Treatment of PD-L1 negative or low expressing cancers with anti-ICOS antibodies

Abstract

The present invention relates to compositions and methods for the treatment of cancer, particularly difficult to treat cancers. More specifically, the present invention relates to compositions and methods for the treatment of PD-L1 negative or PD-L1 low expressing cancers using modulators of ICOS, such as anti-ICOS antibodies, alone or in combination with other agents, such as anti-PD-L1 antibodies.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to compositions and methods for the treatment of cancer, particularly difficult to treat cancers. More specifically, the present invention relates to compositions and methods for the treatment of PD-L1 negative or PD-L1 low expressing cancers using anti-ICOS antibodies alone or in combination with other agents, such as anti-PD-L1 antibodies. [Background technology]

[0002] 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.

[0003] 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].

[0004] (Patent Document 1) described anti-ICOS antibodies and proposed their use to activate T cells and treat 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-CTL4 and anti-PD1 therapy alone. A similar lack of strong therapeutic benefit was shown in mice bearing transplanted EMT6 cells.

[0005] (Patent Document 2) 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.

[0006] Programmed death-1 (PD-1) is a 50-55 kDa type I transmembrane receptor that is a member of the CD28 family. PD-1 is involved in the regulation of T cell activation and is expressed in T cells, B cells, and myeloid cells. Two ligands for PD-1, PD ligand 1 (PD-L1) and ligand 2 (PD-L2), have been identified and have costimulatory functions.

[0007] Programmed cell death 1 ligand 1 (PD-L1), also known as cluster of differentiation (CD274) or B7 homolog 1 (B7-H1), is a member of the B7 family that modulates activation or inhibition of the PD-1 receptor. The open reading frame of PD-L1 encodes a putative type 1 transmembrane protein of 290 amino acids, which contains two extracellular Ig domains (an N-terminal V-like domain and an Ig C-like domain), a hydrophobic transmembrane domain, and a 30 amino acid cytoplasmic tail. The 30 amino acid intracellular (cytoplasmic) domain does not contain obvious signaling motifs, but does have a potential site for protein kinase C phosphorylation.

[0008] The complete amino acid sequence for PD-L1 is found in the NCBI Reference Sequence: NP_054862.1 (SEQ ID NO: 1), which references many journal articles, including, for example, (Non-Patent Document 4). The PD-L1 gene is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, chickens, and zebrafish. The mouse form of PD-L1 retains 69% amino acid identity with the human form of PD-L1 and also shares a conserved structure.

[0009] In humans, PD-L1 is expressed in many immune cell types, including activated and anergized / exhausted T cells, naive and activated B cells, as well as myeloid dendritic cells (DCs), monocytes and mast cells.It is also expressed in non-immune cells, including pancreatic islets, liver Kupffer cells, vascular endothelium, and selected epithelia, such as airway epithelium and renal tubular epithelium, where its expression is enhanced during inflammatory episodes.PD-L1 expression is also found at increased levels in many tumors, including, but not limited to, breast cancer (including but not limited to triple-negative breast cancer and inflammatory breast cancer), ovarian cancer, cervical cancer, colon cancer, colorectal cancer, lung cancer, including non-small cell lung cancer, kidney cancer, including renal cell carcinoma, gastric cancer, esophageal cancer, bladder cancer, hepatocellular carcinoma, squamous cell carcinoma of the head and neck (SCCHN) and pancreatic cancer, melanoma and uveal melanoma.

[0010] PD-1 / PD-L1 signaling is thought to play an important nonredundant function within the immune system by negatively regulating T cell responses. This regulation is involved in T cell development in the thymus, regulation of chronic inflammatory responses, and maintenance of both peripheral tolerance and immune privilege. Upregulation of PD-L1 appears to allow cancer to evade the host immune system, and in many cancers, expression of PD-L1 is associated with reduced survival and unfavorable prognosis. Therapeutic monoclonal antibodies capable of blocking the PD-1 / PD-L1 pathway can enhance antitumor immune responses in patients with cancer. Published clinical data suggest a correlation between clinical response and tumor membrane expression of PD-L1 ((Non-Patent Document 16), (Non-Patent Document 17)), and a strong correlation between lack of clinical response and lack of membrane-localized PD-L1 protein ((Non-Patent Document 16), (Non-Patent Document 17)). Therefore, PD-L1 expression on tumors or tumor-infiltrating leukocytes (18) is a candidate molecular marker for use in patient selection for immunotherapy, e.g., immunotherapy using anti-PD-L1 antibodies. Enrichment of patients based on surface expression of PD-L1 can significantly enhance the clinical success of treatment with drugs that target the PD-1 / PD-L1 pathway. Tumor-infiltrating CD8+ There is also evidence of an ongoing immune response, such as T cells, or the presence of traces of cytokine activation, such as IFNγ.

[0011] Further evidence of PD-L1 expression and correlation with disease will emerge from the numerous ongoing clinical trials. Atezolizumab is the furthest along, with recent data from a Phase II trial demonstrating the role of PD-L1 in metastatic urothelial carcinoma and NSCLC, specifically in the tumor microenvironment. + It has shown therapeutic efficacy in patients with immune cells (see Non-Patent Document 19; Non-Patent Document 20). Recent results from a Phase III study of 1225 patients with NSCLC showed improved survival in patients receiving atezolizumab compared to chemotherapy, regardless of tumor expression of PD-L1 (Non-Patent Document 21).

[0012] Patent document 3 describes exemplary anti-ICOS antibodies. Patent document 4 describes exemplary anti-PD-L1 antibodies.

[0013] PD-L1 expression is often used as a predictive marker for whether a tumor will respond to treatments such as PD-L1 antibodies. PD-L1 acts as a "brake on the immune system" in a negative feedback loop to modulate the immune response. Its presence in tumors, albeit as an inhibitory signal, is therefore an indication of an anti-tumor immune response. PD-L1 negative tumors are immunologically "cold" and their PD-L1 negative status indicates that the cells are not exposed to inflammation. In general, higher PD-L1 expression is associated with higher inflammation, and these PD-L1 high tumors are more likely to respond to immunotherapy because there are existing immune cells that can "see" and attack the tumor. Current anti-PD-L1 antibodies approved for treatment are only approved for PD-L1 expressing tumors. It was previously thought that PD-L1 low expressing tumors were less likely to respond to immunotherapy such as anti-ICOS antibodies, anti-PD-L1 antibodies, or a combination of anti-ICOS and anti-PD-L1 antibodies.

Prior technical literature

[0014]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Non-licensed literature

[0015]

Non-licensed literature 1

Non-licensed Document 2

Non-licensed Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

[0016] Summary of the Invention The present inventors have discovered that immunotherapy can successfully treat PD-L1 negative or low PD-L1 expressing tumors. More specifically, the present inventors have discovered that PD-L1 negative or low PD-L1 expressing tumors can be successfully treated with an inhibitor of ICOS (e.g., an anti-ICOS antibody or an antigen-binding fragment thereof) or a combination of an ICOS inhibitor (e.g., an anti-ICOS antibody or an antigen-binding fragment thereof) and a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody or an antigen-binding fragment thereof or an anti-PD-1 antibody or an antigen-binding fragment thereof). These treatments are surprising because it was not previously thought possible to treat PD-L1 negative or low PD-L1 expressing cancers with immunotherapy, in particular immunotherapy involving the administration of a PD-L1 inhibitor, e.g., an anti-PD-L1 antibody, and / or involving the administration of an ICOS inhibitor, e.g., an anti-ICOS antibody. The present invention provides a surprising new mechanism for the treatment of cancer, including difficult to treat cancers such as those with low levels of PD-L1 expression on tumor cells and tumor-infiltrating lymphocytes, or PD-L1 negative cancers.

[0017] 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.

[0018] 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).

[0019] 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.

[0020] The present invention uses an antibody that binds to human ICOS. The antibody targets 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.

[0021] Exemplary anti-ICOS antibodies include STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, the sequences of which are set forth herein.

[0022] The present invention provides a method of treating cancer in a patient, wherein the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression, comprising administering to the patient a modulator of ICOS.

[0023] The invention also provides a method of treating cancer in a patient who has previously received treatment for cancer, where the previous treatment for cancer was administration of a PD-L1 inhibitor and the patient has not responded to the previous treatment or has stopped responding to the previous treatment, comprising administering to the patient a modulator of ICOS.

[0024] The present invention also provides an ICOS modulator for use in the treatment of cancer in a patient, wherein the patient has a PD-L1 negative tumour or a tumour with low PD-L1 expression.

[0025] The present invention also provides an ICOS modulator for use in the treatment of cancer in a patient, wherein the patient has previously received treatment for cancer, and wherein the patient has not responded to the previous treatment, or has stopped responding to the previous treatment, and wherein the previous treatment for cancer was a PD-L1 inhibitor.

[0026] The invention also provides the use of an ICOS modulator in the manufacture of a medicament for the treatment of cancer in a patient, where the patient has a PD-L1 negative tumour or a tumour with low PD-L1 expression.

[0027] The invention also provides the use of an ICOS modulator in the manufacture of a medicament for the treatment of cancer in a patient, wherein the patient has previously received treatment for the cancer, and wherein the patient has not responded to the previous treatment, or has stopped responding to the previous treatment, and wherein the previous treatment for the cancer was a PD-L1 inhibitor.

[0028] Generally, the modulator of ICOS is an ICOS agonist. The modulator of ICOS is an anti-ICOS antibody. In a preferred embodiment, the modulator of ICOS is an agonist anti-ICOS antibody.

[0029] In some embodiments, the method or use may include combination therapy with a PD-L1 inhibitor, for example an anti-PD-L1 antibody or / and an anti-PD-1 antibody that prevents the binding of PD-L1 to PD-1.

[0030] The anti-ICOS antibodies used in the present invention are those that compete for binding to human ICOS with an antibody (e.g., a human IgG1 or scFv) comprising the heavy and light chain complementarity determining regions (CDRs) of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, and optionally with an antibody comprising the VH and VL domains of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.

[0031] Anti-ICOS antibodies according to the invention may comprise one or more CDRs of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 (e.g. all six CDRs, or a set of HCDRs and / or LCDRs, of any such antibodies), or a variant thereof as described herein.

[0032] The anti-ICOS antibody may comprise an antibody VH domain comprising CDRs HCDR1, HCDR2 and HCDR3, and an antibody VL domain comprising CDRs LCDR1, LCDR2 and LCDR3, where HCDR3 is the HCDR3 of an antibody selected from STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or comprises an HCDR3 with 1, 2, 3, 4 or 5 amino acid changes thereof. HCDR2 may comprise the HCDR2 of the selected antibody, or comprises an HCDR2 with 1, 2, 3, 4 or 5 amino acid changes thereof. HCDR1 may comprise the HCDR1 of the selected antibody, or comprises an HCDR1 with 1, 2, 3, 4 or 5 amino acid changes thereof.

[0033] The anti-ICOS antibody may comprise a VL domain of an antibody comprising CDRs HCDR1, HCDR2 and HCDR3, and a VL domain of an antibody comprising CDRs LCDR1, LCDR2 and LCDR3, where LCDR3 is the LCDR3 of an antibody selected from STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, or comprises an LCDR3 having one, two, three, four or five amino acid changes thereof. LCDR2 may comprise the LCDR2 of the selected antibody, or comprises an LCDR2 having one, two, three, four or five amino acid changes thereof. LCDR1 may comprise the LCDR1 of the selected antibody, or comprises an LCDR1 having one, two, three, four or five amino acid changes thereof.

[0034] Anti-ICOS antibodies are a VH domain of an antibody comprising the complementarity determining regions HCDR1, HCDR2 and HCDR3, and The VL domain of an antibody comprising the complementarity determining regions LCDR1, LCDR2 and LCDR3 where: the heavy chain complementarity determining region is that of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises the heavy chain complementarity determining region of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 with 1, 2, 3, 4 or 5 amino acid changes; and / or The light chain complementarity determining region is that of antibody STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or comprises the light chain complementarity determining region of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 with 1, 2, 3, 4 or 5 amino acid changes.

[0035] The anti-ICOS antibody comprises a set of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein: HCDR1 is HCDR1 of STIM003, HCDR2 is HCDR2 of STIM003, HCDR3 is the HCDR3 of STIM003, or or a VH domain comprising a set of HCDRs with 1, 2, 3, 4, 5 or 6 amino acid changes.

[0036] The anti-ICOS antibody comprises a set of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein: LCDR1 is the LCDR1 of STIM003, LCDR2 is the LCDR2 of STIM003, LCDR3 is the LCDR3 of STIM003, or or a VL domain comprising a set of LCDRs with 1, 2, 3 or 4 amino acid changes.

[0037] The amino acid changes (e.g., substitutions) are at any residue position in the CDR. Examples of amino acid changes are shown in Figures 10, 11 and 12, which show alignments of variant sequences of anti-ICOS antibodies. Thus, the amino acid changes in the CDRs of STIM003 are substitutions of the residues present at the corresponding positions of antibody CL-74570 or antibody CL-71642 shown in Figure 11.

[0038] Examples of amino acid alterations in the CDRs of STIM003 are substitutions at the following residue positions as defined according to IMGT: In HCDR1, a substitution at position 28 of IMGT, optionally a conservative substitution, such as V28F. In HCDR2, a substitution at position 59, 63 and / or 64 of IMGT. Optionally, the substitution at position 59 is N59I, the substitution at position 63 is G63D, and / or the substitution at position 64 is D64N and / or D64S. In HCDR3, a substitution at position 106, 108, 109 and / or 112 of IMGT. Optionally, the substitution at position 106 is R106A, the substitution at position 108 is F108Y, the substitution at position 109 is Y109F, and / or the substitution at position 112 is H112N. In LCDR1, a substitution at position 36, for example, R36S. In LCDR3, substitutions at positions 105, 108 and / or 109. Optionally, the substitution at position 105 is H105Q, the substitution at position 108 is D108G, and / or the substitution at position 109 is M109N or M109S.

