EA044112B1 - ANTIBODIES TO TIM3 AND THEIR APPLICATION - Google Patents

Abstract

The invention provides antibodies that bind to T-cell immunoglobulin-mucin domain-containing protein 3 (TIM3). The use of these antibodies for therapeutic purposes, such as cancer treatment, is also envisaged. Further provided are cells that produce antibodies, polynucleotides encoding regions of the heavy and/or light chains of antibodies, and vectors containing polynucleotides encoding regions of the heavy and/or light chains of antibodies.

Description

This application claims benefit to US Provisional Patent Application No. 62/362,541, filed July 14, 2016, and 62/459,499, filed February 15, 2017, each of which is incorporated by reference in its entirety.

Link to the list of sequences submitted electronically via EFS-Web

The contents of the electronically submitted sequence listing in an ASCII text file (name: 3338_052PC02_SeqListing.txt; size: 779837 bytes and creation date: July 10, 2017) filed with the application are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), is a type I transmembrane protein that functions as a key regulator of immune responses. TIM3 was initially identified on activated IFN-γ-producing T cells (eg, type 1 helper CD4+ T cells and cytotoxic CD8+ T cells) and was shown to induce T cell death or exhaustion upon binding to galectin-9. More recent studies have shown that TIM3 expression is also important in regulating the activity of many innate immune cells (eg, macrophages, monocytes, dendritic cells, mast cells, and natural killer cells). See Han G et al., Front Immunol. 4:449 (2013).

Like many inhibitory receptors (eg, PD-1 and CTLA-4), TIM3 expression has been associated with many types of chronic diseases, including cancer. TIM3+ T cells have been found in patients with advanced melanoma, non-small cell lung cancer, or follicular B cell non-Hodgkin's lymphoma. And the presence of regulatory TIM3+ T cells has been described as an effective indicator of lung cancer progression. See Anderson AC. Cancer Immunol Res. 2: 393-8 (2014).

Several potential ligands for TIM3 have been identified: galectin-9, HMGB1, semaphorin-4A, CEACAM-1, ILT-4, and phosphatidylserine (PtdSer or PS). PS is an important component of the cell membrane and is typically localized on the inner surface of cell membranes. But when a cell undergoes apoptosis, PS is redistributed and exposed on the outer membrane. This redistribution is also observed in many tumor cell lines. See Riedl S et al., Biochim Biophys Acta. 1808: 2638-2645 (2011). TIM3 binding to PS may be critical for phagocytosis and cross-presentation. See Nakayama M et al., Blood. 113: 3821-30 (2009).

Studies have shown a close relationship between TIM3 and the inhibitory receptor PD-1. For example, many tumor-specific T cells express both PD-1 and TIM3, and these T cells have been shown to be more dysfunctional compared to T cells that express only PD-1 or TIM3. See Fourcade J et al., J Exp Med. 207: 2175-2186 (2010).

Accordingly, agents that target TIM3 and methods of using such agents are highly desirable for developing new cancer immunotherapies and improving conventional cancer immunotherapies.

Brief Disclosure of the Present Invention

Provided herein are isolated antibodies, such as monoclonal antibodies, particularly human (eg, monoclonal) antibodies, that specifically bind to TIM3 and have the desired functional properties. These properties include, for example, high binding affinity to human TIM3, binding to monkey TIM3 (e.g., cynomolgus TIM3), and the ability to stimulate immune responses, e.g., antigen-specific T cell responses such as tumor-carrying or virus-carrying (virus-infected) ) subject, and detect TIM3 protein in the sample.

In one aspect, isolated antibodies or antigen-binding portions thereof that bind to TIM3 exhibit at least one of the following properties:

(a) binding to soluble and/or membrane-bound human TIM3;

(b) binding to soluble and/or membrane-bound cynomolgus TIM3;

(c) inducing or stimulating an immune response;

(d) inducing or stimulating T cell activation, eg, Th1 cell activation (as evidenced, for example, by increased secretion and/or proliferation of cytokines);

(e) inducing or stimulating the proliferation of T cells (eg, CD4 ⁺ , CD8 + T cells, Th1 cells or TILs), for example, in a co-culture assay such as described in the examples;

(f) inducing or stimulating the production of IFN-γ by T cells, for example, Th1 cells or tumor infiltrating lymphocytes (TILs), such as TILs from human kidney, lung, pancreatic or breast tumors, as defined, for example, in the described in example analysis;

(g) blocking or inhibiting the binding of human TIM3 to PtdSer, as determined, for example, in the assay described in the examples;

- 1 044112 (h) lack of internalization or inhibition of cell surface TIM3 upon binding to

TIM3 on cells;

(i) binding to the extracellular domain of human TIM3 (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) or (d)

WTSRYWLNGDFR (SEQ ID NO: 297);

(j) competing with or cross-blocking binding to human TIM3 of a TIM3 binding antibody described herein (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, or any of TIM3.2-TIM3.18), as defined, for example, as described herein in example analysis;

(k) binding to human TIM3, but not to human TIM3 containing an amino acid substitution of one or more of the following amino acid residues: L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84 , G86, D87, R89, D104, R111, Q113, G116, M118 and D120, as numbered in SEQ ID NO: 286 (Fig. 20);

(l) binding to human TIM3 regions ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373), as determined by HDX-MS;

(m) the presence of heavy chain and/or light chain variable regions interacting with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64 , N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120 and optionally T70 and/or IL12 as determined by x-ray crystallography, and/or (n) competition or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

In some embodiments, anti-TIM3 antibodies or antigen-binding portions thereof stimulate an antitumor immune response, such as an antigen-specific T-cell response. In other embodiments, anti-TIM3 antibodies or antigen-binding portions thereof increase the production of cytokines (eg, IFN-γ) in TIM3-expressing T cells and/or increase T cell proliferation. In some embodiments, anti-TIM3 antibodies or antigen-binding portions thereof do not bind to Fc receptors.

In certain embodiments, anti-TIM3 antibodies or antigen-binding portions thereof bind to soluble human TIM3 with a KD of 10 nM or less as measured by Biacore, bind to membrane-bound human TIM3 with a KD of 1 nM or less as measured by the Scatchard assay, bind to soluble TIM3 cynomolgus monkey with a KD of 100 nM or less, as measured by Biacore, binds to membrane-bound human TIM3 with an EC ₅₀ of 1 μg/ml or less, as measured by flow cytometry, binds to membrane-bound human TIM3 with an EC ₅₀ of 0.1 μg/ml, or less, as measured by flow cytometry, bound to membrane-bound cynomolgus TIM3 with an EC ₅₀ of 1 μg/ml or less, as measured by flow cytometry, bound to membrane-bound cynomolgus TIM-3 with a KD of 1 nM or less, as measured by Scatchard assay .

The present invention relates to isolated antibodies or antigen binding portions thereof that bind to human TIM3 and comprise heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein the heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 126, SEQ ID NO : 127, SEQ ID NO: 128 and SEQ ID NO: 129.

In some embodiments, the heavy chain CDR1 comprises X1, X2, X3, X4, Y, X5, and X6, and wherein X1 is S or absent, X2 is R or absent, X3 is S, R, or D, X4 is Y or H, X5 is W or M, and X6 is G, N, S, or H. In other embodiments, the heavy chain CDR1 comprises X1, Y, Y, M, and X2, and wherein X1 is S or D and X2 is H or S. In some embodiments, the heavy chain CDR1 comprises R, X1, Y, W, and X2, wherein X1 is H or Y and X2 is N or S.

In one embodiment, the heavy chain CDR2 comprises X1, I, X2, X3, X4, G, X5, X6, X7, X8, Y, X9, X10, X11, X12, X13, and X14, and wherein X1 is S, Y , I or F, X2 is Y, H, N or S, X3 is Y, P, G, T or S, X4 is S, T, R or G, X5 is F, S or D, X6 is S, T or I, X7 is I or none, X8 is Y, N or I, X9 is N, Q, S or A, X10 is P, S, Q or D, X11 is S or K, X12 is L, F or V, X13 is K or Q and X14 is S or G. In another embodiment, the heavy chain CDR2 contains Y, I, H, Y, X1, G, S, T, N, Y, N, X2, S, L, K, and S, and wherein X1 is S or T and X2 is S or P. In some embodiments, the heavy chain CDR2 comprises F, I, S, X1, X2, G, S, X3, I, Y, Y, A, D, S, V, K and G, and wherein X1 is G, T or S, X2 is G or S and X3 is T or I. According to In other embodiments, the heavy chain CDR2 comprises I, I, N, P, R, G, D, S, I, I, Y, A, Q, K, F, Q, and G.

- 2 044112

In certain embodiments, anti-TIM3 antibodies or antigen binding portions thereof comprise a light chain CDR1 comprising SEQ ID NO: 64 or SEQ ID NO: 65, a light chain CDR2 comprising SEQ ID NO: 66 or SEQ ID NO: 67, and/or a CDR3 light chain containing SEQ ID NO: 68,

SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

Provided herein are isolated antibodies or antigen binding portions thereof that bind to human TIM3 and comprise heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3, wherein (a) the heavy chain CDR1 is selected from the group consisting of SEQ ID NO : 41, SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44 and SEQ ID NO: 45;

(b) the heavy chain CDR2 is selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 122; SEQ ID NO: 123; SEQ ID NO: 124 and SEQ ID NO: 125;

(c) the heavy chain CDR3 is selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 126; SEQ ID NO: 127; SEQ ID NO: 128 and SEQ ID NO: 129;

(d) the light chain CDR1 contains SEQ ID NO: 64 or SEQ ID NO: 65;

(e) the light chain CDR2 contains SEQ ID NO: 66 or SEQ ID NO: 67, and (f) the light chain CDR3 contains SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

Provided herein are isolated antibodies or antigen binding portions thereof that bind to human TIM3 and contain:

(a1) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively ;

(a2) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 122, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a3) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 123, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a4) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 124, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a5) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 126, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a6) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 127, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a7) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 128, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a8) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 129, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a9) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 122, 128, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a1 0) CDR1, CDR2 and CDR3 of the heavy chain variable region, which contain SEQ ID NO: 41, 122, 126, respectively, and CDR1, CDR2 and CDR3 of the light chain variable region, which contain SEQ ID NO: 64, 66, 68 , respectively;

(b1) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 42, 47, 54, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 69, respectively;

(b2) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 42, 125, 54, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 69, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 43, 48 and 55, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 69, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 44, 49 and 56, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 68, respectively;

- 3 044112 (e) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 45, 50 and 57, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO:

64, 66 and 69, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 50 and 57, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 71, respectively;

(g) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 50 and 57, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 65, 67 and 70, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 51 and 58, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 68, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 52 and 59, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 69, respectively.

The present invention provides isolated antibodies or antigen binding portions thereof that bind to human TIM3 and comprise heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85% , at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about is 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 35, 36, 37, 38, 39, 40, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 and 364, and/or wherein the light chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 60, 61, 62 and 63.

Provided herein are isolated antibodies or antigen binding portions thereof that bind to human TIM3 and compete for binding to human TIM3 with a reference antibody comprising VH and VL, wherein VH and VL are selected from the group consisting of:

(a) VH containing the amino acid sequence presented in SEQ ID NO: 34, and VL containing the amino acid sequence presented in SEQ ID NO: 60;

(b) VH containing the amino acid sequence presented in SEQ ID NO: 35, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(c) VH containing the amino acid sequence presented in SEQ ID NO: 36, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(d) VH containing the amino acid sequence presented in SEQ ID NO: 37, and VL containing the amino acid sequence presented in SEQ ID NO: 60;

(e) VH containing the amino acid sequence presented in SEQ ID NO: 38, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(f) VH containing the amino acid sequence presented in SEQ ID NO: 38, and VL containing the amino acid sequence presented in SEQ ID NO: 62;

(g) VH containing the amino acid sequence presented in SEQ ID NO: 38, and VL containing the amino acid sequence presented in SEQ ID NO: 63;

(h) VH containing the amino acid sequence presented in SEQ ID NO: 39, and VL containing the amino acid sequence presented in SEQ ID NO: 60;

(i) VH containing the amino acid sequence presented in SEQ ID NO: 40, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(j) VH containing the amino acid sequence shown in SEQ ID NO: 121, and

VL containing the amino acid sequence shown in 63, respectively;

(k) VH containing the amino acid sequence presented in SEQ ID NO: 120, and VL containing the amino acid sequence presented in 61, respectively;

(l) VH containing the amino acid sequence presented in SEQ ID NO: 112, and VL containing the amino acid sequence presented in 60, respectively;

(m) VH containing the amino acid sequence presented in SEQ ID NO: 113, and VL containing the amino acid sequence presented in 60, respectively;

(n) VH containing the amino acid sequence presented in SEQ ID NO: 114, and VL containing the amino acid sequence presented in 60, respectively;

(o) VH containing the amino acid sequence presented in SEQ ID NO: 115, and VL containing the amino acid sequence presented in 60, respectively;

- 4 044112 (p) VH containing the amino acid sequence presented in SEQ ID NO: 116, and

VL containing the amino acid sequence represented in 60, respectively;

(q) VH containing the amino acid sequence set forth in SEQ ID NO: 117, and

VL containing the amino acid sequence represented in 60, respectively;

(r) VH containing the amino acid sequence presented in SEQ ID NO: 118, and VL containing the amino acid sequence presented in 60, respectively;

(s) VH containing the amino acid sequence presented in SEQ ID NO: 119, and VL containing the amino acid sequence presented in 60, respectively; and (t) VH containing the amino acid sequence presented in SEQ ID NO: 364, and VL containing the amino acid sequence presented in 60, respectively.

In one embodiment, isolated anti-TIM3 antibodies or antigen-binding portions thereof bind to TIM3 at the same epitope as the reference antibody.

In other embodiments, the isolated anti-TIM3 antibodies or antigen-binding portions thereof comprise VH and VL selected from the group consisting of:

(a) VH containing the amino acid sequence presented in SEQ ID NO: 34, and VL containing the amino acid sequence presented in SEQ ID NO: 60;

(b) VH containing the amino acid sequence presented in SEQ ID NO: 35, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(c) VH containing the amino acid sequence presented in SEQ ID NO: 36, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(d) VH containing the amino acid sequence presented in SEQ ID NO: 37, and VL containing the amino acid sequence presented in SEQ ID NO: 60;

(e) VH containing the amino acid sequence presented in SEQ ID NO: 38, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(f) VH containing the amino acid sequence presented in SEQ ID NO: 38, and VL containing the amino acid sequence presented in SEQ ID NO: 62;

(g) VH containing the amino acid sequence presented in SEQ ID NO: 38, and VL containing the amino acid sequence presented in SEQ ID NO: 63;

(h) VH containing the amino acid sequence presented in SEQ ID NO: 39, and VL containing the amino acid sequence presented in SEQ ID NO: 60;

(i) VH containing the amino acid sequence presented in SEQ ID NO: 40, and VL containing the amino acid sequence presented in SEQ ID NO: 61;

(j) VH containing the amino acid sequence presented in SEQ ID NO: 121, and VL containing the amino acid sequence presented in 63, respectively;

(k) VH containing the amino acid sequence presented in SEQ ID NO: 120, and VL containing the amino acid sequence presented in 61, respectively;

(l) VH containing the amino acid sequence presented in SEQ ID NO: 112, and VL containing the amino acid sequence presented in 60, respectively;

(m) VH containing the amino acid sequence presented in SEQ ID NO: 113, and VL containing the amino acid sequence presented in 60, respectively;

(n) VH containing the amino acid sequence presented in SEQ ID NO: 114, and VL containing the amino acid sequence presented in 60, respectively;

(o) VH containing the amino acid sequence presented in SEQ ID NO: 115, and VL containing the amino acid sequence presented in 60, respectively;

(p) VH containing the amino acid sequence presented in SEQ ID NO: 116, and VL containing the amino acid sequence presented in 60, respectively;

(q) VH containing the amino acid sequence presented in SEQ ID NO: 117, and VL containing the amino acid sequence presented in 60, respectively;

(r) VH containing the amino acid sequence presented in SEQ ID NO: 118, and VL containing the amino acid sequence presented in 60, respectively;

(s) VH containing the amino acid sequence presented in SEQ ID NO: 119, and VL containing the amino acid sequence presented in 60, respectively; and (t) VH containing the amino acid sequence presented in SEQ ID NO: 364, and VL containing the amino acid sequence presented in 60, respectively.

In certain embodiments, anti-TIM3 antibodies or antigen-binding portions thereof are selected from the group consisting of IgG1, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, anti-TIM3 antibodies or antigen-binding portions thereof comprise an IgG1 Fc without effector functions that contains the following mutations: L234A, L235E, G237A, and optionally A330S and P331S. In other embodiments, anti-TIM3 antibodies or antigen-binding portions thereof comprise a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 130-133. In certain embodiments, the anti-TIM3 antibodies or antigen-binding portions thereof are human

- 5 044112 or humanized antibody.

In certain embodiments, anti-TIM3 antibodies or antigen-binding portions thereof specifically bind to human TIM3 and comprise a1) heavy and light chain sequences comprising SEQ ID NO: 301 (or 302) and 29, respectively;

a2) heavy and light chain sequences containing SEQ ID NO: 1 (or 8) and 29, respectively;

a3) heavy and light chain sequences containing SEQ ID NO: 15 (or 22) and 29, respectively;

a4) heavy and light chain sequences containing SEQ ID NO: 303 (or 304) and 29, respectively;

a5) heavy and light chain sequences containing SEQ ID NO: 72 (or 82) and 29, respectively;

a6) heavy and light chain sequences containing SEQ ID NO: 92 (or 102) and 29, respectively;

a7) heavy and light chain sequences containing SEQ ID NO: 305 (or 306) and 29, respectively;

a8) heavy and light chain sequences containing SEQ ID NO: 73 (or 83) and 29, respectively;

a9) heavy and light chain sequences containing SEQ ID NO: 93 (or 103) and 29, respectively;

a10) heavy and light chain sequences containing SEQ ID NO: 307 (or 308) and 29, respectively;

a11) heavy and light chain sequences containing SEQ ID NO: 74 (or 84) and 29, respectively;

a12) heavy and light chain sequences containing SEQ ID NO: 94 (or 104) and 29, respectively;

a13) heavy and light chain sequences containing SEQ ID NO: 309 (or 310) and 29, respectively;

a14) heavy and light chain sequences containing SEQ ID NO: 75 (or 85) and 29, respectively;

a15) heavy and light chain sequences containing SEQ ID NO: 95 (or 105) and 29, respectively;

a16) heavy and light chain sequences containing SEQ ID NO: 311 (or 312) and 29, respectively;

a17) heavy and light chain sequences containing SEQ ID NO: 76 (or 86) and 29, respectively;

a18) heavy and light chain sequences containing SEQ ID NO: 96 (or 106) and 29, respectively;

a19) heavy and light chain sequences containing SEQ ID NO: 313 (or 314) and 29, respectively;

a20) heavy and light chain sequences containing SEQ ID NO: 77 (or 87) and 29, respectively;

a21) heavy and light chain sequences containing SEQ ID NO: 97 (or 107) and 29, respectively;

a22) heavy and light chain sequences containing SEQ ID NO: 315 (or 316) and 29, respectively;

a23) heavy and light chain sequences containing SEQ ID NO: 78 (or 88) and 29, respectively;

a24) heavy and light chain sequences containing SEQ ID NO: 98 (or 108) and 29, respectively;

a25) heavy and light chain sequences containing SEQ ID NO: 317 (or 318) and 29, respectively;

a26) heavy and light chain sequences containing SEQ ID NO: 79 (or 89) and 29, respectively;

a27) heavy and light chain sequences containing SEQ ID NO: 99 (or 109) and 29, respectively;

a28) heavy and light chain sequences containing SEQ ID NO: 319 (or 320) and 29, respectively;

a29) heavy and light chain sequences containing SEQ ID NO: 349 (or 350) and 29, respectively;

a30) heavy and light chain sequences containing SEQ ID NO: 351 (or 352) and 29, respectively;

a31) heavy and light chain sequences containing SEQ ID NO: 353 (or 354) and 29, respectively;

(b1) heavy and light chain sequences containing SEQ ID NO: 321 (or 322) and 30, respectively;

(b2) heavy and light chain sequences containing SEQ ID NO: 2 (or 9) and 30, respectively;

(b3) heavy and light chain sequences containing SEQ ID NO: 16 (or 23) and 30, respectively;

(b4) heavy and light chain sequences containing SEQ ID NO: 323 (or 324) and 30, respectively;

(b5) heavy and light chain sequences containing SEQ ID NO: 80 (or 90) and 30, respectively;

(b6) heavy and light chain sequences containing SEQ ID NO: 100 (or 110) and 30, respectively;

(b7) heavy and light chain sequences containing SEQ ID NO: 325 (or 326) and 30, respectively;

(c1) heavy and light chain sequences containing SEQ ID NO: 327 (or 328) and 30, respectively;

(c2) heavy and light chain sequences containing SEQ ID NO: 3 (or 10) and 30, respectively;

(c3) heavy and light chain sequences containing SEQ ID NO: 17 (or 24) and 30, respectively;

(c4) heavy and light chain sequences containing SEQ ID NO: 329 (or 330) and 30, respectively;

(d1) heavy and light chain sequences containing SEQ ID NO: 331 (or 332) and 29, respectively;

(d2) heavy and light chain sequences containing SEQ ID NO: 4 (or 11) and 29, respectively;

(d3) heavy and light chain sequences containing SEQ ID NO: 18 (or 25) and 29, respectively;

(d4) heavy and light chain sequences containing SEQ ID NO: 333 (or 334) and 29, respectively;

(e11) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 32, respectively;

(e12) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 33, respectively;

(e13) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 33, respectively;

(e2) heavy and light chain sequences containing SEQ ID NO: 5 (or 12) and 33, respectively;

(e3) heavy and light chain sequences containing SEQ ID NO: 19 (or 26) and 33, respectively;

(e4) heavy and light chain sequences containing SEQ ID NO: 337 (or 338) and 33, respectively;

(e5) heavy and light chain sequences containing SEQ ID NO: 81 (or 91) and 33, respectively;

(e6) heavy and light chain sequences containing SEQ ID NO: 101 (or 111) and 33, respectively;

(e7) heavy and light chain sequences containing SEQ ID NO: 339 (or 340) and 33, respectively;

(f1) heavy and light chain sequences containing SEQ ID NO: 341 (or 342) and 29, respectively;

(f2) heavy and light chain sequences containing SEQ ID NO: 6 (or 13) and 29, respectively;

(f3) heavy and light chain sequences containing SEQ ID NO: 20 (or 27) and 29, respectively;

(f4) heavy and light chain sequences containing SEQ ID NO: 343 (or 344) and 29, respectively;

(g1) heavy and light chain sequences containing SEQ ID NO: 345 (or 346) and 30, respectively;

(g2) heavy and light chain sequences containing SEQ ID NO: 7 (or 14) and 30, respectively;

(g3) heavy and light chain sequences containing SEQ ID NO: 21 (or 28) and 30, respectively, or (g4) heavy and light chain sequences containing SEQ ID NO: 347 (or 348) and 30, respectively.

In other embodiments, anti-TIM3 antibodies or antigen-binding portions thereof have one or more of the following properties:

(1) binding to soluble human TIM3, for example, with a K _D of 10 nM or less (for example, from 0.01 nM to 10 nM), for example, as measured by Biacore, for example, as described in the examples;

(2) binding to soluble cynomolgus TIM3, eg, with a K _D of 100 nM or less (eg, 0.01 nM to 100 nM), eg, as measured by Biacore, eg, as described in the Examples;

(3) binding to membrane-bound human TIM3, e.g., with an EC50 of 1 μg/ml or less (e.g., 0.01 μg/ml to 1 μg/ml), e.g., as measured by flow cytometry (e.g., as described in the Examples );

(4) binding to membrane-bound human TIM3, eg, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(5) binding to membrane-bound cynomolgus TIM3, e.g., with an EC ₅₀ of 20 μg/ml or less (e.g., 0.01 μg/ml to 20 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(6) binding to membrane-bound cynomolgus TIM3, for example, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), for example, as measured by the Scatchard assay, for example, as described in the examples;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) enhancing the proliferation of TIM3-expressing T cells (eg, Th1 cells or TILs), for example, as described in the examples;

(8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, for example, as described in the Examples;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay, for example, as described in the Examples;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297), for example, as described in the examples;

(12) the presence of reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118 and D120 (as numbered in SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3, for example, as described in the examples;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17 and TIM3.18, for example, as described in the examples;

(14) binding to human TIM3 regions 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS, for example, as described in the Examples;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (e.g., described in the Examples; SEQ ID NO: 286 (Fig. 20)) and/or (16) (a) the presence of reduced binding to human TIM3, which has 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58 , P59, F61, E62, C63, R11, D120 and optionally, D104 and Q113 (SEQ ID NO: 286 (FIG. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3; (b) binding to 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ QIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373) as determined by HDX-MS as described in the Examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

Provided herein are bispecific molecules comprising an anti-TIM3 antibody coupled to a molecule having a second binding specificity.

- 8 044112

Provided herein are nucleic acids encoding heavy and/or light chain variable regions of anti-TIM3 antibodies or antigen-binding portions thereof, expression vectors containing the nucleic acid molecules, and cells transformed with the expression vectors.

Provided herein are immunoconjugates containing the anti-TIM3 antibodies described herein coupled to an agent.

Provided herein are compositions comprising anti-TIM3 antibodies or antigen-binding portions thereof, bispecific molecules or immunoconjugates described herein, and a carrier. Also provided herein are kits containing anti-TIM3 antibodies or antigen-binding portions thereof, bispecific molecules or immunoconjugates described herein, and instructions for use.

Provided herein is a method of producing anti-TIM3 antibodies or antigen-binding portions thereof, comprising expressing the anti-TIM3 antibody or antigen-binding portion thereof in a cell and isolating the antibody or antigen-binding portion thereof from the cell.

Provided herein is a method of stimulating an antigen-specific T cell response comprising contacting the T cell with an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecules, or immunoconjugates described herein such that the antigen-specific T cell response is stimulated (e.g., by inhibiting negative effects of TIM3 on cells, such as T cells).

Provided herein is a method of activating or co-stimulating a T cell, e.g., an effector T cell (e.g., a Th1 cell), comprising contacting the cell, e.g., an effector T cell, with an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecules, or immunoconjugates , described herein, and CD3, wherein the effector T cell is activated or co-stimulated (eg, by inhibiting the negative effects of TIM3 on cells, eg, T cells).

Provided herein is a method of increasing IFN-γ production and/or proliferation of a T cell, e.g., a Th1 cell or TIL, comprising contacting the T cell with an effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecules, or immunoconjugates described herein document.

Provided herein is a method of increasing the production of IFN-γ in T cells in a subject, comprising administering to the subject an effective amount of an anti-TIM3 antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein to increase the production of IFN-γ from T cells. .

Provided herein is a method of stimulating TIL activity in a subject comprising administering to the subject a therapeutically effective amount of an anti-TIM3 antibody or antigen-binding portion thereof described herein such that the TILs proliferate or secrete a cytokine, such as IFN-γ.

Provided herein are methods of stimulating NK cells (e.g., by increasing the cytotoxic activity of NK cells) and/or macrophages or other antigen-presenting cells in a subject, comprising administering to the subject an effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule, or immunoconjugate described in this document. For example, the anti-TIM3 antibody described herein can increase the secretion of IL-12 by antigen-presenting cells contacted by the anti-TIM3 antibody.

Provided herein is a method of stimulating an immune response in a subject, comprising administering to the subject an anti-TIM3 antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein such that an immune response is stimulated in the subject. In certain embodiments, the subject has a tumor and an immune response against the tumor is stimulated.

Provided herein is a method of inhibiting the growth of tumors or reducing the size of tumors in a subject, comprising administering to the subject an anti-TIM3 antibody or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate described herein such that the growth of a tumor in the subject is inhibited.

Provided herein is a method of treating cancer, for example, by immunotherapy, comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TIM3 antibody or antigen-binding portion thereof, bispecific molecule or immunoconjugate described herein to treat cancer. In some embodiments, the cancer is selected from the group consisting of: bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer , colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasms, lymphoma, leukemia, myeloma, sarcoma, associated cancer virus and any combinations thereof. In some embodiments, cancer

- 9 044112 is a metastatic cancer, refractory cancer or recurrent cancer. In some embodiments, the cancer is a cold tumor.

In certain embodiments, the methods described herein further provide one or more additional anti-TIM3 antibody therapeutics, such as an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, and/or an anti-TIM3 antibody. to PD-L1.

Provided herein is a method for detecting the presence of TIM3 protein in a sample by contacting the sample with an anti-TIM3 antibody or antigen-binding portion thereof under conditions that allow the formation of a complex between the antibody or antigen-binding portion thereof and TIM3 and detecting the formation of the complex.

Embodiments

Embodiment 1. An isolated antibody (e.g., a human antibody) or antigen-binding portion thereof that binds to human T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), wherein the antibody or antigen-binding portion thereof comprises heavy chain CDR1, CDR2, and CDR3 and light chain CdR1, CDR2 and CDR3, wherein (a) heavy chain CDR1 is selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42; SEQ ID NO: 43; SEQ ID NO: 44 and SEQ ID NO: 45;

(b) the heavy chain CDR2 is selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47; SEQ ID NO: 48; SEQ ID NO: 49; SEQ ID NO: 50; SEQ ID NO: 51; SEQ ID NO: 52; SEQ ID NO: 122; SEQ ID NO: 123; SEQ ID NO: 124 and SEQ ID NO: 125;

(c) the heavy chain CDR3 is selected from the group consisting of SEQ ID NO: 53, SEQ ID NO: 54; SEQ ID NO: 55; SEQ ID NO: 56; SEQ ID NO: 57; SEQ ID NO: 58; SEQ ID NO: 59; SEQ ID NO: 126; SEQ ID NO: 127; SEQ ID NO: 128 and SEQ ID NO: 129;

(d) the light chain CDR1 contains SEQ ID NO: 64 or SEQ ID NO: 65;

(e) the light chain CDR2 contains SEQ ID NO: 66 or SEQ ID NO: 67, and (f) the light chain CDR3 contains SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71.

Embodiment 2. An isolated antibody or antigen-binding portion thereof that binds to human TIM3 and contains:

(a1) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a2) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 122, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a3) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 123, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a4) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 124, 53, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a5) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 126, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a6) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 127, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a7) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 128, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a8) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 46, 129, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively ;

(a9) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 41, 122, 128, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 68, respectively;

(a1 0) CDR1, CDR2 and CDR3 of the heavy chain variable region, which contain SEQ ID NO: 41, 122, 126, respectively, and CDR1, CDR2 and CDR3 of the light chain variable region, which contain SEQ ID NO: 64, 66, 68 , respectively;

(b1) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 42, 47, 54, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 69, respectively;

(b2) CDR1, CDR2 and CDR3 of the heavy chain variable region, which contain SEQ ID NO: 42,

- 10 044112

125, 54, respectively, and CDR1, CDR2 and CDR3 of the light chain variable region, which contain

SEQ ID NO: 64, 66, 69, respectively;

(c) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO: 43, 48 and 55, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NO:

64, 66 and 69, respectively;

(d) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 44, 49 and 56, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 68, respectively;

(e) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 50 and 57, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 69, respectively;

(f) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 50 and 57, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 71, respectively;

(g1) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 50 and 57, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 65, 67 and 70, respectively;

(g2) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 45, 50, 57, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 71, respectively;

(g3) heavy chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 45, 50, 57, respectively, and light chain variable region CDR1, CDR2 and CDR3, which contain SEQ ID NO: 64, 66, 69, respectively;

(h) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 51 and 58, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 68, respectively;

(i) heavy chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 45, 52 and 59, respectively, and/or light chain CDR1, CDR2 and CDR3 sequences containing SEQ ID NOs: 64, 66 and 69, respectively.

Embodiment 3. The antibody or antigen-binding portion thereof of Embodiment 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from group consisting of SEQ ID NO: 34, 35.36, 37.38, 39, 40, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 and 364, and/or lung variable region chain contains an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60, 61, 62 and 63.

Embodiment 4. The antibody or antigen-binding portion thereof of any one of embodiments 1-3, wherein the antibody or antigen-binding portion thereof comprises an IgG1 Fc without effector functions that contains the following mutations: L234A, L235E, G237A, and optionally A330S and P331S.

Embodiment 5. The antibody or antigen-binding portion thereof of any of the preceding embodiments, comprising a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 263-266.

Embodiment 6. An antibody or antigen-binding portion thereof according to any of the preceding embodiments, wherein the antibody or antigen-binding portion thereof is a human or humanized antibody.

Embodiment 7. The antibody of any one of embodiments 1-6, wherein the antibody comprises:

(a1) heavy and light chain sequences containing SEQ ID NO: 301 (or 302) and 29, respectively;

(a2) heavy and light chain sequences containing SEQ ID NO: 1 (or 8) and 29, respectively;

(a3) heavy and light chain sequences containing SEQ ID NO: 15 (or 22) and 29, respectively;

(a4) heavy and light chain sequences containing SEQ ID NO: 303 (or 304) and 29, respectively;

- 11 044112 (a5) heavy and light chain sequences containing SEQ ID NO: 72 (or 82) and 29, respectively;

(a6) heavy and light chain sequences containing SEQ ID NO: 92 (or 102) and 29, respectively;

(a7) heavy and light chain sequences containing SEQ ID NO: 305 (or 306) and 29, respectively;

(a8) heavy and light chain sequences containing SEQ ID NO: 73 (or 83) and 29, respectively;

(a9) heavy and light chain sequences containing SEQ ID NO: 93 (or 103) and 29, respectively;

(a10) heavy and light chain sequences containing SEQ ID NO: 307 (or 308) and 29, respectively;

(a11) heavy and light chain sequences containing SEQ ID NO: 74 (or 84) and 29, respectively;

(a12) heavy and light chain sequences containing SEQ ID NO: 94 (or 104) and 29, respectively;

(a13) heavy and light chain sequences containing SEQ ID NO: 309 (or 310) and 29, respectively;

(a14) heavy and light chain sequences containing SEQ ID NO: 75 (or 85) and 29, respectively;

(a15) heavy and light chain sequences containing SEQ ID NO: 95 (or 105) and 29, respectively;

(a16) heavy and light chain sequences containing SEQ ID NO: 311 (or 312) and 29, respectively;

(a17) heavy and light chain sequences containing SEQ ID NO: 76 (or 86) and 29, respectively;

(a18) heavy and light chain sequences containing SEQ ID NO: 96 (or 106) and 29, respectively;

(a19) heavy and light chain sequences containing SEQ ID NO: 313 (or 314) and 29, respectively;

(a20) heavy and light chain sequences containing SEQ ID NO: 77 (or 87) and 29, respectively;

(a21) heavy and light chain sequences containing SEQ ID NO: 97 (or 107) and 29, respectively;

(a22) heavy and light chain sequences containing SEQ ID NO: 315 (or 316) and 29, respectively;

(a23) heavy and light chain sequences containing SEQ ID NO: 78 (or 88) and 29, respectively;

(a24) heavy and light chain sequences containing SEQ ID NO: 98 (or 108) and 29, respectively;

(a25) heavy and light chain sequences containing SEQ ID NO: 317 (or 318) and 29, respectively;

(a26) heavy and light chain sequences containing SEQ ID NO: 79 (or 89) and 29, respectively;

(a27) heavy and light chain sequences containing SEQ ID NO: 99 (or 109) and 29, respectively;

(a28) heavy and light chain sequences containing SEQ ID NO: 319 (or 320) and 29, respectively;

(a29) heavy and light chain sequences containing SEQ ID NO: 349 (or 350) and 29, respectively;

(a30) heavy and light chain sequences containing SEQ ID NO: 351 (or 352) and 29, respectively;

(a31) heavy and light chain sequences containing SEQ ID NO: 353 (or 354) and 29, respectively;

(b1) heavy and light chain sequences containing SEQ ID NO: 321 (or 322) and 30, respectively;

(b2) heavy and light chain sequences containing SEQ ID NO: 2 (or 9) and 30, respectively;

(b3) heavy and light chain sequences containing SEQ ID NO: 16 (or 23) and 30, respectively;

(b4) heavy and light chain sequences containing SEQ ID NO: 323 (or 324) and 30, respectively;

- 12 044112 (b5) heavy and light chain sequences containing SEQ ID NO: 80 (or 90) and 30, respectively;

(b6) heavy and light chain sequences containing SEQ ID NO: 100 (or 110) and 30, respectively;

(b7) heavy and light chain sequences containing SEQ ID NO: 325 (or 326) and 30, respectively;

(c1) heavy and light chain sequences containing SEQ ID NO: 327 (or 328) and 30, respectively;

(c2) heavy and light chain sequences containing SEQ ID NO: 3 (or 10) and 30, respectively;

(c3) heavy and light chain sequences containing SEQ ID NO: 17 (or 24) and 30, respectively;

(c4) heavy and light chain sequences containing SEQ ID NO: 329 (or 330) and 30, respectively;

(d1) heavy and light chain sequences containing SEQ ID NO: 331 (or 332) and 29, respectively;

(d2) heavy and light chain sequences containing SEQ ID NO: 4 (or 11) and 29, respectively;

(d3) heavy and light chain sequences containing SEQ ID NO: 18 (or 25) and 29, respectively;

(d4) heavy and light chain sequences containing SEQ ID NO: 333 (or 334) and 29, respectively;

(e1.1) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 32, respectively;

(e1.2) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 33, respectively;

(e1.3) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 31, respectively;

(e2) heavy and light chain sequences containing SEQ ID NO: 5 (or 12) and 33, respectively;

(e3) heavy and light chain sequences containing SEQ ID NO: 19 (or 26) and 33, respectively;

(e4) heavy and light chain sequences containing SEQ ID NO: 337 (or 338) and 33, respectively;

(e5) heavy and light chain sequences containing SEQ ID NO: 81 (or 91) and 33, respectively;

(e6) heavy and light chain sequences containing SEQ ID NO: 101 (or 111) and 33, respectively;

(e7) heavy and light chain sequences containing SEQ ID NO: 339 (or 340) and 33, respectively;

(f1) heavy and light chain sequences containing SEQ ID NO: 341 (or 342) and 29, respectively;

(f2) heavy and light chain sequences containing SEQ ID NO: 6 (or 13) and 29, respectively;

(f3) heavy and light chain sequences containing SEQ ID NO: 20 (or 27) and 29, respectively;

(f4) heavy and light chain sequences containing SEQ ID NO: 343 (or 344) and 29, respectively;

(g1) heavy and light chain sequences containing SEQ ID NO: 345 (or 346) and 29, respectively;

(g2) heavy and light chain sequences containing SEQ ID NO: 7 (or 43) and 30, respectively;

(g3) heavy and light chain sequences comprising SEQ ID NOs: 21 (or 28) and 30, respectively, or (g4) heavy and light chain sequences comprising SEQ ID NOs: 347 (or 348) and 30, respectively, wherein the antibody specifically binds to human TIM3.

Embodiment 8. The antibody or antigen-binding portion thereof of any one of embodiments 1-7, wherein the antibody or antigen-binding portion thereof has one or more of the following properties:

(1) binding to soluble human TIM3, eg, with a KD of 10 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by Biacore;

- 13 044112 (2) binding to soluble cynomolgus TIM3, for example, with a K _D of 100 nM or less (for example, from 0.01 nM to 100 nM), for example, as measured by Biacore;

(3) binding to membrane-bound human TIM3, eg, an EC ₅₀ of 1 μg/ml or less (eg, 0.01 μg/ml to 1 μg/ml), for example, as measured by flow cytometry;

(4) binding to membrane-bound human TIM3, for example, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), for example, as measured by the Scatchard assay;

(5) binding to membrane-bound cynomolgus TIM3, for example, with an EC ₅₀ of 20 μg/ml or less (eg, 0.01 μg/ml to 20 μg/ml), for example, as measured by flow cytometry;

(6) binding to membrane-bound cynomolgus TIM3, for example, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), for example, as measured by the Scatchard assay;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) increased proliferation of TM3-expressing T cells (eg, Th1 cells or TILs);

(8) stimulation of T cell proliferation in a mixed lymphocyte reaction (MLR) assay;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297);

(12) the presence of reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118 and D120 are substituted with a different amino acid relative to binding to wild-type human TIM3;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8, TIM3.10, TM3.11, TM3.12, TM3.13, TM3.14, TM3.15, TM3.16, TM3.17 and TM3.18;

(14) binding to human TM3 regions ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following human TM3 amino acids: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (e.g., as described in the Examples ; numbering according to SEQ ID NO: 286 (Fig. 20)); and/or (16) (a) the presence of reduced binding to human TM3, in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120 and , optionally, D104 and Q113 (numbered at SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TM3; (b) binding to ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ RIQIPGMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373) as determined by HDX-MS as described in the Examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3.

Embodiment 9. A bispecific molecule comprising an antibody according to any of the preceding embodiments linked to a molecule having a second binding specificity.

Embodiment 10. A nucleic acid encoding a heavy and/or light chain variable region of an antibody or an antigen binding portion thereof according to any one of embodiments 1-8.

Embodiment 11. A cell transformed with the nucleic acid of Embodiment 10.

Embodiment 12. An immunoconjugate comprising the antibody of any one of embodiments 1-8 coupled to an agent.

Embodiment 13. A composition comprising an antibody or antigen-binding portion thereof, a bispecific molecule or immunoconjugate according to any one of embodiments 1-9 and 12 and a carrier.

Embodiment 14. A kit containing the antibody or antigen-binding portion thereof, or bispecific molecule, or immunoconjugate according to any of embodiments 1-9 and 12, and instructions for use.

Embodiment 15. A method of stimulating, enhancing, or modulating an immune response in a subject in need thereof, or treating cancer in a subject in need thereof, comprising administering an anti- 14 044112 body or an antigen-binding portion thereof, a bispecific molecule, or an immunoconjugate, according to any one of Embodiments 1-9 and 12, wherein an antigen-specific T cell response is stimulated, wherein an effector T cell is activated or co-stimulated, wherein production of IFN-γ in the T cell is increased, wherein the number of T cells is increased, wherein TIL activity is stimulated, wherein tumor size in the subject is reduced, wherein tumor growth in the subject or any combination thereof is inhibited upon administration.

Other features and advantages of the present disclosure will be apparent from the following detailed description and examples, which are to be considered limiting.

Brief description of graphic materials

In fig. 1A shows the nucleotide sequence (SEQ ID NO: 167) and amino acid sequence (SEQ ID NO: 34) of the mature heavy chain variable region (VH) of anti-TIM3 monoclonal antibody 13A3. CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 46) and CDR3 (SEQ ID NO: 53) are delineated, and germline V, D and J derivatives are indicated.

In fig. 1B shows the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 13A3. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are delineated, and germline V and J derivatives are indicated.

In fig. 1C shows the nucleotide sequence (SEQ ID NO: 167) and amino acid sequence (SEQ ID NO: 34) of the heavy chain VH region of anti-TIM3 monoclonal antibody 13A3 with signal sequence (SEQ ID NOs: 274 and 269, respectively), and nucleotide sequence (SEQ ID NO: 193) and the amino acid sequence (SEQ ID NO: 60) of the light chain VL region of anti-TIM3 monoclonal antibody 13A3 with signal sequence (SEQ ID NOs: 273 and 268, respectively).

In fig. 2A shows the nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 35) of the mature heavy chain variable region (VH) of anti-TIM3 monoclonal antibody 8B9. CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 47) and CDR3 (SEQ ID NO: 54) are delineated, and germline V, D and J derivatives are indicated.

In fig. 2B shows the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 8B9. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated, and germline V and J derivatives are indicated.

In fig. 2C shows the nucleotide sequence (SEQ ID NO: 168) and amino acid sequence (SEQ ID NO: 35) of the heavy chain VH region of anti-TIM3 monoclonal antibody 8B9 with signal sequence (SEQ ID NO: 274 and 269, respectively), and the nucleotide sequence ( SEQ ID NO: 194) and the amino acid sequence (SEQ ID NO: 61) of the light chain VL region of anti-TIM3 monoclonal antibody 8B9 with signal sequence (SEQ ID NOs: 273 and 268, respectively).

In fig. 3A shows the nucleotide sequence (SEQ ID NO: 169) and amino acid sequence (SEQ ID NO: 36) of the mature heavy chain variable region (VH) of anti-TIM3 monoclonal antibody 8C4. CDR1 (SEQ ID NO: 43), CDR2 (SEQ ID NO: 48) and CDR3 (SEQ ID NO: 55) are delineated, and germline V, D and J derivatives are indicated.

In fig. 3B shows the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 8C4. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated, and germline V and J derivatives are indicated.

In fig. 3C shows the nucleotide sequence (SEQ ID NO: 169) and amino acid sequence (SEQ ID NO: 36) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 8C4 with signal sequence (SEQ ID NO: 274 and 269, respectively), and the nucleotide sequence ( SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of the anti-TIM3 monoclonal antibody 8C4 with signal sequence (SEQ ID NOs: 273 and 268, respectively).

In fig. 4A shows the nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 37) of the mature heavy chain variable region (VH) of anti-TIM3 monoclonal antibody 17C3. CDR1 (SEQ ID NO: 44), CDR2 (SEQ ID NO: 49) and CDR3 (SEQ ID NO: 56) are delineated, and germline V, D and J derivatives are indicated.

In fig. 4B shows the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 17C3. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are delineated, and germline V and J derivatives are indicated.

In fig. 4C shows the nucleotide sequence (SEQ ID NO: 170) and amino acid sequence (SEQ ID NO: 37) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 17C3 with signal sequence (SEQ ID NO: 272 and 267, respectively), and the nucleotide sequence 15 044112 valency (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the light chain VL region of the anti-TIM3 monoclonal antibody 17C3 with signal sequence (SEQ ID NO: 273 and

268, respectively).

In fig. 5A shows the nucleotide sequence (SEQ ID NO: 171) and amino acid sequence (SEQ ID NO: 38) of the mature heavy chain variable region (VH) of anti-TIM3 monoclonal antibody 9F6. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 50) and CDR3 (SEQ ID NO: 57) are delineated, and germline V, D and J derivatives are indicated.

In fig. 5B shows the nucleotide sequence (SEQ ID NO: 195) and amino acid sequence (SEQ ID NO: 62) of the VK1 mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 9F6. CDR1 (SEQ ID NO: 65), CDR2 (SEQ ID NO: 67) and CDR3 (SEQ ID NO: 70) are delineated, and germline V and J derivatives are indicated.

In fig. 5C shows the nucleotide sequence (SEQ ID NO: 196) and amino acid sequence (SEQ ID NO: 63) of the VK2 mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 9F6. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 71) are delineated, and germline V and J derivatives are indicated.

In fig. 5D shows the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the VK3 mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 9F6. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated, and germline V and J derivatives are indicated.

In fig. 5E shows the nucleotide sequence (SEQ ID NO: 171) and amino acid sequence (SEQ ID NO: 38) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 9F6 with signal sequence (SEQ ID NOs: 275 and 270, respectively), and the nucleotide sequences ( SEQ ID NO: 195, 196 and 194, respectively) and amino acid sequences (SEQ ID NO: 62, 63 and 61, respectively) of the light chain VL region VK1, VK2 and VK3 of anti-TIM3 monoclonal antibody 9F6 with signal sequence (SEQ ID NO: 276 and 271, respectively).

In fig. 6A shows the nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 39) of the mature heavy chain variable region (VH) of anti-TIM3 monoclonal antibody 3G4. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 51) and CDR3 (SEQ ID NO: 58) are delineated, and germline V, D and J derivatives are indicated.

In fig. 6B shows the nucleotide sequence (SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 3G4. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 68) are delineated, and germline V and J derivatives are indicated.

In fig. 6C shows the nucleotide sequence (SEQ ID NO: 172) and amino acid sequence (SEQ ID NO: 39) of the heavy chain VH region of anti-TIM3 monoclonal antibody 3G4 with signal sequence (SEQ ID NOs: 275 and 270, respectively), and the nucleotide sequence ( SEQ ID NO: 193) and amino acid sequence (SEQ ID NO: 60) of the light chain VL region of anti-TIM3 monoclonal antibody 3G4 with signal sequence (SEQ ID NOs: 273 and 268, respectively).

In fig. 7A shows the nucleotide sequence (SEQ ID NO: 173) and amino acid sequence (SEQ ID NO: 40) of the mature heavy chain variable region (VH) of anti-TIM3 monoclonal antibody 17C8. CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 52) and CDR3 (SEQ ID NO: 59) are delineated, and germline V, D and J derivatives are indicated.

In fig. 7B shows the nucleotide sequence (SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the mature light chain (VL) variable region of anti-TIM3 monoclonal antibody 17C8. CDR1 (SEQ ID NO: 64), CDR2 (SEQ ID NO: 66) and CDR3 (SEQ ID NO: 69) are delineated, and germline V and J derivatives are indicated.

In fig. 7C shows the nucleotide sequence (SEQ ID NO: 173) and amino acid sequence (SEQ ID NO: 40) of the heavy chain VH region of the anti-TIM3 monoclonal antibody 17C8 with signal sequence (SEQ ID NO: 275 and 270, respectively), and the nucleotide sequence ( SEQ ID NO: 194) and amino acid sequence (SEQ ID NO: 61) of the light chain VL region of the anti-TIM3 monoclonal antibody 17C8 with signal sequence (SEQ ID NOs: 273 and 268, respectively).

In fig. 8A shows a sequence alignment of the heavy chain variable region (VH) of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8. The complementarity determining regions (CDRs) are circled.

In fig. 8B lists the SEQ ID NOs for the VH regions, each of the CDRs, and antibody mutants thereof.

In fig. 9A shows a sequence alignment of the light chain variable region (VL) of monoclonal antibodies 13A3, 8B9, 8C4, 17C3, 9F6VK1, 9F6VK2, 9F6VK3, 3G4 and 17C8. The complementarity determining regions (CDRs) are circled.

In fig. 9B lists the SEQ ID NOs for the VL regions and each of the antibody CDRs.

In fig. Figure 10 shows the sequence alignment of the mature full-length heavy chain (HC).

