Use of anti-ICOS antibodies

Abstract

Therapeutic uses and dosing regimens of anti-ICOS antibodies or antigen-binding fragments thereof for modulating the ratio between regulatory T cells and effector T cells, for stimulating a patient's immune system, and / or for treating tumors or cancer, either as monotherapy or in combination therapy, e.g., with anti-PD-L1 antibodies or antigen-binding fragments thereof.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 190,016, filed May 18, 2021, the entire disclosure of which is hereby incorporated by reference herein.

[0002] The contents of the electronically submitted sequence listing in an ASCII text file (Name: 728466-SA9-642PC_SL_ST25.txt; Size: 290.6KB; Created: May 17, 2022) are incorporated herein by reference in their entirety.

[0003] 1.2. Field of the invention The present invention relates to compositions comprising anti-ICOS antibodies (including full-length antibodies or antigen-binding fragments thereof) for stimulating immune responses, particularly T-cell responses, in mammals. The present invention also relates to the medical use of such compositions in immuno-oncology, including anti-tumor therapy by promoting anti-tumor T-cell responses in patients, as well as the use of the compositions in other diseases and conditions in which it is of therapeutic benefit to modulate the balance between effector T cells and regulatory T cells in favor of effector T-cell activity, for example, by stimulating effector T cells and / or by depleting regulatory T cells. In some embodiments, the present invention relates to anti-ICOS antibodies as monotherapy. In other embodiments, the present invention relates to anti-ICOS antibodies as part of a combination therapy, for example, a combination therapy further comprising an anti-PD-L1 antibody (including full-length antibodies or antigen-binding fragments thereof). The present invention also relates to dosages and / or frequency of anti-ICOS antibodies (as monotherapy or as part of a combination therapy) that are surprisingly effective in stimulating mammalian immune responses, for example, anti-tumor T-cell responses, in subjects. [Background technology]

[0004] 1.3.Background ICOS (inducible T-cell costimulator) is a member of the CD28 gene family that was first identified in 1999 and is involved in regulating immune responses, particularly humoral immune responses [1]. It is a 55 kDa transmembrane protein that exists as a disulfide-linked homodimer with two differentially glycosylated subunits. ICOS is exclusively expressed on T lymphocytes and is found on various T cell subunits. It is present at low levels on naive T lymphocytes, but its expression is rapidly induced upon immune activation and upregulated in response to proinflammatory stimuli such as TCR engagement and costimulation by CD28 [2, 3]. ICOS plays a role in the late stages of T cell activation, memory T cell formation, and importantly, in regulating humoral responses through T cell-dependent B cell responses [4, 5]. Intracellularly, ICOS binds to PI3K and activates the kinases phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB). Activation of ICOS prevents cell death and upregulates cell metabolism. In the absence of ICOS (ICOS knockout) or in the presence of anti-ICOS neutralizing antibodies, there will be suppression of the proinflammatory response.

[0005] ICOS binds to ICOS ligands expressed on B cells and antigen-presenting cells (APCs) [6, 7]. As a costimulatory molecule, it serves to regulate TCR-mediated immune and antibody responses to antigens. The expression of ICOS on T regulatory cells is important because it suggests that this cell type plays a negative role in the immune surveillance of cancer cells - there is emerging evidence for this in ovarian cancer [8]. Importantly, ICOS expression has been reported to be higher on intratumoral regulatory T cells (Tregs) compared to CD4+ and CD8+ effector cells present in the tumor microenvironment. Depletion of Tregs using antibodies with Fc-mediated cell effector functions has demonstrated strong antitumor efficacy in preclinical models [9]. Increasing evidence implicates ICOS in antitumor effects in both animal models and patients treated with immune checkpoint inhibitors. In ICOS- or ICOSL-depleted mice, the antitumor effect of anti-CTLA4 therapy is reduced

[10] , whereas in normal mice, ICOS ligand increases the efficacy of anti-CTLA4 treatment in melanoma and prostate cancer

[11] . Furthermore, in humans, a retrospective study of patients with advanced melanoma showed increased levels of ICOS after ipilimumab (anti-CTLA4) treatment

[12] . In addition, ICOS expression is upregulated in bladder cancer patients treated with anti-CTLA4

[13] . It has also been observed that in cancer patients treated with anti-CTLA4 therapy, the bulk of tumor-specific IFNγ-producing CD4 T cells are ICOS positive, but a sustained increase in ICOS-positive CD4 T cells correlates with survival [12, 13, 14].

[0006] WO2016 / 120789 described anti-ICOS antibodies and proposed their use for activating T cells and treating cancer, infectious diseases and / or sepsis. Several murine anti-ICOS antibodies were generated, a subset of which were reported to be agonists of the human ICOS receptor. Antibody "422.2" was selected as the lead anti-ICOS antibody and humanized to generate a human "IgG4PE" antibody designated "H2L5". H2L5 was reported to have an affinity of 1.34 nM for human ICOS and 0.95 nM for cynomolgus ICOS, to induce cytokine production in T cells, and to upregulate T cell activation markers in conjunction with CD3 stimulation. However, mice bearing implanted human melanoma cells were reported to show only minimal tumor growth delay or increased survival when treated with H2L5 hIgG4PE compared to control treatment groups. The antibody also did not produce significant additional inhibition of tumor growth in combination experiments with ipilimumab (anti-CTLA-4) or pembrolizumab (anti-PD-1) compared to ipilimumab or pembrolizumab monotherapy. Finally, in mice bearing transplanted colon cancer cells (CT26), low doses of a murine cross-reactive surrogate of H2L5 combined with a murine surrogate of ipilimumab or pembrolizumab only slightly improved overall survival compared to anti-CTLA4 and anti-PD1 therapy alone. A similar lack of strong therapeutic benefit was shown in mice bearing transplanted EMT6 cells.

[0007] WO2016 / 154177 described further examples of anti-ICOS antibodies. These antibodies were reported to be agonists of CD4+ T cells, including effector CD8+ T cells (TEff), and to deplete T regulatory cells (TReg). The selective effect of the antibodies on TEff versus TReg cells was described, whereby the antibodies could preferentially deplete TReg while having minimal effect on TEff, which expresses lower levels of ICOS. Anti-ICOS antibodies were proposed for use in the treatment of cancer, and combination therapy with anti-PD-1 or anti-PD-L1 antibodies was described. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Hutloff A et al. ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28. Nature. 1999 Jan 21;397(6716):263-6. [Non-Patent Document 2] Beier KC et al. Induction, binding specificity and function of human ICOS. Eur J Immunol. 2000 Dec;30(12):3707-17. [Non-Patent Document 3] Coyle AJ et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity. July 2000;13(1):95-105. [Non-Patent Document 4] Dong C et al. The ICOS co-stimulatory receptor is essential for T-cell activation and function. Nature. January 4, 2001; 409(6816): pp. 97 - 101.

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

[0009] 1.4. Overview of the invention Antibodies against ICOS that act to increase effector T cell activity represent a therapeutic approach in immuno-oncology and in other medical situations where CD8+ T cell responses are beneficial, including various diseases and conditions and vaccination regimens. In many diseases and conditions involving immune components, a balance exists between effector T cells (TEff) that display CD8+ T cell immune responses and regulatory T cells (TReg) that suppress immune responses by downregulating TEff. The present invention relates to antibodies that modulate this TEff / TReg balance in favor of effector T cell activity. Antibodies that trigger depletion of ICOS highly positive regulatory T cells have the net effect of relieving TEff suppression, thus promoting effector T cell responses. An additional or complementary mechanism for anti-ICOS antibodies is to stimulate effector T cell responses via agonistic activity at the ICOS receptor level.

[0010] The relative expression of ICOS on effector T cells (TEff) compared to regulatory T cells (TReg), and the relative activity of these cell populations, influence the overall effect of anti-ICOS antibodies in vivo. The postulated mechanism of action combines agonism of effector T cells with depletion of ICOS-positive regulatory T cells. Different and even opposite effects on these two different T cell populations are achievable due to their different levels of ICOS expression. Dual manipulation of the respective variable and constant regions of anti-ICOS antibodies can provide molecules that show a net positive effect on effector T cell responses by influencing the CD8 / TReg ratio. The antigen-binding domain of the agonist antibody, which activates the ICOS receptor, is combined with the constant (Fc) region of the antibody, which promotes down-regulation and / or clearance of highly expressing cells to which the antibody binds. Effector positive constant regions are used to recruit cellular effector functions against target cells (TReg), for example, to promote antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). Thus, antibodies act to promote effector T cell activation and to downregulate immunosuppressive T regulatory cells. Since ICOS is more highly expressed on TReg than on TEff, a therapeutic balance is achieved by promoting Teff function while depleting TReg, resulting in a net increase in T cell immune responses (e.g., antitumor responses or other therapeutically beneficial T cell responses).

[0011] Several preclinical and clinical studies have shown a strong positive correlation between a high effector T cell to Treg cell ratio in the tumor microenvironment (TME) and overall survival. In ovarian cancer patients, the CD8:T-reg cell ratio has been reported to be an indicator of favorable clinical outcomes

[15] . Similar observations were made in metastatic melanoma patients after receiving ipilimumab

[16] . Preclinical studies have also shown that a high effector cell:T-reg ratio in the TME is associated with antitumor responses

[43] .

[0012] The present invention provides antibodies that bind to human ICOS, including those that surprisingly have efficacy at low doses. The antibodies target the extracellular domain of ICOS, thereby binding to T cells expressing ICOS. Examples of antibodies are provided that are designed to have an agonistic effect on ICOS, as shown by their ability to increase IFNγ expression and secretion, thus enhancing the function of effector T cells. As mentioned, anti-ICOS antibodies are also engineered to deplete the cells to which they bind, which should have the effect of preferentially downregulating regulatory T cells, boosting the suppressive effect of these cells on effector T cell responses, and thus promoting the overall effector T cell response. Regardless of their mechanism of action, it is empirically demonstrated that anti-ICOS antibodies according to the present invention stimulate T cell responses and have anti-tumor effects in vivo, as shown in the examples. By selection of the appropriate antibody format, such as one that contains a constant region with the desired level of Fc effector function, or, where appropriate, one that is absent of such effector function, anti-ICOS antibodies are tailored for use in a variety of medical contexts, including the treatment of diseases and conditions in which an effector T cell response is beneficial and / or suppression of regulatory T cells is desired.

[0013] Exemplary antibodies include STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, the sequences of which are provided herein.

[0014] In some embodiments, the present invention provides a method for treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses in a subject in need thereof, comprising administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof that binds to the extracellular domain of human and / or mouse ICOS, wherein the anti-ICOS antibody or antigen-binding fragment thereof is administered to the subject at a dose of about 0.8 mg to 240 mg.

[0015] In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof for use in the method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein (a) HCDR1, HCDR2 and HCDR3 are selected from the group consisting of SEQ ID NO: 363, sequence (b) HCDR1, HCDR2 and HCDR3 have at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:364 and SEQ ID NO:365, and LCDR1, LCDR2 and LCDR3 have at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:370, SEQ ID NO:371, SEQ ID NO:372; (b) HCDR1, HCDR2 and HCDR3 have at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:377, SEQ ID NO:378 and SEQ ID NO:379. and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386; (c) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:391, SEQ ID NO:392 and SEQ ID NO:393, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:398, SEQ ID NO:399, SEQ ID NO:399; (d) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:405, SEQ ID NO:406 and SEQ ID NO:407, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:412, SEQ ID NO:413, SEQ ID NO:414;(e) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:419, SEQ ID NO:420 and SEQ ID NO:421, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:426, SEQ ID NO:427, SEQ ID NO:428; (f) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:435, SEQ ID NO:436 and SEQ ID NO:437, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:442, SEQ ID NO:443, SEQ ID NO:444; (g) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO: 449, SEQ ID NO: 450 and SEQ ID NO: 451, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO: 456, SEQ ID NO: 457, SEQ ID NO: 458; (h) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO: 463, SEQ ID NO: 464 and SEQ ID NO: 465, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472;(i) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:477, SEQ ID NO:478 and SEQ ID NO:479, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:484, SEQ ID NO:485, SEQ ID NO:486, or (j) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:491, SEQ ID NO:492 and SEQ ID NO:493, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500. In some embodiments, (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 363, HCDR2 comprises the amino acid sequence of SEQ ID NO: 364, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 365, LCDR1 comprises the amino acid sequence of SEQ ID NO: 370, LCDR2 comprises the amino acid sequence of SEQ ID NO: 371, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 372; (b) HCDR1 comprises the amino acid sequence of SEQ ID NO: 377, HCDR2 comprises the amino acid sequence of SEQ ID NO: 378, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 379, LCDR1 comprises the amino acid sequence of SEQ ID NO: 384, LCDR2 comprises the amino acid sequence of SEQ ID NO: 385, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 386. (c) HCDR1 comprises the amino acid sequence of SEQ ID NO: 391, HCDR2 comprises the amino acid sequence of SEQ ID NO: 392, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 393, LCDR1 comprises the amino acid sequence of SEQ ID NO: 398, LCDR2 comprises the amino acid sequence of SEQ ID NO: 399, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 400; (d) HCDR1 comprises the amino acid sequence of SEQ ID NO: 405, HCDR2 comprises the amino acid sequence of SEQ ID NO: 406, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 407, LCDR1 comprises the amino acid sequence of SEQ ID NO: 412, LCDR2 comprises the amino acid sequence of SEQ ID NO: 413, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 414;(e) HCDR1 comprises the amino acid sequence of SEQ ID NO: 419, HCDR2 comprises the amino acid sequence of SEQ ID NO: 420, HCDR3 comprises the amino acid sequence of SEQ ID NO: 421, LCDR1 comprises the amino acid sequence of SEQ ID NO: 426, LCDR2 comprises the amino acid sequence of SEQ ID NO: 427, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 428; (f) HCDR1 comprises the amino acid sequence of SEQ ID NO: 435, HCDR2 comprises the amino acid sequence of SEQ ID NO: 436, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 437. (g) HCDR1 comprises the amino acid sequence of SEQ ID NO: 449, HCDR2 comprises the amino acid sequence of SEQ ID NO: 450, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 451, LCDR1 comprises the amino acid sequence of SEQ ID NO: 456, LCDR2 comprises the amino acid sequence of SEQ ID NO: 457, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 458; h) HCDR1 comprises the amino acid sequence of SEQ ID NO: 463, HCDR2 comprises the amino acid sequence of SEQ ID NO: 464, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 465, LCDR1 comprises the amino acid sequence of SEQ ID NO: 470, LCDR2 comprises the amino acid sequence of SEQ ID NO: 471, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 472; (i) HCDR1 comprises the amino acid sequence of SEQ ID NO: 477, HCDR2 comprises the amino acid sequence of SEQ ID NO: 478, and HCDR3 comprises the amino acid sequence of SEQ ID NO: 479, LCDR1 comprises the amino acid sequence of SEQ ID NO:484, LCDR2 comprises the amino acid sequence of SEQ ID NO:485, and LCDR3 comprises the amino acid sequence of SEQ ID NO:486; or (j) HCDR1 comprises the amino acid sequence of SEQ ID NO:491, HCDR2 comprises the amino acid sequence of SEQ ID NO:492, and HCDR3 comprises the amino acid sequence of SEQ ID NO:493, LCDR1 comprises the amino acid sequence of SEQ ID NO:498, LCDR2 comprises the amino acid sequence of SEQ ID NO:499, and LCDR3 comprises the amino acid sequence of SEQ ID NO:500;

[0016] In another embodiment, a method for treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses comprises administering to a subject an anti-ICOS antibody or antigen-binding fragment thereof comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 405, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 406, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 407, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 412, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 413, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 414.

[0017] In another embodiment, a method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses comprises administering to a subject an anti-ICOS antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein (a) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 366, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 373; (b) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 380, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 387; (c) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 394. (d) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 408, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 415; (e) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 422. (f) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 429, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 429; (f) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 438, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 445;(g) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 452, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 459; (h) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 467, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 473; i) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 481 and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 488; or (j) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 494 and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 501. In some embodiments, (a) the VH domain comprises the amino acid sequence of SEQ ID NO: 366, and the VL domain comprises the amino acid sequence of SEQ ID NO: 373; (b) the VH domain comprises the amino acid sequence of SEQ ID NO: 380, and the VL domain comprises the amino acid sequence of SEQ ID NO: 387; (c) the VH domain comprises the amino acid sequence of SEQ ID NO: 394, and the VL domain comprises the amino acid sequence of SEQ ID NO: 401; (d) the VH domain comprises the amino acid sequence of SEQ ID NO: 408, and the VL domain comprises the amino acid sequence of SEQ ID NO: 415. (e) the VH domain comprises the amino acid sequence of SEQ ID NO: 422 and the VL domain comprises the amino acid sequence of SEQ ID NO: 429; (f) the VH domain comprises the amino acid sequence of SEQ ID NO: 438 and the VL domain comprises the amino acid sequence of SEQ ID NO: 445; (g) the VH domain comprises the amino acid sequence of SEQ ID NO: 452 and the VL domain comprises the amino acid sequence of SEQ ID NO: 459; (h) the VH domain comprises the amino acid sequence of SEQ ID NO: 467 and the VL domain comprises the amino acid sequence of SEQ ID NO: 473;(i) the VH domain comprises the amino acid sequence of SEQ ID NO: 480 and the VL domain comprises the amino acid sequence of SEQ ID NO: 487; or (j) the VH domain comprises the amino acid sequence of SEQ ID NO: 494 and the VL domain comprises the amino acid sequence of SEQ ID NO: 501;

[0018] In another embodiment, a method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses comprises administering to a subject an anti-ICOS antibody or antigen-binding fragment thereof comprising a VH domain comprising a sequence having at least 95% sequence identity to SEQ ID NO: 408 and a VL domain comprising a sequence having at least 95% sequence identity to SEQ ID NO: 415. In some embodiments, the VH domain comprises the amino acid sequence of SEQ ID NO: 408 and the VL domain comprises the amino acid sequence of SEQ ID NO: 415.

[0019] In another embodiment, a method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses comprises administering to a subject an anti-ICOS antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein (a) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 368 and the light chain comprises at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 375. (b) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 385 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 389; (c) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 396 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 403. (d) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 410 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 417; (e) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 424 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 432. (f) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:440 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:447; (g) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:454 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:461;(h) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 468 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 475; (i) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 482 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 489; or (j) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 496 and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 503. In some embodiments, (a) the heavy chain comprises the amino acid sequence of SEQ ID NO: 368 and the light chain comprises the amino acid sequence of SEQ ID NO: 375; (b) the heavy chain comprises the amino acid sequence of SEQ ID NO: 382 and the light chain comprises the amino acid sequence of SEQ ID NO: 389; (c) the heavy chain comprises the amino acid sequence of SEQ ID NO: 396 and the light chain comprises the amino acid sequence of SEQ ID NO: 403; (d) the heavy chain comprises the amino acid sequence of SEQ ID NO: 410 and the light chain comprises the amino acid sequence of SEQ ID NO: 417; (e) the heavy chain comprises the amino acid sequence of SEQ ID NO: 424 and the light chain comprises the amino acid sequence of SEQ ID NO: 432. (f) the heavy chain comprises the amino acid sequence of SEQ ID NO:440 and the light chain comprises the amino acid sequence of SEQ ID NO:447; (g) the heavy chain comprises the amino acid sequence of SEQ ID NO:454 and the light chain comprises the amino acid sequence of SEQ ID NO:461; (h) the heavy chain comprises the amino acid sequence of SEQ ID NO:468 and the light chain comprises the amino acid sequence of SEQ ID NO:475; (i) the heavy chain comprises the amino acid sequence of SEQ ID NO:482 and the light chain comprises the amino acid sequence of SEQ ID NO:489; or (j) the heavy chain comprises the amino acid sequence of SEQ ID NO:496 and the light chain comprises the amino acid sequence of SEQ ID NO:503;

[0020] In another embodiment, a method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses comprises administering to a subject an anti-ICOS antibody or antigen-binding fragment thereof comprising a heavy chain comprising a sequence having at least 95% sequence identity to SEQ ID NO: 410 and a light chain comprising a sequence having at least 95% sequence identity to SEQ ID NO: 417. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 410 and the light chain comprises the amino acid sequence of SEQ ID NO: 417.

[0021] In another embodiment, the method comprises administering an anti-ICOS antibody that is a human IgG1 antibody.

[0022] In another embodiment, the method comprises administering KY1044.

[0023] In another embodiment, the method includes administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of about 0.5 mg to about 10 mg. In some embodiments, the method includes administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of about 0.8 mg to about 8 mg. In some embodiments, the method includes administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of less than about 8 mg (e.g., a dose of 7.5 mg or less, a dose of 7 mg or less). In some embodiments, the method includes administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of about 0.8 mg to about 2.4 mg. In some embodiments, the method includes administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of about 2.4 mg to about 8 mg.

[0024] In another embodiment, the method comprises administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of about 0.8 mg. In some embodiments, the method comprises administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of about 2.4 mg. In some embodiments, the method comprises administering to the subject an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) at a dose of about 8 mg.

[0025] In another embodiment, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered to the subject every 2-6 weeks, for example, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks or every 6 weeks. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered every 3 weeks. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered every 6 weeks. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered monthly.

[0026] In another embodiment, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered once. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered more than once. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered for at least 6 months, e.g., 6 months, 12 months or more than 12 months.

[0027] In another embodiment, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered as a monotherapy. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044) is administered in a combination therapy. For example, in some embodiments, the method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses further comprises administering a second therapeutic agent to the subject.

[0028] In another embodiment, the second therapeutic agent comprises an anti-PD-L1 antibody or antigen-binding fragment thereof. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered to the subject at a dose of about 1200 mg.

[0029] In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered to the subject every 2-6 weeks, for example, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered every 3 weeks. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered every 6 weeks. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered monthly.

[0030] In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered once. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered more than once. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered for at least 6 months, such as 6 months, 12 months or more than 12 months. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof is co-administered with the anti-ICOS antibody or antigen-binding fragment thereof to the subject every 3 weeks.

[0031] In another embodiment, an anti-PD-L1 antibody or antigen-binding fragment thereof (e.g., atezolizumab) is administered to a subject in alternating doses with an anti-ICOS antibody or antigen-binding fragment thereof (e.g., KY1044), e.g., the anti-PD-L1 antibody or antigen-binding fragment thereof is administered every 3 weeks and the anti-ICOS antibody or antigen-binding fragment thereof is administered every 6 weeks.

[0032] In another embodiment, the method comprises treating a tumor. In some embodiments, the method comprises treating a cancer. In some embodiments, the cancer comprises advanced and / or metastatic cancer. In some embodiments, the cancer comprises triple-negative breast cancer, head and neck squamous cell carcinoma, penile cancer, pancreatic cancer, non-small cell lung cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, melanoma, renal cell carcinoma, and / or cervical cancer.

[0033] Pharmaceutical compositions comprising the antibodies are also provided.

[0034] ICOS knockout animals were used to generate cross-reactive antibodies. Notably, strong titers were obtained in ICOS knockout mice, and highly functional antibodies were isolated from the antibody repertoire, including the desired cross-reactive antibodies. See WO2018 / 029474A2, which is incorporated herein by reference in its entirety.

[0035] Exemplary embodiments of the invention are illustrated in the drawings, the following description, and the appended claims.

[0036] 1.5. Brief description of the drawings Certain aspects and embodiments of the present invention will now be described in more detail with reference to the accompanying drawings. [Brief description of the drawings]