[0039] The anti-ICOS antibody used in the present invention may comprise a framework region of the VH and / or VL domain that corresponds to a human germline gene segment sequence. For example, it may comprise one or more of the framework regions of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. The one or more framework regions are FR1, FR2, FR3 and / or FR4.

[0040] As described in Example 2, Table E12-1 shows the human germline V, D and J gene segments that recombine to generate the VH domains of these antibodies, and Table E12-2 shows the human germline V and J gene segments that recombine to generate the VL domains of these antibodies. The VH and VL domains of the antibodies used in the present invention can be based on these V(D)J segments.

[0041] The antibody used in the present invention is (i) derived from the recombination of a human heavy chain V gene segment, a human heavy chain D gene segment, and a human heavy chain J gene segment, The V segment is IGHV1-18 (e.g., V1-18 * 01), IGVH3-20 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) or IGVH2-5 (e.g., V2-5 * 10) and; The D gene segment is IGHD6-19 (e.g., IGHD6-19 * 01), IGHD3-10 (for example, IGHD3-10 * 01) or IGHD3-9 (e.g., IGHD3-9 * 01) and / or The J gene segment is IGHJ6 (e.g., IGHJ6 * 02), IGHJ4 (e.g., IGHJ4 *02) or IGHJ3 (e.g., IGHJ3 * 02) or (ii) framework regions FR1, FR2, FR3 and FR4, wherein FR1 is a human germline V gene segment IGHV1-18 (e.g., V1-18), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 01), IGVH3-20 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) or IGVH2-5 (e.g., V2-5 * 10) and aligned. FR2 is selected from the group consisting of human germline V gene segment IGHV1-18 (e.g., V1-18), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 01), IGVH3-20 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) or IGVH2-5 (e.g., V2-5 * 10) and aligned. FR3 is selected from the group consisting of human germline V gene segment IGHV1-18 (e.g., V1-18), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 01), IGVH3-20 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) or IGVH2-5 (e.g., V2-5 * 10) aligned with, and / or FR4 is selected from the group consisting of human germline J gene segment IGJH6 (e.g., JH6), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 02), IGJH4 (e.g., JH4 * 02) or IGJH3 (e.g., JH3 * 02) and align with It may comprise the VH domain of an antibody.

[0042] The FR1, FR2 and FR3 of a VH domain typically align with the same germline V gene segment. Thus, for example, an antibody can be constructed using the human heavy chain V gene segment IGHV3-20 (e.g., VH3-20 * d01), a human heavy chain D gene segment and a human heavy chain J gene segment IGJH4 (e.g., JH4 * The antibody can include a VH domain derived from recombination of human germline V gene segment IGHV3-20 (e.g., IGVH3-20) with up to 1, 2, 3, 4, or 5 amino acid changes. The antibody can include framework regions FR1, FR2, FR3, and FR4 of the VH domain, where FR1, FR2, and FR3 are derived from human germline V gene segment IGHV3-20 (e.g., IGVH3-20) with up to 1, 2, 3, 4, or 5 amino acid changes. * d01), and FR4 is aligned with a human germline J gene segment IGHJ4 (e.g., IGHJ4 * 02). The alignment is exact, but in some cases, one or more residues may be mutated from the germline, resulting in amino acid substitutions, or in rarer cases, deletions or insertions.

[0043] The antibody used in the present invention is (i) derived from the recombination of a human light chain V gene segment and a human light chain J gene segment, The V segment is IGKV2-28 (e.g., IGKV2-28 * 01), IGKV3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and / or The J gene segment is IGKJ4 (e.g., IGKJ4 * 01), IGKJ2 (e.g., IGKJ2 * 04), IGLJ3 (e.g., IGKJ3 * 01) or IGKJ1 (e.g., IGKJ1 *01) or (ii) framework regions FR1, FR2, FR3 and FR4, wherein FR1 is selected from the group consisting of human germline V gene segment IGKV2-28 (e.g., IGKV2-28), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 01), IGKV3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned. FR2 is selected from the group consisting of human germline V gene segment IGKV2-28 (e.g., IGKV2-28), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 01), IGKV3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned. FR3 is selected from the group consisting of human germline V gene segment IGKV2-28 (e.g., IGKV2-28), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 01), IGKV3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and / or FR4 is a human germline J gene segment IGKJ4 (e.g., IGKJ4), optionally with 1, 2, 3, 4 or 5 amino acid changes. * 01), IGKJ2 (e.g., IGKJ2 * 04), IGKJ3 (e.g., IGKJ3 * 01) or IGKJ1 (e.g., IGKJ1 * 01) and align with It may comprise the VL domain of an antibody.

[0044] The FR1, FR2 and FR3 of a VL domain typically align with the same germline V gene segment. Thus, for example, an antibody can be made using the human light chain V gene segment IGKV3-20 (e.g., IGKV3-20 * 01) and human light chain J gene segment IGKJ3 (e.g., IGKJ3 * The antibody can include a VL domain derived from recombination of human germline V gene segment IGHV3-20 (e.g., IGKV3-20), with up to 1, 2, 3, 4, or 5 amino acid changes. The antibody can include framework regions FR1, FR2, FR3, and FR4 of the VL domain, where FR1, FR2, and FR3 are derived from human germline V gene segment IGHV3-20 (e.g., IGKV3-20), with up to 1, 2, 3, 4, or 5 amino acid changes. * 01), and FR4 is aligned with human germline J gene segment IGKJ3 (e.g., IGKJ3 * 01). The alignment is exact, but in some cases, one or more residues may be mutated from the germline, resulting in amino acid substitutions, or in rarer cases, deletions or insertions.

[0045] The antibody used in the present invention may comprise a VH domain of an antibody which is the VH domain of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 or has an amino acid sequence at least 90% identical to the VH domain sequence of the STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 antibody. The amino acid sequence identity is at least 95%.

[0046] The antibody may comprise a VL domain of an antibody which is the VL domain of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 or has an amino acid sequence at least 90% identical to the VL domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 antibody. The amino acid sequence identity is at least 95%.

[0047] The VH domain of an antibody having the HCDRs of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or a variant of these CDRs, can be paired with the VL domain of an antibody having the LCDRs of the same antibody, or a variant of these CDRs. Similarly, the VH domain of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or a variant of that VH domain, can be paired with the VL domain of the same antibody, or a variant of the VL domain of the same antibody.

[0048] For example, the antibody can comprise the VH domain of antibody STIM001 and the VL domain of STIM001. In another example, the antibody can comprise the VH domain of antibody STIM002 and the VL domain of STIM002. In another example, the antibody can comprise the VH domain of antibody STIM003 and the VL domain of STIM003.

[0049] The antibody can include a constant region, optionally a human heavy and / or light chain constant region. An exemplary isotype is IgG, e.g., human IgG1.

[0050] 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]

[0051] [Figure 1] Dual action mechanism of agonistic anti-ICOS antibody KY1044 (also known as STIM003). (A) Untreated tumor before KY1044 treatment. (i) Suppression of effector T cells, (ii) Regulatory T cell-mediated immune evasion. (B) Tumor and ICOS agonism. (iii) Remaining ICOSHI regulatory T cells. (iv) Stimulated ICOSLOW effector T cells expressing IFNγ. (C) Tumor and ICOSH I Treg depletion. (v) Killed ICOSHI regulatory T cells. This figure shows the possibility for dual action mechanism (agonism and depletion). One aspect of dual action mechanism is ICOS Teff agonism, which increases activation (cytokine production) of Teff cells, as shown in (B). Another aspect of dual action mechanism is ICOS Treg depletion, which releases inhibition of Teff cells, as shown in (C). [Diagram 2] Overview of study design for clinical trials. Preferred indications: all prospective patient populations with e.g., NSCLC, HNSCC, HCC, melanoma, cervical cancer, gastric / esophageal cancer, renal cancer, pancreatic cancer, and TNBC. Dosing: Q3W IV (**=n=21 enrolled, n=20 treated). (i) KY1044 and enriched pool. (ii) KY1044 + atezolizumab and enriched pool. Phase 1 dose escalation (completed) - dose escalation of KY1044 single agent, and - dose escalation of KY1044 in combination with atezolizumab. Phase 1 enriched cohort (ongoing). Phase 2 expansion (ongoing) - selected indications where antitumor activity was observed in Phase 1. [Diagram 3] Staining of cells for PD-L1 expression. [Figure 4]CD8 low vs. high threshold based on median. (A) PD-L1+ immune infiltrate in the TME and CD8+ in the TME. (B) PD-L1+ on tumor cells in the TME and CD8+ in the TME. Each panel (A) and (B) is divided into 4 quadrants: Q1=CD8 low / PD-L1 high; Q2=hot tumor and PD-L1 high; Q3=cold tumor; Q4=CD8 high / PD-L1 low. PR=partial response, CR=complete response, SD=stable disease, PD=progressive disease. [Figure 5-1] Effect of anti-ICOS treatment using KY1044 in patient A. (A) Information table for patient A. (B) TME analysis (determined by IHC) at screen C2D8 (cycle 2, day 8), (i) minimal effect on CD8+ T cells. (ii) depletion of ICOS+ Tregs. (iii) 73.6-fold improvement in CD8+ / ICOS+ Treg ratio. (C) PD-L1 expression (determined by IHC) in the TME at screen C2D8, (iv) 0% PD-L1+ tumor cells. (v) low PD-L1+ immune infiltrate in the TME. (D) Baseline PBMC analysis (determined by chip cytometry), (vi) low CD4 cells among T cells; anti-CD8 cell %. (vii) average T cells; above average monocytes. (viii) above average % ICOS+ cells. (E) Longitudinal PBCM and ICOS RO analysis (determined by chip cytometry) (pre-dose, cycle 1 day 8, cycle 2 day 1 (pre-dose) and cycle 2 day 8). (ix) No depletion of peripheral CD4 memory cells. (x) No free ICOS in peripheral CD4 MEM. [Figure 5-2] Continued from Figure 5-1. [Figure 6] IHC analysis for patient A at screening and C2D8 after treatment with KY1044. (A) Depletion of ICOS+ Tregs from 75.97 to 0.7 (cells / mm2) at screening and C2D8. (B) Presence of CD8+ cells at 227.27 and 154.11 (cells / mm2) at screening and C2D8. (C) 0% PD-L1+ tumors at both time points, 1% and 0% PD-L1+ infiltrates at screening and C2D8. [Figure 7-1]Effect of anti-ICOS treatment with KY1044 in patient B. (A) Information table for patient B. (B) TME analysis (determined by IHC). (i) Mean density of CD8+ T cells at screening. (ii) Very low density of ICOS+ Tregs at screening. (iii) Very high ratio of CD8+ / ICOS+ Tregs at screening. (C) PD-L1 expression in the TME (determined by IHC). (iv) 0% PD-L1+ tumor cells at screening. (v) Low PD-L1+ immune infiltration in the TME. (D) Longitudinal PBMC and ICOS RO analysis in patient B (determined by chip cytometry). (vi) No depletion of peripheral CD4 memory cells. (vii) No free ICOS in peripheral CD4 MEM. This patient achieved stable disease after treatment with KY1044. [Figure 7-2] Continued from Figure 7-1. [Figure 8] IHC analysis for patient B at screening. (A) Very low ICOS+ Treg density at screening 0.07 (cells / mm2). (B) High CD8+ cell density at screening 98.65 (cells / mm2). (C) 0% PD-L1+ tumors at screening, 0% PD-L1+ infiltrates at screening. [Figure 9-1] Patient Case Study - Patient C. Results for Patient C after treatment with KY1044 showing reduction in target lesion size at C3D8 and C10D1. Patient C Information: Age / Gender / Diagnosis = 59 y / o / Male / HPV positive metastatic squamous cell carcinoma of the head / neck. PD-L1 Status at Screening (SP263): (%TC / %IC) = 3 / 2. Allocation = KY1044 8.0 mg + Atezolizumab 1, 200 mg Q3W. (A) Number of prior therapies vs treatments in this study. 5-FU = fluorouracil, PD = disease progression, PR = partial response, ** = patient maintained a PR response at data cutoff for this figure (16 Dec 2020). (B) Change in target lesions from baseline. (C) Baseline (June 2020). (D) Cycle Day 3 / 8 (August 2020). [Figure 9-2] Continued from Figure 9-1. [Figure 10]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 11] 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 12] 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Detailed Description ICOS The anti-ICOS antibody used in the present 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.

[0053] PD-L1 Many tumor cells express cancer-specific surface molecules that can serve as diagnostic and / or therapeutic antibody targets. Examples of cell surface proteins expressed by tumor molecules that are useful as biomarkers include, for example, members of the B7 family of proteins, major histocompatibility complex molecules (MHC), cytokines, and growth factor receptors, such as receptors for epidermal growth factor (EGFR). The B7 family is a group of proteins that are members of the immunoglobulin (Ig) superfamily of cell surface proteins that bind to receptors on lymphocytes and regulate immune responses. The family includes transmembrane or glycosylphosphatidylinositol (GPI)-linked proteins characterized by extracellular Ig-like domains (IgV and IgC domains related to the variable and constant domains of immunoglobulins). All members have a short cytoplasmic domain. There are seven known members of the B7 family: B7-1, B7-2, PD-L1 (B7-H1), PD-L2, B7-H2, B7-H3, and B7-H4.

[0054] The complete amino acid sequence for PD-L1 can be found in NCBI Reference Sequence: NP 054862.1, which references many journal articles, including, for example, Dong, H. et al. (1999), "PD-L1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion," Nat. Med. 5(12), pp. 1365-1369, the disclosures of which are incorporated herein by reference in their entirety. The amino acid sequence of PD-L1 contains a 30 amino acid long cytoplasmic domain that is unique to PD-L1 and shows little homology to other molecules, including other B7 family members.