- 16 044112 monoclonal antibody TIM3.5 (13A3) and its illustrative variants: TIM3.13 (D101E), TIM3.14 (P102V), TIM3.15 (P102Y), TIM3.16 (P102L), TIM3.17 (N60Q/ P102Y), TIM3.18 (N60Q/D101E), TIM3.10 (N60Q), TIM3.11 (N60S) and TIM3.12 (N60A). The VH region of each heavy chain is underlined.

In fig. 11 shows a sequence alignment of the mature full-length HC monoclonal antibody 9F6 and its exemplary variant TIM3.7 (A108T). The VH region of each heavy chain is underlined.

In fig. 12 shows a sequence alignment of the mature full-length monoclonal 8B9 HC and its exemplary variant TIM3.8 (S61P). The VH region of each heavy chain is underlined.

In fig. 13 lists the SEQ ID NOs of full-length heavy and light chains, variable regions and CDRs derived from hybridoma antibodies (13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8) and recombinant (TIM3.2-TIM3.18) antibodies to human TIM3. The isotype of the heavy and light chains is also indicated. N.p. refers to the name hybridoma. The heavy and light chains that are shown in FIG. 13 can be derived from its elements, for example, variable and constant regions, which are disclosed herein. If SEQ ID NO does not appear in a given column on the second or third page of the table, it appears in that column on the preceding page or the page preceding it.

In fig. 14A-14B show binding curves and EC ₅₀ values of anti-TIM3 antibodies to human TIM-3 transfected CHO cells (FIG. 14A) and activated human T cells (FIG. 14B).

In fig. 15A-15B show binding curves and EC ₅₀ values of anti-TIM3 antibodies to the TIM3-transfected CHO cell line (FIG. 15A), and activated cynomolgus T cells (FIG. 15B).

In fig. 16 shows anti-TIM3 activity (at various antibody concentrations) in stimulating IFN-γ production from tumor-infiltrating leukocytes (TILs) in renal cell carcinoma (RCC). The 8 columns for each antibody represent different antibody concentrations as indicated.

In fig. 17A-17B show anti-TIM3 activity (at various antibody concentrations) in stimulating IFN-γ production from lung cancer TILs (FIG. 17A, IFN-γ ELISA; FIG. 17B, intracellular IFN-γ staining). In fig. 17A, a separate column for each antibody represents the different antibody concentrations as indicated. In fig. 17B, the top panel shows CD4+ cells and the bottom panel shows CD8+ cells. TIM3 content was measured using 8B9 (X-axis).

In fig. 18 shows antibodies to TIM-3 (ie antibodies 13A3 and 3G4) upon stimulation of IFN-γ secretion from TILs isolated from various tissues in the presence of CHO-OKT3 cells.

In fig. 19 shows cross-blocking of anti-TIM-3 antibodies on activated human T cells.

In fig. 20 shows the amino acid residues that are required for the binding of anti-TIM3 monoclonal antibodies such as 13A3, 3G4, 17C3 and 8B9 to human TIM3. The signal sequence and transmembrane domains are underlined.

In fig. 21A-21B show that certain anti-TIM3 antibodies block the interaction between human TIM3 and the PS liposome. In fig. 21A shows a schematic diagram of a phosphatidylserine (PS)-hTIM3 tandem blocking assay. In fig. 21B shows the blocking of hTIM3-Fc binding to the PS liposome by certain anti-TIM3 antibodies, as measured by the PS-hTIM3 tandem blocking assay shown in FIG. 21A.

In fig. 22 shows a summary of the functional activity of various anti-TIM3 antibodies (eg, TIM3.5, TIM3.4, TIM3.2, TIM3.9, 9F6, TIM3.8 and TIM3.6). Data are presented for the binding assay, T cell assay, TIL assay, and PS-TIM3 blocking assay.

In fig. 23 provides a list of all SEQ ID numbers with a description of the sequences represented by the SEQ ID numbers.

In fig. 24A-24B show the antitumor activity of combined administration of anti-PD1 and anti-TIM3 antibodies in a mouse model of CT26 colorectal tumor. In fig. 24A shows tumor volume at various time points after tumor implantation in mice (n=10/group) treated with (i) control IgG (upper left panel), (ii) anti-TIM3 antibody PMT3-23 alone (upper right panel), (iii ) RMP1-14 anti-PD1 antibody alone (lower left panel) and (iv) combination of RMT3-23 anti-TIM3 and RMP1-14 anti-PD1 antibodies (lower right panel). In fig. 24B shows the average tumor volume versus time (days after tumor implantation) in mice treated with (i) anti-TIM3 antibody RMT3-23 only, (ii) anti-TIM3 antibody AbM only, (iii) anti-PD1 antibody RMP1-14 only. , (iv) a combination of RMT3-23 anti-TIM3 and RMP1-14 anti-PD1 antibodies, (v) a combination of Ab M anti-TIM3 and RMP1-14 anti-PD1 antibodies, and (vi) an isotype control antibody.

In fig. 25 shows a list of common hTIM-3 peptides that were used to map anti-TIM3 antibody epitopes (13A3 and 3G4) using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Each bar represents a peptic peptide. The circled residues (i.e., N99, T145, and N172) indicate glycosylation sites.

- 17 044112

In fig. 26 shows the human TIM3 binding regions of anti-TIM3 antibodies (13A3 and 3G4) identified using HDX-MX. The top panel shows the binding region of the anti-TIM3 antibody 13A3. The lower panel shows the binding region of the anti-TIM4 antibody 3G4.

In fig. 27A-27B show the results of the TIM3.18.IgG1.3 Scatchard assay for CHO cells ectopically expressing human or cynomolgus TIM3. In fig. 27A shows a standard curve of ¹²⁵ I antibody to TIM3. In fig. 27B shows the amount of TIM3.18.IgG1.3 antibody bound to human (left panel) and cynomolgus (right panel) TIM3-expressing CHO cells.

In fig. 28 shows the results of Scatchard's TIM3.18.IgG1.3 assay for activated Th1 cells from two donors (left and right panels).

In fig. 29A and 29B show increased proliferation of Fab TIM3.18.IgG1.3 and TIM3.18.IgG1.3 Th1 T cells in a polarized Th1/irradiated CHO-OKT3 coculture assay. In fig. 29A shows Th1 cell proliferation observed with various concentrations of TIM3.18.IgG1.3, 13A3 (13A3-g4) or without antibodies or isotopic control antibodies (hIgG1.1 and hIgG4). In fig. 29B shows the proliferation of Th1 cells observed with various concentrations of Fab TIM3.18.IgG1.3 or without antibody or IgG1.3 isotype control antibody.

In fig. 30 shows that anti-TIM3 antibody TIM3.18.IgG1.3 enhances Thl T cell proliferation in a polarized Th1/irradiated CHO-OKT3-PD-L1 co-culture assay in combination with nivolumab.

In fig. 31 shows that the anti-TIM3 antibody TIM3.18.IgG1.3 enhances interferon-γ secretion in renal cell carcinoma tumor-infiltrating lymphocytes (TILs) stimulated with irradiated CHO-OKT3 cells.

In fig. 32 shows that the anti-TIM3 antibody TIM3.18.IgG1.3 enhances the secretion of interferon-γ by TILs stimulated by irradiated CHO-OKT3 cells in breast cancer.

In fig. 33 shows the expression of CD163, CD206 and TIM3 on M0 macrophages that were used in the AlloMLR (mixed lymphocyte culture reaction) assay, the results of which are shown in FIG. 34.

In fig. 34 shows cell proliferation in an AlloMLR assay performed in the presence of anti-TIM3 antibody TIM3.18.IgG1.3, isotype control, or in the absence of antibody.

Fig. 35 is a strip diagram of the structure of the TIM3:TIM3.18 Fab complex as determined by crystallography. The Fab fragment is shown in light gray and TIM3 in dark gray.

In fig. 36 shows the structure of the TIM3:TIM3.18 Fab complex as determined by crystallography. A Fab fragment is shown as a strip chart. Images of TIM3 are shown with a white surface with contacting Fab residues depicted in dark grey.

Fig. 37 is a diagram of the assay that was used to measure potential internalization by anti-TIM3 antibodies.

In fig. 38 shows that anti-TIM3 antibodies 13A3 (lower left panel) and some of their variants (D101E - upper left panel; N60Q - upper right panel) do not trigger receptor-mediated internalization (ie, TIM3).

In fig. 39A and 39B are strip charts depicting epitopes of anti-TIM3 antibodies 13A3 (FIG. 39A) and 3G4 (FIG. 39B). The amino acid sequences of the epitopes for each antibody are presented below the strip chart. The different patterns identify specific regions of anti-TIM3 antibodies that correspond to specific epitopes.

Detailed Description of This Disclosure

In order that the present description may be more easily understood, certain terms are first defined. Additional definitions are set forth in the detailed description.

It should be noted that singular object refers to one or more objects; for example, by nucleotide sequence is meant one or more nucleotide sequences. As such, the terms one or more and at least one may be used interchangeably herein.

In addition, and/or when used herein should be understood as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term and/or, when used in a phrase such as A and/or B, is intended to include A and B, A or B, A (separately) and B (separately). Likewise, the term and/or when used in a phrase such as A, B and/or C is intended to cover each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separately) and C (separately).

It is understood that wherever aspects are described herein in terms of containing, similar aspects described in terms of consisting of and/or consisting essentially of are also provided.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999,

- 18 044112

Academic Press and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provides one skilled in the art with a general vocabulary of many of the terms used in the present disclosure.

Units, prefixes and symbols are designated in their accepted Systeme International de Unites (SI) form. Numeric ranges include numbers that define a range. Unless otherwise noted, nucleotide sequences are written from left to right in a 5'-3' orientation. Amino acid sequences are written from left to right in amino-carboxy orientation. The headings set forth herein are not intended to limit various aspects of the present disclosure, which may be obtained by reference to the description as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the full specification.

The term approximately is used herein to mean about, around, about, or in areas. When an approximate term is used in combination with a numeric range, it modifies that range by extending the boundaries above and below the specified numeric values. Typically, a term approximation may vary the numerical value above and below the stated value by deviation, for example, 10 percent, up or down (above or below).

As used herein, the term T-cell immunoglobulin-mucin domain-containing protein 3, or TIM3, refers to a receptor that is a member of the T-cell immunoglobulin-mucin domain (TIM) family of proteins. The primary ligand for TIM3 includes phosphatidylserine (TIM3-L). TIM3 is also called hepatitis A virus cellular receptor 2 (HAVCR2), T cell immunoglobulin mucin receptor 3, TIM-3, TIMD3, TIMD-3, kidney injury molecule 3, KIM-3, and CD366. The term TIM3 includes any variants or isoforms of TIM3 that are naturally expressed by cells. Accordingly, the antibodies described herein may cross-react with TIM3 from non-human species (eg, cynomolgus TIM3). Alternatively, the antibodies may be specific to human TIM3 and not cross-reactive with other species. TIM3 or any of its variants and isoforms can be isolated from cells or tissues that naturally express them, or can be produced recombinantly using methods well known in the art and/or described herein.

Two isoforms of human TIM3 have been identified. Isoform 1 (accession number NP_116171; SEQ ID NO: 286) consists of 301 amino acids and is a canonical sequence. Isoform 2 (accession number AAH20843; SEQ ID NO: 287) consists of 142 amino acids and is soluble. It lacks amino acid residues 143-301, which encode the transmembrane domain, cytoplasmic domain and part of the extracellular domain of TIM3. Amino acid residues 132-142 also differ from the canonical sequence described above.

Below are the amino acid sequences of the two known human TIM3 isoforms.

(A) Human TIM3 isoform 1 (accession number NP_116171; SEQ ID NO: 286; encoded by the nucleotide sequence having accession number NM_032782.4; SEQ ID NO: 288; Fig. 20):

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECG

NWLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLV

IKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDL

RDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEКIQNLSLISLANLPPSGLANAVAE

GIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFAMP (B) Human TIM3 isoform 2 (accession number AAH20843; SEQ ID NO: 287; encoded by the nucleotide sequence having accession number BC020843.1; SEQ ID NO: 289):

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECG

NWLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLV

IKPGEWTFACHLYE

The signal sequence of isoforms 1 and 2 corresponds to amino acids 1-21 (underlined). Thus, mature isoforms 1 and 2 consist of amino acids 22 to 301 or 142, respectively. The extracellular mature domain of human TIM3 consists of amino acids 22-202 SEQ ID NO: 286 and is characterized by the amino acid sequence:

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWLN

GDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFP

RMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIG (SEQ Ш NO:

290).

The cynomolgus TIM3 protein consists of the following amino acid sequence (including the signal sequence):

- 19 044112

MFSHLPFDCVLLLLLLLLTRSSEVEYIAEVGQNAYLPCSYTPAPPGNLVPVCWGKGACPVFDCS NWLRTENRDVNDRTSGRYWLKGDFHKGDVSLTIENVTLADSGVYCCRIQIPGIMNDEKHNLKL WIKPAKVTPAPTLQRDLTSAFPRMLTTGEHGPAETQTPGSLPDVNLTQIFTLTNELRDSGATI RTAIYIAAGISAGL ALALIFGALIFKWYSHSKEKTQNLSLISLANIΡPSGLANAVAEGIRSEEN IYTIEEDVYEVEEPNEYYCYVSSGQQPSQPLGCRFAMP (SEQ ID NO: 360)

The term antibody refers, according to one embodiment, to a protein containing at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2 and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a single domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are more conserved called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with the antigen. Antibody constant regions can mediate binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not contain a C-terminal lysine. Unless otherwise indicated herein, amino acids in the variable regions are numbered using the Kabat numbering system and amino acids in the constant regions are numbered using the EU system.

An IgG antibody as used herein, such as human IgG1, IgG2, IgG3, and IgG4 antibodies, in certain embodiments is characterized by the structure of a naturally occurring IgG antibody, i.e. it contains the same number of heavy and light chains and disulfide bonds as a natural IgG antibody of the same subclass. For example, an IgGl, IgG2, IgG3, or IgG4 anti-TIM3 antibody consists of two heavy chains (HC) and two light chains (LC), the two heavy chains and the light chains being linked by the same number and arrangement of disulfide bridges that occur naturally. antibodies IgG1, IgG2, IgG3 and IgG4, respectively (unless the antibody has been mutated to modify disulfide bridges).

Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (KD) of 10 ^-5 to 10 ^-11 M or less. In general, any KD greater than about 10 ^-4 M is considered to indicate nonspecific binding. As used herein, an antibody that specifically binds an antigen refers to an antibody that binds to an antigen and substantially identical antigens with high affinity, meaning a KD of 10 ^-7 M or less, 10 ^-8 M or less, 5x10 ^-9 M or less or from 10 ^-8 M to 10 ^-10 M or less, but does not bind with high affinity to unrelated antigens. An antigen is substantially identical to a given antigen if it exhibits a high degree of sequence identity to that antigen, for example, if it exhibits at least 80% sequence identity, at least 90%, at least 95%, at least 97 % or at least 99%, relative to the sequence of a given antigen. For example, an antibody that specifically binds to human TIM3 may, in some embodiments, also cross-react with TIM3 antigens from certain primate species (e.g., cynomolgus TIM3), but may not cross-react with TIM3 antigens from other species or with an antigen different from TIM3.

The immunoglobulin can be of any commonly known isotype, including, but not limited to, IgA, secretory IgA, IgG and IgM. The IgG isotype is divided into subclasses in specific species: IgG1, IgG2, IgG3 and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice. In certain embodiments, the anti-TIM3 antibodies described herein are of the IgG1 subtype. Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by no more than a few amino acids. Antibody includes by way of example both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies and fully synthetic antibodies.

As used herein, the term antigen-binding moiety of an antibody refers to one or more antibody fragments that retain the ability to specifically bind

- 20 044112 to interact with an antigen (for example, human TIM3). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term antigen binding portion of an antibody, such as the anti-TIM3 antibody described herein, include (i) a Fab fragment (papain digest fragment) or similar monovalent fragment consisting of the _VL , VH, LC, and CH1 domains ; (ii) an F(ab')2 fragment (pepsin cleavage fragment) or similar divalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the V _L and V H domains of one arm of the antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a V _H domain; (vi) a dedicated complementarity determining region (CDR); and (vii) a combination of two or more dedicated CDRs, which may optionally be connected by a synthetic linker. In addition, although the two domains of the Fv fragment, _VL and _VH , are encoded by separate genes, they can be combined using recombinant methods with the help of a synthetic linker that allows them to be formed as a single protein chain in which the VL and VH regions form pair, forming monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 5879-5883). Such single chain antibodies are also intended to be included within the term antigen binding portion of an antibody. These antibody fragments are prepared using conventional techniques known to those skilled in the art, and the fragments are tested for suitability in the same manner as intact antibodies. Antigen-binding moieties can be obtained by recombinant DNA methods or by enzymatic or chemical digestion of intact immunoglobulins.

A bispecific or bifunctional antibody is an artificial hybrid antibody containing two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by various methods, including fusion of hybridomas or binding of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).

As used herein, the term monoclonal antibody refers to an antibody from a population of essentially homogeneous antibodies, i.e. the individual antibodies contained in a population are substantially similar and bind to the same epitope(s) (e.g., antibodies exhibit a single binding specificity and affinity), except for possible variations that may arise during production of the monoclonal antibody, such variations being usually present in small quantities. The modifier monoclonal indicates the nature of the antibody being produced from an essentially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. The term human monoclonal antibody refers to an antibody from a population of essentially homogeneous antibodies that exhibit binding specificity and that contain variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma that includes a B cell derived from a transgenic non-human animal, such as a transgenic mouse, characterized by having a genome containing a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

As used herein, the term recombinant human antibody includes all human antibodies that are produced, expressed, created or isolated by recombinant methods, such as (a) antibodies isolated from an animal (eg, mouse) that is transgenic or transchromosomal for immunoglobulin genes human or hybridoma-derived hybridomas, (b) antibodies isolated from a host cell transformed to express an antibody, for example, from a transfectome, (c) antibodies isolated from a recombinant human combinatorial antibody library, and (d) antibodies obtained expressed created or isolated by any other means that involve splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences that are encoded by germline genes but include subsequent rearrangements and mutations that occur, for example, during antibody maturation.

As is known in the art (see, for example, Lonberg (2005) Nature Biotech. 23(9): 1117-1125), the variable region contains an antigen binding domain, which is encoded by various genes that are rearranged to form an antibody specific for a foreign antigen . In addition to rearrangement, the variable region can be further modified by multiple changes to a single amino acid (called somatic mutation or hypermutation) to increase the affinity of the antibody for a foreign antigen. The constant region will change with further response to the antigen (i.e., isotype switching). Consequently, rearranged and somatically mutated nucleic acid molecules that encode the light chain and

- 21 044112 heavy chain immunoglobulin polypeptide in response to an antigen may not be characterized by sequence identity with the parent nucleic acid molecules, but instead will be substantially identical or similar (ie, characterized by at least 80% identity).

A human antibody (HuMAb) refers to an antibody containing variable regions in which both framework regions and CDR regions are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Anti-TIM3 antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term human antibody is not intended to include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The terms human antibodies and fully human antibodies are used interchangeably.

A humanized antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domains of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains have been replaced with amino acids from human immunoglobulins, while some, most, or all of the amino acids in one or more CDR regions are unchanged. Minor additions, deletions, insertions, substitutions or modifications of amino acids are acceptable as long as they do not abolish the ability of the antibody to bind to a specific antigen. The humanized antibody retains antigen specificity similar to that of the parent antibody.

A chimeric antibody refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.

As used herein, the term isotype refers to a class of antibodies (eg, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies) that are encoded by heavy chain constant region genes.

The term allotype refers to naturally occurring variants within a particular isotype group that differ in several amino acids (see, for example, Jefferis et al. (2009) mAbs 1:1). Antibodies to TIM3 described herein can be of any allotype. The antibodies used herein, referred to as IgG1f, IgGl.1f or IgG1.3f isotype, are IgG1, IgG1.1 without effector functions and IgG1.3 without effector functions, respectively, allotype f, i.e. containing 214R, 356E and 358M according to the EU index, as in Kabat, as shown, for example, in SEQ ID NO: 3.

The phrases antigen-recognizing antibody and antigen-specific antibody are used interchangeably herein with the term antibody that specifically binds an antigen.

As used herein, isolated antibody is intended to mean an antibody that is substantially free of other proteins and cellular material.

As used herein, an antibody that inhibits the binding of TIM3-L to TIM3 refers to an antibody that inhibits the binding of TIM3 to its ligand, e.g., phosphatidylserine, for example, in binding assays using transfected CHO cells with human TIM3 or TIM3-expressing activated T cells. human cells, with an EC ₅₀ of about 1 μg/ml or less, for example, about 0.9 μg/ml or less, about 0.85 μg/ml or less, about 0.8 μg/ml or less, about 0.75 µg/ml or less, about 0.7 µg/ml or less, about 0.65 µg/ml or less, about 0.6 µg/ml or less, about 0.55 µg/ml or less, about 0.5 µg/ml or less, about 0.45 µg/ml or less, about 0.4 µg/ml or less, about 0.35 µg/ml or less, about 0.3 µg/ml or less, about 0.25 μg/ml or less, about 0.2 μg/ml or less, about 0.15 μg/ml or less, about 0.1 μg/ml or less, or about 0.05 μg/ml or less, as known herein methods in the art, such as the FACS-based binding assays described herein.

Effector function refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the biochemical event that occurs as a result thereof. Exemplary effector functions include C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR-mediated effector functions such as ADCC and antibody-dependent cell phagocytosis (ADCP), and cell surface receptor downregulation (e.g. B cell receptor; BCR). Such effector functions typically require an Fc region to be combined with a binding domain (eg, an antibody variable domain).

- 22 044112

The Fc receptor or FcR is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to IgG antibody contain receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII and FcyRIV in mice; FcyRIA, FcyRIIA and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor. Various properties of human FcyRs are known in the art. Most types of innate effector cells coexpress one or more activating FcyR and an inhibitory FcyRIIB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a with respect to the types of activating Fc receptors it binds to.

The term Fc region (fragment crystallization region) or Fc domain or Fc refers to the C-terminal region of an antibody heavy chain that mediates binding of immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g. effector cells) or with the first component (C1q) of the classical complement system. Thus, the Fc region contains the antibody constant region, excluding the first immunoglobulin constant region domain (eg, CH1 or CL).

In the IgG, IgA and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second (CH2) and third (CH3) constant domains of the two heavy chains of the antibody; The Fc regions of IgM and IgE contain three heavy chain constant domains (CH 2-4 domains) in each polypeptide chain. For IgG, the Fc region contains the immunoglobulin domains CH2 and CH3 and the hinge between the domains CH1 and CH2. Although the definition of the boundaries of the immunoglobulin heavy chain Fc region may vary, as defined herein, the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 for IgG1, V222 for IgG2, L221 for IgG3, and P224 for IgG4 to the carboxy terminus of the heavy chain , and the numbering corresponds to the Eu index, as in Kabat. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain in the Fc region, i.e. it extends from amino acid 341 to amino acid 447 or 446 (if the C-terminal lysine residue is missing) or 445 (if the C-terminal glycine and lysine residues are missing) of IgG. As used herein, the Fc region may be the native Fc sequence, including any allotype or Fc variant (eg, a non-naturally occurring Fc). Fc may also refer to this region alone or in the context of an Fc-containing protein polypeptide, such as an Fc region-containing binding protein, also referred to as an Fc fusion protein (eg, antibody or immunoadhesion).

A native sequence Fc region or native sequence Fc region contains an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. Human native sequence Fc regions include the human native sequence IgG1 Fc region; human IgG2 Fc region with native sequence; the native sequence human IgG3 Fc region and the native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. Native sequence Fc includes various Fc allotypes (see, e.g., Jefferis et al. (2009) mAbs 1:1).

The term epitope or antigenic determinant refers to a site on an antigen (eg, TIM3) to which an immunoglobulin or antibody specifically binds, for example, as determined by the particular method used to identify it. Epitopes can be formed either from contiguous amino acids (usually a linear epitope) or from non-contiguous amino acids located adjacent by tertiary protein folding (usually a conformational epitope). Epitopes formed from contiguous amino acids are generally, but not always, retained when exposed to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost when exposed to denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays in which overlapping or adjacent peptides (eg, from TIM3) are examined for reactivity with a given antibody (eg anti-TIM3 antibody). Methods for determining the spatial conformation of epitopes include methods in the art and methods described herein, for example, X-ray crystallography, antigenic mutation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

The term epitope mapping refers to the process of identifying molecular determinants for antibody-antigen recognition.

- 23 044112

The term binds to the same epitope with reference to two or more antibodies means that the antibodies bind to the same segment of amino acid residues, as determined by this method. Methods for determining whether antibodies bind to the same epitope on TIM3 as the antibodies described herein include, for example, epitope mapping methods such as crystal X-ray diffraction analysis of antigen:antibody complexes, which provides atomic separation of the epitope and mass hydrogen-deuterium exchange spectrometry (HDXMS). Other methods monitor the binding of an antibody to fragments of an antigen or mutated variations of an antigen, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered to be indicative of an epitope component. In addition, computational combinatorial epitope mapping methods can also be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies containing the same VH and VL or the same CDR1, 2 and 3 sequences are expected to bind to the same epitope.

Antibodies that compete with another antibody for binding to a target refer to antibodies that inhibit (partially or completely) the binding of another antibody to a target. Do two antibodies compete with each other for binding to the target, i.e. whether and to what extent one antibody inhibits the binding of another antibody to a target can be determined using known competition experiments, such as the BIACORE® surface plasmon resonance (SPR) assay. In some embodiments, the antibody competes with and inhibits the binding of another antibody to the target by at least 50, 60, 70, 80, 90, or 100%. The level of inhibition or competition may vary depending on which antibody is the blocking antibody (ie, a cold antibody that is first incubated with the target). Competitive assays can be performed as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi: 10.1101/pdb.prot4277 or in Chapter 11 Using Antibodies by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies cross-compete if the antibodies block each other in at least both ways at least 50%, i.e. regardless of whether one or the other antibody contacts the antigen first in a competition experiment.

Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include: competition for binding to TIM3-expressing T cells, for example, using flow cytometry such as described in the examples. Other methods include: SPR (eg, BIACORE®), direct or indirect radioimmunoassay (RIA), direct or indirect enzyme-linked immunosorbent assay (EIA), competitive sandwich assay (see Stahli et al., Methods in Enzymology 9: 242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137: 3614 (1986)); solid-phase direct labeled assay, solid-phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct labeled RIA using the 1-125 tag (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung et al., Virology 176: 546 (1990)) and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

As used herein, the terms specific binding, selective binding, selectively binding, and specifically binding refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, antibody (i) binds with an equilibrium dissociation constant (K _D ) of less than about 10 ^-7 M, for example, less than about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even lower when determined through, for example, surface plasmon resonance (SPR) technology in the BIACORE® 2000 instrument using a predefined antigen, for example, recombinant human TIM3 as an analyte and an antibody as a ligand, or a Scatchard assay for antibody binding to antigen-positive cells, and (ii) binds to a predetermined antigen with an affinity that is at least two times greater than its binding affinity to a nonspecific antigen (eg, BSA, casein) other than the predetermined antigen or a closely related antigen. Accordingly, an antibody that specifically binds to human TIM3 refers to an antibody that binds to soluble or cell-bound human TIM3 with a K _D of 10 ^-7 M or less, such as less than about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even lower. An antibody that cross-reacts with cynomolgus TIM3 refers to an antibody that binds to cynomolgus TIM3 with a KD of 10 ^-7 M or less, for example, less than about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even below. In some embodiments, such antibodies that do not cross-react with TIM3 from a non-human species exhibit substantially undetectable binding to these proteins in standard binding assays.

As used herein, the term k _assoc or k _a is intended to refer to the rate of association of a particular antibody-antigen interaction, whereas as used herein

- 24 044112 document, the term k _dis or k _d is intended to indicate the rate of dissociation of a particular antibody-antigen interaction. As used herein, the term K _D is intended to refer to the dissociation constant, which is obtained from the ratio of k _d to k _a (ie kd/ka) and is expressed as molar concentration (M). K _D values for antibodies can be determined using methods well known in the art. Available methods for determining the K _D of an antibody include surface plasmon resonance, a biosensor system such as the BIACORE® system, or flow cytometry and Scatchard analysis.

As used herein, the term high affinity for an IgG antibody refers to an antibody having a K _D of 10 ^-8 M or less, 10 ^-9 M or less, or 10 ^-10 M or less for the target antigen. However, high affinity binding may vary for other antibody isotypes. For example, high affinity binding for the IgM isotype refers to an antibody having a K _D of 10 ^-10 M or less or 10 ^-8 M or less.

The term EC ₅₀ in the context of an in vitro or in vivo assay using an antibody or an antigen-binding fragment thereof refers to the concentration of the antibody or antigen-binding portion thereof that induces a response that is 50% of the maximum response, i.e. is midway between the maximum response and the original value. As used herein, the term naturally occurring, when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses), that can be isolated from a source in nature, and that has not been intentionally altered by humans in a laboratory is naturally occurring.

The term polypeptide refers to a chain containing at least two consecutively linked amino acid residues with no upper limit on chain length. One or more amino acid residues in a protein may contain a modification, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A protein may contain one or more polypeptides.

The term nucleic acid molecule as used herein is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded and may be a cDNA.

The term conservative amino acid substitutions refers to substitutions of an amino acid residue with an amino acid residue characterized by a similar side chain. Families of amino acid residues characterized by similar side chains have been identified 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), non-polar 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). In some embodiments, a predicted nonessential amino acid residue in an anti-TIM3 antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying nucleotide and amino acid conservative substitutions that do not abolish antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12 (10): 879-884 (1999) and Burks et al. Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

For nucleic acids, the term substantial homology indicates that two nucleic acids or their designated sequences, when optimally aligned and compared, are identical with corresponding nucleotide insertions or deletions on at least about 80% of the nucleotides, on at least about 90 % to 95% or at least about 98 to 99.5% of nucleotides. Alternatively, significant homology exists when the segments will hybridize under selective hybridization conditions with the complement of the chain.

For polypeptides, the term substantial homology indicates that the two polypeptides or their designated sequences, when optimally aligned and compared, are identical with corresponding amino acid insertions or deletions of at least about 80% amino acids, at least about 90 to 95% or at least about 98 to 99.5% amino acids.

The percentage identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of two sequences. Comparing sequences and determining the percent identity between two sequences can be done using a mathematical algorithm like

- 25 044112 is described in the non-limiting examples below.

The percentage identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com), using the NWSgapdna.CMP matrix and a gap penalty of 40, 50, 60, 70 or 80 and penalty for extending the gap 1, 2, 3, 4, 5 or 6. The percentage identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)), which was included in the ALIGN program (version 2.0), using the PAM120 residue substitution weight table, a gap extension penalty of 12, and a sequence gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)), which is included in the GAP program in GCG software (available at http://www.gcg.com), using either the Blossum 62 matrix, or RAM250 matrix, and a penalty for opening a pass 16, 14, 12, 10, 8, 6 or 4 and a penalty for extending a pass 1, 2, 3, 4, 5 or 6.

The nucleic acid and protein sequences described herein can further be used as a query sequence to perform searches in public databases, for example, to identify related sequences. Such searches can be performed using the programs NBLAST and XBLAST (version 2.0) Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. Protein BLAST searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain a gap alignment for comparison, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When using the BLAST and Gapped BLAST programs, the default settings of the corresponding programs (for example, XBLAST and NBLAST) may be used. See worldwideweb.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. A nucleic acid is isolated or becomes substantially pure when purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of a chromosome) or proteins, by standard methods including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

Nucleic acids, such as cDNA, can be mutated according to standard methods to provide gene sequences. For coding sequences, these mutations can affect the amino acid sequence at will. In particular, DNA sequences that are substantially homologous to or derived from native V, D, J, constants, switches, and other similar sequences described herein are contemplated (where derived means that the sequence is identical to or modified from another sequence).

As used herein, the term vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a plasmid, which refers to a circular double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as recombinant expression vectors (or simply expression vectors). In general, expression vectors used in recombinant DNA methods are often in the form of plasmids. As used herein, plasmid and vector may be used interchangeably, since plasmid is the most commonly used form of vector. However, other forms of expression vectors are also included, such as viral vectors (eg, replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that perform equivalent functions.

As used herein, the term recombinant host cell (or simply host cell) is intended to mean a cell that contains a nucleic acid that is not naturally present in the cell and may be a cell into which a recombinant expression vector has been introduced . It should be understood that such terms are intended to refer not only to a particular cell of a subject, but also to the progeny of such cell. Since certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the original cell, but are still included within the scope of the term host cell as used herein.

An immune response is what is understood in the art and generally refers to the biological response in a vertebrate to foreign or abnormal cells, such as cancer cells, that protects the body from these agents and the diseases caused by them. The immune response is mediated by the action of one or more cells of the immune system (eg, T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, or neutrophil) and soluble macromolecules produced by any of these cells or the liver ( including antibodies, cytokines and complement), resulting in the selective targeting, binding, damage, destruction and/or removal from the body of vertebrate pathogens, cells or tissues infected with pathogens, cancer or other abnormal cells or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. The immune response includes, for example, the activation or inhibition of a T cell, such as an effector T cell, a Th cell, a CD4+ cell, a CD8+ T cell, or a Treg cell, or the activation or inhibition of any other cell of the immune system, such as an NK cell.

The term immunomodulator or immunoregulator refers to an agent, for example an agent that targets a component of a signaling pathway that may be involved in modulating, regulating or modifying the immune response. A modulating, regulating, or modifying immune response refers to any change in a cell of the immune system or the activity of such a cell (eg, an effector T cell such as a Th1 cell). Such modulation includes stimulation or suppression of the immune system, which may be manifested by an increase or decrease in the number of different types of cells, an increase or decrease in the activity of these cells, or any other changes that may occur in the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may exhibit improved function in the tumor microenvironment. In some embodiments, the immunomodulator targets a molecule on the surface of the T cell. An immunomodulatory target or immunoregulatory target is a molecule, for example a cell surface molecule, that is a target for binding and whose activity is altered as a result of binding of a substance, agent, fragment, compound or molecule. Immunomodulatory targets include, for example, cell surface receptors (immunomodulatory receptors) and receptor ligands (immunomodulatory ligands).

Immunotherapy refers to the treatment of a subject affected by or at risk of contracting or recurrent disease in a manner that involves inducing, enhancing, suppressing, or otherwise altering the immune system or immune response.

Immunostimulatory therapy refers to a treatment that results in an increase (induction or enhancement) of the immune response in a subject, for example, to treat cancer.

Potentiating an endogenous immune response means increasing the effectiveness or activity of an existing immune response in a subject. This increase in efficiency and activity can be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response, or by stimulating mechanisms that enhance the endogenous host immune response.

T effector (T _eff ) cells refer to T cells (eg, CD4+ and CD8+ T cells) with cytolytic activity, as well as T helper (Th) cells, such as Th1 cells, which secrete cytokines and activate and guide other immune cells but do not include regulatory T cells (Treg cells). Some anti-TIM3 antibodies described herein activate Teff cells, such as CD4+ and CD8+ Teff cells and Th1 cells.

The increased ability to stimulate the immune response or immune system may result from increased agonistic activity of T cell costimulatory receptors and/or increased antagonistic activity of inhibitory receptors. An increased ability to stimulate an immune response or immune system may be reflected in a fold increase in EC ₅₀ or maximum activity level in an assay that measures the immune response, such as an assay that measures changes in cytokine or chemokine release, cytolytic activity (determined directly on target cells or indirectly through the detection of CD 107a or granzymes) and proliferation. The ability to stimulate an immune response or immune system activity may be increased by at least 10%, 30%, 50%, 75%, 2-fold, 3-fold, 5-fold or more.

As used herein, the term bound refers to the association of two or more molecules. The bond may be covalent or non-covalent. This connection may also be genetic

- 27 044112 (i.e. recombinantly fused). Such linkages can be achieved using a wide variety of methods known in the art, such as chemical conjugation and recombinant protein production.

As used herein, the term administration refers to the physical administration to a subject of a composition containing a therapeutic agent using any of the various delivery methods and systems known to those skilled in the art. Various routes of administration of the anti-TIM3 antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes, such as by injection or infusion. As used herein, the phrase parenteral administration means methods of administration other than enteral and local administration, typically by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrathoracic injection and infusion, as well as in vivo electroporation. Alternatively, the antibody described herein can be administered by a non-parenteral route, such as the topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically. Administration may also be performed, for example, once, multiple times and/or over one or more extended periods.

As used herein, the term T cell-mediated response refers to a response mediated by T cells, including effector T cells (eg, CD8+ cells) and helper T cells (eg, CD4+ cells). T cell-mediated responses include, for example, cytotoxicity and T cell proliferation.

As used herein, the term cytotoxic T lymphocyte (CTL) response refers to the immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+ T cells.

As used herein, the terms inhibit or block (eg, relating to inhibiting/blocking the binding of TIM3-L to TIM3 on cells) are used interchangeably and cover both partial and complete inhibition/blocking. In some embodiments, the anti-TIM3 antibody inhibits the binding of TIM3-L to TIM3 by at least about 50%, such as about 60, 70, 80, 90, 95, 99, or 100%, determined, for example, as further described herein document. In some embodiments, the anti-TIM3 antibody inhibits the binding of TIM3-L to TIM3 by no more than 50%, such as about 40, 30, 20, 10, 5, or 1%, determined, for example, as further described herein.

As used herein, the phrase inhibits tumor growth includes any measurable reduction in tumor growth, e.g., inhibiting tumor growth by at least about 10%, e.g., at least about 20%, at least about 30%, at least at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99% or 100%.

As used herein, the term cancer refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade nearby tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

As used herein, the terms treat, treatment and treatment refer to any type of intervention or process performed, or administration of an active agent to a subject for the purpose of reversing, alleviating, ameliorating, inhibiting or slowing or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical features associated with the disease, or improve overall survival. The subject being treated may have the disease or may not have the disease (for example, for prophylaxis).

Hematologic malignancies include lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as cancers of the spleen and lymph nodes. Illustrative lymphomas include both B-cell lymphomas (B-cell hematologic cancers) and T-cell lymphomas. B-cell lymphomas include both Hodgkin lymphomas and most non-Hodgkin lymphomas. Non-limiting examples of B-cell lymphomas include diffuse large B-cell lymphoma, follicular lymphoma, mucosal lymphoid tissue lymphoma, small cell lymphocytic lymphoma (overlaps with chronic lymphocytic leukemia), mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal B-28 04411 2 large cell lymphoma, Waldenström's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis. Non-limiting examples of T-cell lymphomas include extranodal T-cell lymphoma, cutaneous T-cell lymphoma, anaplastic large cell lymphoma, and angioimmunoblastic T-cell lymphoma. Hematologic malignancies also include leukemia, such as, but not limited to, secondary leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and acute lymphoblastic leukemia. Hematologic malignancies also include, but are not limited to, myelomas such as multiple myeloma and smoldering multiple myeloma. Other hematologic and/or B-cell or T-cell cancers are included under the term hematologic malignancies.

The term effective dose or effective dosage is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A therapeutically effective amount or therapeutically effective dosage of a drug or therapeutic agent is any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes regression of the disease, as evidenced by a decrease in the severity of symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of impairment or disability due to disease. A therapeutically effective amount or dosage of a drug includes a prophylactically effective amount or prophylactically effective dosage, which is any amount of a drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a relapse of a disease, inhibits development or relapse of the disease. The ability of a therapeutic agent to promote disease regression or inhibit disease progression or recurrence can be assessed using a variety of methods known to the practitioner, for example, in humans during clinical trials, in animal model systems predictive of efficacy in humans, or by analyzing the activity of the agent in in vitro assays. vitro.

By way of example, an anticancer agent is a drug that promotes regression of cancer in a subject. In some embodiments, a therapeutically effective amount of a drug promotes regression of the cancer to the point of elimination. Promoting cancer regression means that administration of an effective amount of a drug, alone or in combination with an antineoplastic agent, results in a decrease in tumor growth or size, tumor necrosis, a decrease in the severity of at least one symptom of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, prevention of a disorder or disability due to disease or other improvement in the patient's symptoms of disease. In addition, the terms effective and efficacy in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological efficacy refers to the ability of a drug to stimulate cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects at the cellular, organ and/or organism level (adverse events) resulting from the use of a drug.

As an example for treating tumors, a therapeutically effective amount or dosage of a drug inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% % compared to untreated subjects. In some embodiments, a therapeutically effective amount or dosage of a drug completely inhibits cell or tumor growth, i.e. inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be assessed using the assays described below. Alternatively, this property of the composition can be assessed by studying the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro using assays known to one skilled in the art. In other embodiments described herein, tumor regression may be observed and continue for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

The term patient includes humans and other mammals who are receiving either prophylactic or therapeutic treatment.

As used herein, the term subject includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject suffering from cancer. The term non-human animal includes all vertebrate animals, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles and

- 29 044112

etc.

The term weight-based dose or dosage, as used herein, means that the dose that is administered to a patient is calculated based on the patient's weight. For example, when a patient weighing 60 kg requires 3 mg/kg of anti-TIM3 antibody, the appropriate amount of anti-TIM3 antibody (ie, 180 mg) to administer can be calculated and used.

The use of the term fixed dose in relation to the method of the present disclosure means that two or more different antibodies in one composition (for example, an anti-TIM3 antibody and a second antibody, for example, PD-1 or an anti-PD-L1 antibody) are present in the composition in certain (fixed) amounts. relationships with each other. In some embodiments, the fixed dose is based on the weight (eg, mg) of the antibody. In some embodiments, the fixed dose is based on the concentration (eg, mg/ml) of the antibodies. In some embodiments, the ratio of the two antibodies (e.g., anti-TIM3 and anti-PD1 or anti-PD-L1) is at least about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, approximately 1:6, approximately 1:7, approximately 1:8, approximately 1:9, approximately 1:10, approximately 1:15, approximately 1:20, approximately 1:30, approximately 1:40, approximately 1:50, approximately 1:60, approximately 1:70, approximately 1:80, approximately 1:90, approximately 1:100, approximately 1:120, approximately 1:140, approximately 1:160, approximately 1:180, approximately 1:200, approximately 200:1, approximately 180:1, approximately 160:1, approximately 140:1, approximately 120:1, approximately 100:1, approximately 90:1, approximately 80:1, approximately 70:1, approximately 60:1, approximately 50:1, approximately 40:1, approximately 30:1, approximately 20:1, approximately 15:1, approximately 10:1, approximately 9:1, approximately 8:1, approximately 7:1, approximately 6:1, about 5:1, about 4:1, about 3:1, or about 2:1 mg of first antibody (eg, anti-TIM3 antibody) to mg of second antibody. For example, a 2:1 ratio of anti-TIM3 antibody to anti-PD-1 antibody, such as nivolumab, would mean that a vial or injection could contain approximately 480 mg of anti-TIM3 antibody and 240 mg of anti-PD-1 antibody, or approximately 2 mg/mL of antibody anti-TIM3 and 1 mg/ml anti-PD-1 antibody.

When used in connection with the methods and dosages described herein, the term constant dose means a dose that is administered to a patient without regard to the weight or body surface area (BSA) of the patient. Therefore, the constant dose is not provided as a mg/kg dose, but as an absolute amount of agent (eg, anti-TIM3 antibody). For example, a 60 kg person and a 100 kg person would receive the same dose of antibody (eg, 480 mg of anti-TIM3 antibody).

As used herein, the terms μg and μM are used interchangeably with μg and μM, respectively.

The various aspects described herein are described in more detail in the following subsections.

I. Antibodies to human TIM3.

Disclosed herein are antibodies, eg fully human antibodies, that are characterized by certain functional characteristics or properties. For example, antibodies specifically bind to human TIM3 and, more specifically, to a specific domain (eg, functional domain) within the extracellular domain of human TIM3. In a specific embodiment, the antibodies specifically bind to a site on TIM3 to which TIM3-L binds. In certain embodiments, the antibodies are antagonistic antibodies, i.e. they inhibit or suppress the T cell inhibitory activity of TIM3 on cells, e.g. In some embodiments, antibodies to TIM3 cross-react with TIM3 from one or more non-human primates, such as cynomolgus TIM3. In some embodiments, the antibodies specifically bind to the extracellular region of human TIM3 and the extracellular region of cynomolgus monkey TIM3. In one embodiment, the antibodies bind to human TIM3 with high affinity.

Anti-TIM3 antibodies described herein exhibit one or more of the following functional properties:

(a) binding to soluble and/or membrane-bound human TIM3;

(b) binding to soluble and/or membrane-bound cynomolgus TIM3;

(c) inducing or stimulating an immune response;

(d) inducing or stimulating T cell activation, for example, Th1 cell activation (as evidenced, for example, by increased secretion and/or proliferation of cytokines);

(e) inducing or stimulating the proliferation of T cells (eg, CD4 ⁺ , CD8+ T cells, Th1 cells or TILs), for example, in a co-culture assay such as those described in the examples;

(f) inducing or stimulating the production of IFN-γ by T cells, for example, Th1 cells or tumor infiltrating lymphocytes (TILs), such as TILs from human kidney, lung, pancreatic or breast tumors, as determined, for example, in an assay described in the examples;

(g) blocking or inhibiting the binding of human TIM3 to PtdSer, as determined by e.g.

- 30 044112 measures, in the analysis described in the examples;

(h) lack of internalization or cell surface suppression of TIM3 upon binding to

TIM3 on cells;

(i) binding to the extracellular domain of human TIM3 (i) CPVFECG (SEQ ID NO: 296); (ii) RIQIPGIMND (SEQ ID NO: 298); (iii) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) or (iv) WTSRYWLNGDFR (SEQ ID NO: 297);

(j) competing with or cross-blocking binding to human TIM3 of a TIM3 binding antibody described herein (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, or any of TIM3.2-TIM3.18), as determined, for example, in an assay, described in the examples;

(k) binding to human TIM3, but not to human TIM3 containing an amino acid substitution of one or more of the following amino acid residues: L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84 , G86, D87, R89, D104, R111, Q113, G116, M118 and D120, as numbered in SEQ ID NO: 286 (Fig. 20); and (l) binding to human TIM3 regions ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS;

(m) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120 and optionally T70 and/or I112 as determined by x-ray crystallography, and/or (n) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

In some embodiments, anti-TIM3 antibodies described herein bind to human TIM3 with high affinity, e.g., a K _D of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less , 10 ^-11 M or less, 10 ^-12 M or less, from 10 ^-12 M to 10 ^-7 M, from 10 ^-11 M to 10 ^-7 M, from 10 ^-10 M to 10 ^-7 M or from 10 ^-9 M to 10 ^-7 M. In some embodiments, the anti-TIM3 antibody binds to soluble human TIM3, e.g., as determined by BIACORE™ (e.g., as described in the Examples), with a KD of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, 10 ^-12 M to 10 ^-7 M, 10 ^-11 M to 10 ^-7 M, 10 ^-10 M to 10 ^-7 M, 10 ^-9 M to 10 ^-7 M, or 10 ^-8 M to 10 ^-7 M. In certain embodiments, the anti-TIM3 antibody binds to bound (eg, cell membrane bound) human TIM3, such as on activated human T cells, e.g., as determined by flow cytometry and Scatchard plot, with a KD of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM) or less, 5x10 ^-10 M or less, 10 ^-10 M or less, from 10 ^-12 M to 10 ^-7 M, from 10 ^-11 M to 10 ^-8 M, from 10 ^-10 M to 10 ^-8 M, from 10 ^-9 M to 10 ^-8 M, 10 ^-11 M to 10 ^-9 M, or 10 ^-10 M to 10 ^-9 M. In some embodiments, the anti-TIM3 antibody binds to bound (e.g., cell membrane bound) human TIM3, such as on activated human T cells, e.g., as determined by flow cytometry, with an EC50 of 10 μg/ml or less, 5 μg/ml or less, 1 μg/ml or less, 0.9 μg/ml or less, 0.8 µg/ml or less, 0.7 µg/ml or less, 0.6 µg/ml or less, 0.5 µg/ml or less, 0.4 µg/ml or less, 0.3 µg/ml or less , 0.2 µg/ml or less, 0.1 µg/ml or less, 0.05 µg/ml or less, or 0.01 µg/ml or less. In some embodiments, anti-TIM3 antibodies described herein bind to cynomolgus TIM3, e.g., a KD of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, 10 ^-12 M or less, from 10 ^-12 M to 10 ^-7 M, from 10 ^-11 M to 10 ^-7 M, from 10 ^-10 M to 10 ^-7 M or from 10 ^-9 M to 10 ^-7 M. In some embodiments, the anti-TIM3 antibody binds to soluble cynomolgus TIM3, e.g., as determined by BIACORE™ (e.g., as described in the Examples), with a KD of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM) or less, 10 ^-10 M or less, from 10 ^-12 M to 10 ^-7 M, from 10 ^-11 M to 10 ^-7 M, from 10 ^-10 M to 10 ^-7 M, 10 ^-9 M to 10 ^-7 M, or 10 ^-8 M to 10 ^-7 M. Antibodies to TIM3 can bind to membrane-bound cynomolgus TIM3, e.g., EC ₅₀ 100 nM or less, 10 nM or less than 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, for example, as measured by flow cytometry (for example, as described in the Examples). In some embodiments, an anti-TIM3 antibody binds to bound (e.g., cell membrane bound) cynomolgus TIM3, such as on activated human T cells, e.g., as determined by flow cytometry and Scatchard plot, with a K _D of 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M (1 nM) or less, 5x10' ¹⁰ M or less, 10 ^-10 M or less, from 10 ^-12 M to 10 ^-7 M, from 10 ^-11 M to 10 ^-8 M, from 10 ^-10 M to 10 ^-8 M, from 10 ^-9 M to 10 ^-8 M, from 10 ^-11 M to 10 ^-9 M or from 10 ^-10 M to 10 ^-9 M .