[0037] [Figure 1] Graph showing A20 tumor volume over time in mice for the study described in Example 1. Each treatment group is represented by a spider plot showing tumor size in individual animals, n=8 per group. For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing was administered on days 8, 11, 15, 18, 22, 25 and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 1) and anti-PD-L1 treatment group (Figure 2), the STIM001 mIgG2a (Figure 3) and STIM003 mIgG2a (Figure 4) treatment groups showed significant inhibition of A20 tumor growth. [Diagram 2] Graph showing A20 tumor volume over time in mice for the study described in Example 1. Each treatment group is represented by a spider plot showing tumor size in individual animals, n=8 per group. For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing was administered on days 8, 11, 15, 18, 22, 25 and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 1) and anti-PD-L1 treatment group (Figure 2), the STIM001 mIgG2a (Figure 3) and STIM003 mIgG2a (Figure 4) treatment groups showed significant inhibition of A20 tumor growth. [Diagram 3]Graph showing A20 tumor volume over time in mice for the study described in Example 1. Each treatment group is represented by a spider plot showing tumor size in individual animals, n=8 per group. For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing was administered on days 8, 11, 15, 18, 22, 25 and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 1) and anti-PD-L1 treatment group (Figure 2), the STIM001 mIgG2a (Figure 3) and STIM003 mIgG2a (Figure 4) treatment groups showed significant inhibition of A20 tumor growth. [Figure 4] Graph showing A20 tumor volume over time in mice for the study described in Example 1. Each treatment group is represented by a spider plot showing tumor size in individual animals, n=8 per group. For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing was administered on days 8, 11, 15, 18, 22, 25 and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 1) and anti-PD-L1 treatment group (Figure 2), the STIM001 mIgG2a (Figure 3) and STIM003 mIgG2a (Figure 4) treatment groups showed significant inhibition of A20 tumor growth. [Diagram 5] Amino acid sequences of the VH (top) and VL (bottom) domains of STIM002 showing residues that differ in the corresponding sequences of STIM001, STIM002B and related antibodies CL-61091, CL-64536, CL-64837, CL-64841 and CL-64912 and / or in the human germline. Sequence numbering is according to IMGT. [Figure 6]Amino acid sequences of the VH (top) and VL (bottom) domains of STIM003 showing the residues that differ in the corresponding sequences of related antibodies CL-71642 and CL-74570 and / or in human germline. The sequence numbering is according to IMGT. The VL domain of antibody CL-71642 obtained from sequencing is shown here without the N-terminal residue. From the alignment, it can be seen that all VH domain sequences include an N-terminal glutamic acid. [Figure 7] Amino acid sequences of the VH (top) and VL (bottom) domains of STIM007 showing residues that differ in the corresponding sequence of STIM008 and / or in the human germline. Sequence numbering is according to IMGT. [Figure 8] Efficacy of STIM003 (anti-ICOS) and AbW (anti-PD-L1) mIgG2a antibodies in the J558 syngeneic model. Each treatment group is represented by a "spider plot" showing the tumor size of individual animals (n=10 or n=8 per group). STIM003 monotherapy demonstrated some efficacy, with 3 out of 8 animals cured of their disease. Similarly, anti-PDL1 was effective in this model, with 6 out of 8 animals cured of their disease by day 37. When combined with anti-PDL1 antibody, STIM003 mIgG2 completely inhibited tumor growth and improved survival of treated animals. For each group, the number of animals cured of their disease is shown at the bottom right of the individual graphs. Days of dosing are indicated by dotted lines (days 11, 15, 18, 22, 25 and 29). [Figure 9A]Quantification of ICOS expression in different TILS cell subtypes in tumor tissue (percentage of positive cells and relative expression / dMFI). (A) % of immune cell subtypes positive for ICOS expression, and (B) ICOS dMFI (relative ICOS expression in ICOS positive cells) of immune cell subtypes in animals treated with saline or anti-PD-L1 or PD-1 surrogate antibodies. Mice were implanted with 100μl of 1x106 viable cells / ml on day 0 (n=7 or n=8). Animals were dosed ip with 130ug of antibody on days 13 and 15. Tissue samples were isolated and analyzed on day 16. CD4+ / FOXP3+ cells were included only for the TReg population (far right of graph) and excluded from "effector" CD4 cells (far left of graph), which are all Foxp3 negative. See Example 3. [Figure 9B] Quantification of ICOS expression in different TILS cell subtypes in tumor tissue (percentage of positive cells and relative expression / dMFI). (A) % of immune cell subtypes positive for ICOS expression, and (B) ICOS dMFI (relative ICOS expression in ICOS positive cells) of immune cell subtypes in animals treated with saline or anti-PD-L1 or PD-1 surrogate antibodies. Mice were implanted with 100μl of 1x106 viable cells / ml on day 0 (n=7 or n=8). Animals were dosed ip with 130ug of antibody on days 13 and 15. Tissue samples were isolated and analyzed on day 16. CD4+ / FOXP3+ cells were included only for the TReg population (far right of graph) and excluded from "effector" CD4 cells (far left of graph), which are all Foxp3 negative. See Example 3. [Figure 10-1]Data from the A20 in vivo efficacy study. Each treatment group is represented by a "spider plot" showing the tumor size of individual animals (n=10 per group). For each group, the number of animals cured of their disease is shown on the individual graphs. For multiple doses, dosing was on days 8, 11, 15, 18, 22 and 25, indicated by dotted lines. For single doses, animals received an IP injection only on day 8. (A) Saline; (B) STIM003 mIgG2a multiple doses; (C) STIM003 mIgG2a single dose. See Example 4. [Figure 10-2] Continued from Figure 10-1. [Figure 11] Kaplan-Meier curves for the study reported in Example 4 using a 60 μg fixed dose of STIM003 mIgG2a. SD=single dose, day 8. MD=multiple doses BIW from day 8. [Figure 12-1] ICOS expression in major T cell subsets (T-reg [CD4+ / FoxP3+], CD4 Eff [CD4+ / FoxP3-] cells and CD8+) from saline-dosed CT26 tumor-bearing animals (n=4 per time point). Phenotyping of immune cells was performed on days 1, 2, 3, 4 and 8 after treatment and stained for ICOS expression in all tissues at all time points. A-D show the percentage of ICOS positive cells at all time points in four different tissues. E-H show ICOS dMFI (relative expression) at all time points in four different tissues. See Example 5. [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 12-4] Continued from Figure 12-3. [Figure 13]FACS analysis demonstrating T-reg depletion in the TME in response to STIM003 mIgG2a antibody. CT-26 tumor-bearing animals were treated with a single dose (6, 60 or 200 μg) of STIM003 on day 12 after tumor cell implantation. Tissues (n=4 per time point) harvested for FACS analysis on days 1, 2, 3, 4 and 8 after treatment. The percentage of T-reg cells (CD4+CD25+Foxp3+) in total tumor (A) and in blood (B) are shown at different time points. See Example 5. [Figure 14-1] Increased CD8:T-reg and CD4 eff:T-reg ratios in response to STIM003 mIgG2a. CT-26 tumor-bearing animals received a single dose (6, 60 or 200 μg) of STIM003 mIgG2a on day 12 after tumor cell implantation. Tissues (n=4 per time point) were harvested for FACS analysis on days 1, 2, 3, 4 and 8 after treatment, and T eff and T-reg ratios were calculated. (A) and (B) CD8:T-reg ratios in tumor and blood, (C) and (D) CD4-eff:T-reg ratios in tumor and blood. See Example 5. [Figure 14-2] Continued from Figure 14-1. [Figure 15-1] STIM003 treatment correlates with increased degranulation and Th1 cytokine production by TILs. Eight days after treatment, TILs were isolated and FACS analysis was performed to detect CD107a expression in CD4 and CD8 T cells (A-B). In parallel, dissociated tumor-derived cells were rested in the presence of brefeldin-A for 4 hours, and cells were stained for T cell markers and permeabilized for intracellular staining to detect IFN-γ and TNF-α (C-H). See Example 5. [Figure 15-2] Continued from Figure 15-1. [Figure 15-3] Continued from Figure 15-2. [Figure 15-4] Continued from Figure 15-3. [Figure 16A]Evidence of KY1044 target engagement in ICOS positive CD4 memory cells (defined as ICOS+CD3+CD4+FoxP3-CD45RA-). Y-axis measures percentage occupancy in CD4 memory cells as a function of sample collection day. Blood samples were collected on cycle 1 day 1 (C1D1), cycle 1 day 8 (C1D8), cycle 2 day 1 (C2D1) and cycle 2 day 8 (C2D8). Dose level 1=0.8 mg. Dose level 2=2.4 mg. Lines connect data points for the same patient. [Figure 16B] Evidence of KY1044 target engagement in ICOS positive CD4 memory cells (defined as ICOS+CD3+CD4+FoxP3-CD45RA-). Y-axis measures percentage occupancy in CD4 memory cells as a function of sample collection day. Blood samples were collected on cycle 1 day 1 (C1D1), cycle 1 day 8 (C1D8), cycle 2 day 1 (C2D1) and cycle 2 day 8 (C2D8). Dose level 3=8mg. Dose level 4=24mg. Dose level 5=80mg. Dose level 6=240mg. Lines connect data points for the same patient. [Figure 17A] KY1044-dependent agonism assessed by measuring circulating cytokine levels. The solid line plot represents the mean, and the shaded area indicates the 95% confidence interval of the ratio between GM-CSF visit and baseline measurements for patients treated with KY1044. The light gray data points are from patients (n=27) receiving lower KY1044 dose levels (0.8 mg and 2.4 mg), which resulted in incomplete receptor occupancy. The dark gray data points are from patients (n=14) receiving higher KY1044 dose levels (8 mg or higher), which resulted in complete receptor occupancy. [Figure 17B]KY1044-dependent agonism assessed by measuring circulating cytokine levels. The solid line plot represents the mean, and the shaded area indicates the 95% confidence interval of the ratio between visit and baseline measurements of TNFα for patients treated with KY1044. The light gray data points are from patients (n=27) receiving lower KY1044 dose levels (0.8 mg and 2.4 mg), which resulted in incomplete receptor occupancy. The dark gray data points are from patients (n=14) receiving higher KY1044 dose levels (8 mg or higher), which resulted in complete receptor occupancy. [Figure 18A] Interim results of the Phase I / II clinical trial regarding duration of treatment. The median duration of treatment for all enrolled patients was 9 weeks. [Figure 18B] Interim results of a Phase I / II clinical trial showing therapy regimen and duration of treatment with respect to partial or complete receptor occupancy. [Figure 18C] Interim results of a Phase I / II clinical trial showing treatment duration on ICOS receptor occupancy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] 1.6. Detailed Description ICOS The antibody according to the invention binds to the extracellular domain of human ICOS. Therefore, the antibody binds to ICOS-expressing T lymphocytes. "ICOS" or "ICOS receptor" refers to human ICOS in this specification, unless the context dictates otherwise. The sequences of human, cynomolgus monkey and mouse ICOS are shown in the attached sequence listing and are available from NCBI as human NCBI ID: NP_036224.1, mouse NCBI ID: NP_059508.2 and cynomolgus monkey GenBank ID: EHH55098.1.

[0039] Cross-reactivity The antibody according to the present invention is preferably cross-reactive, for example binding to the extracellular domain of mouse ICOS and human ICOS. The antibody can bind to other non-human ICOS, including ICOS of primates such as cynomolgus monkeys. Anti-ICOS antibodies intended for therapeutic use in humans must bind to human ICOS, but binding to ICOS of other species does not have direct therapeutic relevance in the human clinical context. Nevertheless, the data herein show that antibodies that bind to both human and mouse ICOS have properties that make them particularly suitable as agonist and depleting molecules. This arises from one or more specific epitopes targeted by the cross-reactive antibody. However, regardless of the underlying theory, cross-reactive antibodies are of high value and are excellent candidates as therapeutic molecules for preclinical and clinical studies.

[0040] As illustrated in the Experimental Examples, the STIM antibodies described herein were generated using Kymouse™ technology in which mice were engineered to lack expression of mouse ICOS (ICOS knockout). ICOS knockout transgenic animals and their use to generate cross-reactive antibodies are further aspects of the invention.

[0041] One way to quantify the degree of species cross-reactivity of an antibody is as the fold difference in its affinity for an antigen or one species compared to the antigen of another species, e.g., the fold difference in affinity for human ICOS versus mouse ICOS. Affinity refers to the equilibrium dissociation constant, K, of the antibody-antigen reaction as determined by SPR using antibodies in the Fab format described elsewhere herein. D Species cross-reactive anti-ICOS antibodies have a fold difference in affinity for binding human and mouse ICOS that is 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, or 5-fold or less. In other words, the K D is the binding K of the extracellular domain of mouse ICOS DThe antibody has a K D If the threshold is met, for example, the K D and the K of mouse ICOS binding D However, if both are less than 10 mM, preferably less than 5 mM, and more preferably less than 1 mM, it can be considered cross-reactive. D is 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. D is 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less.

[0042] An alternative measure of cross-reactivity for binding of human ICOS and mouse ICOS is the ability of an antibody to neutralize ICOS ligand binding to ICOS receptor, for example, in HTRF assay (see Example 8 of U.S. Patent No. 9,957,323). Examples of species cross-reactive antibodies are provided herein, including STIM001, STIM002, STIM002-B, STIM003, STIM005 and STIM006, each of which has been confirmed as neutralizing the binding of human B7-H2 (ICOS ligand) to human ICOS and neutralizing the binding of mouse B7-H2 to mouse ICOS in HTRF assay. Either of these antibodies or their variants are selected when antibody cross-reactivity is desired for human and mouse ICOS. Species cross-reactive anti-ICOS antibody has IC50 for inhibiting the binding of human ICOS to human ICOS receptor within 25-fold, 20-fold, 15-fold, 10-fold or 5-fold of IC50 for inhibiting the binding of mouse ICOS to mouse ICOS receptor, as determined in HTRF assay.An antibody can also be considered cross-reactive when IC50 for inhibiting the binding of human ICOS to human ICOS receptor and IC50 for inhibiting the binding of mouse ICOS to mouse ICOS receptor are both 1mM or less, preferably 0.5mM or less, for example 30nM or less, 20nM or less, 10nM or less.IC50 is 5nM or less, 4nM or less, 3nM or less or 2nM or less.In some cases, IC50 is at least 0.1nM, at least 0.5nM or at least 1nM.

[0043] 1.6.3. Specificity The antibody according to the present invention is preferably specific for ICOS. That is, the antibody binds to its epitope in the ICOS of the target protein (human ICOS, preferably mouse and / or cynomolgus ICOS as mentioned above), but does not show significant binding to molecules that do not present its epitope, including other molecules in the CD28 gene family. The antibody according to the present invention preferably does not bind to human CD28. The antibody preferably does not bind to mouse or cynomolgus CD28 either.

[0044] CD28 co-stimulates T cell responses when engaged by its ligands CD80 and CD86 on professional antigen presenting cells in the context of TCR-mediated antigen recognition. For various in vivo uses of the antibodies described herein, avoidance of binding to CD28 is considered advantageous. The absence of binding of anti-ICOS antibodies to CD28 should allow CD28 to interact with its native ligand and generate the appropriate co-stimulatory signal for T cell activation. In addition, the absence of binding of anti-ICOS antibodies to CD28 avoids the risk of superagonism. Overstimulation of CD28 can induce the proliferation of resting T cells without the usual requirement for recognition of cognate antigen via TCR, which may lead to runaway activation of T cells and resulting cytokine release syndrome, especially in human subjects. Non-recognition of CD28 by the antibodies according to the present invention therefore represents an advantage in terms of their safe clinical use in humans.

[0045] As discussed elsewhere herein, the present invention extends to multispecific antibodies (e.g., bispecific). Multispecific (e.g., bispecific) antibodies can comprise (i) an antibody antigen-binding site for ICOS, and (ii) an additional antigen-binding site (optionally, an antibody antigen-binding site, as described herein) that recognizes another antigen (e.g., PD-L1). The specific binding of each antigen-binding site can be determined. Thus, an antibody that specifically binds ICOS includes an antibody that comprises an antigen-binding site that specifically binds ICOS, where optionally, the antigen-binding site for ICOS is comprised within an antigen-binding molecule that further comprises one or more additional binding sites for one or more other antigens, e.g., a bispecific antibody that binds ICOS and PD-L1.

[0046] 1.6.4.Affinity The affinity of the antibody's binding to ICOS can be determined. The affinity of an antibody for its antigen is determined by the equilibrium dissociation constant, K DThe Kd, Ka and Kd for antibody-antigen binding can be measured using surface plasmon resonance (SPR).

[0047] The antibody according to the present invention has a K of 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less, for the EC domain of human ICOS. D Join with K D is 50 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. D is 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. D teeth 、 At least 0.001 nM, such as at least 0.01 nM or at least 0.1 nM.

[0048] Affinity quantification can be performed using SPR with antibodies in Fab format. A suitable protocol is as follows: 1. Couple anti-human (or other species-matched antibody constant region) IgG to a biosensor chip (e.g., GLM chip), for example, by primary amine coupling; 2. Expose anti-human IgG (or other matched species antibody) to the test antibody, e.g., a test antibody in Fab format, to capture the test antibody on the chip; 3. Passing the test antibody over the capture surface of the chip at a range of concentrations, e.g., 5000 nM, 1000 nM, 200 nM, 40 nM, 8 nM, and 2 nM, as well as 0 nM (i.e., buffer alone); and 4. Determine the binding affinity of the test antibody to the test antigen using SPR at 25°C. The buffer is pH 7.6, 150 mM NaCl, 0.05% detergent (e.g., P20) and 3 mM EDTA. The buffer can optionally contain 10 mM HEPES. HBS-EP can be used as the running buffer. HBS-EP is available from Teknova Inc (California; catalog number H8022).

[0049] Regeneration of the capture surface can be performed with 10 mM glycine at pH 1.7. This removes the captured antibody and allows the surface to be used for another interaction. Binding data can be fitted to a unique 1:1 model using standard techniques, for example, using the model native to ProteOn XPR36™ analysis software.

[0050] A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™ (Bio-Rad®), and KinExA® (Sapidyne Instruments, Inc.).

[0051] As described, affinity can be determined by SPR using antibodies in Fab format, with the antigen coupled to the chip surface and the test antibody passed over the chip in Fab format in solution to determine the affinity of the monomeric antibody-antigen interaction. Affinity can be determined at any desired pH, e.g., pH 5.5 or pH 7.6, and at any desired temperature, e.g., 25°C or 37°C.

[0052] Other methods for measuring antibody binding to ICOS include, for example, fluorescence-activated cell sorting (FACS) using cells with exogenous surface expression of ICOS (e.g., CHO cells) or activated primary T cells expressing endogenous levels of ICOS. Antibody binding to ICOS-expressing cells measured by FACS indicates that the antibody can bind to the extracellular (EC) domain of ICOS.

[0053] ICOS receptor agonism ICOS ligand (ICOSL, also known as B7-H2) is a molecule expressed on the cell surface that binds to the ICOS receptor

[17] . This intercellular ligand-receptor interaction promotes the multimerization of ICOS on the T cell surface, activating the receptor and stimulating downstream signaling in T cells. In effector T cells, activation of this receptor stimulates effector T cell responses.

[0054] Anti-ICOS antibodies act as agonists of ICOS, mimicking and even exceeding this stimulatory effect of the native ICOS ligand on the receptor. Such agonism arises from the ability of the antibody to promote multimerization of ICOS on T cells. One mechanism for this is when the antibody forms an intercellular bridge between ICOS on the T cell surface and a receptor, such as an Fc receptor, on an adjacent cell (e.g., B cell, antigen-presenting cell or other immune cell). Another mechanism is when an antibody with multiple (e.g., two) antigen-binding sites (e.g., two VH-VL domain pairs) crosslinks multiple ICOS receptor molecules, thus promoting multimerization. A combination of these mechanisms may occur.

[0055] Agonism can be tested in an in vitro T cell activation assay using antibodies in soluble form (e.g., immunoglobulin format or other antibody format that includes two spatially separated antigen binding sites, e.g., two VH-VL pairs), either with or without a crosslinker, or using antibodies bound to a solid surface that provides a tethered array of antigen binding sites. Agonism assays can use human ICOS positive T lymphocyte cell lines, e.g., MJ cells (ATCC CRL-8294), as target T cells for activation in such assays. One or more measures of T cell activation can be determined for the test antibody and compared to a reference molecule or negative control to determine whether there is a statistically significant (p<0.05) difference in T cell activation caused by the test antibody compared to the reference molecule or control. One suitable measure of T cell activation is the production of cytokines, e.g., IFNγ, TNFα, or IL-2. Those skilled in the art will include suitable controls as necessary to standardize the assay conditions between the test antibody and the control. A suitable negative control is an antibody of the same format that does not bind ICOS (e.g., an isotype control), e.g., an antibody specific for an antigen not present in the assay system. A significant difference observed for the test antibody compared to the cognate isotype control within the dynamic range of the assay indicates that the antibody acts as an agonist of the ICOS receptor in that assay.

[0056] An agonist antibody, when tested in a T cell activation assay, is defined as one of the following: have a significantly lower EC50 for inducing IFNγ production compared to the control antibody; Induces significantly higher maximal IFNγ production compared to control antibodies; has a significantly lower EC50 for induction of IFNγ production compared to ICOSL-Fc; Induces significantly higher maximal IFNγ production compared to ICOSL-Fc; has a significantly lower EC50 for inducing IFNγ production compared to the reference antibody C398.4A; and / or It induces a significantly higher maximal IFNγ production compared to the reference antibody C398.4A.

[0057] Exemplary in vitro T cell assays include bead binding assays, plate binding assays, and soluble form assays, as disclosed in Examples 13-15 of US Pat. No. 9,957,323.

[0058] A significantly lower or higher value is, for example, at most 0.5-fold difference, at most 0.75-fold difference, at most 2-fold difference, at most 3-fold difference, at most 4-fold difference, or at most 5-fold difference compared to a reference or control value.

[0059] Thus, in one example, an antibody according to the invention has a significantly lower, e.g., at least 2-fold lower, EC50 for induction of IFNγ in an MJ cell activation assay using the antibody in a bead-coupled format compared to a control.

[0060] Bead binding assays use antibodies (and control antibodies, reference antibodies or ICOSL-Fc for control or reference experiments) bound to the surface of beads. Magnetic beads can be used and are commercially available in various types, such as tosyl-activated DYNABEADS M-450 (DYNAL Inc, 5 Delaware Drive, Lake Success, NY11042 product numbers 140.03, 140.04). Beads can be coated or generally coated by dissolving the coating material in carbonate buffer (pH 9.6, 0.2M) or other methods known in the art. The use of beads conveniently allows the quantification of proteins bound to the bead surface to be determined with good accuracy. Standard Fc protein quantification methods can be used for the quantification of coupled proteins on beads. Any suitable method can be used, including DELFIA, ELISA, or other methods, with reference to relevant standards within the dynamic range of the assay.

[0061] The agonism activity of the antibody can also be measured ex vivo in primary human T lymphocytes. The ability of the antibody to induce expression of IFNγ in such T cells indicates ICOS agonism. Preferably, the antibody shows a significant (p<0.05) induction of IFNγ at 5 μg / ml compared to a control antibody in T cell activation assay 1 and / or T cell activation assay 2. As mentioned above, anti-ICOS antibodies can stimulate T cell activation to a higher degree than ICOS-L or C398.4 in such assays. Thus, the antibody can show a significantly (p<0.05) higher induction of IFNγ at 5 μg / ml compared to a control or reference antibody in T cell activation assay 1 or 2. TNFα or IL-2 induction can be measured as a readout of the alternative assay.

[0062] The agonism of anti-ICOS antibodies is attributed to their ability to shift the balance between TReg and TEff cell populations in vivo at the site of pathology, such as the tumor microenvironment, in favor of TEff cells. The ability of the antibodies to enhance tumor cell killing by activated ICOS-positive effector T cells can be determined as discussed elsewhere herein.

[0063] ICOS receptor agonism and therapeutic efficacy at lower doses The present invention is based in part on the discovery that the lower anti-ICOS antibody concentration resulting from administering a lower dose to a subject can improve clinical efficacy compared to the higher anti-ICOS concentration resulting from a higher dose.Surprisingly, as shown by the data presented herein, the anti-ICOS antibody that produces only partial receptor / transient occupancy induces stronger GM-CSF and TNFα signals after treatment compared to the anti-ICOS antibody dose that produces full receptor occupancy.

[0064] Without being limited by theory, anti-ICOS antibodies such as KY1044 act as agonists of ICOS by promoting the multimerization of ICOS on T cells. The ICOS receptor has a tendency to organize as a homodimer. Therefore, antibodies with multiple antigen-binding sites for ICOS can crosslink multiple ICOS receptor molecules, resulting in ligand-induced clustering or multimerization. Such ligand crosslinking is proposed to mediate the avidity effect due to increased stability of the ligand-receptor interaction.

[0065] Multimerization of ICOS receptors depends in part on antibody concentration and receptor stoichiometry. For example, without being limited to theory, if the concentration of antibody is significantly higher than the number of available receptors, this will favor the formation of isolated receptors bound to two different antibodies, resulting in reduced FcγR-dependent stimulation, but if the number of receptors significantly exceeds the number of antibodies present, ligand cross-linking is less likely to occur, resulting in reduced FcγR-dependent stimulation. In some embodiments, equal concentrations of antibody and receptor are present, promoting the formation of multimeric complexes, maximally inducing FcγR-dependent stimulation, resulting in greater release of pro-inflammatory cytokines. Without being limited to theory, high anti-ICOS antibody opsonization results in no clustering and / or less immune synapses, and no costimulation, while low anti-ICOS antibody opsonization improves clustering and results in FcγR-dependent costimulation.

[0066] In some embodiments, the anti-ICOS antibody administered at a dose effective to produce partial ICOS receptor / transient occupancy, improve clustering, and / or improve costimulation comprises the CDRs of KY1044. In another embodiment, the anti-ICOS antibody comprises heavy and light chain variable domains having at least 85%, 90% or 95% sequence identity to the heavy and light chain variable domains of KY1044. In some such embodiments, the heavy and light chain variable domains having at least 85%, 90% or 95% sequence identity to the heavy and light chain variable domains of KY1044 comprise the CDRs of KY1044. In another embodiment, the anti-ICOS antibody comprises the heavy and light chain variable domains of KY1044. In some embodiments, a dose of about 8 mg of the anti-ICOS antibody produces full ICOS receptor occupancy. Thus, in some embodiments, the effective dose of anti-ICOS antibody to produce partial ICOS receptor / transient occupancy, improve clustering, and / or improve costimulation is less than about 8 mg, such as about 7 mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg, about 1 mg, or less than about 1 mg. In one embodiment, the dose of anti-ICOS antibody is about 2.4 mg. In another embodiment, the dose of anti-ICOS antibody is about 0.8 mg.

[0067] In another embodiment, the anti-ICOS antibody administered at a dose effective to produce partial ICOS receptor / transient occupancy, improve clustering, and / or improve costimulation comprises a heavy chain and a light chain having at least 85%, 90% or 95% sequence identity to the heavy chain and the light chain of KY1044. In some such embodiments, the heavy chain and the light chain having at least 85%, 90% or 95% sequence identity to the heavy chain and the light chain of KY1044 comprises the CDRs of KY1044. In another embodiment, the anti-ICOS antibody comprises a heavy chain and a light chain of KY1044. In another embodiment, the anti-ICOS antibody is KY1044. In some embodiments, a dose of about 8 mg of the anti-ICOS antibody produces full ICOS receptor occupancy. Thus, in some embodiments, the effective dose of anti-ICOS antibody to produce partial ICOS receptor / transient occupancy, improve clustering, and / or improve costimulation is less than about 8 mg, such as about 7 mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2 mg, about 1 mg, or less than about 1 mg. In one embodiment, the dose of anti-ICOS antibody is about 2.4 mg. In another embodiment, the dose of anti-ICOS antibody is about 0.8 mg.

[0068] In some embodiments, ICOS+ Treg depletion (reduction of ICOS+FOXP3+ cells) in the tumor is highest at about 8 mg of anti-ICOS antibody (e.g., KY1044). In some embodiments, improvement in the CD8 / ICOS+FOXP3+ Treg ratio in the tumor microenvironment occurs with a plateau of anti-ICOS antibody at doses of about 8 mg or more of anti-ICOS antibody (e.g., KY1044).

[0069] In some embodiments, ICOS agonism is most evident at doses of anti-ICOS antibody (e.g., KY1044) lower than about 8 mg. In some embodiments, the agonist activity of the anti-ICOS antibody (e.g., KY1044) is effective at about 2.4 to 8 mg. In some embodiments, the agonist activity of the anti-ICOS antibody (e.g., KY1044) is effective at about 0.8 to 2.4 mg. In some embodiments, the agonist activity of the anti-ICOS antibody (e.g., KY1044) is effective at about 2.4 mg. In other embodiments, the agonist activity of the anti-ICOS antibody (e.g., KY1044) is effective at about 0.8 mg.

[0070] 1.6.6.T cell-dependent killing Effector T cell function can be determined in a biologically relevant context using an in vitro co-culture assay in which tumor cells are incubated with relevant immune cells to trigger immune cell-dependent killing, and the effect of anti-ICOS antibodies on tumor cell killing by TEff is observed.

[0071] The ability of the antibody to enhance tumor cell killing by activated ICOS positive effector T cells can be determined. The anti-ICOS antibody stimulates significantly higher (p<0.05) tumor cell killing compared to the control antibody. The anti-ICOS antibody can stimulate similar or higher tumor cell killing, such as in an assay compared to a reference molecule, such as ICOS ligand or C398.4 antibody. A similar degree of tumor cell killing can be shown as an assay reading for the test antibody that is less than 2-fold different from that of the reference molecule.

[0072] 1.6.7. ICOS Ligand-Receptor Neutralization Potency The antibody according to the invention inhibits the binding of ICOS to its ligand ICOSL.

[0073] The degree to which an antibody inhibits the binding of ICOS receptor to its ligand is referred to as its ligand-receptor neutralizing potency. Potency is usually expressed as IC50 value in pM unless otherwise stated. In ligand binding studies, IC50 is the concentration that reduces receptor binding by 50% of the maximum specific binding level. IC50 can be calculated by plotting the % specific receptor binding as a function of the logarithm of antibody concentration and fitting a sigmoidal function to the data using a software program such as Prism (GraphPad) to generate IC50 value. Neutralizing potency can be determined in HTRF assay as disclosed in Example 8 of U.S. Pat. No. 9,957,323.

[0074] IC50 values ​​represent the average of multiple measurements, so, for example, IC50 values ​​can be obtained from the results of triplicate experiments and then an average IC50 value can be calculated.

[0075] The antibody has an IC50 in a ligand-receptor neutralization assay of 1 mM or less, for example 0.5 mM or less. The IC50 is 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, or 2 nM or less. The IC50 is at least 0.1 nM, at least 0.5 nM, or at least 1 nM.

[0076] 1.6.8. Antibodies As described in the Examples of U.S. Patent No. 9,957,323, we have isolated and characterized antibodies of particular interest, designated STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009. In various embodiments of the invention, unless the context dictates otherwise, the antibody is selected from any of these antibodies, or from a subset of STIM001, STIM002, STIM003, STIM004 and STIM005. The sequences of each of these antibodies are provided in the accompanying sequence listing, where for each antibody, respectively, the following sequences are set out: a nucleotide sequence encoding the VH domain; an amino acid sequence of the VH domain; a VH CDR1 amino acid sequence, a VH CDR2 amino acid sequence, a VH CDR3 amino acid sequence; a nucleotide sequence encoding the VL domain; an amino acid sequence of the VL domain; a VL CDR1 amino acid sequence, a VL CDR2 amino acid sequence, and a VL CDR3 amino acid sequence. The present invention encompasses anti-ICOS antibodies having the VH and / or VL domain sequences of all antibodies shown in the accompanying sequence listing and / or figures, as well as antibodies comprising the HCDRs and / or LCDRs of these antibodies, and optionally antibodies having the entire heavy and / or light chain amino acid sequences.

[0077] STIM001 comprises a heavy chain variable region (V) of SEQ ID NO: 366, which contains the CDRH1 amino acid sequence of SEQ ID NO: 363, the CDRH2 amino acid sequence of SEQ ID NO: 364, and the CDRH3 amino acid sequence of SEQ ID NO: 365. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 367. STIM001 comprises a light chain variable region (VL) of SEQ ID NO: 373, which contains a CDRL1 amino acid sequence of SEQ ID NO: 370, a CDRL2 amino acid sequence of SEQ ID NO: 371, and a CDRL3 amino acid sequence of SEQ ID NO: 372. L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 374. HThe domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 368 (heavy chain nucleic acid sequence SEQ ID NO: 369). The full length light chain amino acid sequence is SEQ ID NO: 375 (light chain nucleic acid sequence SEQ ID NO: 376).

[0078] STIM002 comprises a heavy chain variable region (V) of SEQ ID NO: 380, which contains the CDRH1 amino acid sequence of SEQ ID NO: 377, the CDRH2 amino acid sequence of SEQ ID NO: 378, and the CDRH3 amino acid sequence of SEQ ID NO: 379. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 381. STIM002 contains a light chain variable region (VL) of SEQ ID NO: 387, which contains a CDRL1 amino acid sequence of SEQ ID NO: 384, a CDRL2 amino acid sequence of SEQ ID NO: 385, and a CDRL3 amino acid sequence of SEQ ID NO: 386. L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 388 or SEQ ID NO: 519. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 382 (heavy chain nucleic acid sequence SEQ ID NO: 383). The full length light chain amino acid sequence is SEQ ID NO: 389 (light chain nucleic acid sequence SEQ ID NO: 390 or SEQ ID NO: 520).

[0079] STIM002-B comprises a heavy chain variable region (V) of SEQ ID NO: 394, which contains the CDRH1 amino acid sequence of SEQ ID NO: 391, the CDRH2 amino acid sequence of SEQ ID NO: 392, and the CDRH3 amino acid sequence of SEQ ID NO: 393. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 395. STIM002-B contains a light chain variable region (VL) of SEQ ID NO: 401, which contains a CDRL1 amino acid sequence of SEQ ID NO: 398, a CDRL2 amino acid sequence of SEQ ID NO: 399, and a CDRL3 amino acid sequence of SEQ ID NO: 400. L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 402. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 396 (heavy chain nucleic acid sequence SEQ ID NO: 397). The full length light chain amino acid sequence is SEQ ID NO: 403 (light chain nucleic acid sequence SEQ ID NO: 404).

