins was observed, indicating that all except 42Y073-2B04-46 bind to similar epitopes of CD96. The data show that, except for 42Y073-2B04-46, all CD96 binding proteins presented herein bind to the same epitope of CD96 or a spatially close epitope, so that the binding of other CD96 binding proteins is inhibited (i.e., they belong to the same epitope box). However, the finding that 42Y073-2B04-46 does not compete with other CD96 binding proteins presented herein indicates that the clone binds to different epitopes of CD96 (and therefore belongs to different epitope boxes). Therefore, together with the data presented in Table 2, it should be understood that the binding and neutralization of human CD155 binding to CHO cells expressing hCD96v2 by the CD96 binding proteins presented herein is not limited to binding to any specific epitope of CD96.

[0328] Further binding affinity studies of CD96 binding proteins and CD96 isoforms

[0329] The binding affinity of 42Y073-86F08-66 (a fully human IgG1 antibody with wild-type Fc, expressed and purified from CHO cells) to recombinant CD96 was determined using a solution equilibrium titration (MSD-SET) assay. The average KD of 42Y073-86F08-66 binding to recombinant human CD96v2 was 20 pM and the average KD of 42Y073-86F08-66 binding to recombinant cynomolgus monkey CD96v2 was 278 pM ( Figure 2 ). In addition, it was demonstrated that the average KD of 42Y073-86F08-66 binding to recombinant murine CD96v2 was 479 pM.

[0330] In a surface plasmon resonance (SPR) assay, 42Y073-86F08-66 did not bind to recombinant human CD155, human CD226, human TIGIT, or human nectin-1, which are homologs in this pathway.

[0331] MSD-SET assay procedure:

[0332] Abstract: MSD-SET (MSD Solution Equilibrium Titration) analysis was used to determine the affinity of these antibodies to human and mouse CD96 proteins at 25°C, as the off-rates were too slow to be measured by BIACORE at this temperature. MSD-SET determines the solution-phase, equilibrium affinity of antibodies. The method relies on measuring free antigen at equilibrium over a series of titrated antibody concentrations.

[0333] program:

[0334] The human CD96 protein of biotinylation of 1.5nM constant concentration is used, and the CD96 protein of cynomolgus monkey biotinylation is used. For human CD96 and cynomolgus monkey CD96, the antibody sample is titrated by one-fifth on the 22-point curve from 6nM. The titrated antibody and CD96 protein are incubated at room temperature for 24h. After 24h, 5nM antibody is coated on the standard binding MSD plate (Meso Scale Discovery, L15XA) for 30 minutes at room temperature. Then the plate is blocked with STARTINGBLOCK blocking buffer (Thermo Scientific, #37542) for 30min under 700rpm shaking, and then washed 3 times with washing buffer. Under 700rpm shaking, the incubated solution is added to the MSD plate for 150s, and then washed 1 time. By incubating on the plate for 3min, the antigen captured on the plate is detected with streptavidin (Meso Scale Discovery, R32AD-1) labeled with SULFOTAG. The plates were washed 3 times with wash buffer and then read on an MSD SECTOR IMAGER instrument using 1x ReadBuffer T with surfactant (Meso Scale Discovery, R92TC-1). The percentage of free antigen was plotted as a function of titrated antibody using GRAPHPAD PRISM software and fitted with a quadratic equation. Binding of CD96 binding protein to HEK cells overexpressing CD96 protein

[0335] Binding of 42Y073-86F08-66 to known human or cynomolgus CD96 isoforms on the cell membrane was determined by flow cytometry using HEK cells transfected with these different isoforms ( Figure 3). Although the maximum binding response to the expressed isoforms varied between experiments (most likely due to variations in expression efficiency), comparable binding activity to these CD96 isoforms has been demonstrated, as shown by the EC50 of 42Y073-86F08-66 (Table 8). Taken together, these data suggest that 42Y073-86F08-66 is able to recognize all membrane forms of CD96 present in humans and cynomolgus monkeys.

[0336] Table 8. Potency of 42Y073-86F08-66 for binding to HEK cells transiently transfected with CD96 isoforms

[0337]

[0338] Binding affinity of CD96 binding protein to native CD96 on human and cynomolgus monkey T cells

[0339] To confirm that 42Y073-86F08-66 binds to native CD96 expressed on primary human and monkey cells, flow cytometry was used to determine the binding of 42Y073-86F08-66 to CD3 in both species. + T cells and subsets (CD4 + or CD8 + 42Y073-86F08-66 binds to human CD4 + and CD8 + T cell binding (EC50 47pm and 45pM, respectively) Figure 4 and Table 9). 42Y073-86F08-66 binds to primary human T cells with greater potency (pM) compared to HEK cells overexpressing CD96 (nM) due to relatively low expression of CD96 on primary cells compared to expression of CD96 following transient transfection of CD96 isoforms under the control of a strong constitutive promoter element.

[0340] Table 9. Efficacy of 42Y073-86F08-66 on primary human T cells (total CD3 + T cells relative to CD4 + or CD8 + Subgroups)

[0341]

[0342] To confirm that 42Y073-86F08-66 binds to CD96 in cynomolgus monkeys, binding to primary monkey T cells was determined. Based on the binding of 42Y073-86F08-66 and the commercially available anti-CD96 antibody 6F9, it was found that cynomolgus monkey T cells express lower levels of CD96 compared to human T cells. Similar observations were made for rhesus macaque T cells. To robustly quantify CD96 target engagement, purified cynomolgus monkey CD3 + T cells were activated for 17 days to upregulate CD96 expression. 42Y073-86F08-66 binding was determined for 3 different activated T cell samples from cynomolgus monkeys and was confirmed in all of these samples. Despite the presence of different maximum signals among the samples, the EC50 was very similar (average 76.6 pM), which is twice that of human T cells (49.9 pM). Figure 5 ).

[0343] Internalization of CD96-binding protein after binding to human T cells and NK cells

[0344] The fate of 42Y073-86F08-66 after binding was determined in human PBMC cultures from 3 donors using imaging cytometry. + T cells, CD8 + Highly punctate staining of 42Y073-86F08-66 was observed on the cell membrane of T cells and NK cells, and this staining pattern was maintained over a 45-hour period ( Figure 6 ). In all three defined cell populations (CD4 + 、CD8 + In NK cells, internalization of 42Y073-86F08-66-PE was slow and not complete over the 45-hour time course. These data suggest that internalization of 42Y073-86F08-66 is slow relative to other antibodies targeting T cell surface receptors. CD96 binding protein inhibits binding of CD96 ligand CD155 to cells expressing CD96

[0345] In primary human T cells, it was shown that pre-complexation of 42Y073-86F08-66 with membrane CD96 inhibited the binding of the latter to its ligand (recombinant human CD155:Fc) (IC50 0.16 nM) ( Figure 7 ).

[0346] Disruption of established CD96:CD155 interactions by CD96-binding proteins

[0347] When CD155:Fc was pre-bound to primary human T cells, 42Y073-86F08-66 could compete to shut down CD155 ( Figure 8 ), indicating that 42Y073-86F08-66 can replace CD155 (the natural ligand of CD96) bound by CD96.

[0348] Fc receptor engagement by CD96 binding protein

[0349] Since 42Y073-86F08-66 is a fully human IgG1 antibody with WT Fc, it is expected to bind to relevant Fc receptors.

[0350] Using a panel of recombinant cell lines (Promega) expressing activating human Fcγ receptor reporter genes (FcγRI, FcγRIIa(R), FcγRIIa(H), FcγRIIIa(V), and FcγRIIIa(F)), we demonstrated that binding of 42Y073-86F08-66 to CD96 on primary human T cells does not result in activation of human Fcγ receptors ( Fig. 9 ). In contrast, a control anti-CD52 antibody (Campath) efficiently elicited FcγR activation signals in all Fcγ receptor-expressing cells tested.

[0351] Importantly, the findings from the reporter gene assay were confirmed by a cell killing assay using primary human cells. In fresh human PBMC cultures containing NK cells, there was no evidence of increased cell death of either CD4 or CD8 T cells in the presence of 42Y073-86F08-66 ( Fig.10 ). In the same experiment, both CD4 and CD8 T cells were efficiently depleted in the presence of anti-CD52 antibody (Campath).

[0352] The first step of the classical pathway of complement activation is mediated by the binding of complement component C1q to cells opsonized with antibodies. The affinity of human complement component C1q binding to 42Y073-86F08-66 was determined using SPR, which was higher than the affinity of the IgG1 WT isotype control (KD 94.4nM). The potential of 42Y073-86F08-66 to induce complement-dependent cytotoxicity (CDC) was studied using primary human T cells as targets.

[0353] In a CDC assay using human serum as a complement source, there was no evidence of depletion of CD4 or CD8 T cells by 42Y073-86F08-66, whereas a control anti-CD52 antibody efficiently mediated depletion of both subsets ( Fig.11). Taken together, these data suggest that the risk of ADCC- or CDC-mediated depletion of CD96-expressing T cells modified with 42Y073-86F08-66 is negligible.

[0354] Example 3

[0355] In vitro efficacy and mechanism studies of CD96 binding proteins

[0356] In vitro efficacy of 42Y073-86F08-66 in a primary pooled human PBMC-MLR assay

[0357] The effect of 42Y073-86F08-66 expressed and purified from CHO cells and HEK cells was tested in a primary PBMC assay without the addition of CD155, anti-CD3 or anti-TIGIT. For this assay, PBMCs from 8 different human donors were mixed together and added to the wells with different antibodies in solution. After 3 days of incubation, IFNγ in the supernatant was measured by using a homogeneous time-resolved fluorescence (HTRF) detection method. In this assay, anti-CD3 is not required because PBMCs are activated by MHC mismatches in different donors. The efficacy of the two CD96 binding proteins (expressed in CHO or HEK cells) is similar ( Fig.12 ). Both appeared to be more effective than Tecentriq (EC50 49pM). In separate repeats of the same experiment, the EC50s for 42Y073-86F08-66(HEK), 42Y073-86F08-66(CHO), and Tecentriq were 22pM, 10pM, and 140pM, respectively ( Fig.12 ). Cell viability was also measured at the end of 3 days, and only Tecentriq showed some reduction in cell viability. Neither the anti-CD96 mAb or isotype control reduced cell viability, consistent with the lack of cell depletion observed in the ADCC assay.

[0358] CD4+ T cells are the major source of IFNγ induced by 42Y073-86F08-66 in a mixed human PBMC-MLR assay

[0359] To address the question of which cell subsets were responsible for the increased secretion of IFNγ observed in the supernatants after 42Y073-86F08-66(HEK) and 42Y073-86F08-66(CHO) treatment in the PBMC assay, cell depletion studies were performed. PBMCs from four different donors were depleted of CD4 + T cells or CD8 +T cells were then pooled for lymphocyte activation and antibody treatment. Depletion was performed using human CD4 or CD8 MicroBeads (Miltenyi), and CD4 was verified by flow cytometry. + or CD8 + Purity of T cell depletion. 98.5%-99.5% CD4 + Cells, 97.2%-100% CD8 depletion from PBMC + Data obtained from T cell-depleted mixed PBMC-MLR assays showed that CD4 + T cells rather than CD8 + T cells are the major subset responsible for induction of IFNγ by 42Y073-86F08-66 (HEK) ( Fig.13 ).

[0360] Flow cytometric studies to understand the mechanism of action of CD96 binding protein

[0361] In order to further understand the mechanism of action of anti-CD96, flow cytometry studies were performed to study the changes in the expression of cell surface receptors and intracellular cytokines in different cell populations in human PBMCs. The same mixed PBMC-MLR assay system was used. PBMCs from 8 donors were mixed together for cell activation by MHC mismatching. After adding anti-CD96 or isotype control antibodies, PBMCs were incubated for 0 days (baseline) or 3 days, and then the cells were fixed and various markers were quantified by flow cytometry. In order to confirm the activity of 42Y073-86F08-66 in this experiment, IFNγ and granzyme B secreted in the supernatant were also measured by MSD and ELISA after the third day. As previously observed, 42Y073-86F08-66 enhanced the release of IFNγ and granzyme B ( Fig.14 No increase in secreted IFNγ or granzyme B was observed using the anti-TIGIT mAb clone 1F4 (Roche, hIgG1-WT).