In some embodiments, anti-TIM3 antibodies described herein stimulate or enhance an immune response, for example, by activating T cells, for example, in a tumor. For example, antibodies to TIM3 can activate or co-stimulate cells, as evidenced, for example, by increased secretion of cytokines (eg, IFN-γ) and/or increased proliferation, which may result from inhibition of TIM3-mediated T cell inhibitory activity. In some embodiments, T cell activation or antibody co-stimulation

- 31 044112 to TIM3 occurs in the presence of CD3 stimulation. In some embodiments, an anti-TIM3 antibody increases IFN-γ secretion by 50%, 100% (i.e., 2-fold), 3-fold, 4-fold, 5-fold or more, optionally from a maximum of 10-fold, 30 times, 100 times, as measured, for example, on primary human T cells and/or T cells expressing human TIM3, such as tumor infiltrating lymphocytes (TILs).

In some embodiments, anti-TIM3 antibodies inhibit the binding of phosphatidylserine to human TIM3 on cells, e.g., CHO cells or activated T cells expressing human TIM3, e.g., with an EC ₅₀ of 10 μg/mL or less, 1 μg/mL or less, from 0.01 µg/ml to 10 µg/ml, 0.1 µg/ml to 10 µg/ml or 0.1 µg/ml to 1 µg/ml.

In some embodiments, anti-TIM3 antibodies described herein bind to an epitope, e.g., a conformational epitope, in the extracellular portion of human TIM3, e.g., in the Ig-like domain of the extracellular region, i.e. with amino acids 22 to 202 from SEQ ID NO: 286 (Fig. 20). In some embodiments, the anti-TIM3 antibody binds to an epitope located within amino acids 22-120 of the extracellular domain of human TIM3 (SEQ ID NO: 286) or 1-99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). In some embodiments, an anti-TIM3 antibody binds to the region, or epitope, within the region consisting of amino acids 58-64 of human TIM3, characterized by SEQ ID NO: 286, which corresponds to amino acid residues 37-43 of mature human TIM3 (CPVFECG, SEQ ID NO: 296 ; see Fig. 20). In some embodiments, an anti-TIM3 antibody binds to the region or epitope within the region consisting of amino acids 111-120 of human TIM3 characterized by SEQ ID NO: 286, which corresponds to amino acid residues 90-99 of mature human TIM3 (RIQIPGIMND, SEQ ID NO: 298; see Fig. 20). In some embodiments, an anti-TIM3 antibody binds to a region or epitope within the region consisting of amino acids 58-64 of human TIM3 characterized by SEQ ID NO: 286 (CPVFECG, SEQ ID NO: 296) or to a region or epitope within the region consisting from amino acids 111-120 of human TIM3, characterized by SEQ ID NO: 286 (RIQIPGIMND, SEQ ID NO: 298; see Fig. 20). In some embodiments, an anti-TIM3 antibody binds to the region, or epitope, within the region consisting of amino acids 78-89 of human TIM3 characterized by SEQ ID NO: 286, which corresponds to amino acid residues 57-83 of mature human TIM3 (WTSRYWLNGDFR, SEQ ID NO : 297; see Fig. 20).

In one embodiment, an anti-TIM3 antibody binds to substantially the same epitope as 13A3, that is, an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, F61, E62, C63, R111, and D120 at SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 in SEQ ID NO: 286 (FIG. 20 ). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues C58, P59, F61, E62, C63, R111, and D120 in SEQ ID NO: 286 is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 in SEQ ID NO: 286 is replaced with another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 3G4, i.e. with an epitope (or human TIM3 region) containing one or more amino acid residues C58, P59, V60, F61, E62, C63, G116 and M118 in SEQ ID NO: 286 (Fig. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 in SEQ ID NO: 286 (FIG. 20 ). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein having one or more amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 in SEQ ID NO: 286 is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In certain embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to the human TIM3 protein, in which one or more amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 in SEQ ID NO: 286 is replaced with another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, an anti-TIM3 antibody binds to substantially the same epitope as 17C3, that is, an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 in SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, V60, F61, E62,

- 32 044112

C63, G64, D104 and G116 in SEQ ID NO: 286 (Fig. 20). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to the human TIM3 protein, wherein one or more of amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 in SEQ ID NO: 286 is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein, in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 in SEQ ID NO: 286 is replaced with another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, an anti-TIM3 antibody binds to substantially the same epitope as 8B9, that is, an epitope (or region of human TIM3) containing one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 in SeQ ID NO: 286 (Fig. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues L48, W78, S80, R81, W83,

L84, G86, D87 and R89 in SEQ ID NO: 286 (Fig. 20). In some embodiments, an anti-TIM3 antibody binds to substantially the same epitope as 8B9, that is, an epitope (or region of human TIM3) containing one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89 and D104 in SEQ ID NO: 286 (FIG. 20). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein having one or more of amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 in SEQ ID NO: 286 (Fig. 20) is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein, in which one or more amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 in SEQ ID NO: 286 (FIG. 20) is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, an anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein, in which one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 in SEQ ID NO: 286 (FIG. 20) is replaced by another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, anti-TIM3 antibodies compete for binding to human TIM3 (or inhibit binding) with anti-TIM3 antibodies containing CDRs or variable regions described herein, for example, such as antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9 , 8С4 and any from TIM3.2 to TIM3.18. In some embodiments, anti-TIM3 antibodies inhibit the binding of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2TIM3.18 to human TIM3 by at least 50, 60, 70, 80, 90, or 100 %. In some embodiments, 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2-TIM3.18 inhibits the binding of anti-TIM3 antibodies to human TIM3 by at least 50, 60, 70, 80, 90, or 100%. In some embodiments, anti-TIM3 antibodies inhibit the binding of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2-TIM3.18 to human TIM3 by at least 50, 60, 70, 80, 90, or 100% and 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2-TIM3.18 inhibits the binding of anti-TIM3 antibodies to human TIM3 by at least 50, 60, 70, 80, 90 or 100 % (for example, they compete in both directions).

In some embodiments, anti-TIM3 antibodies are characterized by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all of the following:

(1) binding to soluble human TIM3, for example, with a KD of 10 nM or less (for example, from 0.01 nM to 10 nM), for example, as measured by Biacore, for example, as described in the examples;

(2) binding to soluble cynomolgus TIM3, eg, with a KD of 100 nM or less (eg, 0.01 nM to 100 nM), eg, as measured by Biacore, eg, as described in the Examples;

(3) binding to membrane-bound human TIM3, e.g., with an EC ₅₀ of 1 μg/ml or less (e.g., 0.01 μg/ml to 1 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(4) binding to membrane-bound human TIM3, eg, with a KD of 1 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(5) binding to membrane-bound cynomolgus TIM3, e.g., with an EC ₅₀ of 20 μg/ml or less (e.g., 0.01 μg/ml to 20 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(6) binding to membrane-bound cynomolgus TIM3, e.g., with a KD of 1 nM or less (e.g., 0.01 nM to 10 nM), e.g., as measured by Scatchard assay, e.g., as

- 33 044112 is described in examples;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) enhancing the proliferation of TIM-3-expressing T cells (eg, Th1 cells or TILs), for example, as described in the examples;

(8) stimulating T cell proliferation in a mixed lymphocyte response (MLR) assay, for example, as described in the Examples;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay, for example, as described in the Examples;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297), for example, as described in the examples;

(12) the presence of reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118 and D120 (as numbered in SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3, for example, as described in the examples;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.18, for example, as described in the examples;

(14) binding to human TIM3 regions ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS, for example, as described in the Examples;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (for example, described in the examples; numbering according to SEQ ID NO: 286 (Fig. 20)); and/or (16) (a) the presence of reduced binding to human TIM3, in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120 and , optionally, D104 and Q113 (numbered at SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3; (b) binding to ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373) as determined by HDX-MS as described in the Examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

Accordingly, it is understood that an antibody that exhibits one or more of these functional properties (e.g., biochemical, immunochemical, cellular, physiological or other biological activity, or the like) as determined in accordance with methodologies known in the art and described herein demonstrates a statistically significant difference in specific activity relative to the activity observed in the absence of the antibody (eg, or when a control antibody of non-target specificity is present). In some embodiments, an anti-TIM3 antibody-induced increase in a measured parameter (e.g., T cell proliferation, cytokine production) in a given assay causes a statistically significant increase of at least 10% in the measured parameter, e.g., at least 20, 30, 40. 50, 60, 70, 80, 90, 95, 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold, and in some embodiments, an antibody described herein can increase a measured parameter, e.g., more than 92, 94, 95, 97, 98, 99, 100% (i.e., 2-fold), 3-fold, 5-fold, or 10-fold relative to the same assay performed in the absence of the antibody . Conversely, an anti-TIM3 antibody-induced decrease in a measured parameter (eg, tumor volume, TIM3-L binding to human TIM3) in this assay results in a statistically significant reduction of at least 10% in the measured parameter, e.g., at least 20, 30, 40 , 50, 60, 70, 80%, or 90, and in some embodiments, an antibody described herein can reduce a measured parameter, for example, by more than 92, 94, 95, 97, 98, or 99% relative to the same assay performed in the absence of an antibody.

Standard assays for assessing the ability of antibodies to bind to TIM3 of various species are known in the art, including, for example, ELISA, Western blots and RIA. Suitable assays are described in detail in the Examples. The binding kinetics (eg, binding affinity) of antibodies can also be assessed using standard assays known in the art, such as the Biacore assay. Assays to evaluate the effect of antibodies on functional properties

- 34 044112

TIM3 (eg, ligand binding, T cell proliferation, cytokine production) are described in more detail below and in the examples.

In some embodiments, anti-TIM3 antibodies are not native antibodies or do not occur in nature. For example, antibodies to TIM3 contain post-translational modifications that differ from antibody modifications that occur naturally, such as having more, less, or a different type of post-translational modification.

In some embodiments, anti-TIM3 antibodies do not have agonist activity, as determined, for example, by cross-linking an anti-TIM3 antibody in CHO-OKT3-CD32:T cell coculture experiments, in which such antibodies do not enhance activity greater than the antibody alone to TIM3. In certain embodiments, anti-TIM3 antibodies block the interaction of TIM3 with its ligand without stimulating agonist activity.

In some embodiments, anti-TIM3 antibodies enhance IL-12 production from LPS-treated monocytes or dendritic cells.

In some embodiments, anti-TIM3 antibodies restore tumor-infiltrating CD8+ T cells that co-express PD-1 and TIM3 through a combined effect, thereby avoiding CD8+ T cell depletion.

II. Exemplary anti-TIM3 antibodies.

Specific anti-TIM3 antibodies described herein are antibodies, for example, monoclonal, recombinant and/or human antibodies having the CDR and/or variable region sequences of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3. 2TIM3.18 isolated and structurally characterized as described herein, as well as antibodies having at least 80% identity (e.g., at least 85% identity, at least 90% identity, at least 95% identity, or at least 99%) with their variable region or CDR sequences. The amino acid sequences of VH from 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 are presented in SEQ ID NO: 34-40, respectively. The VH amino acid sequences of the mutated versions 13A3, 8B9 and 9F6 are presented in SEQ ID NO: 112-121 and 364. The VL amino acid sequences of 13A3, 17C3 and 3G4 are presented in SEQ ID NO: 60. The VL amino acid sequences of 8B9, 8C4 and 17C8 are presented in SEQ ID NO: 61. The VL amino acid sequence of 9F6 is shown in SEQ ID NO: 61, 62 and 63. The VL amino acid sequences of the mutated versions 13A3, 8B9 and 9F6 are the sequences of the corresponding unmutated antibodies. A summary of the SEQ ID NO is provided in FIG. 13.

Accordingly, provided herein are isolated antibodies or an antigen binding portion thereof comprising heavy and light chain variable regions, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-40, 112-121 and 364.

Also provided are isolated antibodies or antigen binding portions thereof comprising heavy and light chain variable regions, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63.

Provided herein are isolated anti-human TIM3 antibodies or antigen-binding portions thereof comprising:

(a) heavy and light chain variable region sequences containing SEQ ID NO: 34 and 60, respectively;

(b) heavy and light chain variable region sequences comprising SEQ ID NO: 35 and 61, respectively;

(c) heavy and light chain variable region sequences comprising SEQ ID NO: 36 and 61, respectively;

(d) heavy and light chain variable region sequences containing SEQ ID NO: 37 and 60, respectively;

(e) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 61, respectively;

(f) heavy and light chain variable region sequences comprising SEQ ID NO: 38 and 62, respectively;

(g) heavy and light chain variable region sequences comprising SEQ ID NOs: 38 and 63, respectively;

(h) heavy and light chain variable region sequences containing SEQ ID NO: 39 and 60, respectively;

(i) heavy and light chain variable region sequences comprising SEQ ID NOs: 40 and 61, respectively;

(j) heavy and light chain variable region sequences comprising SEQ ID NOs: 121 and 63, respectively;

(k) heavy and light chain variable region sequences comprising SEQ ID NO: 120

- 35 044112 and 61, respectively;

(l) heavy and light chain variable region sequences containing SEQ ID NO: 112 and 60, respectively;

(m) heavy and light chain variable region sequences comprising SEQ ID NO: 113 and 60, respectively;

(n) heavy and light chain variable region sequences comprising SEQ ID NO: 114 and 60, respectively;

(o) heavy and light chain variable region sequences comprising SEQ ID NO: 115 and 60, respectively;

(p) heavy and light chain variable region sequences containing SEQ ID NO: 116 and 60, respectively;

(q) heavy and light chain variable region sequences comprising SEQ ID NO: 117 and 60, respectively;

(r) heavy and light chain variable region sequences comprising SEQ ID NO: 118 and 60, respectively;

(s) the heavy and light chain variable region sequences comprising SEQ ID NOs: 119 and 60, respectively, or (t) the heavy and light chain variable region sequences comprising SEQ ID NOs: 364 and 60, respectively.

Antibodies to TIM3 may contain CDR1, CDR2 and CDR3 heavy and light chains from 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 or any of TIM3.2-TIM3.18 or combinations thereof. The amino acid sequences of CDR1 VH from 13A3, 8B9, 8C4 and 17C3 are presented in SEQ ID NO: 41-44, respectively. The CDR1 VH amino acid sequences of 9F6, 3G4 and 17C8 are shown in SEQ ID NO 45. The CDR1 VH amino acid sequence of the mutated 13A3 antibodies (i.e., TIM3.10-TIM3.18) is the same as that of the unmutated 13A3 antibody, i.e. .e. SEQ ID NO: 41. The CDR1 VH amino acid sequence of the mutated 8B9 antibody (i.e., TIM3.8) is the same as that of the unmutated 8B9 antibody, i.e. SEQ ID NO: 42. The CDR1 VH amino acid sequence of the mutated 9F6 antibody (i.e., TIM3.7) is the same as that of the unmutated 9F6 antibody, i.e. SEQ ID NO: 45. The amino acid sequences of CDR2 VH from 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 are presented in SEQ ID NO: 46-52, respectively. The CDR2 VH amino acid sequence of mutated antibodies 13A3 TIM3.10, TIM3.17 and TIM3.18 is presented in SEQ ID NO: 122. The CDR2 VH amino acid sequence of mutated antibodies 13A3 TIM3.11 and TIM3.12 is presented in SEQ ID NO: 123 and 124, respectively. The amino acid sequence CDR2 VH of the mutated antibodies 13A3 TIM3.13 and TIM3.16 is the same as the sequence of the unmutated antibody 13A3, i.e. SEQ ID NO: 46. The amino acid sequence CDR2 VH of the mutated antibody 8B9 (i.e. TIM3.8) is presented in SEQ ID NO: 125. The amino acid sequence CDR2 VH of the mutated antibody 9F6 (i.e. TIM3.7) is the same , like the unmutated antibody 9F6, i.e. SEQ ID NO: 45. The amino acid sequences of CDR3 VH from 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 are presented in SEQ ID NO: 53-59, respectively.

The amino acid sequence CDR3 VH of mutated antibodies 13A3 (i.e. TIM3.10-TIM3.12 is the same as that of the unmutated antibody 13A3, i.e. SEQ ID NO: 53. The amino acid sequence CDR3 VH of mutated antibodies 13A3 TIM3.13 and TIM3.18 is presented in SEQ ID NO: 126. The CDR3 VH amino acid sequence of mutated antibodies 13A3 TIM3.15 and TIM3.17 is presented in SEQ ID NO: 128. The CDR3 VH amino acid sequence of mutated antibodies 13A3 TIM3.14 and TIM3.16 is presented in SEQ ID NO: 127 and 129, respectively The amino acid sequence of the CDR3 VH of the mutated antibody 8B9 (i.e. TIM3.8) is the same as that of the unmutated antibody 8B9, i.e. SEQ ID NO: 54. The amino acid sequence of the CDR3 VH of the mutated antibody 9F6 (i.e. TIM3.7) is the same as the unmutated antibody 9F6, i.e. SEQ ID NO: 57. The amino acid sequences of CDR3 VH from 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 are presented in SEQ ID NO: 53-59, respectively. The amino acid sequences of CDR1 VL from 13A3, 8B9, 8C4, 17C3, 3G4 and 17C8 are presented in SEQ ID NO: 64. The amino acid sequences of CDR1 VL from 9F6 are presented in SEQ ID NO: 64 and 65. The amino acid sequences of CDR2 VL from 13A3, 8B9, 8C4 , 17C3, 3G4 and 17C8 are presented in SEQ ID NO: 66. The amino acid sequences of CDR2 VL from 9F6 are presented in SEQ ID NO: 66 and 67. The amino acid sequences of CDR3 VL from 13A3, 17C3 and 3G4 are presented in SEQ ID NO: 68. Amino acid sequences The VL CDR3s from 8B9, 8C4 and 17C8 are presented in SEQ ID NO: 69. The VL CDR3 amino acid sequences from 9F6 are presented in SEQ ID NOs: 69, 70 and 71. The VL CDR3 amino acid sequences of the mutated antibodies 13A3, 8B9 and 9F6 are the same as as for the corresponding unmutated antibodies. In fig. 13 provides a listing of the SEQ ID NOs for the CDRs of the anti-TIM3 antibodies described herein.

CDR regions are delineated using the Kabat system (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The Kabat system is the most common numbering system for a scheme called the EU index or EU numbering system, which is based on the sequential numbering of the first human IgG1 sequenced (the EU antibody; Edelman et al. 1969). Based on the Kabat numbering scheme disclosed herein, antibody numbering can be converted to other systems known in the art, such as the Chotia, IMGT, Martin (extended Chothia) or AHo numbering scheme.

Considering that each of these antibodies binds to human TIM3 and that antigen binding specificity is provided primarily by CDR1, 2 and 3 regions, CDR1, 2 and 3 VH sequences and CDR1, 2 and 3 VL sequences, such as those in FIG. 13 can be mixed and combined (i.e., CDRs from different antibodies can be mixed and combined, although each antibody must contain CDR1, 2 and 3 VH and CDR1, 2 and 3 VL) to create the other TIM3 binding molecules described in this document. TIM3 binding of such mixed and combination antibodies can be tested using the binding assays described above and in the Examples (eg, ELISA). In some embodiments, when VH CDR sequences are mixed and combined, the CDR1, CDR2, and/or CDR3 sequence from a particular VH sequence is replaced with structurally similar CDR sequence(s). Likewise, when VL CDR sequences are mixed and combined, the CDR1, CDR2 and/or CDR3 sequence from a particular VL sequence is replaced with structurally similar CDR sequence(s). It will be apparent to one skilled in the art that new VH and VL sequences can be created by replacing one or more sequences of the VH and/or VL CDR regions with structurally similar sequences from the CDR sequences described herein for monoclonal antibodies 13A3, 8B9, 8C4 , 17С3, 9F6, 3G4, 17С8 and any of TIM3.2-TIM3.18.

Provided herein are isolated anti-human TIM3 antibodies or antigen-binding portions thereof comprising:

(a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-45;

(b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-52 and 122-125;

(c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-59 and 126-129;

(d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 64-65;

(e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 66-67; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-71;

wherein the antibody specifically binds to human TIM3.

In one embodiment, an anti-human TIM3 antibody comprises heavy and light chain variable regions, wherein the CDR1, CDR2 and CDR3 regions of the heavy chain variable region comprise:

(a) SEQ ID NO: 41, 46, 53;

(b) SEQ ID NO: 42, 47, 54;

(c) SEQ ID NO: 43, 48, 55;

(d) SEQ ID NO: 44, 49, 56;

(e) SEQ ID NO: 45, 50, 57;

(f) SEQ ID NO: 45, 51, 58;

(g) SEQ ID NO: 45, 52, 59;

(h) SEQ ID NO: 41, 122, 53;

(i) SEQ ID NO: 41, 123, 53;

(j) SEQ ID NO: 41, 124, 53;

(k) SEQ ID NO: 41, 46, 126;

(l) SEQ ID NO: 41,46, 127;

(m) SEQ ID NO: 41, 46, 128;

(n) SEQ ID NO: 41, 46, 129;

(o) SEQ ID NO: 41, 122, 128 or (p) SEQ ID NO: 41, 122, 126;

wherein the antibody specifically binds to human TIM3.

In some embodiments, an anti-human TIM3 antibody comprises heavy and light chain variable regions, wherein the light chain variable region CDR1, CDR2, and CDR3 comprise (a) SEQ ID NO: 64, 66, 68;

- 37 044112 (b) SEQ ID NO: 64, 66, 69;

(c) SEQ ID NO: 65, 67, 70 or (d) SEQ ID NO: 64, 66,71;

wherein the antibody specifically binds to human TIM3.

In a specific embodiment, the anti-TIM3 antibody comprises heavy and light chain variable regions, wherein:

(a1) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 46, 53, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a2) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 122, 53, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a3) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 123, 53, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a4) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 124, 53, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a5) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 46, 126, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a6) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 46, 127, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a7) The heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 46, 128, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a8) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 46, 129, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a9) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 122, 128, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(a10) The heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 41, 122, 126, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(b1) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 42, 47, 54, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 69, respectively;

(b2) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 42, 125, 54, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 69, respectively;

(c) the heavy chain variable region CDR1, CDR2 and CDR3 comprise SEQ ID NO: 43, 48, 55, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 69, respectively;

(d) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 44, 49, 56, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively;

(e) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 45, 50, 57, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 69, respectively;

(f) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 45, 50, 57, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 71, respectively;

(g1) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 45, 50, 57, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 65, 67, 70, respectively;

(g2) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 45, 50, 57, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 71, respectively;

(g3) The heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 45, 50, 57, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66,

- 38 044112

69, respectively;

(h) the heavy chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 45, 51, 58, respectively, and the light chain variable region CDR1, CDR2 and CDR3 contain SEQ ID NO: 64, 66, 68, respectively; or (i) the heavy chain variable region CDR1, CDR2 and CDR3 comprise SEQ ID NO: 45, 52, 59, respectively, and the light chain variable region CDR1, CDR2 and CDR3 comprise SEQ ID NO: 64, 66, 69, respectively;

wherein the antibody specifically binds to human TIM3.

A VH domain or one or more of its CDRs described herein can be linked to a constant domain to form a heavy chain, eg, a full-length heavy chain. Similarly, a VL domain or one or more of its CDRs described herein can be linked to a constant domain to form a light chain, eg, a full-length light chain. The full-length heavy chain (except for the C-terminal lysine (K) or C-terminal glycine and lysine (GK), which may be missing) and the full-length light chain combine to form the full-length antibody.

The VH domain described herein can be fused to a human IgG constant domain, for example, IgG1, IgG2, IgG3 or IgG4, which are either naturally occurring or modified, for example, as further described herein. For example, the VH domain may comprise the amino acid sequence of any VH domain described herein fused to a human IgG, such as an IgG1 constant region, such as the following amino acid sequence of a wild-type human IgG1 constant domain:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL

HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK (SEQ ID NO: 291) or that of the allotype variant SEQ ID NO: 291 and has the following amino acid sequences:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP

KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL

HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK (SEQ ID NO: 277;

Allotype-specific amino acid residues are shown in bold and underlined).

The VH domain of an anti-TIM3 antibody may comprise the amino acid sequence of any VH domain described herein fused to a constant region without effector functions, for example, with the following amino acid sequences of a human IgG1 constant domain without effector functions:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP

KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVL

HQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY

PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK (SEQ ID NO: 294;

IgG1.If containing substitutions L234A, L235E, G237A, A330 and P33 IS, which are underlined) or

- 39 044112

HQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

QKSLSLSPGK (SEQ ID NO: 295;

IgG1.3f containing substitutions L234A, L235E and G237A, which are underlined).

For example, the allotype variant IgG1 contains K97R, D239E and/or L241M (underlined and in bold above) and numbered as in SEQ ID NOs: 277, 294 and 295. In the full-length heavy region, for example, 8C4 (SEQ ID NO : 3) and according to EU numbering these amino acid substitutions are numbered K214R, D356E and L358M. In some embodiments, the anti-TIM3 antibody constant region may further comprise one or more mutations or substitutions at amino acids L117, A118, G120, A213, and P214 (underlined above), as numbered in SEQ ID NOs: 277, 294, and 295, or L234, A235, G237, A330 and P331 according to EU numbering. In further embodiments, the constant region of an anti-TIM3 antibody contains one or more mutations or substitutions at amino acids L117A, A118E, G120A, A213S, and P214S of SEQ ID NO: 291 or L234A, L235E, G237A, A330S, and P331S according to EU numbering. The anti-TIM3 antibody constant region may also contain one or more mutations or substitutions L117A, A118E and G120A SEQ ID NO: 291 or L234A, L235E and G237A according to EU numbering

Alternatively, the VH domain of an anti-TIM3 antibody may comprise the amino acid sequence of any VH domain described herein fused to a human IgG4 constant region, such as the following human IgG4 amino acid sequence or variants thereof:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS

LSSWWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPK

DTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQD WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKS LSLSLGK (SEQ ID NO: 292 containing S228P).

The VL domain described herein may be fused to a human kappa or lambda light chain constant domain. For example, the VL domain of an anti-TIM3 antibody may comprise the amino acid sequence of any VL domain described herein fused to the following human IgG1 kappa light chain amino acid sequence:

RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS

TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 278)

In some embodiments, the heavy chain constant region contains a lysine or other amino acid at the C-terminus, for example, it contains the following last amino acids: LSPGK (SEQ ID NO: 279) in the heavy chain. In some embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus and contains, for example, the C-terminal sequence LSPG (SEQ ID NO: 280) or LSP (SEQ ID NO: 281).

The amino acid sequences of the exemplary heavy and light chains correspond to SEQ ID NOs: 1-28, 72-111, 301-354 for the heavy chains and SEQ ID NOs: 29-30 and 32-33 for the light chains.

The present invention relates to isolated antibodies to human TIM3 or antigen-binding portions thereof, comprising:

(a1) heavy and light chain sequences containing SEQ ID NO: 301 (or 302) and 29, respectively;

(a2) heavy and light chain sequences containing SEQ ID NO: 1 (or 8) and 29, respectively;

(a3) heavy and light chain sequences containing SEQ ID NO: 15 (or 22) and 29, respectively;

(a4) heavy and light chain sequences containing SEQ ID NO: 303 (or 304) and 29, respectively;

(a5) heavy and light chain sequences containing SEQ ID NO: 72 (or 82) and 29, respectively;

(a6) heavy and light chain sequences containing SEQ ID NO: 92 (or 102) and 29, respectively;

(a7) heavy and light chain sequences containing SEQ ID NO: 305 (or 306) and 29, respectively;

- 40 044112 (a8) heavy and light chain sequences containing SEQ ID NO: 73 (or 83) and 29, respectively;

(a9) heavy and light chain sequences containing SEQ ID NO: 93 (or 103) and 29, respectively;

(a10) heavy and light chain sequences containing SEQ ID NO: 307 (or 308) and 29, respectively;

(a11) heavy and light chain sequences containing SEQ ID NO: 74 (or 84) and 29, respectively;

(a12) heavy and light chain sequences containing SEQ ID NO: 94 (or 104) and 29, respectively;

(a13) heavy and light chain sequences containing SEQ ID NO: 309 (or 310) and 29, respectively;

(a14) heavy and light chain sequences containing SEQ ID NO: 75 (or 85) and 29, respectively;

(a15) heavy and light chain sequences containing SEQ ID NO: 95 (or 105) and 29, respectively;

(a16) heavy and light chain sequences containing SEQ ID NO: 311 (or 312) and 29, respectively;

(a17) heavy and light chain sequences containing SEQ ID NO: 76 (or 86) and 29, respectively;

(a18) heavy and light chain sequences containing SEQ ID NO: 96 (or 106) and 29, respectively;

(a19) heavy and light chain sequences containing SEQ ID NO: 313 (or 314) and 29, respectively;

(a20) heavy and light chain sequences containing SEQ ID NO: 77 (or 87) and 29, respectively;

(a21) heavy and light chain sequences containing SEQ ID NO: 97 (or 107) and 29, respectively;

(a22) heavy and light chain sequences containing SEQ ID NO: 315 (or 316) and 29, respectively;

(a23) heavy and light chain sequences containing SEQ ID NO: 78 (or 88) and 29, respectively;

(a24) heavy and light chain sequences containing SEQ ID NO: 98 (or 108) and 29, respectively;

(a25) heavy and light chain sequences containing SEQ ID NO: 317 (or 318) and 29, respectively;

(a26) heavy and light chain sequences containing SEQ ID NO: 79 (or 89) and 29, respectively;

(a27) heavy and light chain sequences containing SEQ ID NO: 99 (or 109) and 29, respectively;

(a28) heavy and light chain sequences containing SEQ ID NO: 319 (or 320) and 29, respectively;

(a29) heavy and light chain sequences containing SEQ ID NO: 349 (or 350) and 29, respectively;

(a30) heavy and light chain sequences containing SEQ ID NO: 351 (or 352) and 29, respectively;

(a31) heavy and light chain sequences containing SEQ ID NO: 353 (or 354) and 29, respectively;

(b1) heavy and light chain sequences containing SEQ ID NO: 321 (or 322) and 30, respectively;

(b2) heavy and light chain sequences containing SEQ ID NO: 2 (or 9) and 30, respectively;

(b3) heavy and light chain sequences containing SEQ ID NO: 16 (or 23) and 30, respectively;

(b4) heavy and light chain sequences containing SEQ ID NO: 323 (or 324) and 30, respectively;

(b5) heavy and light chain sequences containing SEQ ID NO: 80 (or 90) and 30, respectively;

(b6) heavy and light chain sequences containing SEQ ID NO: 100 (or 110) and 30, respectively;

(b7) heavy and light chain sequences containing SEQ ID NO: 325 (or 326) and 30, respectively;

- 41 044112 (c1) heavy and light chain sequences containing SEQ ID NO: 327 (or 328) and 30, respectively;

(c2) heavy and light chain sequences containing SEQ ID NO: 3 (or 10) and 30, respectively;

(c3) heavy and light chain sequences containing SEQ ID NO: 17 (or 24) and 30, respectively;

(c4) heavy and light chain sequences containing SEQ ID NO: 329 (or 330) and 30, respectively;

(d1) heavy and light chain sequences containing SEQ ID NO: 331 (or 332) and 29, respectively;

(d2) heavy and light chain sequences containing SEQ ID NO: 4 (or 11) and 29, respectively;

(d3) heavy and light chain sequences containing SEQ ID NO: 18 (or 25) and 29, respectively;

(d4) heavy and light chain sequences containing SEQ ID NO: 333 (or 334) and 29, respectively;

(e1.1) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 32, respectively;

(e1.2) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 33, respectively;

(e1.3) heavy and light chain sequences containing SEQ ID NO: 335 (or 336) and 31, respectively;

(e2) heavy and light chain sequences containing SEQ ID NO: 5 (or 12) and 33, respectively;

(e3) heavy and light chain sequences containing SEQ ID NO: 19 (or 26) and 33, respectively;

(e4) heavy and light chain sequences containing SEQ ID NO: 337 (or 338) and 33, respectively;

(e5) heavy and light chain sequences containing SEQ ID NO: 81 (or 91) and 33, respectively;

(e6) heavy and light chain sequences containing SEQ ID NO: 101 (or 111) and 33, respectively;

(e7) heavy and light chain sequences containing SEQ ID NO: 339 (or 340) and 33, respectively;

(f1) heavy and light chain sequences containing SEQ ID NO: 341 (or 342) and 29, respectively;

(f2) heavy and light chain sequences containing SEQ ID NO: 6 (or 13) and 29, respectively;

(f3) heavy and light chain sequences containing SEQ ID NO: 20 (or 27) and 29, respectively;

(f4) heavy and light chain sequences containing SEQ ID NO: 343 (or 344) and 29, respectively;

(g1) heavy and light chain sequences containing SEQ ID NO: 345 (or 346) and 30, respectively;

(g2) heavy and light chain sequences containing SEQ ID NO: 7 (or 14) and 30, respectively;

(g3) heavy and light chain sequences containing SEQ ID NO: 21 (or 28) and 30, respectively, or (g4) heavy and light chain sequences containing SEQ ID NO: 347 (or 348) and 30, respectively;

wherein the antibody specifically binds to human TIM3.

In certain embodiments, an anti-TIM3 antibody comprises a combination of heavy and light chain sequences set forth herein, such as in the preceding paragraph, wherein the antibody comprises two heavy chains and two light chains and may further comprise at least one disulfide bond linking the two heavy chains. chains together. Antibodies may also contain disulfide bonds linking each of the light chains to each of the heavy chains.

Heavy and light chains containing an amino acid sequence that is at least 99, 98, 97, 96, 95, 90, 85, 80, 75 or 70% identical to any of the heavy or light chains (or variable regions thereof) provided herein ), for example, SEQ ID NOs: 1-33, 72-111 and 301-354, can be used to generate antibodies to human TIM3 having the desired characteristics, such as those further described herein. Exemplary variants are those comprising an allotypic variation, for example, in a constant domain, and/or a mutation in a variable or constant region, such as mutations disclosed herein. Heavy and light chains containing an amino acid sequence that differs by a maximum of 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, or 1 amino acid (by substitution, addition or deletion) from any of the heavy or light chains provided herein (or variable regions thereof) can be used to generate antibodies to human TIM3 having the desired characteristics, such as those further described herein document.

In various embodiments, the antibodies described above exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more than, twelve or more, thirteen or more, fourteen or more, fifteen or more, or all of the following functional properties:

(1) binding to soluble human TIM3, for example, with a K _D of 10 nM or less (for example, from 0.01 nM to 10 nM), for example, as measured by Biacore, for example, as described in the examples;

(2) binding to soluble cynomolgus TIM3, eg, with a K _D of 100 nM or less (eg, 0.01 nM to 100 nM), eg, as measured by Biacore, eg, as described in the Examples;

(3) binding to membrane-bound human TIM3, e.g., with an EC ₅₀ of 1 μg/ml or less (e.g., 0.01 μg/ml to 1 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(4) binding to membrane-bound human TIM3, eg, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(5) binding to membrane-bound cynomolgus TIM3, e.g., with an EC ₅₀ of 20 μg/ml or less (e.g., 0.01 μg/ml to 20 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(6) binding to membrane-bound cynomolgus TIM3, for example, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), for example, as measured by the Scatchard assay, for example, as described in the examples;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) enhancing the proliferation of TM3-expressing T cells (eg, Th1 cells or TILs), for example, as described in the examples;

(8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, for example, as described in the Examples;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay, for example, as described in the Examples;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297), for example, as described in the examples;

(12) reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111 , Q113, G116, M118 and D120 (as numbered in SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3, for example, as described in the examples;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17 and TIM3.18, for example, as described in the examples;

(14) binding to human TIM3 regions 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ WQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS, for example, as described in the Examples;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (for example, described in the examples; numbering according to SEQ ID NO: 286 (Fig. 20)); and/or (16) (a) the presence of reduced binding to human TIM3, in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120 and optional, D104 and Q113 (numbered by SEQ ID NO:

- 43 044112

286 (Fig. 20)) is replaced with a different amino acid relative to binding to wild-type human TIM3; (b) binding to ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ^m QIPGIMNDEKFNLKL ¹²⁷ (SEQ ID

NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373), as determined by HDX-MS, as described in the examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

Such antibodies include, for example, human antibodies, humanized antibodies or chimeric antibodies.

In one embodiment, anti-TIM3 antibodies described herein bind to a conformational epitope.

In one embodiment, anti-TIM3 antibodies described herein bind to amino acid residues within the following region of the extracellular domain of mature human TIM3 (SEQ ID NO: 290):

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWLN

GDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMND (SEQ ID NO: 299),

Corresponding to amino acid residues 1-99 of the extracellular domain of mature human TIM3 (SEQ ID NO: 299) or amino acid residues 22-120 of human TIM3, characterized by the sequence SEQ ID NO: 286.

In one embodiment, anti-TIM3 antibodies described herein bind to amino acid residues within the following region of the human TIM3 mature extracellular domain (SEQ ID NO: 290): CPVFECG (SEQ ID NO: 296), corresponding to amino acid residues 37-43 of the mature extracellular domain Human TIM3 (SEQ ID NO: 290).

In one embodiment, anti-TIM3 antibodies described herein bind to amino acid residues in the following region of the extracellular domain of human mature TIM3 (SEQ ID NO: 290): WTSRYWLNGDFR (SEQ ID NO: 297), corresponding to amino acid residues 57-83 of the extracellular domain of mature TIM3 human (SEQ ID NO: 290).

In one embodiment, anti-TIM3 antibodies described herein bind to amino acid residues in the following region of the extracellular domain of human mature TIM3 (SEQ ID NO: 290): RIQIPGIMND (SEQ ID NO: 298), corresponding to amino acid residues 90-99 of the extracellular domain of mature TIM3 human (SEQ ID NO: 290).

In one embodiment, anti-TIM3 antibodies have the same binding profile to wild-type and mutated human TIM3 as one or more of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, and TIM3.2-TIM3.18. In one embodiment, the anti-TIM3 antibody binds to amino acid residues in the following regions of the extracellular domain of mature human TIM3 (SEQ ID NO: 290): CPVFECG (SEQ ID NO: 296), WTSRYWLNGDFRKGDVSLTIENVTLAD (SEQ ID NO: 297) and/or RIQIPGIMND (SEQ ID NO: 298).

In some embodiments, the anti-TIM3 antibody binds to (1) ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ^m RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) or (2) 40YTPAAPGNLVPVCWGKGACPVFE ⁶² (SEQ ID NO: 369), 66WLRTDERDVNY ⁷⁷ ( SEQ ID NO: 370), 78WTSRYWLNGDFRKGDVSL ⁹⁵ (SEQ ID NO: 371), ¹¹⁰ CRIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 372) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373), as determined, for example, by HDX-MS, as described, for example, in the examples. In some embodiments, an anti-TIM3 antibody interacts with regions of amino acid residues 40-62 and 111-127 of hTIM3, but does not significantly interact with other regions, such as the region that is N-terminal to amino acid residue Y40, a region that is located between amino acid residues E62 and R111, and a region that is C-terminal to amino acid residue L127, as determined by HDX-MS, as described, for example, in the examples.

In some embodiments, an anti-TIM3 antibody has reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118 and D120 (as numbered in SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3 and the antibody binds to (1) ⁴⁹ VPVCWGKGACPVFE ⁶² ( SEQ ID NO: 367) and ¹¹¹ PJQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) or (2) ⁴⁰ YTPAAPGNLVPVCWGKGACPVFE ⁶² (SEQ ID NO: 369), 66WLRTDERDVNY ⁷⁷ (SEQ ID NO: 370), 78WTSRYWLNGDFRKGDVSL ⁹⁵ (SEQ ID NO: 371 ), ¹¹⁰ CRIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 372) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373), as determined by FTOX-MS, as described, for example, in the Examples.

In some embodiments, the anti-TIM3 antibody has the same binding profile to wild-type and mutated human TIM3 as TIM3.18.IgG1.3 or 13A3, i.e. antibody:

(i) binds to (1) ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373) and, for example, but does not significantly bind to ( a) peptides containing sequences located at the N-terminus of the amino acid residue

- 44 044112

49; (b) peptides containing sequences located between amino acid residue and 111 (for example, 78WTSRYWLNGDFRKGDVSL ⁹⁵ (SEQ ID NO: 371)); and (c) peptides containing sequences that are located C-terminal to amino acid residue 127, as determined by FTOX-MS (eg, as described in the examples);

(ii) does not bind to human TIM3 or has significantly reduced binding to human TIM3 having one or more of the following amino acid mutations, as determined, for example, using a yeast surface display method (for example, as described in the Examples): C58, P59, F61, E62, C63, R111, D120, and optionally D104 and Q113 (SEQ ID NO: 286 (FIG. 20)); and/or (iii) contains heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (e.g., as described in the examples; numbering according to SEQ ID NO: 286 (Fig. 20)).

In some embodiments, an anti-TIM3 antibody comprises a heavy chain and a light chain, wherein the heavy chain is selected from the group consisting of SEQ ID NOs: 72-111, 305-354, 325-326, and 339-340, and/or the light chain is selected from group consisting of SEQ ID NO: 29-33.

As further discussed herein, the heavy chain constant region of the anti-TIM3 antibodies described herein can be of any isotype, for example, IgG1, IgG2, IgG3 and IgG4, or combinations thereof and/or modifications thereof. An anti-TIM3 antibody may have effector function or may have reduced or absent effector function. In certain embodiments, anti-TIM3 antibodies comprise a modified heavy chain constant region that provides improved properties of the antibody.

In some embodiments, an anti-TIM3 antibody comprises a heavy chain and a light chain, wherein the heavy chain is selected from the group consisting of SEQ ID NOs: 72-111 and 349-352, and/or the light chain is selected from the group consisting of SEQ ID NOs: 29-33.

III. Antibodies characterized by the presence of specific germline sequences.

In some embodiments, the anti-TIM3 antibody comprises a heavy chain variable region from a particular germline immunoglobulin heavy chain gene and/or a light chain variable region from a particular germline immunoglobulin light chain gene.

As shown herein, human antibodies specific for TIM3 have been produced that contain a heavy chain variable region that is the product of the VH 4-39 gene, the VH 4-59 gene, the VH 1-46 gene, the VH 3-11 gene, human germline VH 4-17 gene, VH 3-10 gene, VH 6-19 gene, VH 613 gene, VH JH5b gene and/or VH JH6b gene. Accordingly, provided herein are isolated monoclonal antibodies or antigen binding portions thereof comprising a heavy chain variable region that is the product of a human germline VH gene selected from the group consisting of: VH 4-39, VH 4-59, VH 1- 46, VH 3-11, VH 4-17, VH 3-10, VH6-19, VH 6-13, VH JH5b, VH JH6b and any combination thereof, or derived from it.

Human TIM3-specific antibodies have been prepared that contain a light chain variable region that is the product of or derived from the human germline VK A27 gene, the JK5 VK gene, the JK4 VK gene, the L18 VK gene, and/or the human germline VK JK1 gene. Accordingly, provided herein are isolated monoclonal antibodies or antigen binding portions thereof comprising a light chain variable region that is the product of a human germline VK gene selected from the group consisting of: VK A27, VK JK5, VK JK4, VK L18, VK JK1 and any combination thereof, or derived from it.

Anti-TIM3 antibodies described herein include antibodies containing a heavy chain variable region that is the product of or derived from one of the above human germline VH genes, and also containing a light chain variable region that is the product of one of of the above human germline VK genes or derived from it, as shown in the figures.

As used herein, a human antibody contains heavy and light chain variable regions that are the product of or derived from a particular germline sequence if the antibody variable regions are derived from a system that uses human germline immunoglobulin genes. Such systems involve immunizing a transgenic mouse carrying human immunoglobulin genes with an antigen of interest or screening a library of human immunoglobulin genes presented on a phage with an antigen of interest. A human antibody that is the product of or derived from a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence. identity (i.e., highest % identity) to the human antibody sequence. A human antibody that is the product of or derived from a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to naturally occurring somatic mutations or the deliberate introduction of site-directed mutations. However, the selected human antibody is typically at least 90% identical in amino acid sequence to the amino acid sequence encoded by the human germline immunoglobulin gene, and contains amino acid residues that identify the human antibody as human compared to the germline immunoglobulin amino acid sequences of other species ( e.g. mouse germline sequences). In certain cases, a human antibody may be at least 95%, or even at least 96, 97, 98, or 99% identical in amino acid sequence to the amino acid sequence encoded by a germline immunoglobulin gene. Typically, a human antibody derived from a specific human germline sequence will exhibit no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, a human antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by a germline immunoglobulin gene.

IV. Homologous antibodies.

Covered herein are antibodies comprising heavy and light chain variable regions containing amino acid sequences that are homologous to the amino acid sequences of the anti-TIM3 antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-TIM3 antibodies described herein.

For example, an isolated anti-TIM3 antibody or antigen-binding portion thereof may comprise a heavy chain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region contains an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 34-40, 112-121 and 364, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 14, 1-5, 1-10, 1-15, 1-20, 1-25 or 1- 50 amino acid changes (i.e., amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-40, 112-121 and 364, wherein the heavy chain variable region optionally comprises CDR sequences of one of anti-TIM3 antibodies described herein;

(b) the light chain variable region contains an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 60-63 or containing 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 amino acid changes (t ie, amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 60-63, wherein the light chain variable region optionally comprises CDR sequences of one of the anti-TIM3 antibodies described herein;

(c) the antibody specifically binds to human TIM3 and (d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the following functional properties:

(1) binding to soluble human TIM3, for example, with a K _D of 10 nM or less (for example, from 0.01 nM to 10 nM), for example, as measured by Biacore, for example, as described in the examples;

(2) binding to soluble cynomolgus TIM3, eg, with a K _D of 100 nM or less (eg, 0.01 nM to 100 nM), eg, as measured by Biacore, eg, as described in the Examples;

(3) binding to membrane-bound human TIM3, e.g., with an EC ₅₀ of 1 μg/ml or less (e.g., 0.01 μg/ml to 1 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(4) binding to membrane-bound human TIM3, eg, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(5) binding to membrane-bound cynomolgus TIM3, e.g., with an EC ₅₀ of 20 μg/ml or less (e.g., 0.01 μg/ml to 20 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(6) binding to membrane-bound cynomolgus TIM3, for example, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), for example, as measured by the Scatchard assay, for example, as described in the examples;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) increased proliferation of ex-

- 46 044112 TIM3-pressing T cells (eg Thl cells or TILs), for example, as described in the examples;

(8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, for example, as described in the Examples;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay, for example, as described in the Examples;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297), for example, as described in the examples;

(12) the presence of reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118 and D120 (as numbered in SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3, for example, as described in the examples;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17 and TIM3.18, for example, as described in the examples;

(14) binding to human TIM3 regions ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS, for example, as described in the Examples;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (e.g., described in examples; SEQ ID NO: 286 (Fig. 20)), and/or (16) (a) the presence of reduced binding to human TIM3, in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120 and optionally D104 and Q113 (SEQ ID NO: 286 (FIG. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3; (b) binding to ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ QIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373) as determined by HDX-MS as described in the Examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

In various embodiments, an antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the functional properties indicated by ( 1) - (16) above. The antibody may be, for example, a human antibody, a humanized antibody, or a chimeric antibody.

An isolated anti-TIM3 antibody or an antigen-binding portion thereof may comprise a heavy chain and a light chain, wherein:

(a) the heavy chain contains an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-28, 72- 111 and 349-352, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 amino acid changes (i.e., amino acid substitutions, additions, or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 128, 72-111, and 349-352, provided that, in some embodiments, if the sequence is a heavy chain sequence without effector functions, mutations resulting in a heavy chain without effector functions are not modified (eg, no modifications are made to R214, A234, E235, A237, S330 and S331) for IgG1.1 constant regions and no modifications are made to R214, A234 and E235 for the IgG1.3 constant regions, optionally the heavy chain variable region comprising the CDR sequences of one of the anti-TIM3 antibodies described herein;

(b) the light chain contains an amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 29-31, or contains 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 amino acid changes (i.e. e. amino acid substitutions, additions or deletions) relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 29-31, wherein the light chain variable region optionally comprises a CDR sequence of one of the anti-TIM3 antibodies described herein;

(c) the antibody specifically binds to human TIM3 and (d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or all of the following functionalities

- 47 044112 new properties:

(1) binding to soluble human TIM3, for example, with a K _D of 10 nM or less (for example, from 0.01 nM to 10 nM), for example, as measured by Biacore, for example, as described in the examples;

(2) binding to soluble cynomolgus TIM3, eg, with a K _D of 100 nM or less (eg, 0.01 nM to 100 nM), eg, as measured by Biacore, eg, as described in the Examples;

(3) binding to membrane-bound human TIM3, e.g., with an EC ₅₀ of 1 μg/ml or less (e.g., 0.01 μg/ml to 1 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(4) binding to membrane-bound human TIM3, eg, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(5) binding to membrane-bound cynomolgus TIM3, e.g., with an EC ₅₀ of 20 μg/ml or less (e.g., 0.01 to 20 μg/ml), e.g., as measured by flow cytometry (e.g., as described in the Examples) ;

(6) binding to membrane-bound cynomolgus TIM3, eg, with a K _D of 1 nM or less (eg, 0.01 to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) enhancing the proliferation of TIM3-expressing T cells (eg, Th1 cells or TILs), for example, as described in the examples;

(8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, for example, as described in the Examples;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay, for example, as described in the Examples;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297), for example, as described in the examples;

(12) the presence of reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118 and D120 (as numbered in SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3, for example, as described in the examples;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17 and TIM3.18, for example, as described in the examples;

(14) binding to human TIM3 regions 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS, for example, as described in the Examples;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (e.g., described in examples; SEQ ID NO: 286 (Fig. 20)), and/or (16) (a) the presence of reduced binding to human TIM3, in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120 and optionally D104 and Q113 (SEQ ID NO: 286 (FIG. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3; (b) binding to 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ QIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373) as determined by HDX-MS as described in the Examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

Also provided are anti-TIM3 antibodies containing CDR1 VH, CDR2 VH, CDR3 VH, CDR1 VL, CDR2 VL and/or CDR3 VL that are different from the corresponding CDRs 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3. 2-TIM3.18 by 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 amino acid changes (i.e. amino acid substitutions, additions or deletions). In some embodiments, the anti-TIM3 antibody contains 1-5 amino acid changes in each of the 1, 2, 3, 4, 5, or 6 CDRs relative to the corresponding sequence in 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3. 2 - TIM3.18. In some embodiments, the anti-TIM3 antibody comprises a total of

- 48 044112 complexity of 1-5 amino acid changes in all CDRs relative to CDRs in 13A3, 3G4, 17C3, 17C8,

9F6, 8B9, 8C4 or any of TIM3.2-TIM3.18.