[0080] STIM003, interchangeably referred to herein as KY1044, comprises a heavy chain variable region (V) of SEQ ID NO: 408, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 405, a CDRH2 amino acid sequence of SEQ ID NO: 406, and a CDRH3 amino acid sequence of SEQ ID NO: 407. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 409 or SEQ ID NO: 521. STIM003 contains a light chain variable region (VL) of SEQ ID NO: 415, which contains a CDRL1 amino acid sequence of SEQ ID NO: 412, a CDRL2 amino acid sequence of SEQ ID NO: 413, and a CDRL3 amino acid sequence of SEQ ID NO: 414. L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 4416. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 410 (heavy chain nucleic acid sequence SEQ ID NO: 411 or SEQ ID NO: 522). The full length light chain amino acid sequence is SEQ ID NO: 417 (light chain nucleic acid sequence SEQ ID NO: 418).

[0081] STIM004 comprises a heavy chain variable region (V) of SEQ ID NO: 422, which contains a CDRH1 amino acid sequence of SEQ ID NO: 419, a CDRH2 amino acid sequence of SEQ ID NO: 420, and a CDRH3 amino acid sequence of SEQ ID NO: 421. H ) amino acid sequence. HThe heavy chain nucleic acid sequence of the domain is SEQ ID NO: 423. STIM004 contains a light chain variable region (VL) of SEQ ID NO: 429, which contains a CDRL1 amino acid sequence of SEQ ID NO: 426, a CDRL2 amino acid sequence of SEQ ID NO: 427, and a CDRL3 amino acid sequence of SEQ ID NO: 428. L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 430 or SEQ ID NO: 431. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 424 (heavy chain nucleic acid sequence SEQ ID NO: 425). The full length light chain amino acid sequence is SEQ ID NO: 432 (light chain nucleic acid sequence SEQ ID NO: 433 or SEQ ID NO: 434).

[0082] STIM005 comprises a heavy chain variable region (V) of SEQ ID NO: 438, which contains a CDRH1 amino acid sequence of SEQ ID NO: 435, a CDRH2 amino acid sequence of SEQ ID NO: 436, and a CDRH3 amino acid sequence of SEQ ID NO: 437. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 439. STIM005 contains a light chain variable region (VL) of SEQ ID NO: 445, which contains a CDRL1 amino acid sequence of SEQ ID NO: 442, a CDRL2 amino acid sequence of SEQ ID NO: 443, and a CDRL3 amino acid sequence of SEQ ID NO: 444. L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 446. HThe domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 440 (heavy chain nucleic acid sequence SEQ ID NO: 441). The full length light chain amino acid sequence is SEQ ID NO: 447 (light chain nucleic acid sequence SEQ ID NO: 448).

[0083] STIM006 comprises a heavy chain variable region (V) of SEQ ID NO: 452, which contains a CDRH1 amino acid sequence of SEQ ID NO: 449, a CDRH2 amino acid sequence of SEQ ID NO: 450, and a CDRH3 amino acid sequence of SEQ ID NO: 451. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 453. STIM006 comprises a light chain variable region (VL) of SEQ ID NO: 459, which contains a CDRL1 amino acid sequence of SEQ ID NO: 456, a CDRL2 amino acid sequence of SEQ ID NO: 457, and a CDRL3 amino acid sequence of SEQ ID NO: 458. L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 460. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 454 (heavy chain nucleic acid sequence SEQ ID NO: 455). The full length light chain amino acid sequence is SEQ ID NO: 461 (light chain nucleic acid sequence SEQ ID NO: 462).

[0084] STIM007 comprises a heavy chain variable region (V) of SEQ ID NO: 466, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 463, a CDRH2 amino acid sequence of SEQ ID NO: 464, and a CDRH3 amino acid sequence of SEQ ID NO: 465. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 467. STIM007 contains a light chain variable region (VL) of SEQ ID NO: 473, which contains a CDRL1 amino acid sequence of SEQ ID NO: 470, a CDRL2 amino acid sequence of SEQ ID NO: 471, and a CDRL3 amino acid sequence of SEQ ID NO: 472. L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 474. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 468 (heavy chain nucleic acid sequence SEQ ID NO: 469). The full length light chain amino acid sequence is SEQ ID NO: 475 (light chain nucleic acid sequence SEQ ID NO: 476).

[0085] STIM008 comprises a heavy chain variable region (V) of SEQ ID NO: 480, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 477, a CDRH2 amino acid sequence of SEQ ID NO: 478, and a CDRH3 amino acid sequence of SEQ ID NO: 479. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 481. STIM008 contains a light chain variable region (VL) of SEQ ID NO: 487, which contains a CDRL1 amino acid sequence of SEQ ID NO: 484, a CDRL2 amino acid sequence of SEQ ID NO: 485, and a CDRL3 amino acid sequence of SEQ ID NO: 486. L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 488. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 482 (heavy chain nucleic acid sequence SEQ ID NO: 483). The full length light chain amino acid sequence is SEQ ID NO: 489 (light chain nucleic acid sequence SEQ ID NO: 490).

[0086] STIM009 comprises a heavy chain variable region (V) of SEQ ID NO: 494, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 491, a CDRH2 amino acid sequence of SEQ ID NO: 492, and a CDRH3 amino acid sequence of SEQ ID NO: 493. H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 495. STIM009 contains a light chain variable region (VL) of SEQ ID NO: 501, which contains a CDRL1 amino acid sequence of SEQ ID NO: 498, a CDRL2 amino acid sequence of SEQ ID NO: 499, and a CDRL3 amino acid sequence of SEQ ID NO: 500. L) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 502. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 496 (heavy chain nucleic acid sequence SEQ ID NO: 497). The full length light chain amino acid sequence is SEQ ID NO: 503 (light chain nucleic acid sequence SEQ ID NO: 504).

[0087] Additional exemplary anti-ICOS antibodies include, but are not limited to, 37A10S713 (also referred to as bopratelimab or JTX-2011) (see, e.g., U.S. Pat. Nos. 10,023,635 and 11,292,840; WO2017070423; WO2016154177); XMAb23104 (also referred to as XmAb104) (see, U.S. Pat. No. 1,0981,992); 314.8 mAb (Icos 314-8) (WO2014033327A1, WO2012131004A2; US Pat. No. 11180556), JMab-136 (also referred to as IC009) (see, for example, WO2008137915; US Pat. No. 9193789, US20110243929A1) and ICOS.33 IgGlf S267E (US Pat. No. 10898556). Antibodies against ICOS and methods of use in treating diseases are also described in WO2019222188A1 and US Pat. No. 11292840. Antibodies against ICOS are also disclosed in EP1374902, EP1374901 and EP1125585. Agonistic antibodies against ICOS are also disclosed in US20210340250A1; WO2018222711A2; WO2021209356A1; WO2016120789; US20160215059A1; and WO2012131004A2.

[0088] The heavy and light chain sequences of 37A10S713 are disclosed as SEQ ID NOs:611-612. 37A10S713 heavy chain: (SEQ ID NO: 611) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMDWVRQAPGKGLVWVSNIDEDGSITEYSPFVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRWGRFGFDSWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 37A10S713 light chain: (SEQ ID NO: 612) DIVMTQSPDSLAVSLGERATINCKSSQSLLSGSFNYLTWYQQKPGQPPKLLIFYASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHHHYNAPPTFGPGTKVD IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0089] In one embodiment, the ICOS binding protein is vopratelimab. In one embodiment, the ICOS binding protein is JTX-2011.

[0090] The heavy and light chain sequences of XMAb23104 are disclosed as SEQ ID NOs:613-614. XmAb23104 heavy chain: (SEQ ID NO: 613) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGETIYAQKFQGRVTMTRDTSISTAYMELSSLRSEDTAVYYCARTYYYDTSGYYHDAFD VWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSSLGTQTYICNVNHKPSDTKVDKKVEPK SCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVKHEDPEVKFNWYVDGVEVHNAKTKPREEEYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCDVSGFYPSDIAVEWESDGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWEQGDVFSCSVLHEALHSHYTQKSLSLSPGK XmAb23104 light chain: (SEQ ID NO: 614) DIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK / RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0091] The sequences of the heavy and light chain variable regions of the 314.8 mAb are disclosed as SEQ ID NOs:615-616. 314.8 mAb heavy chain variable region: (SEQ ID NO: 615) MGWRCIILFLVSTATGVHSQVQLQQPGTELMKPGASVKLSCKASGYTFTTYWMHWVKQRPGQGLEWIGEIDPSDSYVNYNQNFKGKATLTVDKSSSTAYIQLSSLTSEDSAVYFCARSPDYYGTSLAWFDYWGQGTLVTVST 314.8 mAb light chain variable region: (SEQ ID NO: 616) MRCLAEFLGLLVLWIPGVIGDIVMTQAAPSVPVTPGESVSISCRSSKSPLHSNGNIYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTTFTLKISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK

[0092] The sequences of the heavy and light chain variable regions of JMab-136 are disclosed as SEQ ID NOs: 617-618. JMab-136 heavy chain variable region: (SEQ ID NO: 617) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPHSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARTYYYDSSGYYHDAFDIWGQGTMVTVSS JMab-136 light chain variable region: (SEQ ID NO: 618) DIQMTQSPSSVSASVGDRVTITCRASQGISRLLAWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPWTFGQGTKVEIK

[0093] The heavy and light chain sequences of ICOS.33 IgGlf S267E are disclosed as SEQ ID NOs: 619-620. ICOS.33 IgGlf S267E heavy chain: sequence number 619. EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMHWVRQAPGKGLEWVGVIDTKSFNYATYYSDLVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTATIAVPYYFDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG ICOS.33 IgGlf S267E light chain: sequence number 620. DIQMTQSPSSLSASVGDRVTITCQASQDISNYLSWYQQKPGKAPKLLIYYTNLLAEGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYYNYRTFGPGTKVDIKR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0094] The term "antibody" refers to (full length) antibodies, and antigen-binding fragments thereof. Antibodies according to the invention are molecules comprising immunoglobulins or immunoglobulin domains, whether naturally occurring or partially or wholly synthetically produced. Antibodies are IgG, IgM, IgA, IgD or IgE molecules, or antigen-specific (antigen-binding) antibody fragments thereof (including but not limited to Fab, F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies, multispecific antibodies in closed conformation, disulfide-linked scfv, diabodies), whether derived from any species that naturally produces antibodies or produced by recombinant DNA technology; whether isolated from serum, B cells, hybridomas, transfectomas, yeast or bacteria. Antibodies can be humanized using routine techniques. The term antibody encompasses any polypeptide or protein comprising an antigen-binding site of an antibody. The antigen-binding site (paratope) is the part of an antibody that binds to its target antigen (ICOS) and is complementary to its epitope.

[0095] The term "epitope" refers to the region of an antigen that binds to an antibody. Epitopes are defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments can have specific three-dimensional structural characteristics and / or specific charge characteristics.

[0096] An antigen-binding site is a polypeptide or domain that contains one or more CDRs of an antibody and can bind to an antigen. For example, the polypeptide contains CDR3 (e.g., HCDR3). For example, the polypeptide contains CDR1 and CDR2 (e.g., HCDR1 and HCDR2) or CDR1-CDR3 (e.g., HCDR1-HCDR3) of the variable domain of an antibody.

[0097] The antigen-binding site of an antibody is provided by one or more antibody variable domains. In some examples, an antibody binding site is provided by a single variable domain, such as a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site comprises a VH / VL pair, or two or more of such pairs. Thus, an antibody antigen-binding site can comprise a VH and a VL.

[0098] An antibody is a whole immunoglobulin including the constant region, or an antibody fragment, e.g., an antigen-binding fragment of an antibody. An antibody fragment is a portion of an intact antibody, e.g., including the antigen-binding and / or variable regions of the intact antibody. Examples of antibody fragments include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH or VL domain (Ward et al., (1989) Nature 341:544-546; which is incorporated herein by reference in its entirety); and (vi) An isolated complementarity determining region (CDR) that retains specific antigen-binding functionality.

[0099] A further example of an antibody is the H2 antibody, which comprises a dimer of heavy chains (5'-VH-(optional hinge)-CH2-CH3-3') and lacks light chains.

[0100] Single chain antibodies (e.g., scFv) are commonly used fragments. Multispecific antibodies are formed from antibody fragments. The antibodies of the present invention can use any such format as desired.

[0101] Optionally, the immunoglobulin domain of the antibody is fused or conjugated to additional polypeptide sequences and / or labels, tags, toxins or other molecules. The immunoglobulin domain of the antibody can be fused or conjugated to one or more different antigen-binding regions to provide a molecule that can bind to a second antigen in addition to ICOS. The antibody of the present invention is a multispecific antibody, e.g., a bispecific antibody, comprising (i) an antibody antigen-binding site for ICOS, and (ii) a further antigen-binding site (optionally an antibody antigen-binding site as described herein) that recognizes another antigen (e.g., PD-L1).

[0102] An antibody usually comprises an antibody VH and / or VL domain. The isolated VH and VL domains of an antibody are also part of the present invention. The variable domains of an antibody are the portions of the light and heavy chains of an antibody that comprise the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2 and CDR3) and framework regions (FRs). Thus, within each of the VH and VL domains, there are CDRs and FRs. The VH domain comprises a set of HCDRs, and the VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to the method used in the present invention, the amino acid positions assigned to CDRs and FRs are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to the IMGT nomenclature. An antibody can comprise an antibody VH domain, comprising VH CDR1, CDR2 and CDR3, and a framework. Alternatively or additionally, it can comprise an antibody VL domain, comprising VL CDR1, CDR2 and CDR3, and a framework. Examples of antibody VH and VL domains and CDRs according to the present invention are as listed in the attached sequence listing, which forms part of this disclosure. The CDRs shown in the sequence listing are defined according to the IMGT system

[18] . The VH and VL sequences, CDR sequences, sets of CDRs, and sets of HCDRs and sets of LCDRs disclosed herein all represent aspects and embodiments of the present invention. As described herein, a "set of CDRs" comprises CDR1, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3. Unless otherwise stated, a "set of CDRs" includes HCDRs and LCDRs.

[0103] The antibody of the invention may comprise one or more CDRs described herein, e.g., CDR3, and optionally also CDR1 and CDR2 to form a set of CDRs. The CDR or set of CDRs is any of the CDRs or set of CDRs of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or a variant thereof described herein.

[0104] The present invention provides an antibody comprising the HCDR1, HCDR2 and / or HCDR3 of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, and / or the LCDR1, LCDR2 and / or LCDR3, e.g., a set of CDRs, of any of these antibodies. The antibody may comprise a set of VH CDRs of one of these antibodies. Optionally, it may also comprise a set of VL CDRs of one of these antibodies, with the VL CDRs being from the same or different antibody as the VH CDRs.

[0105] Also provided by the invention are VH domains which comprise a disclosed set of HCDRs and / or VL domains which comprise a disclosed set of LCDRs.

[0106] Typically, the VH domain is paired with the VL domain to provide the antigen-binding site of the antibody, although, as discussed further below, the VH or VL domain alone can be used to bind to the antigen. The VH domain of STIM003 can be paired with the VL domain of STIM003, resulting in the formation of an antigen-binding site of the antibody that includes both the VH and VL domains of STIM003. Similar embodiments are provided for other VH and VL domains disclosed herein. In other embodiments, the VH of STIM003 is paired with a VL domain other than the VL of STIM003. Light chain promiscuity is well established in the art. Again, similar embodiments are provided by the present invention for other VH and VL domains disclosed herein.

[0107] Thus, the VH of any of antibodies STIM001, STIM002, STIM003, STIM004 and STIM005 can be paired with the VL of any of antibodies STIM001, STIM002, STIM003, STIM004 and STIM005. Additionally, the VH of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 can be paired with the VL of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.

[0108] An antibody can comprise one or more CDRs, e.g., a set of CDRs, within an antibody framework. The framework regions are of human germline gene segment sequences. Thus, the antibody is a human antibody with a VH domain that comprises a set of HCDRs in a human germline framework. Usually, the antibody also has a set of LCDRs, e.g., a VL domain that comprises a human germline framework. An antibody "gene segment", e.g., a VH gene segment, a D gene segment, or a JH gene segment, refers to an oligonucleotide having a nucleic acid sequence from which a portion of an antibody is derived, e.g., a VH gene segment is an oligonucleotide that comprises a nucleic acid sequence corresponding to a polypeptide of a VH domain from FR1 to a portion of CDR3. Human V, D, and J gene segments recombine to produce a VH domain, and human V and J segments recombine to produce a VL domain. D domain or D region refers to the various domains or regions of an antibody chain. J domain or J region refers to the connecting domain or region of an antibody chain. Although somatic mutations result in antibody VH or VL domains with framework regions that do not exactly match or align with the corresponding gene sequences, sequence alignment can be used to identify the closest gene segments and therefore the particular combination of gene segments from which the VH or VL domain is derived. When aligning an antibody sequence with a gene sequence, the amino acid sequence of the antibody can be aligned with the amino acid sequence encoded by the gene segment, or the nucleotide sequence of the antibody can be aligned directly with the nucleotide sequence of the gene segment.

[0109] Alignments of the VH and VL domain sequences of the STIM antibody to related antibodies and to human germline sequences are shown in FIG. 5, FIG. 6 and FIG.

[0110] The antibodies of the invention can be human antibodies or chimeric antibodies comprising human variable regions and non-human (e.g., murine) constant regions. The antibodies of the invention have, for example, human variable regions and, optionally, also human constant regions.

[0111] Thus, the antibody may optionally comprise a constant region or a portion thereof, such as the constant region or a portion thereof of a human antibody. For example, the VL domain may be bound at its C-terminus to the light chain kappa or lambda constant domain of an antibody. Similarly, the VH domain of an antibody may be bound at its C-terminus to all or a portion of the immunoglobulin heavy chain constant region (e.g., CH1 domain or Fc region) from any antibody isotype, such as IgG, IgA, IgE and IgM, and any subclass of isotype, such as IgG1 or IgG4.

[0112] Examples of human heavy chain constant regions are shown in Table S1.

[0113] Alternatively, the constant region of the antibody of the present invention is a non-human constant region. For example, when the antibody is produced in a transgenic animal (examples of which are described elsewhere herein), a chimeric antibody is produced that contains a human variable region and a non-human (host animal) constant region. Some transgenic animals produce fully human antibodies. Others have been engineered to produce antibodies that contain chimeric heavy chains and fully human light chains. When antibodies contain one or more non-human constant regions, their immunogenicity is thereby reduced, so these can be replaced with human constant regions to provide antibodies that are more suitable for administration to humans as therapeutic compositions.

[0114] Digestion of an antibody with the enzyme papain results in two identical antigen-binding fragments, also known as "Fab" fragments, with the "Fc" fragment having no antigen-binding activity but the ability to crystallize. "Fab" as used herein refers to a fragment of an antibody that contains one constant domain and one variable domain of each of the heavy and light chains. The term "Fc region" is used herein to define the C-terminal region of the heavy chain of an immunoglobulin, including native sequence Fc regions and variant Fc regions. "Fc fragment" refers to the carboxy-terminal portions of both H chains that are held together by disulfides. The effector functions of an antibody are determined by the sequences in the Fc region, which is also recognized by the Fc receptor (FcR) found on certain types of cells. Digestion of an antibody with the enzyme pepsin results in the F(ab')2 fragment, in which the two arms of the antibody molecule remain linked and contain two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link antigens.

[0115] "Fv" as used herein refers to the minimum fragment of an antibody that retains both antigen recognition and antigen binding sites. This region consists of a dimer of one heavy chain and one light chain variable domain in tight non-covalent or covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0116] The antibodies disclosed herein can be modified to increase or decrease serum half-life. In one embodiment, one or more of the following mutations are introduced to increase the biological half-life of the antibody: T252L, T254S, or T256F. Biological half-life can also be increased by modifying the CH1 domain or CL region of the heavy chain constant region to contain a salvage receptor binding epitope taken from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022, the modifications described therein being incorporated herein by reference. In another embodiment, the Fc hinge region of the antibody or antigen-binding fragment of the present invention is mutated to decrease the biological half-life of the antibody or fragment. One or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment, such that the antibody or fragment has impaired Staphylococcus protein A (SpA) binding compared to the SpA binding of the native Fc-hinge domain. Other methods of increasing serum half-life are known to those skilled in the art. Thus, in one embodiment, the antibody or fragment is PEGylated. In another embodiment, the antibody or fragment is fused to an albumin binding domain, such as an albumin binding single domain antibody (dAb). In another embodiment, the antibody or fragment is PASylated (i.e., genetic fusion of a polypeptide sequence composed of PAS (XL-Protein GmbH), which forms an uncharged random coil structure with a large hydrodynamic volume). In another embodiment, the antibody or fragment is XTENylated® / rPEGylated (i.e., genetic fusion of an imprecise repeat peptide sequence (Amunix, Versartis) to a therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e., genetically fused to an ELP repeat sequence (PhaseBio)). These various half-life extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, the fusions of which are incorporated herein by reference, e.g., those in Tables 2 and 6.

[0117] The antibody can have a modified constant region that increases stability. Thus, in one embodiment, the heavy chain constant region comprises a Ser228Pro mutation. In another embodiment, the antibody and fragment disclosed herein comprises a heavy chain hinge region that is modified to change the number of cysteine ​​residues. This modification can be used to facilitate the assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0118] 1.6.9. Fc effector functions, ADCC, ADCP and CDC As discussed above, anti-ICOS antibodies are provided in various isotypes and with different constant regions. Examples of heavy chain constant region sequences for human IgG antibodies are shown in Table S1. The Fc region of an antibody primarily determines its effector function in terms of Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity and antibody-dependent cellular phagocytosis (ADCP) activity. These "cellular effector functions", distinct from effector T cell functions, include recruitment of Fc receptor-bearing cells to the site of target cells, resulting in the killing of antibody-bound cells. In addition to ADCC and CDC, the ADCP mechanism

[19] represents a means of depleting antibody-bound T cells and thus targeting high ICOS-expressing TRegs for depletion.

[0119] The cellular effector functions ADCC, ADCP and / or CDC are also exhibited by antibodies lacking an Fc region. The antibody can contain multiple different antigen binding sites, one directed to ICOS and another directed to a target molecule whose engagement induces ADCC, ADCP and / or CDC, for example an antibody comprising two scFv regions joined by a linker, where one scFv can engage an effector cell.

[0120] The antibodies according to the invention are those that exhibit ADCC, ADCP and / or CDC. Alternatively, the antibodies according to the invention lack ADCC, ADCP and / or CDC activity. In either case, the antibodies according to the invention comprise or optionally lack an Fc region that binds to one or more types of Fc receptors. The use of different antibody formats, as well as the presence or absence of FcR binding and cellular effector functions, allows the antibodies to be tailored for use, in particular for therapeutic purposes as discussed elsewhere herein.

[0121] A preferred antibody format for some therapeutic applications uses a wild-type human IgG1 constant region. The constant region is an effector-capable IgG1 constant region, optionally with ADCC and / or CDC and / or ADCP activity. A preferred wild-type human IgG1 constant region sequence is SEQ ID NO: 340 (IGHG1 * 01). Further examples of human IgG1 constant regions are shown in Table S1.

[0122] To test candidate therapeutic antibodies in mouse models of human disease, an effector-positive mouse constant region, such as mouse IgG2a (mIgG2a), is included in place of the effector-positive human constant region.

[0123] The constant region can be engineered for enhanced ADCC and / or CDC and / or ADCP.

[0124] The potency of Fc-mediated effects can be enhanced by engineering the Fc domain by a variety of established techniques. Such methods increase the affinity for certain Fc receptors, thus creating a diverse profile of potential enhanced activation. This can be achieved by modification of one or several amino acid residues

[20] . Human IgG1 constant regions containing specific mutations or altered glycosylation at residue Asn297 (e.g., N297Q, EU index numbering) have been shown to enhance binding to Fc receptors. Exemplary mutations are one or more of the residues selected from 239, 332 and 330 for human IgG1 constant regions (or equivalent positions in other IgG isotypes). The antibody can thus comprise a human IgG1 constant region with one or more mutations independently selected from N297Q, S239D, I332E and A330L (EU index numbering). The triple mutation (M252Y / S254T / T256E) can be used to enhance binding to FcRn, and other mutations that affect FcRn binding are discussed in Table 2 of

[21] , any of which can be used in the present invention.

[0125] Increased affinity for Fc receptors can also be achieved by altering the native glycosylation profile of the Fc domain, for example, by creating under fucosylated or defucosylated variants

[22] . Nonfucosylated antibodies retain the trimannosyl core structure of the complex type N-glycan of Fc, without fucose residues. These glycoengineered antibodies, lacking the core fucose residue from the Fc N-glycan, exhibit stronger ADCC than their fucosylated counterparts due to enhanced FcγRIIIa binding ability. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to Fc-gamma RIII

[23] . Thus, the antibody can comprise a human IgG heavy chain constant region that is a variant of the wild-type human IgG heavy chain constant region, where the variant human IgG heavy chain constant region binds to a human Fcγ receptor selected from the group consisting of FcyRIIB and FcyRIIA with a higher affinity than the wild-type human IgG heavy chain constant region binds to the human Fcγ receptor. The antibody can comprise a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, where the variant human IgG heavy chain constant region binds to human FcγRIIB with a higher affinity than the wild-type human IgG heavy chain constant region binds to human FcγRIIB. The variant human IgG heavy chain constant region is a variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (EU index numbering system).In another embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of S267E and L328F; P238D and L328E; P238D, and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G and A330R; P238D, E233D, G237D, H268D, P271G and A330R; G236D and S267E; S239D and S267E; V262E, S267E and L328F; and V264E, S267E and L328F (EU index numbering system). Enhanced CDC can be achieved by amino acid changes that increase affinity for C1q, the first component of the classical complement activation cascade

[24] . Another approach is to create a chimeric Fc domain made from human IgG1 and human IgG3 segments that exploits the higher affinity of IgG3 for C1q

[25] . The antibodies of the present invention can include mutated amino acids at residues 329, 331 and / or 322 to alter C1q binding and / or reduce or abolish CDC activity. In another embodiment, the antibody or antibody fragment disclosed herein can contain an Fc region with mutations at residues 231 and 239, whereby amino acids are replaced to alter the antibody's ability to fix complement. In one embodiment, the antibody or fragment has a constant region that includes one or more mutations selected from E345K, E430G, R344D and D356R, in particular a double mutation including R344D and D356R (EU index numbering system).

[0126] WO2008 / 137915 described an anti-ICOS antibody with a modified Fc region with enhanced effector function. The antibody was reported to mediate enhanced ADCC activity compared to the level of ADCC activity mediated by a parent antibody containing VH and VL domains and a wild-type Fc region. The antibody according to the present invention can use such a variant Fc region with the effector function described therein.

[0127] The ADCC activity of an antibody may be determined in an assay such as that disclosed in WO2008 / 137915. The ADCC activity of an anti-ICOS antibody may be determined in vitro using an ICOS positive T cell line as described in Example 10 of US Patent No. 9,957,323. The ADCC activity of an anti-PD-L1 antibody may be determined in vitro in an ADCC assay using PD-L1 expressing cells.

[0128] For certain applications (such as in vaccination contexts), it is preferable to use an antibody without Fc effector function. The antibody is provided without constant region or without Fc region, and examples of such antibody formats are described elsewhere herein. Alternatively, the antibody can have a constant region with zero effectors. The antibody can have a heavy chain constant region that does not bind to Fcγ receptors, for example, the constant region contains a Leu235Glu mutation (i.e., the wild type leucine residue is mutated to a glutamic acid residue). Another optional mutation for the heavy chain constant region is Ser228Pro, which increases stability. The heavy chain constant region can be an IgG4 that contains both the Leu235Glu mutation and the Ser228Pro mutation. This "IgG4-PE" heavy chain constant region is effector-free.

[0129] An alternative effector null human constant region is a non-functional IgG1. A non-functional IgG1 heavy chain constant region can contain alanine at positions 235 and / or 237 (EU index numbering), for example, an IgG1 heavy chain constant region containing L235A and / or G237A mutations ("LAGA"). * 01 array.

[0130] The variant human IgG heavy chain constant region may comprise one or more amino acid mutations that reduce the affinity of the IgG for human FcγRIIIA, human FcγRIIA or human FcγRI. In one embodiment, FcγRIIB is expressed on a cell selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells and activated T cells. In one embodiment, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises a set of amino acid mutations selected from the group consisting of S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L and I332E; S298A, E333A and K334A; G236A, S239D and I332E; and F243L, R292P, Y300L, V305I and P396L (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises an S239D, A330L or I332E amino acid mutation (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region comprises S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the variant human IgG heavy chain constant region is a variant human IgG1 heavy chain constant region comprising S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the antibody or fragment comprises an afucosylated Fc region. In another embodiment, the antibody or fragment is defucosylated. In another embodiment, the antibody or fragment is underfucosylated.