[0055] PD-1 The complete amino acid sequence for PD-1 can be found in UniProt accession number Q9UMF3.

[0056] ICOS Modulators The ICOS modulator used in the present invention can be any suitable ICOS modulator.Generally, the ICOS modulator is an ICOS agonist.In some embodiments, the ICOS modulator is an anti-ICOS antibody.In a preferred embodiment, the ICOS modulator is an agonist anti-ICOS antibody.

[0057] ICOS modulators (eg, agonistic anti-ICOS antibodies) are capable of depleting ICOS+ T cells, in particular ICOS+ Tregs.

[0058] In some embodiments, ICOS modulators are multispecific (such as bispecific), i.e., they specifically bind to multiple (e.g., two) different antigens. In some embodiments, ICOS modulators are multispecific antibodies (e.g., bispecific antibodies) that specifically bind to ICOS and PD-L1 or PD-1. In some embodiments, ICOS modulators are multispecific antibodies (e.g., bispecific antibodies) that specifically bind to ICOS and are ICOS agonists, and that specifically bind to PD-L1 or PD-1 and are PD-L1 or PD-1 antagonists.

[0059] PD-L1 inhibitors The PD-L1 inhibitors used in the present invention generally inhibit the binding of PD-L1 to PD-1 (or the binding of PD-1 to PD-L1). The PD-L1 inhibitors are anti-PD-L1 or anti-PD-1 binding molecules. In some embodiments, the PD-L1 or PD-1 inhibitors are anti-PD-L1 or anti-PD-1 antibodies, respectively. Generally, the PD-L1 inhibitors are antagonists of PD-L1, such as antagonist anti-PD-L1 or anti-PD-1 antibodies.

[0060] Combination of ICOS modulators and PD-L1 inhibitors In some embodiments, the present invention uses a combination of an ICOS modulator and a PD-L1 inhibitor. The ICOS modulator and the PD-L1 inhibitor are for simultaneous, separate or sequential administration. In some embodiments, the ICOS modulator is an anti-ICOS antibody (e.g., an agonist anti-ICOS antibody), and the PD-L1 inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody. In some embodiments, the ICOS modulator is an IgG1 anti-ICOS antibody, and the PD-L1 inhibitor is an IgG1 anti-PD-L1 antibody or an IgG1 anti-PD-1 antibody.

[0061] Cross-reactivity The antibodies used in the present invention are preferably cross-reactive, for example binding to the extracellular domain of mouse ICOS and human ICOS. The antibodies 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 has no 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 antibodies. 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. The anti-PD-L1 and / or anti-PD-1 antibodies used in the present invention can also exhibit cross-reactivity.

[0062] The STIM antibodies described herein were generated using Kymouse™ technology in which mice have been 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.

[0063] 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 D The 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.

[0064] 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 WO2018 / 029474). 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.

[0065] specificity The antibody used 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.

[0066] 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.

[0067] 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.

[0068] Some antibodies used in the present invention specifically bind to PD-L1 or PD-1, i.e., the antibody binds to its epitope on the target protein PD-L1 or PD-1 (human PD-L1 or PD-1, preferably mouse and / or cynomolgus monkey PD-L1 or PD-1), but does not exhibit significant binding to molecules where that epitope is not present.

[0069] affinity The affinity of the antibody binding to ICOS (or to another antigen, such as PD-L1 or PD-1) can be determined. The affinity of the antibody for its antigen is determined by the equilibrium dissociation constant, K D The Kd, Ka and Kd for antibody-antigen binding can be measured using surface plasmon resonance (SPR).

[0070] The antibody used in the present invention has a K of 10 mM or less, preferably 5 mM or less, and 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.

[0071] 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).

[0072] 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.

[0073] Various SPR devices are known, such as Biacore™, ProteOn XPR36™ (Bio-Rad®) and KinExA® (Sapidyne Instruments, Inc.). A working example of SPR can be found in Example 7 of WO2018 / 029474.

[0074] As described, affinity can be determined by SPR using an antibody in Fab format, with the antigen coupled to the chip surface and the test antibody passing 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, for example, pH 5.5 or pH 7.6, and at any desired temperature, for example, 25°C or 37°C. As reported in Example 7 of WO2018 / 029474, the antibody according to the present invention bound to human ICOS with an apparent affinity of less than 2nM, as determined by SPR using an antibody in monovalent (Fab) format.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] In vitro T cell assays include the bead binding assay of Example 13 of WO2018 / 029474, the plate binding assay of Example 14 of WO2018 / 029474, and the soluble form assay of Example 15 of WO2018 / 029474.

[0081] 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.

[0082] 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.

[0083] 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 as described in Example 13 of WO2018 / 029474 or generally 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 with reference to relevant standards within the dynamic range of the assay. DELFIA is exemplified in Example 13 of WO2018 / 029474, but ELISA or other methods can be used.

[0084] 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. Two T cell activation assays using primary cells are described herein - see T cell activation assay 1 and T cell activation assay 2 in Example 2 of WO2018 / 029474. 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 in such assays to a higher degree than ICOS-L or C398.4. 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 surrogate assay readouts.

[0085] 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.

[0086] PD-L1 or PD-1 receptor antagonism PD-L1 or PD-1 inhibitors can act as PD-L1 or PD-1 antagonists, i.e., they block the binding of PD-L1 to PD-1 (or PD-1 to PD-L1).

[0087] T cell dependent death 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.

[0088] 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.

[0089] ICOS Ligand-Receptor Neutralization Potency The antibodies used in the present invention inhibit the binding of ICOS to its ligand, ICOSL.

[0090] The degree to which an antibody inhibits the binding of ICOS receptor to its ligand is referred to as its ligand-receptor neutralization 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 an IC50 value. Neutralization potency can be determined in a HTRF assay. A detailed working example of the HTRF assay for ligand-receptor neutralization potency is shown in Example 8 of WO2018 / 029474.

[0091] 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.

[0092] 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.

[0093] antibody As described in more detail in the Examples of WO2018 / 029474, 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.

[0094] 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. 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: 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).

[0095] 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. 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: 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).

[0096] 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).

[0097] STIM003 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).

[0098] 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. H The 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).

[0099] 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. HThe 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. 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: 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).

[0100] 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. 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 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).

[0101] 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. 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: 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).

[0102] 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).

[0103] 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).

[0104] An antibody according to the invention is an immunoglobulin or molecule comprising an immunoglobulin domain, whether natural or partially or wholly synthetically produced. An antibody may be an IgG, IgM, IgA, IgD or IgE molecule, or an antigen-specific antibody fragment thereof (including but not limited to Fab, F(ab')), whether derived from any species which naturally produces antibodies or whether produced by recombinant DNA technology; whether isolated from serum, B cells, hybridomas, transfectomas, yeast or bacteria. 2, Fv, disulfide-linked Fv, scFv, single domain antibodies, multispecific antibodies in closed conformation, disulfide-linked scfv, diabodies). Antibodies can be humanized using routine techniques. The term antibody encompasses any polypeptide or protein that contains an antibody antigen-binding site. The antigen-binding site (paratope) is the part of an antibody that binds to its target antigen (ICOS) and is complementary to that epitope.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] An antibody may be a whole immunoglobulin, including the constant region, or an antibody fragment. An antibody fragment is a portion of an intact antibody, including, for example, the antigen-binding region and / or the variable region 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.

[0109] 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.

[0110] 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.

[0111] 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).

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] Alignments of the VH and VL domain sequences of the STIM antibody to related antibodies and to human germline sequences are shown in FIG. 10, FIG. 11 and FIG.

[0120] 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.

[0121] 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.

[0122] Examples of human heavy chain constant regions are shown in Table S1.

[0123] 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.

[0124] 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.

[0125] "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.

[0126] 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 antibody's biological half-life: T252L, T254S, or T256F. Biological half-life can be increased by the heavy chain constant region CH, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022, the modifications described therein being incorporated herein by reference. 1 By modifying the domain or CL region to the CH of the Fc region of IgG 2 It is also increased by containing a salvage receptor binding epitope taken from two loops of the Fc-hinge domain. In another embodiment, the Fc hinge region of the antibody or antigen-binding fragment of the invention is mutated to reduce the biological half-life of the antibody or fragment. Mutation of one or more amino acids is performed in the CH of the Fc-hinge fragment. 2 -CH 3The antibody or fragment is introduced into the domain interface region, such that the antibody or fragment has impaired Staphylococcus protein A (SpA) binding compared to the native Fc-hinge domain's SpA binding. 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., genetic fusion to an ELP repeat sequence (PhaseBio)). These various half-life extending fusions are described in more detail in Strohl, BioDrugs (2015) 29:215-239, which fusions, e.g., those in Tables 2 and 6, are incorporated herein by reference.

[0127] 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.

[0128] The above details are applicable to any ICOS modulator or PD-L1 inhibitor that is an antibody.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] The constant region can be engineered for enhanced ADCC and / or CDC and / or ADCP.

[0135] 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.

[0136] 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).

[0137] 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 VK 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.

[0138] The ADCC activity of the antibody can be determined in the assays described herein. The ADCC activity of the anti-ICOS antibody can be determined in vitro using an ICOS positive T cell line as described in Example 10 of WO2018 / 029474. The ADCC activity of the anti-PD-L1 antibody can be determined in vitro in an ADCC assay using PD-L1 expressing cells.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] Creating and modifying 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] .

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] Further aspects of the invention are the use of such non-human mammals for producing antibodies against ICOS, as well as methods for producing antibodies, or heavy and / or light chain variable domains of antibodies, in such mammals.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] There are many reasons why it is desirable to create variants, including optimizing antibody sequences for large-scale manufacturing, facilitating purification, enhancing stability, or improving suitability for inclusion in desired pharmaceutical formulations. 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 (potentially creating a variant containing all naturally occurring amino acids at this position, with 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 lead candidates are selected and optimized for manufacturing and clinical development, it is generally desirable to change their 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.

[0158] 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.

[0159] 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.

[0160] 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).

[0161] 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.

[0162] 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.

[0163] 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).

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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:

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] The invention also provides methods that include using the nucleic acids described herein in an expression system to express an antibody.

[0179] therapeutic use The antibodies described herein can be used in methods of treatment of the human or animal body with therapy, particularly in the treatment of cancer in patients, where the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression. The antibodies can find use in increasing effector T cell responses, which is beneficial for a range of diseases or conditions, including treating cancer or solid tumors, and in vaccination situations. Increased Teff responses can be achieved using antibodies that modulate the balance or ratio between Teff and Treg to favor Teff activity.

[0180] 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.

[0181] Generally speaking, the present invention relates to the treatment of cancers that are PD-L1 negative or exhibit low PD-L1 expression. In particular, the present invention relates to the treatment of cancer in patients with tumors that are PD-L1 negative or exhibit low PD-L1 expression. The method includes administering to the patient a modulator of ICOS. The tumor cells and / or tumor-associated immune cells are PD-L1 negative or exhibit low PD-L1 expression.

[0182] In some embodiments, the patient has or has a tumor sample that is tested for PD-L1 expression. This testing can be done in a screen, i.e. the patient is screened for PD-L1 expression prior to treatment with an ICOS modulator. In some embodiments, the present invention relates to a method of selecting a patient for treatment for cancer, where the patient is selected for treatment with an anti-ICOS antibody, and optionally an anti-PD-L1 antibody, regardless of the PD-L1 expression status in a tumor sample from the patient, and optionally the tumor sample from the patient is determined to be PD-L1 negative or low expressing, or the PD-L1 expression status of the tumor sample from the patient is not determined prior to selecting the patient for treatment. Given that the present invention further provides a method of treating tumors that do not express PD-L1 or that low express PD-L1, screening for PD-L1 expression may not be necessary. The method may optionally further include administering to the patient an ICOS modulator (e.g., an anti-ICOS antibody, such as an agonistic anti-ICOS antibody) and optionally a PD-L1 inhibitor (such as an anti-PD-L1 or anti-PD-1 antibody).

[0183] In some embodiments, the cancer is associated with infectious agents. The cancer is a virus-induced cancer. In some embodiments, the virus associated with the virus-induced cancer is selected from HBV, HCV, HPV (such as cervical cancer, oropharyngeal cancer), and EBV (such as Burkitt's lymphoma, gastric cancer, Hodgkin's lymphoma, other EBV-positive B-cell lymphoma, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease). In some embodiments, the cancer is selected from the group consisting of head and neck squamous cell carcinoma, cervical cancer, anogenital cancer, and oropharyngeal cancer.

[0184] In some embodiments, the patient has or has a tumor sample that is tested for HPV. In some embodiments, the tumor is HPV positive. In some embodiments, the tumor is HPV negative. This test can be done at screening, i.e. the patient is screened for HPV prior to treatment with ICOS modulators, or the HPV status of the tumor can be determined from the patient's medical history data. In some embodiments, the method further comprises determining the HPV status of the tumor. An "HPV positive" tumor is considered to be associated with or derived from HPV infection. An "HPV negative" tumor is considered to be not associated with or derived from HPV infection. In some embodiments, the tumor cells are PD-L1 negative or show low PD-L1 expression, and the tumor is HPV (human papilloma virus) positive.

[0185] In some embodiments, the patient is tested for infection, for example, HPV, HBV, HCV or EBV infection. In some embodiments, the patient is tested for HPV infection. In some embodiments, the patient has or has HPV infection. Determining whether the patient has or has HPV infection is by using tests known in the art, for example, testing cells taken from a sample from the patient, or DNA analysis of a sample taken from the patient. In some embodiments, the method further comprises determining the HPV status of the patient.