[0362] To understand which cell populations contribute to the increased IFNγ by 42Y073-86F08-66, intracellular staining of IFNγ was measured in different cell populations by flow cytometry. Even in the group treated with the IgG1 isotype control, IFNγ was increased on day 3 compared to day 0. + CD4 + 、CD8 + The expression and frequency of NK cell subsets were significantly increased (data not shown), indicating that lymphocyte activation ( Fig.15Treatment with 42Y073-86F08-66 further significantly increased all three immune subsets (i.e., CD4 + 、CD8 + and NK cells) in IFNγ + The frequency of cells ( Fig.15 Anti-TIGIT antibody (clone 1F4) increased IFNγ only in the NK cell population + The frequency of cells.

[0363] Additionally, CD96 expression was significantly increased in all three cell populations following cell activation at day 3 compared to day 0, a profile typically observed for checkpoint proteins. Anti-CD96 antibody treatment significantly reduced CD4 + 、CD8 + CD96 MFI and CD96 in NK cells + Although the reduction in CD96 expression was more pronounced in the group treated with 42Y073-86F08-66, treatment with the Fc-null 86F08-66-LAGA antibody also resulted in a reduction in CD96 profiles ( Figure 16-Figure 17 ). Anti-TIGIT ab clone 1F4 showed no effect. The apparent reduction in CD96 expression after 42Y073-86F08-66 treatment could be due to competition between the anti-CD96 detection antibody and the therapeutic Ab, indicating receptor occupancy by the therapeutic antibody, or it could be due to internalization of the CD96 receptor after 42Y073-86F08-66 binding. The detection antibody used in the analysis was the commercial Ab clone 6F9 (BD Biosciences).

[0364] CD226 is one of the major activating receptors in NK cells, however, its role in T cells is not well established. To gain a more detailed understanding of the mechanism, we focused on characterizing CD226 in a PBMC-MLR assay. + NK cell population. Upregulation of CD96 expression after cell activation on day 3 increased CD226 + CD96 + Treatment with 42Y073-86F08-66 resulted in an increase in CD226 NK cell frequencies on day 3 compared to both isotype controls (5.91% / 46.4%=0.127) and 86F08-66 IgG1 LAGA Fc null antibody (13.8% / 47.2%=0.29). + Single positive relative to CD226 + CD96 +The proportion of double-positive NK cells was higher (26.2% / 25.2% = 1), which may be due to target occupancy and / or internalization of CD96 ( Fig.18 Since CD226 is an activating receptor in the axis and CD96 is a putative checkpoint receptor in the same axis, CD226 + Single positive cells can be compared to CD226 + CD96 + Double-positive cells represent more activated NK cells.

[0365] On day 3 after 42Y073-86F08-66 treatment, 42Y073-86F08-66 treatment resulted in the expression of + IFNγ in NK cells + Cells and GrzB + No effect was observed using the Fc-null IgG1-LAGA lead antibody (86F08-66-LAGA).

[0366] Overall, 42Y073-86F08-66 treatment resulted in increased expression of IFNγ in total NK cells at day 3 compared to isotype control Ab or Fc-null IgG1-LAGA lead antibody (86F08-66-LAGA). + G JZ + Double positive cells are more ( Fig.19 ).

[0367] CD155-PBMC assay for screening CD96 binding proteins

[0368] To determine the inhibitory effect of plate-bound CD155-Fc on IFNγ production in human PBMCs, round-bottom 96-well non-TC plates (#351177) were coated overnight at 4°C with rhCD155-Fc (Cat#9174-CD-050, R&D Systems) at different doses and blocked for 30 min at room temperature with AIM-V medium (Cat#12055-091, Therrmo Fisher) containing 5% BSA (cat#9576, Sigma). PBMCs (Cat#70025, Stemcell Technologies) were pretreated with CD96 binding protein, anti-TIGIT mAb (Cat#MAB7898, R&D Systems) or isotype control for 10 min at room temperature and incubated at 2×10 5Cells / well were added to the wells and cultured for 3 days. The supernatant was harvested and stored at -20°C for measurement of IFNγ and granzyme B by MSD or ELISA. The inhibitory effect of plate-bound CD155-Fc on IFNγ production in human PMBCs is shown in Fig. 20 CD96 binding protein appears to alleviate CD155-Fc-mediated IFNγ inhibition relative to IgG1-WT isotype control antibody ( Fig.26 Additional assays were performed to determine the ability of CD96 binding protein to mitigate CD155-Fc-mediated inhibition of TNFα in the presence of anti-TIGIT mAb ( Fig. 27 Further assays performed in the presence and absence of anti-TIGIT mAb demonstrated that the ability of CD96 binding protein to alleviate the inhibition of IFNγ production by CD155-Fc was evident in the presence and absence of anti-TIGIT mAb ( Fig.28 ).

[0369] Activity of CD96 binding protein in human tumor infiltrating lymphocyte (TIL) assay

[0370] The tumor microenvironment (TME) is immunosuppressive, and tumor infiltrating lymphocytes (TILs) found in tumors are often immunologically dysfunctional or "exhausted". A primary human TIL assay was developed to evaluate the potential therapeutic effects of 42Y073-86F08-66 in vitro. Fresh primary resected tumors were mechanically and enzymatically dissociated into single cell suspensions containing tumor cells and TILs. TILs in the cell suspension were mildly activated with suboptimal doses of soluble anti-CD3 (clone HIT3a) and seeded in triplicate into 96-well ultra-low attachment round-bottom sphere formation plates. Antibodies were then added to the wells, and IFNγ was measured in the supernatant after 6 days. 42Y073-86F08-66 was evaluated in this assay at 3 concentrations (10, 2, and 0.5 μg / mL) alone or in combination with anti-PD-1 (Keytruda / pembrolizumab) (10 μg / mL) or anti-TIGIT (100 μg / mL). Anti-PD-1 alone was also tested as a positive control for induction of IFNγ above anti-CD3 alone. Appropriate isotype controls were also included.

[0371] A total of 6 tumors (4 endometrial and 2 kidney) were tested in this assay. For 4 endometrial tumors, anti-PD-1 and anti-CD3 treatment together induced IFNγ levels comparable to anti-CD3 alone, indicating that anti-CD3 stimulation may be too high and may require further technical development. In one of the two renal tumors (sample 5001063), anti-PD-1 treatment increased (by one-way ANOVA, p<0.05) IFNγ production higher than anti-CD3 alone. In addition, anti-PD-1+42Y073-86F08-66 (10 μg / mL) combination treatment significantly (by one-way ANOVA, p<0.001) increased IFNγ levels to 7764pg / mL, higher than 10μg / mL of 42Y073-86F08-66 alone (599pg / ml), anti-PD-1 alone (2692pg / mL), or the simple additive effect of both ( Fig.21 ). For the second renal tumor (sample 1002273), anti-PD-1 treatment did not enhance IFNγ production above anti-CD3 stimulation alone. These tumors were not pre-screened for PD-1 axis expression or CD96 axis expression. The complexity of these TIL assays is widely recognized, and variation in the response of patient tumors to anti-PD-1 treatment has been observed. This is not surprising given that the overall response rate to anti-PD-1 (Keytruda) is less than 25% in unselected patients.

[0372] Example 4

[0373] In vivo efficacy and mechanism studies

[0374] Bioluminescent imaging of CD96 binding proteins in a NK cell-dependent B16F10 melanoma lung colonization model

[0375] NK cells are part of the innate lymphoid cell family and play a significant role in controlling early tumor growth and metastatic spread through cytotoxic activity and the release of inflammatory cytokines. A commonly used in vivo model to study NK cell-dependent anticancer activity is the B16F10 melanoma lung colonization model. This model is also described in a CD96 cancer-related publication (Blake SJ et al. 2016). We decided to use the same model to study the in vivo activity of 42Y073-86F08-66 (produced in HEK cells). In order to measure the efficacy of anti-CD96 mAbs in controlling lung metastasis in real time and in a more quantitative manner, instead of using conventional B16F10 melanoma cells, B16F10 cells encoding red firefly luciferase (RFluc) were used to image the luciferase signal from the lung (indicating tumor burden) in vivo.

[0376] In vivo bioluminescence imaging was performed on days 0 (approximately 15 minutes after cell injection), 7, 10, 14, 17, and 20 following tail vein administration of approximately 500,000 B16F10 RFluc metastatic melanoma cells. (Initial cell colonization in the lungs occurred as early as 15 minutes after injection from previous model development studies.) Dosing frequency was twice weekly ( Fig. 22 ). After in vivo imaging on day 20, mice were harvested for ex vivo bioluminescence imaging of mouse lungs.

[0377] To further evaluate the role of specific immune cell types (CD4+, CD8+, and NK cells) in this system, established antibody treatments were used to deplete CD4+ cells, CD8+ cells, NK cells, or both CD4+ and CD8+ cells. Subsequent flow cytometric analysis confirmed the presence of CD4+ cells. + / CD8 + Depletion of NK cells as well as T cells in the depletion group.

[0378] Bioluminescence imaging on day 14 showed a significant increase in the signal in the lungs of the NK cell depletion group ( Fig.23 ). When the lung signal (p / s) was lower, there were fewer metastases. In fact, 19 of 20 mice did not survive to the end of the study, likely due to excessive lung tumor burden. This data clearly supports the key role of NK cells in this model, which is primarily responsible for suppressing lung metastasis.

[0379] In vivo bioluminescence imaging performed on day 14 while NK-depleted mice were still alive showed that for the NK-depleted group, 42Y073-86F08-66 (produced in HEK cells) treatment significantly reduced lung metastasis compared with isotype control treatment (*P<0.05)( Fig.24 , A). This trend was also observed for the non-depleted group, but was not statistically significant. At day 20, for the non-depleted group, lung signaling was reduced in the 42Y073-86F08-66 treated group, but was not statistically significant (ns). For the T cell depleted group, 42Y073-86F08-66 significantly reduced (**P<0.01) lung signaling compared to the isotype control ( Fig.24 , B). When the lung signal (p / s) was lower, there were fewer metastases. Digital images of the lungs after mice were sacrificed on day 20 confirmed the effect of 42Y073-86F08-66 ( Fig.25 ).

[0380] Example 5

[0381] Summary of efficacy data and mechanism

[0382] Evidence for the activity of 42Y073-86F08-66 on T cells

[0383] Human: CD96 is expressed at readily detectable levels on CD4 and CD8 T cells in PBMCs and the tumor microenvironment. In TILs, CD96 is found on CD8, CD4 Teff cells, and Tregs in addition to NK cells. Consistent with its expression pattern, flow cytometric analysis showed that 42Y073-86F08-66 treatment increased IFNγ in a mixed PBMC-MLR assay. + CD4 + and CD8 + Percentage of T cells. Depletion studies showed a significant loss of IFNγ release by 42Y073-86F08-66 in the mixed PBMC-MLR assay when CD4 T cells were depleted. However, CD8 depletion was not very effective in this particular assay.

[0384] Mice: In an in vivo setting, for the B16F10 lung colonization assay, when NK-depleted mice were still alive at day 14 after tumor cell injection, 42Y073-86F08-66 treatment still showed a statistically significant reduction in lung metastasis when NK cells were depleted compared to isotype controls, likely through activation of T cells.