In some embodiments, an anti-TIM3 antibody comprises VH and VL CDRs consisting of 13A3 amino acids, wherein one or more amino acids in one or more CDRs are those of one of the other anti-TIM3 antibodies disclosed herein.

For example, in some embodiments, an anti-TIM3 antibody comprises a CDR1 VH containing one or more amino acid modifications to SRSYYWG (SEQ ID NO: 41), and may contain, for example, the following degenerate sequence: X ₁ X ₂ X ₃ X ₄ YX ₅ X ₆ (SEQ ID NO: 282), where X ₁ represents any amino acid, for example, S or the absence thereof; X ₃ represents any amino acid, for example, R or the absence thereof; X ₃ is any amino acid, for example S, R or D; X4 is any amino acid, for example Y or H; X ₅ is any amino acid, for example W or M, and X ₆ is any amino acid, for example G, N, S or H.

In some embodiments, the anti-TIM3 antibody comprises a CDR2 VH containing one or more amino acid modifications to SIYYSGFTYYNPSLKS (SEQ ID NO: 46) and may contain, for example, the following degenerate sequence: X ₁ IX ₂ X ₃ X ₄ GX ₅ X ₆ X ₇ X ₈ YX ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ (SEQ ID NO: 283), where X ₁ represents any amino acid, for example, S, Y, I or F; X ₂ represents any amino acid, for example, Y, H, N or S; X ₃ is any amino acid, for example Y, P, G, T or S; X4 is any amino acid, for example S, T, R or G; X ₅ represents any amino acid, for example F, S or D; X ₆ is any amino acid, for example S, T or I; X ₇ represents any amino acid, for example, I or the absence thereof; X ₈ represents any amino acid, for example, Y, N or I; X9 is any amino acid, for example N, Q, S or A; X ₁₀ is any amino acid, for example P, S, Q or D; X _p is any amino acid, for example S or K; X ₁₂ is any amino acid, for example L, F or V; X ₁₃ is any amino acid, such as K or Q, and X ₁₄ is any amino acid, such as S or G.

In some embodiments, the anti-TIM3 antibody comprises a CDR3 VH containing one or more amino acid modifications to GGPYGDYAHWFDP (SEQ ID NO: 53), and may contain, for example, the following degenerate sequence:

X1IX2X3X4GX5X6X7X8YX9X10X11X12X13X14X15X16X17X18 (SEQ ID NO: 284), where X ₁ represents any amino acid, for example, D, E or the absence thereof; X2 is any amino acid, for example F, G or lack thereof; X3 is any amino acid, for example Y or the absence thereof; X4 is any amino acid, for example G, S or lack thereof; X ₅ represents any amino acid, for example G, T or S; X ₆ is any amino acid, for example G or S; X7 is any amino acid, for example N, W or lack thereof; X8 is any amino acid, for example Y, S, E or the absence thereof; X ₉ represents any amino acid, for example, Y or the absence thereof; X ₁₀ is any amino acid, for example P or Y; X _n represents any amino acid, for example, D or the absence thereof; X ₁₂ represents any amino acid, for example, Y or the absence thereof; X ₁₃ represents any amino acid, for example A or the absence thereof; X ₁₄ represents any amino acid, for example, H or the absence thereof; X ₁₅ represents any amino acid, for example, W or lack thereof; X ₁₆ represents any amino acid, for example F or M; X ₁₇ is any amino acid, for example D or E, and X ₁₈ is any amino acid, for example P, I, V, Y or L.

In some embodiments, the anti-TIM3 antibody comprises a CDR3 VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-59 and 126-129.

In some embodiments, the anti-TIM3 antibody comprises a CDR1 VL comprising the amino acid sequence set forth in SEQ ID NO: 64 or SEQ ID NO: 65.

In some embodiments, the anti-TIM3 antibody comprises a CDR2 VL comprising the amino acid sequence set forth in SEQ ID NO: 66 or SEQ ID NO: 67.

In some embodiments, the anti-TIM3 antibody comprises a CDR3 VL containing one or more amino acid modifications to QQYGSSPIT (SEQ ID NO: 68), and may contain, for example, the following degenerate sequence: QQX ₁ X ₂ SX ₃ X ₄ X ₅ T (SEQ ID NO: 285), where X ₁ represents any amino acid, for example, F or Y; X ₂ represents any amino acid, for example N or G; X3 is any amino acid, for example Y or S; X4 is any amino acid, for example P or the absence thereof; X ₅ is any amino acid, such as I, R or L.

Antibodies having sequence homology to those of 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 8C4 or any of TIM3.2-TIM3.18, for example, the VH and VL regions in SEQ ID NO: 34-40, 112 -121 or 364 and SEQ ID NO: 60-63, respectively, or the heavy and light chains of SEQ ID NO: 1-28, 72-111 or 349-352 and SEQ ID NO: 29-33, respectively, or CDR, may be obtained by mutagenesis (for example, site-49 044112 site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding SEQ

ID NO: 167-173 and/or SEQ ID NO: 193-196 or SEQ ID NO: 134-161 and/or SEQ ID NO: 162-166, followed by testing the encoded altered antibody for retained function (i.e.

functions presented in (1) - (16) above) using the functional assays described herein.

V. Antibodies with conservative modifications.

Antibodies to TIM3 may contain a heavy chain variable region containing the sequences CDR1, CDR2 and CDR3, and a light chain variable region containing the sequences CDR1, CDR2 and CDR3, wherein one or more of these CDR sequences contain certain amino acid sequences based on those described herein document antibodies to TIM3 (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2-TIM3.18), or conservative modifications thereof, and wherein the antibodies retain the desired functional properties of the antibodies described herein to TIM3. Accordingly, an isolated anti-TIM3 antibody or antigen-binding portion thereof may comprise a heavy chain variable region comprising the sequences CDR1, CDR2 and CDR3 and a light chain variable region comprising the sequences CDR1, CDR2 and CDR3, wherein:

(a) the CDR3 sequence of the heavy chain variable region contains an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NO: 53-59 and 126-129 and conservative modifications thereof, for example, 1, 2, 3, 4, 5, 1 -2, 1-3, 1-4 or 1-5 conservative amino acid substitutions, wherein the heavy chain variable region optionally comprises the CDR sequences of one of the anti-TIM3 antibodies described herein;

(b) the light chain variable region CDR3 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequence SEQ ID NO: 68-71 and conservative modifications thereof, for example, 1, 2, 3, 4, 5, 1-2, 1 -3, 1-4 or 1-5 conservative amino acid substitutions, wherein the light chain variable region optionally contains CDR sequences of one of the anti-TIM3 antibodies described herein;

(c) the antibody specifically binds to human TIM3 and (d) the antibody exhibits 1, 2, 3, 4, 5, 6, 7, 8, 9, or all of the following:

(1) binding to soluble human TIM3, for example, with a K _D of 10 nM or less (for example, from 0.01 nM to 10 nM), for example, as measured by Biacore, for example, as described in the examples;

(2) binding to soluble cynomolgus TIM3, eg, with a K _D of 100 nM or less (eg, 0.01 nM to 100 nM), eg, as measured by Biacore, eg, as described in the Examples;

(3) binding to membrane-bound human TIM3, e.g., with an EC ₅₀ of 1 μg/ml or less (e.g., 0.01 μg/ml to 1 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(4) binding to membrane-bound human TIM3, eg, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(5) binding to membrane-bound cynomolgus TIM3, e.g., with an EC ₅₀ of 20 μg/ml or less (e.g., 0.01 μg/ml to 20 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(6) binding to membrane-bound cynomolgus TIM3, for example, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), for example, as measured by the Scatchard assay, for example, as described in the examples;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) enhancing the proliferation of TIM3-expressing T cells (eg, Th1 cells or TILs), for example, as described in the examples;

(8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, for example, as described in the Examples;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay, for example, as described in the Examples;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297), for example, as described in the examples;

(12) the presence of reduced binding to human TIM3, in which one or more amino acids L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113, G116, M118 and D120 (as numbered in SEQ ID NO: 286 (FIG. 20)) are replaced by another amino acid

- 50 044112 regarding binding to wild-type human TIM3, for example, as described in the examples;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8,

TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17 and TIM3.18, for example, as described in the examples;

(14) binding to human TIM3 regions ⁴⁹ VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS, for example, as described in the Examples;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (e.g., described in examples; SEQ ID NO: 286 (Fig. 20)), and/or (16) (a) the presence of reduced binding to human TIM3, in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120 and optionally D104 and Q113 (SEQ ID NO: 286 (FIG. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3; (b) binding to 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ⁿ¹ QIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and 119NDEKFNLKL ¹²⁷ (SEQ ID NO: 373), as determined by HDX-MS, as described in the examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

In one embodiment, the heavy chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NOs: 46-52 and 122-125 and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions; and the light chain variable region CDR2 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 66-67 and conservative modifications thereof, for example, 1, 2, 3, 4, 5, 1-2, 1-3 , 1-4 or 1-5 conservative amino acid substitutions.

In another embodiment, the heavy chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 41-45 and conservative modifications thereof, e.g., 1, 2, 3, 4, 5, 1-2, 1-3, 1-4 or 1-5 conservative amino acid substitutions; and the light chain variable region CDR1 sequence comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 64-65 and conservative modifications thereof, for example, 1, 2, 3, 4, 5, 1-2, 1-3 , 1-4 or 1-5 conservative amino acid substitutions.

In various embodiments, an antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the functional properties represented by ( 1) - (16) above. Such antibodies may be, for example, human antibodies, humanized antibodies or chimeric antibodies.

Conservative amino acid substitutions can also be made in or in addition to portions of antibodies other than the CDR. For example, conservative amino acid modifications can be made in the framework region or in the Fc region. The variable region or heavy or light chain may contain 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25 or 1-50 conservative amino acid substitutions relative to the sequences of the anti-TIM3 antibodies provided herein. In certain embodiments, an anti-TIM3 antibody comprises a combination of a conservative and a non-conservative amino acid modification.

VI. Antibodies that bind to the same epitope or compete for binding.

Also provided are antibodies that compete for binding to human TIM3 with one or more specific anti-TIM3 antibodies described herein (e.g., antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 and/or TIM3.2-TIM3.18 ). Such competitive antibodies can be identified based on their ability to competitively inhibit the binding to human TIM3 of one or more monoclonal antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 and/or TIM3.2-TIM3.18 in standard TIM3 binding assays. For example, standard ELISA assays or competitive ELISA assays can be used in which recombinant human TIM3 protein is immobilized on a plate, various concentrations of unlabeled first antibody are added, the plate is washed, labeled second antibody is added, and the amount of label is measured. If increasing concentrations of unlabeled (first) antibody (also called blocking antibody) inhibit the binding of labeled (second) antibody, the first antibody is said to inhibit the binding of the second antibody to the target on the plate, or is said to compete for binding with the second antibody. Additionally or alternatively, the Biacore assay can be used to assess the ability of antibodies to compete. The ability of the test antibody to inhibit binding of the anti-TIM3 antibody described herein to TIM3 demonstrates that the test antibody can compete with the antibody for binding to human TIM3.

Accordingly, provided herein are anti-TIM3 antibodies that inhibit the binding of anti-TIM3 antibodies described herein to TIM3 on cells, e.g., activated T cells, by at least 50, 55, 60, 65, 70, 75, 80. 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% and/or whose binding to human TIM3 on cells, e.g. activated T cells, is inhibited by at least 50, 55 , 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, for example, as measured by ELISA or FACS, for example, using an assay described in the next paragraph.

An exemplary competition experiment to determine, for example, whether a first antibody blocks the binding of (i.e., competes with) a second antibody can be performed as described in the Examples or as follows: Activated human T cells are prepared as follows: peripheral blood mononuclear cells (PBMC) are isolated from human whole blood using a Ficoll gradient and activated with 10 μg/ml phytohemagglutinin (PHA-L) (USBiol # P3370-30) and 200 IU/ml recombinant IL-2 (Peprotech # 200-02) in within 3 days. Activated T cells are resuspended in FACS buffer (PBS with 5% fetal bovine serum) and seeded at 105 sample cells per well in a 96-well plate. The plate is placed on ice followed by the addition of unconjugated first antibody at concentrations ranging from 0 to 50 μg/ml (titrated three times, starting with the highest concentration of 50 μg/ml). An unrelated IgG can be used as an isotype control for the first antibody and added at the same concentrations (titrate three times, starting with the highest concentration of 50 μg/ml). A sample preincubated with unlabeled secondary antibody at 50 μg/mL can be included as a positive control for complete blocking (100% inhibition), and a sample without antibody in the primary incubation can be used as a negative control (no competition; 0 inhibition %). After 30 minutes of incubation, a labeled, for example biotinylated, second antibody is added at a concentration of 2 μg/ml per well without washing. Samples are incubated for an additional 30 minutes on ice. Unbound antibodies are removed by washing cells with FACS buffer. The cell-bound second antibody is detected using a label detector, such as RE-conjugated streptavidin (Invitrogen, catalog number S21388) to detect biotin. Samples were collected on a FACS Calibur flow cytometer (BD, San Jose) and analyzed using FLOWJO® software (Tree Star, Inc, Ashland, OR). Results can be reported as % inhibition (ie, subtracting from 100% the amount of label at each concentration divided by the amount of label obtained without blocking antibody). Typically, the same experiment is then performed in reverse order, that is, the first antibody is the second antibody and the second antibody is the first antibody.

In certain embodiments, the antibody at least partially (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%) or completely (100%) blocks the binding of another antibody to its target, e.g. , human TIM3, or parts thereof, and whether inhibition occurs when one or the other antibody is the first antibody. The first and second antibodies cross-block each other's binding to the target when the antibodies compete with each other in both directions, i.e. in competition experiments in which the first antibody is added first, and in competition experiments in which the second antibody is added first.

In some embodiments, anti-TIM3 antibodies bind to the same epitope as the anti-TIM3 antibodies described herein (e.g., 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, and/or TIM3.2-TIM3.18), for example, as determined by a given epitope mapping method. Methods for identifying antibodies that bind to the same epitope on TIM3 as the anti-TIM3 antibodies described herein include, for example, epitope mapping methods such as X-ray crystal analysis of antigen:antibody complexes, which provides atomic resolution of the epitope. Other methods monitor the binding of an antibody to fragments of an antigen or mutated variations of an antigen, where loss of binding due to modification of an amino acid residue in the antigen sequence is often considered indicative of an epitope component (see FIG. 20). In addition, computational combinatorial methods for epitope mapping can also be used. The methods may also rely on the ability of the antibody of interest to affinity isolate specific short peptides (in native three-dimensional form or denatured form) from combinatorial phage display peptide libraries. The peptides are then considered as a guide to identify the epitope corresponding to the antibody used to screen the peptide library. Computational algorithms have also been developed for epitope mapping and have been shown to map conformational discontinuous epitopes.

Antibodies that compete for or bind to the same epitope as the anti-TIM3 antibodies described herein can be identified using methods known in the art. For example, mice can be immunized with human TIM3 as described herein, hybridomas can be generated, and the resulting monoclonal antibodies can be screened for the ability to compete with the antibody described herein for binding to human TIM3. Mice can also be immunized with a smaller fragment of TIM3 containing the epitope to which the antibody binds. An epitope or region containing an epitope can be localized, for example, by screening for binding to a series of overlapping peptides spanning TIM3. Alternatively, the method of Jespers et al., Biotechnology 12:899, 1994 can be used to direct the selection of antibodies containing the same epitope and therefore having similar properties to the anti-TIM3 antibody described herein. Using phage display, first the anti-TIM3 antibody heavy chain is paired with a repertoire of (human) light chains to select a TIM3-binding antibody, and then the new light chain is paired with a repertoire of (human) heavy chains to select a TIM3-binding (human) antibody having that the same epitope or epitope region as the anti-TIM3 antibody described herein. Alternatively, variants of the antibodies described herein can be obtained by mutagenesis of cDNA encoding the heavy and light chains of the antibody.

Alanine scanning mutagenesis, as described in Cunningham and Wells (1989) Science 244: 1081-1085, or some other form of point mutagenesis of amino acid residues in TIM3 can also be used to obtain the binding characteristics of an anti-TIM3 antibody.

The binding characteristics of a specific antibody can also be determined by assessing the binding of the antibody to peptides containing TIM3 fragments, such as undenatured or denatured fragments. A series of overlapping peptides spanning the TIM3 sequence (e.g., human TIM3) can be synthesized and screened for binding, e.g., in a direct ELISA, a competition ELISA assay (where the peptide is assessed for its ability to prevent antibody binding to TIM3 bound to a microplate well ) or on a chip.

Binding characteristics of anti-TIM3 antibodies can also be obtained using MS-based protein fingerprinting, such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP). HDX-MS can be carried out, for example, as described in WO 2015/18735 and Wei et al. (2014) Drug Discovery Today 19:95, methods of which are specifically incorporated herein by reference. FPOP can be performed as described, for example, in Hambley and Gross (2005) J. American Soc. Mass Spectrometry 16:2057, the methods of which are specifically incorporated herein by reference.

The binding characteristics of anti-TIM3 antibodies can also be obtained by structural methods such as X-ray crystal structure determination (e.g. WO 2005/044853), molecular modeling and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the H-D exchange rate of labile amide hydrogens in TIM3 when free and complexed with the antibody of interest (Zinn-Justin et al. (1992) Biochemistry 31, 11335-11347; Zinn-Justin et al. (1993) Biochemistry 32, 6884-6891).

With regard to X-ray crystallography, crystallization can be performed using any of the known methods in the art (for example, Giege et al. (1994) Acta Crystallogr. D50: 339-350; McPherson (1990) Eur. J Biochem. 189: 1 -23), including microvolume (e.g. Chayen (1997) Structure 5: 1269-1274), pendant drop method by vapor diffusion (e.g. McPherson (1976) J. Biol. Chem. 251: 6300-6303), culture and dialysis. It is desirable to use a protein preparation having a concentration of at least about 1 mg/ml or from about 10 mg/ml to about 20 mg/ml. Crystallization can best be achieved in a precipitant solution containing 1,000 to 20,000 polyethylene glycol (PEG; average molecular weight ranging from about 1,000 to about 20,000 Da), about 5,000 to about 7,000 Da, or about 6,000 Da, with concentrations ranging from about 10% to approximately 30% (w/v). It may also be desirable to include a protein stabilizing agent, such as glycerol, at a concentration of from about 0.5% to about 20%. A suitable salt, such as sodium chloride, lithium chloride or sodium citrate, may also be desired in the precipitant solution at a concentration ranging from about 1 mM to about 1000 mM. The precipitant is buffered to a pH of from about 3.0 to about 5.0. The specific buffers used in the precipitant solution may vary and are well known in the art (Scopes, Protein Purification: Principles and Practice, Third ed., (1994) Springer-Verlag, New York). Examples of useful buffers include, but are not limited to, HEPES, Tris, MES and acetate. Crystals can be grown at a wide range of temperatures, including 2, 4, 8 and 26°C.

Antibody:antigen crystals can be studied using well-known x-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Blundell & Johnson (1985) May. Enzymol. 114 & 115, N. W. Wyckoff et al., eds., Academic Press; US Patent Application Publication No. 2004/0014194) and BUSTER (Bricogne (1993) Acta Cryst. D49: 37-60 Bricogne (1997) Meth. Enzymol. 276A: 361-423, Carter & Sweet, eds.; Roversi et al. (2000) Acta Cryst. D56: 1313-1323), the disclosures of which are incorporated herein by reference in their entirety.

Antibodies to TIM3 can bind to the same epitope as any of the antibodies to TIM3 that have

- 53 044112 the amino acid sequences described herein, as determined using an epitope mapping method, such as the method described herein.

Antibodies that bind to human TIM3 and, optionally, cynomolgus TIM3 with binding characteristics similar to the anti-TIM3 antibodies described herein and determined by one of the methods used in the examples are included herein.

In certain embodiments, anti-TIM3 antibodies described herein bind to an epitope, e.g., a conformational epitope, in the extracellular portion of human TIM3, e.g., in the Ig-like domain or the IgV domain of the extracellular region, i.e. amino acids 22 to 130 of SEQ ID NO: 286 (FIG. 20). In some embodiments, the anti-TIM3 antibody binds to an epitope located at amino acids 22-120 of the extracellular domain of human TIM3 (SEQ ID NO: 286) or 1-99 of mature human TIM3 (SEQ ID NO: 290) (see Examples). In some embodiments, an anti-TIM3 antibody binds to a region or epitope within the region consisting of amino acids 58-64 of human TIM3 characterized by SEQ ID NO: 286, which corresponds to amino acid residues 37-43 of mature human TIM3 (CPVFECG, SEQ ID NO: 296 ; see Fig. 20). In some embodiments, an anti-TIM3 antibody binds to a region or epitope within the region consisting of amino acids 111-120 of human TIM3 characterized by SEQ ID NO: 286, which corresponds to amino acid residues 90-99 of mature human TIM3 (RIQIPGIMND, SEQ ID NO: 298 ; see Fig. 20). In some embodiments, an anti-TIM3 antibody binds to a region or epitope within the region consisting of amino acids 58-64 of human TIM3, characterized by SEQ ID NO: 286 (CPVFECG, SEQ ID NO: 296), and the region consisting of amino acids 111-120 human TIM3, characterized by SEQ ID NO: 286 (RIQIPGIMND, SEQ ID NO: 298; see Fig. 20). In some embodiments, an anti-TIM3 antibody binds to a region or epitope within the region consisting of amino acids 78-89 of human TIM3 characterized by SEQ ID NO: 286, which corresponds to amino acid residues 57-83 of mature human TIM3 (WTSRYWLNGDFR, SEQ ID NO: 297 ; see Fig. 20).

In one embodiment, the anti-TIM3 antibody binds to substantially the same epitope as 13A3. In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 from SEQ ID NO: 286 (FIG. 20 ). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues C58, P59, F61, E62, C63, R111, and D120 of SEQ ID NO: 286 is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues C58, P59, F61, E62, C63, D104, R111, Q113, and D120 of SEQ ID NO: 286 is replaced with another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 3G4. In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO: 286 (FIG. 20 ). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein having one or more amino acid residues C58, P59, V60, F61, E62, C63, G116, and M118 of SEQ ID NO: 286 is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues C58, P59, V60, F61, E62, C63, D104, G116, and M118 of SEQ ID NO: 286 is replaced with another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 17C3. In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 of SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) containing one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO: 286 (FIG. 20 ). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to the human TIM3 protein, which has one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, and G116 from SEQ ID NO: 286 are replaced with another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues C58, P59, V60, F61, E62, C63, G64, D104, and G116 of SEQ ID NO: 286 is replaced with another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 8B9. In some embodiments, the anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO: 286 (FIG. 20). In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO: 286 (FIG. 20 ). In some embodiments, the anti-TIM3 antibody binds to substantially the same epitope as 8B9. In some embodiments, an anti-TIM3 antibody binds to an epitope (or region of human TIM3) comprising one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO: 286 (FIG. 20 ). In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein having one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, and R89 of SEQ ID NO: 286 is replaced by another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues L48, W78, S80, R81, W83, L84, G86, D87, and R89 of SEQ ID NO: 286 is replaced with another amino acid, for example, in a non-conservative amino acid substitution. In some embodiments, the anti-TIM3 antibody does not bind significantly, or binds only with significantly reduced binding affinity, to a human TIM3 protein in which one or more amino acid residues L48, W78, S80, R81, W83, G86, D87, R89, and D104 of SEQ ID NO: 286 (FIG. 20) is replaced by another amino acid, for example, in a non-conservative amino acid substitution.

In some embodiments, anti-TIM3 antibodies compete for binding to human TIM3 (or inhibit binding) with anti-TIM3 antibodies containing CDRs or variable regions described herein, such as those of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9 , 8С4 and any of TIM3.2 to TIM3.18. In some embodiments, anti-TIM3 antibodies inhibit the binding of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2TIM3.18 to human TIM3 by at least 50, 60, 70, 80, 90, or 100 %. In some embodiments, 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2-TIM3.18 inhibits the binding of anti-TIM3 antibodies to human TIM3 by at least 50, 60, 70, 80, 90, or 100%. In some embodiments, anti-TIM3 antibodies inhibit the binding of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2-TIM3.18 to human TIM3 by at least 50, 60, 70, 80, 90, or 100%, and 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or any of TIM3.2-TIM3.18 inhibit the binding of anti-TIM3 antibodies to human TIM3 by at least 50, 60, 70, 80, 90 or 100% (for example, they compete in both directions).

VII. Engineered and modified antibodies.

Also provided are engineered and modified antibodies that can be produced using an antibody containing one or more VH and/or VL sequences disclosed herein as starting material for constructing a modified antibody, wherein the modified antibody may have altered properties relative to the original antibody. antibody. An antibody can be constructed by modifying one or more residues in one or both variable regions (ie, VH and/or VL), for example, one or more CDR regions and/or one or more framework regions. Additionally or alternatively, an antibody can be constructed by modifying residues in the constant region(s), for example, to change the effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. For this reason, amino acid sequences within the CDR are more diverse between individual antibodies than sequences outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that incorporate the sequences

- 55 044112

A CDR from a specific naturally occurring antibody grafted onto framework sequences from another antibody with different properties (see, for example, Riechmann, L. et al. (1998) Nature 332: 323

327; Jones, P. et al. (1986) Nature 321: 522-525; Queen, S. et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 1002910033; US Patent No. 5,225,539 to Winter and US Patent No. 5,530,101; 5585089; 5693772 and

6180370 issued by Queen et al.).

Accordingly, another embodiment described herein provides an isolated monoclonal antibody or antigen binding portion thereof comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-45 , SEQ ID NO: 4652, 122-125 and SEQ ID NO: 53-59, 126-129, respectively, and a light chain variable region comprising the sequences CDR1, CDR2 and CDR3 containing an amino acid sequence selected from the group consisting of SEQ ID NO: 64-65, SEQ ID NO: 66-67 and SEQ ID NO: 68-71, respectively. Thus, such antibodies contain the CDR VH and VL sequences of monoclonal antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8c4 or any of TIM3.2-TIM3.18, but may contain different framework sequences from these antibodies.

Such framework sequences can be obtained from publicly available DNA databases or published references that include germline antibody gene sequences. For example, germline DNA sequences for the human heavy and light chain variable region genes can be found in the human germline sequence database VBase (available online at www.mrc-cpe.cam.ac.uk/vbase) as well as in Kabat ,E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) The Repertoire of Human Germline V _H Sequences Reveals about Fifty Groups of V _H Segments with Different Hypervariable Loops /. Mol. Biol. 227:776-798 and Cox, JPL et al. (1994) A Directory of Human Germ-line V _H Segments Reveals a Strong Bias in their Usage Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.

In some embodiments, the framework sequences for use in the anti-TIM3 antibodies described herein are those that are structurally similar to the framework sequences used in the anti-TIM3 antibodies described herein. CDR1, 2 and 3 VH sequences and CDR1, 2 and 3 VL sequences can be grafted onto framework regions that are identical to the sequence that is found in the germline immunoglobulin gene from which the framework sequence is derived, or CDR sequences can be grafted onto framework regions , which contain one or more mutations compared to germline sequences. For example, it has been found that in some cases it is useful to mutate residues in framework regions to maintain or enhance the antigen-binding ability of an antibody (see, for example, US Pat. Nos. 5,530,101; 5,585,989; 5,693,662 and 6,180,370 to Queen et al.).

The engineered anti-TIM3 antibodies described herein include antibodies in which modifications have been made to framework residues within VH and/or VL, for example, to improve the properties of the antibody, for example, mutations at amino acid 107 in 9F6. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to mutate one or more framework residues back to the original form into the corresponding germline sequence. More specifically, an antibody that has undergone a somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the framework sequences of the antibody with the germline sequences from which the antibody is derived. To return the framework sequences to their germline configuration, somatic mutations can be mutated to the original germline sequence, for example, using site-directed mutagenesis or PCR-mediated mutagenesis. Such antibodies mutated to the original species should also be covered. Another type of framework modification involves mutation of one or more residues within the framework region or even within one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as deimmunization and is described in more detail in US Patent Publication No. 20030153043 issued to Carr et al.

Another type of variable region modification is carried out to mutate amino acid residues in the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutation(s), and the effect on antibody binding or other functional property of interest can be assessed in in vitro or in vivo assays as described herein and presented in the Examples. In some embodiments, conservative modifications are introduced (such as

- 56 044112 discussed above). Mutations can be amino acid substitutions, additions or deletions. In addition, typically no more than one, two, three, four or five residues in the CDR region are changed.

Accordingly, isolated anti-TIM3 monoclonal antibodies or antigen-binding portions thereof are also provided comprising a heavy chain variable region comprising:

(a) a CDR1 VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-45, or an amino acid sequence characterized by one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 41-45;

(b) a CDR2 VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 46-52 and 122-125, or an amino acid sequence characterized by one, two, three, four or five amino acid substitutions, deletions or additions at compared to SEQ ID NOs: 46-52 and 122-125;

(c) a CDR3 VH region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 53-59 and 126-129, or an amino acid sequence characterized by one, two, three, four or five amino acid substitutions, deletions or additions at compared to SEQ ID NOs: 53-59 and 126-129;

(d) a CDR1 VL region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-65, or an amino acid sequence characterized by one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 64-65;

(e) a CDR2 VL region containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-67, or an amino acid sequence characterized by one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 66-67; and (f) a CDR3 VL region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 68-71, or an amino acid sequence characterized by one, two, three, four or five amino acid substitutions, deletions or additions compared to SEQ ID NO: 68-71.

Methionine residues in the CDR of antibodies can be oxidized, leading to potential chemical degradation and subsequent reduction in antibody activity. Accordingly, anti-TIM3 antibodies are also provided that contain one or more methionine residues in the heavy and/or light chain CDRs replaced by amino acid residues that are not subject to oxidative degradation. In one embodiment, the methionine residues in the CDR of antibodies 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4, or any of TIM3.2-TIM3.18 are replaced with amino acid residues that are not subject to oxidative degradation.

Similarly, deamidation sites can be removed from anti-TIM3 antibodies, especially in the CDR.

The TIM3 variable regions described herein may be linked (eg, covalently linked or fused) to an Fc, such as an IgG1, IgG2, IgG3, or IgG4 Fc, which may be of any allotype or isoallotype, for example, for IgG1: G1m, G1m1( a), G1m2(x), G1m3(f), G1m17(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v) and for K: Km, Km1, Km2, Km3 (see, for example, Jefferies et al. (2009) mAbs 1:1).

In some embodiments, the anti-TIM3 variable regions described herein are associated with an Fc with no or largely no effector functions, such as IgG1.

In general, variable regions described herein may be linked to an Fc containing one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. In addition, an antibody described herein may be chemically modified (eg, one or more chemical moieties may be attached to the antibody) or may be modified to alter its glycosylation to alter one or more functional properties of the antibody. Each of these embodiments is described in more detail below. The numbering of residues in the Fc region corresponds to the EU index according to Kabat.

The Fc region encompasses domains derived from the immunoglobulin constant region, including a fragment, analog, variant, mutant or derivative of the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4 and other classes such as IgA, IgD, IgE and IgM. An immunoglobulin constant region is defined as a naturally occurring or synthetic polypeptide homologous to the C-terminal region of an immunoglobulin and may include a CH1 domain, a hinge domain, a CH2 domain, a CH3 domain, or a CH4 domain, alone or in combination.

- 57 044112

Ig molecules interact with several classes of cellular receptors. For example, IgG molecules interact with three classes of antibody class-specific Fcy receptors (FcyRs)

IgG, namely FcyRI, FcyRII and FcyRIII. Important sequences for IgG binding to FcyR receptors have been reported to be in the CH2 and CH3 domains. The half-life of an antibody in serum depends on the ability of that antibody to bind to the Fc receptor (FcR).

In some embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion, and/or insertion) relative to the original Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to create a variant ) to provide the desired structural features and/or biological activity.

Typically, variants of the constant region or portions thereof, such as the CH1, CL, hinge, CH2, or CH3 domains, may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations and/or the most greater than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation, or 1-10 or 1-5 mutations, or contain an amino acid sequence that is at least about 75, 80, 85, 90 , 95, 96, 97, 98, or 99% identical to that of the corresponding wild-type region or domain (CH1, CL, hinge, CH2, or CH3 domain, respectively), provided that the heavy chain constant region containing the particular variant retains the necessary biological activity.

For example, modifications can be made to the Fc region to produce an Fc variant that (a) has increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased or decreased complement-mediated cytotoxicity (CDC), (c) increased or decreased affinity for C1q and/or (d) increased or decreased affinity for the Fc receptor compared to the parent Fc. Such Fc region variants will typically contain at least one amino acid modification in the Fc region. It is believed that combining amino acid modifications is particularly desirable. For example, a variant Fc region may include two, three, four, five, etc. substitutions, for example, of certain Fc region provisions identified herein.

The variant Fc region may also contain a sequence change in which amino acids involved in the formation of a disulfide bond are removed or replaced by other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell that are used to produce the anti-TIM3 antibodies described herein. Even when cysteine residues are removed, single-chain Fc domains can still form a dimeric Fc domain that is held together non-covalently. In other embodiments, the Fc region may be modified to make it more compatible with the selected host cell. For example, the PA sequence near the N-terminus of a typical native Fc region that can be recognized by a digestive enzyme in E. coli, such as proline iminopeptidase, can be deleted. In other embodiments, one or more glycosylation sites in the Fc domain may be deleted. Residues that are typically glycosylated (eg, asparagine) can induce a cytolytic response. Such residues may be removed or replaced with non-glycosylated residues (eg alanine). In other embodiments, sites involved in interaction with complement, such as the C1q binding site, may be removed from the Fc region. For example, the human IgG1 EKK sequence may be removed or replaced. In certain embodiments, sites that affect Fc receptor binding may be removed, preferably sites other than salvage receptor binding sites. In other embodiments, the Fc region may be modified to remove the ADCC site. ADCC sites are known in the art; see for example Molec. Immunol. 29 (5): 633-9 (1992) regarding ADCC sites in IgG1. Specific examples of variant Fc domains are disclosed, for example, in international application publications WO 97/34631 and WO 96/32478.

In one embodiment, the Fc hinge region is modified such that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. This approach is further described in US Pat. No. 5,677,425 issued to Bodmer et al. The number of cysteine residues in the Fc hinge region is changed, for example, to facilitate the assembly of light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain boundary region of the Fc hinge fragment such that the antibody exhibits impaired staphylococcal protein A (SpA) binding relative to the SpA binding of the native Fc hinge domain. This approach is described in more detail in US Pat. No. 6,166,545 issued to Ward et al.

In still other embodiments, the Fc region is altered by replacing at least one amino acid residue with another amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acids

- 58044112 residues 234, 235, 236, 237, 297, 318, 320, 322, 330 and/or 331 may be replaced by another amino acid residue such that the antibody has altered affinity for the effector ligand, but retains the antigen-binding ability of the original antibody. The effector ligand for which the affinity is changed may be, for example, an Fc receptor or a complement component C1. This approach is described in more detail in US Patent Nos. 5,624,821 and 5,648,260 issued to Winter et al.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 may be replaced by another amino acid residue such that the antibody exhibits altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in more detail in US patent No. 6194551 issued to Idusogie et al.

In another example, one or more amino acid residues at amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT publication WO 94/29351 issued by Bodmer et al.

In yet another example, the Fc region can be modified to reduce antibody-dependent cellular cytotoxicity (ADCC) and/or to reduce affinity for the Fcy receptor by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278,

280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320,

322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389,

398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439. Illustrative substitutions include

236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D and 332E. Exemplary embodiments include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F7324T. Other modifications to enhance FcyR-complement interaction include, but are not limited to, substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L . These and other modifications are discussed in Strohl, 2009, Current Opinion in Biotechnology 20: 685691.

Fc modifications that increase binding to the Fcy receptor include amino acid modifications at any one or more amino acid positions 238, 239, 248, 249, 252.

254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295,296,

298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, 388,389,

398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, the numbering of residues in the Fc region being the same as according to the EU index as in Rabat (WO 00/42072).

Other Fc modifications that can be made to the Fc are modifications that reduce or eliminate binding to FcyR and/or complement proteins, thereby reducing or eliminating Fc-mediated effector functions such as ADCC, ADCP and CDC. Exemplary modifications include, without limitation, substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, 328, 330, and/or 331 (e.g., 330 and 331), numbered according to the EU suffix . Illustrative substitutions include, without limitation, 234A, 235E, 236R, 237A, 267R, 269R, 325L, 328R, 330S and 331S (eg, 330S and 331S), numbered according to the EU suffix. The Fc variant may contain 236R/328R. Other modifications to reduce the interaction of FcyR and complement include substitutions of 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330s, 331S, 220S, 226S, 229S, 238S, 233P and 234V, as well as deletion glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are discussed in Strohl, 2009, Current Opinion in Biotechnology 20: 685-691.

Optionally, the Fc region may contain a non-naturally occurring amino acid residue at additional and/or alternative positions known to one of ordinary skill in the art (see, for example, US Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCX Patent Publications WO 00/42072; WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO 05/07 0963; WO 05/040217, WO 05/092925 and WO 06/0201 14).

Fc variants that enhance affinity for the inhibitory receptor FcyRIIb can also be used. Such variants may provide an Fc fusion protein with immunomodulatory activities associated with FcyRIIb cells, including, for example, B cells and monocytes. In one embodiment, Fc variants provide selectively improved affinity for FcyRIIb for one or more activating receptors. Modifications to alter binding to FcyRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, 330, 331, and 332 according to the EU index. Exemplary substitutions to enhance the affinity of FcyRIIb include, but are not limited to, 234A, 234D, 234E, 234F, 234W, 235D, 235E, 235F, 235R, 235Y, 236D, 236N, 237A, 237D, 237N, 239D, 2 39E, 266M, 267D , 267M, 267M, 267E, 268D, 268E, 327D, 327E, 328F, 328 W, 328Y, 330S, 331S and 332E. Illustrative substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W and

- 59 044112

328Y. Other Fc variants to enhance binding to FcyRIIb include 235Y/267E, 236D/267E,

239D/268D, 239D/267E, 267E/268D, 267E/268E and 267E/328F.

The affinity and binding properties of the Fc region for its ligand can be determined by various in vitro assay methods (biochemical or immunoassays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (eg, BIACORE assay) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (eg, gel filtration). These and other methods may use a label on one or more components of interest and/or use a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

In certain embodiments, the antibody is modified to increase its biological half-life. Different approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region for FcRn. For example, one or more of the following residues may be mutated: 252, 254, 256, 433, 435, 436, as described in US Pat. No. 6,277,375. Specific illustrative substitutions include one or more of the following: T252L, T254S and/or T256F. Alternatively, to increase the biological half-life, the antibody can be modified in the CH1 or CL region to contain a salvage receptor binding epitope taken from the two loops of the CH2 domain of the IgG Fc region, as described in US Patent Nos. 5,869,046 and 6,121,022 to Presta et al. Other exemplary variations that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 2591, 308F, 428L, 428M, 434S, 43411, 434F, 434Y and 434X1. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hintone et al. 2004, J. Biol. Chem. 279 (8): 6213-6216, Hintone et al. 2006 Journal of Immunology 176: 346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al., Journal of Biological Chemistry, 2 001, 276 (9): 6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433R, 433S, 4331, 433P, 4 33Q, 434H, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311S (Dall'Acqua et al. Journal of Immunology, 2002, 169: 5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281: 23514-23524). Other modifications to modulate FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

In some embodiments, hybrid IgG isotypes with specific biological characteristics may be used. For example, an IgG1/IgG3 hybrid variant can be constructed by replacing the IgG1 positions in the CH2 and/or CH3 region with amino acids from IgG3 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody can be constructed that includes one or more substitutions, for example, 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R and 436F. In other embodiments described herein, an IgG1/IgG2 hybrid variant can be constructed by replacing the IgG2 positions in the CH2 and/or CH3 region with amino acids from IgG1 at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody can be constructed that contains one or more substitutions, for example, one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (relating to the glycine insertion at position 236) and 327A.

In addition, the binding sites on human IgG1 for FcyRI, FcyRII, FcyRIII and FcRn have been mapped, and variants with improved binding have been described (see Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604) . Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcyRIII. In addition, the following combination mutants have been shown to improve FcyRIIIa binding: T256A/S298A, S298A/E333A, S298A/K224A, and S298A/E333A/K334A, which have been shown to exhibit enhanced FcyRIIIa binding and ADCC activity (Shields et al. , 2001). Other IgG1 variants with highly enhanced binding to FcyRIIIa have been identified, including variants with the S239D/I332E and S239D/I332E/A330L mutations, which showed the greatest increase in affinity for FcyRIIIa, decreased FcyRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al ., 2006). Introduction of triple mutations in antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2/neu-specific), rituximab (CD20-specific) and cetuximab (EGFR-specific) resulted in significantly increased ADCC activity in vitro, and the S239D/I332E variant demonstrated an increased ability to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgG1 mutants containing the L235V, F243L, R292P, Y300L, and P396L mutations were identified that showed enhanced binding to FcyRIIIa and concomitantly increased ADCC activity in transgenic mice expressing human FcyRIIIa in models of B-cell malignancies and breast cancer (Stavenha- 60 044112 gen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that can be used include: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L, L235V/F243L/R292P/Y300L/P396L and

M428L/N434S.

In certain embodiments, an Fc is selected that exhibits reduced binding to FcyR. An exemplary Fc, for example, an IgG1 Fc with reduced binding to FcyR, contains the following three amino acid substitutions: L234A, L235E and G237A.

In certain embodiments, an Fc is selected that is characterized by reduced complement fixation. An exemplary Fc, such as the reduced complement fixation IgG1 Fc, contains the following two amino acid substitutions: A330S and P331S.

In certain embodiments, an Fc is selected that has essentially no effector function, i.e. it is characterized by reduced binding to FcyR. and decreased complement fixation. An exemplary Fc, for example, the IgG1 Fc, which does not contain effectors, contains the following five mutations: L234A, L235E, G237A, A330S and P331S.

When using an IgG4 constant domain, it may include the S228P substitution, which mimics the hinge sequence in IgG1 and thereby stabilizes IgG4 molecules.

In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody (ie, an antibody without glycosylation) can be produced. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such modifications of carbohydrates can be accomplished, for example, by changing one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions may be made that result in the removal of one or more glycosylation sites of the variable region backbone, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in more detail in US Patent Nos. 5,714,350 and 6,350,861 issued to Co et al.

Glycosylation of the constant region on N297 can be prevented by mutating residue N297 to another residue, eg N297A, and/or by mutating an adjacent amino acid, eg 298, thereby reducing glycosylation on N297.

Additionally or alternatively, an antibody may be produced that is characterized by an altered glycosylation pattern, such as a hypofucosylated antibody containing reduced amounts of fucosyl residues, or an antibody containing an increased amount of GlcNac structures at branch points. These altered glycosylation patterns have been shown to enhance the anti-ADCC potency of antibodies. Such modifications of carbohydrates can be accomplished, for example, by expressing an antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which the recombinant anti-TIM3 antibodies described herein are expressed to thereby produce the altered glycosylation antibody. For example, European patent EP 1176195 issued to Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT publication WO 03/035835 issued by Presta describes a variant of the CHO cell line, Led 3 cells, with a reduced ability to attach fucose to Asn(297) bound carbohydrates, which also leads to hypofucosylation of antibodies expressed in this host cell ( see also Shields, R. L. et al (2002) J Biol Chem 277: 26733-26740). PCT publication WO 99/54342 issued by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (eg, beta(1,4)-M-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in engineered cell lines exhibit increased amounts of GlcNac structures at branch points, resulting in increased antibody ADCC activity (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).

Another modification of the anti-TIM3 antibodies described herein is pegylation. The antibody may be pegylated, for example, to increase the biological (eg, serum) half-life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. In some embodiments, the PEGylation is carried out through an acylation reaction or an alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term polyethylene glycol is intended to cover any of the forms of PEG that have been used to derivatize other proteins, such as mono(CI-CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for PEGylation of proteins are known in the art and can be applied to the anti-TIM3 antibodies described herein. See, for example, EP 0154316 issued by Nishimura et al., and EP 0401384 issued by Ishikawa et al.

In some embodiments, an anti-TIM3 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is selected from the group consisting of SEQ ID NOs: 263-266.

VIII. Physical properties of antibodies.

Antibodies to TIM3, such as those described herein, have some or all of the physical characteristics of the specific antibodies to TIM3 described herein, such as those described in the examples.

The anti-TIM3 antibodies described herein may contain one or more glycosylation sites in the light or heavy chain variable region. Such glycosylation sites may result in increased antibody immunogenicity or changes in antibody pK due to altered antigen binding (Marshall et al., (1972) Appy Rev Biochem 41:673-702; Gala and Morrison (2004) J. Immunol 172:5489-94; Wallick et al., (1988) J Exp Med 168: 1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al., (1985) Nature 316:452-7; Mimura et al., (2000) ) Mol Immunol 31:697–106). Glycosylation is known to occur in motifs containing the sequence N-X-S/T. In some cases, the anti-TIM3 antibody does not contain variable region glycosylation. This can be achieved either by selecting antibodies that do not contain a glycosylation motif in the variable region, or by mutating residues in the glycosylation region.

In certain embodiments, anti-TIM3 antibodies described herein do not contain asparagine isomerism sites. Deamidation of asparagine can occur in the N-G or D-G sequences and lead to the formation of an isoaspartic acid residue, which introduces a kink in the polypeptide chain and reduces its stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pi), which is typically in the pH range of 6 to 9.5. The pi value for an IgG1 antibody is typically in the pH range of 7-9.5, and the pi value for an IgG4 antibody is typically in the pH range of 6-8. It has been suggested that antibodies with pi outside the normal range may exhibit some unfolding and instability under in vivo conditions. Thus, an anti-TIM3 antibody may contain a pi value that is in the normal range. This can be achieved either by selecting antibodies with pi in the normal range or by mutating charged surface residues.

Each antibody will have a characteristic melting point, with higher melting points indicating greater overall stability in vivo (Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71). Typically, T _M i (initial unfolding temperature) may be above 60°C, above 65°C, or above 70°C. The melting point of an antibody can be measured using differential scanning calorimetry (Chen et al., (2003) Pharm Res 20: 1952-60; Ghirlando et al., (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. ., (2002) J Chromatogr Sci 40:343-9).

In one embodiment, antibodies are selected that do not degrade rapidly. Antibody degradation can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

In another embodiment, antibodies are selected that have minimal aggregation effects that could result in triggering an undesirable immune response and/or altered or adverse pharmacokinetic properties. Typically, antibodies are acceptable with an aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured by several methods including size exclusion column (SEC), high performance liquid chromatography (HPLC) and light scattering.

In some embodiments, an anti-TIM3 antibody has a combination of the structures and properties described in sections (I), (II), (III), (IV), (V), (VI), (VII), (VIII), and (IX ) higher. In one embodiment, the anti-TIM3 antibody cross-competes with antibodies 13A3, 17C3, 8B9, 8C4, 3G4, 17C8, and 9F6, as described in sections I and/or VI, derived from a germline sequence, as described in section III, contains conserved mutations , as described in Section V, and/or has homology to the anti-TIM3 antibodies in Sections I and II, as described in Section IV, in combination with one or more functional properties described elsewhere herein.

IX. Methods for constructing antibodies.

As discussed above, anti-TIM3 antibodies having the VH and VL sequences disclosed herein can be used to create new anti-TIM3 antibodies by modifying the VH and/or VL sequences or the constant region(s) attached thereto. Thus, in another aspect described herein, the structural features of an anti-TIM3 antibody described herein are used to create structurally related anti-TIM3 antibodies that retain at least one functional property of the anti-TIM3 antibodies described herein, e.g., binding to human TIM3 and cynomolgus TIM3. For example, one or more

- 62 044112 CDR regions 17C3, 8B9, 8C4, 3G4, 17C8, 9F6, 13A3 or any of TIM3.2-TIM3.18 can be recombinantly combined with known framework regions and/or other CDRs to create additional, recombinantly engineered antibodies to TIM3 described herein as discussed above. Other types of modifications include those described in the previous section. The starting material for the construction method is one or more VH and/or VL sequences provided herein, or one or more CDR regions. To create an engineered antibody, it is not necessary to actually produce (ie, express as a protein) an antibody having one or more VH and/or VL sequences provided herein or one or more CDR regions thereof. Rather, the information contained in the sequence(s) is used as starting material to create second-generation sequence(s) derived from the original sequence(s), and the second-generation sequence(s) are then prepared and expressed as a protein .

Accordingly, provided herein are methods for producing an anti-TIM3 antibody comprising:

(a) providing: (i) an antibody heavy chain variable region sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 41-45, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 46-52, and 122-125, and/or a CDR3 sequence selected from the group consisting of SEQ ID NOs: 53-59 and 126-129; and (ii) an antibody light chain variable region sequence comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 64 and 65, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 66 and 67, and/or a sequence CDR3 selected from the group consisting of SEQ ID NO: 68-71;

(b) altering at least one amino acid residue in the antibody heavy chain variable region sequence and/or the antibody light chain variable region sequence to create at least one altered antibody sequence, and (c) expressing the altered antibody sequence as a protein.