[0186] In some embodiments, the present invention relates to a treatment of cancer in a patient who has previously undergone a treatment for cancer, the previous treatment for cancer being administration of a PD-L1 inhibitor, the patient has not responded to the previous treatment or has ceased to respond to the previous treatment, and comprises administering an ICOS modulator inhibitor to the patient. In other words, the cancer is refractory or characterized as refractory to PD-L1 inhibitor treatment. In some embodiments, the cancer is refractory or characterized as refractory to PD-L1 immunotherapy (e.g., anti-PD-L1 antibody or anti-PD-1 antibody treatment). In some embodiments, the patient has previously undergone PD-L1 inhibitor treatment as a sole immunotherapy. In general, the cancer is or is characterized as a PD-L1 negative cancer or a cancer that shows low PD-L1 expression. Thus, the present invention includes the use of ICOS modulators as a second-line or further-line treatment.

[0187] In some embodiments, the present invention relates to the treatment of patients with cancer who have previously been administered a kinase inhibitor (in addition to or instead of a PD-L1 inhibitor). In some embodiments, the patient has undergone surgical treatment (e.g., complete or partial tumor resection) and / or radiation therapy and / or chemotherapy for the cancer. The chemotherapy is docetaxel, fluorouracil, cisplatin, paclitaxel and / or nab-paclitaxel. The cancer is refractory or characterized as refractory to one or all of the previous treatments or has stopped responding to the previous treatments. In some embodiments, the cancer is refractory or characterized as refractory to PD-L1 inhibitor monotherapy treatment. In some embodiments, the cancer is refractory or characterized as refractory to treatment with a PD-L1 inhibitor as the sole immunotherapeutic agent. In some embodiments, the cancer is refractory or characterized as refractory to treatment with nivolumab.

[0188] In some embodiments, the method includes determining the level of PD-L1 expression. This can be performed in a tumor sample from a patient. In some embodiments, the method includes obtaining a tumor sample from a patient. In some embodiments, the method can be performed on a tumor sample previously obtained from a patient. The tumor sample is any suitable sample, for example a tumor tissue sample, such as a tumor biopsy. The tumor sample is a sample of tumor cells.

[0189] Once a determination is made that the cancer is PD-L1 negative or exhibits low PD-L1 expression, an ICOS modulator (such as an agonistic anti-ICOS antibody) may be administered or the patient may be recommended for such treatment, or a report may be made making a patient recommendation for such treatment (and thus the invention extends to providing such reports).

[0190] Determining PD-L1 expression status (i.e., whether a cancer or tumor is PD-L1 negative or exhibits low PD-L1 expression) can be determined by any suitable means. In some embodiments, determining PD-L1 expression status can be performed on a tumor sample (e.g., a tumor biopsy). In some embodiments, PD-L1 expression status can be determined by immunohistochemistry (IHC). The sample is prepared for analysis using IHC, e.g., slicing and fixation.

[0191] The sample is analyzed to determine the number of cells in the tumor sample that express PD-L1 (e.g., the number of tumor cells and / or tumor-associated immune cells). In some embodiments, the method determines the ratio (e.g., percentage) of tumor cells in the tumor sample that express PD-L1 to tumor cells in the tumor sample that do not express PD-L1. In some embodiments, the method determines the ratio (e.g., percentage) of tumor cells in the tumor sample and tumor-associated immune cells in the tumor sample that express PD-L1 to the total number of tumor cells and tumor-associated immune cells in the sample.

[0192] Generally speaking, the number of tumor cells and / or tumor-associated immune cells in a tumor sample that express PD-L1 is considered to be representative of the tumor as a whole.

[0193] Generally, for PD-L1 negative tumors (i.e., tumors that do not express PD-L1), 0% of the cells (i.e., tumor cells and / or tumor-associated immune cells) express PD-L1.

[0194] The difference in cutoff points can be used to determine whether a cancer or tumor is a "low" PD-L1 expressing tumor. In some embodiments, a cancer or tumor is considered a low PD-L1 expressing tumor if about 25% or less of the tumor cells (in the tumor tissue sample or sampled tumor cells) express PD-L1. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the tumor cells (in the tumor tissue sample or sampled tumor cells) express PD-L1 at the cutoff. In some embodiments, a cancer or tumor is considered a low PD-L1 expressing tumor if about 25% or less of the tumor associated immune cells (in the tumor tissue sample or sampled tumor cells) express PD-L1. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the tumor-associated immune cells (in the tumor tissue sample or sampled tumor cells) express PD-L1 at a cutoff. In some embodiments, a cancer or tumor is considered a low PD-L1 expressing tumor if about 25% or less of the tumor cells and tumor-associated immune cells (in the tumor tissue sample or sampled tumor cells) express PD-L1 at a cutoff. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the tumor cells and tumor-associated immune cells (in the tumor tissue sample or sampled tumor cells) express PD-L1 at a cutoff. Generally, any non-tumor associated immune cells present in the tumor sample or tumor cell sample are excluded (e.g., neutrophils).

[0195] PD-L1 expression is calculated or expressed as a percentage. In some embodiments, the percentage of PD-L1 expression (i.e., the percentage of analyzed cells that express PD-L1) is determined according to the following formula: (number of PD-L1 positive tumor cells in a tumor tissue sample or tumor cell sample / total number of tumor cells in a tumor tissue sample or tumor cell sample)×100. In some embodiments, the percentage of PD-L1 expression (i.e., the percentage of analyzed cells that express PD-L1) is determined according to the following formula: (number of PD-L1 positive tumor cells and number of PD-L1 positive tumor-associated immune cells in a tumor tissue sample or tumor cell sample / total number of tumor cells and tumor-associated immune cells in a tumor tissue sample or tumor cell sample)×100. Non-tumor-associated immune cells (e.g., neutrophils) are generally excluded from the calculation.

[0196] The cancer or tumor is a CD8+ cancer or tumor. In some embodiments, at least 50% of the T cells in the tumor are CD8+. The CD8 expression status of the cancer or tumor (i.e., the CD8 expression status of the T cells in the tumor) can be determined by any suitable means, such as, for example, by IHC on a tumor sample or a sample of tumor cells. In some embodiments, including but not limited to, embodiments in which the expression status is determined using IHC performed on a slice of the tumor, the tumor sample or the sample of tumor cells is a 1 mm 2 The cells may contain at least 190 CD8+ T cells per cell.

[0197] The cancer or tumor is an ICOS+ cancer or tumor, i.e., a cancer or tumor that contains ICOS+ immune cells, e.g., T cells (more specifically, ICOS+ Treg cells in the tumor microenvironment). In some embodiments, at least 50% of the T cells (i.e., Treg) in the tumor are ICOS+. The ICOS expression status of the cancer or tumor (i.e., the ICOS expression status of T cells in the tumor) can be determined by any suitable means, e.g., by IHC, such as in a tumor sample or a tumor cell sample. In some embodiments, the patient has an increased level of ICOS+ immune cells (such as ICOS+ regulatory T cells in the TME) after treatment with another therapeutic agent. In some embodiments, the method includes administering a therapeutic agent to the patient, determining that the patient has an increased level of ICOS positive+ immune cells (such as ICOS+ regulatory T cells) after treatment with the agent, and administering a modulator of ICOS (e.g., an anti-ICOS antibody, such as an agonist anti-ICOS antibody) to the patient to reduce the level of ICOS+ regulatory T cells. In some embodiments, the therapeutic agent is IL-2 or an immune modulating antibody (eg, anti-PDL-1, anti-PD-1, or anti-CTLA-4).

[0198] Tumor-associated immune cells are also referred to herein as tumor-infiltrating lymphocytes (TILs), or simply immune cells, in the tumor or in the tumor microenvironment (TME).

[0199] The antibodies disclosed herein, or compositions comprising such antibody molecules or their encoding nucleic acids, are used or provided for use in any such method. The use of the antibodies, or compositions comprising them or their encoding nucleic acids, for the manufacture of a medicament for use in any such method is also envisaged. The method typically includes administering the antibody or composition to a mammal. Suitable formulations and methods of administration are described elsewhere herein.

[0200] The cancer is a solid tumor, such as renal cell carcinoma (possibly renal cell carcinoma, e.g., clear cell renal cell carcinoma), head and neck cancer, melanoma (possibly 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] . By enhancing a patient's immune response against 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.

[0201] 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.

[0202] 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 25 of WO2018 / 029474. Example 20 of WO2018 / 029474 demonstrates the efficacy of exemplary anti-ICOS antibodies in treating tumors derived from cancerous B cells (A20 syngeneic cells) that express ICOS ligand.

[0203] 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.

[0204] Patients can be tested to determine whether their cancer is positive for the expression of a protein of interest (e.g., ICOS ligand, ICOS, FOXP3 and / or CD8), or positive, negative, or low expression of PD-L1, 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 negative for PD-L1 or has low PD-L1 expression are selected for treatment. In some cases, patients whose cancer is characterized as positive for the expression of one, two, or all of such proteins of interest (e.g., ICOS ligand, ICOS, FOXP3 and / or CD8) are also 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.

[0205] Anti-ICOS antibodies also offer hope to patients whose cancers are refractory to treatment with antibodies against immune checkpoint molecules such as CTLA-4, PD-1, PD-L1, CD137, GITR or CD73, but particularly those against cancers that are refractory to PD-L1 inhibitors, or other drugs. These immunotherapies are effective against some cancers, but 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 such other antibodies fail. It is shown herein that animals with 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] .

[0206] Thus, anti-ICOS antibodies can be used in methods of treating cancers that are refractory to PD-L1 inhibitors, such as anti-PD1 or anti-PD-L1 antibodies, but 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 samples (e.g., tumor biopsy samples) for cancer cell death or growth inhibition, and / or in a clinical setting by observing (e.g., using imaging techniques, including MRI) that patients treated with a therapy do not respond to the treatment. Patients whose cancer has been characterized as refractory to such immunotherapeutic treatment are selected for treatment with anti-ICOS antibodies.

[0207] A sample obtained from a patient can be tested in this manner to determine the surface expression of a protein of interest, such as ICOS ligand, ICOS, FOXP3, and / or a target receptor targeted by another therapeutic agent (e.g., an anti-receptor antibody). The lack or loss of surface expression of ICOS ligand, ICOS, FOXP3, and / or the surface expression of 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 surface expression of the target receptor, where optionally the patient has previously been treated with 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.

[0208] Any suitable method can be used to determine whether cancer cells test positive for surface expression of a protein such as ICOS ligand, PD-L1 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 to test positive for ICOS or PD-L1 if at least a certain percentage of cells are labeled, as visualized by cell staining or other label detection. 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, antibody MAB1651 is currently available from R&D systems as a mouse IgG that recognizes human ICOS ligand. To test for PD-L1, one can use the antibody SP263, currently available from Roche, as a rabbit monoclonal primary antibody that recognizes human PD-L1. Detection of ICOS ligand or mRNA levels of PD-L1 or the target receptor of interest are alternative techniques

[27] .

[0209] A further indication that tumors respond to treatment with anti-ICOS antibodies is the presence of Tregs in the tumor microenvironment. Activated Tregs are characterized by high ICOS and high Foxp3 surface expression. The presence of Tregs in tumors, particularly their elevated numbers, provides a further basis for which patients are selected for treatment with anti-ICOS antibodies. Tregs are 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. FACS methods are exemplified in Example 17 and Example 18 of WO2018 / 029474. In some embodiments, treatment with ICOS modulators (and optionally PD-L1 inhibitors) can cause a reduction in tumor size (compared to the size of the tumor at the start of treatment). In some embodiments, treatment with an ICOS modulator (and optionally a PD-L1 inhibitor) can inhibit tumor growth. In some embodiments, treatment with an ICOS modulator (and optionally a PD-L1 inhibitor) can result in stable disease. Stable disease can be considered when a tumor does not grow in size by more than 20% from the start of treatment and does not decrease in size by more than 30% from the start of treatment. In some embodiments, treatment with an ICOS modulator (and optionally a PD-L1 inhibitor) can prolong patient survival and / or delay disease progression.

[0210] ICOS modulators, such as anti-ICOS antibodies, can be used to treat cancers associated with infectious agents, such as virus-induced cancers, e.g., cancers caused by infection with a virus. This category includes head and neck squamous cell carcinoma, cervical cancer, Merkel cell carcinoma, etc. 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.

[0211] 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.

[0212] In some embodiments, the cancer is liver cancer, renal cell carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, diffuse large B-cell lymphoma, breast cancer, penile cancer, pancreatic cancer or esophageal cancer. In some embodiments, the liver cancer is hepatocellular carcinoma. In some embodiments, the head and neck cancer is metastatic squamous cell carcinoma. In some embodiments, the breast cancer is triple-negative breast cancer. The present invention is particularly relevant to solid cancers.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] The present invention also provides an ICOS modulator for use in treating cancer in a patient, the patient having a PD-L1 negative tumor or a tumor with low PD-L1 expression. The present invention also provides an ICOS modulator for use in treating cancer in a patient, the patient having previously undergone treatment for cancer, the patient having failed to respond to the previous treatment or has ceased to respond to the previous treatment, the previous treatment for cancer being a PD-L1 inhibitor. The present invention also provides the use of an ICOS inhibitor modulator in the manufacture of a medicament for treating cancer in a patient, the patient having a PD-L1 negative tumor or a tumor with low PD-L1 expression. The present invention also provides the use of an ICOS inhibitor in the manufacture of a medicament for treating cancer in a patient, the cancer being refractory to PD-L1 inhibitor treatment or characterized as refractory to PD-L1 inhibitor treatment. The present invention also provides the use of an ICOS inhibitor modulator in the manufacture of a medicament for the treatment of cancer in a patient, the patient having previously undergone a treatment for cancer, the patient having failed to respond to the previous treatment or has ceased to respond to the previous treatment, the previous treatment for cancer being a PD-L1 inhibitor. In some embodiments, the ICOS modulator is for use in combination with a PD-L1 inhibitor. In some embodiments, the ICOS modulator is an agonist anti-ICOS antibody. In some embodiments, the ICOS modulator is a bispecific antibody that is an anti-ICOS agonist and an anti-PD-L1 antagonist, or a bispecific antibody that is an anti-ICOS agonist and an anti-PD-1 antagonist. Generally, the cancer (such as a solid cancer) is a PD-L1 negative cancer or a cancer with low PD-L1 expression.