[0385] Evidence for NK cell activity

[0386] Human: CD96 is expressed at readily detectable levels on NK cells in PBMCs as well as in the tumor microenvironment. In fact, among TIL subsets, the highest levels of CD96 expression were found on NK cells. CD226 is one of the major activating receptors for NK cells, and strong NK activity target validation data for CD96 has also been reported in the literature. In vitro, intracellular cytokine staining studies using flow cytometry showed that IFNγ was expressed after 42Y073-86F08-66 treatment in a mixed PBMC-MLR assay. + G JZ + NK cells increase.

[0387] Mice: In an in vivo setting, using a well-established model of high NK cell dependency, the B16F10 lung colonization model, 42Y073-86F08-66 was able to significantly inhibit lung metastasis upon depletion of both CD4 and CD8 T cells, strongly suggesting NK activity.

[0388] Example 6: Anti-CD96 mAb - Non-clinical toxicology studies

[0389] GSK6097608B (hereinafter referred to as GSK6097608, which is the same as 42Y073-86F08-66) is a monoclonal antibody (mAb) targeting cluster of differentiation (CD) 96 and is being developed for the treatment of cancer. Intravenous (IV) dose range and 4-week repeated dose toxicity studies were performed using anti-CD96 mAb in cynomolgus monkeys and BALB / c mice. In addition, single-dose PK and PD studies were performed in monkeys. In vitro CRAs and combined CRAs with dotalizumab (anti-PD-1 mAb) were performed in human blood samples. Evaluation of the binding profile was performed using human microarrays with subsequent confirmatory binding assays. Preliminary immunohistochemistry (IHC) studies were performed using selected human and cynomolgus monkey tissues.

[0390] BALB / c mice and cynomolgus monkeys were selected as nonclinical species to evaluate the safety profile of anti-CD96 mAbs. Since anti-CD96 mAbs cross-react with human and monkey CD96 receptors with similar affinity, monkeys were considered appropriate nonclinical species to evaluate the potential toxicity of anti-CD96 mAbs. Based on the results of initial PK and efficacy studies, as well as in vitro binding of anti-CD96 mAbs to murine splenocytes and cytokine production by prestimulating murine splenocytes with CD3 / CD28 and incubating with anti-CD96 mAbs, additional nonclinical safety studies were performed in BALB / c mice.

[0391] Anti-CD96 mAbs were well tolerated in monkey toxicology studies following 4 weeks of dosing up to 100 mg / kg / week. In all studies, CD96 receptor occupancy (RO) or CD8 + T cells, CD4 + Target engagement on both T cells and NK cells was maintained. A low incidence of low-titer anti-drug antibodies (ADA) that did not affect receptor binding or systemic exposure was observed. There was no change in the number of circulating cells expressing CD96 in tissues (including primary and secondary immune tissues), and there were no histopathological findings, indicating a low risk of fragment crystallizable (Fc)-dependent depletion of effector cells expressing CD96. Anti-CD96 mAb was tolerated at 100 mg / kg / week for 4 weeks in mice; however, a lower dose of 10 mg / kg / week caused immune-mediated allergic reactions due to ADA or circulating immune complexes after the third week of dosing.

[0392] In vitro CRAs were performed using PBMCs isolated from 5 male and 5 female healthy donors to evaluate anti-CD96 mAb alone, dotalimab alone, and the combination of anti-CD96 mAb and dotalimab. Compared with medium controls, there was minimal increase in interleukin (IL)-10 (immunomodulatory anti-inflammatory cytokine) in most donors treated with each antibody, and mild to moderate treatment-related increases in IL-6 and / or tumor necrosis factor alpha (TNF-α) for 1 donor in each of the anti-CD96 mAb alone and dotalimab alone conditions. These levels were lower than the anti-CD3 and anti-CD28 positive controls that induce pan-cytokine responses. Overall, the combined treatment did not increase cytokine responses; however, 1 donor with increases in IL-6 and TNF-α under the anti-CD96 mAb alone condition was also more sensitive to the combined treatment. The results suggest that the overall risk of cytokine release syndrome (CRS) is low, but there may be individual participants with cytokine-induced responses.

[0393] Based on tolerability and the absence of relevant adverse findings, the no observed adverse effect level (NOAEL) was established at 100 mg / kg / week (the highest dose tested) in monkeys (sex average 4-week mean area under the curve) [AUC]0-168h: 580 mg·h / mL [range: 483 to 647 mg·h / mL] and Cmax: 5.54 mg / mL [range: 5.10 to 6.54 mg / mL]) and mice (sex average 4-week mean AUC0-168h: 319 mg·h / mL and Cmax: 2.98 mg / mL).

[0394] Example 7: First in Human Study of Anti-CD96 mAb as Monotherapy and in Combination with Dotarimab (Also Known as TSR042)

[0395] GSK6097608B (hereinafter referred to as GSK6097608, which is the same as 42Y073-86F08-66) is a monoclonal antibody (anti-CD96 mAb) targeting cluster of differentiation (CD) 96, which is being developed for the treatment of cancer. Engagement of the related receptor CD155 with CD96 acts as a "shutdown switch" or immune checkpoint to downregulate the immune response. The CD96 antagonist antibody is developed to block the CD96:CD155 inhibitory axis and increase the anti-tumor activity of T cells and natural killer (NK) cells. The first-in-human (FTIH) study evaluates the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary clinical activity of GSK6097608 as a monotherapy and in combination with dotalimumab. Dutarimab (also known as TSR 042) is an investigational humanized mAb of the immunoglobulin (Ig) G4 (IgG4) isotype; it has high affinity for binding to programmed cell death protein 1 (PD-1), resulting in inhibition of binding to programmed death ligand (PD-L) 1 and PD-L2. Based on the anti-tumor activity observed with other antibodies of the same class, Dutarimab is expected to show clinical activity in a broad spectrum of cancers.

[0396] Based on evidence supporting molecular interactions between pathways and the utility of addressing different immune populations, GSK6097608 may not only work synergistically with PD-(L)1 (programmed cell death protein 1 and / or programmed death ligand 1) inhibition but also benefit patients who are refractory or resistant to current T-cell-based therapies.

[0397] Scientific Rationale for Combination with Anti-PD-1

[0398] Despite the therapeutic benefit of blocking the immune checkpoint pathways PD-(L)1 and CTLA-4 in multiple tumor types, the majority of patients do not respond to monotherapy with checkpoint inhibitors, and strategies to increase their activity through combination approaches are being actively explored. The rationale for combining an anti-CD96 mAb (GSK6097608) with an agent designed to block the PD-(L)1 pathway (dotarimab, an anti-PD-1 mAb) is based on evidence supporting molecular interactions between the pathways, the utility of addressing distinct immune populations, and therapeutic complementarity between intervention strategies.

[0399] At a mechanistic level, PD-1 signaling has been shown to dephosphorylate the intracellular domain of CD226, attenuating the potential for costimulation following CD96-mediated redirection of CD155. Of note, CD96 expression is upregulated in melanoma tissue following nivolumab (anti-PD-1) treatment, suggesting a CD96 inhibitory axis as a possible adaptive resistance mechanism to PD-1 blockade. Reciprocal upregulation of pathway components may also be observed with CD96 blockade, as GSK6097608-mediated induction of interferon gamma (IFNγ) has the potential to upregulate PD-L1 expression. Overall, these observations suggest that co-blockade of CD96 and PD-(L)1 may be necessary to achieve an effective antitumor immune response.

[0400] Clinical experience with dotalimumab

[0401] Dutarimab is currently being developed as a monotherapy for patients with recurrent or advanced solid tumors, including endometrial cancer (microsatellite stable and microsatellite instability high [MSI-H] tumors), non-small cell lung cancer (NSCLC), and non-endometrial MSI-H solid tumors and cancers with polymerase epsilon mutations. In addition, Dutarimab is being developed as a combination therapy with other therapeutic agents for patients with advanced solid tumors (including melanoma, NSCLC, and colorectal cancer) or advanced or metastatic cancers (including endometrial ovarian cancer, triple-negative breast cancer, and urothelial carcinoma).

[0402] As of January 21, 2019, there are 4 ongoing Phase 1 studies, 2 ongoing Phase 2 studies, and 1 ongoing Phase 3 study using dotalimumab.

[0403] The safety and tolerability of dotarimab have been evaluated in more than 627 participants with advanced cancer who have received at least 1 dose of dotarimab. Of the 335 participants treated with dotarimab monotherapy in FTIH Study 4010 01-001 (GARNET), 93.7% reported at least 1 treatment-emergent adverse event (TEAE), with fatigue, nausea, and diarrhea being the most frequently reported events. TEAEs ≥ Grade 3 related to the study intervention were reported in 36 participants (10.7%). Most TEAEs ≥ Grade 3 related to the study intervention occurred in only 2 participants, with the exception of fatigue (6 participants), increased alanine aminotransferase (ALT) (4 participants), anemia (4 participants), increased aspartate aminotransferase (AST) (3 participants), and increased lipase (3 participants). Serious adverse events (SAEs) occurred in 106 participants (31.6%); in 21 of these participants, these SAEs were considered related to the study intervention. All SAEs related to the study intervention occurred in 1 participant, except pneumonia (4 participants), dyspnea (2 participants), pyrexia (2 participants), and maculopapular rash (2 participants). Twenty-three participants (6.9%) receiving dotalimumab monotherapy experienced at least 1 immune-related adverse event (irAE) of grade ≥3 severity; for 18 of the 23 participants with grade ≥3 irAEs, the adverse event (AE) was assessed by the investigator as related to the study intervention. Most grade ≥3 irAEs were reported in ≤2 participants. Grade ≥3 irAEs reported in >2 participants were increased AST (4 participants), increased ALT (4 participants), increased lipase (4 participants), and rash (3 participants).

[0404] Of the 292 participants treated with dotalimumab in combination with other therapeutic agents, 94.5% reported at least 1 TEAE, with fatigue, nausea, and dyspnea being the most frequently reported events. SAEs occurred in 108 participants (37.0%); in 20 of these 108 participants, the SAE was related to the study intervention.

[0405] Based on safety data from human experience and available nonclinical pharmacology and toxicology information, dotarizumab has demonstrated an acceptable clinical and nonclinical safety profile that appears consistent with the safety experience with the approved mAb PD-1 inhibitors pembrolizumab and nivolumab.

[0406] Based on the observed antitumor activity of other antibodies in the same class, dotarimab is expected to show clinical activity in a broad spectrum of cancers. Preliminary efficacy data from 15 participants with MSI-H endometrial cancer and 24 NSCLC participants who had at least 1 tumor assessment were presented at the American Association for Cancer Research Annual Meeting in April 2018. Investigators used the immune-related response evaluation criteria in solid tumors (irRECIST) to assess responses. In the 15 participants with MSI-H endometrial cancer, the overall response rate was 47% and consisted of all partial responses (PR); 20% had stable disease (SD) and 33% had progressive disease. In the 24 participants with NSCLC, the ORR was 29%, consisting of all PRs; 42% had SD and 17% had progressive disease. Thus, in participants with advanced and recurrent cancer, preliminary efficacy data from participants with NSCLC treated with dolimab appear comparable to efficacy reported with other PD-(L)1 inhibitors, such as pembrolizumab (ORR: 19%) [Herbst, 2016].

[0407] Goals and End Points

[0408] This FTIH, open-label, dose-escalation study evaluated the safety, tolerability, PK, PD, and preliminary clinical activity of GSK6097608 as monotherapy (Arm A) or in combination with dometamine (Arm B) in participants with locally advanced, recurrent, or metastatic solid tumors; the study was used to define the recommended phase 2 dose (RP2D).

[0409] Table 10 - Research summary:

[0410]

[0411]

[0412] Abbreviations: ADA = antidrug antibodies; AE = adverse event; AUC = area under the curve; Cmax = maximum concentration; Cmin = minimum concentration; DCR = disease control rate; DLT = dose-limiting toxicity; DNA = deoxyribonucleic acid; DoR = duration of response; IHC = immunohistochemistry; iRECIST = modified Response Evaluation Criteria in Solid Tumors, version 1.1, for immune-based therapies; IV = intravenous; ORR = overall response rate; PD = pharmacodynamics; PK = pharmacokinetics; RNA = RNA; RECIST 1.1 = Response Evaluation Criteria in Solid Tumors, version 1.1; RP2D = recommended phase 2 dose; SAE = serious adverse event; t1 / 2 = half-life; TTP = time to progression; TTR = time to response.