[0131] An antibody can have a heavy chain constant region that does not induce cellular effector function, i.e., does not mediate ADCC, CDC or ADCP activity, but binds to one or more types of Fc receptors, and such a constant region is incapable of binding to a particular Fc receptor responsible for triggering ADCC, CDC or ADCP activity.

[0132] 1.6.10. Generation and modification of antibodies Methods for identifying and preparing antibodies are well known. Antibodies are generated using transgenic mice (e.g., Kymouse™, Velocimouse®, Omnimouse®, Xenomouse®, HuMab Mouse® or MeMo Mouse®), rats (e.g., Omnirat®), camels, sharks, rabbits, chickens or other non-human animals immunized with ICOS or a fragment thereof, or a synthetic peptide containing the ICOS sequence motif of interest, optionally followed by humanization of the constant and / or variable regions to generate human or humanized antibodies. In certain instances, display technologies such as yeast, phage or ribosome display can be used, as will be apparent to those skilled in the art. For example, standard affinity maturation using display technologies can be performed in a further step after isolation of antibody leads from transgenic animals, phage display libraries or other libraries. Representative examples of suitable techniques are described in US20120093818 (Amgen, Inc.), which is incorporated by reference in its entirety, e.g., the methods set forth in paragraphs

[0309] to

[0346] .

[0133] Immunization of ICOS knockout non-human animals with human ICOS antigens facilitates the generation of antibodies that recognize both human and non-human ICOS. As described herein and illustrated in the Examples, ICOS knockout mice can be immunized with cells expressing human ICOS to stimulate the production of antibodies to human and mouse ICOS in mice, which are harvested and tested for binding to human and mouse ICOS. In this way, cross-reactive antibodies can be selected, which can be screened for other desirable properties described herein. The method of generating antibodies to an antigen (e.g., a human antigen) by immunizing an animal with an antigen whose expression of an endogenous antigen (e.g., an endogenous mouse antigen) has been knocked out in the animal can be performed in an animal capable of generating antibodies comprising a human variable domain. The genome of such an animal can be engineered to include human or humanized immunoglobulin loci that encode human variable region gene segments, and optionally endogenous or human constant regions. Recombination of the human variable region gene segments results in human antibodies, which have either non-human or human constant regions. If the antibody is intended for in vivo use in humans, the non-human constant regions are then replaced by human constant regions. Such methods and knockout transgenic animals are described in WO2013 / 061078.

[0134] Generally, Kymouse™, VELOCIMMUNE® or other mice or rats (optionally ICOS knockout mice or rats as mentioned) can be loaded with the antigen of interest, and lymphoid cells (such as B cells) are harvested from the mouse that express the antibody. The lymphoid cells can be fused with a myeloma cell line to prepare an immortalized hybridoma cell line, which is screened and selected to identify a hybridoma cell line that produces an antibody specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and linked to constant regions of the desired isotype of heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibodies or variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0135] First, a high affinity chimeric antibody having a human variable region and a mouse constant region is isolated. The antibody is characterized and selected for desired characteristics, including affinity, selectivity, agonism, T cell-dependent killing, neutralizing potency, epitope, etc. The mouse constant region is optionally replaced with a desired human constant region to create a fully human antibody of the present invention, such as wild-type or modified IgG1 or IgG4 (e.g., SEQ ID NOs: 751, 752, 753 in US2011 / 0065902, which is incorporated herein by reference in its entirety). Since the selected constant region varies according to the specific use, high affinity antigen binding and target specificity characterize the residues in the variable region.

[0136] Therefore, in a further aspect, the present invention provides a transgenic non-human mammal having a genome comprising a human or humanized immunoglobulin locus, wherein the mammal does not express ICOS. The mammal is, for example, a knockout mouse or rat, or other experimental animal species. A transgenic mouse, such as Kymouse™, contains human heavy and light chain immunoglobulin loci inserted at the corresponding endogenous mouse immunoglobulin loci. A transgenic mammal according to the present invention may contain such targeted insertions, or may contain human heavy and light chain immunoglobulin loci or immunoglobulin genes inserted randomly into its genome, inserted at a locus other than the endogenous Ig locus, or provided on an additional chromosome or chromosomal fragment.

[0137] Further aspects of the invention are the use of such non-human mammals for producing antibodies against ICOS, as well as methods of producing antibodies, or heavy and / or light chain variable domains of antibodies, in such mammals.

[0138] A method for producing antibodies that bind the extracellular domain of human and non-human ICOS includes providing a transgenic non-human mammal having a genome that includes a human or humanized immunoglobulin locus, wherein the mammal does not express ICOS; (a) immunizing a mammal with a human ICOS antigen (e.g., with cells expressing human ICOS or with purified recombinant ICOS protein); (b) isolating the antibody produced by the mammal; (c) testing the antibody for its ability to bind to human ICOS and non-human ICOS; and (d) selecting one or more antibodies that bind to both human and non-human ICOS.

[0139] Testing for the ability to bind to human ICOS and non-human ICOS can be performed using surface plasmon resonance, HTRF, FACS or any other method described herein. Optionally, the binding affinity to human and mouse ICOS is determined. The affinity of binding to human ICOS and mouse ICOS, or the affinity fold difference, can be determined, and antibodies that present cross-reactive species are selected in this way (the affinity threshold and fold difference used as selection criteria are exemplified elsewhere herein). The neutralization potency of the antibody for inhibiting human and mouse ICOS ligand binding to human and mouse ICOS receptors, respectively, or the neutralization potency fold difference can alternatively be determined, for example, in HTRF assay, as a method of screening for cross-reactive antibodies. Again, the threshold and fold difference that can be used as selection criteria are exemplified elsewhere herein.

[0140] The method can include testing the antibody for its ability to bind to non-human ICOS from the same species as the immunized mammal or from a different species. Thus, if the transgenic mammal is a mouse (e.g., Kymouse™), the antibody is tested for its ability to bind to mouse ICOS. If the transgenic mammal is a rat, the antibody is tested for its ability to bind to rat ICOS. However, this is also useful for determining the cross-reactivity of the isolated antibody to the non-human ICOS of another species. Thus, the antibody made in goat can be tested for its binding to rat or mouse ICOS. Optionally, the binding to goat ICOS can be determined instead or in addition.

[0141] In other embodiments, the transgenic non-human mammal is immunized with non-human ICOS, optionally ICOS of the same mammalian species, instead of human ICOS (e.g., ICOS knockout mice are immunized with mouse ICOS). The affinity of the isolated antibodies for binding to human ICOS and non-human ICOS is then determined in the same manner, and antibodies that bind to both human and non-human ICOS are selected.

[0142] Nucleic acids encoding the antibody heavy and / or light chain variable domains of a selected antibody can be isolated. Such nucleic acids encode the entire antibody heavy and / or light chains, or the variable domains without the associated constant regions. As mentioned, the coding nucleotide sequences can be obtained directly from mouse antibody-producing cells, or B cells can be immortalized or fused to create hybridomas expressing the antibodies, and the coding nucleic acids obtained from such cells. Optionally, the nucleic acids encoding the variable regions are then conjugated to nucleotide sequences encoding human heavy and / or light chain constant regions to provide nucleic acids encoding human antibody heavy and / or light chains, e.g., nucleic acids encoding antibodies comprising both heavy and light chains. As described elsewhere herein, this process is particularly useful when the immunized mammal produces chimeric antibodies with non-human constant regions, which are preferably replaced with human constant regions to produce antibodies that are less immunogenic when administered to humans as pharmaceuticals. The provision of a particular human isotype constant region is also important in determining the effector functions of the antibody, and a number of suitable heavy chain constant regions are discussed herein.

[0143] As described herein, other alterations to the nucleic acids encoding the antibody heavy and / or light chain variable domains can be made, eg, mutating residues and making variants.

[0144] The isolated (optionally mutated) nucleic acid is introduced into a host cell, e.g., a CHO cell, as discussed. The host cell is then cultured under conditions for expression of the antibody or antibody heavy and / or light chain variable domains of any desired antibody format. Some possible antibody formats, e.g., whole immunoglobulins, antigen-binding fragments, and other designs, are described herein.

[0145] Amino acid sequence variants of the variable domains of either the VH and VL domains or CDRs, whose sequences are specifically disclosed herein, may be used in accordance with the present invention, as discussed.

[0146] There are many reasons why it is desirable to create variants, including improving the antibody sequence for large-scale production, facilitating purification, enhancing stability, or improving suitability for inclusion in a desired pharmaceutical formulation. Protein engineering can be performed at one or more target residues in an antibody sequence, for example, to replace an amino acid with an alternative amino acid (possibly creating a variant containing all naturally occurring amino acids at this position, with the possible elimination of Cys and Met), and to monitor the effect on function and expression to determine the best substitution. In some cases, it is not desirable to replace a residue with Cys or Met, or to introduce these residues into the sequence, for example, because the formation of new intramolecular or intermolecular cysteine-cysteine ​​bonds would create manufacturing difficulties. When a lead candidate is selected and modified for manufacturing and clinical development, it is generally desirable to change its antigen-binding properties as little as possible, or at least retain the affinity and potency of the parent molecule. However, variants are also created to modulate important antibody characteristics, such as affinity, cross-reactivity, or neutralization potency.

[0147] The antibody can comprise a set of H and / or L CDRs of any of the disclosed antibodies with one or more amino acid mutations within the disclosed set of H and / or L CDRs. The mutations are amino acid substitutions, deletions, or insertions. Thus, for example, there are one or more amino acid substitutions within the disclosed set of H and / or L CDRs. For example, there are up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations, e.g., substitutions, within the set of H and / or L CDRs. For example, there are up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions, within the HCDR3, and / or there are up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions, within the LCDR3. The antibody can comprise an HCDR, a set of LCDRs, or a set of six (H and L) CDRs as shown for any STIM antibody herein, or a set of CDRs with one or two conservative substitutions.

[0148] One or more amino acid mutations are optionally made in the framework regions of the VH or VL domains of the antibodies disclosed herein. For example, one or more residues that differ from the corresponding human germline segment sequences are reverted to germline. The human germline gene segment sequences corresponding to the VH and VL domains of the exemplary anti-ICOS antibodies are shown in Table E12-1, Table E12-2 and Table E12-3, and the alignments of the VH and VL domains of the antibodies with the corresponding germline sequences are shown in the figures.

[0149] The antibody may comprise a VH domain having at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with any of the VH domains of the antibodies shown in the attached sequence listing, and / or a VL domain having at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with any of the VL domains of the antibodies. Algorithms that can be used to calculate the percent identity of two amino acid sequences include, for example, BLAST, FASTA or Smith-Waterman algorithms, for example, using default parameters. Particular variants may contain one or more amino acid alterations (addition, deletion, substitution and / or insertion of amino acid residues).

[0150] The modification can be made in one or more framework regions and / or one or more CDRs.Variants can be provided by CDR mutagenesis in some cases.Modifications usually do not cause loss of function, so that the antibody comprising such modified amino acid sequence retains the ability to bind to ICOS.This retains the same quantitative binding ability as the antibody that does not undergo modification, for example, as measured in the assay described herein.The antibody comprising such modified amino acid has improved ability to bind to ICOS.

[0151] The alterations include replacing one or more amino acid residues with non-naturally occurring or non-standard amino acids, modifying one or more amino acid residues to non-naturally occurring or non-standard forms, or inserting one or more non-naturally occurring or non-standard amino acids into the sequence. Examples of the number and location of alterations in the sequences of the present invention are described elsewhere herein. Naturally occurring amino acids include the 20 "standard" L-amino acids, identified by their standard one-letter code as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E. Non-standard amino acids include any other residues that are introduced into the polypeptide backbone or result from the modification of existing amino acid residues. Non-standard amino acids are naturally occurring or non-naturally occurring.

[0152] The term "variant" as used herein refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by the deletion, substitution or addition of one or more amino acids or nucleic acids, but still retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include changes in which an amino acid is replaced with a different naturally occurring amino acid residue. Such substitutions are classified as "conservative", in which an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar characteristics, either in terms of polarity, side chain functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention are also "non-conservative", in which an amino acid residue present in a peptide is replaced with an amino acid having different properties, for example, a naturally occurring amino acid from a different group (e.g., replacing a charged or hydrophobic amino acid with alanine), or alternatively, a naturally occurring amino acid is replaced with a non-conventional amino acid. In some embodiments, the amino acid substitution is conservative. Also included within the term variant, when used in reference to a polynucleotide or polypeptide, is a polynucleotide or polypeptide that can be altered in primary, secondary, or tertiary structure compared to a reference polynucleotide or polypeptide, respectively (e.g., compared to a wild-type polynucleotide or polypeptide).

[0153] In some embodiments, "synthetic variants," "recombinant variants," or "chemically modified" polynucleotide or polypeptide variants isolated or created using methods well known in the art can be used. "Modified variants" can include conservative or non-conservative amino acid changes, as described below. The polynucleotide changes result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Some embodiments of use include insertion variants, deletion variants, or substitution variants with amino acid substitutions, including insertions and substitutions of amino acids and other molecules that are not normally present in the peptide sequence on which the variant is based, such as, but not limited to, the insertion of ornithine, which is not normally present in human proteins. The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of a polypeptide that does not substantially alter the activity of the polypeptide. For example, a conservative substitution refers to the replacement of an amino acid residue with a different amino acid residue that has similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.). Conservative amino acid substitutions include leucine and isoleucine or valine, aspartic acid and glutamic acid, or threonine and serine. Conservative substitution tables that provide functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W) (see also Creighton, Proteins, W.H. Freeman and Company (1984), which is incorporated by reference in its entirety).In some embodiments, individual substitutions, deletions or additions that change, add or delete a single amino acid or a small percentage of amino acids can also be considered "conservative substitutions" if the change does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1-5 amino acids. The choice of conservative amino acid can be based on the location of the amino acid to be substituted in the peptide, for example, whether the amino acid is on the exterior of the peptide and exposed to the solvent, or on the interior and not exposed to the solvent.

[0154] The amino acid to be substituted for the existing amino acid can be selected based on the location of the existing amino acid, including its exposure to solvent (i.e., whether the amino acid is exposed to solvent or is present on the outer surface of the peptide or polypeptide, compared to an amino acid located inside that is not exposed to solvent).The selection of such conservative amino acid substitutions is well known in the art, for example, as disclosed in Dordo et al., J.MoI Biol, 1999, 217, 721-739 and Taylor et al., J.Theor.Biol.119(1986);205-218 and S.French and B.Robson, J.Mol.Evol.19(1983) 171. Thus, suitable conservative amino acid substitutions can be selected for amino acids on the exterior of the protein or peptide (i.e., amino acids exposed to the solvent), for example, but not limited to, the following substitutions can be used: Y for F, T for S or K, P for A, E for D or Q, N for D or G, R for K, G for N or A, T for S or K, D for N or E, I for L or V, F for Y, S for T or A, R for K, G for N or A, K for R, A for S, K or P.

[0155] In alternative embodiments, conservative amino acid substitutions can also be selected that are preferably encompassed for amino acids in the interior of the protein or peptide, for example, conservative substitutions that are suitable for amino acids that are in the interior of the protein or peptide (i.e., amino acids not exposed to solvent) can be used, for example, but not limited to, the following conservative substitutions can be used: Y with F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T, and A with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also encompassed within the term variant.

[0156] The present invention includes methods for producing antibodies containing VH and / or VL domain variants of the VH and / or VL domains of the antibodies shown in the attached sequence listing. Such antibodies include (i) providing an antibody VH domain which is an amino acid sequence variant of the VH domain of a parent antibody via addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the VH domain of the parent antibody, wherein the VH domain of the parent antibody is the VH domain of any of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or a VH domain comprising the heavy chain complementarity determining regions of any of those antibodies; (ii) optionally combining the VH domain thus provided with a VL domain to provide a VH / VL combination; and (iii) testing the VH domains or VH / VL domain combinations thus provided to identify antibodies having one or more desired characteristics. The method includes:

[0157] Desired characteristics include binding to human ICOS, binding to mouse ICOS, and binding to other non-human ICOS, such as cynomolgus monkey ICOS. Antibodies with equal or higher affinity to human and / or mouse ICOS can be identified. Other desired characteristics include increasing effector T cell function indirectly through depletion of immunosuppressive Tregs, or directly through activation of ICOS signaling in T effector cells. Identifying an antibody with desired characteristics includes identifying an antibody with functional characteristics described herein, such as its affinity, cross-reactivity, specificity, ICOS receptor agonism, neutralization potency and / or promotion of T cell-dependent killing, any of which can be determined in the assay described herein.

[0158] Where a VL domain is included in the method, the VL domain is any of the VL domains of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or a variant provided via addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the parent VL domain, wherein the parent VL domain is any of the VL domains of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or a VL domain comprising the light chain complementarity determining region of any of those antibodies.

[0159] The method of making a variant antibody optionally includes generating a copy of the antibody or a combination of VH / VL domains. The method may further include expressing the resulting antibody. Optionally, it is possible to generate nucleotide sequences corresponding to the VH and / or VL domains of the desired antibody in one or more expression vectors. Suitable methods of expression, including recombinant expression in a host cell, are set forth in detail herein.

[0160] 1.6.11. Encoding Nucleic Acids and Methods of Expression Isolated nucleic acids can provide the encoding antibodies according to the invention. The nucleic acid can be DNA and / or RNA. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof, can encode the antibody.

[0161] The present invention provides a construct in the form of a plasmid, vector, transcription or expression cassette, which comprises at least one of the above polynucleotides.Exemplary nucleotide sequences are included in the sequence listing.Reference to the nucleotide sequence shown herein includes the DNA molecule with the specified sequence, and includes the RNA molecule with the specified sequence with U replaced by T, unless the context otherwise requires.

[0162] The present invention also provides a recombinant host cell comprising one or more nucleic acids encoding the antibody. Methods for producing the encoded antibody include expression from the nucleic acid, for example, by culturing a recombinant host cell containing the nucleic acid. The antibody thus obtained can be isolated and / or purified using any suitable technique, and then used as required. Methods for production can include formulating the product into a composition comprising at least one additional component, such as a pharma- ceutically acceptable excipient.

[0163] Systems for cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems, and transgenic plants and animals.

[0164] The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is Escherichia coli. Expression in eukaryotic organisms in culture is also available to those skilled in the art as a production option. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 rat myeloma cells, human fetal kidney cells, human fetal retina cells, etc.

[0165] The vector can contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as necessary. The nucleic acid encoding the antibody can be introduced into the host cell. The nucleic acid can be introduced into eukaryotic cells by various methods, including calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or baculovirus for insect cells. The introduction of nucleic acid in host cells, particularly eukaryotic cells, can use virus- or plasmid-based systems. The plasmid system can be maintained episomally or integrated into the host cell or into an artificial chromosome. Integration is either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include calcium chloride transformation, electroporation, and transfection using bacteriophage. The introduction is followed by expression of the nucleic acid, eg, by culturing host cells under conditions for expression of the gene, and then optionally isolating or purifying the antibody.

[0166] The nucleic acids of the invention are integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

[0167] The invention also provides methods that include using the nucleic acids described herein in an expression system to express an antibody.

[0168] 1.6.12. Therapeutic Use The antibody (e.g., full-length antibody or its antigen-binding fragment) described herein can be used in the method of therapeutic treatment of human or animal body.The antibody finds use in increasing effector T cell response, which is beneficial for a range of diseases or conditions, including treating cancer or solid tumors, and in the context of vaccination.Increased Teff response can be achieved by using the antibody to modulate the balance or ratio between Teff and Treg to favor Teff activity.

[0169] Anti-ICOS antibodies can be used to deplete regulatory T cells and / or increase effector T cell responses in a patient and can be administered to a patient to treat a disease or condition amenable to therapy by depleting regulatory T cells and / or increasing effector T cell responses.

[0170] The antibody of the invention, or a composition comprising such an antibody molecule or its encoding nucleic acid, is used or provided for use in any such method. The use of the antibody, or a composition comprising it or its encoding nucleic acid, for the manufacture of a medicament for use in any such method is also envisaged. The method typically involves administering the antibody or composition to a mammal. Suitable formulations and methods of administration are described elsewhere herein.

[0171] It is also envisaged that the therapeutic use of the antibody is the treatment of cancer. The cancer is a solid tumor, such as renal cell carcinoma (optionally renal cell carcinoma, e.g. clear cell renal cell carcinoma), head and neck cancer, melanoma (optionally malignant melanoma), non-small cell lung cancer (e.g. adenocarcinoma), bladder cancer, ovarian cancer, cervical cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, testicular germ cell carcinoma, or a metastasis of a solid tumor, such as those listed, or it is a liquid hematological tumor, such as lymphoma (Hodgkin's lymphoma or non-Hodgkin's lymphoma, e.g. diffuse large B-cell lymphoma, DLBCL, etc.) or leukemia (e.g. acute myeloid leukemia). Anti-ICOS antibodies can enhance tumor shedding in melanoma, head and neck cancer and non-small cell lung cancer, as well as other cancers with a medium to high mutation load

[26] . In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is triple-negative breast cancer. In some embodiments, the cancer is head and neck squamous cell carcinoma. In some embodiments, the cancer is penile cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is an aggressive cancer. In some embodiments, the cancer is a metastatic cancer.

[0172] In some embodiments, treatment with one or more doses of an anti-ICOS antibody or antigen-binding fragment thereof results in a partial anti-tumor response. In some embodiments, treatment with one or more doses of an anti-ICOS antibody or antigen-binding fragment thereof results in a complete anti-tumor response. In some embodiments, treatment with one or more doses of an anti-ICOS antibody or antigen-binding fragment thereof and one or more doses of an anti-PD-L1 antibody or antigen-binding fragment thereof results in a partial anti-tumor response. In some embodiments, treatment with one or more doses of an anti-ICOS antibody or antigen-binding fragment thereof and one or more doses of an anti-PD-L1 antibody or antigen-binding fragment thereof results in a complete anti-tumor response.

[0173] In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof comprises the CDR sequence of KY1044. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof comprises the CDR sequence of KY1044. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof comprises the heavy and light chain variable domain sequences of KY1044. In some embodiments, the anti-ICOS antibody or antigen-binding fragment thereof comprises the heavy and light chain sequences of KY1044. In some embodiments, the anti-ICOS antibody is KY1044. In some embodiments, the anti-PD-L1 antibody is atezolizumab. In some embodiments, treatment with KY1044 promotes the efficacy of the anti-PD-L1 antibody (e.g., atezolizumab).

[0174] By enhancing a patient's immune response to their neoplastic lesion, immunotherapy using anti-ICOS antibodies offers the prospect of durable cure or long-term remission, potentially even in the setting of end-stage disease.

[0175] Cancer is a diverse group of diseases, but anti-ICOS antibodies offer the possibility of treating a range of different cancers by utilizing the patient's own immune system, which has the potential to kill any cancer cells by recognizing mutated or overexpressed epitopes that distinguish cancer cells from normal tissues. By modulating the Teff / Treg balance, anti-ICOS antibodies can enable and / or promote immune recognition and killing of cancer cells. Thus, although anti-ICOS antibodies are useful therapeutic agents for a wide variety of cancers, there are certain categories of cancers for which anti-ICOS therapy is particularly suitable and / or effective when other therapeutic agents are not.

[0176] One such group is cancers that are positive for ICOS ligand expression. Cancer cells can acquire ICOS ligand expression, as described for melanoma

[27] . Expression of ICOS ligand provides cells with a selective advantage, as the surface expressed ligand binds ICOS on Tregs, promoting the expansion and activation of Tregs, thereby suppressing the immune response to cancer. Cancer cells expressing ICOS ligand will depend for their survival on this suppression of the immune system by Tregs, and will therefore be vulnerable to treatment with anti-ICOS antibodies that target Tregs. This also applies to cancers that originate from cells that naturally express ICOS ligand. The continued expression of ICOS ligand by these cells again provides a survival advantage through immune suppression. Cancers that express ICOS ligand are derived from antigen-presenting cells such as B cells, dendritic cells and monocytes, and are liquid hematological tumors such as those listed herein. Interestingly, these types of cancers have also been shown to have high ICOS and FOXP3 expression (TCGA data) - see Example 6. Example 1 herein demonstrates the efficacy of an exemplary anti-ICOS antibody in treating tumors derived from cancerous B cells (A20 syngeneic cells) that express the ICOS ligand.

[0177] Thus, anti-ICOS antibodies can be used in methods of treating cancers that are positive for the expression of ICOS ligand. Furthermore, the cancers to be treated with anti-ICOS antibodies according to the invention are cancers that are positive for the expression of ICOS and / or FOXP3, and optionally also express ICOS ligand.

[0178] Patients can be tested to determine whether their cancer is positive for the expression of a protein of interest (e.g., ICOS ligand, ICOS and / or FOXP3), for example, by taking a test sample (e.g., tumor biopsy) from the patient and determining the expression of the protein of interest. Patients whose cancer is characterized as positive for the expression of one, two or all of such proteins of interest are selected for treatment with anti-ICOS antibodies. As discussed elsewhere herein, anti-ICOS antibodies can be used as a monotherapy or in combination with one or more other therapeutic agents.

[0179] Anti-ICOS antibodies also offer hope to patients whose cancers are refractory to treatment with antibodies or other drugs against immune checkpoint molecules such as CTLA-4, PD-1, PD-L1, CD137, GITR or CD73. While these immunotherapies are effective against some cancers, in some cases the cancers do not respond and become unresponsive to continued treatment with the antibodies. In common with antibodies against immune checkpoint inhibitors, anti-ICOS antibodies modulate the patient's immune system, but nevertheless, they succeed where other such antibodies fail. It is shown herein that animals bearing A20 B-cell lymphoma treated with anti-ICOS antibodies had reduced tumor growth, shrunk tumors and actually cleared tumors from the body, whereas treatment with anti-PD-L1 antibodies was no better than controls. The A20 cell line has also been reported to be resistant to anti-CTLA-4

[28] .

[0180] Thus, anti-ICOS antibodies can be used in methods of treating cancers that are refractory to treatment with one or more immunotherapies, such as anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CD137 antibodies, anti-GITR antibodies, or anti-CD73 antibodies (any or all of them). A cancer is characterized as refractory to treatment with an antibody or other drug if treatment with the antibody or drug does not significantly reduce the growth of the cancer, for example, if the tumor continues to grow or is not reduced in size, or if the tumor resumes its growth after a period of response. Non-response to a therapeutic agent is determined ex vivo by testing a sample (e.g., a tumor biopsy sample) for cancer cell death or growth inhibition, and / or in a clinical setting by observing (e.g., using imaging techniques, including MRI) that a patient treated with a therapy does not respond to the treatment. Patients whose cancers are characterized as refractory to treatment with such immunotherapies are selected for treatment with anti-ICOS antibodies.

[0181] In addition, anti-ICOS antibodies can be used to treat B cell derived cancers that are resistant to treatment with anti-CD20 antibodies. Anti-ICOS antibodies are indicated for cancers that have not responded to or have become resistant to therapy with anti-CD20 antibodies such as rituximab. Anti-ICOS antibodies can be used as a second-line (or additionally or in addition) treatment for such cancers. Anti-CD20 antibody resistant cancers are B cell cancers, for example B cell lymphomas, such as diffuse large B cell lymphoma. Cancer resistance to anti-CD20 can be determined ex vivo by testing samples (e.g., tumor biopsy samples) for cancer cell killing or growth inhibition by anti-CD20 antibodies, and / or in a clinical setting by observing that patients treated with anti-CD20 antibodies do not respond to treatment. Alternatively, or in addition, the cancer (e.g., a tumor biopsy sample) can be tested to assess expression of CD20, where the absence or low levels of CD20 expression indicates loss of sensitivity to an anti-CD20 antibody.

[0182] A sample obtained from a patient can be tested in this way to determine the surface expression of a protein of interest, for example, ICOS ligand, ICOS, FOXP3, and / or a target receptor targeted by another therapeutic agent (e.g., an anti-receptor antibody). The target receptor is CD20 (targeted by anti-CD20 antibody therapy, such as rituximab), or another receptor, such as PD1, EGFR, HER2, or HER3. The lack or loss of surface expression of ICOS ligand, ICOS, FOXP3, and / or the target receptor is an indication that the cancer is susceptible to anti-ICOS antibody therapy. An anti-ICOS antibody can be provided for administration to a patient whose cancer is characterized by the lack or loss of surface expression of ICOS ligand, ICOS, FOXP3, and / or the target receptor, where optionally the patient has previously been treated with anti-CTLA4, anti-PD1, anti-PD-L1, or an antibody against the target receptor, and has not responded or has ceased to respond to the antibody treatment, for example, as measured by continued or resumed cancer cell growth, e.g., an increase in tumor size.