[0218] Combination therapy It is beneficial to combine anti-ICOS antibody with such immune modulators to enhance its therapeutic effect.In particular, the present invention relates in some embodiments to a combination of ICOS modulators (anti-ICOS antibodies, such as agonistic anti-ICOS antibodies) and PD-L1 inhibitors, i.e., PD-1 or PD-L1 binding agents (such as anti-PD-L1 or anti-PD-1 antibodies) that inhibit the binding of PD-L1 to PD-1.In the combination therapy, ICOS modulators and PD-L1 inhibitors are administered simultaneously, separately or sequentially.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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 196175, US7932358, US8389690, WO02070010, EP1286668, EP1374901, US7438905, US7438905, 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.

[0225] 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.

[0226] 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) 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 or other antibody can include a total of four doses administered intravenously over a period of 90 minutes every three weeks.

[0227] 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.

[0228] 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.

[0229] As discussed in Example 22 of WO2018 / 029474, treatment with anti-PD-L1 antibodies, particularly those with effector-positive Fc, does not appear to increase ICOS expression in Teff cells. This is advantageous when administering such antibodies in combination with effector-positive anti-ICOS antibodies, where increased ICOS expression in 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 take advantage of 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 the 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 studied previously - see Figure S6C in ref.

[30] (supplementary material), where it was reported that treatment with CTLA-4 and / or anti-PD-1 antibodies increased the percentage of CD4+ Tregs expressing ICOS. Note that 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 anti-ICOS antibodies, and that the effect of anti-ICOS antibodies is enhanced under conditions where there is high differential expression of ICOS on Tregs versus Teffs.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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 high dose (HD) or low dose (LD) IL-2.

[0235] 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.

[0236] 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.

[0237] 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 optimize 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.

[0238] 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.

[0239] 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].

[0240] 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.

[0241] 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

[0242] 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).

[0243] Therefore, 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.

[0244] 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.

[0245] 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.

[0246] The present invention also provides a combination of an ICOS modulator and a PD-L1 inhibitor for use in treating cancer in a patient, where the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression.The present invention also provides a combination of an ICOS modulator and a PD-L1 inhibitor for use in treating cancer in a patient, where the patient has previously received treatment for cancer, where the patient has not responded to the previous treatment or has stopped responding to the previous treatment, where the previous treatment for cancer was a PD-L1 inhibitor.The present invention also provides a modulator of ICOS and an inhibitor of PD-L1 for use in treating cancer in a patient, where the patient has a PD-L1 negative cancer or a cancer with low PD-L1 expression. The present invention also provides a modulator of ICOS and an inhibitor of PD-L1 for use in the treatment of cancer in a patient, where the patient has previously undergone treatment for cancer, and where the patient has not responded to or has stopped responding to the previous treatment, and where the previous treatment for cancer was a PD-L1 inhibitor.The present invention also provides the use of a combination of an ICOS modulator and a PD-L1 inhibitor in the manufacture of a medicament for the treatment of cancer in a patient, where the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression.The present invention also provides the use of a combination of an ICOS modulator and a PD-L1 inhibitor in the manufacture of a medicament for the treatment of cancer in a patient, where the patient has not responded to or has stopped responding to the previous treatment, and where the patient has previously undergone treatment for cancer, and where the previous treatment for cancer was a PD-L1 inhibitor. The invention also provides the use of a modulator of ICOS and an inhibitor of PD-L1 in the manufacture of a medicament for the treatment of cancer in a patient, wherein the patient has a PD-L1 negative cancer or a cancer with low PD-L1 expression.The present invention also provides the use of a modulator of ICOS and an inhibitor of PD-L1 in the manufacture of a medicament for the treatment of cancer in a patient, the patient having previously undergone treatment for cancer, the previous treatment for cancer being a PD-L1 inhibitor. In some embodiments, the ICOS modulator is for use in combination with a PD-L1 inhibitor. In some embodiments, the ICOS modulator is an agonist anti-ICOS antibody. In some embodiments, the ICOS modulator is a bispecific antibody that is an anti-ICOS agonist and an anti-PD-L1 antagonist, or a bispecific antibody that is an anti-ICOS agonist and an anti-PD-1 antagonist. Generally, the cancer (such as a solid cancer) is a PD-L1 negative cancer or a cancer with low PD-L1 expression.

[0247] Antibodies against 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.

[0248] 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. HThe 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. 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: 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).

[0249] 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. HThe 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. 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: 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).

[0250] 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). H1D05 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).

[0251] 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. LThe 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).

[0252] 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). H 1D05 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).

[0253] 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).

[0254] 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. 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. 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).

[0255] 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. 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).

[0256] 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). LThe 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. 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: 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).

[0257] 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. 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: 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).

[0258] 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 the domain is SEQ ID NO: 89. 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: 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).

[0259] 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. 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: 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).

[0260] 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. 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: 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).

[0261] 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. 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 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).

[0262] 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. 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: 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).

[0263] 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. 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 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).

[0264] 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. 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 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).

[0265] 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. 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 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).

[0266] 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. 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 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).

[0267] 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. 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 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).

[0268] In some embodiments, the anti-PD-L1 antibody is atezolizumab (Roche), avelumab (Merck), durvalumab / Medi4736 (Medimmune), KN035, CK-301, AUNP12, CA-170, BMS-936559 / MDX-1105 (BMS), FAZ-053 M7824, ABBV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504, and those disclosed in WO2017 / 220990, WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801, WO2016 / 1116 No. 45, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 16 No. 0792, No. WO2016 / 022630, No. WO2016 / 007235, No. WO2015 / 179654, No. WO2015 / 173267, No. WO2015 / 1 No. 81342, No. WO2015 / 109124, No. WO2015 / 112805, No. WO2015 / 061668, No. WO2014 / 159562, No. WO2014 / 165082, WO2014 / 100079, WO2014 / 055897, WO2013 / 181634, WO2013 / 173223, WO20 the anti-PD-L1 antibody is selected from the group consisting of any of the PD-L1 antibodies disclosed in WO2013 / 079174, WO2012 / 145493, WO2011 / 066389, WO2010 / 077634, WO2010 / 036959, WO2010 / 089411, or WO2007 / 005874.

[0269] Antibodies to PD-1 In some embodiments, the present invention relates to anti-PD-L1 antibodies, such as pembrolizumab, nivolumab, cemiplimab, JTX-401, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), dostallimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224 and AMP-514, MEDI-0680 / AMP514, PDR001, lambrolizumab, BMS-936558, REGN2810, BGB-A317, BGB-108, PDR-001, SHR-1210, JS-001, JNJ-63723283, AGEN-2034, PF-06801591, genolimuzumab, MGA-012 (INCMGA00012), IBI-308, BCD-100, TSR-042 from the group consisting of ANA011, AUNP-12, KD033, MCLA-134, mDX400, muDX400, STI-A1110, AB011, 244C8, 388D4, XCE853, or pidilizumab / CT-011, or from the group consisting of the compounds described in WO2015 / 112800 and US2015 / 0203579 (Tables 1 to 3 Nos. 9,394,365, 5,897,862 and 7,488,802, WO2017 / 087599 (including antibodies SSI-361 and SHB-617), WO2017 / 079112, WO2017 / 071625 (including deposit C2015132, hybridoma LT004, and antibodies 6F5 / 6 F5(Re), 6F5H1 L1 and 6F5 H2L2), WO2017 / 058859 (including PD1AB-1 to PD1AB-6), WO2017 / 058115 (including 67D9, c67D9 and hu67D9), WO2017 / 055547 (including 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375 and 13112.15380), WO2017 / 040790 (AGEN2033w, AGEN2034w, AGEN2046w, AGEN2047w,AGEN2001w and AGEN2002w), WO2017 / 025051 and WO2017 / 024515 (including 1.7.3 hAb, 1.49.9 hAb, 1.103.11 hAb, 1.103.11-v2 hAb, 1.139.15 hAb and 1.153.7 hAb), WO2017 / 025016 and WO2017 / 024465 (including Antibody A to Antibody I), WO2017 / 020858 and WO2017 / 020291 (including 1.4.1, 1.14.4, 1.20.15 and 1.46.11), WO2017 / 019896 and WO2015 / 112900 and US2015 / 0210769 (including BAP049-hum01 to BAP049-hum16 and BAP049-clone-A to BAP049-clone-E), WO2017 / 019846 (PD-1 mAb 1 to PD-1 mAb 15), WO2017 / 016497 (including MHC723, MHC724, ​​MHC725, MHC728, MHC729, m136-M13, m136-M19, m245-M3, m245-M5 and m136-M14), WO2016 / 201051 (including antibody EH12.2H7, antibody hPD-1 mAb2, antibody hPD-1 mAb7, antibody hPD-1 mAb9, antibody hPD-1 No. WO2016 / 197497 (including DFPD1-1 to DFPD1-13), No. WO2016 / 197367 (including 2.74.15 and 2.74.15.hAb4 to 2.74.15.hAb8), No. WO2016 / 196173 (including the antibodies in Table 5, and Figures 1 to 5), No. WO2016 / 127179 (including R3A1, R3A2, R4B3 and R3D6), No. WO2016 / 077397 ( including the antibodies described in Table 1 of Example 9), WO2016 / 106159 (including the murine antibodies in Table 3 of Example 2, and the humanized antibodies in Tables 7, 8 and 9 of Example 3), WO2016 / 092419 (including C1, C2, C3, EH12.1, mAb7-G4, mAb15-G4, mAb-AAA, mAb15-AAA), WO2016 / 068801 (including clone A3 and variants thereof, as well as other antibodies described in Figures 1-4),WO2016 / 014688 (including 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4, and 7A4D, as well as the humanized antibodies of Examples 9 / 10), WO2016 / 015685 (including 10F8, BA08-1, BA-08-2, and 15H6), WO2015 / 091911, and WO2015 / 091910 (including the humanized antibodies of Examples 2, 3, and 4). No. WO2015 / 091914 (including the anti-canine PD-1 antibodies in Table 3), No. WO2015 / 085847 (including mAb005, H005-1 to H005-4), No. WO2015 / 058573 (including cAB7), No. WO2015 / 036394 (including LOPD180), No. WO2015 / 035606 (including Table 1 in Example 2, Tables 14, 15 and 16, and the antibodies in Tables 20, 21 and 22 of Example 11), WO2014 / 194302 (including GA2, RG1B3, RG1H10, RG2A7, RG2H10, SH-A4, RG4A6, GA1, GB1, GB6, GH1, A2, C7, H7, SH-A4, SH-A9, RG1H11 and RG6B), WO2014 / 179664 (including 9A2, 10B11, 6E9, APE1 922, APE1923, APE1924, APE1950, APE1963 and APE2058), WO2014 / 206107 (including clones 1, 10, 11, 55, 64, 38, 39, 41 and 48), WO2012 / 135408 (including h409A11, h409A16 and h409A17), WO2012 / 145493 (including antibodies 1E3, 1E8, 1H3 and h1H3 Var 1 to h1H3 Var 14), WO2011 / 110621 (including antibody 949 and modified versions disclosed in Figures 1 to 11), WO2011 / 110604 (including antibody 948 and modified versions disclosed in Figures 3 to 11), WO2010 / 089411 (including CNCM Deposit Nos. 1-4122, 1-4080 or 1-4081), WO2010 / 036959 (including the antibodies in Table 1 of Example 1), WO2010 / 029435 and WO2010 / 029434 (including clones 2, 10 and 19), WO2008 / 156712 (hPD-1.08A,hPD-1.09A, h409A11, h409A16 and h409A17, and the antibodies described in Example 2, Table H, Example 4 and Table IV), WO2006 / 121168 (including clones 17D8, 4H1, 5C4, 4A11, 7D3, 5F4 and 2D3), WO2004 / 004771 and WO2004 / 056875 (including PD1-17, PD1-28, PD1-33, PD1-35, PD1-F2 and the Abs described in Table 1).

[0270] Antibody-drug conjugates Anti-ICOS antibodies can be used as a carrier of cytotoxic agents to target Tregs. As reported in Example 18 of WO2018 / 029474, Tregs are located in the tumor microenvironment (TME) where ICOS is strongly expressed. 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).

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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).

[0275] 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.

[0276] 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.

[0277] 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; Immunocytokines are motif X 1 GSGX 2 YGX 3 X 4 FD (SEQ ID NO: 609) (wherein X 1 , X 2 and X 3 are independently any amino acid, and X 4 is either present or absent, and if present, is any amino acid H Includes the domain.

[0278] 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.

[0279] The IL-2 is human wild-type or variant IL-2.

[0280] 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.

[0281] 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.

[0282] 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.

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.).

[0292] 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).

[0293] 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).

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] As shown by the in vivo studies described in the Examples of WO2018 / 029474, anti-ICOS antibodies are effective over a range of doses. Pharmacodynamic studies are reported in Example 24 of WO2018 / 029474.

[0303] The anti-ICOS antibody may be administered in one of the following ranges per dose: Approximately 10μg / kg body weight ~ approximately 100mg / kg body weight, Approximately 50μg / kg body weight to approximately 5mg / kg body weight, Approximately 100μg / kg body weight ~ approximately 10mg / kg body weight, Approximately 100μg / kg body weight ~ approximately 20mg / kg body weight, About 0.5 mg / kg body weight to about 20 mg / kg body weight, or Less than about 5 mg / kg body weight, for example, less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, or less than 1 mg / kg of antibody.

[0304] An optimal therapeutic dose is 0.1-0.5 mg / kg in humans, for example, about 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg or 0.5 mg / kg. For fixed dosing in adult humans, a suitable dose is 8-50 mg, or 8-25 mg, for example, 15 mg or 20 mg.

[0305] 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 patient at intervals of 4-6 weeks, for example, every 4 weeks, every 5 weeks, or every 6 weeks. Optionally, the anti-ICOS antibody can be administered to the patient once a month, or less frequently, for example, every 2 months or every 3 months. Thus, the method of treating a patient can include administering a single dose of the anti-ICOS antibody to the patient 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.