[0413] Overall Design

[0414] This is an FTIH, open-label, non-randomized, multicenter study designed to investigate the safety, tolerability, PK, PD, and preliminary clinical activity of escalating doses of GSK6097608 administered IV as monotherapy (Arm A) or in combination with dotarizumab (Arm B) to participants with locally advanced, recurrent, or metastatic solid tumors.

[0415] GSK6097608 administered IV once every 3 weeks (Q3W) was evaluated as a monotherapy at escalating doses (Group A) under the guidance of the Dose Escalation Committee (DEC). To further evaluate PK and PD, additional participants (up to 15 participants) participated in the PK / PD group with RP2D in the following tumor types: non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) or other tumor types defined based on emerging nonclinical and / or clinical data. For dose exploration, additional participants (up to 15 participants / group) participated in the PK / PD group at previously defined dose levels. These additional participants helped to assess safety and preliminary anticancer activity as well as the overall PK / PD data profile.

[0416] Once a tolerable GSK6097608 dose with sufficient drug exposure is identified based on PK data, participation in the combination group (Group B) can begin. In Group B, the escalating dose of GSK6097608 is evaluated using a fixed dosing regimen of dotalimumab. To further evaluate PK and PD, additional participants (up to 15 participants) participate in the PK / PD group with RP2D in the following tumor types: NSCLC, HNSCC, or other tumor types defined based on emerging nonclinical and / or clinical data. For dose exploration, in addition to the group treated with RP2D, participants (up to 15 participants / group) participate in the PK / PD group at a previously defined dose level.

[0417] These additional participants will help assess safety and preliminary anticancer activity as well as the overall PK / PD data profile.

[0418] Disease status assessments at screening and during study intervention visits were performed by the investigator according to Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1) and modified RECIST based on immunotherapy (iRECIST). The decision to discontinue treatment due to disease progression was based on iRECIST; however, some secondary and exploratory anticancer activity analyses were based on RECIST 1.1. Scans were collected centrally and stored to allow for the option of centralized review.

[0419] analyze

[0420] After each dose cohort, the Neuenschwander continuous reassessment method (N-CRM) was used to guide monotherapy and combination therapy dose escalation. Dose escalation decisions were primarily based on dose-limiting toxicity (DLT); however, the overall context of clinical safety assessments, PK, and PD data was considered. Informal statistical assumptions were tested, and the analysis was descriptive.

[0421] This was a 2-group, open-label, interventional study.

[0422] Number of participants

[0423] Approximately 100 participants will be recruited to receive the study intervention. The total number of participants to be recruited is an estimate and depends on the number required to fully characterize the DLT profile and determine the RP2D.

[0424] Intervention groups and duration

[0425] In Group A (monotherapy), GSK6097608 was administered IV to participants over approximately 30 minutes under the medical supervision of the investigator or an appropriately qualified designee.

[0426] In Group B (combination therapy), GSK6097608 was administered IV first. Dotarimab was then administered IV within 30 minutes after the end of the GSK6097608 infusion. Both study interventions were administered under medical supervision of the investigator or designee.

[0427] The study consists of 3 periods: screening (assessment up to 28 days before the first dose), treatment (until disease progression, unacceptable toxicity, death, or withdrawal of consent), and treatment cessation and follow-up (90 days). The total duration of study participation begins with the signing of the informed consent form (ICF) and continues until the final protocol-defined follow-up assessment period. The maximum duration of study participation depends on the participant's study intervention duration, with an approximate duration of up to 2 years.

[0428] Tumor Imaging and Disease Assessment

[0429] Lesion assessment methods and timing, disease assessment, disease progression, and response criteria were performed according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). RECIST 1.1 was used to assess disease burden (target and non-target lesions determined) at screening and as the primary measure of tumor response endpoints. Investigators used iRECIST to assess tumor response and progression and make treatment decisions.

[0430] Sequence Listing

[0431] The Kabat-defined CDRs are underlined.

[0432] 42Y073-86F08-1 variable light chain amino acid sequence

[0433] SEQ ID NO: 1

[0434] DIQLTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[0435] 42Y073-86F08-1 variable heavy chain amino acid sequence

[0436] SEQ ID NO: 2

[0437] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GIIPIFGTASYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMDV WGQGTTVTVSS

[0438] 42Y073-86F08-1 variable light chain nucleic acid sequence

[0439] SEQ ID NO: 3

[0440] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCA CTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0441] 42Y073-86F08-1 variable heavy chain nucleic acid sequence

[0442] SEQ ID NO: 4

[0443] CAGGTGCAGCTGGTGCAGAGCGGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGACCTTCAGCAGCTACGCCATCAGCTTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTTTCGGCACCGCCAGCTACGCC CAGAAGTTCCAGGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0444] 42Y073-86F04-3 variable light chain amino acid sequence

[0445] SEQ ID NO: 5

[0446] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVEIK

[0447] 42Y073-86F04-3 variable heavy chain amino acid sequence

[0448] SEQ ID NO: 6

[0449] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS YNAIS WVRQAPGQGLEWMG GIIPIMGTARYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGESGMDV WGQGTTVTVSS

[0450] 42Y073-86F04-3 variable light chain nucleic acid sequence

[0451] SEQ ID NO: 7

[0452] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0453] 42Y073-86F04-3 Variable heavy chain nucleic acid sequence

[0454] SEQ ID NO: 8

[0455] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCTACAACGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTATGGGCACCGCCAGGTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0456] 42Y073-86F04-4 Variable light chain amino acid sequence

[0457] SEQ ID NO: 9

[0458] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVEIK

[0459] 42Y073-86F04-4 variable heavy chain amino acid sequence

[0460] SEQ ID NO: 10

[0461] QVQLVQSGAEVKKPGSSVKVSCKASGGTFE SEAIS WVRQAPGQGLEWMG GIIPIFGRARYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGESGMDV WGQGTTVTVSS

[0462] 42Y073-86F04-4 variable light chain nucleic acid sequence

[0463] SEQ ID NO: 11

[0464] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCAC TGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0465] 42Y073-86F04-4 variable heavy chain nucleic acid sequence

[0466] SEQ ID NO: 12

[0467] CAGGTGCAGCTGGTGCAGAGCGGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGACCTTCGAGAGCGAGGCCATCAGCTTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTTTCGGCAGGGCCAGG TACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0468] 42Y073-86F04-5 variable light chain amino acid sequence

[0469] SEQ ID NO: 13

[0470] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVEIK

[0471] 42Y073-86F04-5 variable heavy chain amino acid sequence

[0472] SEQ ID NO: 14

[0473] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SHAIS WVRQAPGQGLEWMG GIIPIFGRGKYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGESGMDV WGQGTTVTVSS

[0474] 42Y073-86F04-5 variable light chain nucleic acid sequence

[0475] SEQ ID NO: 15

[0476] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0477] Variable heavy chain nucleic acid sequence of 42Y073-86F04-5

[0478] SEQ ID NO: 16

[0479] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCAGCCACGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTTTCGGCAGGGGCAAGTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0480] Variable light chain amino acid sequence of 42Y073-86F04-6

[0481] SEQ ID NO: 17

[0482] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVEIK

[0483] 42Y073-86F04-6 variable heavy chain amino acid sequence

[0484] SEQ ID NO: 18

[0485] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS GHAIS WVRQAPGQGLEWMG GIIPIFGRARYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGESGMDV WGQGTTVTVSS

[0486] 42Y073-86F04-6 variable light chain nucleic acid sequence

[0487] SEQ ID NO: 19

[0488] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCAC TGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0489] 42Y073-86F04-6 variable heavy chain nucleic acid sequence

[0490] SEQ ID NO: 20

[0491] CAGGTGCAGCTGGTGCAGAGCGGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGACCTTCAGCGGCCACGCCATCAGCTTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTTTCGGCAGGGCCAGG TACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0492] 42Y073-86F04-18 variable light chain amino acid sequence

[0493] SEQ ID NO: 21

[0494] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVEIK

[0495] 42Y073-86F04-18 variable heavy chain amino acid sequence

[0496] SEQ ID NO: 22

[0497] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SRAIS WVRQAPGQGLEWMG GIIPIMGTARYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGESGMDV WGQGTTVTVSS

[0498] 42Y073-86F04-18 variable light chain nucleic acid sequence

[0499] SEQ ID NO: 23

[0500] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0501] 42Y073-86F04-18 Variable heavy chain nucleic acid sequence

[0502] SEQ ID NO: 24

[0503] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCAGCAGGGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTATGGGCACCGCCAGGTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0504] 42Y073-86F04-33 Variable light chain amino acid sequence

[0505] SEQ ID NO: 25

[0506] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVEIK

[0507] 42Y073-86F04-33 variable heavy chain amino acid sequence

[0508] SEQ ID NO: 26

[0509] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAIS WVRQAPGQGLEWMG GILPIFGRANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGESGMDV WGQGTTVTVSS

[0510] 42Y073-86F04-33 variable light chain nucleic acid sequence

[0511] SEQ ID NO: 27

[0512] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCAC TGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0513] 42Y073-86F04-33 variable heavy chain nucleic acid sequence

[0514] SEQ ID NO: 28

[0515] CAGGTGCAGCTGGTGCAGAGCGGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGACCTTCAGCAGCTACGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCCTGCCCATTTTCGGCAGGGCCAAC TACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0516] 42Y073-86F04-88 variable light chain amino acid sequence

[0517] SEQ ID NO: 29

[0518] DIQMTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVEIK

[0519] 42Y073-86F04-88 variable heavy chain amino acid sequence

[0520] SEQ ID NO: 30

[0521] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS SSAIS WVRQAPGQGLEWMG GIIPIFGRANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGESGMDV WGQGTTVTVSS

[0522] 42Y073-86F04-88 variable light chain nucleic acid sequence

[0523] SEQ ID NO: 31

[0524] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0525] 42Y073-86F04-88 Variable heavy chain nucleic acid sequence

[0526] SEQ ID NO: 32

[0527] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCAGCAGCGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTTTCGGCAGGGCCAACTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0528] 42Y073-86F08-3 Variable light chain amino acid sequence

[0529] SEQ ID NO: 33

[0530] DIQLTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[0531] 42Y073-86F08-3 variable heavy chain amino acid sequence

[0532] SEQ ID NO: 34

[0533] QVQLVQSGAEVKKPGSSVKVSCKASGGTFV NYAIS WVRQAPGQGLEWMG GIIPALGTANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMDV WGQGTTVTVSS

[0534] 42Y073-86F08-3 variable light chain nucleic acid sequence

[0535] SEQ ID NO: 35

[0536] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCA CTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0537] 42Y073-86F08-3 variable heavy chain nucleic acid sequence

[0538] SEQ ID NO: 36

[0539] CAGGTGCAGCTGGTGCAGAGCGGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCGTGAACTACGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCGCCCTGGGCACCGCCAACTACGCC CAGAAGTTCCAGGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0540] 42Y073-86F08-4 variable light chain amino acid sequence

[0541] SEQ ID NO: 37

[0542] DIQLTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[0543] 42Y073-86F08-4 variable heavy chain amino acid sequence

[0544] SEQ ID NO: 38

[0545] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS HYA WVRQAPGQGLEWMG NIIPIFGTAGYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMDV WGQGTTVTVSS

[0546] 42Y073-86F08-4 variable light chain nucleic acid sequence

[0547] SEQ ID NO: 39

[0548] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0549] Variable heavy chain nucleic acid sequence of 42Y073-86F08-4