[0183] Any suitable method can be used to determine whether cancer cells test positive for surface expression of a protein such as ICOS ligand, CD20, or other target receptors listed herein. A typical method is immunohistochemistry, where a sample of cells (e.g., a tumor biopsy sample) is contacted with an antibody against the protein of interest, and antibody binding is detected using a labeling reagent, typically a secondary antibody that recognizes the Fc region of the first antibody and has a detectable label, such as a fluorescent marker. A sample is declared positive for the test if at least 5% of the cells are labeled, as visualized by cell staining or other label detection. Optionally, a higher cutoff, such as 10% or 25%, can be used. Antibodies are generally used in excess. Reagent antibodies against the molecule of interest are available or can be made by direct methods. To test for ICOS ligand, the antibody MAB1651 is currently available from R&D systems as a mouse IgG that recognizes human ICOS ligand. To test for CD20 expression, rituximab can be used. Detection of mRNA levels of ICOS ligands or target receptors of interest is an alternative technique

[27] .

[0184] A further indication that tumor responds to treatment with anti-ICOS antibody is the presence of Treg in the tumor microenvironment. Activated Treg is characterized by high ICOS and high Foxp3 surface expression. The presence of Treg in tumor, particularly the increase in number, provides a further basis for which patients are selected for treatment with anti-ICOS antibody. Treg is detected ex vivo in tumor biopsy samples, for example, by immunohistochemistry (assay for co-expression of both Foxp3 and ICOS using antibodies against target proteins followed by detection of labeling, as described above), or by single cell dispersion of samples for use in FACS using labeled antibodies against ICOS and Foxp3.

[0185] Anti-ICOS antibodies can be used to treat cancers associated with infectious agents, such as virus-induced cancers. This category includes head and neck squamous cell carcinoma, cervical cancer, and Merkel cell carcinoma. Viruses associated with cancer include HBV, HCV, HPV (cervical cancer, oropharyngeal cancer), and EBV (Burkitt's lymphoma, gastric cancer, Hodgkin's lymphoma, other EBV-positive B-cell lymphomas, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease). The International Agency for Research on Cancer (Monograph 100B) has identified the main cancer sites associated with the following infectious agents: Stomach: Helicobacter pylori Liver: Hepatitis B virus, Hepatitis C virus (HCV), Clonorchis sinensis, Clonorchis liver fluke Cervical: Human papillomavirus (HPV) with or without HIV Anogenital (penis, vulva, vagina, anus): HPV with or without HIV Nasopharynx: Epstein-Barr virus (EBV) Oropharyngeal: HPV with or without tobacco or alcohol use Kaposi's sarcoma: Human herpesvirus type 8 with or without HIV Non-Hodgkin's lymphoma: H. pylori, EBV with or without HIV, HCV, human T-cell lymphotropic virus type 1 Hodgkin's Lymphoma: EBV with or without HIV Bladder: Schistosoma haematobium.

[0186] The antibodies according to the invention may be used to treat cancers associated with or induced by any of these infectious agents, such as those defined above.

[0187] Stimulation of effector T cell responses also contributes to immunity against and / or recovery from infectious diseases in patients. Therefore, anti-ICOS antibodies can be used to treat infectious diseases by administering the antibodies to patients.

[0188] Infectious diseases include those caused by pathogens, such as bacterial, fungal, viral or protozoan pathogens, and the treatment is to promote the immune response in patients against pathogen infection.An example of bacterial pathogens is tuberculosis.An example of viral pathogens is hepatitis B and HIV.An example of protozoan pathogens is Plasmodium species that cause malaria, such as Plasmodium falciparum.

[0189] The antibody can be used to treat infectious diseases, such as infectious diseases caused by any pathogen listed herein. The infectious disease is a persistent or chronic infectious disease. The infectious disease is local or systemic. Prolonged contact between the pathogen and the immune system leads to immune system exhaustion or resistance development (e.g., manifested by increased levels of Treg and Treg:Teff balance tilted in favor of Treg), and / or immune evasion by the pathogen through evolution and modification of presented pathogen antigens. These functions reflect a process similar to that thought to occur in cancer. Anti-ICOS antibodies offer a therapeutic approach to treat infectious diseases caused by pathogens, such as chronic infectious diseases, by modulating the Treg:Teff ratio in favor of TEff and / or other effects described herein.

[0190] The treatment is of a patient who has been diagnosed with an infectious or infectious disease. Alternatively, the treatment is prophylactic and is administered to a patient to prevent contracting the disease, e.g., as a vaccine, as described elsewhere herein.

[0191] It has also been proposed that immune responses, particularly IFNγ-dependent systemic immune responses, are beneficial for the treatment of Alzheimer's disease and other CNS pathologies that share a neuroinflammatory component

[29] . WO2015 / 136541 proposed the treatment of Alzheimer's disease using anti-PD-1 antibodies. Anti-ICOS antibodies, optionally in combination with one or more other immune modulators (e.g., antibodies against PD-1), can be used in the treatment of Alzheimer's disease or other neurodegenerative diseases.

[0192] Combination Therapy Treatment with immune modulating antibodies such as anti-CTLA4, anti-PD1 or anti-PDL1, especially those with Fc effector function, can create an environment in which further depletion of ICOS, which is highly expressed by immune suppressive cells, is beneficial. It is beneficial to combine anti-ICOS antibodies with such immune modulators to enhance their therapeutic effect.

[0193] Patients treated with immunomodulatory antibodies (e.g., anti-PDL-1, anti-PD-1, anti-CTLA-4) will particularly benefit from treatment with anti-ICOS antibodies. One reason for this is that immunomodulatory antibodies increase the number of ICOS-positive Tregs (e.g., intratumoral Tregs) in patients. This effect is also observed with certain other therapeutic agents, such as recombinant IL-2. Anti-ICOS antibodies can reduce and / or reverse the surge or rise in ICOS+ Tregs (e.g., intratumoral Tregs) resulting from treatment of the patient with another therapeutic agent. Patients selected for treatment with anti-ICOS antibodies are therefore patients who have already been treated with a first therapeutic agent, which is an antibody (e.g., an immunomodulatory antibody) or other agent (e.g., IL-2) that increases the number of ICOS+ Tregs in the patient.

[0194] Immune modulators that can be combined with anti-ICOS antibodies include antibodies against either PDL1 (e.g., avelumab), PD-1 (e.g., pembrolizumab or nivolumab) or CTLA-4 (e.g., ipilimumab or tremelimumab). Anti-ICOS antibodies can be combined with pidilizumab. In other embodiments, anti-ICOS antibodies are not administered in combination with anti-CTLA-4 antibodies and / or are optionally administered in combination with a therapeutic antibody that is not an anti-CTLA-4 antibody.

[0195] For example, an anti-ICOS antibody can be used in combination therapy with an anti-PDL1 antibody. Preferably, the anti-ICOS antibody mediates ADCC, ADCP and / or CDC. Preferably, the anti-PDL1 antibody mediates ADCC, ADCP and / or CDC. An example of such combination therapy is the administration of an anti-ICOS antibody and an anti-PDL1 antibody, where both antibodies have effector-positive constant regions. Thus, both the anti-ICOS antibody and the anti-PDL1 antibody can mediate ADCC, CDC and / or ADCP. The selection of Fc effector functions and constant regions is described in detail elsewhere herein, but as an example, an anti-ICOS human IgG1 can be combined with an anti-PD-L1 human IgG1. An anti-ICOS antibody and / or an anti-PD-L1 antibody can be combined with a wild-type human IgG1 constant region. Alternatively, the effector-positive constant region of the antibody is one that has been engineered for enhanced effector function, for example, enhanced CDC, ADCC and / or ADCP. Examples of antibody constant regions containing wild-type human IgG1 sequences and mutations that alter effector function are discussed in detail elsewhere herein.

[0196] Anti-PDL1 antibodies with which the anti-ICOS antibody can be combined include: an anti-PDL1 antibody, optionally as an effector-positive human IgG1, that inhibits binding of PD-1 to PDL1 and / or inhibits PDL1; ·Anti-PD-1 antibodies that block the binding of PD-1 to PDL1 and / or PDL2; Avelumab, a human IgG1 antibody that inhibits PD-1 binding to PDL-1. See WO2013 / 079174; Durvalumab (or "MEDI4736"), a variant human IgG1 antibody with mutations L234A, L235A and 331. See WO2011 / 066389; Atezolizumab, a variant human IgG1 antibody with the mutations N297A, D356E and L358M. See US2010 / 0203056; BMS-936559, a human IgG4 antibody containing the mutation S228P. See WO2007 / 005874.

[0197] In some embodiments, the anti-PD-L1 antibody comprises atezolizumab.In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0198] Numerous additional examples of anti-PD-L1 antibodies are disclosed herein and others are known in the art. Characterization data for many of the anti-PD-L1 antibodies listed herein are published in US 9,567,399 and US 9,617,338, both of which are incorporated herein by reference. Exemplary anti-PD-L1 antibodies have VH and / or VL domains that include the HCDRs and / or LCDRs of any of 1D05, 84G09, 1D05 HC variant 1, 1D05 HC variant 2, 1D05 HC variant 3, 1D05 HC variant 4, 1D05 LC variant 1, 1D05 LC variant 2, 1D05 LC variant 3, 411B08, 411C04, 411D07, 385F01, 386H03, 389A03, 413D08, 413G05, 413F09, 414B06 or 416E01, as set forth in US 9,567,399 or US 9,617,338. The antibody may comprise the VH and VL domains of any of these antibodies, and optionally may comprise heavy and / or light chains having the heavy and / or light chain amino acid sequences of any of these antibodies. The VH and VL domains of these anti-PD-L1 antibodies are further described elsewhere herein.

[0199] Further examples of anti-PD-L1 antibodies include KN-035, CA-170, FAZ-053, M7824, ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504, or those described in WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801 No., WO2016 / 111645, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, W No. O2016 / 160792, No. WO2016 / 022630, No. WO2016 / 007235, No. WO2015 / 179654, No. WO2015 / 173267, WO20 No. 15 / 181342, No. WO2015 / 109124, No. WO2015 / 112805, No. WO2015 / 061668, No. WO2014 / 159562, No. WO2014 / No. 165082, No. WO2014 / 100079, No. WO2014 / 055897, No. WO2013 / 181634, No. WO2013 / 173223, No. WO2013 / 079 The anti-ICOS antibody has a VH and / or VL domain that includes the HCDRs and / or LCDRs of the anti-PD-L1 antibody disclosed in any of the following publications: WO2012 / 145493, WO2011 / 066389, WO2010 / 077634, WO2010 / 036959, WO2010 / 089411, and WO2007 / 005874. The antibody may include the VH and VL domains of any of these antibodies, and may optionally include heavy and / or light chains with the heavy and / or light chain amino acid sequences of any of these antibodies. The anti-ICOS antibody used in combination therapy with anti-PD-L1 is an antibody of the invention disclosed herein. Alternatively, the anti-ICOS antibody may include the CDRs, or the VH and / or VL domains of the anti-ICOS antibody disclosed in any of the following publications: WO2016154177, US2016304610 - for example, any of antibodies 7F12, 37A10, 35A9, 36E10, 16G10, 37A10S713, 37A10S714, 37A10S715, 37A10S716, 37A10S717, 37A10S718, 16G10S71, 16G10S72, 16G10S73, 16G10S83, 35A9S79, 35A9S710, or 35A9S89; WO16120789, US2016215059 - e.g., antibodies known as 422.2 and / or H2L5; WO14033327, EP2892928, US2015239978 - for example, the antibody known as 314-8 and / or the antibody produced from hybridoma CNCM I-4180; WO12131004, EP2691419, US9376493, US20160264666 - for example the antibody Icos145-1 and / or antibodies produced by the hybridoma CNCM I-4179; WO10056804 - for example, the antibody JMAb 136 or "136"; WO9915553, EP1017723B1, US7259247, US7132099, US7125551, US7306800, US7722872, WO05103086, EP1740617, US8318905, US8916155 - for example, antibodies MIC-944 or 9F3; WO983821, US7932358B2, US2002156242, EP0984023, EP1502920, US7030225, US7045615, US7279560, US7226909, US7 No. 196175, No. US7932358, No. US8389690, No. WO02070010, No. EP1286668, No. EP1374901, No. US7438905, No. US7438905, No. WO0187981, EP1158004 No. 6,803,039, US7,166,283, US7,988,965, WO0115732, EP1125585, US7,465,445, US7,998,478 - for example, any JMAb antibody, such as JMAb-124, JMAb-126, JMAb-127, JMAb-128, JMAb-135, JMAb-136, JMAb-137, JMAb-138, JMAb-139, JMAb-140, JMAb-141, for example, JMAb136; WO2014 / 089113 - for example, antibody 17G9; WO12174338; US2016145344; WO11020024, EP2464661, US2016002336, US2016024211, US8840889; No. US8497244.

[0200] The anti-ICOS antibody optionally comprises the CDRs of 37A10S713 disclosed in WO2016154177. It can comprise the VH and VL domains of 37A10S713, and can optionally have the heavy and light chains of the 37A10S713 antibody.

[0201] The combination of an anti-ICOS antibody and an immune modulator provides increased therapeutic efficacy compared to monotherapy, allowing a lower dose of the immune modulator to achieve therapeutic benefit. Thus, for example, an antibody (e.g., an anti-PD-L1 antibody, optionally ipilimumab or atezolizumab) used in combination with an anti-ICOS antibody is dosed at 3 mg / kg, rather than the more usual dose of 10 mg / kg. The dosing regimen for the anti-PD-L1 antibody or other antibody can include a total of four doses administered intravenously over a period of 90 minutes every three weeks.

[0202] Anti-ICOS antibodies can be used to increase the sensitivity of tumors to treatment with anti-PD-L1 antibodies, which is recognized as a reduction in the dose at which anti-PD-L1 antibodies show therapeutic benefit. Thus, anti-ICOS antibodies can be administered to patients to reduce the effective dose of anti-PD-L1 antibodies to treat cancer or tumors in the patient. Administration of anti-ICOS antibodies can reduce the recommended or required dosage of anti-PD-L1 antibodies for the patient, for example, by 75%, 50%, 25%, 20%, 10% or less, compared to the dosage when anti-PD-L1 antibodies are administered without anti-ICOS. Patients are treated by administration of anti-ICOS antibodies and anti-PD-L1 antibodies in the combination therapy described herein.

[0203] The benefit of combining anti-PD-L1 with anti-ICOS extends to a reduction in the dosage of each agent when compared to its use as a monotherapy. Anti-PD-L1 antibodies can be used to reduce the dose at which anti-ICOS antibodies exhibit therapeutic benefit, and thus can be administered to patients to reduce the effective dose of anti-ICOS antibodies to treat cancer or tumors in the patient. Thus, anti-PD-L1 antibodies can reduce the recommended or required dosage of anti-ICOS antibodies administered to the patient, for example, by 75%, 50%, 25%, 20%, 10% or less, compared to the dosage when anti-ICOS antibodies are administered without anti-PD-L1. Patients are treated by administration of anti-ICOS antibodies and anti-PD-L1 antibodies in the combination therapy described herein.

[0204] As discussed elsewhere herein, treatment with anti-PD-L1 antibodies, particularly those with effector-positive Fc, does not appear to increase ICOS expression on Teff cells. This is advantageous when administering such antibodies in combination with effector-positive anti-ICOS antibodies, where increased ICOS expression on Teffs would unnecessarily make these cells more susceptible to depletion by anti-ICOS antibodies. In combination with anti-PD-L1, therefore, anti-ICOS therapy can exploit the differential expression of ICOS on Teffs compared to Tregs and preferentially target ICOS-high Tregs for depletion. This, in turn, has the net effect of relieving suppression of Teffs and promoting effector T cell responses in patients. The effect of targeting immune checkpoint molecules on the expression of ICOS on T cells has also been previously studied - see Figure S6C in ref.

[30] (supplementary material), where treatment with CTLA-4 and / or anti-PD-1 antibodies was reported to increase the percentage of CD4+ Tregs expressing ICOS. The effect of a therapeutic agent on ICOS expression in Tregs and Teffs is a factor in selecting an appropriate agent for use in combination with an anti-ICOS antibody, noting that the effect of anti-ICOS antibodies is enhanced under conditions where there is high differential expression of ICOS on Tregs versus Teffs.

[0205] As described herein, a single dose of anti-ICOS antibody is sufficient to provide a therapeutic effect, especially in combination with other therapeutic agents such as anti-PD-L1 antibodies. In tumor therapy, the underlying reason for this single dose benefit is that anti-ICOS antibody mediates its effect, at least in part, by sufficiently resetting or altering the tumor microenvironment to make the tumor more susceptible to immune attack and / or the effects of other immune modulators such as those mentioned. Resetting of the tumor microenvironment is triggered, for example, by depletion of ICOS-positive tumor-infiltrating T-regs. Thus, for example, a patient is treated with a single dose of anti-ICOS antibody, followed by one or more doses of anti-PD-L1 antibody. Over the course of the treatment, for example, six months or a year, the anti-ICOS antibody can be administered in a single dose, while the other agent, for example, anti-PD-L1 antibody, can optionally be administered multiple times over the course of the treatment, preferably with at least one such dose administered after treatment with anti-ICOS antibody.

[0206] Further examples of combination therapies include combinations of anti-ICOS antibodies with: - Antagonists of the adenosine A2A receptor ("A2AR inhibitors"); - CD137 agonists (e.g. agonist antibodies); - Antagonists of indoleamine-2,3 dioxygenase ("IDO inhibitors"), an enzyme that catalyzes the degradation of tryptophan. IDO is an immune checkpoint that is activated in dendritic cells and macrophages and contributes to immune suppression / tolerance.

[0207] Anti-ICOS antibodies can be used in combination therapy with IL-2 (e.g., recombinant IL-2, such as aldesleukin). IL-2 can be administered in high doses (HD). A typical HD IL-2 therapy involves bolus injections of more than 500,000 IU / kg, e.g., 600,000 or 720,000 IU / kg, per cycle of therapy, where 10-15 such bolus injections are given at intervals of between 5-10 hours, e.g., up to 15 bolus injections are given every 8 hours, with therapy cycles repeated approximately every 14-21 days for up to 6-8 cycles. HD IL-2 therapy has been successful in treating tumors, particularly melanoma (e.g., metastatic melanoma) and renal cell carcinoma, but its use is limited by the highly toxic IL-2, which causes severe adverse effects.

[0208] Treatment with high doses of IL-2 has been shown to increase the population of ICOS-positive Tregs in cancer patients

[31] . This increase in ICOS+ Tregs after the first cycle of HD IL-2 therapy was reported to correlate with worse clinical outcomes, with higher numbers of ICOS+ Tregs correlating with worse prognosis. The IL-2 variant F42K has been proposed as an alternative therapy to avoid this undesirable increase in ICOS+ Treg cells

[32] . However, an alternative approach would be to exploit the increase in ICOS+ Tregs by using antibodies according to the present invention as second-line therapeutic agents.

[0209] It is beneficial to combine IL-2 therapy with anti-ICOS antibodies, taking advantage of the ability of anti-ICOS antibodies to target TRegs that highly express ICOS, inhibiting these cells and improving the prognosis of patients receiving IL-2 therapy. Concurrent administration of IL-2 and anti-ICOS antibodies can increase response rates while avoiding or reducing adverse events in the treated patient population. The combination allows the use of lower doses of IL-2 compared to IL-2 monotherapy, and can reduce the risk or level of adverse events resulting from IL-2 therapy while retaining or enhancing clinical benefits (e.g., reduced tumor growth, reduced solid tumor shedding and / or reduced metastasis). In this way, the addition of anti-ICOS can improve the treatment of patients receiving IL-2, regardless of whether they are receiving high-dose (HD) or low-dose (LD) IL-2.

[0210] Thus, one aspect of the present invention provides a method of treating a patient by administering an anti-ICOS antibody to the patient, where the patient is also treated with IL-2, e.g., HD IL-2. Another aspect of the present invention is an anti-ICOS antibody for use in treating a patient, where the patient is also treated with IL-2, e.g., HD IL-2. The anti-ICOS antibody can be used as a second-line therapy. Thus, the patient is a patient treated with IL-2, e.g., a patient who has undergone at least one cycle of HD IL-2 therapy, and has an increased level of ICOS+ Tregs. Assays can be performed on a sample of cancer cells, e.g., a tumor biopsy sample, using immunohistochemistry or FACS as described elsewhere herein to detect cells positive for ICOS, Foxp3, ICOSL, and optionally one or more additional markers of interest. The method can include determining patients with increased levels of ICOS+ Tregs after IL-2 treatment (e.g., in peripheral blood or in tumor biopsies), where the increased levels indicate that the patient will benefit from treatment with anti-ICOS antibodies. The increase in Tregs is compared to control (untreated) individuals or to the patient before IL-2 therapy. Such patients with elevated Tregs represent a group that will not benefit from continued IL-2 treatment alone, but will provide therapeutic benefit to a combination of anti-ICOS antibodies and IL-2 therapy, or treatment with anti-ICOS antibodies alone. Thus, after a positive determination that the patient has increased levels of ICOS+ Tregs, anti-ICOS antibodies and / or additional IL-2 therapy can be administered. Treatment with anti-ICOS antibodies selectively targets and depletes ICOS+ Tregs compared to other T cell populations in such patients. This provides a therapeutic effect by relieving the immune suppression mediated by these cells, thereby enhancing the activity of Teff against target cells, e.g., tumor cells or infected cells.

[0211] Combination therapy with anti-ICOS antibodies and IL-2 can be used for any of the therapeutic indications described herein, in particular to treat tumors, such as melanomas, such as metastatic melanomas, or renal cell carcinomas. Thus, in one example, the patient treated with anti-ICOS antibodies is a patient presenting with metastatic melanoma and being treated with IL-2, such as HD IL-2 therapy or LD IL-2 therapy.

[0212] In general, when an anti-ICOS antibody is administered to a patient undergoing treatment with a first therapeutic agent (e.g., an immune modulator antibody) or another agent (e.g., IL-2), the anti-ICOS antibody can be administered a minimum period after administration of the first therapeutic agent, for example, 24 hours, 48 ​​hours, 72 hours, 1 week or 2 weeks. The anti-ICOS antibody can be administered within 2, 3, 4 or 5 weeks after administration of the first therapeutic agent. This does not preclude additional administration of either agent at any time, but it is desirable to minimize the number of treatments administered to facilitate compliance for patients and reduce costs. Rather, the relative timing of administration is selected to enhance their combined effect, with the first therapeutic agent creating an immunological environment (e.g., ICOS+Treg elevation, or antigen release, discussed below) in which the effect of the anti-ICOS antibody is particularly favorable. Thus, the sequential administration of the first therapeutic agent and then the anti-ICOS antibody allows time for the first agent to act, creating in vivo conditions in which the anti-ICOS antibody can demonstrate its enhanced effect. Various dosing regimens, including simultaneous or sequential combination treatment, are described herein and can be utilized as needed.If the first therapeutic agent increases the number of ICOS+ Tregs in the patient, the treatment regimen for the patient can include determining that the patient has an increased number of ICOS+ Tregs, and then administering an anti-ICOS antibody.

[0213] As mentioned, the use of anti-ICOS antibodies in combination therapy can provide the advantage of reducing the effective dose of therapeutic agents and / or the adverse effects of therapeutic agents that increase ICOS+ Tregs in patients. Further therapeutic benefits can also be achieved by selecting a first therapeutic agent that causes release of antigens from target cells by "immune cell death" and administering the first therapeutic agent in combination with the anti-ICOS antibody. As mentioned, administration of the anti-ICOS antibody is then followed by administration of the first therapeutic agent, with the administration of the two agents being separated by a certain time frame as discussed above.

[0214] Immune cell death is a recognized mode of cell death, in contrast to apoptosis, which is characterized by the release of ATP and HMGB1 from the cell and exposure of calreticulin on the cell membrane [33, 34].

[0215] Immune cell death in target tissue or target cell promotes phagocytosis of cells by antigen-presenting cells, resulting in the presentation of antigen from target cells, which in turn induces antigen-specific Teff cells. Anti-ICOS antibodies can increase the magnitude and / or duration of Teff responses by acting as agonists of ICOS on Teff cells. In addition, anti-ICOS antibodies cause the depletion of antigen-specific Tregs when Fc effector function is effective (e.g., human IgG1 antibodies). Thus, by either or a combination of both of these effects, the balance between Teff and Treg cells is modulated in favor of enhancing Teff activity. The combination of anti-ICOS antibodies with treatments that induce immune cell death in target tissues or cell types, for example, in tumor or cancer cells, thereby promotes immune responses in patients against target tissues or cells, representing a form of vaccination in which vaccine antigens arise in vivo.

[0216] Thus, one aspect of the invention is a method of treating cancer in a patient by in vivo vaccination of the patient against cancer cells. Another aspect of the invention is an anti-ICOS antibody for use in such a method. The anti-ICOS antibody is treating the patient with a therapy that induces immune cell death of cancer cells, resulting in presentation of the antigen to antigen-specific effector T cells; and Administering anti-ICOS antibodies to patients wherein the anti-ICOS antibody enhances antigen-specific effector T cell responses against cancer cells. The method can be used in

[0217] Treatments that induce immune cell death include radiation (e.g., ionizing irradiation of cells using UVC light or gamma rays), chemotherapeutic agents (e.g., oxaliplatin, anthracyclines such as doxorubicin, idarubicin or mitoxantrone, BK channel agonists such as phloretin or pimaric acid, bortezomib, cardiac glycosides, cyclophosphamide, GADD34 / PP1 inhibitors and mitomycin, PDT and hypericin, polyinosinic-polycytidylic acid, 5-fluorouracil, gemcitabine, gefitinib, erlotinib, or thapsigargin and cisplatin), and antibodies against tumor-associated antigens. A tumor-associated antigen is any antigen that is overexpressed by tumor cells compared to non-tumor cells of the same tissue, e.g., HER2, CD20, EGFR. Suitable antibodies include Herceptin (anti-HER2), Rituximab (anti-CD20) or Cetuximab (anti-EGFR).

[0218] Therefore, in some embodiments, it is advantageous to combine the anti-ICOS antibody with one or more such treatments. Optionally, the anti-ICOS antibody is administered to a patient who has already undergone such treatment. The anti-ICOS antibody can be administered a period of time after the immune cell death-inducing treatment, for example, 24 hours, 48 ​​hours, 72 hours, one week or two weeks, for example, 24-72 hours after the treatment. The anti-ICOS antibody can be administered within 2, 3, 4 or 5 weeks after the treatment. Other regimens for combination therapy are discussed elsewhere herein.

[0219] Although "in vivo vaccination" is described above, tumor cells can also be treated to induce immune cell death ex vivo, and the cells are then reintroduced into the patient. Rather than administering an agent or treatment that induces immune cell death directly to the patient, the treated tumor cells are administered to the patient. Treatment of the patient can follow the dosing regimens described above.

[0220] As already mentioned, a single dose of anti-ICOS antibody is sufficient to provide therapeutic benefit. Therefore, in the method of treatment described herein, anti-ICOS antibody is optionally administered as a single dose. A single dose of anti-ICOS antibody can deplete Tregs in patients with subsequent beneficial effects in diseases such as cancer. It has previously been reported that transient ablation of Tregs has anti-tumor effects, including reducing tumor progression, treating established tumors and metastases, and prolonging survival, and that it can enhance the therapeutic effect of tumor irradiation

[35] . Administration of a single dose of anti-ICOS can provide such Treg depletion and can be used to enhance the effect of other therapeutic approaches used in combination, such as radiation therapy.

[0221] Antibodies to PD-L1 Antibodies to PD-L1 for use in combination with anti-ICOS antibodies can include the antigen-binding site of any anti-PD-L1 antibody, whether as separate therapeutic agents or in a multispecific antibody as described herein. Numerous examples of anti-PD-L1 antibodies are disclosed herein, and others are known in the art. Characterization data for many of the anti-PD-L1 antibodies listed herein are published in US9,567,399 and US9,617,338, both of which are incorporated herein by reference.

[0222] 1D05 comprises a heavy chain variable region (V) of SEQ ID NO: 33, which comprises the CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 34. 1D05 contains the light chain variable region (VL) of SEQ ID NO: 43, which contains the CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 44. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 35 (heavy chain nucleic acid sequence SEQ ID NO: 36). The full length light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0223] 84G09 is a heavy chain variable (V) polypeptide of SEQ ID NO: 13, which comprises the CDRH1 amino acid sequence of SEQ ID NO: 7 (IMGT) or SEQ ID NO: 10 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 8 (IMGT) or SEQ ID NO: 11 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 9 (IMGT) or SEQ ID NO: 12 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 14. 84G09 contains the light chain variable region (VL) of SEQ ID NO: 23, which contains the CDRL1 amino acid sequence of SEQ ID NO: 17 (IMGT) or SEQ ID NO: 20 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 18 (IMGT) or SEQ ID NO: 21 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 19 (IMGT) or SEQ ID NO: 22 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 24. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full-length heavy chain amino acid sequence is SEQ ID NO: 15 (heavy chain nucleic acid sequence SEQ ID NO: 16). The full-length light chain amino acid sequence is SEQ ID NO: 25 (light chain nucleic acid sequence SEQ ID NO: 26).