[0306] As discussed in Example 11c of WO2018 / 029474, a comparable therapeutic effect can be obtained using either one or multiple doses of anti-ICOS antibody, which is the result of a single dose of the antibody being effective in resetting the tumor microenvironment. A physician can tailor the administration regimen of the anti-ICOS antibody to the disease and the patient being treated, taking into account the state of the disease and any other therapeutic agents or treatment strategies (e.g., surgery, radiation therapy, etc.) with which the anti-ICOS antibody is combined. In some embodiments, an effective dose of the anti-ICOS antibody is administered more frequently than once a month, such as, for example, once every 3 weeks, once every 2 weeks, or once every week. Treatment with the 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.

[0307] 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.

[0308] 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.

[0309] Morphological assays for anti-ICOS antibodies as potential therapeutic agents 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.

[0310] 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.

[0311] 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.

[0312] 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.

[0313] 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.

[0314] 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

[0315] Experimental Example

[0316] Example 1 - Study Background and Design KY1044 (also known as STIM003) is a fully human IgG1 anti-ICOS (inducible T cell costimulator) antibody designed to stimulate Teff and deplete ICOS-high Tregs in the tumor microenvironment. ICOS is a key costimulatory receptor on effector T cells (Teff) that also promotes tumor growth due to its high expression in regulatory T cells (TReg). KY1044 targets ICOS and inhibits ICOS high Depleting Tregs and ICOS lowIt is a fully human IgG1 that acts via a dual mechanism of action (MoA) by stimulating Teff (Sainson RCA, Thotakura AK, Kosmac M et al. An Antibody Targeting ICOS Increases Intratumoral Cytotoxic to Regulatory T-cell Ratio and Induces Tumor Regression. Cancer Immunology Research. 2020;8(12):1568-1582) - see Figure 1. KY1044-CT01 (ClinicalTrials.gov Identifier: NCT03829501) is a first-in-human study evaluating the safety, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary antitumor activity of KY1044 as a single agent and in combination with atezolizumab in patients with advanced / metastatic malignancies. Using blood samples and tumor biopsies longitudinally, we aim to correlate KY1044 target engagement levels with pharmacodynamic (PD) properties (e.g., dual MoA) in the tumor microenvironment (TME) and circulation. The study will consist of a Phase 1 dose escalation and enrichment cohort, as well as a Phase 2 part.

[0317] Research purpose: Main: To characterize the safety and tolerability of KY1044 as a single agent and in combination with atezolizumab, and to identify recommended doses for future studies. Secondary: To evaluate the preliminary antitumor activity of KY1044 as a single agent and in combination with atezolizumab. · To characterize the PK profile of KY1044 as a single agent and in combination with atezolizumab. Exploratory: To evaluate the pharmacodynamic effects of KY1044 as a single agent and in combination with atezolizumab in tumor tissue and peripheral blood.

[0318] method Important inclusion criteria: Histologically documented advanced / metastatic malignancies with measurable disease by RECIST 1.1 (non-measurable disease is only accepted in Phase I). Prior therapy with an anti-PD-(L)1 inhibitor is permitted, provided that any toxicity attributable to the prior anti-PD-L1 directed therapy did not lead to discontinuation of therapy. Eastern Cooperative Oncology Group performance status 0 or 1. Must have a site of disease suitable for biopsy and be a candidate for tumor biopsy according to the guidelines of the treating institution.

[0319] Important exclusion criteria: Symptomatic CNS metastases or CNS metastases requiring local CNS-directed therapy. ·Severe hypersensitivity reactions to other monoclonal antibodies or excipients. Laboratory values ​​outside protocol-defined ranges for renal and liver function or hematological parameters. ·Clinically significant cardiac disease and / or QT prolongation. · Documented medical history of acute autoimmune disease or autoimmune disease. ·Systemic corticosteroid therapy or any immunosuppressive therapy (≥10 mg / day prednisone or equivalent). Presence of CTCAE v5 ≥ grade 2 toxicity attributable to prior anticancer therapy.

[0320] Figure 2 outlines the study design.

[0321] result PD-L1 expression in the tumor microenvironment (TME) was assessed in tumor and immune cells using the anti-PD-L1 antibody SP263 (see Figure 3). Figure 4 shows PD-L1 immune cell expression in the TME and CD8+ T cells (baseline).

[0322] The effect of treatment on three patients (Patients A, B, and C) with PD-L1 negative or low PD-L1 expressing tumors was evaluated. The results for Patient A are shown in Figures 5 and 6. The results for Patient B are shown in Figures 7 and 8. Despite having low PD-L1 expression, both patients achieved stable disease (tumors that were neither growing nor shrinking) after treatment.

[0323] [Table 1]

[0324] The results for patient C are shown in Figure 9. This patient responded well, with significant reductions in lesion size from baseline at C3D8 (3rd cycle, day 8) and C10D1 (10th cycle, day 1).

[0325] HPV status When the HPV status of the tumour was available, this was recorded as part of the enrolled patient's medical / tumour history.

[0326] An "HPV positive" tumor is considered to be associated with or resulting from HPV infection. An "HPV negative" tumor is considered to be not associated with or resulting from HPV infection.

[0327] Testing for tumor HPV status is known in the art.Testing can include viral DNA detection by polymerase chain reaction or in situ hybridization, or HPV RNA detection by reverse transcription polymerase chain reaction or in situ hybridization.Testing for HPV status can be performed on tissue biopsy, fine needle aspiration biopsy specimen, blood sample, or saliva sample, depending on the patient and tumor type.

[0328] The effect of treatment on five patients (patients D-H) was evaluated. The results are shown below. Despite having low PD-L1 expression on tumor cells and immune infiltrates, the patients achieved partial responses (PR). Three patients (patients E, G, and H) were also documented to have HPV-positive tumors.

[0329] Without being bound by theory, a favorable outcome may be attributed to patients having properties of HPV-positive tumor cells, such as a reduced rate of invasion, or an improved immune response due to a pre-existing immune response to the virus.

[0330] [Table 2]

[0331] conclusion The study concluded: Partial and transient receptor occupancy was observed up to dose level 2 (2.4 mg), and complete and prolonged receptor occupancy was observed at dose levels 3 (8 mg) and above. No significant depletion of ICOS+ T cells in the periphery. KY1044 reduced ICOS+ Tregs and improved the ratio of CD8 to ICOS+ Tregs in the tumor microenvironment (dose-dependent, plateauing at dose level 3 [8 mg]). Signs of antitumor activity (PR / CR) were observed in both PD-L1 low and PD-L1 high tumors.

[0332] Example 2: Antibody sequence analysis The framework regions of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 were compared to human germline gene segments to identify the closest matches. See Tables E12-1 and E12-2.

[0333] [Table 3]

[0334] [Table 4]

[0335] Additional antibody sequences were obtained by next generation sequencing of PCR amplified antibody DNA from additional ICOS-specific cells selected from immunized mice as described in Example 3 of WO2018 / 029474. This identified several antibodies that could be grouped into clusters with STIM001, STIM002 or STIM003 based on their heavy and light chain v and j gene segments and CDR3 length. CL-61091 clustered with STIM001; CL-64536, CL-64837, CL-64841 and CL-64912 clustered with STIM002; CL-71642 and CL-74570 clustered with STIM003. Sequence alignments of the VH and VL domains of the antibodies are shown in Figures 10-12.

[0336] [Table 5]

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Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS. Blood. 2000 Oct. 15; 96(8):2808-13. 8 Conrad C, Gilliet M. Plasmacytoid dendritic cells and regulatory T cells in the tumor microenvironment: A dangerous liaison. Oncoimmunology. 2013 May 1; 2(5):e2388. 9 Simpson et al., Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J. Exp. Med. 210(9):1695-1710 2013 10 Fu T, He Q, Sharma P. The ICOS / ICOSL pathway is required for optimal antitumor responses mediated by anti-CTLA-4 therapy. Cancer Res. 2011 Aug. 15; 71(16):5445-54. 11 Fan X, Quezada S A, Sepulveda M A, Sharma P, Allison J P. Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy. J Exp Med. 2014 Apr. 7; 211(4):715-25. 12 Carthon, B. C., et al. Preoperative CTLA-4 blockade: Tolerability and immune monitoring in the setting of a presurgical clinical trial. Clin. Cancer Res. 16:2861-2871. 13 Liakou C I, et al. CTLA-4 blockade increases IFNgamma-producing CD4+ICOShi cells to shift the ratio of effector to regulatory T cells in cancer patients. Proc Natl Acad Sci USA. 2008 Sep. 30; 105(39):14987-92. 14 Vonderheide, R. H., et al. 2010. Tremelimumab in combination with exemestane in patients with advanced breast cancer and treatment-associated modulation of inducible costimulator expression on patient T cells. Clin. Cancer Res. 16:3485-3494. 15 Preston C C, et al., The ratios of CD8+ T cells to CD4+CD25+FOXP3+ and FOXP3- T cells correlate with poor clinical outcome in human serous ovarian cancer. PLoS One November 14; 8(11):e80063. 16 Hodi F S, et al., Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. PNAS 2008 Feb. 26; 105(8):3005-10 17 Chattopadhyay et al., Structural Basis of Inducible Costimulatory Ligand Function: Determination of the Cell Surface Oligomeric State and Functional Mapping of the Receptor Binding Site of the Protein, J. Immunol. 177(6):3920-3929 2006 18 Lefranc M P, IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev Comp Immunol. 27(1):55-77 2003 19 Gul et al., “Antibody-Dependent Phagocytosis of Tumor Cells by Macrophages: A Potent Effector Mechanism of Monoclonal Antibody Therapy of Cancer”, Cancer Res., 75(23), Dec. 1, 2015 20 Lazar et al., 2006, Proc. Natl. Acad. Sci. U.S.A., March 14; 103(11):4005-10 21 Dall et al., Immunol 2002; 169:5171-5180 22 Natsume et al., 2009, Drug Des. Devel. Ther., 3:7-16 or by Zhou Q., Biotechnol. Bioeng., 2008, February 15, 99(3):652-65) 23 Shields et al., 2001, J. Biol. Chem., March 2; 276(9):6591-604) 24 Idusogie et al., J. Immunol., 2001, 166:2571-2575 25 Natsume et al., 2008, Cancer Res., 68: 3863-3872 26 Alexandrov L B, et al. Signatures of mutational processes in human cancer. Nature. 2013 Aug. 22; 500(7463):415-21 27 Martin-Orozco et al., Melanoma Cells Express ICOS Ligand to Promote the Activation and Expansion of T-Regulatory Cells, Cancer Research 70(23):9581-9590 2010 28 Houot et al., Therapeutic effect of CD137 immunomodulation in lymphoma and its enhancement by Treg depletion, Blood 114:3431-3438 2009 29 Curran et al., PD01 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumours, PNAS 107(9):4275-4280 2010 30 Sim et al., IL-2 therapy promotes suppressive ICOS+ Treg expansion in melanoma patients, J Clin Invest 2014 31 Sim et al., IL-2 variant circumvents ICOS+ regulatory T cell expansion and promotes NK cell activation, Cancer Immunol Res 2016 32 Kroemer et al. Immunologic Cell Death in Cancer Therapy, Ann Rev Immunol. 31:51-72 2013 33 Galluzzi, Zitvogel & Kroemer Canc. Imm. Res. 4:895-902 2016 34 Bos et al., Transient regulatory T cell ablation deters oncogene-driven breast cancer and enhances radiotherapy, J Exp Med 210(11):2434-2446 2013 35 Sato et al., Spatially selective depletion of tumor-associated regulatory T cells with near-infrared photoimmunotherapy, Science Translational Medicine 8(352) 2016 37 Crotty S. T follicular helper cell differentiation, function, and roles in disease. Immunity. 2014 Oct. 16; 41(4):529-42. 37 Shields et al. (2002) JBC 277:26733

[0338] Sequence Antibody STIM001 VH domain nucleotide sequence: SEQ ID NO: 367 CAGGTTCAGGTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTTCCACCTTTGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAATGGATGGGATGGATCAGCGCTTACAATGGTGACACAAACTATGCACAGAATCTCCAGGGCAGAGTCATCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTTTATTACTGTGCGAGGAGCAGTGGCCACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO:366 QVQVVQSGAEVKKPGASVKVSCKASGYTFSTFGITWVRQAPGQGLEWMGWISAYNGDTNYAQNLQGRVIMTTDTSTSTAYMELRSLRSDDTAVYYCARSSGHYYYYGMDVWGQGTTVTVSS VH CDR1 amino acid sequence: GYTFSTFG SEQ ID NO: 363 VH CDR2 amino acid sequence: ISAYNGDT SEQ ID NO: 364 VH CDR3 amino acid sequence: ARSSGHYYYYGMDV SEQ ID NO: 365 VL domain nucleotide sequence: SEQ ID NO:374 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGAATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTTTTTGGGT TCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCACCAGAGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAATCTCTACAAACTCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA VL domain amino acid sequence: SEQ ID NO:373 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNEYNYLDWYLQKPGQSPQLLIFLGSNRASGVPDRFSGSGSGTDFTLKITRVEAEDVGIYYYCMQSLQTPLTFGGGTKVEIK VL CDR1 amino acid sequence: QSLLHSNEYNY SEQ ID NO: 370 VL CDR2 amino acid sequence: LGS SEQ ID NO: 371 VL CDR3 amino acid sequence: MQSLQTPLT SEQ ID NO: 372