Standard molecular biology techniques can be used to obtain and express the altered antibody sequence. In some embodiments, the antibody encoded by the altered antibody sequence(s) is an antibody that retains one, some, or all of the functional properties of the anti-TIM3 antibodies described herein, which include:

(1) binding to soluble human TIM3, for example, with a KD of 10 nM or less (for example, from 0.01 nM to 10 nM), for example, as measured by Biacore, for example, as described in the examples;

(2) binding to soluble cynomolgus TIM3, eg, with a KD of 100 nM or less (eg, 0.01 nM to 100 nM), eg, as measured by Biacore, eg, as described in the Examples;

(3) binding to membrane-bound human TIM3, e.g., with an EC ₅₀ of 1 μg/ml or less (e.g., 0.01 μg/ml to 1 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(4) binding to membrane-bound human TIM3, eg, with a K _D of 1 nM or less (eg, 0.01 nM to 10 nM), eg, as measured by the Scatchard assay, eg, as described in the Examples;

(5) binding to membrane-bound cynomolgus TIM3, e.g., with an EC ₅₀ of 20 μg/ml or less (e.g., 0.01 μg/ml to 20 μg/ml), e.g., as measured by flow cytometry (e.g., as described in examples);

(6) binding to membrane-bound cynomolgus TIM3, for example, with a KD of 1 nM or less (eg, 0.01 nM to 10 nM), for example, as measured by the Scatchard assay, for example, as described in the examples;

(7) inducing or enhancing T cell activation (eg, by blocking or reducing the inhibitory effect of TIM3), as evidenced by (i) increased production of IFN-γ in TIM3-expressing T cells (eg, Th1 cells or TILs) and/ or (ii) enhancing the proliferation of TIM-3-expressing T cells (eg, Th1 cells or TILs), for example, as described in the examples;

(8) stimulating T cell proliferation in a mixed lymphocyte reaction (MLR) assay, for example, as described in the Examples;

(9) inhibition of phosphatidylserine binding to TIM3, for example, as measured by a PS-hTIM3 tandem blocking assay, for example, as described in the Examples;

(10) lack of internalization or downregulation of cell surface TIM3 upon binding to TIM3 on cells;

(11) binding to one of the following regions of the extracellular domain of human TIM3 (SEQ ID NO: 290): (a) CPVFECG (SEQ ID NO: 296); (b) RIQIPGIMND (SEQ ID NO: 298); (c) CPVFECG and RIQIPGIMND (SEQ ID NO: 296 and 298, respectively) and (d) WTSRYWLNGDFR (SEQ ID NO: 297), for example, as described in the examples;

- 63 044112 (12) the presence of reduced binding to human TIM3, in which one or more amino acids

L48, C58, P59, V60, F61, E62, C63, G64, W78, S80, R81, W83, L84, G86, D87, R89, D104, R111, Q113,

G116, M118 and D120 (as numbered in SEQ ID NO: 286 (Fig. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3, for example, as described in the examples;

(13) competition in one or both directions for binding to human TIM3 with an antibody containing the VH and VL domains of any of 13A3, 3G4, 17C3, 17C8, 9F6, 8B9, 8C4 or TIM3.7, TIM3.8, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.18, for example, as described in the examples;

(14) binding to human TIM3 regions 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367) and 111RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368), as determined by HDX-MS, for example, as described in the Examples;

(15) the presence of heavy chain and/or light chain variable regions that interact with at least 5, 10, 15, 20 or all of the following amino acids of human TIM3: P50, V51, C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120, and optionally T70 and/or I112, as determined by x-ray crystallography (e.g., described in the Examples; numbering according to SEQ ID NO: 286 (Fig. 20)); and/or (16) (a) the presence of reduced binding to human TIM3, in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids C58, P59, F61, E62, C63, R111, D120 and optionally, D104 and Q113 (SEQ ID NO: 286 (FIG. 20)) are replaced with a different amino acid relative to binding to wild-type human TIM3; (b) binding to 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and ¹¹⁹ NDEKFNLKL ¹²⁷ (SEQ ID NO: 373) as determined by HDX-MS as described in the Examples; and/or (c) competing with or cross-blocking binding to human TIM3 13A3 or TIM3.18.IgG1.3, for example, as described in the Examples.

The modified antibody may exhibit one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all of the functional properties represented by (1) - (16) above. The functional properties of the modified antibodies can be assessed using standard assays available in the art and/or described herein, such as those presented in the examples (eg, ELISA, FACS).

In certain embodiments of the methods for constructing anti-TIM3 antibodies described herein, mutations may be introduced randomly or selectively along all or a portion of the coding sequence of the anti-TIM3 antibody, and the resulting modified anti-TIM3 antibodies may be screened for binding activity and/or other functional properties as described herein. Mutation methods have been described in the art. For example, PCT publication WO 02/092780 issued by Short describes methods for generating and screening antibody mutations using saturation mutagenesis, artificial ligation assembly, or a combination thereof. Alternatively, PCT publication WO 03/074679 issued by Lazar et al. describes methods of using computer-assisted screening methods to optimize the physicochemical properties of antibodies.

X. Nucleic acid molecules.

Another aspect described herein relates to nucleic acid molecules that encode anti-TIM3 antibodies described herein. The nucleic acids may be present in whole cells, in a cell lysate, or in partially purified or substantially pure form. A nucleic acid is isolated or substantially pure when it is purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA that is associated with isolated DNA in nature) or standard proteins methods including alkali/SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis and others well known in the art. See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acid described herein may be, for example, DNA or RNA and may or may not contain intronic sequences. In certain embodiments, the nucleic acid is a cDNA molecule.

The nucleic acids described herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (eg, hybridomas obtained from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibody produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning methods. For antibodies obtained from an immunoglobulin gene library (eg, using phage display methods), the nucleic acid encoding the antibody can be retrieved from the library.

Certain nucleic acid molecules described herein are molecules encoding the VH and VL sequences of antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4, 17C8 or

- 64 044112 any of the antibodies from TIM3.2 to TIM3.18. Exemplary DNA sequences encoding VH sequences 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 are provided in SEQ ID NOs: 167-173, 245254 and 359. Exemplary DNA sequences encoding VL 13A3 sequences 17C3 and 3G4 are provided in SEQ ID NO: 193. Exemplary DNA sequences encoding VL 8B9, 8C4 and 17C8 sequences are provided in SEQ ID NO: 194. Exemplary DNA sequences encoding VL 9F6 sequences are presented in SEQ ID NO: 194-196. Exemplary DNA sequences encoding heavy chain sequences 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 are provided in SEQ ID NOs: 134-161, 205-244 and 355-358. Exemplary DNA sequences encoding light chain sequences 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 are provided in SEQ ID NOs: 162-166.

Exemplary nucleic acids encoding the mature VH and VL domains of antibodies 13A3.IgG1.1 and 13A3.IgG1.3 (the same variable region) are provided as SEQ ID NOs: 167 and 193, respectively. Exemplary nucleic acids encoding the mature heavy chains of antibodies 13A3.IgG1.1 and 13A3.IgG1.3 are shown as SEQ ID NOs: 134 and 148, respectively, and an exemplary nucleic acid encoding the mature light chain of antibodies 13A3.IgG1.1 and 13A3.IgG1.3, provided as SEQ ID NO: 162.

Exemplary nucleic acids encoding the mature VH and VL domains of antibodies 8B9.IgG1.1 and 8B9.IgG1.3 (the same variable region) are provided as SEQ ID NOs: 168 and 194, respectively. Exemplary nucleic acids encoding the mature heavy chains of the antibodies 8B9.IgG1.1 and 8B9.IgG1.3 are provided as SEQ ID NOs: 135 and 149, respectively, and an exemplary nucleic acid encoding the mature light chain of the antibodies 8B9.IgG1.1 and 8B9.IgG1.3, provided as SEQ ID NO: 163.

Exemplary nucleic acids encoding the mature VH and VL domains of antibodies 8C4.IgG1.1 and 8C4.IgG1.3 (the same variable region) are provided as SEQ ID NOs: 169 and 194, respectively. Exemplary nucleic acids encoding the mature heavy chains of the antibodies 8C4.IgG1.1 and 8C4.IgG1.3 are provided as SEQ ID NOs: 136 and 150, respectively, and an exemplary nucleic acid encoding the mature light chain of the antibodies 8C4.IgG1.1 and 8C4.IgG1.3, provided as SEQ ID NO: 163.

Exemplary nucleic acids encoding the mature VH and VL domains of antibodies 17C3.IgG1.1 and 17C3.IgG1.3 (the same variable region) are provided as SEQ ID NOs: 170 and 193, respectively. Exemplary nucleic acids encoding the mature heavy chains of antibodies 17C3.IgG1.1 and 17C3.IgG1.3 are shown as SEQ ID NOs: 137 and 151, respectively, and an exemplary nucleic acid encoding the mature light chain of antibodies 17C3.IgG1.1 and 17C3.IgG1.3, provided as SEQ ID NO: 162.

Exemplary nucleic acids encoding the mature VH and VL domains of antibodies 9F6.IgG1.1 and 9F6.IgG1.3 (the same variable region) are provided as SEQ ID NOs: 171 and 197, respectively. Exemplary nucleic acids encoding the mature heavy chains of the antibodies 9F6.IgG1.1 and 9F6.IgG1.3 are provided as SEQ ID NOs: 138 and 152, respectively, and an exemplary nucleic acid encoding the mature light chain of the antibodies 9F6.IgG1.1 and 9F6.IgG1.3, provided as SEQ ID NO: 166.

Exemplary nucleic acids encoding the mature VH and VL domains of antibodies 3G4.IgG1.1 and 3G4.IgG1.3 (the same variable region) are provided as SEQ ID NOs: 172 and 193, respectively. Exemplary nucleic acids encoding the mature heavy chains of the antibodies 3G4.IgG1.1 and 3G4.IgG1.3 are provided as SEQ ID NOs: 139 and 153, respectively, and an exemplary nucleic acid encoding the mature light chain of the antibodies 3G4.IgG1.1 and 3G4.IgG1.3, provided as SEQ ID NO: 162.

Exemplary nucleic acids encoding the mature VH and VL domains of antibodies 17C8.IgG1.1 and 17C8.IgG1.3 (the same variable region) are provided as SEQ ID NOs: 173 and 194, respectively. Exemplary nucleic acids encoding the mature heavy chains of the antibodies 17C8.IgG1.1 and 17C8.IgG1.3 are provided as SEQ ID NOs: 140 and 154, respectively, and an exemplary nucleic acid encoding the mature light chain of the antibodies 17C8.IgG1.1 and 17C8.IgG1.3, provided as SEQ ID NO: 163.

The above exemplary nucleic acids may further comprise a signal peptide set forth in SEQ ID NOs: 267-267 and 361. Nucleotide sequences encoding these signal peptides are set forth in SEQ ID NOs: 272-276, 362, and 363.

The nucleic acid molecules described herein may be modified to remove specific sequences, such as restriction enzyme recognition sequences, or for codon optimization.

The method of obtaining 13A3 IgG1.1, 8B9 IgG1.1, 8C4 IgG1.1, 17C3 IgG1.1, 9F6 IgG1.1, 3G4 IgG1.1, 17C8 IgG1.1 and/or TIM3.2-TIM3.18 IgG1.1 can provide for the expression of heavy chains and light chains in a cell line containing nucleotide sequences encoding heavy and light chains with a signal peptide, for example, for 13A3 IgG1.1, SEQ ID NO: 269 and 268, respectively. Way

- 65 044112 obtaining 13A3 IgG1.3, 8B9 IgG1.3, 8C4 IgG1.3, 17C3 IgG1.3, 9F6 IgG1.3, 3G4 IgG1.3 and/or 17C8 IgG1.3 may involve the expression of the heavy chain and light chains in the cell line containing nucleotide sequences encoding heavy and light chains with a signal peptide, for example, for 13A3 IgG1.3, SEQ ID NO: 274 and 273, respectively. Host cells containing these nucleotide sequences are included herein.

Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or an scFv gene. In these manipulations, a DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term operably linked, as used in this context, is intended to mean that two DNA fragments are joined so that the amino acid sequences encoded by the two DNA fragments remain in frame.

The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant regions (hinge, CH1, CH2 and/or CH3). Human heavy chain constant region gene sequences are known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments covering these regions can be obtained using standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, such as an IgG1 region. For a Fab heavy chain gene, the DNA encoding VH can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region, CL. Human light chain constant region gene sequences are known in the art (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments covering these regions can be obtained using standard PCR amplification. The light chain constant region may be a kappa or lambda constant region.

To create the scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker, for example encoding the amino acid sequence (Gly ₄ -Ser) ₃ , so that the VH and VL sequences can be expressed as a continuous single-stranded protein with VL and VH regions connected by a flexible linker (see, for example, Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879- 5883; McCafferty et al., (1990) Nature 348:552-554).

Also provided herein are nucleic acid molecules encoding VH and VL sequences that are homologous to those of antibodies 17C3, 8B9, 8C4, 3G4, 17C8, 9F6, 13A3 and any of the antibodies TIM3.2 to TIM3.18. Exemplary nucleic acid molecules encode VH and VL sequences that are at least 70% identical, for example, at least 75%, at least 80%, at least 85%, at least 90%, in are at least 95% or at least 99% identical to the nucleic acid molecules encoding the VH and VL sequences of 17C3, 8B9, 8C4, 3G4, 17C8, 9F6, 13A3 or any of the antibodies TIM3.2 to TIM3.18. Also provided herein are nucleic acid molecules with conservative substitutions (ie, substitutions that do not change the resulting amino acid sequence when the nucleic acid molecule is translated), for example, for codon optimization.

Also provided are nucleic acids encoding the VH and/or VL regions of anti-TIM3 antibodies, such as the anti-TIM3 antibodies described herein, wherein the nucleic acids contain a nucleotide sequence that is at least about 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to any of the nucleotide sequences encoding the VH and/or VL regions of the anti-TIM3 antibodies described herein.

Also provided are nucleic acids encoding the heavy chain and/or light chain of anti-TIM3 antibodies, such as the anti-TIM3 antibodies described herein, wherein the nucleic acids contain a nucleotide sequence that is at least about 75, 80, 85, 90, 95 , 96, 97, 98 or 99% identical to any of the nucleotide sequences encoding the heavy and/or light chains of the anti-TIM3 antibodies described herein.

XI. Antibody production.

The anti-TIM3 monoclonal antibodies described herein can be produced using various known methods, such as the standard somatic cell hybridization method described by Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle other methods can be used

- 66 044112 production of monoclonal antibodies, for example, viral or oncogenic transformation of B-lymphocytes, phage display method using human antibody gene libraries.

The preferred animal system for producing hybridomas is the murine system. The production of hybridoma in mice is a very well established procedure. Immunization protocols and methods for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg, mouse myeloma cells) and fusion procedures are also known.

The chimeric or humanized anti-TIM3 antibodies described herein can be derived from the murine monoclonal antibody sequence prepared as described above. DNA encoding the immunoglobulin heavy and light chains can be obtained from a mouse hybridoma of interest and engineered to contain non-mouse (eg, human) immunoglobulin sequences using standard molecular biology techniques. For example, to create a chimeric antibody, mouse variable regions can be linked to human constant regions using methods known in the art (see, for example, US Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody, mouse CDR regions can be inserted into a human scaffold using methods known in the art (see, for example, US Pat. No. 5,225,539 issued to Winter, and US Pat. Nos. 5,530,101; 5,585,809; 5,693,772 and 6,180,370 issued Queen et al.).

In one embodiment, the anti-TIM3 antibodies described herein are human monoclonal antibodies. Such human monoclonal antibodies directed against TIM3 can be produced using transgenic or transchromosomal mice carrying parts of the human immune system rather than the murine system. These transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively, and are collectively referred to herein as human Ig mice.

HUMAB-MOUSE® (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged heavy (μ and γ) and light chain sequences of human immunoglobulins together with targeted mutations that inactivate endogenous μ and κ chain loci (see, e.g. , Lonberg, et al (1994) Nature 368 (6474): 856-859). Accordingly, mice exhibit reduced expression of murine IgM or K, and in response to immunization, introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human monoclonal IgGK (Lonberg, N. et al. (1994) supra , reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93 and Harding, F. and Lonberg , N. (1995) Ann. NY. Acad. Sci. 764:536546). The generation and use of HuMab mice and the genomic modifications that such mice carry are further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al., (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117–123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al. (1994) Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6: 579-591 and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-851. See additional US patents No. 5545806; 5569825; 5625126; 5633425; 5789650; 5877397; 5661016; 5814318; 5874299 and 5770429; all issued by Lonberg and Kay; US Patent No. 5,545,807 to Surani et al.; PCT Publications No. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all issued by Lonberg and Kay; and PCT Publication No. WO 01/14424 issued by Korman et al.

In certain embodiments, anti-TIM3 antibodies described herein are produced using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as KM mice, are described in detail in PCT publication WO 02/43478 issued by Ishida et al.

In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-TIM3 antibodies described herein. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used; such mice are described, for example, in US patent No. 5939598; 6075181; 6114598; 6150584 and 6162963 issued by Kucherlapati et al.

In addition, alternative animal transchromosomal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-TIM3 antibodies described herein. For example, mice carrying both the human heavy chain transchromosome and the human light chain transchromosome, called TC mice, can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-727. In addition, bovines carrying human heavy and light chain transchromosomes have been described in the present art (Kuroiwa et al. (2002) Nature Biotechnology 20:889-894) and can be used for

- 67 044112 obtaining the anti-TIM3 antibodies described herein.

Additional mouse systems described in the art for producing human antibodies, such as human anti-TIM3 antibodies, include (i) the VELOCLMMUNE® mouse (Regeneron Pharmaceuticals, Inc.), in which the endogenous mouse heavy and light chain variable regions have been replaced by homologous recombination into human heavy and light chain variable regions operably linked to murine endogenous constant regions such that chimeric antibodies (human V/mouse C) are produced in mice and then subsequently converted into fully human antibodies using standard recombinant DNA techniques; and (ii) a MEMO® mouse (Merus Biopharmaceuticals, Inc.), in which the mouse contains unrearranged human heavy chain variable regions but one rearranged human light chain variable region. Such mice and their use for the production of antibodies are described, for example, in WO 2009/15777, US 2010/0069614, WO 2011/072204, WO 2011/097603, WO 2011/163311, WO 2011/163314, WO 2012/148873, US 20 12 /0070861 and US 2012/0073004.

The anti-human TIM3 monoclonal antibodies described herein can also be generated using phage display methods to screen human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are known in the art. See, for example: US patents No. 5223409; 5403484 and 5571698 issued by Ladner et al.; US Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; US Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al. and US Patent No. 5,887,993; 6521404; 6544731; 6555313; 6582915 and 6593081 issued by Griffiths et al.

The human monoclonal antibodies to TIM3 described herein can also be generated using SCID mice in which human immune cells have been reconstituted such that a human antibody response can be produced upon immunization. Such mice are described, for example, in US patent No. 5479996 and 5698767 issued by Wilson et al.

XI.A. Immunization.

To obtain fully human antibodies to TIM3, transgenic or transchromosomal mice containing human immunoglobulin genes (eg, HCol2, HCo7, or KM mice) can be immunized with a purified or enriched preparation of TIM3 antigen and/or cells expressing TIM3 or a fragment thereof, as described for other antigens, for example, in the publications of Lonberg et al., (1994) Nature 368 (6474): 856-859; Fishwild et al., (1996) Nature Biotechnology 14: 845-851 and WO 98/24884. Alternatively, mice can be immunized with DNA encoding human TIM3 or a fragment thereof. In some embodiments, the mice may be 6-16 weeks old after the first infusion. For example, a purified or enriched preparation (5-50 μg) of recombinant TIM3 antigen can be used to immunize mice with HuMAb intraperitoneally. In cases where immunization with a purified or enriched TIM3 antigen preparation does not result in antibodies, mice can also be immunized with TIM3-expressing cells, such as a cell line, to stimulate immune responses. Exemplary cell lines include the TIM3-overexpressing stable cell lines CHO and Raji.

Cumulative experience with various antigens has shown that HuMAb transgenic mice respond best when initially immunized intraperitoneally (IP) or subcutaneously (SC) with antigen in Ribi adjuvant, followed by IP/SC immunization every two weeks (up to a total of 10) with antigen in Ribi adjuvant . The immune response can be monitored during the immunization protocol using plasma samples obtained using retro-orbital blood draws. Plasma can be screened by ELISA and FACS (as described below), and mice with sufficient human TIM3 immunoglobulin titers can be used for fusions. Mice can be immunized intravenously with antigen 3 days before sacrifice and removal of the spleen and lymph nodes. It is expected that 2-3 fusions may be required for each immunization. Typically, 6 to 24 mice are immunized for each antigen. As a rule, strains HCo7, HCol2 and KM are used. In addition, both HCo7 and HCol2 transgenes can be propagated together in a single mouse having two different human heavy chain transgenes (HCo7/HCol2).

XI.B. Generation of hybridomas producing monoclonal antibodies to TIM3.

To create hybridomas producing the anti-human TIM3 monoclonal antibodies described herein, splenocytes and/or lymph node cells from immunized mice can be isolated and fused with an appropriate immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, single-cell suspensions of spleen lymphocytes from immunized mice can be fused to non-secreting mouse Sp2/0 myeloma cells (ATCC, CRL 1581) with PEG. Cells can be seeded into a flat bottom microplate followed by incubation in selective media. After a few weeks, the cells can be cultured in the medium. Individual wells can then be screened by ELISA for human IgM and IgG monoclonal antibodies. When extensive hybridoma growth occurs, the environment can usually be observed

- 68 044112 in 10-14 days. Antibody-secreting hybridomas can be replicated, re-screened, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. Stable subclones can then be cultured in vitro to produce small amounts of antibodies in tissue culture medium for characterization.

To purify human monoclonal antibodies, selected hybridomas can be grown in two-liter monoclonal antibody purification flasks. Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, N.J.). Eluted IgG can be tested by gel electrophoresis and high performance liquid chromatography to ensure purity. The buffer solution can be replaced with PBS and the concentration can be determined from OD280 using an extinction coefficient of 1.43. Monoclonal antibodies can be aliquoted and stored.

XI.C. Generation of transfectomes producing monoclonal antibodies to TIM3.

Antibodies can be produced in a host cell transfectome using, for example, a combination of recombinant DNA methods and gene transfection methods, as is well known in the art (Morrison S. (1985) Science 229: 1202).

For example, to express antibodies or antibody fragments, DNA encoding partial or full-length light and heavy chains can be prepared by standard molecular biology techniques (eg, PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest), and the DNA can be inserted into expression vectors so that the genes are operably linked to transcriptional and translational control sequences. In this context, the term operably linked is intended to mean that the antibody gene is ligated into the vector such that the transcriptional and translational control sequences within the vector perform their intended function in regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene may be inserted into a separate vector, or both genes may be inserted into the same expression vector. Antibody genes are inserted into the expression vector(s) by standard methods (eg, ligation of complementary restriction sites on the gene fragment and the antibody vector, or blunt end ligation if restriction sites are not present). The light and heavy chain variable regions of the anti-TIM3 antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant regions and light chain constant regions of the desired isotype, so that the VH segment is functional associated with the CH segment(s) in the vector, and the V _L segment will be operably associated with the C _L segment in the vector.

Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

In exemplary embodiments, the following human antibody heavy and light chain signal peptides may be used: MDWTWRVFCLLAVAPGAHS (SEQ ID NO: 267); METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268); MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 269); MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270); MDMRVPAQLLGLLLWLPGARC (SEQ ID NO: 271) or MRAWIFFLLCLAGRALA (SEQ ID NO: 361). In a specific embodiment, the signal sequence used to express any of the anti-TIM3 antibodies described herein is SEQ ID NO: 361. The heavy and light chains of the anti-TIM3 antibodies can be expressed with the corresponding signal sequence that has been associated with each chain in hybridoma from which they were cloned. Below are the signal sequences of the various anti-TIM3 antibodies that are present in the hybridoma from which they were cloned, these signal sequences can be used to express the same antibody or a different antibody:

(i) amino acid sequence of the VH 13A3 signal sequence:

MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 269);

(ii) the nucleic acid sequence of the VH 13A3 signal sequence: ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCC (SEQ ID NO: 274);

(iii) amino acid sequence of the VL 13A3 signal sequence:

METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268);

(iv) nucleic acid sequence of the VL 13A3 signal sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ

- 69 044112

ID NO: 273);

(v) amino acid sequence of the VH 8B9 signal sequence:

MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 269);

(vi) nucleic acid sequence of the VH 8B9 signal sequence:

ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCC (SEQ ID NO: 274);

(vii) amino acid sequence of the VL 8B9 signal sequence:

METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268);

(viii) nucleic acid sequence of the VL8B9 signal sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACC GGA (SEQ ID NO: 273);

(ix) amino acid sequence of the VH 8C4 signal sequence:

MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 269);

(x) nucleic acid sequence of the VH 8C4 signal sequence:

ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCC (SEQ ID NO: 274);

(xi) amino acid sequence of the VL 8C4 signal sequence:

METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268);

(xii) nucleic acid sequence of the VL 8C4 signal sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 273);

(xiii) amino acid sequence of the VH 17C3 signal sequence:

MDWTWRVFCLLAVAPGAHS (SEQ ID NO: 267);

(xiv) nucleic acid sequence of the VH 17C3 signal sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 272);

(xv) amino acid sequence of the VL 17C3 signal sequence:

METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268);

(xvi) nucleic acid sequence of the VL 17C3 signal sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 273);

(xvii) amino acid sequence of the VH 9F6 signal sequence:

MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270);

(xviii) nucleic acid sequence of the VH 9F6 signal sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 275);

(xix) amino acid sequence of the VL1 9F6 signal sequence:

MDMRVTAQLLGLLLLWLPGARC (SEQ ID NO: 271);

(xx) nucleic acid sequence of the VL1 9F6 signal sequence:

ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTGGCTCCCAGGT GCCAGATGT (SEQ ID NO: 276);

(xxi) amino acid sequence of the VL2 and VL3 9F6 signal sequence:

METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268);

(xxii) 9F6 VL2 and VL3 signal sequence nucleic acid sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 273);

(xxiii) amino acid sequence of the VH 3G4 signal sequence:

MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270);

(xxiv) VH 3G4 signal sequence nucleic acid sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 275);

(xxv) amino acid sequence of the VL 3G4 signal sequence:

METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268);

(xxvi) VL 3G4 signal sequence nucleic acid sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 273);

(xxvii) amino acid sequence of the VH 17C8 signal sequence:

MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 270);

(xxviii) VH 17C8 signal sequence nucleic acid sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ ID NO: 275);

(xxix) amino acid sequence of the VL 17C8 signal sequence:

- 70 044112

METPAQLLFLLLLWLPDTTG (SEQ ID NO: 268);

(xxx) nucleic acid sequence of VL 17C8 signal sequence:

ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGA (SEQ

ID NO: 273).

In another embodiment, the heavy and light chains of anti-TIM3 antibodies (eg, TIM3.2-TIM3.18) can be engineered with signal sequences that differ from those present in the hybridomas from which they were cloned. Examples of such sequences include, but are not limited to, the following:

(i) the nucleic acid sequence of the signal sequence for the heavy chain: ATGAGGGCTTGGATCTTCTTTCTGCTCTGCCTGGCCGGGAGAGCGCTCGCA (SEQ ID NO: 362);

(ii) the nucleic acid sequence of the signal sequence for the light chain: ATGAGGGCTTGGATCTTCTTTCTGCTCTGCCTGGCCGGGCGCGCCTTTGGCC (SEQ ID NO: 363);

(iii) the amino acid sequence of the signal sequence for the heavy and light chains: MRAWIFFLLCLAGRALA (SEQ ID NO: 361).

In addition to antibody chain genes, recombinant expression vectors may carry regulatory sequences that control expression of antibody chain genes in a host cell. The term regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control transcription or translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will appreciate that the design of an expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (eg , adenovirus major late promoter (AdMLP) and polyomas. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or P-globin promoter, can be used. In addition, regulatory elements consisting of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8: 466-472).

In addition to antibody chain genes and regulatory sequences, recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US Patent Nos. 4,399,216, 4,634,665 and 5,179,017, all issued to Axel et al). For example, typically, a selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, to the host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the peo gene (for G418 selection).

To express the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell by standard methods. The various forms of the term transfection are intended to cover a wide range of methods typically used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, for example, electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

Although it is theoretically possible to express the anti-TIM3 antibodies described herein in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells and most preferably mammalian host cells is most preferred since such eukaryotic cells and especially mammalian cells are more likely to than prokaryotic cells, assemble and secrete a properly folded and immunologically active antibody. It has been reported that prokaryotic expression of antibody genes is inefficient for the production of active antibodies in high yield (Boss, M.A. and Wood, C.R. (1985), Immunology Today 6: 12-13).

Some mammalian host cells for expressing the recombinant anti-TIM3 antibodies described herein include Chinese hamster ovary cells (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220), used with a DHFR selectable marker, for example, as described in R.J. Kaufman and P.A. Sharp (1982), Mol. Biol. 759:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to permit expression of the antibody in the host cells or, more preferably, secretion of the antibody in the culture medium in which host cells are grown. Antibodies can be isolated from the culture medium using standard protein purification methods.

XII. Analyzes.

Anti-TIM3 antibodies described herein can be assayed for binding to human TIM3, for example, using a standard ELISA assay. Briefly, microtiter plates are coated with purified TIM3 and then blocked with bovine serum albumin. Antibody dilutions (eg, dilutions of plasma from mice immunized with TIM3) are added to each well and incubated. The plates are washed and incubated with a secondary reagent (eg, for human antibodies, human Fc IgG specific goat polyclonal reagent) conjugated to horseradish peroxidase (HRP). After washing, the plates can be developed and analyzed using a spectrophotometer. Serum from immunized mice can then be further screened by flow cytometry for binding to a cell line expressing human TIM3, but not to a control cell line that does not express TIM3.

Briefly, the binding of anti-TIM3 antibodies can be assessed by incubating TIM3-expressing CHO cells with anti-TIM3 antibody. Cells can be washed and binding can be detected using anti-human IgG antibodies. Flow cytometric analysis can be performed using FACScan flow cytometry (Becton Dickinson, San Jose, CA). The mice that develop the highest titers can be used for fusions.

The ELISA assay described above can be used to screen for antibodies and thus hybridomas that produce antibodies that exhibit positive reactivity with the TIM3 immunogen. Hybridomas that produce antibodies that bind with high affinity to TIM3 can then be subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the original cells (by ELISA) can then be selected for cell banking and antibody purification.

To purify antibodies to TIM3, selected hybridomas can be grown to purify monoclonal antibodies. Supernatants can be filtered and concentrated before affinity chromatography. Eluted IgG can be tested by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer solution can be replaced and the concentration can be determined. Monoclonal antibodies can be aliquoted and stored.

To determine whether selected anti-TIM3 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Biotinylated monoclonal antibody binding can be detected using a streptavidin-labeled probe. Competition assays using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using TIM3-coated ELISA plates as described above.

To determine the isotype of purified antibodies, an isotyping ELISA assay can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, microtiter plate wells can be coated with 1 μg/mL anti-human immunoglobulin antibody overnight at 4°C. After blocking with 1% BSA, the plates are reacted with 1 μg/ml or less of the monoclonal antibodies of interest or purified isotype controls at ambient temperature for one to two hours. The wells can then be reacted with either human IgG1 or human IgM specific alkaline phosphatase-conjugated probes. The plates were developed and analyzed as described above.

Flow cytometry can be used to study the binding of monoclonal antibodies to living cells expressing TIM3, as described in the Examples. Briefly, cell lines expressing membrane-bound TIM3 (grown under standard growth conditions) were mixed with various concentrations of monoclonal antibodies in PBS containing 0.1% BSA at 4°C for 1 hour. After washing, the cells are reacted with fluorescein-labeled anti-IgG antibody under the same conditions as the primary antibody staining. Samples can be analyzed with a FACScan instrument using light and side scatter properties to gate individual cells and determine the binding of labeled antibodies. An alternative analysis using fluorescence microscopy analysis (in addition to or instead of) flow cytometry may be used. Cells can be stained exactly as described above and examined using fluorescence microscopy. This method allows the visualization of individual cells, but may have reduced sensitivity depending on the density of the antigen.

Antibodies to TIM3 can be further tested for reactivity with TIM3 antigen using Western blotting. Briefly, cell extracts from cells expressing TIM3 can be prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked with 20% mouse serum and probed with the monoclonal antibodies of interest. IgG binding can be detected using anti-IgG alkaline phosphatase and demonstrated using BCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, MO).

Methods for analyzing the binding affinity, cross-reactivity and binding kinetics of various anti-TIM3 antibodies include standard assays known in the art, for example, the BIACORE™ surface plasmon resonance (SPR) assay using the BIACORE™ 2000 SPR instrument (Biacore AB, Uppsala, Sweden).

To determine the biological activity of anti-TIM3 antibodies (which can be used, for example, to compare different anti-TIM3 antibodies), various assays, such as those described herein, can be used:

(1) T cell activation assays, such as assays using purified T cells derived from human donor PBMCs. Assays can be performed on all T cells or subsets thereof, eg Th1 cells, cytotoxic T cells, Treg cells, CD4+ T cells, CD8+ T cells, provided they express TIM3. Activation can be measured by determining the level of secretion of certain cytokines, such as interferon-γ or IL-2, or the level of T cell proliferation. Without limitation to a specific mechanism of action, binding of anti-TIM3 antibodies to TIM3 on T cells may prevent TIM3 from binding to a TIM3 ligand (putative TIM3 ligands include galectin-9, HMGB1, semaphorin-4A, CEACAM-1, ILT-4, and phosphatidylserine) and thereby prevent TIM3-mediated T cell signaling, thereby preventing negative regulation of T cells by TIM3. Exemplary assays, including Th1 assays, TIL assays, and mixed lymphocyte culture reactions (MLRs), are presented in the Examples;

(2) assays measuring stimulation of macrophages, such as M1 or M2 macrophages; and (3) assays measuring the secretion of myeloid-associated cytokines, such as TNFa, Π-1β, GM-CSF, IL-6, IL-2, IL-10, CCL2, CCL3, CCL4, or CCL5 from TIM3-positive myeloid cells. cells. In a certain embodiment, anti-TIM3 antibodies stimulate the secretion of TNFa, IL-1e, GM-CSF, IL-6, and IL-2 and/or inhibit the secretion of IL-10, CCL2, CCL3, CCL4, or CCL5 from TIM3 positive myeloid cells.

In general, any method for testing the biological activity of an agent that inhibits immune responses can be used to characterize the biological activity of anti-TIM3 antibodies, such as those described in the literature (including patents and patent applications) relating to TIM3.

XIII. Immunoconjugates, antibody derivatives and diagnostics.

The anti-TIM3 antibodies described herein can be used for diagnostic purposes, including specimen testing and in vivo imaging, and for this purpose, the antibody (or binding fragment thereof) can be conjugated to a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable detectable tags include radioisotopes for whole-body imaging, and radioisotopes, enzymes, fluorescent tags, and other suitable antibody tags for specimen testing.

Detectable labels that may be associated with any anti-TIM3 antibody described herein can be any of the various types currently used in the in vitro diagnostic field, including particulate labels, including metal sols such as colloidal gold, isotopes such as I ¹²⁵ or Tc ⁹⁹ , presented, for example, with a peptide chelating agent such as N2S2, N ₃ S or N ₄ , chromophores, including fluorescent markers, luminescent markers, phosphorescent markers and the like, as well as enzyme tags that convert a given substrate into a detectable marker, and polynucleotide markers that are detected after amplification, for example, using polymerase chain reaction. Suitable enzyme tags include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label may be the enzyme alkaline phosphatase, determined by measuring the presence or production of chemiluminescence following the conversion of 1,2-dioxetane substrates such as adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3, 2'(5'-chloro)tricyclo{3.3.1.13,7}decane}-4-yl)phenylphosphate (CSPD), as well as CDP and CDP-STAR® or other luminescent substrates well known to those skilled in the art, e.g. , chelates of suitable lanthanides such as terbium(III) and europium(III).The means of detection is determined by the label chosen.The appearance of the label or its reaction products may be visible to the naked eye in the case where the label contains particles and accumulates in appropriate quantities, or with using instruments such as spectrophotometer, luminometer, fluorimeter, etc., all in accordance with standard practice.

In some embodiments, conjugation methods result in bonds that are substantially (or substantially) non-immunogenic, such as peptide (ie, amide), sulfide, (sterically hindered), disulfide, hydrazone, and ester bonds. These bonds are almost non-immunogenic and show reasonable stability in serum (see e.g.

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Senter, P. D., Curr. Opin. Chem. Biol. 13 (2009) 235-244; WO 2009/059278; WO 95/17886).

Depending on the biochemical nature of the fragment and antibody, different conjugation strategies can be used. In the case where the fragment is naturally occurring or recombinant from 50-500 amino acids, standard procedures are described in textbooks describing the chemistry of the synthesis of protein conjugates, which can be easily performed by one skilled in the art (see, for example, Hackenberger, S.R. R., and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In one embodiment, the maleimide moiety is reacted with a cysteine residue in the antibody or fragment. This is a particularly suitable coupling chemistry when, for example, a Fab or Fab' fragment of an antibody is used. Alternatively, in one embodiment, the connection is made to the C-terminus of the antibody or fragment. C-terminal modification of a protein, for example a Fab fragment, can be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

In general, site-specific reaction and covalent binding rely on the conversion of a naturally occurring amino acid into an amino acid with a reactivity that is orthogonal to the reactivity of other functional groups present. For example, a specific cysteine in the context of a rare sequence can be enzymatically converted to an aldehyde (see Frese, M. A., and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modification by exploiting the specific enzymatic reactivity of certain enzymes with a natural amino acid in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al. Chem Biol. 15 (2008) 128-136, and protease-catalyzed C-N bond formation is used by Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site-specific reaction and covalent binding can also be achieved by selectively reacting terminal amino acids with suitable modifying reagents.

The reactivity of N-terminal cysteine with benzonitriles (see Ren, H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent binding.

Native chemical ligation may also depend on C-terminal cysteine residues (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

US Pat. No. 6,437,095 B1 describes a conjugation method that is based on a faster reaction of a cysteine at a site with negatively charged amino acids with a cysteine located at a site with positively charged amino acids.

The fragment may also be a synthetic peptide or a peptide mimic. In the case where the polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity may be included during such synthesis (see, for example, de Graaf, A. J. et al., Bioconjug. Chem. 20 (2009) 1281-1295). Since there is a wide variety of orthogonal functional groups that can be introduced into a synthetic peptide, conjugation of such a peptide to a linker is standard chemistry.

To obtain a monolabeled polypeptide, the conjugate with a 1:1 stoichiometry can be separated by chromatography from other conjugation byproducts. This procedure can be facilitated by using a dye labeled with a member of the binding pair and a charged linker. By using this type of labeled and highly negatively charged member of the binding pair, monoconjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides that carry more than one linker, since the difference in charge and molecular weight can be used for separation. A fluorescent dye may be useful to purify the complex from unbound components, such as a labeled monovalent binder.

In one embodiment, the moiety attached to the anti-TIM3 antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety.

Anti-TIM3 antibodies described herein can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC). Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In an ADC, the antibody and therapeutic agent are preferably conjugated through a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. In other embodiments, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 300), Ala-Asn -Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. ADCs can be prepared as described in US Pat. No. 7,087,600; 6989452 and 7129261; publications according to PCT WO 02/096910; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312 and WO 08/103693; US Patent Publications 20060024317; 20060004081 and 20060247295.

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Antibodies to TIM3, such as those described herein, can also be used to detect TIM3, such as human TIM3, such as human TIM3, in tissues or tissue samples. Antibodies can be used, for example, in an ELISA assay or in flow cytometry. In some embodiments, the anti-TIM3 antibody is contacted with cells, eg, cells in a tissue, for a time suitable for specific binding, and then a reagent, eg, an antibody that detects the anti-TIM3 antibody, is added. Illustrative analyzes are presented in the examples. The anti-TIM3 antibody may be a fully human antibody, or it may be a chimeric antibody, such as an antibody containing human variable regions and mouse constant regions or a portion thereof. Exemplary methods for detecting TIM3, e.g., human TIM3, in a sample (cell or tissue sample) involve (1) contacting the sample with an anti-TIM3 antibody for a time sufficient to ensure specific binding of the anti-TIM3 antibody to TIM3 in the sample and (2) contacting the sample with a detection reagent, for example an antibody, that specifically binds to an anti-TIM3 antibody, such as the Fc region of the anti-TIM3 antibody, to thereby detect TIM3 bound by the anti-TIM3 antibody. Washing steps may be included after incubation with the antibody and/or detection reagent. Antibodies to TIM3 for use in these methods do not need to be associated with a label or detection means, since a separate detection means can be used.

Other uses for anti-TIM3 antibodies, for example as monotherapy or combination therapy, are presented elsewhere herein, for example in the section relating to combination therapy.

XIV. Bispecific molecules.

Anti-TIM3 antibodies described herein can be used to generate bispecific molecules. The anti-TIM3 antibody or antigen-binding portions thereof may be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to produce a bispecific molecule that binds to at least two different binding sites or molecules -targets. For example, an anti-TIM3 antibody may be coupled to an antibody or scFv that specifically binds to any protein that may be used as potential targets for combination therapy, such as the proteins described herein (e.g., anti-PD-1, PD- L1, GITR or LAG-3). An antibody described herein may in fact be produced by or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term bispecific molecule as used herein. To create a bispecific molecule described herein, an antibody described herein can be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules, such as another antibody, an antibody fragment, peptide or binding mimetic, so that a bispecific molecule is obtained.

Accordingly, provided herein are bispecific molecules comprising at least one first binding specificity for TIM3 and a second binding specificity for a second target epitope. According to an embodiment described herein, in which the bispecific molecule is multispecific, the molecule may further include a third binding specificity.

In one embodiment, the bispecific molecules described herein comprise as binding specificity at least one antibody or antibody fragment, including, for example, Fab, Fab', F(ab')2, Fv or single chain Fv (scFv). The antibody may also be a light or heavy chain dimer or any minimal fragment thereof, such as an Fv or single chain construct, as described in US Pat. No. 4,946,778 to Ladner et al.

Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules described herein are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules described herein can be prepared by conjugating the binding specificities of the components using methods known in the art. For example, each binding specificity of a bispecific molecule can be prepared separately and then conjugated to each other. When the binding specificities are proteins or peptides, various binding or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, Nsuccinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), phenylenedimaleimide (oPDM), N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP) and sulfosuccinimidyl-4-(M-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu , M. A. et al (1985) Proc. Natl. Acad. Sci. USA 82:8648). other methods

- 75 044112 include the methods described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al.

(1985) Science 229:81-83 and Glennie et al. (1987) J. Immunol. 139: 2367-2375. Some conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the binding specificities are antibodies, they can be conjugated through a sulfhydryl bond at the C-terminal hinge regions of the two heavy chains. In a particular embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly applicable when the bispecific molecule is an mAbxmAb, mAbxFab, mAbx(scFv) ₂ , FabxF(ab') ₂ fusion protein or a χFab ligand. The bispecific antibody may comprise an antibody containing an scFv at the C-terminus of each heavy chain. A bispecific molecule described herein may be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may contain at least two single-chain molecules. Methods for producing bispecific molecules are described, for example, in US patent No. 5260203; US Patent No. 5455030; US Patent No. 4881175; US Patent No. 5132405; US patent No. 5091113; US Patent No. 5477786; US patent No. 5013653; US Patent No. 5258988 and US Patent No. 5482858.

The binding of bispecific molecules to their specific targets can be confirmed using methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (eg, growth inhibition) or Western blot analysis. Each of these assays typically detects the presence of protein-antibody complexes of particular interest using a labeled reagent (eg, antibody) specific for the complex of interest.

XV. Compositions.

Additionally provided are compositions, for example pharmaceutical compositions, comprising one or a combination of anti-TIM3 antibodies or a combination with antibodies to other targets, or antigen-binding moiety(s) thereof, described herein, formulated together with a pharmaceutically acceptable carrier. Such compositions may include one or a combination (eg, two or more different) antibodies or immunoconjugates or bispecific molecules described herein. For example, a pharmaceutical composition described herein may contain a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on a target antigen or that have complementary activities.

In some embodiments, the composition contains an anti-TIM3 antibody at a concentration of at least 1, 5, 10, 50, 100, 150, 200, 1-300 mg/ml, or 100-300 mg/ml.

The pharmaceutical compositions described herein can also be administered in a combination mode of treatment, i.e. in combination with other means. For example, a combination treatment may include an anti-TIM3 antibody described herein in combination with at least one other antitumor and/or immunomodulatory, such as T cell stimulating (eg, activating) agent. Examples of therapeutic agents that can be used in a combination treatment are described in more detail below in the section on the use of anti-TIM3 antibodies described herein.

In some embodiments, the therapeutic compositions disclosed herein may include other compounds, drugs, and/or agents used to treat cancer. Such compounds, drugs and/or agents may include, for example, chemotherapeutic drugs, small molecule drugs or antibodies that stimulate an immune response to the cancer. In some cases, the therapeutic compositions may include, for example, one or more of an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-OX40 antibody (also known as CD134, TNFRSF4, ACT35, and/or TXGP1L ), anti-CD137 antibodies, anti-LAG-3 antibodies, anti-GITR antibodies, or any combination thereof.

As used herein, the term pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retarding agents, and the like, which are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active substance, i.e. the antibody, immunoconjugate, or bispecific molecule may be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound.

The pharmaceutical compounds described herein may include one or

- 76 044112 several pharmaceutically acceptable salts. A pharmaceutically acceptable salt refers to a salt that retains the desired biological activity of the parent compound and does not cause any undesirable toxicological effects (see, for example, Berge, S. M., et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphoric and the like, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids , phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., as well as from non-toxic organic amines such as N,N'-dibenzylethylenediamine, Nmethylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

The pharmaceutical composition described herein may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) fat-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc. and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper flow can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain additional substances such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be achieved both by the sterilization procedures above and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and means for pharmaceutically active substances is known in the art. Unless any conventional carrier or agent is incompatible with the active compound, it is intended for use in the pharmaceutical compositions described herein. The pharmaceutical composition may or may not contain a preservative. Additional active compounds may be included in the compositions.

Therapeutic compositions generally must be sterile and stable under conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper flow can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. In many cases, the compositions may include in the composition isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate and gelatin salts.

Sterile injection solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above ingredients, as required, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains the basic dispersion medium and the required other ingredients listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, some preparation methods are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredient from its pre-sterile-filtered solution.

The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will vary depending on the subject being treated and the particular route of administration. The amount of active ingredient that can be combined with the carrier material to form a unit dosage form will generally be that amount of the composition that produces a therapeutic effect. Typically, of one hundred percent, this amount will be from about 0.01% to about ninety-nine percent of the active ingredient, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient in combination with a pharmaceutically acceptable carrier.

Dosing regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased as indicated, depending on the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier. The description of the unit dosage forms described herein is dictated by and is directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of preparing such an active compound for the treatment of sensitivity in individuals.

For administration of an anti-TIM3 antibody, such as that described herein, the dosage ranges from about 0.0001 to 100 mg/kg and, more typically, from 0.01 to 5 or 10 mg/kg host body weight. For example, dosages may be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, or 10 mg/kg body weight, or in the range of 1-10 mg/kg . An exemplary treatment regimen is administered once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every 3 months, or once every 3-6 months. Exemplary dosing regimens for the anti-TIM3 antibody described herein include 1 mg/kg body weight or 3 mg/kg body weight via intravenous administration, with the antibody administered using one of the following dosing regimens: (i) every four weeks for six dosed injections, then every three months; (ii) every three weeks; (iii) 3 mg/kg body weight once, then 1 mg/kg body weight every three weeks.

The anti-TIM3 antibody can be administered at a constant dose (continuous dose regimen). In other embodiments, the anti-TIM3 antibody may be administered at a fixed dose with another antibody. In certain embodiments, the anti-TIM3 antibody is administered at a dose based on body weight.

In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered falls within specified ranges. The antibody is usually administered several times. The intervals between single doses may be, for example, a week, a month, three months or a year. The intervals may also be irregular, as indicated by measuring the level of antibodies in the patient's blood to the target antigen. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of approximately 1-1000 μg/ml, and in some methods, approximately 25-300 μg/ml.

The anti-TIM3 antibody can be administered with another antibody in a different antibody dosing regimen. For example, an anti-TIM3 antibody can be administered with an anti-PD-1 antibody such as nivolumab (OPDIVO®) every two weeks as an intravenous infusion over 60 minutes until disease progression or unacceptable toxicity. Anti-TIM3 antibody can be administered with pembrolizumab (KEYTRUDA®) every 3 weeks as an intravenous infusion over 30 minutes until disease progression or unacceptable toxicity. Anti-TIM3 antibody can be administered with atezolizumab (TECENTRIQ™) every 3 weeks as an intravenous infusion over 60 or 30 minutes until disease progression or unacceptable toxicity.

The antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. For therapeutic applications it is sometimes necessary

- 78 044112 relatively high dose at relatively short intervals until disease progression decreases or stops and the patient shows partial or complete improvement in disease symptoms. After this, the patient can be prescribed a prophylactic regimen.

The actual dosage levels of the active ingredients in the pharmaceutical compositions described herein can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration without being toxic to the patient. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular compositions described herein or their ester, salt or amide, route of administration, time of administration, rate of elimination of the particular compound, time of administration, duration of treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated and similar factors well known in medicine.

A therapeutically effective dosage of an anti-TIM3 antibody described herein may result in a reduction in the severity of symptoms of a disease, an increase in the frequency and duration of periods without symptoms of a disease, or the prevention of impairment or disability due to a disease. In the context of cancer, a therapeutically effective dose may result in increased survival, such as overall survival, and/or prevention of further worsening of physical symptoms associated with cancer. Symptoms of cancer are well known in the art and include, for example, unusual features of a mole, a change in the appearance of a mole including asymmetry, border, color and/or diameter, a newly pigmented area of skin, an abnormal mole, a darkened area under the nail, lumps in the breast, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, difficulty eating, loss of appetite, chronic cough, worsening shortness of breath, coughing up blood, blood in the urine, blood during bowel movements, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal fullness, flatulence, abdominal fluid, vaginal bleeding, constipation, bloating, colon perforation, acute peritonitis (infection, fever, pain), pain, vomiting blood, heavy sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastases, lung metastases, bladder metastases, liver metastases, bone metastases, metastases in the kidneys and metastases in the pancreas, difficulty swallowing, etc.

A therapeutically effective dose may prevent or delay the onset of cancer, which may be desirable when early or preliminary signs of the disease are present. Laboratory tests used in cancer diagnosis include chemistry (including TIM3 measurements), hematology, serology and radiology. Accordingly, any clinical or biochemical assay that monitors any of the above can be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer. One skilled in the art will be able to determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration chosen.

The composition described herein can be administered by one or more routes of administration, using one or more of a variety of methods known in the art. As will be appreciated by one skilled in the art, the route and/or method of administration will vary depending on the desired results. Routes of administration of anti-TIM3 antibodies described herein may include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as by injection or infusion. As used herein, the phrase parenteral administration means methods of administration other than enteral and local administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrathoracic injection and infusion.