[0224] 1D05 HC variant 1 comprises a heavy chain variable (V) variant of SEQ ID NO: 47, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H 1D05 HC variant 1 has a light chain variable region (VL) of SEQ ID NO: 43, which contains a CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 44. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0225] 1D05 HC variant 2 comprises a heavy chain variable (V) variant of SEQ ID NO: 48, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H 1D05 HC variant 2 has a light chain variable region (VL) of SEQ ID NO: 43, which contains a CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 44. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0226] 1D05 HC variant 3 comprises a heavy chain variable (V) variant of SEQ ID NO: 49, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H1D05 HC variant 3 has a light chain variable region (VL) of SEQ ID NO: 43, which contains a CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 44. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0227] 1D05 HC variant 4 comprises a heavy chain variable (V) variant of SEQ ID NO: 342, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H 1D05 HC variant 4 has a light chain variable region (VL) of SEQ ID NO: 43, which contains a CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L ) amino acid sequence.L The light chain nucleic acid sequence of the domain is SEQ ID NO: 44. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0228] 1D05 LC variant 1 comprises a heavy chain variable (V) variant of SEQ ID NO: 33, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of domain is SEQ ID NO: 34. 1D05 LC variant 1 comprises a light chain variable region (VL) of SEQ ID NO: 50, which contains a CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat) and a CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L The CDRL2 sequence of 1D05 LC variant 1 has the amino acid sequence of V L From the sequence, as defined by the Kabat or IMGT system. HThe domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, or SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532, or SEQ ID NO:534. L The domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 35 (heavy chain nucleic acid sequence SEQ ID NO: 36).

[0229] 1D05 LC variant 2 comprises a heavy chain variable (V) domain of SEQ ID NO: 33, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 34. 1D05 LC variant 2 comprises a light chain variable region (VL) of SEQ ID NO: 51, which contains a CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L ) amino acid sequence. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 35 (heavy chain nucleic acid sequence SEQ ID NO: 36).

[0230] 1D05 LC variant 3 comprises a heavy chain variable (V) domain of SEQ ID NO: 33, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of domain is SEQ ID NO: 34. 1D05 LC variant 3 comprises a light chain variable region (VL) of SEQ ID NO: 298, which contains a CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat) and a CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat). L The CDRL2 sequence of 1D05 LC variant 3 has the amino acid sequence of V L From the sequence, as defined by the Kabat or IMGT system. L The light chain nucleic acid sequence of the domain is SEQ ID NO: 44. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, or SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532, or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 35 (heavy chain nucleic acid sequence SEQ ID NO: 36). The full length light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0231] 411B08 comprises a heavy chain variable (V) domain of SEQ ID NO: 58, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 52 (IMGT) or SEQ ID NO: 55 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 53 (IMGT) or SEQ ID NO: 56 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 54 (IMGT) or SEQ ID NO: 57 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 59. 411B08 contains a light chain variable region (VL) of SEQ ID NO: 68, which contains a CDRL1 amino acid sequence of SEQ ID NO: 62 (IMGT) or SEQ ID NO: 65 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 63 (IMGT) or SEQ ID NO: 66 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 64 (IMGT) or SEQ ID NO: 67 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO:69. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 60 (heavy chain nucleic acid sequence SEQ ID NO: 61). The full length light chain amino acid sequence is SEQ ID NO: 70 (light chain nucleic acid sequence SEQ ID NO: 71).

[0232] 411C04 contains the heavy chain variable (V) sequence of SEQ ID NO: 78, which comprises the CDRH1 amino acid sequence of SEQ ID NO: 72 (IMGT) or SEQ ID NO: 75 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 73 (IMGT) or SEQ ID NO: 76 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 74 (IMGT) or SEQ ID NO: 77 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 79. 411C04 contains a light chain variable region (VL) of SEQ ID NO: 88, which contains a CDRL1 amino acid sequence of SEQ ID NO: 82 (IMGT) or SEQ ID NO: 85 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 83 (IMGT) or SEQ ID NO: 86 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 84 (IMGT) or SEQ ID NO: 87 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 89. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 80 (heavy chain nucleic acid sequence SEQ ID NO: 81). The full length light chain amino acid sequence is SEQ ID NO: 90 (light chain nucleic acid sequence SEQ ID NO: 91).

[0233] 411D07 comprises a heavy chain variable (V) domain of SEQ ID NO: 98, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 92 (IMGT) or SEQ ID NO: 95 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 93 (IMGT) or SEQ ID NO: 96 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 94 (IMGT) or SEQ ID NO: 97 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 99. 411D07 contains a light chain variable region (VL) of SEQ ID NO: 108, which contains the CDRL1 amino acid sequence of SEQ ID NO: 102 (IMGT) or SEQ ID NO: 105 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 103 (IMGT) or SEQ ID NO: 106 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 104 (IMGT) or SEQ ID NO: 107 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 109. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 100 (heavy chain nucleic acid sequence SEQ ID NO: 101). The full length light chain amino acid sequence is SEQ ID NO: 110 (light chain nucleic acid sequence SEQ ID NO: 111).

[0234] 385F01 comprises a heavy chain variable (V) domain of SEQ ID NO: 118, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 112 (IMGT) or SEQ ID NO: 115 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 113 (IMGT) or SEQ ID NO: 116 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 114 (IMGT) or SEQ ID NO: 117 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of domain is SEQ ID NO: 119. 385F01 contains a light chain variable region (VL) of SEQ ID NO: 128, which contains a CDRL1 amino acid sequence of SEQ ID NO: 122 (IMGT) or SEQ ID NO: 125 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 123 (IMGT) or SEQ ID NO: 126 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 124 (IMGT) or SEQ ID NO: 127 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 129. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 120 (heavy chain nucleic acid sequence SEQ ID NO: 121). The full length light chain amino acid sequence is SEQ ID NO: 130 (light chain nucleic acid sequence SEQ ID NO: 131).

[0235] 386H03 is a heavy chain variable (V) polypeptide of SEQ ID NO: 158, which comprises the CDRH1 amino acid sequence of SEQ ID NO: 152 (IMGT) or SEQ ID NO: 155 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 153 (IMGT) or SEQ ID NO: 156 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 154 (IMGT) or SEQ ID NO: 157 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of domain is SEQ ID NO: 159. 386H03 contains a light chain variable region (VL) of SEQ ID NO: 168, which contains a CDRL1 amino acid sequence of SEQ ID NO: 162 (IMGT) or SEQ ID NO: 165 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 163 (IMGT) or SEQ ID NO: 166 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 164 (IMGT) or SEQ ID NO: 167 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 169. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 160 (heavy chain nucleic acid sequence SEQ ID NO: 161). The full length light chain amino acid sequence is SEQ ID NO: 170 (light chain nucleic acid sequence SEQ ID NO: 171).

[0236] 389A03 is a heavy chain variable (V) polypeptide of SEQ ID NO: 178, which comprises the CDRH1 amino acid sequence of SEQ ID NO: 172 (IMGT) or SEQ ID NO: 175 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 173 (IMGT) or SEQ ID NO: 176 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 174 (IMGT) or SEQ ID NO: 177 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of domain is SEQ ID NO: 179. 389A03 contains a light chain variable region (VL) of SEQ ID NO: 188, which contains a CDRL1 amino acid sequence of SEQ ID NO: 182 (IMGT) or SEQ ID NO: 185 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 183 (IMGT) or SEQ ID NO: 186 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 184 (IMGT) or SEQ ID NO: 187 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 189. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 180 (heavy chain nucleic acid sequence SEQ ID NO: 181). The full length light chain amino acid sequence is SEQ ID NO: 190 (light chain nucleic acid sequence SEQ ID NO: 191).

[0237] 413D08 comprises a heavy chain variable (V) domain of SEQ ID NO: 138, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 132 (IMGT) or SEQ ID NO: 135 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 133 (IMGT) or SEQ ID NO: 136 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 134 (IMGT) or SEQ ID NO: 137 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of domain is SEQ ID NO: 139. 413D08 contains a light chain variable region (VL) of SEQ ID NO: 148, which contains a CDRL1 amino acid sequence of SEQ ID NO: 142 (IMGT) or SEQ ID NO: 145 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 143 (IMGT) or SEQ ID NO: 146 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 144 (IMGT) or SEQ ID NO: 147 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 149. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 140 (heavy chain nucleic acid sequence SEQ ID NO: 141). The full length light chain amino acid sequence is SEQ ID NO: 150 (light chain nucleic acid sequence SEQ ID NO: 151).

[0238] 413G05 contains the heavy chain variable (V) amino acid sequence of SEQ ID NO: 244, which comprises the CDRH1 amino acid sequence of SEQ ID NO: 238 (IMGT) or SEQ ID NO: 241 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 239 (IMGT) or SEQ ID NO: 242 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 240 (IMGT) or SEQ ID NO: 243 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 245. 413G05 contains a light chain variable region (VL) of SEQ ID NO: 254, which contains a CDRL1 amino acid sequence of SEQ ID NO: 248 (IMGT) or SEQ ID NO: 251 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 249 (IMGT) or SEQ ID NO: 252 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 250 (IMGT) or SEQ ID NO: 253 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 255. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 246 (heavy chain nucleic acid sequence SEQ ID NO: 247). The full length light chain amino acid sequence is SEQ ID NO: 256 (light chain nucleic acid sequence SEQ ID NO: 257).

[0239] 413F09 comprises a heavy chain variable (V) domain of SEQ ID NO: 264, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 258 (IMGT) or SEQ ID NO: 261 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 259 (IMGT) or SEQ ID NO: 262 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 260 (IMGT) or SEQ ID NO: 263 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 265. 413F09 contains a light chain variable region (VL) of SEQ ID NO: 274, which contains a CDRL1 amino acid sequence of SEQ ID NO: 268 (IMGT) or SEQ ID NO: 271 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 269 (IMGT) or SEQ ID NO: 272 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 270 (IMGT) or SEQ ID NO: 273 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 275. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 266 (heavy chain nucleic acid sequence SEQ ID NO: 267). The full length light chain amino acid sequence is SEQ ID NO: 276 (light chain nucleic acid sequence SEQ ID NO: 277).

[0240] 414B06 comprises a heavy chain variable (V) domain of SEQ ID NO: 284, which comprises a CDRH1 amino acid sequence of SEQ ID NO: 278 (IMGT) or SEQ ID NO: 281 (Kabat), a CDRH2 amino acid sequence of SEQ ID NO: 279 (IMGT) or SEQ ID NO: 282 (Kabat), and a CDRH3 amino acid sequence of SEQ ID NO: 280 (IMGT) or SEQ ID NO: 283 (Kabat). H ) region amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 285. 414B06 contains a light chain variable region (VL) of SEQ ID NO: 294, which contains a CDRL1 amino acid sequence of SEQ ID NO: 288 (IMGT) or SEQ ID NO: 291 (Kabat), a CDRL2 amino acid sequence of SEQ ID NO: 289 (IMGT) or SEQ ID NO: 292 (Kabat), and a CDRL3 amino acid sequence of SEQ ID NO: 290 (IMGT) or SEQ ID NO: 293 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 295. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 286 (heavy chain nucleic acid sequence SEQ ID NO: 287). The full length light chain amino acid sequence is SEQ ID NO: 296 (light chain nucleic acid sequence SEQ ID NO: 297).

[0241] 416E01 contains a heavy chain variable region (V) of SEQ ID NO: 349, which contains the CDRH1 amino acid sequence of SEQ ID NO: 343 (IMGT) or SEQ ID NO: 346 (Kabat), the CDRH2 amino acid sequence of SEQ ID NO: 344 (IMGT) or SEQ ID NO: 347 (Kabat), and the CDRH3 amino acid sequence of SEQ ID NO: 345 (IMGT) or SEQ ID NO: 348 (Kabat). H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 350. 416E01 contains a light chain variable region (VL) of SEQ ID NO: 359, which contains the CDRL1 amino acid sequence of SEQ ID NO: 353 (IMGT) or SEQ ID NO: 356 (Kabat), the CDRL2 amino acid sequence of SEQ ID NO: 354 (IMGT) or SEQ ID NO: 357 (Kabat), and the CDRL3 amino acid sequence of SEQ ID NO: 355 (IMGT) or SEQ ID NO: 358 (Kabat). L ) amino acid sequence. L The light chain nucleic acid sequence of domain is SEQ ID NO: 360. H The domain can be combined with any of the heavy chain constant region sequences described herein, for example, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:340, SEQ ID NO:524, SEQ ID NO:526, SEQ ID NO:528, SEQ ID NO:530, SEQ ID NO:532 or SEQ ID NO:534. LThe domains can be combined with any of the light chain constant region sequences described herein, for example, SEQ ID NO: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. The full length heavy chain amino acid sequence is SEQ ID NO: 351 (heavy chain nucleic acid sequence SEQ ID NO: 352). The full length light chain amino acid sequence is SEQ ID NO: 361 (light chain nucleic acid sequence SEQ ID NO: 362).

[0242] In some embodiments, the anti-PD-L1 antibody comprises atezolizumab.In some embodiments, the anti-PD-L1 antibody is atezolizumab.

[0243] 1.6.15. Antibody-Drug Conjugates Anti-ICOS antibodies can be used as a carrier of cytotoxic agents to target Tregs. Tregs located in the tumor microenvironment (TME) strongly express ICOS (see US Pat. No. 9,957,323). ICOS is more strongly expressed in intratumoral Tregs than in intratumoral Teffs or peripheral Tregs. Therefore, anti-ICOS antibodies labeled with toxic drugs or prodrugs preferentially target Tregs in the TME to deliver toxic payloads and selectively inhibit those cells. Such targeting of cytotoxic agents provides an additional route to remove the immunosuppressive effect of Tregs, thereby altering the Treg:Teff balance in favor of Teff activity, and can be used as an alternative to or in combination with any one or more of the other therapeutic approaches discussed herein (e.g., Fc effector-mediated inhibition of Tregs, agonism of effector T cells).

[0244] Thus, the present invention provides an anti-ICOS antibody conjugated to a cytotoxic drug or prodrug. In the case of a prodrug, the prodrug can be activated in the TME or other target site of therapeutic activity to produce a cytotoxic drug. Activation is in response to a trigger, such as photoactivation, for example, using near-infrared light to activate the light absorber conjugate

[36] . The spatially selective activation of the prodrug, in combination with high ICOS expression in intratumoral Tregs, further enhances the cytotoxic effect of the antibody-drug conjugate to provide a cytotoxic effect that is highly selective for these cells.

[0245] For use in antibody-drug conjugates, the cytotoxic drug or prodrug is preferably non-immunogenic and non-toxic during the circulation of the antibody-drug conjugate in blood (dormant or inactive). Preferably, the cytotoxic drug (or prodrug, if activated) is potent, e.g., two out of four molecules of the drug are sufficient to kill the target cell. The photoactivatable prodrug is a silicapthalocyanine dye (IRDye 700 DX), which induces lethal damage to cell membranes after exposure to near-infrared light. Cytotoxic drugs include antimitotic agents such as monomethyl auristatin E, and microtubule inhibitors such as maytansine derivatives, e.g., mertansine, DM1, emtansine.

[0246] Conjugation of the drug (or prodrug) to the antibody is usually via a linker. The linker is a cleavable linker, such as a disulfide, hydrazone or peptide linkage. A cathepsin-cleavable linker can be used, so that the drug is released by cathepsin in tumor cells. Alternatively, a non-cleavable linker can be used, such as a thioether linkage. Additional attachment groups and / or spacers can also be included.

[0247] The antibody in the antibody drug conjugate is an antibody fragment, such as Fab'2 or other antigen-binding fragment described herein, as the small size of such fragments can assist in penetration into tissue sites (e.g., solid tumors).

[0248] The anti-ICOS antibody according to the present invention is provided as an immunocytokine. The anti-ICOS antibody can also be administered together with an immunocytokine in combination therapy. Several examples of antibodies are described herein for use in combination therapy with anti-ICOS, and any of these (e.g., anti-PD-L1 antibodies) are provided as an immunocytokine for use in the present invention. Immunocytokine includes antibody molecules conjugated to a cytokine, such as IL-2. Anti-ICOS:IL-2 conjugates and anti-PD-L1:IL-2 conjugates are therefore further aspects of the present invention.

[0249] The IL-2 cytokine has activity at the high (αβγ) affinity IL-2 receptor and / or the intermediate affinity (αβ) IL-2 receptor. The IL-2 used in the immunocytokine is human wild-type IL-2 or a variant IL-2 cytokine with one or more amino acid deletions, substitutions or additions, for example, an IL-2 with a deletion of 1-10 amino acids at the N-terminus. Other IL-2 variants include the mutations R38A or R38Q.

[0250] An example of an anti-PD-L1 immunocytokine comprises an immunoglobulin heavy chain and an immunoglobulin light chain, where the heavy chain comprises, from N-terminus to C-terminus: a) V containing CDRH1, CDRH2 and CDRH3 H domain; and b) heavy chain constant region; Including, The light chain is, from N-terminus to C-terminus, c) a V comprising CDRL1, CDRL2 and CDRL3 L domain; d) Light chain constant region (C L ); e) optionally a linker (L); and f) IL-2 cytokine; Including, V H Domains and V L The domain is composed of an antigen-binding site that specifically binds to human PD-L1; The immunocytokine is a V antigen comprising a CDRH3 that contains the motif X1GSGX2YGX3X4FD (SEQ ID NO: 609), where X1, X2 and X3 are independently any amino acid, and X4 is either present or absent and, if present, is any amino acid. H Includes the domain.

[0251] The VH and VL domains are the VH and VL domains of any of the anti-PD-L1 antibodies listed herein, for example the VH and VL domains of 1D05.

[0252] The IL-2 is human wild-type or variant IL-2.

[0253] Vaccination Anti-ICOS antibodies are provided in vaccine compositions or co-administered with vaccine preparations. ICOS is involved in T follicular helper cell formation and germinal center reactions

[37] . Therefore, agonistic ICOS antibodies have potential clinical utility as molecular adjuvants to enhance vaccine efficacy. The antibodies can be used to increase the protective efficacy of a number of vaccines, such as those against hepatitis B, malaria, and HIV.

[0254] In the context of vaccination, anti-ICOS antibodies generally lack Fc effector functions and therefore do not mediate ADCC, CDC or ADCP. The antibodies are provided in formats that lack Fc regions or have effector-free constant regions. Optionally, anti-ICOS antibodies can have heavy chain constant regions that bind to one or more types of Fc receptors but do not induce ADCC, CDC or ADCP activity or exhibit lower ADCC, CDC and ADCP activity compared to wild-type human IgG1. Such constant regions may not be able to bind to, or may bind with lower affinity to, a particular Fc receptor responsible for triggering ADCC, CDC or ADCP activity. Alternatively, if cellular effector functions are acceptable or desired in the context of vaccination, anti-ICOS antibodies can include heavy chain constant regions that are positive for Fc effector functions. Any of the IgG1, IgG4 and IgG4.PE formats can be used, for example, for anti-ICOS antibodies in vaccination regimens, and other examples of suitable isotypes and antibody constant regions are set out in more detail elsewhere herein.

[0255] 1.6.17. Formulation and Administration Antibodies may be monoclonal or polyclonal, but are preferably provided as monoclonal antibodies for therapeutic use. They are optionally provided as part of a mixture of other antibodies, including antibodies of different binding specificities.

[0256] The antibodies and encoding nucleic acids according to the present invention are usually provided in isolated form. Thus, the antibodies, VH and / or VL domains, and nucleic acids are provided purified from their natural environment or their production environment. Isolated antibodies and isolated nucleic acids are free or substantially free from other polypeptides or nucleic acids with which they are naturally associated, e.g., found in vivo or in the environment in which they are prepared if such preparation is by recombinant DNA technology in vitro (e.g., cell culture). Optionally, an isolated antibody or nucleic acid is (1) free from at least some other proteins with which it would normally be found, (2) substantially free from other proteins from the same source, e.g., from the same species, (3) expressed by cells from a different species, (4) separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated, or (6) not naturally occurring.

[0257] The antibodies or nucleic acids are formulated with a diluent or adjuvant, and further isolated for practical purposes, e.g., they are mixed with a carrier when used to coat microtiter plates for use in immunoassays, and mixed with a pharma- ceutically acceptable carrier or diluent when used in therapy. As described elsewhere herein, other active ingredients are also included in the therapeutic preparation. Antibodies are either naturally glycosylated in vivo or by heterologous eukaryotic systems such as CHO cells, or they are aglycosylated (e.g., when produced by expression in prokaryotic cells). The present invention encompasses antibodies with modified glycosylation patterns. In some applications, modifications to remove undesirable glycosylation sites are useful, or, for example, removal of fucose moieties can increase ADCC function

[38] . In other applications, modifications of galactosylation can be made to modify CDC.

[0258] Typically, an isolated product will constitute at least about 5%, at least about 10%, at least about 25% or at least about 50% of a given sample. The antibody is substantially free of proteins or polypeptides or other contaminants found in its natural or production environment that would interfere with therapeutic, diagnostic, prophylactic, research or other uses.

[0259] An antibody can be identified, separated and / or recovered from components of its production environment (e.g., natural or recombinant). An isolated antibody is free of association with all other components from its production environment, e.g., such that the antibody has been isolated to an FDA approvable or approved standard. Contaminating components of its production environment, e.g., those resulting from recombinant transfected cells, are typically materials that would interfere with research, diagnostic or therapeutic uses for the antibody, including enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody, e.g., as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver staining. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody or its encoding nucleic acid will be prepared by at least one purification step.

[0260] The present invention provides therapeutic compositions comprising the antibodies described herein. Therapeutic compositions comprising nucleic acids encoding such antibodies are also provided. Coding nucleic acids are described in more detail elsewhere herein and include DNA and RNA, e.g., mRNA. In the therapeutic methods described herein, the use of nucleic acids encoding antibodies and / or cells containing such nucleic acids can be used as an alternative (or in addition) to compositions comprising the antibodies themselves. Cells containing nucleic acids encoding antibodies, optionally with the nucleic acid stably integrated into the genome, thus become medicines for therapeutic use in patients. Nucleic acids encoding anti-ICOS antibodies can be introduced into human B-lymphocytes, optionally derived from the intended patient and modified ex vivo. Optionally, memory B-cells are used. Administration of cells containing coding nucleic acids to a patient provides a reservoir of cells capable of expressing anti-ICOS antibodies, which can provide therapeutic benefits over a longer period of time compared to administration of isolated nucleic acids or isolated antibodies.

[0261] The compositions can contain suitable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like. Numerous suitable formulations can be found in a formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipids (cationic or anionic) containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311. The compositions can include antibodies or nucleic acids in combination with medical injection buffers and / or adjuvants.

[0262] The antibodies or their encoding nucleic acids are formulated for the desired route of administration to the patient, for example, in liquid (optionally aqueous) for injection. A variety of delivery systems are known and can be used to administer the pharmaceutical composition of the present invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Formulating antibodies for subcutaneous administration typically requires concentrating them into smaller volumes compared to intravenous preparations. The high potency of the antibodies according to the present invention lends itself to their use at doses low enough to make subcutaneous formulations practical, which represents an advantage compared to less potent anti-ICOS antibodies.

[0263] The compositions can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0264] Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (see Langer (1990) Science 249:1527-1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

[0265] In certain circumstances, pharmaceutical compositions can be delivered in controlled release systems. In one embodiment, pumps can be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, the controlled release system can be placed in close proximity to the target of the composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138, 1984).

[0266] The injectable preparations can include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations can be prepared by publicly known methods. For example, the injectable preparations can be prepared by dissolving, suspending or emulsifying, for example, the above-described antibody or its salt in a sterile aqueous medium or oily medium that is usually used for injections. As the aqueous medium for injection, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, and the like, can be used in combination with a suitable solubilizing agent such as alcohol (e.g., ethanol), polyhydric alcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)], and the like. As the oily medium, for example, sesame oil, soybean oil, and the like can be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, and the like. The injections thus prepared can be filled into suitable ampoules. The pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. It is envisioned that the treatment is not limited to clinical use. Thus, subcutaneous injection using a needleless device is also advantageous. For subcutaneous delivery, a pen delivery device easily has application in the delivery of the pharmaceutical composition of the present invention. Such a pen delivery device is reusable or disposable. Reusable pen delivery devices generally utilize a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device is sold pre-filled with the pharmaceutical composition held in a reservoir in the device. Once the reservoir is empty of pharmaceutical composition, the entire device is discarded.Many reusable pen and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIKT™ (Sanofi-Aventis, Frankfurt, Germany). Examples of disposable pen delivery devices having application in subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not necessarily limited to, the SOLOSTAR™ pen (Sanofi-Aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly).

[0267] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms with unit doses suitable for adapting the dose of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form of unit dose; particularly, in the form of injection, the antibody is contained in about 5 to about 100 mg, and for other dosage forms, in about 10 to about 250 mg.

[0268] The antibody, nucleic acid or composition comprising it is contained in a medical container such as a vial, a syringe, an IV container or an injection device.In some examples, the antibody, nucleic acid or composition is in vitro and in a sterile container.In some examples, a kit is provided that comprises the antibody, packaging and instructions for use in the therapeutic method described herein.

[0269] One aspect of the present invention is a composition comprising the antibody or nucleic acid of the present invention and one or more pharma- ceutically acceptable excipients, examples of which are listed above. "Pharmaceutically acceptable" refers to being approved or approvable by the regulatory agency of the U.S. Federal or state government, or being listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, including humans. A pharma-ceutically acceptable carrier, excipient, or adjuvant can be administered to a patient together with a drug, such as any antibody or antibody chain described herein, and is non-toxic and does not destroy its pharmacological activity when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0270] In some embodiments, the anti-ICOS antibody is the sole active ingredient in the composition according to the invention. Thus, the composition consists of the antibody or of the antibody and one or more pharma- ceutically acceptable excipients. However, the composition according to the invention optionally comprises one or more additional active ingredients. A detailed description of the agents to be combined with the anti-ICOS antibody is provided elsewhere herein. Optionally, the composition contains multiple antibodies (or encoding nucleic acids) in a combined preparation, e.g., in a single formulation containing the anti-ICOS antibody and one or more other antibodies. Other therapeutic agents that may be desirable to administer together with the antibody or nucleic acid according to the invention include analgesics. Any such agent or combination of agents, whether in a combined or separate preparation, may be administered in combination with or provided in a composition having the antibody or nucleic acid according to the invention. The antibody or nucleic acid according to the invention may be administered separately and sequentially, or simultaneously, and optionally in a combined preparation, with another therapeutic agent or agents such as those mentioned.

[0271] Anti-ICOS antibody for use in certain therapeutic indications can be combined with accepted standard therapy.Therefore, for anti-cancer treatment, antibody therapy can be used in treatment regimens that also include chemotherapy, surgery and radiation therapy.Radiation therapy is delivered directly to affected tissue or systemically, in a single dose or in divided doses.

[0272] The compositions can be administered separately or simultaneously. Separate administration refers to two compositions administered at different times, for example, at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. The compositions can also be administered 24 hours apart, or even longer apart. Alternatively, two or more compositions can be administered simultaneously, for example, less than 10 minutes or less than 5 minutes apart. Compositions administered simultaneously can, in some embodiments, be administered as a mixture, with or without similar or different sustained release mechanisms for each of the components.

[0273] The antibodies and their encoding nucleic acids can be used as therapeutic agents. The patient herein is generally a mammal, typically a human. The antibodies or nucleic acids can be administered to the mammal, for example, by any of the routes of administration mentioned herein.

[0274] Administration is usually in a "therapeutically effective amount", which is an amount that produces the desired effect for which it is administered, sufficient to show benefit to the patient. The exact amount depends on the purpose of the treatment and is ascertained by those skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). Prescription of treatment, such as determining dosage, is within the responsibility of general practitioners and other physicians and depends on the severity and / or progression of the symptoms of the disease being treated. The therapeutically effective amount or suitable dose of an antibody or nucleic acid can be determined by comparing its in vitro activity and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice and other test animals to humans are known.

[0275] As shown by the in vivo studies described in the Examples herein, anti-ICOS antibodies are effective at a range of doses, including surprisingly low doses.Surprisingly, in light of these preclinical studies, low doses of anti-ICOS antibodies (e.g., KY1044) per body weight or low fixed doses of anti-ICOS antibodies (e.g., KY1044) are effective in producing partial or complete anti-tumor activity in human patients across a variety of cancers.

[0276] An anti-ICOS antibody (e.g., a full-length antibody or an antigen-binding fragment thereof) can be administered to a subject in one of the following per-dose values ​​or ranges: Approximately 10μg / kg body weight ~ approximately 3mg / kg body weight, Approximately 10μg / kg body weight ~ approximately 1mg / kg body weight, Approximately 10μg / kg body weight ~ approximately 0.3mg / kg body weight, Approximately 10 μg / kg body weight to approximately 0.1 mg / kg body weight, or Approximately 10μg / kg body weight to approximately 30μg / kg body weight.