[0339] Antibody STIM002 VH domain nucleotide sequence: SEQ ID NO:381 CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAG AAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO: 380 QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSS VH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO: 377 VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO: 378 VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO: 379 VL domain nucleotide sequence: 388 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGT TCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGGACCAAGCTGGAGATCAAA Corrected STIM002 VL domain nucleotide sequence: SEQ ID NO:519 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGT TCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCAGTTTTGGCCAGGGGGACCAAGCTGGAGATCAAA VL domain amino acid sequence: SEQ ID NO:387 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNYLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLSFGQGTKLEIK VL CDR1 amino acid sequence: QSLLHSDGYNY SEQ ID NO: 384 VL CDR2 amino acid sequence: LGS SEQ ID NO: 385 VL CDR3 amino acid sequence: MQALQTPLS SEQ ID NO:386

[0340] Antibody STIM002-B VH domain nucleotide sequence: SEQ ID NO:395 CAGGTTCAACTGGTGCAGTCTGGAGGTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTTTCAGCTGGGTGCGACAGGCCCCTGGACAAGGACTAGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAG AAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCTTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGATCTACGTATTTCTATGGTTCGGGGACCCTCTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO:394 QVQLVQSGGEVKKPGASVKVSCKASGYTFTSYGFSWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARSTYFYGSGTLYGMDVWGQGTTVTVSS VH CDR1 amino acid sequence: GYTFTSYG SEQ ID NO: 391 VH CDR2 amino acid sequence: ISAYNGNT SEQ ID NO: 392 VH CDR3 amino acid sequence: ARSTYFYGSGTLYGMDV SEQ ID NO: 393 VL domain nucleotide sequence: SEQ ID NO:402 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTGATGGATACAACTGTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGT TCTACTCGGGCCTCCGGGTTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGTGCAGTTTTGGCCAGGGGGACCAAGCTGGAGATCAAA VL domain amino acid sequence: SEQ ID NO: 401 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSDGYNCLDWYLQKPGQSPQLLIYLGSTRASGFPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPCSFGQGTKLEIK VL CDR1 amino acid sequence: QSLLHSDGYNC SEQ ID NO: 398 VL CDR2 amino acid sequence: LGS SEQ ID NO: 399 VL CDR3 amino acid sequence: MQALQTPCS SEQ ID NO: 400

[0341] Antibody STIM003 VH domain nucleotide sequence: SEQ ID NO:409 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGARTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGAC TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA Corrected STIM003 VH domain nucleotide sequence: SEQ ID NO:521 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGAGTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGGCGACACAGATTATTCAGAC TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTACAAATGAATAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTTCTATGGTTCGGGGAGTTATTATCACGTTCCTTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO: 408 EVQLVESGGGVVRPGGSLRLSCVASGVTFDDYGMSWVRQAPGKGLEWVSGINWNGGDTDYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDFYGSGSYYHVPFDYWGQGILVTVSS VH CDR1 amino acid sequence: GVTFDDYG SEQ ID NO: 405 VH CDR2 amino acid sequence: INWNGGDT SEQ ID NO:406 VH CDR3 amino acid sequence: ARDFYGSGSYYHVPFDY SEQ ID NO: 407 VL domain nucleotide sequence: SEQ ID NO:416 GAAATTGTGTTGACGCAGTCTCCAGGGACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGAAGCTACTTAGCCTGGTACCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCGATGGGTCTGGGACAGACTTCACTCTCTCCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCACCAGTATGATATGTCACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA VL domain amino acid sequence: SEQ ID NO:415 EIVLTQSPGTLSLSPGERATLSCRASQSVSRSYLAWYQQKRGQAPRLLIYGASSRATGIPDRFSGDGSGTDFTLSISRLEPEDFAVYYCHQYDMSPFTFGGPGTKVDIK VL CDR1 amino acid sequence: QSVSRSY SEQ ID NO:412 VL CDR2 amino acid sequence: GAS SEQ ID NO:413 VL CDR3 amino acid sequence: HQYDMSPFT SEQ ID NO:414

[0342] Antibody STIM004 VH domain nucleotide sequence: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGTGTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGACTCACCTTTGATGATTATGGCATGAGCTGGGTCCGCCAAGTTCCAGGGAAGGGGCTGGAGTGGGTCTCTGGTATTAATTGGAATGGTGATAACACAGATTATGCAGAC TCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCTTGTATTACTGTGCGAGGGATTACTATGGTTCGGGGAGTTATTATAACGTTCCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA Sequence number 423 VH domain amino acid sequence: EVQLVESGGGVVRPGGSLRLSCAASGLTFDDYGMSWVRQVPGKGLEWVSGINWNGDNTDYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARDYYGSGSYYNVPFDYWGQGTLVTVSS SEQ ID NO: 422 VH CDR1 amino acid sequence: GLTFDDYG SEQ ID NO: 419 VH CDR2 amino acid sequence: INWNGDNT SEQ ID NO: 420 VH CDR3 amino acid sequence: ARDYYGSGSYYNVPFDY SEQ ID NO: 421 VL domain nucleotide sequence: SEQ ID NO:431 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCAG CAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAGGTAGTTCACCATTCACTTCGGCCCTGGGACCAAAGTGGATATCAAA The VL domain amino acid sequence encoded by the above VL domain nucleotide sequence. Corrected VL domain nucleotide sequence: SEQ ID NO: 430 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATATATGGTGCATCCA GCAGGGCCACTGGCATCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGAAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTAGGTAGTTCACCATTCTTCGGCCCTGGGACCAAAGTGGATATCAAA Corrected VL domain amino acid sequence: SEQ ID NO: 432 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIRRLEPEDFAVYYCQQYGSSPFFGPGTKVDIK VL CDR1 amino acid sequence: QSVSSSY SEQ ID NO: 426 VL CDR2 amino acid sequence: GAS SEQ ID NO: 427 VL CDR3 amino acid sequence: QQYGSSPF SEQ ID NO: 428

[0343] Antibody STIM005 VH domain nucleotide sequence: SEQ ID NO:439 CAGGTTCAGTTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTAATAGTTATGGTATCATCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGTTCACAATGGTAACACAAACTGTGCACAG AAGCTCCAGGGTAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGAACTGACGACACGGCCGTGTATTACTGTGCGAGAGCGGGTTACGATATTTTGACTGATTTTTCCGATGCTTTTGATATCTGGGGCCACGGGACAATGGTCACCGTCTCTTCA VH domain amino acid sequence: SEQ ID NO: 438 QVQLVQSGAEVKKPGASVKVSCKASGYTFNSYGIIWVRQAPGQGLEWMGWISVHNGNTNCAQKLQGRVTMTTDTSTSTAYMELRSLRTDDTAVYYCARAGYDILTDFSDAFDIWGHGTMVTVSS VH CDR1 amino acid sequence: GYTFNSYG SEQ ID NO: 435 VH CDR2 amino acid sequence: ISVHNGNT SEQ ID NO: 436 VH CDR3 amino acid sequence: ARAGYDILTDFSDAFDI SEQ ID NO: 437 VL domain nucleotide sequence: SEQ ID NO:446 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAACATTAATAACTTTTTAAATTGGTATCAGCAGAAAGAAGGGAAAGGCCCTAAGCTCCTGATCTATGCAGCATCCAGT TTGCAAAGAGGGATACCATCAACGTTCAGTGGCAGTGGATCTGGGACAGACTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACATCTGTCAACAGAGCTACGGTATCCCGTGGGTCGGCCAAGGGACCAAGGTGGAAATCAAA VL domain amino acid sequence: SEQ ID NO: 445 DIQMTQSPSSLSASVGDRVTITCRASQNINNFLNWYQQKEGKGPKLLIYAASSLQRGIPSTFSGSGSGTDFTLTISSLQPEDFATYICQQSYGIPWVGQGTKVEIK VL CDR1 amino acid sequence: QNINNF SEQ ID NO:442 VL CDR2 amino acid sequence: AAS SEQ ID NO: 443 VL CDR3 amino acid sequence: QQSYGIPW SEQ ID NO: 444

[0344] Antibody STIM006 VH domain nucleotide sequence: SEQ ID NO:453 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTTCATGAGCTGGATCCGCCAGGCGCCAGGGAAGGGGCTGGAGTGGATTTCATACATTAGTTCTAGTGGTAGTACCATATACTACGCAGACTCTGTG AGGGGCCGATTCACCATCTCCAGGGACAACGCCAAGTACTCACTGTATCTGCAAATGAACAGCCTGAGATCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATCACTACGATGGTTCGGGGATTTATCCCCTCTACTATTACGGTTTGGACGTCTGGGGCCAGGGGACCACGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO: 454 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYFMSWIRQAPGKGLEWISYISSSGSTIYYADSVRGRFTISRDNAKYSLYLQMNSLRSEDTAVYYCARDHYDGSGIYPLYYYYGLDVWGQGTTVTVSS VH CDR1 amino acid sequence: GFTFSDYF SEQ ID NO: 449 VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO: 450 VH CDR3 amino acid sequence: ARDHYDGSGIYPLYYYYGLDV SEQ ID NO: 451 VL domain nucleotide sequence: SEQ ID NO:460 ATTGTGATGACTCAGTCTCCACTCTCCCTACCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTATTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTT CTTATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGCAGTTTTGGCCAGGGGGACCACGCTGGAGATCAAA VL domain amino acid sequence: SEQ ID NO: 459 IVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDYYLQKPGQSPQLLIYLGSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRSFGQGTTLEIK VL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO: 456 VL CDR2 amino acid sequence: LGS SEQ ID NO: 457 VL CDR3 amino acid sequence: MQALQTPRS SEQ ID NO: 458

[0345] Antibody STIM007 VH domain nucleotide sequence: SEQ ID NO:467 CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTACTGGAGTGGGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGC CCATCTCTGAAGAGCAGACTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO: 466 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSS VH CDR1 amino acid sequence: GFSLSTTGVG SEQ ID NO: 463 VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO: 464 VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO: 465 VL domain nucleotide sequence: SEQ ID NO:474 GAAATTGTATTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAG GGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCACCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAAC VL domain amino acid sequence: SEQ ID NO: 473 EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNWPLTFGGGTKVEIK VL CDR1 amino acid sequence: QSVTNY SEQ ID NO: 470 VL CDR2 amino acid sequence: DAS SEQ ID NO: 471 VL CDR3 amino acid sequence: QHRSNWPLT SEQ ID NO: 472

[0346] Antibody STIM008 VH domain nucleotide sequence: SEQ ID NO:481 CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAGACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTGGAGTGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCAGTCATTTATTGGGATGATGATAAGCGCTACAGC CCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTTCTGTACACACGGATATGGTTCGGCGAGTTATTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO: 480 QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAVIYWDDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYFCTHGYGSASYYHYGMDVWGQGTTVTVSS VH CDR1 amino acid sequence: GFSLSTSGVG SEQ ID NO: 477 VH CDR2 amino acid sequence: IYWDDDK SEQ ID NO: 478 VH CDR3 amino acid sequence: THGYGSASYYHYGMDV SEQ ID NO: 479 VL domain nucleotide sequence: SEQ ID NO:488 GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTACCAACTACTTAGCCTGGCACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACA GGGCCACTGGCATCCCAGCCAGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA VL domain amino acid sequence: SEQ ID NO: 489 EIVLTQSPATLSLSPGERATLSCRASQSVTNYLAWHQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK VL CDR1 amino acid sequence: QSVTNY SEQ ID NO: 484 VL CDR2 amino acid sequence: DAS SEQ ID NO: 485 VL CDR3 amino acid sequence: QQRSNWPLT SEQ ID NO: 486

[0347] Antibody STIM009 VH domain nucleotide sequence: SEQ ID NO:495 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCAAGCCTGGAGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCATACATTAGTAGTAGTGGTAGTACCATATACTACGCAGACTCTG TGAAGGGCCGATTCACCATCTCCAGGGACAACGCCAAGAACTCACTGTATCTGCAAATTAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATTTTTACGATATTTTGACTGATAGTCCGTACTTCTACTACGGTGTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA VH domain amino acid sequence: SEQ ID NO:494 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQINSLRAEDTAVYYCARDFYDILTDSPYFYYGVDVWGQGTTVTVSS VH CDR1 amino acid sequence: GFTFSDYY SEQ ID NO: 491 VH CDR2 amino acid sequence: ISSSGSTI SEQ ID NO: 492 VH CDR3 amino acid sequence: ARDFYDILTDSPYFYYGVDV SEQ ID NO: 493 VL domain nucleotide sequence: SEQ ID NO:502 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTGGGT TCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA VL domain amino acid sequence: SEQ ID NO:501 DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPRTFGQGTKVEIK VL CDR1 amino acid sequence: QSLLHSNGYNY SEQ ID NO: 498 VL CDR2 amino acid sequence: LGS SEQ ID NO: 499 VL CDR3 amino acid sequence: MQALQTPRT SEQ ID NO:500

[0348] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

Table 6-14

Table 6-15

Table 6-16

Table 6-17

Table 6-18

Table 6-19

Table 6-20

Table 6-21

Table 6-22

Table 6-23

Table 6-24

Table 6-25

Table 6-26

Table 6-27

Table 6-28

Table 6-29

Table 6-30

Table 6-31

Table 6-32

Table 6-33

Table 6-34

Table 6-35

Table 6-36

Table 6-37

Table 6-38

Table 6-39

Table 6-40

Table 6-41

Table 6-42

Table 6-43

Table 6-44

Table 6-45

Table 6-46

Table 6-47

Table 6-48

Table 6-49

Table 6-50

Table 6-51

Table 6-52

Table 6-53

Table 6-54

Table 6-55

Table 6-56

Table 6-57

Table 6-58

[0349]

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

Table 7-21

Table 7-22

Table 7-23

Table 7-24

Table 7-25

Table 7-26

Table 7-27

Table 7-28

Table 7-29

Table 7-30

Table 7-31

Table 7-32

Table 7-33

[0350]

Table 8

[0351]

Table 9

[0352]

Table 10

[0353]

Table 11

[0354]

Table 12

[0355]

Table 13

[0356]

Table 14

[0357]

Table 15

[0358]

Table 16-1

Table 16-2

Table 16-3

[0359] [Table 17-1] [Table 17-2] [Table 17-3]