Alternatively, an antibody described herein can potentially be administered by a non-parenteral route, such as a topical, epidermal or mucosal route, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be formulated with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for preparing such compositions are patented or well known to those skilled in the art. Look,

- 79 044112 e.g. Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc.,

New York, 1978.

Therapeutic compositions can be administered using medical devices known in the art. For example, according to a specific embodiment, a therapeutic composition described herein can be administered using a needleless subcutaneous injection device, such as the devices disclosed in US Pat. No. 5,399,163; 5383851; 5312335; 5064413; 4941880; 4,790,824 or 4,596,556. Examples of well-known implants and modules for use with the anti-TIM3 antibodies described herein include: US Pat. No. 4,487,603, which discloses an implantable microinfusion pump for controlled rate drug dosing; US Pat. No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; US Pat. No. 4,447,233, which discloses a drug infusion pump for delivering a drug at a precise infusion rate; US Patent No. 4,447,224, which discloses an implantable variable-flow infusion device for continuous drug delivery; US Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments, and US Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems and modules are known to those skilled in the art.

In certain embodiments, anti-TIM3 antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if necessary, for example in brain cancer), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, US Pat. No. 4,522,811; 5374548 and 5399331. Liposomes may contain one or more groups that are selectively transported to specific cells or organs, thereby improving targeted drug delivery (see, for example, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685) . Exemplary targeting moieties include folate or biotin (see, for example, US Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBSLett. 346: 123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4:273.

XVI. Applications and methods.

The antibodies, antibody compositions and methods described herein have numerous beneficial properties in vitro and in vivo, including, for example, enhancing the immune response, for example, by inhibiting (or antagonizing) TIM3 (eg, signaling) or detecting TIM3. In one embodiment, the anti-TIM3 antibodies described herein are human antibodies. For example, anti-TIM3 antibodies described herein can be administered to cells in culture in vitro or ex vivo or to human subjects, for example, in vivo, to enhance immunity in various diseases. Accordingly, provided herein are methods of modifying the immune response in a subject, comprising administering to the subject an anti-TIM3 antibody or an antigen-binding portion thereof described herein such that the immune response in the subject is modified. In some embodiments, the response is enhanced, stimulated, or activated.

Subjects suitable for the present methods include human patients in whom an enhanced immune response would be desired. The methods are particularly suitable for treating human patients having a disorder that can be treated by enhancing an immune response (eg, a T cell-mediated immune response, eg, an antigen-specific T cell response). In a specific embodiment, the methods are particularly suitable for treating cancer in vivo. To achieve antigen-specific immune enhancement, the anti-TIM3 antibodies described herein may be administered together with the antigen of interest, or the antigen may already be present in the subject being treated (eg, a subject harboring a tumor or virus). When anti-TIM3 antibodies are administered along with another agent, both may be administered separately or simultaneously.

Also covered are methods for detecting the presence of human TIM3 antigen in a sample or measuring the amount of human TIM3 antigen, comprising contacting the sample and a control sample with a monoclonal antibody, e.g., a human monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to human TIM3, under conditions that allow formation of a complex between an antibody or part thereof and human TIM3. Complex formation is then detected, with the difference in complex formation between the sample compared to the control sample indicating the presence of human TIM3 antigen in the sample. In addition, the anti-TIM3 antibodies described herein can be used to purify human TIM3 using immunoaffinity purification.

Given the ability of the anti-TIM3 antibodies described herein to stimulate or co-stimulate T cell responses, e.g., antigen-specific T cell responses, such as inhibiting the negative effects of TIM3 presented herein, methods for in vitro and in vivo use of those described herein are provided herein. document anti-TIM3 antibodies to stimulate, enhance, or activate antigen-specific T cell responses, such as antitumor T cell responses. In some embodiments, CD3 stimulation is also provided (eg, by co-incubation with a cell expressing membrane CD3), which stimulation may be provided at the same time, before or after stimulation with an anti-TIM3 antibody. For example, methods for stimulating an antigen-specific T cell response are provided herein by contacting said T cell with an anti-TIM3 antibody described herein and optionally an anti-CD3 antibody such that an antigen-specific T cell response is stimulated.

Any suitable indicator of antigen-specific T-cell response can be used to measure the antigen-specific T-cell response. Non-limiting examples of such suitable indicators include increased T cell proliferation in the presence of an antibody and/or increased production of cytokines in the presence of an antibody. In some embodiments, production of interleukin-2 and/or interferon-γ by an antigen-specific T Cell is stimulated.

T cells that can be enhanced or co-stimulated by anti-TIM3 antibodies include CD4+ T cells and CD8+ T cells. The T cells may be Teff cells, eg, CD4 ⁺ Teff cells, CD8+ Teff cells, T helper (Th) cells (eg, Th1 cells), or T cytotoxic (Tc) cells.

Further covered are methods of stimulating an immune response (eg, an antigen-specific T-cell response) in a subject, comprising administering an anti-TIM3 antibody described herein to the subject such that an immune response (eg, an antigen-specific T-cell response) in the subject is stimulated. In some embodiments, the subject is a tumor carrier and an immune response against the tumor is stimulated. The tumor may be a solid tumor or a liquid tumor, such as a hematologic malignancy. In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is not immunogenic. In certain embodiments, the tumor is PD-L1 positive. In some embodiments, the tumor is PD-L1 negative. The subject may also be a carrier of the virus, and an immune response against the virus is stimulated.

Further provided are methods of inhibiting the growth of tumor cells in a subject, comprising administering to the subject an anti-TIM3 antibody described herein such that tumor growth in the subject is inhibited. Methods of treating a viral infection in a subject are also provided, comprising administering to the subject an anti-TIM3 antibody described herein such that the viral infection in the subject is treated.

In some embodiments, the anti-TIM3 antibody is administered to a subject as an additional treatment. Treatment of subjects with cancer with an anti-TIM3 antibody may result in prolonged survival, such as long-term long-term response, compared with the current standard of care; long-term survival of at least 3, 6, 9 months, 1, 2, 3, 4, 5, 10 or more years or disease-free survival of at least 3, 6, 9 months, 1, 2, 3, 4 , 5 or 10 or more years. In certain embodiments, treatment of a subject having cancer with an anti-TIM3 antibody prevents recurrence of the cancer or delays recurrence of the cancer, for example, 3, 6, 9 months, 1, 2, 3, 4, 5, or 10 or more years. Treatment with anti-TIM3 antibodies can be used as first, second or third line treatment.

Treatment of a subject with cancer with an anti-TIM3 antibody described herein, e.g., TIM3.18.IgG1, may result in, for example, stable disease, partial response, prolonged overall survival, prolonged disease-free survival, or improved progression-free survival.

In certain embodiments, an anti-TIM3 antibody described herein is not significantly toxic. For example, an anti-TIM3 antibody is not significantly toxic to a human organ, such as one or more of the liver, kidney, brain, lung, and heart, as determined, for example, in clinical trials. In some embodiments, the anti-TIM3 antibody does not cause a significant adverse immune response, such as autoimmunity or inflammation.

In some embodiments, treatment of a subject with a TIM3 antagonist (e.g., an anti-TIM3 antibody described herein) does not result in excessive stimulation of the immune system.

- 81 044112 system to such an extent that the subject's immune system then attacks the subject itself (eg, an autoimmune response) or leads, for example, to anaphylaxis. Thus, in some embodiments, anti-TIM3 antibodies do not cause anaphylaxis.

In some embodiments, treatment of a subject with an anti-TIM3 antibody described herein, e.g., an antibody containing the CDRs or variable regions of 13A3 or a variant thereof (e.g., as described herein), or other anti-TIM3 antibodies described herein, does not cause significant inflammatory reactions, for example, immune-mediated pneumonitis, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis or renal dysfunction, immune-mediated hypophysitis, immune-mediated hypothyroidism and hyperthyroidism or other mediated adverse reactions. In some embodiments, an anti-TIM3 antibody containing CDRs or 13A3 variable regions or a variant thereof (e.g., as described herein) causes fewer inflammatory responses, e.g., immune-mediated pneumonitis, immune-mediated colitis, immune-mediated hepatitis, immune-mediated nephritis, or immune-mediated renal dysfunction hypophysitis, immune-mediated hypothyroidism and hyperthyroidism, anaphylaxis or other immune-mediated adverse reactions than other anti-TIM3 antibodies. In some embodiments, treatment of a subject with an anti-TIM3 antibody described herein, e.g., an antibody containing the CDRs or variable regions of 13A3 or a variant thereof (e.g., as described herein), or other anti-TIM3 antibodies described herein, does not cause significant cardiac disorders, such as ventricular arrhythmia; visual disturbances, such as iridocyclitis; infusion reactions; increased amylase, increased lipase; Nervous system disorders such as dizziness, peripheral and sensory neuropathy; diseases of the skin and subcutaneous tissue, for example, rash, itching, exfoliative dermatitis, erythema multiforme, vitiligo or psoriasis; respiratory, thoracic and mediastinal disorders, such as cough; fatigue; nausea; decreased appetite; constipation; arthralgia or diarrhea.

In some embodiments, an anti-TIM3 antibody provides a synergistic anti-tumor effect in combination with another cancer treatment, such as a compound that stimulates the immune system (e.g., an immuno-oncology agent), such as a compound described herein or a target modulating compound described herein .

The use of human antibodies, as opposed to chimeric or humanized antibodies, may result in decreased anti-drug antibody (ADA) levels. Accordingly, anti-human TIM3 antibodies described herein, e.g., TIM3.18.IgG1.3, may have a lower ADA compared to anti-TIM3 antibodies that are not human antibodies (e.g., relatively humanized or chimeric anti-TIM3 antibodies) .

These and other methods described herein are discussed in more detail below. XVI.A. Cancer.

Inhibition of TIM3 by anti-TIM3 antibodies may enhance the immune response to cancer cells in a patient with cancer. Provided herein are methods of treating a subject characterized by the presence of cancer, comprising administering to the subject an anti-TIM3 antibody described herein such that the subject is treated, for example, such that the growth of cancerous tumors is inhibited or reduced and/or the tumor regresses, and/or that long-term survival is achieved. Antibody to TIM3 can be used alone to inhibit the growth of malignant tumors. Alternatively, an anti-TIM3 antibody may be used in combination with another agent, such as another immunogenic agent, a conventional anti-cancer agent, or another antibody described below.

Accordingly, provided herein are methods of treating cancer, for example, by inhibiting the growth of tumor cells in a subject, comprising administering to the subject a therapeutically effective amount of an anti-TIM3 antibody described herein, eg, TIM3.2, TIM3.4, TIM3.5, TIM3. 6, 9F6, 8B9, TIM3.9, TIM3.10, TIM3.11, TIM3.12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.17, TIM3.18, TIM3.7 and TIM3.8 containing a wild-type IgG constant region or a constant region having reduced effector function, such as IgG1.1 or IgG1.3, or an antigen-binding portion thereof. The antibody may be an anti-human TIM3 antibody (such as any anti-human TIM3 antibody described herein). Cancers whose growth can be inhibited using the antibodies of the present invention include cancers that typically respond to immunotherapy and those that typically do not respond to immunotherapy. Types of cancer that can be treated also include TIM3-positive cancers. The cancer may be a cancer with solid tumors or blood malignancies (liquid tumors). Non-limiting examples of cancers treated include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous cell non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (eg, clear cell carcinoma), ovarian cancer, liver cancer,

- 82 044112 colorectal cancer, endometrial cancer, kidney cancer (for example, renal cell carcinoma (RCC)), prostate cancer (for example, hormone-refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cancer cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon cancer and head and neck cancer (or carcinoma), stomach cancer, germ cell tumor, childhood sarcoma, sinonasal tumor, melanoma (eg, metastatic malignant melanoma, such such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer systems, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis carcinoma, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal tumor , brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmental cancers, including asbestos-related cancers, virus-related cancers, or viral-related cancers ( for example, human papillomavirus (HPV-related or HPV-derived tumors)), as well as hematological malignancies originating from one of the two main blood cell lines, i.e. myeloid cell line (which produces granulocytes, red blood cells, platelets, macrophages and mast cells) or lymphoid cell line (which produces B, T, NK and plasma cells) such as all types of leukemia, lymphoma and myeloma, such as acute, chronic, lymphocytic and/or myelogenous leukemias such as acute leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), undifferentiated AML (UM), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cell maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7) , isolated granulocytic sarcoma and chloroma; lymphomas such as Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies such as B-cell lymphomas, T-cell lymphomas, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma (mucosal associated lymphoid tissue (MALT) ) lymphoma, anaplastic (eg, Ki 1+) large cell lymphoma, T-cell lymphoma/adult leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, T - lymphoblastic leukemia from progenitor cells, T-lymphoblastic and lymphoma/leukemia (T-Lbly/T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative pathology, true histiocytic lymphoma, primary lymphoma of the central nervous system, primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), hematopoietic tumors of lymphoid origin, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, B-lymphoma flipper leukemia from progenitor cells, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sezary syndrome) and lymphoplasmacytoid lymphoma (LPL) with Waldenström's macroglobulinemia; myelomas such as IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma and multiple myelomas, chronic lymphocytic leukemia (CLL), villous cell lymphoma; hematopoietic tumors of myeloid origin, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; seminoma, teratocarcinoma, tumors of the central and peripheral nervous system, including astrocytoma, schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscaroma and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid cancer and teratocarcinoma, hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors, including, but not limited to, T-cell disorders , such as T-cell and prolymphocytic leukemia (T-PLL), including small cell and cerebriform types; large granular lymphocytic leukemia (LGL) T cell type; a/d T-NHL hepatosplenic lymphoma; peripheral/postthymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; head or neck cancer, kidney cancer, colorectal cancer, thyroid cancer; acute myeloid lymphoma, as well as any combination of these types of cancer. The methods described herein can also be used to treat metastatic cancer, inoperable, resistant cancers (eg, cancers refractory to previous immunotherapy, such as with a CTLA-4 or PD-1 blocking antibody), and/or recurrent cancers.

In some embodiments, an anti-TIM3 antibody is administered to patients having cancer that has shown an inadequate response or has progressed with prior treatment, such as prior treatment with an immuno-oncology or immunotherapeutic drug.

- 83 044112 medicinal drug, or patients characterized by the presence of a cancer that is resistant or resistant, or inherently resistant or resistant (for example, resistant to a PD-1 pathway antagonist), or in which a resistant or resistant state is acquired. For example, subjects who do not respond or insufficiently respond to a first treatment or who see disease progression after treatment, such as treatment with an anti-PD-1 antibody, can be treated by administering an anti-TIM3 antibody alone or in combination with another treatment (eg, anti-PD-1 antibody). PD-1 treatment).

In some embodiments, the anti-TIM3 antibody is administered to patients who have not previously received (ie, been treated with) an immuno-oncology agent, such as a PD-1 pathway antagonist.

In some embodiments, a method of treating cancer in a subject comprises first determining whether the subject is TIM3 positive, e.g., has cancer cells or TILs that express TIM3, and if the subject has TIM3 positive cancer or TIL cells , then administering an anti-TIM3 antibody, such as that described herein, to the subject. A method of treating a subject having cancer with an anti-TIM3 antibody may comprise administering to the subject having cancer cells or TIL cells that express TIM3 a therapeutically effective amount of an anti-TIM3 antibody. Also provided herein are methods for predicting whether a subject will respond to treatment with an anti-TIM3 antibody, wherein the methods include determining the level of TIM3 in cancer cells or TIL cells of the subject, and if the cancer cells or TIL cells of the subject are TIM3 positive, then the subject , will respond to treatment with anti-TIM3 antibody.

In certain embodiments, a method of treating cancer in a subject comprises first determining whether the subject is PD-L1 or PD-1 positive, e.g., has cancer cells or TILs that express PD-L1 or PD-1, and if the subject has PD-L1 or PD-1 positive cancer cells or TIL cells, then the subject is then administered an anti-TIM3 antibody (and optionally a PD-1 or PD-L1 antagonist), such as those described herein. A method of treating a subject having cancer with an anti-TIM3 antibody (and optionally an anti-PD1 or PD-L1 antagonist) may comprise administering to the subject who has cancer cells or TIL cells that express PD-L1 or PD-1 a therapeutically effective amount of an anti-TIM3 antibody. TIM3 (and possibly a PD-1 or PD-L1 antagonist). Also provided herein are methods for predicting whether a subject will respond to treatment with an anti-TIM3 antibody (and optionally a PD-1 or PD-L1 antagonist), the methods comprising determining the PD-L1 or PD-1 content of cancers or TILs. cells of the patient, and if the subject's cancer or TIL cells are PD-L1- or PD-1 positive, then the subject is likely to respond to treatment with an anti-TIM3 antibody (and optionally a PD-1 or PD-1 antagonist).

Antibody to TIM3 can be administered as part of standard treatment. Antibody to TIM3 can be administered as a maintenance treatment, for example, a treatment intended to prevent the occurrence or recurrence of tumors.

Anti-TIM3 antibody can be administered with other treatment, such as radiation, surgery or chemotherapy. For example, additional treatment with an anti-TIM3 antibody may be given when there is a risk of micrometastases and/or to reduce the risk of relapse.

Antibody to TIM3 can be administered as monotherapy or as the only immunostimulating treatment. Antibodies to TIM3, such as anti-TIM3, can also be combined with an immunogenic agent such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules), cells and cells transfected with genes encoding immunostimulatory cytokines (He et al (2004) J Immunol 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as gp100 peptides, MAGE, Trp-2, MART1 and/or tyrosinase antigens, or tumor cells transfected to express the cytokine GM-CSF (discussed further below) .

In humans, some tumors have been shown to be immunogenic, such as melanomas. By lowering the threshold for T cell activation through TIM3 inhibition, tumor responses in the host can be activated, allowing treatment of non-immunogenic tumors or tumors with limited immunogenicity.

An anti-TIM3 antibody, such as the anti-TIM3 antibody described herein, can be combined with a vaccination protocol. Many experimental tumor vaccination strategies have been developed (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring: 60-62; Logothetis, C, 2000, ASCO Educational Book Spring: 300-302; Khayat, D 2000, ASCO Educational Book Spring: 414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023- 3043 in DeVita et al (eds.), 1997, Cancer: Principles and Practice of Oncology, Fifth Edition). In one of these strategies, the vaccine is prepared using autologous or allogeneic tumor cells. These cell-based vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. Was shown,

- 84 044112 that GM-CSF is a potent activator of antigen presentation for antitumor vaccination (Dranoff et al. (1993) Proc. Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large-scale gene expression patterns in various tumors has led to the identification of so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10: 281-7). In many cases, these tumor-specific antigens are differentiation antigens expressed in tumors and in the cell from which the tumor arose, for example, melanocyte antigens gp100, MAGE and Trp-2 antigens. More importantly, many of these antigens can be shown to be targets of tumor-specific T cells found in the host. TIM3 inhibition can be used in combination with a collection of recombinant tumor-expressed proteins and/or peptides to generate an immune response to these proteins. These proteins are generally regarded by the immune system as self-antigens and are therefore tolerant to them. The tumor antigen may include the protein telomerase, which is required for chromosome telomere synthesis and which is expressed in more than 85% of human cancers and only in a limited number of somatic tissues (Kim et al. (1994) Science 266): 20112013). A tumor antigen may also be a neoantigen expressed in cancer cells due to somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (ie bcr-abl in the Philadelphia chromosome) or an idiotype from B cell tumors.

Other cancer vaccines may include proteins from viruses implicated in human cancers, such as human papillomaviruses (HPV), hepatitis viruses (HBV and HCV), and Kaposi's sarcoma herpesvirus (KHSV). Another form of tumor-specific antigen that can be used in combination with TIM3 inhibition is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain protein fragments from tumor cells, and these HSPs are very effective when delivered to antigen presenting cells to elicit tumor immunity (Suot & Srivastava (1995) Science 269: 1585-1588; Tamura et al. (1997) Science 278: 117-120 ).

Dendritic cells (DCs) are potent antigen-presenting cells that can be used to initiate antigen-specific responses. DCs can be generated ex vivo and loaded with various protein and peptide antigens, as well as tumor cell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs can also be genetically transduced to express these tumor antigens. DCs have also been fused directly to tumor cells for immunization purposes (Kugler et al. (2000) Nature Medicine 6:332-336). As a vaccination modality, DC immunization can be effectively combined with TIM3 inhibition to promote stronger antitumor responses.

TIM3 inhibition can also be combined with standard cancer treatments (eg, surgery, radiation, and chemotherapy). TIM3 inhibition can be effectively combined with chemotherapy regimens. In these cases, the dose of the chemotherapy reagent administered can be reduced (Mokyr et al. (1998) Cancer Research 58: 5301-5304). An example of such a combination is an anti-TIM3 antibody in combination with decarbazine for the treatment of melanoma. Another example of such a combination is an anti-TIM3 antibody in combination with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale for the combined use of TIM3 inhibition and chemotherapy is that cell death, which is a consequence of the cytotoxic effects of most chemotherapeutic compounds, should result in increased levels of tumor antigen in the antigen presentation pathway. Other combination treatments that may synergize with TIM3 inhibition through cell death include radiation, surgery, and endocrine shutdown. Each of these protocols creates a source of tumor antigen in the host. Angiogenesis inhibitors can also be combined with TIM3 inhibition. Inhibition of angiogenesis results in the death of tumor cells that can feed tumor antigen into the host antigen presentation pathway.

The anti-TIM3 antibodies described herein can also be used in combination with bispecific antibodies that target Fca or Fcy receptor-expressing effector cells to tumor cells (see, for example, US Pat. Nos. 5,923,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, bispecific antibodies to Fc receptor/antitumor antigen (eg, Her-2/neu) have been used to target macrophages to tumor sites. This targeting may more effectively activate tumor-specific responses. The T cell branch of these responses will be enhanced by TIM3 inhibition. Alternatively, the antigen can be delivered directly to DCs using bispecific antibodies that bind to the tumor antigen and a dendritic cell-specific cell surface marker.

Tumors evade host immune surveillance through a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins that are expressed by tumors and that are immunosuppressive. These include, in particular, TGF-β (Kehrl et al. (1986) J. Exp. Med. 163: 1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13: 198-200 ) And

Fas ligand (Hahne et al. (1996) Science 274: 1363-1365). Antibodies to each of these entities can be used in combination with antibodies to TIM3 to counteract the effects of the immunosuppressive agent and stimulate host tumor immune responses.

Other antibodies that activate host immune sensitivity can be used in combination with anti-TIM3 antibodies. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Antibodies to CD40 can effectively replace T helper cell activity (Ridge et al. (1998) Nature 393: 474-478) and can be used in combination with antibodies to TIM3. Activation of antibodies to T cell costimulatory molecules such as CTLA-4 (eg, US Pat. No. 5,811,907), OX-40 (Weinberg et al. (2000) Immunol. 164: 2160-2169), 41BB (Melero et al. ( 1997) Nature Medicine 3: 682-685 (1997) and ICOS (Hutloff et al. (1999) Nature 397: 262266) may also provide increased levels of T cell activation. PD1 or PD-L1 inhibitors may also be used in combination with an antibody to TIM3 Another combination is provided elsewhere herein.

Currently, bone marrow transplantation is used to treat various tumors of hematopoietic origin. While graft-versus-host disease is a consequence of this treatment, therapeutic benefit may be derived from graft-versus-tumor disease. Inhibition of TIM3 can be used to improve the efficiency of donor-specific tumor T cells.

There are also several experimental treatment protocols that involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells to recipients to stimulate antigen-specific T cells against the tumor (Greenberg & Riddell (1999) Science 285: 546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-TIM3 antibodies can increase the frequency and activity of adoptively transferred T cells.

XVI.B. Infectious diseases.

The methods described herein may also be used to treat patients who have been exposed to certain toxins or pathogens. Accordingly, another aspect described herein provides a method of treating an infectious disease in a subject, comprising administering to the subject an antibody to TIM3 or an antigen-binding portion thereof such that the subject is treated for the infectious disease. Additionally or alternatively, the antibody may be a chimeric or humanized antibody.

Similar to its use in tumors as discussed above, antibody-mediated TIM3 inhibition can be used alone or adjunctive in combination with vaccines to stimulate immune responses to pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly applicable include pathogens for which there is currently no effective vaccine or pathogens for which conventional vaccines are less than completely effective. These include, but are not limited to, HIV, hepatitis (A, B and C), influenza, herpes, giardiasis, malaria, leishmania, Staphylococcus aureus, Pseudomonas aeruginosa. Inhibition of TIM3 may be useful against established infections with agents such as HIV, which are altered antigens during infections. These new epitopes are recognized as foreign during the administration of anti-human TIM3 antibodies, thereby inducing a strong T cell response.

Some examples of pathogenic viruses that cause infections that can be treated by the methods described herein include HIV, hepatitis (A, B or C), herpes virus (eg, VZV, HSV-1, HAV-6, HSV-II and CMV, Epstein-Bar virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, Coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, virus dengue, papillomavirus, molluscan virus, poliovirus, rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections that can be treated by the methods described herein include chlamydia, rickettsial bacteria, mycobacterium, staphylococcus, streptococcus, pneumococcus, meningococcus and gonococcus, Klebsiella, Protea, serratia, Pseudomonas aeruginosa, Legionella, diphtheria, salmonella, mycobacterium, cholera, tetanus, botulism, anthrax, plague, leptospirosis and Lyme disease bacteria.

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

Some examples of pathogenic parasites that cause infections that are treatable by the methods described herein include Entamoeba histolytica, Balantidium coli,

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Naegleriafowleri, Acanthamoeba sp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and

Nippostrongylus brasiliensis.

In all of the above methods, TIM3 inhibition may be combined with other forms of immunotherapy, such as those described herein, such as cytokine treatment (eg, interferons, GM-CSF, G-CSF, IL-2) or a bispecific antibody treatment that provides improved presentation of tumor antigens (see, for example, Holliger (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123).

XVI. C. Autoimmune reactions.

Antibodies to TIM3 can provoke and enhance autoimmune responses. Indeed, the induction of antitumor responses using tumor and peptide vaccines indicates that many antitumor responses involve autoimmune reactivity (van Elsas et al. (2001) J. Exp. Med. 194:481-489; Overwijk, et al. (1999) ; Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987; Hurwitz, (2000), supra; Rosenberg & White (1996) J. Immunother Emphasis Tumor Immunol 19(1):81-4) Therefore, it may be possible to consider the use of anti-TIM3 antibodies in combination with various self-proteins to develop vaccination protocols to effectively elicit immune responses against these self-proteins to treat the disease. For example, Alzheimer's disease involves inappropriate accumulation of the Ae peptide in amyloid deposits in the brain; anti-amyloid antibody responses are capable of clear these amyloid deposits (Schenk et al., (1999) Nature 400: 173-177).

Other self-proteins can also be used as targets, such as IgE for the treatment of allergies and asthma and TNF-α for rheumatoid arthritis. Finally, antibody responses to various hormones can be induced by the use of anti-TIM3 antibodies. Neutralizing antibody responses to reproductive hormones can be used for contraception. Neutralizing antibody responses to hormones and other soluble factors that are essential for the growth of certain tumors can also be considered as possible targets for vaccination.

Similar methods as described above for the use of antibodies to TIM3 can be used to induce therapeutic autoimmune responses to treat patients characterized by inappropriate accumulation of other autoantigens, such as amyloid deposits, including Alzheimer's disease Ae, cytokines such as TNF-α and IgE.

XVI.D. Vaccines.

The anti-TIM3 antibodies described herein can be used to stimulate antigen-specific immune responses by co-administrating the anti-TIM3 antibody with an antigen of interest (eg, a vaccine). Accordingly, provided herein are methods of enhancing an immune response to an antigen in a subject, comprising administering to the subject: (i) an antigen and (ii) an antibody to TIM3 or an antigen-binding portion thereof such that the immune response to an antigen is enhanced in a subject. The antibody may be a human anti-human TIM3 antibody (such as any of the human anti-TIM3 antibodies described herein). Additionally or alternatively, the antibody may be a chimeric or humanized antibody. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen from a pathogen. Non-limiting examples of such antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) discussed above, or antigens from viruses, bacteria or other pathogens described above.

In some embodiments, a peptide or fusion protein containing an epitope to which an anti-TIM3 antibody binds is used as a vaccine instead of or in addition to an anti-TIM3 antibody.

Suitable routes of administration of the antibody compositions (eg, human monoclonal antibodies, multispecific and bispecific molecules and immunoconjugates) described herein, in vivo and in vitro, are well known in the art and can be selected by those skilled in the art. For example, antibody compositions can be administered by injection (eg, intravenously or subcutaneously). Suitable dosages of the molecules used will depend on the age and weight of the subject, as well as the concentration and/or composition of the antibodies.

As described previously, the anti-TIM3 antibodies described herein can be administered in conjunction with one or more other therapeutic agents, for example, a cytotoxic agent, a radiotoxic agent, or an immunosuppressant. The antibody may be associated with the agent (as an immunocomplex) or may be administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the agent, or may be used in conjunction with other known treatments, such as an anticancer treatment such as radiation. Such therapeutic agents include, but are not limited to, antineoplastic agents such as doxorubicin (Adriamycin), cisplatin, bleomycin sulfate, carmustine, chlorambucil, dacarbazine, and cyclophosphamide hydroxyurea, which themselves are effective only at levels that are toxic or subtoxic to the patient . Cisplatin is administered intravenously at a dose of 100 mg/ml once every four weeks, and adriamycin is administered intravenously at a dose of 60-75 mg/ml once every 21 days. Coadministration of the anti-TIM3 antibodies or antigen-binding fragments thereof described herein with chemotherapeutic agents provides two antitumor agents that act through distinct mechanisms that are cytotoxic to human tumor cells. Such co-administration may overcome problems associated with the development of drug resistance or changes in the antigenicity of tumor cells, making them inactive with the antibody.

Also included within the scope described herein are kits containing antibody compositions (eg, human antibodies, bispecific or multispecific molecules, or immunoconjugates) described herein and instructions for use. The kit may further contain at least one additional reagent or one or more additional anti-human TIM3 antibodies described herein (e.g., a human antibody having complementary activity that binds to an epitope on a TIM3 antigen different from the first human antibody). Kits typically include a label indicating the intended use of the kit's contents. The term label includes any written or recorded material supplied on or with the set, or otherwise accompanying the set.

XVI.E. Combined treatment methods.

In addition to the combination treatments presented above, anti-TIM3 antibodies, such as those described herein, can also be used in a combination treatment, for example, for the treatment of cancer, as described below.

Provided herein are combination therapy methods in which an anti-TIM3 antibody is co-administered with one or more additional agents, e.g., small molecule drugs, antibodies, or antigen-binding moieties thereof, that are effective in stimulating immune responses, thereby further enhancing, stimulating or activate immune responses in the subject.

Typically, an anti-TIM3 antibody, such as that described herein, can be combined with (i) an agonist of a stimulatory (e.g., costimulatory) molecule (e.g., a receptor or ligand) and/or (ii) an antagonist of an inhibitory signal or molecule (e.g., a receptor or ligand) on immune cells such as T cells, both of which lead to enhanced immune responses such as antigen-specific T cell responses. In certain aspects, the immuno-oncology agent is (i) an agonist of a stimulatory (including co-stimulatory) molecule (e.g., a receptor or ligand) or (ii) an antagonist of an inhibitory (including co-inhibitory) molecule (e.g., a receptor or ligand) on cells, e.g. inhibit the activation of T cells, or those that participate in innate immunity, for example, NK cells, and wherein the immuno-oncology agent enhances innate immunity. Such immuno-oncology agents are often referred to as immune checkpoint regulators, such as an immune checkpoint inhibitor or an immune checkpoint stimulator.

In some embodiments, the anti-TIM3 antibody is administered with an agent that targets a stimulatory or inhibitory molecule that is a member of the immunoglobulin superfamily (IgSF). For example, anti-TIM3 antibodies, such as those described herein, can be administered to a subject using an agent that targets a member of the IgSF family to enhance the immune response. For example, an anti-TIM3 antibody can be administered with an agent that targets (or specifically binds to) a member of the B7 family of membrane-bound ligands, which includes B7-1, B7-2, B7-H1 (PD-L1), B7DC (PD- L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA) and B7-H6 or co-stimulatory or co-inhibitory receptor or specific ligand binding to a member of the B7 family.

Anti-TIM3 antibody can also be administered with an agent that targets a member of the TNF and TNFR family of molecules (ligands or receptors), such as CD40 and CD40L, OX-40, OX40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 , TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR/Fn 14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTpR, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDA1, EDA2, TNFR1, lymphotoxin a/TNFp, TNFR2, TNFa, LTpR, lymphotoxin a 1 β2, FAS, FASL, RELT, TROY and NGFR (see, for example, Tansey (2009 ) Drug Discovery Today o0:1).

T cell responses can be stimulated by a combination of antibodies to TIM3 having variable regions, for example, TIM3.2, TIM3.4, TIM3.5, TIM3.6, 9F6, 8B9, TIM3.9, TIM3.10, TIM3.11, TIM3 .12, TIM3.13, TIM3.14, TIM3.15, TIM3.16, TIM3.16, TIM3.17, TIM3.18, TIM3.7 and TIM3.8 and one or more of the following:

(1) An antagonist (inhibitor or blocking agent) of a protein that inhibits T cell activation (eg, immune checkpoint inhibitors), such as CTLA-4, PD-1, PD-L1, PD-L2, GITR and LAG-3, galectin 9, SEACAM-1, BTLA, CD69, galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1 and TIM-4; and/or

- 88 044112 (2) A protein agonist that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, GITR, ICOS, ICOS-L, OX40, OX40L , CD70, CD27, CD40, DR3 and CD28H.

Exemplary agents that modulate one of the above proteins and can be combined with anti-TIM3 antibodies, such as those described herein, for the treatment of cancer include: YERVOY® (ipilimumab) or tremelimumab (for CTLA-4), galiximab (for B7.1), BMS936558 (for PD-1), MK-3475 (for PD-1), atezolizumab (TECENTRIQ®), AMP224 (for B7DC), BMS936559 (for B7-H1), MPDL3280A (for B7-H1) , MEDI-570 (for ICOS), AMG557 (for V7N2), MGA271 (for V7N3), IMP321 (for LAG-3), BMS-663513 (for CD137), PF-05082566 (for CD137), CDX-1127 (for CD27), anti-OX40 (Providence Health Services), huMAbOX40L (for OX40L), atacicept (for TACI), CP-870893 (for CD40), lucatumumab (for CD40), dacetuzumab (for CD40), muromonab-CD3 (for CD3 ); antibodies to GITRMK4166, TRX518, Medil873, INBRX-110, LK2-145, GWN-323, GITRL-Fc or any combination thereof.

Other molecules that can be combined with anti-TIM3 antibodies to treat cancer include inhibitory receptor antagonists on NK cells or activating receptor agonists on NK cells. For example, anti-TIM3 antibodies can be combined with KIR antagonists (eg, lirilumab).

T cell activation is also regulated by soluble cytokines, and antibodies to TIM3 can be administered to a subject, for example, having cancer, with cytokine antagonists that inhibit T cell activation or cytokine agonists that stimulate T cell activation.

In some embodiments, anti-TIM3 antibodies may be used in combination with (i) antagonists (or inhibitors or blocking agents) of IgSF family or B7 family proteins or TNF family proteins that inhibit T cell activation, or cytokine antagonists that inhibit T cell activation. cells (e.g. IL-6, IL-10, TGF-β, VEGF; immunosuppressive cytokines) and/or (ii) agonists of stimulatory receptors of the IgSF family, B7 family or TNF family, or cytokines that stimulate T cell activation, for stimulating the immune response, for example to treat proliferative diseases such as cancer.

Still other combination therapies include agents that inhibit or deplete macrophages or monocytes, including, but not limited to, CSF1R antagonists, such as CSF-1R antagonist antibodies, including RG7155 (WO11/70024, WO11/ 107553, WO11/131407, WO01/87699, WO01/119716, WO13/132044) or FPA-008 (WO11/140249; WO13169264; WO14/036357).

Antibodies to TIM3 can also be administered with agents that inhibit TGF-β signaling.

Additional agents that can be combined with anti-TIM3 antibody include agents that enhance tumor antigen presentation, such as dendritic cell vaccines, cell vaccines secreting GM-CSF, CpG oligonucleotides and imiquimod, or treatments that enhance the immunogenicity of tumors. cells (for example, anthracyclines).

Still other treatments that can be combined with an anti-TIM3 antibody include treatments that deplete or block Treg cells, such as an agent that specifically binds to CD25.

Another treatment that can be combined with an anti-TIM3 antibody is a treatment that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase, or nitric oxide synthetase.

Another class of agents that can be used with an anti-TIM3 antibody include agents that inhibit adenosine formation, such as CD73 inhibitors, or inhibit the adenosine A2A receptor.

Other treatments that can be combined with an anti-TIM3 antibody to treat cancer include treatments that reverse/prevent T cell anergy or exhaustion and treatments that trigger innate immune activation and/or inflammation at the tumor site.

Other treatments that can be combined with an anti-TIM3 antibody to treat cancer include treatments that block IL-8, such as HuMax-IL8.

An anti-TIM3 antibody can be combined with more than one immuno-oncology agent and can, for example, be combined with a combinatorial approach that targets multiple elements of the immune pathway, such as one or more of the following: a treatment that enhances tumor antigen presentation (eg, a vaccine from dendritic cells, cell vaccines secreting GM-CSF, CpG oligonucleotides, imiquimod); a treatment that inhibits immune downregulation, for example, by inhibiting the CTLA-4 and/or PD1/PD-L1/PDL2 pathway and/or depleting or blocking Tregs or other immunosuppressive cells; a treatment that stimulates positive immune regulation, for example, with agonists that stimulate the CD-137, OX-40 and/or CD40 or GITR pathway and/or stimulate T cell effector function; a treatment that systemically increases the frequency of anti-tumor T cells; a treatment that depletes or inhibits Tregs, such as Tregs in a tumor, for example, using a CD25 antagonist (eg, daclizumab) or by depleting anti-CD25 granules ex vivo; a treatment that affects the function of myeloid suppressor cells in a tumor; a treatment that enhances the immunogenicity of tumor cells (eg, anthracyclines); adoptive transfer of T cells or NK cells, including genetically modified cells, for example, cells modified with chimeric antigen receptors (CAR-T treatment method); a treatment that inhibits a metabolic enzyme such as indoleamine dioxygenase (IDO), dioxygenase, arginase or nitric oxide synthetase; a treatment that reverses/prevents anergy or T cell depletion; a treatment method that triggers innate immune activation and/or inflammation at the tumor site; administering immunostimulatory cytokines or blocking immunorepressive cytokines.

The anti-TIM3 antibodies described herein can be used in conjunction with one or more agonistic agents that ligate positive co-stimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists, and one or more agents that systemically increase the frequency of anti-tumor T cells. agents that overcome various immunosuppressive pathways in the tumor microenvironment (eg, block inhibitory receptor engagement (eg, PD-L1/PD-1 interaction), deplete or inhibit Tregs (eg, using an anti-CD25 monoclonal antibody (eg, daclizumab) (or by depleting anti-CD25 beads), inhibit metabolic enzymes such as IDO, or reverse/prevent anergy or T cell exhaustion), and agents that trigger innate immune activation and/or inflammation at tumor sites.

In some embodiments, an anti-TIM3 antibody is administered to a subject along with a BRAF inhibitor if the subject is positive for the BRAF V600 mutation.

Suitable PD-1 antagonists for use in the combination treatment method described herein include, but are not limited to, ligands, antibodies (eg, monoclonal antibodies and bispecific antibodies), and multivalent agents. In one embodiment, the PD-1 antagonist is a fusion protein, for example, an Fc fusion protein such as AMP-244. In one embodiment, the PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody.

An exemplary anti-PD-1 antibody is nivolumab (BMS-936558) or an antibody that contains the CDRs or variable regions of one of the antibodies 17D8, 2D3, 4H1, 5C4, 7D3, 5F4 and 4A11 described in WO 2006/121168. In some embodiments, the anti-PD-1 antibody is MK-3475 (lambrolizumab), described in WO2012/145493; AMP-514, described in WO 2012/145493, or PDROOl. Other known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009/014708, WO 03/099196, WO 2009/114335, WO 2011/066389, WO 2011/161699, WO 2012/145493 , US Patent Nos. 7635757 and 8217149 and US Patent Publication No. 2009/0317368. Any of the anti-PD-1 antibodies disclosed in WO2013/173223 may also be used. An anti-PD-1 antibody that competes for binding and/or binds to the same epitope on PD-1 as one of these antibodies can also be used in combination treatments.

In some embodiments, the anti-PD-L1 antibody useful for the combination treatment is BMS-936559 (referred to as 12A4 in WO 2007/005874 and US Pat. No. 7,943,743) or an antibody that contains CDRs or variable regions 3G10, 12A4, 10A5 , 5F8, 10H10, IB 12, 7H1, 11E6, 12B7 and 13G4, which are described in PCT publication WO 07/005874 and US patent No. 7943743. In a certain embodiment, the anti-PD-L1 antibody is MEDI4736 (also known as durvalumab and B7-H1), MPDL3280A (also known as atezolizumab and RG7446), MSB0010718C (also known as avelumab; WO2013/79174) or rHigM12B7. Any of the anti-PD-L1 antibodies disclosed in WO2013/173223, WO2011/066389, WO2012/145493, US Patent Nos. 7,635,757 and 8,217,149, and US Publication No. 2009/145493 may also be used. Antibodies to PD-L1 that compete and/or bind to the same epitope as either of these antibodies can also be used in combination treatments.

In some embodiments, an anti-TIM3 antibody of the present disclosure may be used with a CTLA-4 antagonist, such as an anti-CTLA-4 antibody. In one embodiment, the anti-CTLA-4 antibody is an antibody selected from the group: YERVOY® (ipilimumab or the 10D1 antibody described in PCT publication WO 01/14424), tremelimumab (formerly ticilimumab, CP-675206), a monoclonal antibody, or an anti-CTLA-4 antibody. CTLA-4 described in any of the following publications: WO 98/42752; WO 00/37504; US Patent No. 6207156; Hurwitz et al. (1998) Pro. Natl. Acad. Sci. USA 95(17): 10067-10071; Camacho et al. (2004) J. Clin. Oncology 22(145): Abstract No. 2505 (antibody CP675206); and Mokyr et al. (1998) Cancer Res. 58:5301–5304. Any of the anti-CTLA-4 antibodies disclosed in WO2013/173223 may also be used.

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In other embodiments, an anti-TIM3 antibody of the present disclosure is used in combination with a LAG3 antagonist. Examples of anti-LAG3 antibodies include antibodies containing the CDRs or variable regions of antibodies 25F7, 26H10, 25E3, 8B7, 11F2 or 17E5, which are described in US Patent Publications No. US2011/0150892, WO10/19570 and WO2014/008218. In one embodiment, the anti-LAG-3 antibody is BMS-986016. Other anti-LAG-3 antibodies known in the art that may be used include IMP731 and IMP321, described in US 2011/007023, WO08/132601 and WO09/44273.

Antibodies to LAG-3 that compete and/or bind to the same epitope as any of these antibodies can also be used in combination treatments.

In some embodiments, an anti-TIM3 antibody of the present disclosure may be administered in combination with a CD137 agonist (4-1BB), such as an anti-CD137 agonist antibody. Suitable anti-CD137 antibodies include, for example, urelumab or PP-05082566 (WO12/32433).

In other embodiments, an anti-TIM3 antibody may be administered in combination with an OX40 agonist, such as an OX40 agonist antibody. Suitable anti-OX40 antibodies include, for example, MEDI-6383, MEDI-6469 or MOXR0916 (RG7888; WO06/029879).

In one embodiment, the anti-TIM3 antibody is administered in combination with a CD40 agonist, such as an anti-CD40 agonist antibody. In some embodiments, the immuno-oncology agent is a CD40 antagonist, such as an anti-CD40 antagonist antibody. Suitable anti-CD40 antibodies include, for example, lucatumumab (HCD122), dacetuzumab (SGN-40), CP-870893 or Chi Lob 7/4.

In one embodiment, the anti-TIM3 antibody is administered in combination with a CD27 agonist, such as an anti-CD27 agonist antibody. Suitable anti-CD27 antibodies include, for example, varlilumab (CDX-1127).

In some embodiments, an anti-TIM3 antibody is administered together with an anti-GITR antibody, for example, an antibody characterized by CDR 6C8 sequences, for example, a humanized antibody containing CDR 6C8, as described, for example, in WO2006/105021; an antibody containing the CDR of the anti-GITR antibody described in WO 2011/028683; an antibody containing the CDR of an anti-GITR antibody described in JP2008278814, an antibody containing a CDR of an anti-GITR antibody described in WO2015/031667, WO2015/187835, WO2015/184099, WO2016/054638, WO2016/057841, or

WO2016/057846 or other anti-GITR antibody described or referenced herein.

In other embodiments, an anti-TIM3 antibody is administered in combination with MGA271 (up to B7H3) (WO11/109400).

In some embodiments, the anti-TIM3 antibody is administered in combination with a KIR antagonist, such as lirilumab.

In other embodiments, an anti-TIM3 antibody is administered in combination with an IDO antagonist. Suitable IDO antagonists include, for example, INCB-024360 (WO2006/122150, WO07/75598, WO08/36653, WO08/36642), indoximob, NLG-919 (WO09/73620, WO09/1156652, WO11/56652, WO1 2/ 142237) or F001287.

In yet other embodiments, an anti-TIM3 antibody is administered in combination with a Toll-like receptor agonist, eg, a TLR2/4 agonist (eg, Bacillus Calmette-Guerin); a TLR7 agonist (eg, hiltonol or imiquimod); a TLR7/8 agonist (eg, resiquimod) or a TLR9 agonist (eg, CpG7909).

In one embodiment, anti-TIM3 is administered in combination with a TGF-β inhibitor, such as GC1008, LY2157299, TEW7197, or IMC-TR1.

The anti-TIM3 antibodies and combination therapies described herein may also be used in combination with other well-known therapies selected for their particular utility against the indication being treated (eg, cancer). Combinations of anti-TIM3 antibodies described herein can be used sequentially with known pharmaceutically acceptable agent(s).

For example, the anti-TIM3 antibodies and combination therapies described herein may be used in combination (e.g., concomitantly or separately) with additional treatments such as radiation and/or chemotherapy, e.g., camptothecin (CPT-11), 5fluorouracil (5 -FU), cisplatin, doxorubicin, irinotecan, paclitaxel, gemcitabine, cisplatin, paclitaxel, carboplatin-paclitaxel (Taxol), doxorubicin or camptothecin + apo21/TRAIL (6X combo), one or more proteasome inhibitors (for example, bortezomib or MG132), one or more Bcl-2 inhibitors (eg, BH3I-2' (bcl-xl inhibitor), indoleamine dioxygenase-1 inhibitor (eg, INCB24360, indoximod, NLG-919 or F001287), AT-101 (R-(-) derivative gossypol), ABT-263 (small molecule), GX-15-070 (obatoclax) or MCL-1 (myeloid leukemia cell differentiation protein-1 antagonists), iAP (inhibitor of apoptosis protein) (e.g. smac7, smac4, small molecule smac mimetics , synthetic peptides smac (see. Fuldae et al., Nat Med 2002;8:80815), ISIS23722 (LY2181308) or AEG-35156 (GEM-640)), HDAC inhibitors (histone deacetylase), anti-CD20 antibodies (eg, rituximab), angiogenesis inhibitors (eg, bevacizumab ), antian

- 91 044112 hyogenic agents targeting VEGF and VEGFR (eg Avastin), synthetic triterpenoids (see Hyer et al., Cancer Research 2005; 65:4799-808), c-FLIP (FLICE cell inhibitory protein) modulators (eg , natural and synthetic ligands PPARy (peroxisome proliferator-activated receptor γ), 5809354 or 5569100), kinase inhibitors (eg, sorafenib), trastuzumab, cetuximab, temsirolimus, mTOR inhibitors such as rapamycin and temsirolimus, bortezomib, JAK2 inhibitors, HSP90 inhibitors, PI3K-AKT inhibitors, lenalidomide, GSK3P inhibitors, IAP inhibitors and/or genotoxic drugs.

The anti-TIM3 antibodies and combination therapies described herein may further be used in combination with one or more anti-proliferative cytotoxic agents. Classes of compounds that can be used as antiproliferative cytotoxic agents include, but are not limited to, the following.

Alkylating agents (including, but not limited to, nitrogen mustard, ethylene imine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine, cyclophosphamide (CYTOXAN®), phosphamide, melphalan, chlorambucil, pipobromane, triethylene melamine, triethylene thiophosphoramine, busulfan, carmustine, lomustine, streptozocin, dacarbazine and temozolomide.

Antimetabolites (including, but not limited to, folate antagonists, pyrimidine analogues, purine analogues and adenosine deaminase inhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin and gemcitabine.

Suitable antiproliferative agents for combination with anti-TIM3 antibodies include, but are not limited to, taxanes, paclitaxel (paclitaxel is commercially available as TAXOL™), docetaxel, discodermolide (DDM), dictyostatin (DCT), peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, deoxyepothilone B1, [17]dehydrodeoxyepothilone B, [18]dehydrodeoxyepothilone B, C12,13-cyclopropyl-epothyldosilothilone A, C6-C8 bridged epothilone A, trans-9,10 -dehydroepothilone D, cis-9,10-dehydroepothilone D, 16desmethylepothilone B, epothilone BIO, discoderomolide, patupilone (EPO-906), KOS-862, KOS-1584, ZKEPO, ABJ-789, XAA296A (discodermolide), TZT-1027 (soblidotin), ILX-651 (tasidotin hydrochloride), halichondrin B, eribulin mesylate (E-7389), hemiasterlin (HTI-286), E-7974, Cyptophycin, LY-355703, maytansinoid immunoconjugates (DM-1), MKC- 1, ABT-751, T1-38067, T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312, eleutherobin, 17-beta-acetoxy-2-ethoxy-6-oxo-B -homoestra1,2,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl(+)-discodermolides and cryptotilone 1 in addition to other microtubulin stabilizing agents , known in the present technical field.