[0277] For a fixed dose in an adult human, a suitable dose is about 10 mg or less, 9 mg or less, or about 8 mg or less, for example, about 8 mg, about 7 mg, about 6 mg, about 5 mg, about 4 mg, about 3 mg, about 2.4 mg, about 2 mg, about 1 mg, about 0.8 mg, or about 0.5 mg, or any value therebetween. In some embodiments, the subject is administered about 0.5 to 10 mg of anti-ICOS antibody (e.g., KY1044) per dose. In some embodiments, the subject is administered about 0.5 to 8 mg of anti-ICOS antibody (e.g., KY1044) per dose. In some embodiments, the subject is administered about 0.8 to 8 mg of anti-ICOS antibody (e.g., KY1044) per dose. In some embodiments, the subject is administered about 0.8 to 2.4 mg of anti-ICOS antibody (e.g., KY1044) per dose.

[0278] In some embodiments, the subject is administered about 8 mg of an anti-ICOS antibody (e.g., KY1044) per dose. In some embodiments, the subject is administered about 2.4 mg of an anti-ICOS antibody (e.g., KY1044) per dose. In some embodiments, the subject is administered about 0.8 mg of an anti-ICOS antibody (e.g., KY1044) per dose.

[0279] In the methods of treatment described herein, one or more doses can be administered. In some cases, a single dose is effective to achieve benefits over a long period of time. Thus, the method can include administering a single dose of the antibody, its encoding nucleic acid, or composition. Alternatively, multiple doses can be administered, usually sequentially and separated by a period of days, weeks, or months. The anti-ICOS antibody can be repeatedly administered to the subject at intervals of 2-6 weeks, for example, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the anti-ICOS antibody is administered to the subject every 3 weeks. In some embodiments, the anti-ICOS antibody is administered to the subject every 6 weeks. In some embodiments, the KY1044 is administered to the subject every 3 weeks. In some embodiments, the KY1044 is administered to the subject every 6 weeks. Optionally, the anti-ICOS antibody can be administered to the subject once a month, or less frequently, for example, every 2 months or every 3 months. Thus, methods of treating a patient can include administering a single dose of an anti-ICOS antibody to the subject and not repeating the administration for at least 1 month, at least 2 months, at least 3 months, and optionally not repeating the administration for at least 12 months.

[0280] In some embodiments, the anti-ICOS antibody, e.g., KY1044, is administered to the subject for at least 6 months. In some embodiments, the anti-ICOS antibody, e.g., KY1044, is administered to the subject for 6 months. In some embodiments, the anti-ICOS antibody, e.g., KY1044, is administered to the subject for at least 12 months. In some embodiments, the anti-ICOS antibody, e.g., KY1044, is administered to the subject for 12 months. In some embodiments, the anti-ICOS antibody, e.g., KY1044, is administered to the subject for more than 12 months.

[0281] In some embodiments, KY1044 is administered to the subject every three weeks for at least six months, eg, six months, twelve months, or more than twelve months.

[0282] The same therapeutic effect can be obtained using either one dose or multiple doses of anti-ICOS antibody, which is the result of a single dose of antibody being effective in resetting the tumor microenvironment.The physician can tailor the administration regimen of anti-ICOS antibody to the disease and the patient being treated, taking into account the disease state and any other therapeutic agent or treatment strategy (e.g., surgery, radiation therapy, etc.) with which anti-ICOS antibody is combined.In some embodiments, an effective dose of anti-ICOS antibody is administered more frequently than once a month, such as once every 3 weeks, once every 2 weeks, or once every week.Treatment with anti-ICOS antibody can include multiple doses administered over a period of at least one month, at least six months, or at least one year.The multiple doses can be the same or different.

[0283] As used herein, the terms "treat", "treatment", "treating" or "amelioration" refer to therapeutic treatment, where the purpose is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a disease is reduced or stopped. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation of symptoms, or at least the slowing down of progression or worsening, compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or alleviation of disease state, remission (whether partial or total), and / or reduction in mortality. The term "treatment" of disease also includes providing relief of disease symptoms or side effects (including palliative treatment). For treatment to be effective, complete cure is not intended. The method may also include cure in certain embodiments. In the context of the present invention, treatment is a preventive treatment.

[0284] In some embodiments, "treating" includes treating a disease or condition suitable for therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) response. As used herein, such diseases or conditions include, but are not limited to, tumors and / or cancers. In some embodiments, the cancer is progressive and / or metastatic cancer.

[0285] As used herein, "about" with respect to dosages in mg refers to plus or minus 0.1 mg of the stated value when the stated value is less than 1.5 mg, and to plus or minus 0.5 mg of the stated value when the stated value is at least 1.5 mg.

[0286] 1.6.18.T cell therapy WO2011 / 097477 describes the use of anti-ICOS antibodies to generate and expand T cells by contacting a population of T cells with a first agent (e.g., anti-CD3 antibody) that provides a primary activation signal and a second agent (e.g., anti-ICOS antibody) that activates ICOS, optionally in the presence of a Th17 polarizing agent, such as IL-1β, IL-6, neutralizing anti-IFNγ and / or anti-IL-4. The anti-ICOS antibodies described herein can be used in such methods to provide a T cell population. A population of cultured and expanded T cells with therapeutic activity (e.g., anti-tumor activity) can be generated. As described in WO2011 / 097477, such T cells can be used therapeutically in a method of treating patients by immunotherapy.

[0287] 1.6.19. Morphological Assays for Anti-ICOS Antibodies as Potential Therapeutics It was observed that when candidate therapeutic anti-ICOS antibodies were coupled to a solid phase and contacted with ICOS-expressing T cells, they were able to induce morphological changes in the cells. Upon addition of ICOS+ T cells to wells first coated with anti-ICOS antibody, the cells were seen to change from their initial round shape to adopt a spindle-like shape and stretch and adhere to the antibody-coated surface. This morphological change was not observed with control antibodies. This effect was also found to be dose-dependent, with faster and / or more pronounced shape changes occurring as the concentration of antibody on the surface increased. The shape change provides a surrogate indicator of T cells binding to ICOS and / or agonism by anti-ICOS antibodies. The assay can be used to identify antibodies that promote multimerization of ICOS on the T cell surface. Such antibodies are indicative of therapeutic candidate agonist antibodies. Advantageously, the visual indication provided by this assay is a simple way to screen antibodies or cells, especially in large numbers. The assay can be automated to be performed in high throughput systems.

[0288] Thus, one aspect of the invention is an assay for selecting antibodies that bind to ICOS, and optionally for selecting ICOS agonist antibodies, the assay comprising: Providing an array of antibodies immobilized (attached or attached) to a substrate in a test well; adding ICOS-expressing cells (e.g., activated primary T cells or MJ cells) to test wells; Observing cell morphology; detecting a change in cell shape from round to flattened against the substrate within the well; where the change in shape indicates that the antibody is an antibody that binds to ICOS, optionally an ICOS agonist antibody; and Selecting antibodies from the test wells.

[0289] The assay can be carried out in parallel, for example in a 96-well plate format, with multiple test wells, each containing a different antibody for testing. The substrate is preferably the inner surface of the well. This provides a two-dimensional surface against which the flattening of cells is observed. For example, the bottom and / or walls of the well can be coated with the antibody. The antibody is anchored to the substrate via the constant region of the antibody.

[0290] A negative control can be included, such as an antibody known not to bind ICOS, preferably an antibody that does not bind to an antigen on the surface of the ICOS-expressing cells used. The assay can include quantifying the degree of morphological change, and, if multiple antibodies are tested, selecting an antibody that induces a greater morphological change than one or more of the other tested antibodies.

[0291] The selection of the antibody can include expressing a nucleic acid encoding the antibody present in the test well of interest, or expressing an antibody that includes the CDR or antigen-binding domain of the antibody. The antibody can optionally be reformatted to provide, for example, an antibody that includes the antigen-binding domain of the selected antibody, for example, an antibody fragment or an antibody that includes a different constant region. The selected antibody is preferably provided with a human IgG1 constant region, or other constant regions described herein. The selected antibody can be further formulated into a composition that includes one or more additional components, and suitable pharmaceutical formulations are discussed elsewhere herein.

[0292] Various further aspects and embodiments of the present invention will be apparent to those of skill in the art in view of the present disclosure. All documents cited herein, including the published U.S. counterparts of any referenced patent or patent application, are incorporated herein by reference in their entirety. EXAMPLES

[0293] 1.7. Experimental Examples The generation, characterization and performance of anti-ICOS antibodies were previously disclosed in US Pat. No. 9,957,323.

[0294] 1.8. Example 1: Monotherapy Efficacy of Anti-ICOS Abs Against A20 Tumor Growth in Mice The anti-ICOS antibodies STIM001 mIgG2a and STIM003 mIgG2a each demonstrated robust anti-tumor efficacy when used as monotherapy in vivo in the murine A20 syngeneic model.

[0295] Materials and Methods Efficacy studies were performed in BALB / c mice using the subcutaneous A20 reticulum cell sarcoma model (ATCC, TIB-208). The A20 cell line is a BALB / c B cell lymphoma line derived from a spontaneous reticulum cell neoplasm found in an aged BALB / cAnN mouse. This cell line has been reported to be positive for ICOSL.

[0296] BALB / c mice were supplied by Charles River UK at >18 grams and housed under specific sterile conditions. A total of 5x10e5 A20 cells (passage number below P20) were injected subcutaneously into the right flank of mice. A20 cells were passaged in vitro, washed twice with PBS, and resuspended in RPMI supplemented with 10% fetal bovine serum. Cell viability was confirmed to be >85% at the time of tumor cell injection. Antibody or isotype administration began 8 days after tumor cell injection unless otherwise stated.

[0297] STIM001 and STIM003 anti-ICOS antibodies were generated in a mouse IgG2a isotype format. A mouse cross-reactive anti-PD-L1 antibody (AbW) was also generated in the same isotype format (mouse IgG2a). STIM001, STIM003 and anti-PD-L1 antibodies were dosed intraperitoneally (IP) at 200μg of each antibody twice a week starting on day 8 after tumor cell implantation (3 weeks of dosing between days 8-29). Animal weights and tumor volumes were measured three times a week from the day of tumor cell injection. Tumor volumes were calculated by use of the modified ellipsoid formula 1 / 2 (length x width 2). Mice continued on the study until their tumors reached an average diameter of 12mm. The experiment was stopped on day 43 after tumor cell implantation. Tumor growth was monitored and compared to tumors from animals treated with an isotype control (mIgG2a) antibody. Treatment groups are shown in Table E20 below.

[0298] [Table 1]

[0299] 1.8.2.Results Monotherapy administration of either STIM001 or STIM003 (mIgG2a) in the A20 tumor model produced a complete antitumor response (Figure 3, Figure 4). All animals administered either STIM001 or STIM003 were cured of disease. This contrasts with the results of the isotype control and PD-L1 mIgG2a groups (Figure 1, Figure 2). In rare cases, tumor regression was observed for some animals in the isotype control (spontaneous regression) and anti-PDL-1 groups, but treatment with anti-ICOS antibodies produced significantly higher efficacy. At the end of the study, 3 of 8 control animals and 2 of 8 anti-PDL-1 treated animals were tumor-free. However, all animals treated with either STIM001 or STIM003 were tumor-free at the end of the study (8 of 8 mice in both groups), indicating 100% cure using anti-ICOS antibodies.

[0300] 1.9. Example 2: Robust anti-tumor efficacy in vivo in the J558 myeloma syngeneic model for the combination of anti-ICOS and anti-PD-L1 antibodies The anti-ICOS antibody STIM003 mIgG2a and the anti-PD-L1 antibody AbW mIgG2a were administered individually and in combination in the J558 tumor model, a syngeneic mouse model of myeloma. The anti-ICOS antibody was found to inhibit tumor growth when administered as monotherapy or in combination with anti-PD-L1.

[0301] Materials and Methods Antitumor efficacy studies were performed in Balb / c mice using subcutaneous J558 plasmacytoma:myeloma cell line (ATCC, TIB-6). Balb / c mice were supplied by Charles River UK at 6-8 weeks of age and >18 g and housed under specific pathogen-free conditions. A total of 5 × 10 6 Cells (passage number below P15) were injected subcutaneously (100 μl) into the right flank of mice. Unless otherwise stated, animals were randomized based on tumor size and treatment was initiated 11 days after tumor injection. J558 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice with PBS, and resuspended in DMEM supplemented with 10% fetal bovine serum. Cell viability was confirmed to be greater than 90% at the time of tumor cell injection.

[0302] Treatment was initiated when tumors reached a mean volume of approximately 140mm^3. Animals were then assigned to four groups with similar mean tumor sizes (see Table E-21 for dosing groups). Both antibodies, which are mouse cross-reactive, were IP dosed twice a week for three weeks starting on day 11 (after tumor cell implantation) unless an animal had to be removed from the study due to welfare (rare) or tumor size (Figure 8). As a control, a group of animals (n=10) was dosed at the same time using saline solution. For the combination group, both STIM003 and anti-PDL1 antibody were IP dosed simultaneously at 60μg and 200μg (in 0.9% saline), respectively. Tumor growth was monitored over a period of 37 days and compared to tumors from saline-treated animals. Animal weights and tumor volumes were measured three times a week from the day of tumor cell injection. Tumor volumes were calculated using a modified ellipsoid formula 1 / 2 (length x width) = 1.0×1.0×1.0 mm^2. 2 ) was used. Mice were randomly assigned to receive a 12 mm 3 The study was continued until a mean diameter of 100 mm was reached or, in rare cases, the occurrence of tumor ulceration was observed (well-being).

[0303] [Table 2]

[0304] 1.9.2.Results J558 syngeneic tumors were highly aggressive and all animals (n=10) in the saline control group had to be removed from the study by day 21 due to tumor size. Both anti-STIM003 mIgG2a and anti-PDL1 mIgG2a demonstrated good efficacy as monotherapy in this model with 37.5% and 75% of animals cured of disease, respectively. Importantly, the combination of the two antibodies led to rejection of the plasma cell tumor in 100% of animals by day 37. The data are shown in Figure 8.

[0305] 1.10. Example 3: Administration of anti-PD1 increases ICOS expression in TILs more significantly than anti-PD-L1 antibody.

[0306] Pharmacodynamic studies were performed in animals bearing established CT26 tumors to evaluate the effect of treatment with anti-PD-L1 or anti-PD-1 antibodies on ICOS expression in subsets of tumor-infiltrating lymphocytes (TILs). The following antibodies were compared: Anti-PD-L1 AbW mIgG1 [limited effector function] ·Anti-PD-L1 AbW mIgG2a [has effector function] · Anti-PD-L1 10F9.G2 Rat IgG2b [has effector function] · Anti-PD1 antibody RMT1-14 rat IgG2a [effector null].

[0307] Tumors from treated mice were isolated, dissociated into single cells and stained for CD45, CD3, CD4, CD8, FOXP3 and ICOS.

[0308] Materials and Methods Rat anti-PD-1 RMP1-14 IgG2a (BioXCell; Catalog No.: BE0146), rat anti-PD-L1 10F9.G2 IgG2b (Bio-Legend; Catalog No.: 124325) and anti-PD-L1 AbW mIgG1 and mIgG2a were tested in the CT26 tumor model by ip dosing at 130 μg on days 13 and 15 after tumor cell implantation. On day 16, animals were culled and mouse tumors were harvested for FACS analysis. Tumors were dissociated and homogenized using a Mouse Tumor Dissociation Kit (Miltenyi Biotec). The resulting cell suspension was clarified through a 70 μM filter, pelleted, and resuspended in FACS buffer at 2 million cells / well in a 96-well plate. Cell suspensions were incubated with anti-16 / 32 mAb (eBioscience) and stained with FACS antibodies specific for CD3 (17A2), CD45 (30-F11), CD4 (RM4-5), CD8 (53-6.7) and ICOS (7E.17G9), all from eBioscience Ltd. Cells were also stained with LiveDead Yellow fixable viability dye (Life technologies). For intracellular staining of Foxp3, samples were fixed, permeabilized and stained with an antibody specific for Foxp3 (eBioscience, FJK-16s). Samples were resuspended in PBS and data were acquired on an Attune flow cytometer (Invitrogen) and analyzed using FlowJo V10 software (Treestar).

[0309] 1.10.2.Results Treatment with anti-PD1 and anti-PD-L1 antibodies resulted in only a slight increase in the percentage of CD8 cells and T Regs expressing ICOS at the time points measured. However, in response to anti-PD1 rat IgG2a, a clear and significant (over saline-treated group) increase in ICOS expression (increased dMFI) was observed on the surface of ICOS+ve CD8 cells. It was also noted that ICOS expression was upregulated in CD4 effector and CD4 T Reg cells, but this did not reach statistical significance. This anti-PD1 antibody induced a marked increase in ICOS expression on CD8 effector cells that was barely seen with anti-PD-L1 mIgG2a. Similarly, when comparing different formats of anti-PD-L1 antibodies, it was observed that in a portion of treated animals, the antibody with the lowest effector function (mIgG1) was associated with higher ICOS expression in effector CD8 and CD4 cells when compared to antibodies with effector function (mIgG2a and ratIgG2b), which was rarely seen. Please refer to Figure 9.

[0310] Increased ICOS expression in effector CD8 / CD4 T cells has the effect of making these cells more susceptible to depletion by anti-ICOS antibodies (e.g., in treatment of mice with STIM003 mIgG2a). Antibodies that show lower ICOS induction in effector CD8 and CD4 T cells are preferred for use in combination with anti-ICOS antibodies. Data from this study indicate that anti-PD-L1 effector positive antibodies are particularly suitable for combination with anti-ICOS effector positive antibodies, mirroring the anti-tumor efficacy observed when anti-PDL1 mIgG2a is combined with STIM003 mIgG2a reported in other Examples herein.

[0311] 1.11. Example 4: Potent antitumor efficacy of a single dose of anti-ICOS antibody monotherapy in vivo in a syngeneic B cell lymphoma model This experiment confirms the antitumor efficacy of STIM003 mIgG2a as monotherapy. Strong antitumor efficacy was demonstrated after short exposure to STIM003 mIgG2a.

[0312] Materials and Methods Efficacy studies were performed in BALB / c mice using a subcutaneous A20 reticulum cell sarcoma model (ATCC number CRL-TIB-208). BALB / c mice were supplied by Charles River UK at 6-8 weeks of age and >18g and housed under specific sterile conditions. A total of 5x10E5 A20 cells (passage number below P20) were injected subcutaneously into the right flank of the mice. Treatments were initiated 8 days after tumor cell injection as shown in the table below. A20 cells were passaged in vitro by using TrypLE™ Express Enzyme (Thermofisher), washed twice with PBS, and resuspended in RPMI supplemented with 10% fetal bovine serum. Cell viability was confirmed to be greater than 85% at the time of tumor cell injection. STIM003 mIgG2a was used either as a single dose (SD) of 60 μg (equivalent to 3 mg / kg for a 20 g animal) or multiple doses (MD, twice weekly for 3 weeks) of 60 μg. Antitumor efficacy observed in response to the two schedules was compared to that of saline "treated" animals (MD, twice weekly for 3 weeks). Antibodies were dosed intraperitoneally (IP) at 1 mg / ml in 0.9% saline. Animal body weights and tumor volumes were measured three times a week from the day of tumor cell injection. Tumor volumes were calculated using a modified ellipsoid formula 1 / 2 (length × width) = 1.0 × 1.0 mm. 2 ) was used to calculate the tumor size. Mice were continued on study until their tumors reached a mean diameter of 12 mm or, in rare cases, when the development of tumor ulceration was observed (welfare).

[0313] [Table 3]

[0314] 1.11.2.Results Both multiple and single dose STIM003 mIgG2a resulted in strong and significant monotherapy antitumor efficacy as indicated by the number of animals without signs of tumor growth at the endpoint (day 41). SD resulted in cure of disease in 7 of 10 animals injected with lymphoblastic A20 B cells, while multiple doses resulted in cure of 9 of 10 animals. All animals in the saline treatment group had to be removed from the study by day 40 due to tumor size. See Figure 10.

[0315] Humane endpoint survival statistics were calculated from Kaplan-Meier curves (Figure 11) using GraphPad Prism V7.0. This approach was used to determine whether treatment was associated with improved survival. Hazard ratio (Mantel-Haenszel) values ​​and their associated P values ​​(log-rank Mantel-Cox) are shown in the table below.

[0316] [Table 4]

[0317] 1.12. Example 5: Time- and dose-dependent effects of anti-ICOS antibodies in CT-26 tumor-bearing animals This example shows the results of a pharmacodynamic study evaluating the effect of anti-ICOS antibodies on immune cells in CT-26 tumor-bearing mice. T and B cell subtypes from different tissues were analyzed by FACS after a single dose of STIM003 mIgG2a.

[0318] 1.12.1. Method CT-26 tumor-bearing animals were dosed ip with 200 μg, 60 μg or 6 μg saline or STIM003 on day 12 after tumor cell implantation. Tumor tissue, blood, tumor-draining lymph nodes (TDLN) and spleens were harvested on days 1, 2, 3, 4 and 8 after treatment. Tumors were dissociated into single cell suspensions using a mouse tumor dissociation kit (Miltenyi Biotec). Spleen tissue was dissociated using gentle MACS dissociation and red blood cells were lysed using RBC lysis buffer. Tumor-draining lymph nodes were mechanically disaggregated into single cell suspensions. The resulting cell suspensions were clarified through either a 70 μM or 40 μM filter depending on the tissue, and the cells were then washed twice in RMPI complete medium and finally resuspended in ice-cold FACS buffer. Total blood was collected into plasma tubes, red blood cells were lysed using RBC lysis buffer, cells were washed twice in RMPI complete medium, and finally resuspended in ice-cold FACS buffer. Single cell suspensions from all tissues were distributed into 96 deep-well plates for FACS analysis. Cells were stained with Live Dead Fixable Yellow viability dye (Life technologies). Cell suspensions were incubated with anti-CD16 / CD32 mAb (eBioscience) and stained with FACS antibodies specific for CD3 (17A2), CD45 (30-F11), CD4 (RM4-5), CD8 (53-6.7), CD25 (PC61.5), ICOSL (HK5.3), B220 (RA3-6B2), Ki-67 (SolA15), CD107a (eBio1D4B), IFN-γ (XMG1.2), TNF-α (MP6-XT22), Foxp3 (FJK-16s) and ICOS (7E.17G9), all from eBioscience Ltd. For cytokine readings by FACS, single cell suspensions from tumors were plated in 24-well plates in the presence of Brefeldin-A for 4 h. For intracellular staining, samples were fixed, permeabilized, and stained with specific antibodies. Samples were finally resuspended in PBS and data were acquired on an Attune flow cytometer (Invitrogen) and analyzed using FlowJo V10 software (Treestar).

[0319] The results are presented and discussed below.

[0320] 1.12.2. ICOS expression is high on intratumoral T-regs in the CT26 model When the percentage of tumor infiltrating lymphocytes (TILs) expressing ICOS was compared to the percentage of immune cells in the spleen, blood and TDLN, we demonstrated that more immune cells expressed ICOS in the CT-26 tumor microenvironment versus other tissues. More importantly, the percentage of ICOS-positive T-reg cells in all tissues and at all time points was higher than the percentage of CD4 or CD8 effector T cells positive for ICOS. Importantly, the dMFI (relative expression) for ICOS also followed a similar ranking of expression, with intratumoral T-regs being highly positive for ICOS expression versus other TIL subtypes. Interestingly, within the time frame of this experiment, ICOS + There was no significant change in the percentage of TIL. Similar results were also seen in spleen and TDLN. On the other hand, in blood, ICOS expression is relatively stable in T effector cells, but increases on T-reg during the course of the experiment. The data demonstrated that more cells expressed ICOS in tumor microenvironment, and these positive cells also expressed more ICOS molecules on their surface. More importantly, Treg in TIL is highly positive for ICOS. See Figure 12.

[0321] 1.12.3.Strong depletion of intratumoral T-reg cells in response to STIM003 administration In response to STIM003 mIgG2a antibody, there was a strong and rapid depletion of T-reg cells (CD4+CD25+Foxp3) in the TME. Since T-regs have high ICOS expression compared to other T cell subsets, it is expected that anti-ICOS antibodies with effector function would preferentially deplete these cells. At lower doses of STIM003 (6 μg, corresponding to 0.3 mg / kg for a 20 g animal), there was a continued depletion of T-regs, and by day 3, most T-regs were depleted from the TME. Interestingly, by day 8, T-reg cells repopulate the TME, then reaching levels slightly above those observed in saline-treated animals. The repopulation of T-reg cells at lower doses contributes to the increased infiltration of CD4 T cells in the TME, as evidenced by the observed increase in Ki-67+ CD4 T-cells. At doses higher than 6 μg, there was a long-term depletion of T-reg cells, as shown by the complete T Reg depletion by the last time point analyzed in this study (day 8). Meanwhile, at all doses, there was a transient depletion of T-reg cells in the blood. Importantly, by day 8, all treated animals had similar (or higher for the 6 μg dose) levels of T-reg cells in the blood when compared to saline-treated animals. The data are shown in FIG. 13. Notably, and similar to previously published data for CTLA-4 antibody depletion, there was no significant change in the percentage of T-reg cells in spleen or TDLN tissues, suggesting that T-reg cells are protected from depletion in these organs.

[0322] In summary, robust depletion of T-reg cells in the TME was achieved in the CT-26 model at doses as low as 6 μg per animal. However, a dose of 60 μg resulted in prolonged depletion for up to 8 days after STIM003 mIgG2a injection. This was not improved by using a higher dose (200 μg).

[0323] 1.12.4.STIM003 mIgG2a increased CD8:T Reg and CD4:T Reg ratios The effect of STIM003 on the T-eff:T-reg ratio is shown in FIG.

[0324] STIM003 mIgG2a increased not only the CD8:T-reg ratio but also the CD4 eff:T-reg ratio. All treatment doses were associated with increased T-eff and T-reg ratios, but the mid-dose of 60 μg (equivalent to 3 mg / kg for 20 g animals) was associated with the highest ratios by day 8 post-treatment.

[0325] Interestingly, at the 6 μg dose, the ratios were high until day 4, but by day 8 after treatment, they were consistent with those of saline-treated animals. This can be explained by the repopulation of Tregs observed for this dose until day 8 after treatment. On the other hand, at the 60 or 200 μg doses, the ratio of Teff to T-regs remained high at all time points. This is explained by the prolonged depletion of Tregs at these doses. Notably, at the higher dose (200 μg), despite the prolonged Treg depletion, there was only a moderate improvement in the ratio until day 8. This is indicative of the ICOS at high concentrations of STIM003. INT This can be explained by a partial depletion of effector cells.

[0326] In summary, the data demonstrated TReg depletion and increased effector:T reg ratios at all tested doses. However, the 60 μg (approximately 3 mg / kg) dose achieved both long-term depletion of T-regs and the highest T-eff and T-reg ratios, which would be associated with the most favorable immune context for initiating anti-tumor immune responses. Interestingly, a similar pattern was observed in blood, with the intermediate dose of 60 μg associated with the highest T-eff and T-reg ratios. Importantly, in blood, improved ratios were observed at the earliest time point (between day 3 and day 4).

[0327] 1.12.5. Activation of effector cells in response to STIM003 Surface expression of CD107a on tumor-infiltrating T effector cells was previously identified as a reliable marker for cells achieving and exhibiting cytotoxic activity

[39] . In this study, we used this marker to confirm that STIM003, in addition to depleting T-regs, is able to stimulate the cytotoxic activity of effector T cells in the TME. Interestingly, at day 8 after treatment, there was an increase in surface expression of CD107a in both the CD4 and CD8 effector T cell compartments at all doses of STIM003. Furthermore, this upregulation of CD107a expression on the surface of both CD4 and CD8 T cells appeared to plateau when animals were dosed with 60 μg, as no improvement was seen with a dose of 200 μg.

[0328] To further demonstrate the activation of effector cells in the TME, cytokine release by CD4 and CD8 TILs was analyzed by FACS. As expected, and consistent with the in vitro agonism data shown in the previous examples herein, all doses of STIM003 mIgG2a promoted the production of proinflammatory cytokines IFN-γ and TNF-α by effector CD4 and CD8 T cells. The induction of proinflammatory cytokine production appeared to be high at the 60 μg dose. Indeed, 60 μg of STIM003 significantly increased cytokine production by CD4 T cells. A similar trend was seen for the production of proinflammatory cytokines IFN-γ and TNF-α by effector CD8 T cells in the TME. The data are shown in Figure 15.

[0329] Taken together, all doses of STIM003 led to T cell activation in the TME as indicated by (1) the presence of the degranulation marker CD107a on their surface and (2) the production of Th1 cytokines (IFNγ and TNFα) by T cells, indicating that STIM003 has a strong impact on the immune landscape in the TME, playing a dual role in depleting Treg cells and stimulating the killing activity of T effector cells.