[0550] SEQ ID NO: 40

[0551] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCGAGTACGCCATCCACTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCAACATCATCCCCATTTTCGGCACCGCCGGCTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0552] Variable light chain amino acid sequence of 42Y073-86F08-8

[0553] SEQ ID NO: 41

[0554] DIQLTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[0555] 42Y073-86F08-8 variable heavy chain amino acid sequence

[0556] SEQ ID NO: 42

[0557] QVQLVQSGAEVKKPGSSVKVSCKASGGTFW LYAIS WVRQAPGQGLEWMG GIIPQLGTANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMDV WGQGTTVTVSS

[0558] 42Y073-86F08-8 variable light chain nucleic acid sequence

[0559] SEQ ID NO: 43

[0560] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCA CTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0561] 42Y073-86F08-8 variable heavy chain nucleic acid sequence

[0562] SEQ ID NO: 44

[0563] CAGGTGCAGCTGGTGCAGAGCGGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGACCTTCTGGCTGTACGCCATCAGCTTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCCAGCTGGGCACCGCCAACTACGCC CAGAAGTTCCAGGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0564] 42Y073-86F08-17 variable light chain amino acid sequence

[0565] SEQ ID NO: 45

[0566] DIQLTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[0567] 42Y073-86F08-17 variable heavy chain amino acid sequence

[0568] SEQ ID NO: 46

[0569] QVQLVQSGAEVKKPGSSVKVSCKASGGTFR EYAIS WVRQAPGQGLEWMG GIIPVFGTANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMDV WGQGTTVTVSS

[0570] 42Y073-86F08-17 variable light chain nucleic acid sequence

[0571] SEQ ID NO: 47

[0572] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0573] 42Y073-86F08-17 Variable heavy chain nucleic acid sequence

[0574] SEQ ID NO: 48

[0575] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGGGAGTACGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCGTGTTCGGCACCGCCAACTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0576] 42Y073-86F08-22 Variable light chain amino acid sequence

[0577] SEQ ID NO: 49

[0578] DIQLTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[0579] 42Y073-86F08-22 variable heavy chain amino acid sequence

[0580] SEQ ID NO: 50

[0581] QVQLVQSGAEVKKPGASVKVSCKASGYTFD SYAMH WVRQAPGQGLEWMG GIIPIFGTAWYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMDV WGQGTTVTVSS

[0582] 42Y073-86F08-22 variable light chain nucleic acid sequence

[0583] SEQ ID NO: 51

[0584] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCA CTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0585] 42Y073-86F08-22 variable heavy chain nucleic acid sequence

[0586] SEQ ID NO: 52

[0587] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCTACACCTTCGACAGCTACGCCATGCACTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATTTTCGGCACCGCCTGGTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0588] 42Y073-86F08-47 Variable light chain amino acid sequence

[0589] SEQ ID NO: 53

[0590] DIQLTQSPSSLSASVGDRVTITC RASQSISSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[0591] 42Y073-86F08-47 Variable heavy chain amino acid sequence

[0592] SEQ ID NO: 54

[0593] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS QUR WVRQAPGQGLEWMG VIIPIFGKANYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMDV WGQGTTVTVSS

[0594] 42Y073-86F08-47 Variable light chain nucleic acid sequence

[0595] SEQ ID NO: 55

[0596] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0597] 42Y073-86F08-47 Variable heavy chain nucleic acid sequence

[0598] SEQ ID NO: 56

[0599] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCCAGTACGCCATCCACTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGATGGGCGTGATCATCCCCATTTTCGGCAAGGCCAACTACGCCCAGAAGTTCCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGCGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGGGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0600] 42Y073-2B04-46 Variable light chain amino acid sequence

[0601] SEQ ID NO: 57

[0602] EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASCRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQLDNWPIT FGGGTKVEIK

[0603] 42Y073-2B04-46 variable heavy chain amino acid sequence

[0604] SEQ ID NO: 58

[0605] QVQLQQWGAGLLKPSETLSLTCAVYGGSFR RYYWS WIRQPPGKGLEWIG EIDGWGSTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR GGSVDFWSSGSDYYYYMDV WGKGATVTVSS

[0606] 42Y073-2B04-46 variable light chain nucleic acid sequence

[0607] SEQ ID NO: 59

[0608] GAGATCGTGCTGACCCAGAGCCCCGCAACCCTGTCCCTGAGCCCCGGCGAAAGGGCCACTCTGAGCTGCAGGGCCAGCCAGAGCGTGAGCAGCTACCTCGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCTAGGCTGCTGATCTACGACGCCAGCAAGA GGGCCACCGGCATTCCCGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTCTACTACTGCCAGCAGCTGGACAACTGGCCCATCACCTTCGGGGGCGGCACCAAGGTGGAGATCAAG

[0609] 42Y073-2B04-46 variable heavy chain nucleic acid sequence

[0610] SEQ ID NO: 60

[0611] CAGGGTGCAGCTGCAGCAGTGGGGCGCCGGACTGCTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCGCCGTGTACGGCGGGTCCTTCAGGAGGTACTACTGGAGCTGGATCAGGCAGCCCCCCGGCAAAGGCCTGGAGTGGATCGGCGAGATCGACGGCTGGGGCAGCACCAACTACAACCCCAGCCTC AAGAGCAGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTATTGCGCCAGGGGCGGCAGCGTGGACTTCTGGAGCGGCAGCGACTACTACTACATGGACGTGTGGGGCAAGGGCGCCACCGTCACCGTGAGCAGC

[0612] 42Y073-1A01-85 variable light chain amino acid sequence

[0613] SEQ ID NO: 61

[0614] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSLYPPRT FGGGTKVEIK

[0615] 42Y073-1A01-85 variable heavy chain amino acid sequence

[0616] SEQ ID NO: 62

[0617] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYPMH WVRQAPGQGLEWMG IINPSGGFTSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR ETAYYTTKGNWFDP WGQGTLVTVSA

[0618] 42Y073-1A01-85 variable light chain nucleic acid sequence

[0619] SEQ ID NO: 63

[0620] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCCTGTACCCCCCCAGGACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0621] 42Y073-1A01-85 Variable heavy chain nucleic acid sequence

[0622] SEQ ID NO: 64

[0623] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCGCCAGCGTGAAGGTCAGCTGCAAGGCCTCCGGGTACACCTTCACCAGCTACCCCATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTCGAGTGGATGGGCATCATCAACCCCAGCGGAGGCTTCACCAGCTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACAAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTATTACTGCGCAAGGGAGACCGCCTACTACACCACCAAGGGCAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0624] 42Y073-1A01-97 Variable light chain amino acid sequence

[0625] SEQ ID NO: 65

[0626] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSLYPPRT FGGGTKVEIK

[0627] 42Y073-1A01-97 variable heavy chain amino acid sequence

[0628] SEQ ID NO: 66

[0629] QVQLVQSGAEVKKPGASVKVSCKASGYTFT RAAMH WVRQAPGQGLEWMG INPAGGYTSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR ETAYYTTKGNWFDP WGQGTLVTVSS

[0630] 42Y073-1A01-97 variable light chain nucleic acid sequence

[0631] SEQ ID NO: 67

[0632] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCAC TGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCCTGTACCCCCCCAGGACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0633] 42Y073-1A01-97 variable heavy chain nucleic acid sequence

[0634] SEQ ID NO: 68

[0635] CAGGTGCAGCTGGTGCAGAGCGGCCGAAGTGAAAAAGCCCGGCGCCAGCGTGAAGGTCAGCTGCAAGGCCTCCGGGTACACCTTTCACCAGGGCCGCCATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTCGAGTGGATGGGCATCATCAACCCCGCCGGAGGCTACACCAGCTACGCCC AGAAGTTTCCAGGGCAGGGTGACCATGACAAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTATTACTGCGCAAGGGAGACCGCCTACTACACCACCAAGGGCAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCT

[0636] 42Y073-1A01-100 variable light chain amino acid sequence

[0637] SEQ ID NO: 69

[0638] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSLYPPRT FGGGTKVEIK

[0639] 42Y073-1A01-100 variable heavy chain amino acid sequence

[0640] SEQ ID NO: 70

[0641] QVQLVQSGAEVKKPGASVKVSCKASGYTFT TYRMH WVRQAPGQGLEWMG IINPSGGLTQYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR ETAYYTTKGNWFDP WGQGTLVTVSS

[0642] 42Y073-1A01-100 variable light chain nucleic acid sequence

[0643] SEQ ID NO: 71

[0644] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCCTGTACCCCCCCAGGACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0645] 42Y073-1A01-100 Variable heavy chain nucleic acid sequence

[0646] SEQ ID NO: 72

[0647] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCGCCAGCGTGAAGGTCAGCTGCAAGGCCTCCGGGTACACCTTCACCACCTACAGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTCGAGTGGATGGGCATCATCAACCCCAGCGGAGGCCTGACCCAGTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACAAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTATTACTGCGCAAGGGAGACCGCCTACTACACCACCAAGGGCAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCT

[0648] 42Y073-1A01-103 Variable light chain amino acid sequence

[0649] SEQ ID NO: 73

[0650] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSLYPPRT FGGGTKVEIK

[0651] 42Y073-1A01-103 variable heavy chain amino acid sequence

[0652] SEQ ID NO: 74

[0653] QVQLVQSGAEVKKPGASVKVSCKASGYTFT AYQMH WVRQAPGQGLEWMG IINPAGWTSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR ETAYYTTKGNWFDP WGQGTLVTVSS

[0654] 42Y073-1A01-103 variable light chain nucleic acid sequence

[0655] SEQ ID NO: 75

[0656] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCAC TGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCCTGTACCCCCCCAGGACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0657] 42Y073-1A01-103 variable heavy chain nucleic acid sequence

[0658] SEQ ID NO: 76

[0659] CAGGTGCAGCTGGTGCAGAGCGGCCGAAGTGAAAAAGCCCGGCGCCAGCGTGAAGGTCAGCTGCAAGGCCTCCGGGTACACCTTCACCGCCTACCAGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTCGAGTGGATGGGCATCATCAACCCCGCCGGAGGCTGGACCAGCTACGCCC AGAAGTTTCCAGGGCAGGGTGACCATGACAAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTATTACTGCGCAAGGGAGACCGCCTACTACACCACCAAGGGCAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCT

[0660] 42Y073-1A01-126 variable light chain amino acid sequence

[0661] SEQ ID NO: 77

[0662] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSLYPPRT FGGGTKVEIK

[0663] 42Y073-1A01-126 variable heavy chain amino acid sequence

[0664] SEQ ID NO: 78

[0665] QVQLVQSGAEVKKPGASVKVSCKASGYTFT RYNMH WVRQAPGQGLEWMG WINPAGSTSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR ETAYYTTKGNWFDP WGQGTLVTVSA

[0666] 42Y073-1A01-126 variable light chain nucleic acid sequence

[0667] SEQ ID NO: 79

[0668] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGAGACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCCTGTACCCCCCCAGGACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0669] 42Y073-1A01-126 Variable heavy chain nucleic acid sequence

[0670] SEQ ID NO: 80

[0671] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCGCCAGCGTGAAGGTCAGCTGCAAGGCCTCCGGGTACACCTTCACCAGGTACAACATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTCGAGTGGATGGGCTGGATCAACCCCGCCGGAGGCAGCACCAGCTACGCCCAGAAGTTCCAGGGCAGGGTGACCATGACAAGGGACACCAGCACCAGCACCGTGTACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTATTACTGCGCAAGGGAGACCGCCTACTACACCACCAAGGGCAACTGGTTCGACCCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0672] 42Y073-1A01-191 Variable light chain amino acid sequence

[0673] SEQ ID NO: 81

[0674] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSLYPPRT FGGGTKVEIK

[0675] 42Y073-1A01-191 variable heavy chain amino acid sequence

[0676] SEQ ID NO: 82

[0677] QVQLVQSGAEVKKPGASVKVSCKASGYTFT KYRMH WVRQAPGQGLEWMG IINPQGGSYAQKFQG RVTMTRDTTSTVYMELSSLRSEDTAVYYCAR ETAYYTTKGNWFDP WGQGTLVTVSA