In cases where it is desired to obtain quiescent aberrantly proliferative cells in combination with or prior to treatment with the anti-TIM3 antibodies described herein, the patient may also be administered hormones and steroids (including synthetic analogues), such as 17a-ethinyl estradiol , diethylstilbestrol, testosterone, prednisone, fluoxymesterone, dromostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, thiamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flute amide, toremifene, ZOLADEX®. When using the methods or compositions described herein, other agents used to modulate tumor growth or metastasis in the clinical setting, such as antimimetics, may also be administered as desired.

In some embodiments, the combination of an anti-TIM3 antibody and a second agent discussed herein may be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions with the anti-TIM3 antibody and the second agent in a pharmaceutically acceptable carrier. In another embodiment, the combination of an anti-TIM3 antibody and a second agent may be administered sequentially. The administration of the two agents can begin at times separated by, for example, 30, 60, 90, 120 minutes; 3, 6, 12, 24, 36, 48 hours; 3, 5, 7 days or one or more weeks, or administration of the second agent may begin, for example, after 30, 60, 90, 120 minutes; 3, 6, 12, 24, 36, 48 hours; 3, 5, 7 days or one or several weeks after the introduction of the first remedy.

In some embodiments, the anti-tumor antibody that can be combined with an anti-TIM3 antibody and/or a second agent includes RITUXAN® (rituximab), HERCEPTIN® (trastuzumab), BEXXAR® (tositumomab), ZEVALIN® (ibritumomab), CAMPRATH® ( alemtuzumab), LYMPHOCIDE® (eprutuzumab), AVASTIN® (bevacizumab) and TARCEVA® (erlotinib) or any combination thereof. In other embodiments, the second antibody useful for a combination treatment with an anti-TIM3 antibody may be an antibody-drug conjugate.

In another embodiment, an anti-TIM3 antibody, alone or in combination with another agent, is used simultaneously or sequentially with bone marrow transplantation to treat various tumors of hematopoietic origin.

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Provided herein are methods of modifying an adverse event associated with treatment of a hyperproliferative disease (eg, cancer) with an immunostimulatory agent by administering an anti-TIM3 antibody to a subject with or without a second agent. For example, the methods described herein provide a method of reducing the incidence of immunostimulating therapeutic antibody-induced colitis or diarrhea by administering a non-absorbable steroid to a patient. As used herein, a non-absorbable steroid is a glucocorticoid that exhibits extensive first-pass metabolism such that after metabolism in the liver, the bioavailability of the steroid is low, i.e. less than about 20%. In one embodiment described herein, the non-absorbable steroid is budesonide. Budesonide is a topical glucocorticosteroid that is extensively metabolized, primarily by the liver, after oral administration. ENTOCORT EC® (Astra-Zeneca) is a pH- and time-sensitive oral formulation of budesonide designed to optimize drug delivery to the ileum and throughout the colon. ENTOCORT EC® is approved in the United States for the treatment of mild to moderate Crohn's disease involving the ileum and/or ascending colon. In other embodiments, the anti-TIM3 antibody in combination with a non-absorbable steroid may be further combined with a salicylate. Salicylates include 5-ASA agents, such as sulfasalazine (AZULFIDINE® Pharmacia & Up John); olsalazine (DJPENTUM®, Pharmacia & Up John); balsalazide (COLAZAL®, Salix Pharmaceuticals, Inc.) and mesalamine (ASACOL®, Procter & Gamble Pharmaceuticals; PENTASA®, Shire US; CANASA®, Axcan Scandipharm, Inc.; ROWASA®, Solvay).

Table 1

SEQ ID Description Sequences 1 IgG heavy chain 1.If TIM3.5 (13A3) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 93 044112

YTQKSLSLSPGK* 2 Heavy chain IgG 1.If 8B9 QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNSSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK* 3 Heavy chain IgG 1.If TIM3.6 (8C4) QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIG YIHYTGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATD TGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK* 4 Heavy chain IgG 1.If TIM3.2 (17СЗ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG IINPRGDSIIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DFYGSGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV P.S. YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 5 IgG heavy chain 1.If 9F6 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 6 IgG heavy chain 1.If TIM3 .4 (3G4) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISTSGSIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EGYSSSWSYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAP SVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN

- 94 044112

HYTQKSLSLSPGK* 7 Heavy chain IgGi.If TIM3.9 (17C8) S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 8 Heavy chain (without terminal K) IgGi. If TIM3.5 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 9 Heavy chain (without Terminal K) IgGi. If 8В9 QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNSSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG* 10 Heavy chain (without terminal K) IgGi. If TIM3.6 (8С4) QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIG YIHYTGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATD TGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG* AND Heavy chain (without terminal K) IgGi. If TIM3.2 (17NW) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG IINPRGDSIIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DFYGSGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV P.S. YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 95 044112

YTQKSLSLSPG* 12 Heavy chain (without terminal K) IgGl.lf9F6 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 13 Heavy chain (without terminal K) IgGl.lf TIM3.4 (3G4) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISTSGSIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EGYSSSWSYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAP SVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG* 14 Heavy chain (without terminal K) IgGl.lf TIM3.9 (17C8) S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 15 IgG1.3f heavy chain TIM3.5 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 16 Heavy chain IgG1.3f 8B9 QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNSSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL

- 96 044112

SLSPGK* 17 Heavy chain IgG1.3f TIM3.6 (8C4) QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIG YIHYTGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATD TGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK* 18 Heavy chain IgG1.3f TIM3.2 (17СЗ) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG IINPRGDSIIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DFYGSGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV P.S. YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 19 Heavy chain IgG1.3f 9F6 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 20 Heavy chain IgG1.3f TIM3.4 (3G4) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISTSGSIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EGYSSSWSYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAP SVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 21 Heavy chain IgG1.3f TIM3.9 (17C8) S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 97 044112

YTQKSLSLSPGK* 22 Heavy chain (without terminal K) IgG1.3f TIM3.5 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 23 Heavy chain (without terminal K) IgG1.3f 8B9 QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNSSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG* 24 Heavy chain (without terminal K) IgG1.3f TIM3.6 (8С4) QVQLQESGPGLVKPSETLSLTCTVSGGSISRYYWSWIRQPPGKGLEWIG YIHYTGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATD TGYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG* 25 Heavy chain (without terminal K) IgG1.3f TIM3.2 (17NW) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGLEWMG IINPRGDSIIYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DFYGSGNYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTV P.S. YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 26 Heavy chain (without terminal K) IgG1.3f9F6 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTAVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 98 044112

YTQKSLSLSPG* 27 Heavy chain (without terminal K) IgG1.3f TIM3.4 (3G4) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISTSGSIIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR EGYSSSWSYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAP SVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG* 28 Heavy chain (without terminal K) IgG1.3f TIM3.9 (17С8) S SLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 72 IgG 1.If Heavy Chain (N60Q) TIM3.10 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 73 IgG 1.If Heavy Chain (N60S) TiM3.li (13A3) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 74 IgG 1.If Heavy Chain (N60A) TIM3.12 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYAPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 99 044112

YTQKSLSLSPGK* 75 Heavy chain IgGi.If (DIO IE) TIM3.13 (13A3) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 76 Heavy chain IgGi.If (Pl 02 V) TIM3.14 (13A3) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 77 Heavy chain IgGi.If (P102Y) TIM3.15 (13A3) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 78 Heavy chain IgGi.If (P102L) TIM3.16 (13A3) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 79 Heavy chain IgGi.If (N60Q/P102 Y)TIM3.17 (13A3) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 100 044112

YTQKSLSLSPGK* 349 Heavy chain IgG 1.If (N60Q/D101 E) TIM3.18 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 80 Heavy chain IgG 1.If (S61P) TIM3.8 (8B9) QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK* 81 Heavy chain IgG 1.If (A108T) TIM3.7 (9F6) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 82 Heavy chain (without terminal K) IgGl.lf (N60Q) TIM3.10 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 83 Heavy chain (without terminal K) IgGl.lf (N60S) TiM3.li QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 101 044112

(VAZ) YTQKSLSLSPG* 84 Heavy chain (without terminal K) IgGi. If (N60A) TIM3.12 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYAPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 85 Heavy chain (without terminal K) IgGi. If (DIO IE) TIM3.13 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 86 Heavy chain (without terminal K) IgGi. If (Pl 02 V) TIM3.14 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 87 Heavy chain (without terminal K) IgGi. If (P102Y) TIM3.15 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 88 Heavy chain (without terminal K) IgGi. If QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK

- 102 044112

(P102L) TIM3.16 (VAZ) TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 89 Heavy chain (without terminal K) IgGl.lf (N60Q/P102 Y)TIM3.17 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 350 Heavy chain (without terminal K) IgGl.lf (N60Q/D101 E) TIM3.18 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 90 Heavy chain (without terminal K) IgGl.lf (S61P) TIM3.8 (8B9) QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG* 91 Heavy chain (without terminal K) IgGl.lf (A108T) TIM3.7 (9F6) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV P.S. YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 92 IgG1.3f heavy chain (N60Q) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS

- 103 044112

TIM3.10 (VAZ) SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 93 IgG1.3f heavy chain (N60S) TIM3.11 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 94 IgG1.3f heavy chain (N60A) TIM3.12 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYAPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 95 Heavy chain IgG1.3f (D101E) TIM3.13 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 96 Heavy chain IgG1.3f (Pl 02 V) TIM3.14 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 97 IgG1.3f heavy chain (P102Y) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS

- 104 044112

TIM3.15 (VAZ) SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 98 IgG1.3f heavy chain (P102L) TIM3.16 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 99 Heavy chain IgG1.3f (N60Q/P102 Y)TIM3.17 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 351 Heavy chain IgG1.3f (N60Q/D101 E) TIM3.18 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 100 IgG1.3f heavy chain (S61P) TIM3.8 (8B9) QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK* 101 Heavy chain IgG1.3f (A108T) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS

- 105 044112

TIM3.7 (9F6) SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK* 102 Heavy chain (without terminal K) IgG1.3f (N60Q) TIM3.10 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 103 Heavy chain (without terminal K) IgG1.3f (N60S) TIM3.11 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYSPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 104 Heavy chain (without terminal K) IgG1.3f (N60A) TIM3.12 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYAPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 105 Heavy chain (without terminal K) IgG1.3f (D101E) TIM3.13 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 106 Heavy QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA

- 106 044112

chain (without terminal K) IgG1.3f (Pl 02 V) TIM3.14 (13A3) TGGPYGDYAHWFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 107 Heavy chain (without terminal K) IgG1.3f (P102Y) TIM3.15 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 108 Heavy chain (without terminal K) IgG1.3f (P102L) TIM3.16 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 109 Heavy chain (without terminal K) IgG1.3f (N60Q/P102 Y)TIM3.17 (VAZ) QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 352 Heavy chain (without terminal K) IgG1.3f (N60Q/D101 E) TIM3.18 QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEW IGSIYYSGFTYYQPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCA TGGPYGDYAHWFEPWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH

- 107 044112

(13A3) YTQKSLSLSPG* 110 Heavy chain (without terminal K) IgG1.3f (S61P) TIM3.8 (8B9) QVQLQESGPGLVKPSETLSLTCTVSGGSISRHYWNWIRQPPGKGLEWIG YIHYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARD TGYYGMDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLG TQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPP KPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG* 111 Heavy chain (without terminal K) IgG1.3f (A108T) TIM3.7 (9F6) QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVS FISGGGSTIYYADSVKGRFTISRDNAKNSLFLQMNSLRVEDTAVYYCAR DGYSSGWYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPS SSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPS VFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG* 29 Light chain IgGl TIM3.5 (VAZ), TIM3.2 (17СЗ), TIM3.4 (3G4) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPIT FGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACE VTHQGLSSPVTKSFNRGEC* thirty Light chain IgGl 8B9, TIM3.6 (8C4), TIM3.9 (17C8) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLI YGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLT FGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACE VTHQGLSSPVTKSFNRGEC* 32 Light chain IgGl VK1 9F6 AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIY DASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPRTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEV THQGLSSPVTKSFNRGEC* 134 Heavy chain IgGl.If TIM3.5 CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGAGACGTCCAAGAACC AGTTCTCCCT

- 108044112

(VAZ) GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG TGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GAGAAGAGAG T T GAG C C CAAAT C T T G T GAGAAAAC TCACACATGCCCACCGTGCCCAGC ACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGCCTCTCCCTGTCCCCGGGTAAATGA 135 Heavy chain IgG 1.If 8B9 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTA CTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATCCAT TAGAG T G GAAG WITH AC CAAC TACAAT TCCTCCCT CAAGAG T C GAG T CAC CATAT CAG TAGACAC G T C CAAGAAC CAG TTCTCCCT GAAG C T GAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAT ACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTC CCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG T T GAG C C CAAAT C T T G T GACAAAAC T CACACAT G C C CAC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC

- 109 044112

TCCCTGTCCCCGGGTAAATGA 136 Heavy chain IgGi.If TIM3.6 (8C4) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTTACTA CTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATCCAT TACAC T G G GAG CAC CAAC TACAACCCCTCCCT CAAGAGT C GAG T CAC CATAT CAG TAGACAC G T C CAAGAAC CAG TTCTCCCT GAAG C T GAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGAT ACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG Τ T GAG C C CAAAT C Τ T G T GACAAAAC T CACACAT G C C CAC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGTAAATGA 137 Heavy chain IgGi.If TIM3.2 (17СЗ) CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGAAGGTCTCCTGCAAGGCATCTGGATACACTTTCACCAGCTACTA TATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGA ATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGG GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAG TCTACATGGA GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA GATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC GGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGΤ T GAGCCCAAAT C Τ T GT GACAAAAC TCACACATGCCCACCG TGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCAC CGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAG G T C T C CAACAAAG C C C T C C CAAG CAG CATC GAGAAAAC CAT C T C C

- 110044112

AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 138 Heavy chain IgGi.If 9F6 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCT GTTTCTGCA AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGAGAAGAGAGT T GAGCCCAAAT S T T GT GAGAAAAC TCACACATGCCCACCGTGCC CAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACA ACTACAAGACCAGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTTAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 139 Heavy chain IgGi.If TIM3.4 (3G4) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA T T CAT TAG TAG TAG T G G TAG TAT CATATAC TAG G CAGAC T S T G T GAAG G GCCGATTCACCATCTCCAGGG ACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GAAGGGTATAGCAGCAGCTGGTCCTACTACGGTATGGACGTCTGGG GCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGGCTGC CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT CCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC C CAG CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT C T T GT G ACAA AACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCG

-Ill 044112

TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG CAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCG TGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 140 Heavy chain IgG 1.If TIM3.9 (17C8) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA T CAT TAG TAG TAG T G G TAG TAT SATATAS TAS G CAGAC T S T G T GAAG G GCCGATTCACCATCTCC AGGGACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG C C CAA AT C Τ T G T GACAAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGCCTCTCCCTGTCCCCGGGTAAATGA 141 Heavy chain (without terminal K) IgGl.lf TIM3.5 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG AT T G G GAG TATCTATTATAGTGGGTT SAS STAS TASAAS CCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACA CGTCCAAGAACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC

- 112 044112

CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT GAGCCCAAA T C Τ T GT GACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGC GACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 142 Heavy chain (without Terminal K) IgGi. If 8В9 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTA CTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATSSAT TACAG T G GAAG CAC CAAC TASAATTSSTSSST CAAGAGT C GAG T CAC SATAT CAG TAGACAC G T C CAAGAAC CAG TTTTSSSST GAAG S T GAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAT ACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACT ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG AC TASAT S T G CAAC G T GAAT CACAAGC S CAGCAACACCAGGT GGACA AGAGAG Τ T GAG C C CAAAT C Τ T G T GACAAAAC T CACACAT G C C CAC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCCTCAACAAAGC CCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCA GGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCC TGTCCCCGGGTTGA 143 Heavy chain (without C- CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTTACTA CTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG

- 113 044112

terminal K) IgGl.lf TIM3.6 (8С4) TATATSSAT TASAS T G G GAG CAC SAAS TACAACSSSTSSST CAAGAGT C GAG T CAC SATA CAG TAGAAC G T S CAAGAAC CAG TTSTSSSST GAAG S T GAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGAT ACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGC TAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTA CATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG T T GAG S C CAAAT S T T G T GACAAAAC T CACACAT G C C CAC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC S GAGAAC CACAG G T G TACAC CCTGCCCCCATCCCG GAG GAGAT GAC SA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGTTGA 144 Heavy chain (without terminal K) IgGl.lf TIM3.2 (17СЗ) CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGAAGGTCTCCTGCAAGGCATCTGGATACACTTTCACCAGCTACTA TATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGA ATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGG GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAG TCTACATGGA GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA GATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC GGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACASSAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT

- 114 044112

CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 145 Heavy chain (without terminal K) IgGl.lf9F6 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCT GTTTCTGCA AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG C C CAAAT C Τ T G T GACAAA AAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG AGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 146 Heavy chain (without terminal K) IgGl.lf TIM3.4 (3G4) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA Τ T CAT TAG TAC TAG T G G TAG TAT SATATAS TAC G CAGAC T S T G T GAAG G GCCGATTCACCATCTCC AGGGACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GAAGGGTATAGCAGCAGCTGGTCCTACTACTACGGTATGGACGTCTGGG GCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGG CTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT CCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC C CAG CAACAC CAAG G T G GACAAGAGAG T GAG C C CAAAT S Τ T G T GACAA AACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCG TCAGTCTTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCA

- 115 044112

GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGGGACATCGCCGTGGAGTGGGAGAGCA ATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 147 Heavy chain (without terminal K) IgGi. If TIM3.9 (17С8) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA T T CAT TAG TAG TAG T G G TAG TAT SATATAS TAS G CAGAC T S T G T GAAG G GCCGATTCACCATCTCCAGGG ACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGC CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT G ACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACG TACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 148 Heavy chain IgG1.3f TIM3.5 (HAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCG AACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA

- 116 044112

G C AAC AC C AAG G T G GAC AAGAGAG Τ T GAG C C C AAAT C Τ T G T GAC AAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTC CCC GGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 149 Heavy chain IgG1.3f 8B9 AGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTAC TGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGT ATAT C SAT TACAG T G GAAG CAC CAC TACSAAT TSSTSSSST CAAGAG T C G AG T CAC SAT CAG T AGACAC G T S CAAGAAC CAG TTTSSSST GAAG S T G AGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATA CTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGT CTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAA ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAG Τ T GAG C C CAAAT C Τ T G T GACAAAAAS T CACACAT G C C CAC C G T G C CCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAA AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAAC CACAG G T G TACAC CCTGCCCCCATCCCGG GAG GAGAT GAC CAA GAACCAGGTCAGCCTG ACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAA GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCCCCGGGTA AATGA 150 Heavy chain IgG1.3f TIM3.6 (8C4) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTTACTA CTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATSSAT TASAS T G G GAG CAC SAAS TACSAAS CCSTSSST CAAGAG T C GAG T CAC SAT CAG TAGAC AC G T C CAAGAAC CAG TTTSSSST GAAG C T GAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGAT ACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG

- 117044112

TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTT GGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG Τ T GAG S S CAAAT S Τ T G T GAGAAAAC T CACACAT G S S SAC S G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGG TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAACAAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC TTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGTAAATGA 151 Heavy chain IgG1.3f TIM3.2 (17СЗ) CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGAAGGTCTCCTGCAAGGCATCTGGATACACTTTCACCAGCTACTA TATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGA ATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGG GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAG TCTACATGGA GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA GATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC GGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACAAGGT GGACAAGAGAGΤ T GAGCCCAAAT S Τ T GT GACAAAAC TCACACATGCCC ACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTSTCCAACAAAGSSSTSSAGSSSSCATTSGAGAAAACCATSTSS AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 152 Heavy CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT

- 118 044112

IgG1.3f chain 9F6 CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCTGTTTCTGCA AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACT GTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACA CCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTC CAACAAAGCCCTCCAGCCCCCATCGAGAAAACCATCCCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CC TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 153 Heavy chain IgG1.3f TIM3.4 (3G4) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA T T CAT TAG TAG TAG T G G TAG TAT CATATAC TAG G CAGAC T S T G T GAAG G GCCGATTCACCATCTCCAGGG ACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GAAGGGTATAGCAGCAGCTGGTCCTACTACGGTATGGACGTCTGGG GCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGGCTGC CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT CCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC C CAG CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT C T T GT G ACAA AACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC GTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTСTCCAACAAAGSSCTCCAGSSSSCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT

- 119044112

CAAAGGGCTTTTTTACCCAGCAGCATCCCCCCGGAGGAGGAGGAGGAGGAGGGGGGGGGAGAACAACAACAACAACCAACCCCCCCCCCCCCCCTGACTCTCTCTCTAGCAAGCAAGCAAGCAAGCAAGCAAGCAAGGACAAAGAA GCAGGGTGGCA GCAGGGGGAACGTCTCTCTCCTGATGAGCTGAGCTCTCCAC CACTACGCAAGAAGAGAGAAGAGTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC 154 Heavy chain IgG1.3f TIM3.9 (17C8) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA T CAT TAG TAG TAG T G G TAG TAT SATATAS TAS G CAGAC T S T G T GAAG G GCCGATTCACCATCTCC AGGGACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCT GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG C C CAA AT C Τ T G T GACAAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAAC AGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGCCTCTCCCTGTCCCCGGGTAAATGA 155 Heavy chain (without terminal K) IgG1.3f TIM3.5 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G C AAC AC C AAG G T G GAC AAGAGAG Τ T GAG C C C AAAT C Τ T G T GAC AAA AU TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA

- 120 044112

GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGSSCTCCAGSSSSCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA CACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGA GGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 156 Heavy chain (without terminal K) IgG1.3f 8B9 AGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGAC CCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTAC TGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGT ATATCCAT TAGAGTGGAAGСACCAACTACAATTCCTCCCTCAAGAGTCG AG T CAC CATAT CAG TAGACAC G T C CAAGA AC CAG TTCTCCCT GAAG C T G AGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGATA CTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCGT CTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC TCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGG TGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGA CCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAA GAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGC CCAGCACCT GAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCAA AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGC AGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CA GGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAAC CACAG G T G TAGAC CCTGCCCCCATCCCG GAG GAGAT GAC CAA GAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG AGAACAACTACAAGA CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAA GCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCT CCCTGTCCCCGGGTTGA 157 Heavy chain (without terminal K) IgG1.3f TIM3.6 (8C4) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTTACTA CTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATSSAT TACAC T G G GAG CAC CAAC TACAACCCCTCCCT CAAGAGT C GAG T CAC CATAT CAG TAGACAC G T C CAAGAAC CAG TTCTCCCT GAAG C T GAGCTCTGTGACCGCAGCGGACACGGCCGTGTATTACTGTGCGACAGAT ACGGGCTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACT TCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA

- 121 044112

CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG T T GAG C C C AAAAT C T T G T GAG AAAAC T C AC AC AT G C C CAC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACC CTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTC CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCA AGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 158 Heavy chain (without terminal K) IgG1.3f TIM3.2 (17NW) CAGGTGCAGTTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCT CAGTGAAGGTCTCCTGCAAGGCATCTGGATACACTTTCACCAGCTACTA TATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGA ATAATCAACCCTAGGGGTGATAGCATAATCTACGCACAGAAGTTCCAGG GCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAG TCTACATGGA GCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGA GATTTCTATGGTTCGGGAAACTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC GGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GAGAAGAGAG T T GAG S C CAAAT S T T G T GAGAAAAC TCACA CATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG CG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 159 Heavy chain (without terminal K) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCT GTTTCTGCA

- 122 044112

160

IgG1.3f9F6 AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCGCGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGAC GGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GAGAAGAGAG T T GAG C C CAAAT S T T G T GACAAAAAC TCACACATGCCC ACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCCAACAAAGSSSTSSAGSSSSCATTSGAGAAAACCATSTSS AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA Heavy chain (without terminal K) IgG1.3f TIM3.4 (3G4) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA T T CAT TAG TAG TAG T G G TAG TAT CATATAC TAG G CAGAC T S T G T GAAG G GCCGATTCACCATCTCCAGGG ACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GAAGGGTATAGCAGCAGCTGGTCCTACTACGGTATGGACGTCTGGG GCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATC GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG GCCCTGGGCTGC CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGT CCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCC TCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGC C CAG CAACACCAAGT GGACAAGAGAGT T GAGCCCAAT C T T GT G ACAA AACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCG TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC GTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTСTCCAACAAAGSSCTCCAGSSSSCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCC CATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGGCATCGCCG TGGAGTGGGAGAGCAATGGG CAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACG GCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC

- 123 044112

CACTACACCGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 161 Heavy chain (without terminal K) IgG1.3f TIM3.9 (17С8) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA T T CAT TAG TAG TAG T G G TAG TAT SATATAS TAS G CAGAC T S T G T GAAG G GCCGATTCACCATCTCCAGGG ACAACGCCAAGAACTCACTGTATCTGCA AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGGTATAGCAGTGGCTGGGAGTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAASSASSAAGGT GGACAAGAGAGT T GAGCCAAAAT S T T GT GAC AAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA CCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 205 Heavy chain IgGl.lf (N60Q) TIM3.10 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCASSATACCGTTGACACGTCCAAGA ACCAGTTTSSSST GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCC CGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACASSAAGGT GGACAAGAGAGT T GAGCCAAAAT S T T GT GACAAAAAC TCACACAT GCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC

- 124 044112

AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 206 Heavy chain IgGi.If (N60S) TIM3.11 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT C T T GT GAGAAAAC TCACACATGCCCACCGTGCCCAGC ACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGCCTCTCCCTGTCCCCGGGTAAATGA 207 Heavy chain IgGi.If (N60A) TIM3.12 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACGCACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G C AAC AC C AAG G T G GAC AAGAGAG T T GAG C C C AAAT C T T G T GAC AAAAC TCACACATGC CCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA

- 125 044112

GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT CCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 208 IgG Heavy Chain 1.If (DIO IE) TIM3.13 (13A3) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGAGAAGAGAGΤ T GAGCCCAAAT C Τ T GT GAGAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 209 Heavy chain IgG 1.If (Pl 02 V) TIM3.14 (13A3) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC

- 126 044112

CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT C T T GT GAGAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAG CACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCG TGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGCCTCTCCCTGTCCCCGGGTAAATGA 210 Heavy chain IgG 1.If (P102Y) TIM3.15 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGAGACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTGCC CAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACA ACTACAAGACCAGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTTAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 211 IgG heavy chain 1.If CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG

- 127 044112

(P102L) TIM3.16 (VAZ) ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGG CCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGA ATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG C C CAAAT C Τ T G T GACAAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAG G T C T C CAACAAAG C C C T C C CAAG CAG CATC GAGAAAAC CAT C T C AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG TGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 212 Heavy chain IgG 1.If (N60Q/P102 Y)TIM3.17 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G C AAC AC C AAG G T G GAC AAGAGAG Τ T GAG C C C AAAT C Τ T G T GAC AAAAC T CACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAG G T C T C CAACAAAG C C C T C C CAAG CAG CATC GAGAAAAC CAT C T C AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT

- 128 044112

CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 355 Heavy chain IgGi.If (N60Q/D101 E) TIM3.18 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAATC T T GT GACAAAAC TCACACATGCCCACCGTGCCCAGC ACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGCCTCTCCCTGTCCCCGGGTAAATGA 213 IgGi.If heavy chain (S61P) TIM3.8 (8B9) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTA CTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATCCAT TAGAG T G GAAG CAC CAACTACAATCCCTCCCT CAAGAGT C GAG T GAG CATAT GAG TAGACAC G T C C AAGAAC GAG TTCTCCCT GAAG C T GAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAT ACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCG AACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG T T GAG C C CAAAT C T T G T GACAAAAC T CACACAT G C C C AC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC

- 129 044112

AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCCAACAAAGC CCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA ACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 214 Heavy chain IgGi.If (A108T) TIM3.7 (9F6) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCT GTTTCTGCA AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAASSASSAAGGT GGAGAAGAGAGΤ T GAGCCAAAAT S Τ T GT GAGAAAAC TCACA CATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG CG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 215 Heavy chain (without terminal K) IgGi. If (N60Q) TIM3.10 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGΤTGAGACGTCCAAG AACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTC CCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA

- 130044112

G CAACAC CAAG G T G GACAAGAGAG T T GAG C C CAAAT S T T G T GAGAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAG G T S T S CAACAAAGC S S T S S CAAG CAG SAT S GAGAAAAC SAT S T S S AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 216 Heavy chain (without terminal K) IgGl.lf (N60S) TIM3.11 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG T T GAG S C CAAAT S T T G T GACAAAAAC TCACACAT GCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAG G T S T S CAACAAAGC S S T S S CAAG CAG CAT C GAGAAAAC CAT C T C AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTC CTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 217 Heavy chain (without terminal K) IgGl.lf (N60A) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACGCACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC

- 131 044112

TIM3.12 (VAZ) AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGTGGACAAGAGAGT T GAGCCCCAAAT C T T GT GACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACG TGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTTAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTG ATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 218 Heavy chain (without terminal K) IgGi. If (DIO IE) TIM3.13 (13A3) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGTGGACAAGAGAGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTGCCCAGCAC CTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAA GACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 219 Heavy CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA

- 132 044112

chain (without terminal K) IgGi. If (Pl 02 V) TIM3.14 (13A3) CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCASSATACCGTTGAGACGTCCAAGAACCAGTTTSSSST GAAGCTGAGCTCTGTGACCGCCGCAGACACGG CTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACAAGGT GGACAAGAGAGT T GAGCCCCAAAT S T T GT GACAAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTC CTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG TG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG CT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 220 Heavy chain (without terminal K) IgGi. If (P102Y) TIM3.15 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTGCC CAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA

- 133 044112

AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 221 Heavy chain (without terminal K) IgGi. If (P102L) TIM3.16 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGAGACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCG AACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 222 Heavy chain (without Terminal K) IgGi. If (N60Q/P102 Y)TIM3.17 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTGCC CAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA

- 134044112

GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC CCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC AACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 356 Heavy chain (without terminal K) IgGi. If (N60Q/D101 E) TIM3.18 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGAGACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCG AACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G C AACACCAAGGT GGAC AAGAGAGT T GAGCCCΑΑΆΤ C T T GT GACAAAAC T CACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCA ATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 223 Heavy chain (without terminal K) IgGi. If (S61P) TIM3.8 (8B9) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTA CTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATCCAT TAGAG T G GAAG CAC CAAC TACΑΆΤCCCTCCCT CAAGAGT C GAG T CAC CATAT CAG TAGAC AC G T C CAAGAAC CAG TTCTCCCT GAAG C T GAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAT ACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG G ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA

- 135 044112

CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG AS C TACAT S T G CAAC G T GAAT CACAAGC S CAGCAACASSAAGGT GGACA AGAGAG T T GAG S C CAAAAT S T T G T GACAAAAAS T CACACAT G C C CAC S G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCT TCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTA CAAGTGCAAGGTCTCCAACAAAGC CCTCCCAAGCAGCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGTTGA 224 Heavy chain (without terminal K) IgGl.lf (A108T) TIM3.7 (9F6) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCT GTTTCTGCA AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACASSAAGGT GGACAAGAGAGT T GAGCCAAAAT S T T GT GACAAAAAC TCACACATGCCC ACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAAGCAGCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 225 IgG1.3f heavy chain (N60Q) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACC AGTTCTCCCT

- 136044112

TIM3.10 (VAZ) GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGG TGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GAGAAGAGAG T T GAG C C CAAAT S T T G T GACAAAAAC TCACACATGCCCAC CGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGG AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTTAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 226 IgG1.3f heavy chain (N60S) TIM3.11 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GAGAAGAGAG T T GAG S C CAAAT S T T G T GACAAAAAC TCACACAT GCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC

- 137 044112

TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 227 IgG1.3f heavy chain (N60A) TIM3.12 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACGCACCGTCCCTCA AGAGTCGAGTCASSATACCGTTGAGACGTCCAAGA ACCAGTTTSSSST GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCC CGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCCAAAT S T T GT GAGAAAAC TCACACATGCCC ACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCCAACAAAGSSSTSSAGSSSSCATTSGAGAAAACCATSTSS AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 228 Heavy chain IgG1.3f (D101E) TIM3.B (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCG AACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGGT T GAGCCCAAAT S T T GT GAGAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGSSCTCCAGSSSSCATCGAGAAAACCATCTCC

- 138 044112

AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 229 Heavy chain IgG1.3f (Pl 02 V) TIM3.14 (13A3) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC GGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAGT T GAGCCCAAAT S T T GT GAGAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAA CAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 230 Heavy chain IgG1.3f (P102Y) TIM3.15 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG T T GAG C C CAAAT C T T G T GACAAAAC TCACACATGCCCACCGTGCC CAGCACCTGAAGCCGAAGGGGCCCCGTCA

- 139 044112

GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGA GTACAAGTG GAAGGTCTCCAACAAAGCCCTCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCC CGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 231 IgG1.3f heavy chain (P102L) TIM3.16 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G GAAGACCAAGGT GGACAAGAGAGΤ T GAGCCCAAAT C Τ T GT GACAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCAGSSSSCATCGAGAAAACCATCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGC AG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGCCTCTCCCTGTCCCCGGGTAAATGA 232 Heavy chain IgG1.3f (N60Q/P102 Y)TIM3.17 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGΤTGACACGTCCAAG AACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC

- 140 044112

CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG S S CAAAAT S Τ T G T GAGAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 357 Heavy chain IgG1.3f (N60Q/D101 Ε) ΤΙΜ3.18 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG C C CAAAT C Τ T G T GAGAAAAC TCA CACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 233 IgG1.3f heavy chain CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTA CTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG

- 141 044112

(S61P) TIM3.8 (8B9) TATATSSAT TACAG T G GAAG CAC CAAC TASAATSSTSSSST CAAGAGT C GAG T CAC SATAT CAG TAGACAC G T C CAAGAAC CAG TTCTCCCT GAAG S T GAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAT ACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGC ACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCT GCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG T T GAG C C CAAAT S T T G T GACAAAAAC T CACACAT G C C CAC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC S GAGAAC CACAG G T G TACAC CCTGCCCCCATCCCG GAG GAGAT GAC CA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCCCCGGGTAAATGA 234 Heavy chain IgG1.3f (A108T) TIM3.7 (9F6) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCT GTTTCTGCA AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACASSAAGGT GGACAAGAGAGT T GAGCCAAAAT S T T GT GACAAAAAC TCACACATGCCC ACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCCAACAAAGSSSTSSAGSSSSCATTSGAGAAAACCATSTSS AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT

- 142 044112

CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATGA 235 Heavy chain (without terminal K) IgG1.3f (N60Q) TIM3.10 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACASSAAGGT GGACAAGAGAGT T GAGCCCAAT S T T GT GAGAAAAC TCACACATGCCCACCGTGCC CAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTC CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACA ACTCAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 236 Heavy chain (without terminal K) IgG1.3f (N60S) TIM3.11 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACTCACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAGG T GGACAAGAGAG T T GAGC C CAAAT S T T G T GAGAAAAC TCACACATGCCCACCG TGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG

- 143 044112

TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 237 Heavy chain (without terminal K) IgG1.3f (N60A) TIM3.12 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG AT T G G GAG TATCTATTATAGTGGGTT CAC C TAS TAS G CAC CGTCCCTCA AGAGTCGAGTCACCATATCCGTTGA CACGTCCAAGAACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGG ACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GAGAAGAGAG T T GAG C C CAAAT S T T G T GAC AAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA CCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 238 Heavy chain (without terminal K) IgG1.3f (D101E) TIM3.13 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG AT T G G GAG TATCTATTATAGTGGGTT SAS STAS TASAAS CCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACA CGTCCAAGAACCAGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACT ACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA

- 144 044112

G CAACAC CAAG G T G GACAAGAGAG T T GAG C C CAAAT S T T G T GACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAA GAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCC TCTCCCTGTCCCCGGGTTGA 239 Heavy chain (without terminal K) IgG1.3f (Pl 02 V) TIM3.14 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACGTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACC GGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG T T GAG C C CAAAT S T T G T GACAAAAC TCACACATGCCC ACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCC TCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 240 Heavy chain (without terminal K) IgG1.3f (P102Y) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC

- 145 044112

TIM3.15 (VAZ) AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG CCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG S C CAAAT S Τ T G T GAGAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTC ACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAG CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGG GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 241 Heavy chain (without terminal K) IgG1.3f (P102L) TIM3.16 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACAACCCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACCAG TTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACCTATGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG TGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACAC CAAG G T G GACAAGAGAG Τ T GAG C C CAAAT C Τ T G T GAGAAAAC TCA CACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTC AGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 242 Heavy CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA

- 146 044112

chain (without terminal K) IgG1.3f (N60Q/P102 Y)TIM3.17 (13A3) CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCASSATACCGTTGAGACGTCCAAGAACCAGTTTSSSST GAAGCTGAGCTCTGTGACCGCCGCAGACACGG CTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGACTACTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTG CACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACAAGGT GGACAAGAGAGT GAGCCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCC CCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGG TCTCCAACAAAGSSSTSSCAGGSSSSCATTCGAGAAAACCATSTSS AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 358 Heavy chain (without terminal K) IgG1.3f (N60Q/D101 E) TIM3.18 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCG AACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGGT T GAGCCCAAAT S T T GT GACAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGSSCTCCAGSSSSCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA

- 147 044112

AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 374 Heavy chain (without terminal K) IgG1.3f (N60Q/D101 E)(T168S) TIM3.18 (VAZ) CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGCAGTAGAAG TTACTACTGGGGCTGGATTCGCCAGCCCCCAGGGAAGGGGCTGGAGTGG ATTGGGAGTATCTATTATAGTGGGTTCACCTACTACCAACCGTCCCTCA AGAGTCGAGTCACCATATCCGTTGACACGTCCAAGAACC AGTTCTCCCT GAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTATTGTGCG ACAGGGGGGCCCTACGGTGACTACGCCCACTGGTTCGAACCCTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCG AACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCAAAT S T T GT GAGAAAAC TCACACATGCCCACCGTG CCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCG TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGCCCCTCCAGSSSSCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 243 Heavy chain (without terminal K) IgG1.3f (S61P) TIM3.8 (8B9) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGA CCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTCGTCACTA CTGGAACTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGG TATATCCAT TAGAG T G GAAG CAC CAAC TACAATCCCTCCCT CAAGAGT C GAG T CAC CATAT CAG TAGACAC G T C CAAGAAC CAG TTCTCCCT GAAG C T GAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAT ACTGGGTACTACGGTATGGACATCTGGGGCCAAGGGACCACGGTCACCG TCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTC CTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAG GACTACTTCCC CGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGA CCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTA CTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAGAG T T GAG C C CAAAT C T T G T GAGAAAAC T CACACAT G C C CAC C G T G CCCAGCACCTGAAGCCGAAGGGGCCCCGTCAGTCTTCCTCTTCCCCCCA AAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCG TGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTA

- 148 044112

CGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAG CAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAG GAGATGACCA AGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAG ACCACGCCTCCCGTGCTGGACTCCGACGCTCCTTCTTCCTCTATAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG CTCCGTGATGCATGAGGCTCTGCACAACCACTACACG CAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 244 Heavy chain (without terminal K) IgG1.3f (A108T) TIM3.7 (9F6) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGT CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTA CATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCA TTCATTAGTGGTGGTGGTAGTACCATATACTACGCAGACTCTGTGAAGG GCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCGCT GTTTCTGCA AATGAACAGCCTGAGAGTCGAGGACACGGCTGTGTATTACTGTGCGAGA GATGGCTATAGCAGTGGCTGGTACTACTACGGTATGGACGTCTGGGGCC AAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGT CTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCC CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA CCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA G CAACACCAAGGT GGACAAGAGAGT T GAGCCAAAAT S T T GT GACAAAAAC TCACACATGCCCAC CGTGCCCAGCACCTGAAGCCGAAGGGGCCCCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAG ACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTC ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG CAAGGTCTCCAACAAAGSSCTCCAGSSSSCATCGAGAAAACCATCTCC AAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCT CCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCA GGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTTGA 162 Light chain IgGl TIM3.5 (VAZ), TIM3.2 (17СЗ), TIM3.4 (3G4) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGG AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTA CTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACT GGAGCCTGA AGAT T T T G CAG TGTATTACTGT CAG CAG TATGGTAGCT CAC C GAT CAC C TTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTGGCTGCACCAT CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAAC GCCCTCCAATCGGGTAACTCCCAGGAGAGTG TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT

- 149 044112

GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG 163 IgGi light chain 8B9, TIM3.6 (8C4), TIM3.9 (17C8) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGG AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTA CTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACT GGAGCCTGA AGAT T T T G GAG TGTATTACTGT GAG GAG TATGGTAGCT CAC C T C T CAC T TTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCAT CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATA ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTG TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG 165 Light chain IgGi VK1 9F6 GCCATCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAG ACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTT AGCCTGGTATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTAT GATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTG GAT C T GGGACAGAT T T CAC T C T GAGCAT CAGC AGCC T GCAGCC T GAAGA TTTTGCAACTTATTACTGTCAACAGTTTAATAGTTACCCTCGGACGTTC GGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTG TCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG TGGAAGGTGGATAACGC CCTCCAATCGGGTAACTCCCAGGAGAGTGTCA CAGAGCAGCACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGAC GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTAG 166 Light chain IgGi VK2 9F6 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGG AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTA CTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACT GGAGCCTGA AGAT T T T G GAG TGTATTACTGT CAG GAG TATGGTAGCT CAC T CAC T T T C GGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCATCTG TCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG TGGAAGGTGG ATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCA CAGAGCAGGACAGCAAGCAGCACCTACAGCCTCAGCAGCACCCTGAC GCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGTTAG 164 Light chain IgGi VK3 9F6 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGG AAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTA CTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATC TATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCA GTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACT GGAGCCTGA AGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTCACT TTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTGGCTGCACCAT CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGG AGAGTG TCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG

The practice of the present disclosure will use, unless otherwise indicated, conventional cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology techniques known to those skilled in the art. Such methods are described in detail in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed.

- 150 044112 (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. US Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., NY); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986); Crooks, Antisense drug technology: Principles, strategies and applications, 2 ^nd Ed. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

All references cited above, as well as all references cited herein, are incorporated herein by reference in their entirety.

The following examples are offered as illustrations and not as limitations.

Examples

Example 1. Identification of antibodies to human TIM3.

Human IgG transgenic mice (KM) were immunized with the plasma membrane fraction of human HEK-293 cells transfected with human TIM-3. Lymph node cells from all immunized mice were fused with the SP2/0 fusion partner. Hybridoma supernatants were first screened for the presence of human IgG antibodies using a high-throughput assay. Antigen specificity was then determined by FACS binding on transfected human TIM-3 cells. Briefly, 47 fusions were performed, 3935 IgG-positive clones were identified, of which 448 were identified as hTIM3 positive by ELISA, and of these 126 were found to be hTIM3 FACS positive. Of these, 117 clones (or antibodies) were further analyzed by various methods, including: (1) epitope-specific sorting performed by Biacore; (2) TIM3-specific sorting with a TIM-3 transfected cell line (293-TIM3) to determine EC ₅₀ ; (3) Th1 assays (as further described below) and (4) TIL assays (as further described below). From 117, seven hybridomas expressing fully human anti-human TIM3 antibodies were selected as having the desired characteristics: 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8. The amino acid and nucleotide sequences encoding the antibody variable domains produced by these hybridomas are shown in FIG. 1-7, a SEQ ID NO CDR, variable regions and heavy and light chains, as well as their isotype in FIG. 13 (see lines with names of hybridomas). The hydribome and the antibody it secretes have the same name (for example, 13A3).

Antibodies containing the CDR and/or variable domains of antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 were also expressed recombinantly in host cells. Recombinant antibodies are indicated herein under the names TIM3.2 to TIM3.18. When referring to any of these recombinant antibodies by their names TIM3.2 to TIM3.18, no specific constant region is indicated, i.e. antibodies TIM3.2 to TIM3.18 may have any desired constant region, such as those shown in FIG. 13.

CDRs and variable domains were expressed in the context of the IgG1 constant region without effector functions (allotype f), which contains the substitutions L234A, L235E, G237A, A330S and P331S (IgG1.1f'), and IgG1.3f, an IgG1 constant region without effector functions (allotype f), which contains replacements L234A, L235E, G237A, i.e. differs from IgG1.1f only in that it does not contain the A330S and P331S substitutions. CDRs and variable regions can also be used in the context of IgG4, eg IgG4P (ie IgG4 with S228P substitution). Some CDRs and framework regions of these antibodies have also been mutated. In particular, VHCDR3 from 13A3 and 8B9, VHCDR3 from 13A3 and VHFR4 were mutated. A list of IgG1.1f and IgG1.3f antibodies obtained and other antibodies that can be obtained is presented in FIG. 13, in table. 1 and in the list of sequences. Antibodies expressed recombinantly include those described in the examples below, as well as antibodies 3G4, 8C4, 9F6, 8B9, 17C8, 5D6, which were expressed as IgG1.1f antibodies.

The sequence alignment of the variable regions of the heavy and light chains of antibodies 13A3, 8B9, 8C4, 17C3, 9F6, 3G4 and 17C8 is presented in Fig. 8A and 9A, respectively. Sequence designations for the VH and VL regions are shown in FIG. 8B and 9B, respectively. Sequence alignments of wild-type and mutated 13A3 VH chains are presented in Fig. 10. Sequence alignment of wild-type and mutated 9F6 VH chains is presented in FIG. 11. Sequence alignment of wild-type and mutated 8B9 VH chains is shown in FIG. 12.

Example 2. Characterization of human antibodies to TIM3.

Selected anti-TIM3 antibodies were assayed for binding to TIM3-expressing cells. In fig. 14A shows the binding of various anti-TIM3 antibodies to cells transfected with human TIM3 (FIG. 14A) and to activated anti-CD3/anti-CD28 human T cells (FIG. 14B), as

- 151 044112 determined by flow cytometry. Antibodies were also tested for binding to cynomolgus TIM3 using cells transfected with cynomolgus TIM3 (FIG. 15A) and activated anti-CD3/anti-CD28 cynomolgus T cells (FIG. 15B). In fig. 15A shows that 13A3 has the best binding EC50 for a cell line transfected with cynomolgus TIM3, and is the only anti-TIM3 antibody that reacts with activated cynomolgus T cells.

Example 3: Binding affinity of anti-TIM3 antibodies to human and cynomolgus TIM3 determined by surface plasmon resonance.

The kinetics and affinity of Fab 13A3 fragments for TIM3 relative to human and cynomolgus TIM3 were determined on a Biacore T200 instrument at 37°C in PBS, pH 7.4, supplemented with 0.05% (v/v) Tween-20 , as further described below.

The human TIM3 protein used consisted of the extracellular domain (ECD) of human TIM3 linked to a mouse Fc, thereby forming the dimeric hTIM3 ECD-Fc protein (hTIM3-mFc). This fusion protein was expressed from stably transfected CHO cells and purified from the medium using protein A affinity followed by size exclusion chromatography. The recombinant cynomolgus TIM3 protein used consisted of the extracellular domain of cynomolgus TIM3 followed by a linker and affinity markers, thereby forming the monomeric cynoTIM3 ECD protein (cynoTIM3-MycHisAvi). This fusion protein was expressed from transiently transfected Expi293 cells (Life Tech), and the protein was isolated from the medium and purified using an affinity tag (6x His) followed by size exclusion chromatography.

The amino acid sequence of hTIM3-mFc was as follows:

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWLN

GDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFP

RMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIRIGASVPRDCGCKP

CICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPRE

EQFNS T FRSVSELPIMHQDWLNGKE FKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQ

MAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGN

TFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 375)

The amino acid sequence of supo-TIM3-MycHisAvi was as follows:

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWLN

GDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKSPGGGSGGGSEQKLIS

EEDLGHHHHHHGLNDIFEAQKIEWHE (SEQ ID NO: 376)

Fabs from 13A3 and TIM3.18.IgG1.3 coupled to a histidine tail were used. The amino acid sequence of Fab 6xHis heavy chain (HC) 13A3 was as follows:

QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYNPS

LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFDPWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW

TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGHHHHHH (SEQ ID NO: 365)

The amino acid sequence of Fab 6xHis N60Q D101E heavy chain (HC) 13A3 was as follows:

QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPS

LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKG

PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSW

TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGHHHHHH (SEQ ID NO: 366)

Recombinant Fab 13A3 and TIM3.18.IgG1.3 were generated using transient transfection Expi293 (Life Tech). The expressed Fab contained a heavy chain variable region followed by CH1 from hIgG1 and a light chain variable region followed by the hKappa CL domain. The expressed Fab was secreted into the medium and purified using an affinity marker (6x His).

The anti-mouse antibody capture chip was prepared on a Biacore CM4 S series chip (GE Healthcare Life Sciences, Cat. No. BR-1005-34) using the Biacore Mouse Antibody Capture Kit (Cat. No. BR-1008-38). Human TIM3-mouse Fc fusion protein was captured on flow cells 2 and 3 at two different surface densities. The cynomolgus TIM3-mouse Fc fusion protein was captured on flow cell 4. Flow cell 1 (control capture surface) served as the reference. Recombinantly expressed His-tagged antibody Fab fragments were passed as analytes onto all surfaces in a 3-fold, 6-mer dilution series with a top concentration of 1.0

- 152 044112 µM and a lower concentration of 4.1 nM. The resulting sensorgrams were with two control sources (using flow cell 1 and a buffer control cell) and fitted to a 1:1 Langmuir coupling model with mass transfer. Data from flow cells 2 and 3 were plotted globally.