[0330] 1.12.6. Human Dose Estimation Based on the preclinical efficacy data seen in mice, initial predictions of appropriate clinical doses for human patients can be made based on the corresponding biological surface area (BSA)

[40] .

[0331] For example, taking the dose of anti-ICOS IgG in mice which is 3 mg / kg (60 μg) and following the method of reference

[40] , the corresponding dose for humans is 0.25 mg / kg.

[0332] Using the Mosteller formula, for an individual of 60 kg and 1.70 m, the BSA is 1.68 m 2 Multiplying the mg / kg dose by a factor of 35.7 (60 / 1.68) gives a fixed dose of 15 mg. For an 80 kg individual, the corresponding fixed dose would be 20 mg.

[0333] Doses can be adjusted for human therapy in clinical trials to determine a safe and effective treatment regimen.

[0334] 1.13. Example 6: Bioinformatics Analysis of Data from Tumor Samples One target group of cancers according to the present invention are those cancers associated with relatively high levels of ICOS+ immunosuppressive Tregs.

[0335] To identify cancer types associated with high Treg content, transcriptomic data were obtained from The Cancer Genome Atlas (TCGA) public dataset and analyzed for ICOS and FOXP3 expression levels. TCGA is a large-scale study with catalogued genomic and transcriptomic data accumulated for many different cancer types, including mutations, copy number variations, mRNA and miRNA gene expression, and DNA methylation, along with substantial sample metadata.

[0336] Gene set enrichment analysis (GSEA) was performed as follows: Gene expression RNA-seq data collected as part of the TCGA consortium were downloaded from the UCSC Xena Functional Genomics Browser as log2(normalized_count+1). Non-tumor tissue samples were removed from the dataset, leaving data for 20530 genes from 9732 samples. Gene-level counts were replaced with gene set scores for each sample using an algorithm from

[41] and its implementation in

[42] that calculates enrichment scores for genes within a given gene set. Gene sets of interest were defined as those containing both ICOS and FOXP3. Samples were grouped by primary disease, and ssGSEA scores for each group were compared across the 33 primary disease groups. We found that the disease groups with the highest median scores were the lymphoid neoplasms diffuse large B-cell lymphoma, thymoma, and head and neck squamous cell carcinoma, although diffuse large B-cell lymphoma showed a multimodal distribution of scores, with a subset scoring highly and the remainder scoring below the group median.

[0337] Ranked from highest to lowest ssGSEA scores for ICOS and FOXP3 expression, the top 15 cancer types were: DLBC (n=48) Diffuse large B-cell lymphoma, a type of lymphoid neoplasm THYM(n=120) Thymoma HNSC(n=522) Head and neck squamous cell carcinoma TGCT (n=156) Testicular germ cell tumor STAD(n=415) Gastric adenocarcinoma SKCM (n=473) Cutaneous melanoma CESC (n=305) Cervical squamous cell carcinoma and endocervical adenocarcinoma LUAD(n=517) Lung adenocarcinoma LAML (n=173) Acute myeloid leukemia ESCA (n=185) Esophageal cancer LUSC(n=502) Lung squamous cell carcinoma READ(n=95) Rectal adenocarcinoma COAD(n=288) Colon adenocarcinoma BRCA(n=1104) Invasive breast cancer LIHC (n=373) Hepatocellular carcinoma of the liver

[0338] where n is the number of patient samples for that cancer type in the TCGA dataset. The anti-ICOS antibodies described herein can be used for the treatment of these and other cancers.

[0339] Cancers that are associated with relatively high levels of ICOS+ immunosuppressive Tregs and that additionally express PD-L1 may respond particularly well to treatment with a combination of anti-ICOS and anti-PD-L1 antibodies. Suitable treatment regimens and antibodies for this purpose have already been detailed above.

[0340] Using the TCGA dataset as before, enrichment scores for ICOS and FOXP3 were correlated with expression levels of PD-L1 using Spearman's rank correction and grouped by primary disease indication. p-values ​​were calculated for each group and a p-value of 0.05 (Bonferroni multiple comparisons correction) was taken as statistically significant. The disease groups with the highest correlation between ICOS / FOXP3 and PD-L1 expression were: TGCT (n=156) Testicular germ cell tumor COAD(n=288) Colon adenocarcinoma READ(n=95) Rectal adenocarcinoma BLCA(n=407) Bladder urothelial carcinoma OV(n=308) Ovarian serous cystadenocarcinoma BRCA(n=1104) Invasive breast cancer SKCM (n=473) Cutaneous melanoma CESC (n=305) Cervical squamous cell carcinoma and endocervical adenocarcinoma STAD(n=415) Gastric adenocarcinoma LUAD(n=517) Lung adenocarcinoma

[0341] Patients can be selected for treatment according to the assay that determines that their cancer is associated with ICOS+ immunosuppressive Treg and PD-L1 expression.For the cancer type there is a high correlation score as described above, and it is sufficient to determine that one of ICOS+ immunosuppressive Treg and PD-L1 expression exists (e.g., above threshold).PD-L1 immunohistochemical assay can be used in this situation.

[0342] 1.14. Example 7: Evaluation of additional anti-ICOS antibodies The CL-74570 and CL-61091 antibody sequences were synthesized and expressed in HEK cells in an IgG1 format.

[0343] The functional characterization of these antibodies was performed using HTRF assay as previously described (see, for example, Example 6 of US Pat. No. 9,957,323), with modifications to adapt the assay to use purified IgG1 instead of BCT supernatant. 5 μL of supernatant containing human IgG1 antibody expressed from HEK cells was used instead of BCT supernatant, and the total volume was brought up to 20 μl per well using HTRF buffer as before. Human IgG1 antibody was used as a negative control. Both antibodies showed greater than 5% efficacy for binding to human and mouse ICOS, as calculated using formula 1, and therefore confirmed to test positive in this assay.

[0344] The ability of these antibodies to bind to human and mouse ICOS expressed on the surface of CHO-S cells was further confirmed using a Mirrorball assay. In this assay, 5 μl of supernatant containing anti-ICOS IgG1 was transferred to each well of a 384 Mirrorball black plate (Corning). Anti-ICOS antibody binding was detected by adding 10 μl of goat anti-human 488 (Jackson Immunoresearch) diluted in assay buffer (PBS+1% BSA+0.1% sodium azide) at a concentration of 0.8 mg / ml to all wells.

[0345] For positive control wells, 5 μL of reference antibody diluted at 2.2 μg / mL in assay medium was added to the plate. For negative control wells, 5 μl of hybrid control IgG1 diluted at 2.2 μg / mL in assay medium was added to the plate. 10 μM DRAQ5 (Thermoscientific) was added to the plate using 0.4×10 6 Cells were added at 100 / ml and 5 μl was added to all wells. Plates were incubated at 4 degrees for 2 hours.

[0346] Fluorescence intensity was measured from populations of 500–700 single cells using a Mirrorball plate reader (TTP Labtech) measuring Alexafluor 488 (excitation 493 nM, emission 519 nm). Assay signal was measured as median (FL2) mean intensity.

[0347] Total binding was defined using a reference antibody at an assay concentration of 2.2 μg / mL. Non-specific binding was defined using a hybrid control hIgG1 at an assay concentration of 2.22 μg / mL. Both antibodies showed an efficacy greater than 1 percent and therefore were confirmed to test positive in this assay. Efficacy Percentage = (Sample well - non-specific binding) ×100 (Total binding - Non-specific binding)

[0348] Each of CL-74570 and CL-61091 also demonstrated binding to human and mouse ICOS expressed in CHO-S cells as determined by flow cytometry. FACS screening was performed using purified IgG1 rather than BCT supernatant. Both antibodies showed binding >10-fold above the average of the geometric mean of negative control binding to hICOS, mICOS and WT CHO cells.

[0349] [Table 5]

[0350] 1.15. Example 8: Clinical trial Phase I / II open-label study of KY1044. A phase I / II open-label study of KY1044, an anti-ICOS antibody with a dual mechanism of action, as a single agent and in combination with atezolizumab, was conducted in adult patients with advanced malignancies. Participants included patients with advanced / metastatic malignancies who had measurable disease (non-measurable disease was only allowed in Phase I) as determined by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1) and were eligible if there was no available therapy known to confer clinical benefit for their disease or if they had exhausted all such available options, according to the National Comprehensive Cancer Network (NCCN) guidelines.

[0351] 1.15.1. Method Research group: KY1044 Monotherapy Phase I:KY1044 Monotherapy Dose escalation KY1044 and Atezolizumab Phase I: Combination of KY1044 and Atezolizumab Dose escalation KY1044 monotherapy Phase II:KY1044 monotherapy KY1044 and atezolizumab Phase II: Combination of KY1044 and atezolizumab

[0352] Phase I: Participants with advanced / metastatic malignancies and favorable indications (non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), melanoma, cervical cancer, esophageal cancer, gastric cancer, renal cell carcinoma, pancreatic cancer and triple-negative breast cancer).

[0353] Phase II KY1044 Single Agent: Participants with advanced / metastatic malignancies for which no signs of antitumor activity (complete response (CR), partial response (PR), or durable stable disease (SD) with tumor shrinkage not qualifying for PR) have been seen during dose escalation of KY1044 as a single agent.

[0354] Phase II KY1044 in combination with atezolizumab: Participants with advanced / metastatic malignancies in the following selected indications and / or indications that have shown promising activity in Phase I: NSCLC (anti-PD-(L)1 therapy naïve and pre-treated) Stomach (anti-PD-(L)1 therapy naïve and pre-treated) HNSCC (anti-PD-(L)1 therapy naïve and pre-treated) Esophagus (anti-PD-(L)1 therapy naïve and pre-treated) Cervix (anti-PD-(L)1 therapy naïve and pre-treated) Indications where signs of antitumor activity have been observed in Phase I studies using KY1044 in combination with atezolizumab.

[0355] Patients with progressive / metastatic malignancies received dose escalation of KY1044, either as a single agent or in combination with the anti-PD-L1 antibody atezolizumab at 1200 mg, by IV infusion every 3 weeks until disease progression or unacceptable toxicity. Dose escalation was guided by a modified probability interval design for toxicity. The primary objective was to determine safety, tolerability, and maximum tolerated dose. Cohorts that demonstrated tolerance were subsequently enriched with more subjects. Adverse events (AEs) were classified according to the Common Terminology Criteria for Adverse Events, version 5 (CTCAE v5), and efficacy measurements were performed according to the Response Evaluation Criteria in Solid Tumours, version 1.1 (RECIST v1.1), every 8 weeks for the first 16 weeks, then every 12 weeks.

[0356] Patient Inclusion Criteria: Participants must meet all of the following additional inclusion criteria: 1. Prior therapy with an anti-PD-(L)1 and / or anti-PD-L1 inhibitor is permitted, provided that any toxicity attributable to the prior anti-PD-(L)1 and / or anti-PD-L1 directed therapy did not lead to discontinuation of therapy; 2. Eastern Cooperative Oncology Group (ECOG) performance status 0-1; 3. Life expectancy greater than 12 weeks; and 4. Candidate for tumor biopsy according to site of disease suitable for biopsy and treating institutional guidelines.

[0357] 1.15.3. Patient exclusion criteria: Patients must not have any of the following exclusion criteria: 1. Symptomatic central nervous system (CNS) metastases or CNS metastases requiring local CNS-directed therapy, or the presence of escalating doses of corticosteroids within 2 weeks prior to the first dose of study treatment; 2. History of severe hypersensitivity reactions to other monoclonal antibodies and / or their excipients; 3. Presence of known neutralizing anti-atezolizumab antibodies (for patients previously treated with atezolizumab); 4. Having out-of-range laboratory values: creatinine, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), absolute neutrophil count (ANC), platelet count, hemoglobin; 5. Impaired cardiac function or clinically significant cardiac disease; 6. Known human immunodeficiency virus (HIV), active hepatitis B virus (HBV), or active hepatitis C virus (HCV) infection; 7. Malignant neoplastic disease other than that being treated in this study; 8. Any medical condition that, in the investigator's judgment, prevents participation in the clinical study due to safety concerns, compliance with clinical study procedures, or interpretation of study results; 9. Acute autoimmune disease or documented history of autoimmune disease; 10. Participants who have previously received anti-PD-(L)1 treatment, who have not been adequately treated for skin rash or have not received alternative therapy for endocrine disease, must be excluded; 11. Participants with a history of drug-induced interstitial pneumonia or currently with interstitial pneumonia; 12. Systemic steroid therapy or any immunosuppressive therapy. Topical, inhaled, nasal and ophthalmic steroids are not prohibited; 13. Use of a live attenuated vaccine against an infectious disease within 4 weeks of the first dose of study treatment; 14. Anti-CTLA4, anti-PD-(L)1 treatment within 4 weeks of first dose of study treatment; 15. Prior treatment with anti-CTLA4 antibodies in combination with any other antibodies or drugs that specifically target T cell costimulatory or checkpoint pathways; 16. Presence of Common Terminology Criteria for Adverse Events version 5 (CTCAE v5) ≥ grade 2 toxicity (excluding alopecia, peripheral neuropathy, and hearing toxicity, which are excluded if ≥ grade 3 per CTCAE v5) attributable to previous cancer therapy; 17. Radiation therapy within 2 weeks of the first dose of study treatment, except for palliative radiation therapy to limited sites, such as for the treatment of bone pain or locally painful tumor masses. To allow evaluation of response to treatment, participants enrolled in the Phase II part must have residual, unirradiated, measurable disease; and 18. Pregnant or lactating women.

[0358] Medication KY1044 was administered at doses of 0.8 mg, 2.4 mg, 8 mg, 24 mg, 80 mg, or 240 mg every 3 weeks as a single agent or in combination with 1200 mg of atezolizumab.

[0359] Intermediate Results One hundred and three patients were enrolled in the study (38 patients as monotherapy in six cohorts at doses ranging from 0.8 to 240 mg, and 65 patients as combination with atezolizumab in five cohorts at doses ranging from 0.8 to 80 mg). In the single agent and combination cohorts, respectively, 63% and 55% of patients had received ≥4 prior anticancer therapies. 【0360...

Claims

1. 1. An anti-ICOS antibody or antigen-binding fragment thereof that binds to the extracellular domain of human and / or mouse ICOS for use in a method of treating a disease or condition amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses in a subject in need thereof, the method comprising administering the anti-ICOS antibody or antigen-binding fragment thereof to the subject, wherein the anti-ICOS antibody or antigen-binding fragment thereof is administered to the subject at a dose of about 0.8 mg to 240 mg.

2. The anti-ICOS antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein: (a) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:405, SEQ ID NO:406 and SEQ ID NO:407, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:412, SEQ ID NO:413, SEQ ID NO:414; (b) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NOs: 363, 364 and 365, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NOs: 370, 371, 372; (c) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NOs: 377, 378 and 379, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NOs: 384, 385, 386; (d) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:391, SEQ ID NO:392 and SEQ ID NO:393, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:391, SEQ ID NO:392 and SEQ ID NO:393, (e) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NOs:419, 420 and 421, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NOs:426, 427 and 428; (f) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:435, SEQ ID NO:436 and SEQ ID NO:437, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:442, SEQ ID NO:443, SEQ ID NO:444; (g) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:449, SEQ ID NO:450 and SEQ ID NO:451, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:456, SEQ ID NO:457, SEQ ID NO:458; (h) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:463, SEQ ID NO:464 and SEQ ID NO:465, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472; (i) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:477, SEQ ID NO:478 and SEQ ID NO:479, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:484, SEQ ID NO:485, SEQ ID NO:486, or (j) HCDR1, HCDR2 and HCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:491, SEQ ID NO:492 and SEQ ID NO:493, and LCDR1, LCDR2 and LCDR3 comprise sequences having at least 85%, 90% or 95% sequence identity to the amino acid sequences of SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500; 2. An anti-ICOS antibody or antigen-binding fragment thereof for use according to claim 1.

3. The anti-ICOS antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein: (a) HCDR1 comprises the amino acid sequence of SEQ ID NO:405, HCDR2 comprises the amino acid sequence of SEQ ID NO:406, HCDR3 comprises the amino acid sequence of SEQ ID NO:407, LCDR1 comprises the amino acid sequence of SEQ ID NO:412, LCDR2 comprises the amino acid sequence of SEQ ID NO:413, and LCDR3 comprises the amino acid sequence of SEQ ID NO:414; (b) HCDR1 comprises the amino acid sequence of SEQ ID NO: 363, HCDR2 comprises the amino acid sequence of SEQ ID NO: 364, HCDR3 comprises the amino acid sequence of SEQ ID NO: 365, LCDR1 comprises the amino acid sequence of SEQ ID NO: 370, LCDR2 comprises the amino acid sequence of SEQ ID NO: 371, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 372; (c) HCDR1 comprises the amino acid sequence of SEQ ID NO:377, HCDR2 comprises the amino acid sequence of SEQ ID NO:378, HCDR3 comprises the amino acid sequence of SEQ ID NO:379, LCDR1 comprises the amino acid sequence of SEQ ID NO:384, LCDR2 comprises the amino acid sequence of SEQ ID NO:385, and LCDR3 comprises the amino acid sequence of SEQ ID NO:386; (d) HCDR1 comprises the amino acid sequence of SEQ ID NO: 391, and HCDR2 comprises the amino acid sequence of SEQ ID NO: 3 92, wherein HCDR3 comprises the amino acid sequence of SEQ ID NO:393, LCDR1 comprises the amino acid sequence of SEQ ID NO:398, LCDR2 comprises the amino acid sequence of SEQ ID NO:399, and LCDR3 comprises the amino acid sequence of SEQ ID NO:400; (e) HCDR1 comprises the amino acid sequence of SEQ ID NO:419, HCDR2 comprises the amino acid sequence of SEQ ID NO:420, HCDR3 comprises the amino acid sequence of SEQ ID NO:421, LCDR1 comprises the amino acid sequence of SEQ ID NO:426, LCDR2 comprises the amino acid sequence of SEQ ID NO:427, and LCDR3 comprises the amino acid sequence of SEQ ID NO:428; (f) HCDR1 comprises the amino acid sequence of SEQ ID NO:435, HCDR2 comprises the amino acid sequence of SEQ ID NO:436, HCDR3 comprises the amino acid sequence of SEQ ID NO:437, LCDR1 comprises the amino acid sequence of SEQ ID NO:442, LCDR2 comprises the amino acid sequence of SEQ ID NO:443, and LCDR3 comprises the amino acid sequence of SEQ ID NO:444; (g) HCDR1 comprises the amino acid sequence of SEQ ID NO:449, HCDR2 comprises the amino acid sequence of SEQ ID NO:450, HCDR3 comprises the amino acid sequence of SEQ ID NO:451, LCDR1 comprises the amino acid sequence of SEQ ID NO:456, LCDR2 comprises the amino acid sequence of SEQ ID NO:457, and LCDR3 comprises the amino acid sequence of SEQ ID NO:458; (h) HCDR1 comprises the amino acid sequence of SEQ ID NO:463, HCDR2 comprises the amino acid sequence of SEQ ID NO:464, HCDR3 comprises the amino acid sequence of SEQ ID NO:465, LCDR1 comprises the amino acid sequence of SEQ ID NO:470, LCDR2 comprises the amino acid sequence of SEQ ID NO:471, and LCDR3 comprises the amino acid sequence of SEQ ID NO:472; (i) HCDR1 comprises the amino acid sequence of SEQ ID NO:477, HCDR2 comprises the amino acid sequence of SEQ ID NO:478, HCDR3 comprises the amino acid sequence of SEQ ID NO:479, LCDR1 comprises the amino acid sequence of SEQ ID NO:484, LCDR2 comprises the amino acid sequence of SEQ ID NO:485, and LCDR3 comprises the amino acid sequence of SEQ ID NO:486; or (j) HCDR1 comprises the amino acid sequence of SEQ ID NO:491, HCDR2 comprises the amino acid sequence of SEQ ID NO:492, HCDR3 comprises the amino acid sequence of SEQ ID NO:493, LCDR1 comprises the amino acid sequence of SEQ ID NO:498, LCDR2 comprises the amino acid sequence of SEQ ID NO:499, and LCDR3 comprises the amino acid sequence of SEQ ID NO:500; 3. An anti-ICOS antibody or antigen-binding fragment thereof for use according to claim 1 or 2.

4. The anti-ICOS antibody or antigen-binding fragment thereof comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein: (a) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:408, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:415; (b) the VH domain comprises the amino acid sequence of SEQ ID NO:408 and the VL domain comprises the amino acid sequence of SEQ ID NO:415; (c) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 366, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 373; (d) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 380, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 387; (e) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 394, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 401; (f) the VH domain has at least 85%, 90%, or more amino acid sequence identical to that of SEQ ID NO: 422; %, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 429, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 429; (g) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 438, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 445; (h) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 452, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 459; (i) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 467, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 473; (j) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 481, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 488; (k) the VH domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 494, and the VL domain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 501; (l) the VH domain comprises the amino acid sequence of SEQ ID NO: 366 and the VL domain comprises the amino acid sequence of SEQ ID NO: 373; (m) the VH domain comprises the amino acid sequence of SEQ ID NO: 380 and the VL domain comprises the amino acid sequence of SEQ ID NO: 387; (n) the VH domain comprises the amino acid sequence of SEQ ID NO: 394 and the VL domain comprises the amino acid sequence of SEQ ID NO: 401; (o) the VH domain comprises the amino acid sequence of SEQ ID NO: 408 and the VL domain comprises the amino acid sequence of SEQ ID NO: 415; (p) the VH domain comprises the amino acid sequence of SEQ ID NO: 422 and the VL domain comprises the amino acid sequence of SEQ ID NO: 429; (q) the VH domain comprises the amino acid sequence of SEQ ID NO: 438 and the VL domain comprises the amino acid sequence of SEQ ID NO: 445; (r) the VH domain comprises the amino acid sequence of SEQ ID NO: 452 and the VL domain comprises the amino acid sequence of SEQ ID NO: 459; (s) the VH domain comprises the amino acid sequence of SEQ ID NO: 467 and the VL domain comprises the amino acid sequence of SEQ ID NO: 473; (t) the VH domain comprises the amino acid sequence of SEQ ID NO: 480 and the VL domain comprises the amino acid sequence of SEQ ID NO: 487; or (u) the VH domain comprises the amino acid sequence of SEQ ID NO: 494, and the VL domain comprises the amino acid sequence of SEQ ID NO: 501; An anti-ICOS antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 3.

5. The anti-ICOS antibody or antigen-binding fragment thereof comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, and heavy chain variable (VH) and light chain variable (VL) domains, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO:405; 5. An anti-ICOS antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 4, wherein HCDR2 comprises the amino acid sequence of SEQ ID NO:406, HCDR3 comprises the amino acid sequence of SEQ ID NO:407, LCDR1 comprises the amino acid sequence of SEQ ID NO:412, LCDR2 comprises the amino acid sequence of SEQ ID NO:413, and LCDR3 comprises the amino acid sequence of SEQ ID NO:414, and optionally the VH domain comprises the amino acid sequence of SEQ ID NO:408 and the VL domain comprises the amino acid sequence of SEQ ID NO:

415.

6. The anti-ICOS antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein: (a) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:410, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:417; (b) the heavy chain comprises the amino acid sequence of SEQ ID NO:410 and the light chain comprises the amino acid sequence of SEQ ID NO:417; (c) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 368, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 375; (d) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 385, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 389; (e) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 396, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 403; (f) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:424, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:432; (g) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:440, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:447; (h) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 454, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 461; (i) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:468, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO:475; (j) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 482, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 489; (k) the heavy chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 496, and the light chain comprises a sequence having at least 85%, 90% or 95% sequence identity to the amino acid sequence of SEQ ID NO: 503; (l) the heavy chain comprises the amino acid sequence of SEQ ID NO: 368 and the light chain comprises the amino acid sequence of SEQ ID NO: 375; (m) the heavy chain comprises the amino acid sequence of SEQ ID NO: 382 and the light chain comprises the amino acid sequence of SEQ ID NO: 389; (n) the heavy chain comprises the amino acid sequence of SEQ ID NO: 396 and the light chain comprises the amino acid sequence of SEQ ID NO: 403; (o) the heavy chain comprises the amino acid sequence of SEQ ID NO: 424 and the light chain comprises the amino acid sequence of SEQ ID NO: 432; (p) the heavy chain comprises the amino acid sequence of SEQ ID NO: 440 and the light chain comprises the amino acid sequence of SEQ ID NO: 447; (q) the heavy chain comprises the amino acid sequence of SEQ ID NO: 454 and the light chain comprises the amino acid sequence of SEQ ID NO: 461; (r) the heavy chain comprises the amino acid sequence of SEQ ID NO: 468 and the light chain comprises the amino acid sequence of SEQ ID NO: 475; (s) the heavy chain comprises the amino acid sequence of SEQ ID NO: 482 and the light chain comprises the amino acid sequence of SEQ ID NO: 489; or (t) the heavy chain comprises the amino acid sequence of SEQ ID NO: 496 and the light chain comprises the amino acid sequence of SEQ ID NO: 503; An anti-ICOS antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 5.

7. i) the anti-ICOS antibody is a human IgG1 antibody; and / or ii) the anti-ICOS antibody is KY1044; An anti-ICOS antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 6.

8. the anti-ICOS antibody or antigen-binding fragment thereof is administered to the subject at a dose of about 0.5 mg to about 10 mg; Optionally, the anti-ICOS antibody or antigen-binding fragment thereof comprises: i) from about 0.8 mg to about 8 mg; ii) about 8 mg (e.g., a dose of 7.5 mg or less, a dose of 7 mg or less); iii) about 0.8 mg to about 2.4 mg; iv) about 2.4 mg to about 8 mg; v) about 0.8 mg; vi) about 2.4 mg; or vii) Approximately 8 mg The anti-ICOS antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 7, wherein the anti-ICOS antibody or antigen-binding fragment thereof is administered to a subject at a dose of

9. the anti-ICOS antibody or antigen-binding fragment thereof is administered to the subject every 2 to 6 weeks, e.g., every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks; Optionally, the anti-ICOS antibody or antigen-binding fragment thereof is administered to the subject every three weeks, every six weeks, or every month. An anti-ICOS antibody or an antigen-binding fragment thereof for use according to any one of claims 1 to 8.

10. 10. The anti-ICOS antibody or antigen-binding fragment thereof for use according to any one of claims 1 to 9, wherein the anti-ICOS antibody or antigen-binding fragment thereof is administered to the subject for at least 6 months, such as 6 months, 12 months or more than 12 months.

11. further comprising administering a second therapeutic agent to the subject; Optionally, the second therapeutic agent comprises an anti-PD-L1 antibody or antigen-binding fragment thereof; Optionally, the anti-PD-L1 antibody is atezolizumab; Optionally, the anti-PD-L1 antibody or antigen-binding fragment thereof is administered to the subject at a dose of about 1200 mg. An anti-ICOS antibody or an antigen-binding fragment thereof for use according to any one of claims 1 to 10.

12. the anti-PD-L1 antibody or antigen-binding fragment thereof is administered to the subject every 2 to 6 weeks, e.g., every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks; Optionally, the anti-PD-L1 antibody or antigen-binding fragment thereof is administered to the subject every 3 weeks or every 6 weeks.

12. An anti-ICOS antibody or antigen-binding fragment thereof for use according to claim 11.

13. the anti-PD-L1 antibody or antigen-binding fragment thereof is administered to the subject monthly; 12. The anti-ICOS antibody or antigen-binding fragment thereof for use according to claim 11, optionally wherein the anti-PD-L1 antibody or antigen-binding fragment thereof is administered to the subject for at least 6 months, e.g., 6 months, 12 months or more than 12 months.

14. The anti-PD-L1 antibody or antigen-binding fragment thereof is a) co-administered to a subject with an anti-ICOS antibody or antigen-binding fragment thereof every three weeks; or b) administered to the subject in alternating doses with an anti-ICOS antibody or antigen-binding fragment thereof, e.g., an anti-PD-L1 antibody or antigen-binding fragment thereof is administered every 3 weeks and an anti-ICOS antibody or antigen-binding fragment thereof is administered every 6 weeks; 12. An anti-ICOS antibody or antigen-binding fragment thereof for use according to claim 11.

15. Diseases or conditions amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses include tumors, Optionally, diseases or conditions amenable to therapy by depleting regulatory T cells (Treg) and / or increasing effector T cell (Teff) responses include cancer, Optionally, the cancer comprises advanced and / or metastatic cancer; In some cases, the cancer comprises triple negative breast cancer, head and neck squamous cell carcinoma, penile cancer, pancreatic cancer, non-small cell lung cancer, hepatocellular carcinoma, esophageal cancer, gastric cancer, melanoma, renal cell carcinoma, and / or cervical cancer; An anti-ICOS antibody or an antigen-binding fragment thereof for use according to claims 1 to 14.

Images