[0678] 42Y073-1A01-191 variable light chain nucleic acid sequence

[0679] SEQ ID NO: 83

[0680] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGA

[0681] GACAGGGTGACCATCACCTGCAGGGGCCAGCCAGTCCATCAGCAGCTAC

[0682] CTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC

[0683] TACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGC

[0684] AGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCC

[0685] GAGGACTTCGCCACCTACTACTGCCAGCAGAGCCTGTACCCCCCCAGG

[0686] ACTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0687] 42Y073-1A01-191 variable heavy chain nucleic acid sequence

[0688] SEQ ID NO: 84

[0689] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAAGTGAAAAAGCCCGGCGC

[0690] CAGCGTGAAGGTCAGCTGCAAGGCCTCCGGGTACACCTTCACCAAGTA

[0691] CAGGATGCACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTCGAGTGGAT

[0692] GGGCATCATCAACCCCCAGGGAGGCAGCACCAGCTACGCCCAGAAGTT

[0693] CCAGGGCAGGGTGACCATGACAAGGGACACCAGCACCAGCACCGTGT

[0694] ACATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTATTAC

[0695] TGCGCAAGGGAGACCGCCTACTACACCACCAAGGGCAACTGGTTCGAC

[0696] CCCTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC

[0697] 42Y073-86F08-66 Variable light chain amino acid sequence

[0698] SEQ ID NO: 85

[0699] DIQLTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AAS

[0700] SLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEI

[0701] K

[0702] 42Y073-86F08-66 Variable heavy chain amino acid sequence

[0703] SEQ ID NO: 86

[0704] QVQLVQSGAEVKKPGSSVKVSCKASGGTFV EYAIS WVRQAPGQGLEWM

[0705] G GIIPAFGTAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYCAR G

[0706] AGYYGDKDPMDV WGQGTTVTVSS

[0707] 42Y073-86F08-66 Remote control

[0708] SEQ ID NO:87

[0709] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGA

[0710] GACAGGGTGACCATCACCTGCA

[0711] GGGCCAGCCAGTCCATCAGCAGCTACCTGAACTGGTACCAGCAGAAGC

[0712] CCGGCAAGGCCCCCAAGCTGCT

[0713] GATCTACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAG

[0714] CGGCAGCGGCAGCGGCACCGAC

[0715] TTCACCCTGACCATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTACT

[0716] ACTGCCAGCAGGTGCTGCACACCATCACTTTCGGCGGCGGCACCAAGG

[0717] TGGAGATTAAG

[0718] 42Y073-86F08-66ShaftShares

[0719] SEQ ID NO:88

[0720] CAGGTGCAGCTGGTGCAGAGCGGCGCAGAGGTGAAAAAGCCCGGCAG

[0721] CAGCGTGAAGGTGAGCTGCAAGG

[0722] CCTCCGGCGGGACCTTCGTGGAGTACGCCATCAGCTGGGTGAGGCAGG

[0723] CTCCCGGACAGGGCCTGGAGTG

[0724] GATGGGCGGCATCATCCCCGCCTTCGGCACCGCCCAGTACGCCCAGAA

[0725] GTTCCAGGGAAGGGTCACCATC

[0726] ACCGCCGACGAGAGCACCAGCACCGCCTACATGGAACTCAGCAGCCTG

[0727] AGGAGCGAGGACACCGCCGTGT

[0728] ACTATTGCGCCAGGGGAGCCGGCTACTACGGCGACAAGGACCCCATGG

[0729] ACGTGTGGGGCCAGGGCACCAC CGTGACTGTGAGCAGC

[0730] 42Y073-86F08-16 Variable light chain amino acid sequence

[0731] SEQ ID NO: 89

[0732] DIQLTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AAS

[0733] SLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEI

[0734] K

[0735] 42Y073-86F08-16 Variable heavy chain amino acid sequence

[0736] SEQ ID NO: 90

[0737] QVQLVQSGAEVKKPGSSVKVSCKASGGTFN EYAIS WVRQAPGQGLEWM

[0738] G GIVPVFGTAKYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYCAR

[0739] GAGYYGDKDPMDV WGQGTTVTVSS

[0740] 42Y073-86F08-16 Remote control

[0741] SEQ ID NO:91

[0742] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGA

[0743] GACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTAC

[0744] CTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC

[0745] TACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGC

[0746] AGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCC

[0747] GAGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTT

[0748] TCGGCGGCGGCACCAAGGTGGAGATTAAG

[0749] 42Y073-86F08-16-16

[0750] SEQ ID NO:92

[0751] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAG

[0752] CAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAACGAGTA

[0753] CGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGAT

[0754] GGGCGGCATCGTGCCCGTGTTCGGCACCGCCAAGTACGCCCAGAAGTT

[0755] CCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTA

[0756] CATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTG

[0757] CGCCAGGGGCGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTG

[0758] GGGCCAGGGCACCACCGTGACTGTGAGCAGC

[0759] Variable light chain amino acid sequence of 42Y073-86F04-23

[0760] SEQ ID NO: 93

[0761] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AAS

[0762] SLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKV

[0763] EIK

[0764] Variable heavy chain amino acid sequence of 42Y073-86F04-23

[0765] SEQ ID NO: 94

[0766] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS GYPIS WVRQAPGQGLEWMG