The rates of formation (Ka) and dissociation (KD) of the complex, as well as the overall dissociation constant ( _KD ) are presented in table. 2.

table 2

Kinetics and affinity of binding of antibodies to TIM3 13A3 and TIM3.18.IgG1.3 to human and cynomolgus TIM3 proteins

Ligand Analytes ka (1/Mc) kd(1/c) Kd(hM) Human TIM3 Fab hTIM3Fc/13A3 3.2χ10 ⁶ 6.9χ10' ³ 2.2 TIM3 cynomolgus macaque Fab cynoTIM3Fc/13A3 2.4xl0 ⁶ 5.3 x IO' ² 22 Human TIM3 Fab hTIM3Fc/TIM3.18 3.2xl0 ⁶ 5xl0' ³ 1.6 TIM3 cynomolgus macaque Fab cynoTIM3Fc/TIM3.18 3.4xl0 ⁶ 5.9χ10' ² 17

Experiments with 13A3 were not performed on the same day as experiments with TIM3.18.

Example 4 Binding affinity of anti-TIM3 antibodies to human and cynomolgus TIM3 determined by Scatchard assay.

Antibody TIM3.18.IgG1.3 was radioiodinated with ¹²⁵ I-Na (1 mCi; PerkinElmer catalog NEZ033H001 MS) using IODOGEN® solid phase iodination reagent (1,3,4,6-tetrachloro-3a-6a-diphenylglycuryl; Pierce catalog 28601). Excess iodide was removed using a desalting column (Pierce catalog 43243). Labeled antibody fractions were collected and analyzed for radioactivity on a Wizard 1470 gamma counter (PerkinElmer). The concentration of antibody ¹²⁵ I-TIM3.18.IgG1.3 in each fraction was calculated using a Qubit fluorometer from Invitrogen. Radiological purity was determined by thin layer chromatography of peak proteins and radioactive fractions (Pinestar Technology catalog 151-005).

Radioiodinated TIM3.18.IgG1.3 antibody binding to CHO cells expressing human or cynomolgus TIM3 was demonstrated by incubating CHO cells expressing human or cynomolgus TIM3 with titration of ^{the 125} I-TIM3.18.IgG1.3 antibody. Nonspecific binding was determined by binding in the presence of titration of a 100-fold molar excess of unlabeled antibody and subtracted from the total CPM to calculate specific binding. A linear standard curve of ¹²⁵ I-TIM3.18.IgG1.3 antibody concentration versus CPM was used to extrapolate the specific activity, the maximum nM amount of bound ¹²⁵ ITIM3.18.IgG1.3 antibody and thus calculate the number of receptors per cell.

The results are shown in Fig. 27A and 27B. The standard curve of ¹²⁵ I-TIM3.18.IgG1.3 antibody (FIG. 27A) shows that 1 nM of ¹²⁵ I-labeled antibody equals 81,119.3 cpm. The number of receptors per cell is calculated using the following equation: (Bmax)χ(Avogadro's number)x(assay volume)/number of cells per well. Results show that the TIM3.18.IgG1.3 antibody has an affinity of 0.26-0.48 nM for overexpressed human TIM3 on CHO cells (with 414,720 receptors per cell) and an affinity of 0.36-0.48 nM for overexpressed Java TIM3 macaque (with 235,944 receptors per cell).

A similar assay performed with the ¹²⁵ I-TIM3.18.IgG1.3 antibody on activated human Th1 cells from 2 donors (50,000 cells/well) provided an affinity of 0.125-0.164 nM, despite an almost fourfold difference in the number of receptors per cell between donors ( Fig. 28). Binding of radioiodinated TIM3.18.IgG1.3 to human TIM3 was demonstrated by incubating activated primary human Th1 cells (prepared as described in other examples herein) with a titration of ¹²⁵ I-TIM3.18.IgG1.3. Nonspecific binding was determined by binding in the presence of titration of a 100-fold molar excess of unlabeled antibody and subtracted from the total CPM to calculate specific binding. A linear standard curve of ¹²⁵ ITIM3.18.IgG1.3 concentration versus CPM was used to extrapolate the maximum amount of bound ¹²⁵ I-TIM3.18.IgG1.3 in nM and hence calculate the number of receptors per cell.

Example 5. Lack of cross-reactivity of TIM3.18.IgG1.3 with human TIM1, human TIM4 and mouse TIM3.

After a quick search of the IgV TIM-3 domain against the entire gene bank, the most homologous molecules were TIM1 and TIM4 (45% identity). Selectivity profiling of TIM3.18.IgG1.3 using human TIM1 or TIM4-transfected cell lines by flow cytometry revealed no cross-reactivity with TIM1 or TIM4. Using flow cytometry on mouse TIM3-transfected cells, it was also shown that TIM3.18.IgG1.3 was not cross-reactive to mouse TIM-3-transfected cells.

Example 6 IFN-γ production by tumor infiltrating lymphocytes (TILs) is enhanced by anti-TIM3 antibodies.

To further characterize anti-TIM3 antibodies and identify those most likely to have significant T cell stimulatory activity in vivo, a specific T cell assay was developed. The assay measures the amount of IFN-γ secreted from tumor-infiltrating lymphocytes (TILs) isolated from fresh tumor tissue and incubated in the presence of irradiated CD3-expressing CHO cells (CHO-OKT3 cells), in the presence or absence of anti-TIM3 antibody (or control ). Without wishing to be limited by a specific mechanism of action, secretion of IFN-γ in the presence of a given anti-TIM3 antibody indicates that the antibody inhibits the negative signaling typically provided by TIM3 on TILs and stimulates activation (ie, IFN-γ production) of TILs .

Fresh tumor tissue (including tumor infiltrating lymphocytes (TIL)) from a patient with renal cell carcinoma was obtained by enzymatic digestion as a single-cell suspension (Miltenyi, catalog no. 130-095-929). Cell viability was greater than 80% as determined by FACS analysis. 1.5 105 cells were cocultured for 5 days with 2.5 10 ⁴ irradiated (67,000 RAD for 1 h 20 min; Rad Source irradiator, RS-2000 Biological System). CHO-OKT3 cells in IL-2-containing medium (EL-2 (Peprotech, catalog no. 200-02) at a concentration of 20 IU/ml) in the presence of either isotype control antibody or anti-TIM3 antibody at various concentrations. On day 5 of culture, cell supernatant was collected and IFN-γ content was assessed by ELISA (BD Opteia hlFNy ELISA kit, BD, catalog no. 555152). The results, which are shown in Fig. 16 indicate that anti-TIM3 antibodies 13A3, 3G4, 17C3, 17C8 and 9F6 stimulate IFN-γ production by renal cell carcinoma TIL cells.

Fresh tumor tissue from a lung cancer patient was digested using a Miltenyi enzymatic digestion kit (Miltenyi, catalog no. 130-095-929). The single cell suspension was cocultured with irradiated (67,000 RAD for 1 h 20 min; Rad Source irradiator, RS2000 Biological System) CHO-OKT3 cells in IL-2 containing medium (IL-2 (Peprotech, Cat. No. 200-02) in concentration of 20 IU/ml) in the presence of isotype control antibody or anti-TIM3 antibody at various concentrations. On day 5 of culture, cell supernatant was collected for IFN-γ ELISA analysis (BD Opteia hlFNy ELISA kit, BD, catalog no. 555152). The results, which are shown in Fig. 17A show that the anti-TIM3 antibodies tested (ie, 13A3 and 3G4) stimulate the production of IFN-γ by lung cancer TIL cells.

In addition, on day 3.5 of co-cultivation of a cell suspension from lung cancer tumor tissue with irradiated (67,000 RAD for 1 h 20 min; Rad Source irradiator, RS-2000 Biological System) CHO-OKT3 cells treated with isotype control antibody or antibody to TIM3 in the presence of IL-2, cells were incubated with BD GolgiStop overnight. Cells were then first stained with cell surface markers, CD45, CD4, CD8, TIM3, and PD1, and then fixed and permeabilized using the BD Cytofix/Cytoperm kit, followed by intracellular IFN-γ staining. The results, which are shown in Fig. 17B show that the percentage of cells expressing IFN-γ increases in CD8+ cells (lower panel) after treatment with anti-TIM3 antibody.

In fig. 18 shows pooled data from multiple tumor TIL experiments (performed as described above in this example) in response to anti-TIM-3 antibody clones 13A3 or 3G4 (i.e., each point in the figure represents TIL from a single patient tumor sample, treated with either 13A3 or 3G4). Several renal cell carcinoma (RCC) and lung cancer TILs responded to anti-TIM-3 antibody in stimulating IFN-γ production, while one TIL preparation from a thyroid tumor did not.

Example 7 FACS-based cross-blocking of anti-TIM3 antibodies.

All human T cells were isolated from PBMCs using the Miltenyi T cell purification kit and activated with plate-bound anti-CD3 (1 μg/ml; clone OKT3 anti-CD3, eBioscience, catalog no. 16-0037-85) and soluble anti -CD28 (µg/ml; clone CD28.2 to CD28, BD Biosciences, catalog no. 555725) for 4 days. TIM3 was expressed in >80% of T cells as determined by FACS analysis. T cells were incubated with various anti-TIM3 antibodies for 30 min, followed by incubation with selected biotin-labeled anti-TIM3 antibodies for 30 min and detected with PE-conjugated streptavidin. The results, which are shown in Fig. 19 indicate that antibodies 13A3, 3G4, 17C3, 17C8 and 9F6 are in the same sorting group (group I), i.e. cross-compete with each other, while antibodies 8B9 and 8C4 are in a separate sorting group (group II), i.e. do not compete with antibodies in group I, but compete with each other. Antibodies in sort group I have been shown to have biological activity (see examples), whereas antibodies in sort group II have weaker activity. Two anti-TIM3 antibodies that did not cross-compete with either group I or group II did not appear to have any biological activity. Sorting group I antibodies were also those that interfered with TIM3 binding to PS (as described further herein).

Example 8: Epitope mapping by yeast surface display.

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The nucleotide sequence encoding the extracellular domain of human TIM3 (NM_032782), i.e.

SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNWLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENWLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKWPAPTRQRDFTAAFPPJVILTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDSGATIR IG, (SEQ ID NO: 290), cloned into yeast display of plasmid PDV0023 by ligation into the restriction enzyme sites XhoI and NotI. Low-rate random mutagenesis was performed on the sequence to generate single-point mutations in the TIM3 coding region using the GeneMorph II random mutagenesis kit from Agilent Technologies. A library of 9.8x10 ⁶ clones was created in VWK18gal S. cerevisiae cells. 2x108 library cells were passaged and induced for antibody labeling and cell sorting. Approximately 2x10' induced cells were incubated with 100 mM primary target anti-human TIM3 antibody and 100 nM anti-cMyc antibody (9E10) for 1 hour at 25°C. Cells were washed and then detected with fluorescently labeled goat anti-human IgG-PE secondary antibody and goat anti-mouse IgG-A633 secondary antibody for 45 min at 4°C to detect bound primary antibodies on the cell surface. Labeled cells were sorted on a BD FACSARIA II instrument into yeast culture medium. Cells that were positively labeled with anti-Myc antibody and negatively labeled with anti-human TIM3 antibody were collected. The APC+/PE− cell population was expanded, passaged, and induced for a second round of identical labeling and sorting to enrich for the desired populations. Yeast plasmid DNA was purified from approximately 2 × 10 ⁷ cells from the unselected library and both rounds of selected sorted cells. For each cell population, the TIM3 target sequence was extracted and purified from yeast plasmid DNA by PCR using vector-specific primers that flank the human TIM3 sequence. Target sequence PCR products were subjected to NGS library generation using the Nextera XT Illumina Sequencing DNA Library Kit from Illumina. The resulting libraries were submitted to EA/Q2 Solutions for high-throughput sequencing on the Illumina MiSeq platform with 300 cycles per flow cell. Between 0.5 and 1.0 million sequence reads for each library were compared to the wild-type TIM3 sequence, and mutations at each position along the sequence were tabulated. The difference in mutation frequency at each residue position between the selected runs and the unselected library was calculated and used to determine critical residues for antibody binding. Positions with high mutation rates were examined for surface effects using a structural model of human TIM3 based on the known crystal structures of mouse TIM3 (PDB: 20YP and PDB: 3BIB). High mutation rates, surface-exposed residues are considered part of the epitopes, while high mutation rates, buried residues are considered false positives. False positive residues are generally residues that disrupt either local or intrinsic protein folding and indirectly alter the binding of the antibody to its surface epitope.

In fig. 20 shows the residues that were identified as part of the human TIM3 epitope for each of the antibodies used. Additionally, D104 shows a positive mutation score in all mappings and may represent a structural salt bridge to R81. For the 8B9 epitope, L84 exhibits a high mutation rate, although it appears to be embedded in a structure supporting epitope residues. Q113 shows a low but positive score for 13A3. It likely plays a structural supporting role for the epitope region but exhibits some surface effects.

Example 9. Blocking the TIM3-PtdSer interaction with antibodies to TIM3.

The tandem blocking assay shown in FIG. 21A was used to determine whether anti-TIM3 antibodies inhibit the interaction between human TIM3 and phosphatidylserine (PtSer or PS). Since PS is insoluble in water, PS liposome was prepared for analysis. Briefly, lipids were mixed with methanol/chloroform and then the chloroform was evaporated under a stream of nitrogen and vacuum overnight. Subsequently, the lipids were sonicated using a microtip to completely disperse the lipid to create the liposome. They were then passed through the extruder >10 times to ensure uniform size.

PS liposomes are prepared using PS (L-a-phosphatidylserine (brain, porcine) Avanti Polar Lipids, catalog no. 840032C) suspended in chloroform. The original PS is first diluted in chloroform to the required amount, and the chloroform is evaporated under a stream of nitrogen until liquid is visible. To remove trace amounts of chloroform, the dried PS is placed in a vacuum overnight. The dried PS is then suspended in PBS by spinning and briefly sonicating until the solution becomes cloudy. To create PS liposomes of a certain size, an extruder with a 100 nm filter is used. The suspended PS is loaded into an extruder and passed through a filter at least 10 times. At this point, the PS liposome is diluted in PBS to the required concentration.

In the tandem blocking assay, TIM3 (ECD)-Fc was captured on the Octet biosensor and the antibody

- 155 044112 to TIM3 and PS liposomes bound to the TIM3 protein. When anti-T1M3 binds to a region that blocks PS binding, the PS liposome does not detect binding.

The results, which are shown in Fig. 21B indicate that antibodies 3G4, 13A3, 17C3 and 17C8 inhibit the binding of PtSer to human TIM3, whereas the other 2 anti-TIM3 antibodies, i.e. AbA and AbB do not inhibit PtSer binding to human TIM3. As further described in the Examples, antibodies that inhibit PtSer binding are also the antibodies that have the most potent functional activity (as determined in Th1 and TIL assays).

Example 10: Epitope mapping by HDX-MS of anti-TIM3 antibodies.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to probe hTIM-3 binding epitopes with antibodies 13A3 and 3G4.

HDX-MS probes protein conformation and conformational dynamics in solution by monitoring the rate and extent of exchange of deuterium with backbone amide hydrogen atoms [1, 2]. The level of HDX depends on the solvent availability of backbone amide hydrogen atoms and protein hydrogen bonds. The increase in protein mass in HDX can be accurately measured using MS. When this method is combined with enzymatic digestion, structural features at the peptide level can be resolved, allowing differentiation of peptides with exposed surfaces from those folded inwards or from those sequestered at the protein-protein complex interface. Typically, deuterium labeling and subsequent quenching experiments are performed, followed by enzymatic digestion, peptide separation, and MS analysis.

Before epitope mapping experiments, deuteration-free experiments were performed to obtain a list of common peptides for recombinant human TIM-3 ((hTIM3-ECD (22-200) His-tagged (see Fig. 25); 10 μM, Sino Biological Inc.) and protein complexes of hTIM-3 with Fab antibodies 13A3 and 3G4 (molar ratio 1:1).Samples were injected into a Waters Enzymate BEN pepsin enzyme column (2.1x30 mm) and digested for 3 minutes at 200°C. Cooling chamber of the UPLC system, in which all chromatographic elements were placed, was maintained at a temperature of 0.0 ± 0.1 ° C during the entire measurement time.Injected peptides were captured and desalted for 3 min at a speed of 100 μl/min and then separated after 6 min using an acetonitrile gradient -water 5-40% at a rate of 65 µl/min. The separation column is an ACQUITY UPLC VEN C18 1.0 mmx50.0 column (Waters). Identification of peptic peptides was carried out using a combination of accurate mass analysis and MSE using ProteinLynx Global SERVER 2.5 (Waters) on a Waters HDX-MS system.

For the HDX-MS experiment, 5 µl of each sample (hTIM-3 or hTIM-3 with Fab antibodies 13A3 or 3G4) was diluted in 55 µl D2O2 buffer (10 mM phosphate buffer, D ₂ O ₂ , pH 7.0) to initiate reactions tagging. Reactions were carried out for different periods of time: 1, 10 and 240 min. At the end of each labeling reaction period, the reaction was quenched by adding quenching buffer (100 mM phosphate buffer with 4 M GdnCl and 0.4 M TCEP, pH 2.5, 1:1, v/v). 50 μl of the quenched sample was digested online using the same conditions as in the experiments without deuteration. All comparative experiments were carried out under identical experimental conditions. All experiments were performed in duplicate. The resulting relative deuterium levels were plotted against exchange time using DynamX 3.0™ software (Waters).

As shown in FIG. 25, 97.3% sequence coverage of hTIM-3 was obtained from HDXMS experiments. As shown in FIG. 26, analysis of HDX-MS data of hTIM-3 upon binding to Fab antibodies 13A3 and 3G4 revealed the following discontinuous epitopes:

monoclonal antibody 13A3: 49VPVCWGKGACPVFE ⁶² (SEQ ID NO: 367), ¹¹¹ RIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 368) and its fragment ¹¹⁹ NDEKFNLKL ¹²⁷ ; and monoclonal antibody 3G4: ⁴⁰ YTPAAPGNLVPVCWGKGACPVFE ⁶² (SEQ ID NO: 369), 66WLRTDERDVNY ⁷⁷ (SEQ ID NO: 3 70), ⁷⁸ WTSRYWLNGDFRKGDVSL ⁹⁵ (SEQ ID NO: 371) and ¹¹⁰ CRIQIPGIMNDEKFNLKL ¹²⁷ (SEQ ID NO: 372).

In fig. 26 shows the HDX-MS peptides to which antibodies 13A3 and 3G4 bind, as determined using the HDX-MS protocol described in this example.

Thus, antibody 13A3 interacts with the regions of amino acid residues 49-62 and 111127 of hTIM3, but does not significantly interact with other regions, such as the region that is N-terminal to amino acid residue Y40 or V49, the region that is located between the amino acid residues E62 and R111, and the region that is C-terminal to amino acid residue L127. 13A3 binds to the phosphatidylserine binding loop of the IgV TIM-3 domain.

Example 11. IHC staining for TIM3.18 target with cross-reactivity of human tissues.

Immunohistochemistry (IHC) was performed with 13A3 on frozen sections of human and cynomolgus macaque spleens. In both species, 13A3 (0.5 μg/ml) stained the endothelium of the venous sinusoids. As expected, antibody 3G4, which does not cross-react with cynomolgus TIM-3, stained human but not cynomolgus spleen.

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In a preliminary tissue cross-reactivity assay, FITC-conjugated TIM3.18.IgG1.3 was applied to frozen sections or smears from 20 types of normal human tissues, which include brain, cerebellum, heart, liver, lung, kidney, PBMC, spleen , tonsil, thymus, skin, colon, small intestine, stomach, pancreas, peripheral nerve, pituitary gland, thyroid gland, prostate gland and placenta (1 donor each). Specific staining was observed in a subset of mononuclear cells (MNC) in PBMC, spleen, and tonsils, as well as in epithelial reticular cells or macrophages in the thymus. The deepest staining was in macrophage/DC-like cells, which were observed in every tissue examined, including tissue-specific macrophages (eg, Kupffer cells in the liver, cutaneous macrophages/DC in the skin, and Hofbauer cells in the placenta). At the organ level, the strongest staining was found in the spleen. Apart from small subsets of MNC, strong staining was very frequently observed in splenic endothelial cells in the red pulp. In addition, positive staining was observed in a small subset of cortical tubular epithelial cells in the renal cortex.

Example 12. Antitumor activity of combined antibodies to TIM3 and PD-1 in mice.

Commercial rat antibodies to mouse TIM-3 (RMT3 23) and mouse PD-1 (RMP1-14) (Bio-X-Cell) were evaluated in the CT26 colorectal tumor model. The experimental design was similar to the previously described in vivo study in Ngiow et al. (2011) Cancer Res. 71:3540. Because TIM-3 is expressed relatively late (day 15) in this tumor model, a small volume of tumor cells (2x105) was implanted into the flank of each mouse so that tumor growth would be minimal, allowing time for TIM-3 expression. When tumors became palpable on day 8, mice were randomized into 4 treatment groups of 10 mice each with an average tumor volume of 40 mm ³ . RMT3-23 (anti-TIM-3 antibody) and RMP1-14 (anti-PD-1 antibody) were administered by intraperitoneal injection as single or combination agents (250 μg/injection of each antibody); isotype control was administered at a dose of 500 μg/injection. Each study animal received 250 μg of one antibody or 500 μg of 2 combination antibodies per injection for a total of 3 doses. Tumor size was assessed every two weeks. Mice in each group treated with individual or combined investigational products exhibited antitumor activity, while 2/10 mice in the anti-PD-1 monotherapy group and 6/10 mice in the anti-PD-1 and anti-TIM-3 combination group remained without tumors at the end of the study (Fig. 24A). A previous CT26 study of the same design yielded similar results: 3/10 mice in the anti-PD-1 monotherapy group and 7/10 mice in the combination anti-PD-1 and anti-TIM-3 group were tumor-free. There was little or no antitumor activity when anti-TIM-3 was administered as a single agent.

It should be noted that the EC50 value of RMT3-23 binding to activated murine T cells is 1.7 nM, which is 17 times weaker than the EC50 of TIM3.18 for binding to human TIM-3. Another rat anti-mouse TIM-3 antibody (Ab M), which cross-blocks RMT3-23, has an EC50 of 0.1 nM in binding to activated murine T cells, which is equivalent to the EC50 of TIM3.18. Like RMT3-23, Ab M maps to PS-binding loops of mouse TIM-3. The use of this antibody with the heavy chain constant region mIgG1-D265A (Fc-inert isotype) in the CT26 tumor model demonstrated that it enhances the antitumor response to anti-PD-1 (Fig. 24B).

Example 13 Th1 cell proliferation assay using anti-TIM3 antibody blockade (full-length or Fab).

To further characterize antibodies to TIM3, a specific T cell proliferation assay was developed using in vitro polarized Th1 cells. Polarized Th1 cells were generated by repeated restimulation of naïve CD4+ T cells. These cells were then incubated with irradiated (growth arrested) CHO-OKT3 cells in the presence of anti-TIM3 antibodies (or control) and Th1 cell proliferation was measured.

Naïve CD4 T cells were polarized to Th1 cell-like memory T cells as follows. Naïve CD4 T cells were purified from PBMCs using a naïve CD4 T cell isolation kit from Miltenyi. Cells were cultured for 3-4 days in IMDM/10% FBS at 3.6 x 105 cells/ml in the presence of: CD3/CD28 (80%/20%, respectively) coated beads at a ratio of 1 bead to 1 cell; 10 ng/ml human IL-2; 1 ng/ml human IL-12 and 10,000 ng/ml anti-human IL-4 antibody. After incubation, the cells were collected in a tube, the beads were removed using a magnet, and the cells were returned to culture in a new flask. Recombinant human IL-2 was added to a final concentration of 4 ng/ml, and cells were incubated for an additional 3 days. Cells were then harvested and washed with IX IMDM/10% FBS. Cells were counted, resuspended in IMDM/10% FBS at 4.1 x 105 cells/ml and cultured for 3-4 days in the presence of: CD3/CD28 (80%/20%, respectively) coated beads at a ratio of 1 bead per cell ; 10 ng/ml human IL-2; 1 ng/ml human IL-12 and 10,000 ng/ml anti-human IL-4 antibody. After incubation, the cells were collected in a tube, the beads were removed using a magnet, and the cells were returned to culture in a new flask.

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Recombinant human IL-2 was added to a final concentration of 4 ng/ml, and cells were incubated for an additional 2-3 days. Polarized Th1 cells were then collected and washed 3 times. On the day of analysis, polarized Th1 cells were resuspended in complete medium.

The following reagents were used:

Microparticles Dynabeads M-450 Epoxy Dynal Biotech A.S.A. 140.11 Sodium phosphate buffer at a concentration of 100 mM, pH 8.5 Teknova 0214-250 Clone UCHT-1 to hCD3 function. class eBioscience 16-0038-85 Clone CD28.2 to hCD28 function. class eBioscience 16-0289-85 Recombinant human IL-2 PeproTech, Inc. 200-02 Recombinant human IL-12 PeproTech, Inc. 200-12 Anti-human IL-4 antibody eBioscience 16-7048-85 Iscove's DMEM Mediatech, Inc. 10-016-CM Fetal bovine serum (inactivated by heat clone) SH30071.03

The CHO-OKT3 cell line was grown in shake flasks and irradiated (67,000 RAD for 1 h 20 min; Rad Source irradiator, RS-2000 Biological System) on the day of analysis. Irradiated CHO-OKT3 cells provided T cell stimulation and exposed phosphatidylserine (PS), as confirmed by Annexin V staining. TIM3.18.IgG1.3 or isotype control was titrated from 20 μg/ml by 4-fold serial dilutions, with each condition set to three repetitions. Fab TIM3.18.IgG1.3 was titrated from 53 μg/ml also by 4-fold serial dilution. The Fab fragment TIM3.18.IgG1.3 was the same as that used in the crystallographic experiment (see examples).

Cultures were plated in flat-bottomed 96-well plates treated with TC (Costar) with 1x105 polarized Th1 cells and 2.5x104 irradiated CHO-OKT3 cells (CHO:T cell ratio was 1:4) in 200 µl complete medium per well in the presence of 0.1 μg/ml anti-CD28 (clone CD28.2, BD Biosciences, catalog no. 555725) and incubated for 3 days at 37°C and 5% CO ₂ . The plates were then pulsed with 1 μCi of tritium-labeled thymidine (Perkin Elmer, catalog no. NET027001MC) per well for 16 h, and then cells were collected onto filter plates (Perkin Elmer) for tritium-labeled thymidine incorporation assay to assess proliferation.

The results, which are shown in Fig. 29A and 29B show that the anti-TIM3 antibody TIM3.18.IgG1.3 increased Th1 cell proliferation in a dose-dependent manner in a CHO-OKT3/Th1 cell coculture assay. The overall activity of TIM3.18.IgG1.3 is equivalent to that of its parent antibody 13A3 (IgG4 isotype) (FIG. 29A). The TIM3.18.IgG1.3 Fab fragment also exhibited a dose-dependent induction of proliferation (FIG. 29B) in the CHO-OKT3/Th1 cell assay.

Thus, TIM3.18.IgG1.3 (both full-length and Fab) potentiated Th1 cell activity in a dose-dependent manner in co-culture with irradiated CHO-OKT3 cells. The presence of activity with the Fab fragment indicated that TIM3.18.IgG1.3 acts as an antagonistic antibody and that TIM-3 is an inhibitory receptor for T cell function. Fc cross-linking was not required for TIM3.18.IgG1.3 biological activity.

Example 14 Th1 cell proliferation assay with simultaneous blockade of TIM-3 and PD-1.

This assay was a coculture of irradiated (growth arrested; 67,000 RAD for 1 h 20 min; Rad Source irradiator, RS-2000 Biological System) CHO-OKT3 cells transfected with human PD-L1 (CHO-OKT3-PD-L1) , and Th1 cells at a CHO:T cell ratio of 1:4 in the presence of anti-CD28. The CHO-OKT3-PD-L1 cell line was grown in shake flasks and irradiated on the day of analysis. Polarized Th1 cells were prepared as described in other examples described herein. On the day of analysis, polarized Th1 cells were resuspended in complete medium.

The anti-PD-1 antibody nivolumab was titrated at 10 μg/ml through 10-fold serial dilutions, with each condition performed in triplicate. Anti-TIM3 antibody TIM3.18.IgG1.3 or isotype control was added at a dose of 20 μg/ml.

Cultures were plated in flat-bottomed 96-well plates treated with TC (Costar) with 1x105 Th1 cells and 2.5x104 CHO-OKT3-PD-L1 cells in 200 μl complete medium per well [in the presence of 0.1 μg/ml anti-CD28 (clone CD28.2, BD Biosciences, catalog no. 555725) and incubated for 3 days at 37°C and 5% CO ₂ . The plates were then pulsed with 1 μCi of tritium-labeled thymidine (Perkin Elmer, cat. no. NET027001MC) per well for 16 h, and then cells

- 158 044112 were collected on filter plates (Perkin Elmer) for tritiated thymidine incorporation assay to assess proliferation.

The results, which are shown in Fig. 30 indicate that the anti-PD-1 antibody nivolumab increased Th1 cell proliferation stimulated with CHO-OKT3-PD-L1 cells in a dose-dependent manner, and that proliferation was significantly enhanced in combination with TIM3.18.IgG1.3 . Co-blockade of TIM3 and PD-1 pathways showed additive effects in this assay.

Example 15: IFN-γ release assay from tumor-infiltrating lymphocytes with TIM3.18.IgG1.3 blockade.

For this analysis, fresh tumor tissues were obtained from a surgically removed human renal cell carcinoma specimen or a breast cancer specimen. Tumor infiltrating lymphocytes (TILs) were isolated using an enzymatic dissociation kit (Miltenyi, catalog no. 130-095-929). TILs were supplemented with 20 IU/ml IL-2 (recombinant human IL-2, Peprotech, catalog no. 200-02) and cocultured with irradiated ones (growth arrested; 67,000 RAD for 1 h 20 min; Rad Source irradiator, RS-2000 Biological System) with CHO-OKT3 cells in a CHO:T cell ratio of 1:6. The CHO-OKT3 cell line was grown in shake flasks and irradiated on the day of analysis.

Anti-TIM-3 antibody TIM3.18.IgG1.3 or isotype control was titrated from 20 μg/ml by 4-fold serial dilutions, with each condition performed in triplicate. Cultures were plated in TC-treated flat-bottom 96-well plates (Costar) with 1.5 x 105 T cells and 2.5 x 104 irradiated CHO-OKT3 cells in 200 µl per well in IMDM+5% FBS and 5% human serum AB (Gemini , catalog no. 100-512) and incubated for 5 days at 37°C and 5% CO ₂ . The supernatant was collected from each sample to measure IFN-γ using ELISA (BD Opteia hIFN-γ ELISA kit, BD, catalog no. 555152).

The results, which are shown in Fig. 31 for renal cell carcinoma TILs and FIG. 32 for breast cancer TILs indicate that TIM3.18.IgG1.3 increased IFN-γ production in a dose-dependent manner in a CHO-OKT3/TIL co-culture assay with a 4-fold increase compared to negative controls at higher concentrations of TIM3. 18.IgG1.3 in renal cell carcinoma TIL assay.

Example 16 TIM3.18.IgG1.3 stimulates IFN-γ secretion in an M0:T allogeneic MLR assay.

Isolated CD14+ monocytes from healthy donors were differentiated to the M0 stage in a culture medium containing M-CSF. After day 6 in culture, a significant population of macrophages expressed CD163+ and CD206+ on the cell surface when stained by FACS, consistent with the signature of suppressive macrophages. Using flow cytometry with anti-TIM-3 antibody, TIM-3 was shown to be expressed in M0 macrophages (FIG. 33). These M0 macrophages were then irradiated (5000 RAD for 7 min; Rad Source irradiator, RS-2000 Biological System) and cocultured with total allogeneic donor T cells, and on day 6 after coculture, the mixed cells were pulsed with ³ H -thymidine overnight to assess T cell proliferation.

The results, which are shown in Fig. 34 indicate that TIM3.18.IgG1.3 increased T cell proliferation compared to isotype control.

Example 17. Crystal structure of Fab TIM3.18.IgG1.3 interacting with hTIM3.

The IgV region of hTIM3 was co-crystallized with the Fab fragment of TIM3.18 as follows. The following sequences were used:

- 159 044112 hTim3_IgV:

HHHHHH SAALEVL FQG P GSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNW

LRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKP

A (SEQ IDNO: 377; TIM3 sequence is underlined)

Tim3.18_Fab:

QLQLQESGPGLVKPSETLSLTCTVSGGSISSRSYYWGWIRQPPGKGLEWIGSIYYSGFTYYQPS LKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCATGGPYGDYAHWFEPWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS SW TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCGGHHHHHH (SEQ ID NO: 366)

Tim3.18_kappa:

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFS GSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACE VTHQGLSSPVTKSFNRGEC (SEQ ID NO: 29)

Expression and purification. The histidine-tagged IgV domain of hTim3 was expressed in E. Coli (BL21 DE3) with the pET47K vector. Purification and refolding were performed according to the published protocol for mTim3 (DeKruyff et al. J. Immunology 2010). Fab Tim3.18 was transiently expressed in HEK293 cells and purified through the C-terminal His tag on the heavy chain.

Crystallization of the complex and determination of the structure. The crystal structure of the IgV domain of hTim3 with Fab Tim3.18 differed by up to 1.5A. The Fab:antigen complex was first screened for crystallization conditions using various screens from Hampton Research, and crystal clusters were observed under PEG 3350 conditions with a pH ranging from 6.5 to 5.5. Crystal growth conditions were further optimized to ensure single crystal growth. Single crystals were collected with glycerol as a cryoprotectant and flash frozen in liquid nitrogen. Data acquisition was carried out at IMCA-CAT at APS using a Pilatus-6M detector. Diffraction images were processed using Global Phasing software and phased using the Tim3.18 Fab model. Numerous improvements were carried out using the CCP4 package, Coot, Phenix and Global Phasing software.

The resolved IgV domain of hTim3 corresponds well to the domain of the published structure of hTim3 (PDB: 5F71; worldwideweb.rcsb.org/pdb/explore/explore.do?pdbId=5F71), as well as the structure of mTim3 (PDB: 3KAA; worldwideweb.rcsb.org/pdb /explore/explore.do?structureId=3kaa; Rosemarie et al (2010) J Immunol 184:1918), which was resolved in complex with PS. The PS binding pocket in hTim3 was inferred from these structural alignments. In addition, the location of the PS binding pocket is conserved among human and mouse TIM members (Freeman et al. (2010) Immunol Rev. 235: 172).

Contact residues for TIM3.18 on the hTim3 protein were identified by calculating the difference in available surface area between the hTIM3:TIM3.18 Fab crystal structure and the hTIM3 structure alone (embedded surface method). The hTIM3 residues that show the contact surface area when complexed with Fab TIM3.18 were determined to be part of the contacting residues. The solvent accessible surface of the protein was defined as the locus of the center of the probe sphere (representing a solvent molecule of radius 1.4 Å) as it rolled over the van der Waals surface of the protein. The surface area available to the solvent was calculated by creating surface points on an extended sphere around each atom (at a distance from the center of the atom equal to the sum of the radii of the atom and the probe) and excluding points lying in equivalent spheres associated with neighboring atoms, implemented in the AREAIMOL program (http://www.ccp4.ac.uk/newsletters/newsletter38/03_surfarea.html).

The results, which are shown in Fig. 35 and 36 show that the following amino acids are contacting residues as determined by the above-described method for determining immersed surfaces: P29, V30, C31, P38, V39, F40, E41, C42, G43, N44, V45, V46, L47, R48, T49, D50, E51, D53, R90, Q92, G95, I96, M97, D99 (numbered according to SEQ ID NO: 290, which is the extracellular domain of mature hTIM3) or P50, V51, C52, P59, V60, F61, E62 , C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, Q113, G116, I117, M118, D120 (numbered by SEQ ID NO: 286 (Fig. 20), which is hTIM3 with a signal peptide). These results indicate that the contacting residues of TIM3.18.IgG1.3 on human TIM3 overlap with the PS binding pocket on human TIM3. Specifically, heavy chain CDR2 from TIM3. 18 occupies the PS binding pocket. Additional contacts with the PS binding loops are made by heavy chain CDR1 and CDR3. The structural data obtained here confirm the results obtained in the blocking analysis.

- 160 044112 PS (see examples).

The crystallography results also show that the following amino acid residues of hTIM3 contain an atom that is within 5A of an amino acid residue atom (5A distance method) from Fab TIM3.18: P29 V30, C31, P38, V39, F40, E41, C42, G43, N44, V45, V46, L47, R48, D50, E51, D53, R90, 191, Q92, G95, I96, M97, D99 (numbered according to SEQ ID NO: 290, which is the mature extracellular domain of hTIM3) or P50, V51 , C52, P59, V60, F61, E62, C63, G64, N65, V66, V67, L68, R69, D71, E72, D74, R111, I112, Q113, G116, I117, M118, D120 (numbering according to SEQ ID NO : 286 (Fig. 20), which is hTIM3 with signal peptide). Specific interacting residues of the Fab and hTIM3 proteins are presented in Table. 3.

Table 3

List of human TIM3 residues that interact with Fab residues Remaining T i m3 Antibody Tim3.18 heavy/light chain Residue no.: remainder hyp 29(PRO) N/ 56 (SER) N/ 57 (GLY) n/ 58 (RNE) 30(VAL) n/ 56 (SER) n/ 58 (RNE) 31(CYS) n/ 58 (RNE) n/ 55 (TYR) 38(PRO) n/ 106 (TYR) 39(VAL) n/ 60 (TYR) n/ 106 (TYR) L/ 92 (TYR) L/ 93 (GLY) L/ 94 (SER) L/ 95 (SER)

40(RNE) N/ 49 (TRP) n/ 54 (TYR) n/ 60 (TYR) n/ 106 (TYR) n/ 108 (HIS) L/ 95 (SER) L/ 97 (ILE) 41(GLU) n/ 54 (TYR) n/ 55 (TYR) n/ 56 (SER) n/ 58 (RNE) n/ 60 (TYR) n/ 103 (TYR) 42(CYS) n/ 103 (TYR) n/ 104 (GLY)

- 161 044112

43(GLY) N/ N/ N/ N/ 103 104 105 106 (TYR) (GLY) (ASP) (TYR) 44(ASN) N/ 103 (TYR) N/ 104 (GLY) N/ 105 (ASP) 45(VAL) N/ 32 (ARG) N/ 33 (SER) N/ 55 (TYR) N/ 103 (TYR) N/ 104 (GLY) 46(VAL) N/ 32 (ARG) 47(LEU) N/ 32 (ARG) N/ 35 (TYR) 48(ARG) N/ thirty (SER) N/ 31 (SER) N/ 32 (ARG) N/ 55 (TYR) 50(ASP) N/ 55 (TYR) N/ 56 (SER) 51(GLU) N/ thirty (SER) 53(ASP) N/ 32 (ARG) 90(ARG) N/ 58 (PHE) N/ 60 (TYR) 91(ILE) N/ 58 (PHE) 92(GLN) N/ 57 (GLY) N/ 58 (PHE) N/ 59 (THR) N/ 60 (TYR) 95(GLY) N/ 66 (LYS) 96(ILE) N/ 66 (LYS) 97(MET) N/ 66 (LYS) 99(ASP) N/ 58 (PHE) N/ 60 (TYR)

Comparison of the amino acid residues identified by both methods shows that the residues are essentially the same, with the exception of residue T49, which is identified only by the embedded surface method, and residue I91, which is identified only by the 5 A distance method.

Example 18 Additional characteristics of TIM3.18.IgG1.3.

Biophysical characteristics of TIM3.18.IgG1.3 expressed in CHO cells are presented in Table. 4.

- 162 044112

Table 4

Biophysical characteristics of TIM3.18.IgG1.3

Property Way results Identification Peptide map LC- Deglycosylated MW = 145619 Yes (same as MS/MS was predicted) Deglycosylated, reduced and alkylated HC = 50068 Yes Aglycosylated, reduced and alkylated LC = 23683 Yes >99% sequence and disulfide structure confirmed by peptide mapping and mass spectrometry Purity/homogeneity CE-SDS 95.1% monomer, impurities include 2.9% HHL, 0.9% HL, 0.5% HH, 0.6% LC; non-glycosylated heavy chain 2.1% SEC 98.5% monomer SE-MALS 99.94% (150 kDa), 0.06% (322 kDa) HIC-HPLC 92% main peak, 1% peak before main peak, 7% peak after main peak CE (glycans) GOF (79.3%), GIF (12.2%), G2F (0.7%), Map5 (6.8%), GO (0.9%). cIEF Main peak pl = 8.6, range pl 8.17-8.66 Chemical LC-MS/MS Very low modifications peptide map Thermal stability and DSC (dissolved in reversibility storage buffer) Tsh1 =68.1°C, Tsh2 = 80.3°C, Tsh3=82.6°C Reversibility at 74°C = 96%, at 80°C = 26%

A single N-glycosylation site was confirmed at N297 on the heavy chain with a glycan profile that is consistent with the glycan profile of CHO-expressed IgG1 monoclonal antibodies. TIM3.18.IgG1.3 does not bind to CD16, CD32, or CD64, indicating that it is inert to any Fc-FcR-mediated effector function. TIM3.18.IgG1.3 is characterized by good thermal stability (Tm1=68.1°C, Tm2=80.3°C, Tm3=82.6°C) and thermal reversibility (95.6% at 74°C, 25 .5% at 80°C), which suggests that the molecule retains its structural integrity under heat stress and has robust refolding properties under stress.

The stability characteristics of TIM3.18.IgG1.3 are presented in Table. 5.

Table 5 Stability of TIM3.18.IgG1.3

Property Method(s) results Freezing/warming (1 hour@-80°C, 1 hour@YaTx6) UV, SEC No risk of freeze/thaw stability identified Solubility/concentration profile UV, SEC At least 60 mg/ml

Stability by SEC, DLS, HIC, 12 weeks. @ 40°C = acceleration of “accelerated aging” cIEF, peptide 2%/month in LMW mg/ml 12 weeks. @ 4°C, 25°C and LC mapping - 12 weeks. @ 40°C < 1% acceleration in

40°C as part of the MS/MS HMW platform weeks. @ 40°C = acceleration 18%/month in acid variants

There were no problems with physical stability during freeze-thaw stress (6 cycles) at 50 mg/ml. Forced degradation studies at 50 mg/mL were carried out at temperatures of 4, 25 and 40°C. No chemical modifications were observed in the CDR region over 12 weeks under any conditions.

The potential immunogenicity risk of TIM3.18.IgG1.3 was assessed by in silico methods. In silico iDAB analysis of TIM3.18.IgG1.3 showed few potential HLA-binding sequences in the CDR of this monoclonal antibody, indicating a low risk of inducing a human immune response.

Example 19. PK/PD TIM3.18.IgG1.3 in monkeys.

In a single dose PK/PD and tolerance study, all monkeys were immunized intramuscularly with 2.5 mg of keyhole limpet hemocyanin (KLH) and nonproliferative recombinant adenovirus-5 (Ad5) vectors expressing the Nef and Gag proteins of simian immunodeficiency virus (SIV) (3 x 10 ⁹ of each vector). . After immunization, monkeys were intravenously administered TIM3.18.IgG1.3 at doses of 0 (vehicle), 0.5, 10, or 25 mg/kg (N = 3/group; mixed sex). Serum samples were collected for up to 42 days to assess pharmacokinetics (PK) and anti-drug antibodies (ADA), and blood samples were collected for up to 42 days to assess receptor occupancy. Additional serum samples were reserved for other study endpoints, including soluble TIM-3 levels.

AUC _0-168h was dose proportional from 0.5 to 25 mg/kg. TIM3.18.IgG1.3 demonstrated a T1 _/2 of approximately 2 weeks and a total serum clearance of 0.18 ml/h/kg. The volume of distribution at steady state varied from 68 to 84 ml/kg, suggesting that TIM3.18.IgG1.3 is predominantly located in the extracellular space (Table 6).

Table 6

Pharmacokinetic parameters of TIM3.18.IgG1.3 after intravenous administration to cynomolgus monkeys

Researched Quantity Dose AUC(o-inf) T1/2 CLT Vss tion monkeys (mg/kg) (µMhch) (I) (ml/h/kg) (ml/kg) 3 0.5 NC NC NC NC DT16095 3 10 358 ±90* 337 ±91 0.19 ±0.047 84 ±3.8 3 25 1076±324 321± 104 0.17 ±0.053 68 ±7.4

* Extrapolated AUC exceeded the 20% limit and ranged from 21 to 55%.

Based on PK in cynomolgus monkeys and the allometric scale, the predicted total human serum clearance is 0.10 ml/h/kg and Vss is 88 ml/kg. As a result, the predicted half-life in humans is approximately 26 days.

Example 20 Preliminary cytokine release assay.

To determine whether TIM3.18.IgG1.3 treatment confers a risk of cytokine release syndrome, whole blood from 16 human donors was incubated with 20 μg/ml TIM3.18.IgG1.3 or positive control in solution. A panel of 75 serum cytokines and chemokines was examined for each donor. There was no evidence of increased release of T cell-derived cytokines or chemokines, suggesting a low risk of cytokine release syndrome. In whole blood assays from some donors, increases in IL-1β, IL-6, IL-10, TNF-α, and G-CSF were observed, consistent with the data presented above that TIM-3 blockade increases monocyte- or macrophage-derived production cytokines.

Example 21: TIM3.18.IgG1.3 does not cause receptor downregulation or internalization.

To determine whether 13A3 inhibits or internalizes human TIM3 upon binding to the cell membrane, a fluorescence quenching assay shown in FIG. 37. Results after 3 hours of treatment, which are shown in FIG. 38 indicate that neither the 13A3 antibody nor the D101E or N60Q variants caused a dose-dependent accumulation of intracellular TIM3 antibody in activated donor CD8+ T cells, indicating that the antibody is not internalized.

To determine potential down-regulation, activated donor CD8+ T cells were incubated for 2 hours in the presence of various amounts of 13A3, 13A3.D101.Igl.1f, 13A3.D101E/N60Q.IgG1.1f or control antibody or in the absence of antibody, and the amount of TIM3 per cell surface. The results showed that incubation with anti-TIM3 antibodies did not suppress cell surface TIM3.

Claims (27)

1. An isolated antibody that binds to human T-cell immunoglobulin and mucin domain-containing protein-3 (TIM3), comprising a heavy chain CDR1 containing the amino acid sequence set forth in SEQ ID NO: 41, a heavy chain CDR2 containing the amino acid sequence, presented in SEQ ID NO: 122; Heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NO: 126; Light chain CDR1 containing the amino acid sequence shown in SEQ ID NO: 64; A light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 66, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68. 2. The antibody of claim 1, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH contains the amino acid sequence set forth in SEQ ID NO: 364 and VL contains the amino acid sequence set forth in SEQ ID NO: : 60. 3. An antibody according to claim 1 or 2, wherein the antibody is selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. 4. Antibody according to any one of claims 1-3, containing Fc IgG1 without effector functions. 5. The antibody of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain contains the amino acid sequence set forth in SEQ ID NO: 349 and the light chain contains amino acids - 164 044112 lot sequence shown in SEQ ID NO: 29. 6. The antibody of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 351 and the light chain contains the amino acid sequence set forth in SEQ ID NO: 29. 7. The antibody of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 353 and the light chain contains the amino acid sequence set forth in SEQ ID NO: 29. 8. The antibody of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 350 and the light chain contains the amino acid sequence set forth in SEQ ID NO: 29. 9. The antibody of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 352 and the light chain contains the amino acid sequence set forth in SEQ ID NO: 29. 10. The antibody of claim 1, comprising a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence set forth in SEQ ID NO: 354 and the light chain contains the amino acid sequence set forth in SEQ ID NO: 29. 11. A composition for the treatment of cancer, comprising an antibody according to any one of claims 1 to 10, and a carrier. 12. A bispecific molecule comprising an antibody according to any one of claims 1 to 10, linked to a molecule having a second binding specificity. 13. Nucleic acid encoding the variable region of the heavy and light chain of an antibody according to any one of claims 1 to 10. 14. An expression vector containing a nucleic acid molecule according to claim 13. 15. In vitro host cell transformed with the expression vector according to claim 14. 16. A host cell containing a nucleic acid encoding a light chain variable region and a heavy chain variable region of an antibody according to any one of claims 1 to 10. 17. An immunoconjugate for the treatment of cancer, comprising an antibody according to any one of claims 1 to 10, associated with an agent that contains a detectable label and/or a therapeutic agent. 18. The immunoconjugate of claim 17, wherein the detectable label includes a radioisotope, an enzyme, a fluorescent label, or any combination thereof. 19. An immunoconjugate according to claim 17, wherein the therapeutic agent includes an antimetabolite, an alkylating agent, a DNA minor groove binder, a DNA intercalator, a DNA cross-linker, a histone deacetylase inhibitor, a nuclear export inhibitor, a proteasome inhibitor, a topoisomerase I or II inhibitor, a thermal protein inhibitor shock, a tyrosine kinase inhibitor, an antibiotic, an antimitotic agent, or any combination thereof. 20. A method for producing an antibody to TIM3, comprising expressing the antibody in a cell according to claim 16 and isolating the antibody from the cell. 21. A method of stimulating an immune response in a subject, comprising administering to the subject an antibody according to any one of claims 1 to 10, a bispecific molecule according to claim 12, or an immunoconjugate according to any of claims 17 to 19. 22. A method of inhibiting the growth of tumors or reducing the size of tumors in a subject, comprising administering to the subject an antibody according to any one of claims 1 to 10, a bispecific molecule according to claim 12, or an immunoconjugate according to any one of claims 17 to 19. 23. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an antibody according to any one of claims 1 to 10, a bispecific molecule according to claim 12, or an immunoconjugate according to any one of claims 17 to 19. 24. The method of claim 23, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, cancer pancreas, colorectal cancer, colon cancer, kidney cancer, head and neck cancer, lung cancer, stomach cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, central nervous system neoplasm, lymphoma, leukemia, myeloma , sarcoma and virus-related cancer. 25. The method of claim 23 or 24, wherein the cancer is metastatic cancer, refractory cancer or recurrent cancer. 26. The method according to any one of claims 22-25, further comprising administering one or more additional therapeutic agents. 27. The method of claim 26, wherein the additional therapeutic agent is an anti-PD-1 antibody, an anti-LAG-3 antibody, an anti-CTLA-4 antibody, an anti-GITR antibody, or an anti-PD-L1 antibody.