[0767] GIIPIMGTARYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LL

[0768] GESGMDVWGQGTTVTVSS

[0769] 42Y073-86F04-23 Remote control

[0770] SEQ ID NO:95

[0771] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGA

[0772] GACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTAC

[0773] CTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC

[0774] TACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGC

[0775] AGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCC

[0776] GAGGACTTCGCCACCTACTACTGCCAGCAGCCCTACTTCAGCCCCCCCA

[0777] CTTTCGGCGGCGGCACCAAGGTGGAGATTAAG

[0778] 42Y073-86F04-23-23

[0779] SEQ ID NO:96

[0780] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAG

[0781] CAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCGGCTA

[0782] CCCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGAT

[0783] GGGCGGCATCATCCCCATTATGGGCACCGCCAGGTACGCCCAGAAGTT

[0784] CCAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTA

[0785] CATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTG

[0786] CGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGTGGGGCCAGGGCA

[0787] CCACCGTGACTGTGAGCAGC

[0788] CDRL1 amino acid sequence 1

[0789] SEQ ID NO: 97

[0790] RASQSISSYLN

[0791] CDRL1 amino acid sequence 2

[0792] SEQ ID NO: 98

[0793] RASQSVSSYLA

[0794] CDRL2 amino acid sequence 1

[0795] SEQ ID NO: 99

[0796] AASSLQS

[0797] CDRL2 amino acid sequence 2

[0798] SEQ ID NO: 100

[0799] DASKRAT

[0800] CDRL3 amino acid sequence 1

[0801] SEQ ID NO: 101

[0802] QQVLHTIT

[0803] CDRL3 amino acid sequence 2

[0804] SEQ ID NO: 102

[0805] QQPYFSPPT

[0806] CDRL3 amino acid sequence 3

[0807] SEQ ID NO: 103

[0808] QQLDNWPIT

[0809] CDRL3 amino acid sequence 4

[0810] SEQ ID NO: 104

[0811] QQSLYPPRT

[0812] CDRH1 amino acid sequence 1

[0813] SEQ ID NO: 105

[0814] SYAIS

[0815] CDRH1 amino acid sequence 2

[0816] SEQ ID NO: 106

[0817] YNAIS

[0818] CDRH1 amino acid sequence 3

[0819] SEQ ID NO: 107

[0820] SEAIS

[0821] CDRH1 amino acid sequence 4

[0822] SEQ ID NO: 108

[0823] SHAIS

[0824] CDRH1 amino acid sequence 5

[0825] SEQ ID NO: 109

[0826] GHAIS

[0827] CDRH1 amino acid sequence 6

[0828] SEQ ID NO: 110

[0829] SRAIS

[0830] CDRH1 amino acid sequence 7

[0831] SEQ ID NO: 111

[0832] SSAIS

[0833] CDRH1 amino acid sequence 8

[0834] SEQ ID NO: 112

[0835] NYAIS

[0836] CDRH1 amino acid sequence 9

[0837] SEQ ID NO: 113

[0838] HYA

[0839] CDRH1 amino acid sequence 10

[0840] SEQ ID NO: 114

[0841] LYAIS

[0842] CDRH1 amino acid sequence 11

[0843] SEQ ID NO: 115

[0844] EYAIS

[0845] CDRH1 amino acid sequence 12

[0846] SEQ ID NO: 116

[0847] SYAMH

[0848] CDRH1 amino acid sequence 13

[0849] SEQ ID NO: 117

[0850] QUR

[0851] CDRH1 amino acid sequence 14

[0852] SEQ ID NO: 118

[0853] YYW

[0854] CDRH1 amino acid sequence 15

[0855] SEQ ID NO: 119

[0856] SYPMH

[0857] CDRH1 amino acid sequence 16

[0858] SEQ ID NO: 120

[0859] RAAMH

[0860] CDRH1 amino acid sequence 17

[0861] SEQ ID NO: 121

[0862] TYRMH

[0863] CDRH1 amino acid sequence 18

[0864] SEQ ID NO: 122

[0865] QUR

[0866] CDRH1 amino acid sequence 19

[0867] SEQ ID NO: 123

[0868] RYNMH

[0869] CDRH1 amino acid sequence 20

[0870] SEQ ID NO: 124

[0871] KYRMH

[0872] CDRH1 amino acid sequence 21

[0873] SEQ ID NO: 125

[0874] GYPIS

[0875] CDRH2 amino acid sequence 1

[0876] SEQ ID NO: 126

[0877] GIIPIFGTASYAQKFQG

[0878] CDRH2 amino acid sequence 2

[0879] SEQ ID NO: 127

[0880] GIIPIMGTARYAQKFQG

[0881] CDRH2 amino acid sequence 3

[0882] SEQ ID NO: 128

[0883] GIIPIFGRARYAQKFQG

[0884] CDRH2 amino acid sequence 4

[0885] SEQ ID NO: 129

[0886] GIIPIFGRGKYAQKFQG

[0887] CDRH2 amino acid sequence 5

[0888] SEQ ID NO: 130

[0889] GILPIFGRANYAQKFQG

[0890] CDRH2 amino acid sequence 6

[0891] SEQ ID NO: 131

[0892] GIIPIFGRANYAQKFQG

[0893] CDRH2 amino acid sequence 7

[0894] SEQ ID NO: 132

[0895] GIIPALGTANYAQKFQG

[0896] CDRH2 amino acid sequence 8

[0897] SEQ ID NO: 133

[0898] NIIPIFGTAGYAQKFQG

[0899] CDRH2 amino acid sequence 9

[0900] SEQ ID NO: 134

[0901] GIIPQLGTANYAQKFQG

[0902] CDRH2 amino acid sequence 10

[0903] SEQ ID NO: 135

[0904] GIIPVFGTANYAQKFQG

[0905] CDRH2 amino acid sequence 11

[0906] SEQ ID NO: 136

[0907] GIIPIFGTAWYAQKFQG

[0908] CDRH2 amino acid sequence 12

[0909] SEQ ID NO: 137

[0910] VIIPIFGKANYAQKFQG

[0911] CDRH2 amino acid sequence 13

[0912] SEQ ID NO: 138

[0913] EIDGWGSTNYNPSLKS

[0914] CDRH2 amino acid sequence 14

[0915] SEQ ID NO: 139

[0916] IINPSGGFTSYAQKFQG

[0917] CDRH2 amino acid sequence 15

[0918] SEQ ID NO: 140

[0919] IINPAGGYTSYAQKFQG

[0920] CDRH2 amino acid sequence 16

[0921] SEQ ID NO: 141

[0922] IINPSGGLTQYAQKFQG

[0923] CDRH2 amino acid sequence 17

[0924] SEQ ID NO: 142

[0925] IINPAGGWTSYAQKFQG

[0926] CDRH2 amino acid sequence 18

[0927] SEQ ID NO: 143

[0928] WINPAGGSTSYAQKFQG

[0929] CDRH2 amino acid sequence 19

[0930] SEQ ID NO: 144

[0931] IINPQGGSTSYAQKFQG

[0932] CDRH2 amino acid sequence 20

[0933] SEQ ID NO: 145

[0934] GIIPAFGTAQYAQKFQG

[0935] CDRH2 amino acid sequence 21

[0936] SEQ ID NO: 146

[0937] GIVPVFGTAKYAQKFQG

[0938] CDRH3 amino acid sequence 1

[0939] SEQ ID NO: 147

[0940] GAGYYGDKDPMDV

[0941] CDRH3 amino acid sequence 2

[0942] SEQ ID NO: 148

[0943] LLGESGMDV

[0944] CDRH3 amino acid sequence 3

[0945] SEQ ID NO: 149

[0946] GGSVDFWSGSDYYYYMDV

[0947] CDRH3 amino acid sequence 4

[0948] SEQ ID NO: 150

[0949] ETAYYTTKGNWFDP

[0950] PD-1 antagonist CDRH1

[0951] SEQ ID NO: 151

[0952] SYDMS

[0953] PD-1 antagonist CDRH2

[0954] SEQ ID NO: 152

[0955] TISGGGSYTYYQDSVKG

[0956] PD-1 antagonist CDRH3

[0957] SEQ ID NO: 153

[0958] PYYAMDY

[0959] PD-1 antagonist CDRL1

[0960] SEQ ID NO: 154

[0961] KASQDVGTAVA

[0962] PD-1 antagonist CDRL2

[0963] SEQ ID NO: 155

[0964] WASTLHT

[0965] PD-1 antagonist CDRH3

[0966] SEQ ID NO: 156

[0967] QHYSSYPWT

[0968] PD-1 antagonists as an alternative to CDRL3

[0969] SEQ ID NO: 157

[0970] QHYNSYPWT

[0971] PD-1 antagonist heavy chain variable region

[0972] SEQ ID NO: 158

[0973] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST

[0974] ISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYY

[0975] AMDYWGQGTTVTVSS

[0976] PD-1 antagonist light chain variable region

[0977] SEQ ID NO: 159

[0978] DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWA

[0979] STLHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTK

[0980] LEIK

[0981] PD-1 antagonist monoclonal antibody heavy chain

[0982] SEQ ID NO: 160

[0983] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST

[0984] ISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYY

[0985] AMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

[0986] VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[0987] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV

[0988] TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV

[0989] LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

[0990] TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL

[0991] TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0992] Light chain of PD-1 antagonist monoclonal antibody

[0993] SEQ ID NO: 161

[0994] DIQLTQSPSFLSAYVGDRVTITCKASQDVGTAVAWYQQKPGKAPKLLIYWA

[0995] STLHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHYSSYPWTFGQGTK

[0996] LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL

[0997] QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV

[0998] TKSFNRGEC

[0999] Heavy chain sequence of PD-1 antagonist with N380D modification

[1000] SEQ NO:162

[1001] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST

[1002] ISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYY

[1003] AMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

[1004] VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[1005] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV

[1006] TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV

[1007] LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

[1008] TKNQVSLTCLVKGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSRL

[1009] TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[1010] PD-1 antagonist light chain sequence with N385D modification

[1011] SEQ NO:163

[1012] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST

[1013] ISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYY

[1014] AMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

[1015] VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[1016] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV

[1017] TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV

[1018] LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

[1019] TKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDSDGSFFLYSRL

[1020] TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[1021] PD-1 antagonist heavy chain sequence with N380D and N385D modifications

[1022] SEQ NO:164

[1023] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST

[1024] ISGGGSYTYYQDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPYY

[1025] AMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP

[1026] VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH

[1027] KPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEV

[1028] TCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTV

[1029] LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM

[1030] TKNQVSLTCLVKGFYPSDIAVEWESDGQPEDNYKTTPPVLDSDGSFFLYSRL

[1031] TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[1032] Full-length heavy chain amino acid sequence of 42Y073-86F08-66

[1033] SEQ ID NO: 165

[1034] QVQLVQSGAEVKKPGSSVKVSCKASGGTFV EYAIS WVRQAPGQGLEWMG

[1035] GIIPAFGTAQY

[1036] AQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GAGYYGDKDPMD

[1037] V WGQGTTVTV

[1038] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[1039] VHTFPAVLQ

[1040] SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC

[1041] PPCPAPELL

[1042] GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[1043] NAKTKPREEQ

[1044] YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE

[1045] PQVYTLPPSR

[1046] DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[1047] YSKLTVDKS

[1048] RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1049] Full-length light chain amino acid sequence of 42Y073-86F08-66

[1050] SEQ ID NO: 166

[1051] DIQLTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASS

[1052] LQS GVPS

[1053] RFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIKRTVAAPS

[1054] VFIFPPS

[1055] DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

[1056] STYSLSSTLTL

[1057] SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[1058] Full-length heavy chain DNA sequence of 42Y073-86F08-66

[1059] SEQ ID NO: 167

[1060] CAGGTGCAGCTGGTGCAGAGCGGCGCAGAGGTGAAAAAGCCCGGCAG

[1061] CAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCGTGGAGTA

[1062] CGCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGAT

[1063] GGGCGGCATCATCCCCGCCTTCGGCACCGCCCAGTACGCCCAGAAGTTC

[1064] CAGGGAAGGGTCACCATCACCGCCGACGAGAGCACCAGCACCGCCTAC

[1065] ATGGAACTCAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTATTGC

[1066] GCCAGGGGAGCCGGCTACTACGGCGACAAGGACCCCATGGACGTGTGG

[1067] GGCCAGGGCACCACCGTGACTGTGAGCAGCGCCAGCACCAAGGGCCCC

[1068] AGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACA

[1069] GCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACC

[1070] GTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCC

[1071] GCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACC

[1072] GTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTGAAC

[1073] CACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAG

[1074] CTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGCTG

[1075] GGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGA

[1076] TGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTGAGCC

[1077] ACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGG

[1078] TGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCT

[1079] ACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACG

[1080] GCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCCCCTAT

[1081] CGAGAAAACC

[1082] ATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTG

[1083] CCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGC

[1084] CTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGC

[1085] AACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC

[1086] AGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGC

[1087] AGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCC

[1088] CTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG 42Y073-86F08-66 full-length light chain DNA sequence

[1089] SEQ ID NO: 168

[1090] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGA

[1091] GACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTAC

[1092] CTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC

[1093] TACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCA

[1094] GCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCG

[1095] AGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTT

[1096] CGGCGGCGGCACCAAGGTGGAGATTAAGCGTACGGTGGCCGCCCCCAG

[1097] CGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCC

[1098] AGCGTGGTGTGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTG

[1099] CAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAGAG

[1100] CGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCAC

[1101] CCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTG

[1102] TGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAA

[1103] CCGGGGCGAGTGC

[1104] Full-length amino acid sequence of 42Y073-86F08-16 light chain

[1105] SEQ ID NO: 169

[1106] DIQLTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AASS

[1107] LQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQVLHTIT FGGGTKVEIK

[1108] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[1109] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS

[1110] FNRGEC

[1111] Full-length amino acid sequence of 42Y073-86F08-16 heavy chain

[1112] SEQ ID NO: 170

[1113] QVQLVQSGAEVKKPGSSVKVSCKASGGTFN EYAIS WVRQAPGQGLEWMG

[1114] GIVPVFGTAKYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR GA

[1115] GYYGDKDPMDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL

[1116] VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

[1117] YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK

[1118] DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN

[1119] STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ

[1120] VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

[1121] DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1122] Full-length light chain DNA sequence of 42Y073-86F08-16

[1123] SEQ ID NO: 171

[1124] GACATCCAGCTGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGA

[1125] GACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTAC

[1126] CTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC

[1127] TACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCA

[1128] GCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTCCAGCCCG

[1129] AGGACTTCGCCACCTACTACTGCCAGCAGGTGCTGCACACCATCACTTT

[1130] CGGCGGCGGCACCAAGGTGGAGATTAAG

[1131] CGTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGC

[1132] AGCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCT

[1133] ACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGA

[1134] GCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCC

[1135] ACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAG

[1136] AAGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGC

[1137] CCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[1138] Full-length heavy chain DNA sequence of 42Y073-86F08-16

[1139] SEQ ID NO: 172

[1140]

[1141] Full-length light chain amino acid sequence of 42Y073-86F04-23

[1142] SEQ ID NO: 173

[1143] DIQMTQSPSSLSASVGDRVTITC QUESTIONSYLN WYQQKPGKAPKLLIY AAS

[1144] SLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQPYFSPPT FGGGTKVE

[1145] IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[1146] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK

[1147] SFNRGEC

[1148] Full-length heavy chain amino acid sequence of 42Y073-86F04-23

[1149] SEQ ID NO: 174

[1150] QVQLVQSGAEVKKPGSSVKVSCKASGGTFS GYPIS WVRQAPGQGLEWMG

[1151] GIIPIMGTARYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR LLGE

[1152] SGMDV WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP

[1153] EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN

[1154] HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

[1155] TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS

[1156] VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR

[1157] DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL

[1158] YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1159] Full-length light chain DNA sequence of 42Y073-86F04-23

[1160] SEQ ID NO: 175

[1161] GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCCAGCGTGGGA

[1162] GACAGGGTGACCATCACCTGCAGGGCCAGCCAGTCCATCAGCAGCTAC

[1163] CTGAACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATC

[1164] TACGCCGCAAGCTCACTGCAGAGCGGCGTGCCCTCTAGGTTTAGCGGCA

[1165] GCGGCAGCGGCACCGACTTCACCCTGACCATC

[1166] AGCAGCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCCCT

[1167] ACTTCAGCCCCCCCACTTTCGGCGGCGGCACCAAGGTGGAGATTAAGC

[1168] GTACGGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCA

[1169] GCTGAAGAGCGGCACCGCCAGCGTGGTGTGTCTGCTGAACAACTTCTA

[1170] CCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAG

[1171] CGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCA

[1172] CCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGA

[1173] AGCACAAGGTGTACGCCTGTGAGGTGACCCACCAGGGCCTGTCCAGCC

[1174] CCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

[1175] Full-length heavy chain DNA sequence of 42Y073-86F04-23

[1176] SEQ ID NO: 176

[1177] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCAG

[1178] CAGCGTGAAGGTGAGCTGCAAGGCCTCCGGCGGGACCTTCAGCGGCTA

[1179] CCCCATCAGCTGGGTGAGGCAGGCTCCCGGACAGGGCCTGGAGTGGAT

[1180] GGGCGGCATCATCCCCATTATGGGCACCGCCAGGTACGCCCAGAAGTTC

[1181] CAGGGAAGGGTCACCATCACCGCCGACGAGAGC

[1182] ACCAGCACCGCCTACATGGAACTCAGCAGCCTGAGGAGCGAGGACACC

[1183] GCCGTGTACTATTGCGCCAGGCTGCTGGGCGAGAGCGGCATGGACGTGT

[1184] GGGGCCAGGGCACCACCGTGACTGTGAGCAGCGCCAGCACCAAGGGC

[1185] CCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGC

[1186] ACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTG

[1187] ACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTC

[1188] CCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTG

[1189] ACCGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGTAACGTG

[1190] AACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAA

[1191] GAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCT

[1192] GCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACAC

[1193] CCTGATGATCAGCAGAACCCCCGAGGTGACCTGTGTGGTGGTGGATGTG

[1194] AGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTG

[1195] GAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAG

[1196] CACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTG

[1197] AACGGCAAGGAGTACAAGTGTAAGGTGTCCAACAAGGCCCTGCCTGCC

[1198] CCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCC

[1199] CAGGTGTACACCCTGCCCCTTAGCAGAGATGAGCTGACCAAGAACCAG

[1200] GTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCG

[1201] TGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACC

[1202] CCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGCAAG

[1203] The amino acid sequence of CD155-fc used in the examples (the bold sequence is the mature CD155 ECD (extracellular domain) without the leader sequence, and the rest is the human IgG1 Fc region)

[1204]

[1205] CD155 amino acid sequence (the native leader sequence is underlined, the transmembrane and cytoplasmic parts are in bold)

[1206]

[1207] Human IgG1 Fc region amino acid sequence

[1208] SEQ ID NO: 179

[1209] ASTKGPSVFPLAPSSKSGSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH

[1210] TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[1211] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

[1212] VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK

[1213] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF

[1214] YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF

[1215] SCSVMHEALHNHYTQKSLSLSPGK

[1216] Nucleic acid sequence of human IgG1 Fc region

[1217] SEQ ID NO: 180

[1218] GCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAG

[1219] AGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTAC

[1220] TTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGC

[1221] GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGC

[1222] CTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGGCACCCAGACC

[1223] TACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAG

[1224] AAGGTGGAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGC

[1225] CCTGCCCCCGAGCTGCTGGGAGGCCCCAGCGTGTTCCTGTTCCCCCCCA

[1226] AGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGTG

[1227] TGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGT

[1228] ACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAG

[1229] GAGCAGTACAACAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTG

[1230] CACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAAC

[1231] AAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGC

[1232] CAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCTAGCAGAGATGAG

[1233] CTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCTACC

[1234] CCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAAC

[1235] AACTACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCC

[1236] TGTACAGCAAGCTGACCGTGGACAAGAGCAGATGGCAGCAGGGCAACG

[1237] TGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACCCA

[1238] GAAGAGCCTGAGCCTGTCCCCTGGCAAG

Claims

1. A CD96 binding protein comprising a heavy chain variable region that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:86 and a light chain variable region that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to SEQ ID NO:

85.

2. The CD96 binding protein according to claim 1, comprising the following 6 CDRs: CDRH1 of SEQ ID NO: 115; CDRH2 of SEQ ID NO: 145; and CDRH3 of SEQ ID NO: 147; and CDRL1 of SEQ ID NO: 97; CDRL2 of SEQ ID NO: 99; and CDRL3 of SEQ ID NO:

101.

3. The CD96 binding protein according to claim 1, wherein the binding protein comprises: A VH region that is 100% identical to SEQ ID NO:86; or A VL region that is 100% identical to SEQ ID NO:

85.

4. The CD96 binding protein of any one of claims 1-3, wherein the binding protein comprises: a heavy chain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to the amino acid sequence of SEQ ID NO: 165; or a light chain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to the amino acid sequence of SEQ ID NO:

166.

5. The CD96 binding protein according to any one of claims 1 to 4, wherein the binding protein is an antibody or an antibody fragment, such as a (Fab')2 fragment or a Fab fragment, preferably wherein the antibody is IgG1, IgG2, IgG3 or IgG4; or IgM; IgA, IgE or IgD.

6. The CD96 binding protein according to any one of claims 1 to 5, wherein the binding protein comprises a synthetic polypeptide, a humanized sequence or a chimeric sequence.

7. The CD96 binding protein according to any one of claims 1-6, which is an antibody and comprises an antibody Fc region, wherein the antibody Fc region can bind to an Fcγ receptor and / or can promote IFNγ release, preferably, wherein the Fc region is a wild-type IgG1Fc or a functional variant thereof.

8. A nucleic acid sequence encoding the CD96 binding protein according to any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid sequence according to claim 8.

10. A recombinant host cell comprising a nucleic acid sequence according to claim 8, or an expression vector according to claim 9.

11. A method for producing a C96 binding protein, the method comprising culturing the recombinant host cell according to claim 10 under conditions suitable for expressing the nucleic acid sequence or vector, thereby producing a polypeptide comprising the CD96 binding protein.

12. A cell line engineered to express a CD96 binding protein according to any one of claims 1 to 7.

13. A combination drug or pharmaceutical composition comprising a CD96 binding protein and another therapeutically active agent, wherein the CD96 binding protein comprises: CDRH1 of SEQ ID NO: 115; CDRH2 of SEQ ID NO: 145; and CDRH3 of SEQ ID NO: 147; and CDRL1 of SEQ ID NO: 97; CDRL2 of SEQ ID NO: 99; and CDRL3 of SEQ ID NO:

101.

14. The combination drug or pharmaceutical composition according to claim 13, wherein the CD96 binding protein is the CD96 binding protein according to any one of claims 1-7.

15. A combination drug or pharmaceutical composition according to claim 13 or 14, wherein the other therapeutically active agent is an antitumor agent, preferably wherein the antitumor agent is selected from the group consisting of antimicrotubule agents or antimitotic agents; platinum coordination complexes; alkylating agents; antibiotics; topoisomerase I inhibitors; topoisomerase II inhibitors; antimetabolites; hormones and hormone analogs; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; pro-apoptotic agents; cell cycle signaling inhibitors; proteasome inhibitors; heat shock protein inhibitors; cancer metabolism inhibitors; and cancer gene therapy agents; chemotherapeutic agents; immunomodulators; and immunostimulatory adjuvants.

16. The combination drug or pharmaceutical composition according to claim 15, wherein the anti-microtubule agent or anti-mitotic agent is selected from the group consisting of diterpenes and vinca alkaloids; and / or The platinum coordination complex is selected from the group consisting of cisplatin and carboplatin; and / or The alkylating agent is selected from the group consisting of nitrogen mustards such as cyclophosphamide, melphalan and chlorambucil; alkyl sulfonates such as busulfan; nitrosoureas such as carmustine; and triazenes such as dacarbazine; and / or The antibiotic is selected from the group consisting of: actinomycins such as actinomycin D; anthracyclines such as daunorubicin and doxorubicin; and bleomycin; and / or The topoisomerase I inhibitor is camptothecin; The topoisomerase II inhibitors are epipodophyllotoxins such as etoposide and teniposide; The antimetabolite is selected from the group consisting of fluorouracil, methotrexate, cytarabine, mercaptopurine, thioguanine and gemcitabine; The hormones and hormone analogs are selected from the group consisting of adrenocortical steroids such as prednisone and prednisolone; aminoglutethimide and other aromatase inhibitors such as anastrozole, letrozole, vorozole and exemestane; progestins such as megestrol acetate; estrogens, androgens and antiandrogens such as flutamide, nilutamide, bicalutamide, cyproterone acetate and 5α-reductases such as finasteride and dutasteride; antiestrogens such as tamoxifen, toremifene, raloxifene, droloxifene, iodoxifene and selective estrogen receptor modulators (SERMS); and gonadotropin-releasing hormone (GnRH) and its analogs, which stimulate the release of luteinizing hormone (LH) and / or follicle-stimulating hormone (FSH), LHRH agonists and antagonists such as gonadotropin acetate and leuprolide; and / or The signal transduction pathway inhibitor is selected from the group consisting of inhibitors of receptor tyrosine kinases, non-receptor tyrosine kinases, SH2 / SH3 domain blockers, serine / threonine kinases, phosphatidylinositol-3 kinases, inositol signaling and Ras oncogenes; and / or The cell cycle signaling inhibitor is selected from the group consisting of vorinostat, romidepsin, panobinostat, valproic acid and mocetinostat; and / or The proteasome inhibitor is selected from the group consisting of bortezomib, disulfiram, epigallocatechin gallate, salinosporamide A and carfilzomib; and / or The heat shock protein inhibitor is selected from the group consisting of tanspiramycin and radicicol; and / or The cancer metabolism inhibitor is selected from the group consisting of an LDH-A inhibitor and a fatty acid biosynthesis inhibitor; and / or The immunomodulator is selected from the group consisting of antibodies or other antagonists to CTLA-4, such as ipilimumab; and antibodies or other antagonists to PD-1, such as dotarizumab, nivolumab, pembrolizumab, and cemiplimab; and antibodies or other antagonists to PD-L1, OX-40, LAG3, TIM-3, 41BB, and GITR; and / or The immunostimulatory adjuvant is aminoalkylaminoglucosyl phosphate.

17. The combination drug or pharmaceutical composition according to claim 13 or 14, wherein the other therapeutically active agent is a PD-1 antagonist and / or an anti-TIGIT antibody, preferably wherein the PD-1 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to PD-1 or PD-L1.

18. The combination or pharmaceutical composition according to claim 13 or 14, wherein the other therapeutically active agent is an antibody to ICOS, a poly ADP ribose polymerase (PARP) inhibitor such as niraparib, olaparib, rucaparib and talazoparib, a STING modulating compound, a CD39 inhibitor and an A2a and / or A2a adenosine antagonist.

19. The combination drug or pharmaceutical composition according to claim 13 or 14, wherein the other therapeutically active agent is one or more of the following: abarelix, abemaciclib, abiraterone, afatinib, aflibercept, aldoxorubicin, alectinib, alemtuzumab, arsenic trioxide, asparaginase, axitinib, AZD-9291, belinostat, bendamustine, bevacizumab, blinatumomab, bosutinib, ben toximab, cabazitaxel, cabozantinib, capecitabine, ceritinib, clofarabine, cobimetinib, crizotinib, daratumumab, dasatinib, degarelix, denosumab, dinutuximab, docetaxel, elotuzumab, entinostat, enzalutamide, epirubicin, eribulin, filgrastim, flumatinib, fulvestrant, furquintinib, gemtuzumab ozogamicin, ibrutinib, idelalisib, imatinib Matinib, irinotecan, ixabepilone, ixazomib, lenalidomide, lenvatinib, folinic acid, dichloromethyl diethylamide, nexituzumab, nelarabine, netupitant, nilotinib, obinutuzumab, olaparib, omacetaxine, osimertinib, oxaliplatin, paclitaxel, palbociclib, palonosetron, panitumumab, pegfilgrastim, peginterferon alfa-2b, pemetrexed, plerixafor, pomalidomide , ponatinib, pralatrexate, quizartinib, radium-223, ramucirumab, regorafenib, rolapitant, ricapapab, sipuleucel-T, sonidegi, sunitinib, talimogenelaherparepvec, tipiracil, topotecan, trabectinib, trifluridine, triptorelin, uridine, vandetanib, veliparib, vemurafenib, venetoclax, vincristine, vismodegib, and zoledronic acid.

20. The combination drug or pharmaceutical composition according to claim 17, wherein the PD-1 antagonist comprises: CDRH1 of SEQ ID NO:151; CDRH2 of SEQ ID NO:152; CDRH3 of SEQ ID NO: 153; CDRL1 of SEQ ID NO: 154; CDRL2 of SEQ ID NO: 155; and CDRL3 of SEQ ID NO: 156; Preferably, the PD-1 antagonist comprises: A VH region that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 158; and / or a VL region that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 159; Preferably, the PD-1 antagonist comprises: a heavy chain that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 160; and / or A light chain that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

162.

21. Use of a CD96 binding protein according to any one of claims 1 to 7 or a pharmaceutical composition according to any one of claims 13 to 20 in the preparation of a medicament for treating a solid tumor in a subject in need thereof.

22. The use according to claim 21, wherein the subject is a human subject.

23. The use according to claim 21, wherein the solid tumor is selected from ovarian cancer, lung cancer, gastric cancer, bladder cancer, colorectal cancer, liver cancer, kidney cancer and head and neck squamous cell carcinoma (HNSCC).

24. The use according to claim 23, wherein the lung cancer is NSCLC, the liver cancer is HCC and / or the kidney cancer is RCC.

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