[0360] Clause 1. A method of treating cancer in a patient, wherein the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression, comprising administering to the patient a modulator of ICOS. 2. The method according to item 1, comprising administering to a patient an ICOS modulator and a PD-L1 inhibitor. 3. A method of treating cancer in a patient who has previously received treatment for cancer, wherein the previous treatment for cancer was administration of a PD-L1 inhibitor, and the patient did not respond to the previous treatment or has stopped responding to the previous treatment, and the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression, comprising administering to the patient a modulator of ICOS. 4. The method according to item 3, comprising administering to a patient a modulator of ICOS and an inhibitor of PD-L1. 5. The method according to any one of paragraphs 1 to 4, comprising determining the level of PD-L1 expression in a tumor sample from a patient, and administering an ICOS modulator to the patient if the tumor is a PD-L1 negative tumor or a PD-L1 low expressing tumor. 6. The method according to item 5, comprising administering to the patient a modulator of ICOS and an inhibitor of PD-L1. 7. The method of any one of clauses 2, 4 and 6, wherein the modulator of ICOS and the inhibitor of PD-L1 are administered simultaneously, separately or sequentially. 8. The method according to any one of items 1 to 7, wherein the ICOS modulator is an ICOS agonist. 9. The method according to any one of items 1 to 8, wherein the ICOS agonist is an agonistic anti-ICOS antibody. 10. The method of claim 9, wherein the anti-ICOS antibody is a bispecific antibody that specifically binds to ICOS and PD-L1, or specifically binds to ICOS and PD-1. 11. The method of clause 9, wherein the bispecific antibody is an ICOS agonist and a PD-L1 antagonist, or an ICOS agonist and a PD-1 antagonist. 12. The method according to any one of items 1 to 11, wherein the PD-L1 inhibitor is an anti-PD-L1 binding molecule. 13. The method according to any one of items 1 to 12, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody. 14. The method according to any one of items 1 to 13, wherein the PD-L1 inhibitor inhibits binding of PD-L1 to PD-1. 15. The method according to any one of items 1 to 14, wherein the PD-L1 inhibitor is an antagonistic anti-PD-L1 antibody or an antagonistic ...

Claims

1. An ICOS modulator for use in a method of treating cancer in a patient, wherein the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression, the ICOS modulator comprising administering an ICOS modulator to the patient.

2. An ICOS modulator for use in a method of treating cancer in a patient who has previously received treatment for cancer, wherein the previous treatment for cancer is administration of a PD-L1 inhibitor, the patient has not responded to the previous treatment or the response to the previous treatment has ceased, the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression, the ICOS modulator comprising administering an ICOS modulator to the patient.

3. The method comprises a) determining the level of PD-L1 expression in a tumor sample derived from the patient and, if the tumor is a PD-L1 negative tumor or a PD-L1 low-expression tumor, administering an ICOS modulator to the patient; and / or b) administering an ICOS modulator and an inhibitor of PD-L1 to the patient, optionally, the ICOS modulator and the inhibitor of PD-L1 are administered simultaneously, separately or sequentially, the ICOS modulator for use according to claim 1 or 2.

4. a) The ICOS modulator is an ICOS agonist, optionally, the ICOS agonist is an agonist anti-ICOS antibody, optionally, the anti-ICOS antibody is a bispecific antibody that specifically binds to ICOS and PD-L1 or specifically binds to ICOS and PD-1, optionally, the bispecific antibody is an ICOS agonist and a PD-L1 antagonist or an ICOS agonist and a PD-1 antagonist; b) The PD-L1 inhibitor is an anti-PD-L1 binding molecule, optionally, the PD-L1 inhibitor is an anti-PD-L1 antibody or an anti-PD-1 antibody, optionally, the PD-L1 inhibitor inhibits the binding of PD-L1 to PD-1, optionally, the PD-L1 inhibitor is an antagonist anti-PD-L1 antibody or an antagonist anti-PD-1 antibody; c) The tumor cells are PD-L1 negative or exhibit low PD-L1 expression; d) The tumor contains immune cells, the immune cells are PD-L1 negative or show low PD-L1 expression, optionally the tumor cells are PD-L1 negative or show low PD-L1 expression, and the immune cells are PD-L1 negative or show low PD-L1 expression; e) The cancer is associated with an infectious pathogen, optionally the cancer is a virus-induced cancer, and optionally the virus associated with the virus-induced cancer is selected from HPV (cervical cancer, oropharyngeal cancer), HBV, HCV, and EBV (Burkitt lymphoma, gastric cancer, Hodgkin lymphoma, other EBV-positive B-cell lymphomas, nasopharyngeal cancer, and post-transplant lymphoproliferative disorders), and optionally the cancer is selected from the group consisting of head and neck squamous cell carcinoma, cervical cancer, ano-genital cancer, and oropharyngeal cancer; f) The tumor is positive for HPV (human papillomavirus); and / or g) The patient has been tested for HPV infection and / or the patient has or has had an HPV infection, An ICOS modulator for use according to any one of claims 1 to 3.

5. The method includes the step of determining the HPV status of the patient and / or the step of determining the HPV status of the tumor, and is an ICOS modulator for use according to any one of claims 1 to 4.

6. a) The patient has previously a. been administered a kinase inhibitor; b. received a surgical treatment for cancer (e.g., complete or partial tumor resection) and / or radiotherapy and / or chemotherapy, and optionally the chemotherapy includes docetaxel, fluorouracil, cisplatin, paclitaxel, and / or nab-paclitaxel; and / or c. received a treatment for cancer, and optionally the previous treatment for cancer is the administration of a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody or an anti-PD-1 antibody), and optionally the previous treatment for cancer was the administration of a PD-L1 inhibitor as monotherapy or a PD-L1 inhibitor as a single immunotherapy agent; b) The cancer is a. refractory to PD-L1 inhibitor treatment (e.g., anti-PD-L1 antibody or anti-PD-1 antibody monotherapy); b. treated with PD-L1 inhibitor monotherapy; c. treated with a PD-L1 inhibitor as a single immunotherapy agent; and / or d. treated with nivolumab is refractory to or characterized as refractory; c) the tumor is a CD8+ tumor and / or an ICOS+ tumor; and / or d) The patient has an increase in the level of immune cells (such as ICOS regulatory T cells) after treatment with another therapeutic agent, and optionally, the method comprises administering a therapeutic agent to the patient, determining that the patient has an increase in the level of immune cells (such as ICOS regulatory T cells) after treatment with said agent, and administering a modulator of ICOS (such as an anti-ICOS antibody like an agonist anti-ICOS antibody) to the patient to reduce the level of ICOS regulatory T cells, and optionally, the therapeutic agent is IL-2 or an immunomodulatory antibody (such as anti-PDL-1, anti-PD-1 or anti-CTLA-4). ＋ immune cells (ICOS ＋ regulatory T cells, etc.) having an increase in level, and optionally, the method comprises administering a therapeutic agent to the patient, determining that the patient has an increase in the level of ICOS ＋ immune cells (ICOS ＋ regulatory T cells, etc.) having an increase in level, and administering a modulator of ICOS (such as an anti-ICOS antibody like an agonist anti-ICOS antibody) to the patient to reduce the level of ICOS ＋ regulatory T cells, and optionally, the therapeutic agent is IL-2 or an immunomodulatory antibody (such as anti-PDL-1, anti-PD-1 or anti-CTLA-4). An ICOS modulator for use according to any one of claims 1 to 5. **Claim 7** The method comprises administering a single dose of an ICOS modulator, optionally followed by multiple doses of a PD-L1 inhibitor, an ICOS modulator for use according to any one of claims 1 to 6. **Claim 8** The treatment is a. resulting in a reduction in tumor size; b. inhibiting tumor growth; c. resulting in stable disease; d. prolonging the patient's survival and / or delaying disease progression; e. depleting ICO⁺ immune cells (such as ICO ＋ regulatory T cells, etc.) in the tumor microenvironment; and / or ＋ ​ f. increasing the ratio of CD8+ to ICOS+ immune cells (e.g., the ratio of CD8+ to ICOS+ regulatory T cells) in the tumor microenvironment, An ICOS modulator for use according to any one of claims 1 to 7. **Claim 9** The ICOS modulator is an anti-ICOS antibody, optionally the anti-ICOS antibody is any of the following antibodies, or comprises the VH and VL domains of any of the following antibodies, or comprises the HCDR and / or LCDR of any of the following antibodies: a. KY1044; b. anti-ICOS antibodies described in PCT / GB2017 / 052352, WO2018 / 029474 or US9957323, the contents of which are incorporated herein by reference (e.g., STIM001, STIM002, STIM002B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009); c. anti-ICOS antibodies described in PCT / GB2018 / 053701, WO2019 / 122884, the contents of which are incorporated herein by reference (e.g., STIM017, STIM020, STIM021, STIM022, STIM023, STIM039, STIM040, STIM041, STIM042, STIM043, STIM044, STIM050, STIM051, STIM052, STIM053, STIM054, STIM055, STIM056, STIM057, STIM058, STIM059, STIM060, STIM061, STIM062, STIM063, STIM064, STIM065 or STIM066); d. The anti-ICOS / PD-L1 mAb2 bispecific antibody described in PCT / GB2018 / 053698 and WO2019 / 122882; e. Bopratelimab; f. The anti-ICOS antibody described in WO2016 / 154177 or US2016 / 0304610 (for example, 37A10S713, 7F12, 37A10, 35A9, 36E10, 16G10, 37A10S714, 37A10S715, 37A10S716, 37A10S717, 37A10S718, 16G10S71, 16G10S72, 16G10S73, 16G10S83, 35A9S79, 35A9S710 or 35A9S89); g. The anti-ICOS antibody described in WO2016 / 120789 or US2016 / 0215059 (for example, 422.2 H2L5); h. Antibody C398.4 described in WO2018 / 187613 or US2018 / 0289790 or its humanized antibody, for example, ICOS.33 IgG1f S267E, ICOS.4, ICOS34 G1f, ICOS35 G1f, 17C4, 9D5, 3E8, 1D7a, 1D7b or 2644 (see Table 35 of WO2018187613 for the sequence), for example, antibody BMS-986226 in NCT03251924; i. Antibody JMAb 136, "136", or any other antibody described in WO2010 / 056804; j. Antibody 314-8 described in WO2012 / 131004, WO2014 / 033327 or US2015 / 0239978, the antibody generated from hybridoma CNCM I-4180, or any other anti-ICOS antibody; k. Antibody Icos145-1 described in WO2012 / 131004, US9,376,493 or US2016 / 0264666, the antibody produced by hybridoma CNCM I-4179, or any other antibody; l. The antibody MIC-944 (derived from hybridoma DSMZ 2645), 9F3 (DSMZ 2646) or any other anti-ICOS antibody described in WO99 / 15553, US7,259,247, US7,132,099, US7,125,551, US7,306,800, US7,722,872, WO05 / 103086, US8,318,905 or US8,916,155; m. Anti-ICOS antibodies described in WO98 / 3821, US7,932,358B2, US2002 / 156242, US7,030,225, US7,045,615, US7,279,560, US7,226,909, US7,196,175, US7,932,358, US8,389,690, WO02 / 070010, US7,438,905, US7,438,905, WO01 / 87981, US6,803,039, US7,166,283, US7,988,965, WO01 / 15732, US7,465,445 or US7,998,478 (e.g., JMAb-124, JMAb-126, JMAb-127, JMAb-128, JMAb-135, JMAb-136, JMAb-137, JMAb-138, JMAb-139, JMAb-140 or JMAb-141, e.g., JMAb136); n. Anti-ICOS antibodies described in WO2014 / 08911; o. Anti-ICOS antibodies described in WO2012 / 174338; p. Anti-ICOS antibodies described in US2016 / 0145344; q. Anti-ICOS antibodies described in WO2011 / 020024, US2016 / 002336, US2016 / 024211 or US8,840,889; r. Anti-ICOS antibodies described in US8,497,244; or s. Antibody clones ISA-3 (eBioscience), clone SP98 (Novus Biologicals), clone 1G1, clone 3G4 (Abnova Corporation), clone 669222 (R&D Systems), clone TQ09 (Creative Diagnostics), clone 2C7 (Deng et al Hybridoma Hybromics 2004), clone ISA-3 (eBioscience) or clone 17G9 (McAdam et al J Immunol 2000), An ICOS modulator for use according to any one of claims 1 to 8.

10. The anti-ICOS antibody is an antibody that binds to the extracellular domain of human and / or mouse ICOS, and the antibody comprises a VH domain comprising an amino acid sequence having at least 95% sequence identity to the VH domain of STIM003 of SEQ ID NO: 408, and a VL domain comprising an amino acid sequence having at least 95% sequence identity to the VL domain of STIM003 of SEQ ID NO: 415, Optionally, a) The VH domain comprises a set of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, where a. HCDR1 is STIM003 HCDR1 having the amino acid sequence of SEQ ID NO: 405, b. HCDR2 is STIM003 HCDR2 having the amino acid sequence of SEQ ID NO: 406, c. HCDR3 is STIM003 HCDR3 having the amino acid sequence of SEQ ID NO: 407; and / or The VL domain comprises a set of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, where: d. LCDR1 is STIM003 LCD R1 having the amino acid sequence of SEQ ID NO: 412, e. LCDR2 is STIM003 LCDR2 having the amino acid sequence of SEQ ID NO: 413, f. LCDR3 is STIM003 LCDR3 having the amino acid sequence of SEQ ID NO: 414; b) The VH domain amino acid sequence is SEQ ID NO: 408, and / or the VL domain amino acid sequence is SEQ ID NO: 415; or c) The anti-ICOS antibody is a VH domain of an antibody comprising complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, and An antibody that binds to the extracellular domain of human and / or mouse ICOS and comprises the VL domain of an antibody comprising complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 is the HCDR1 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises an HCDR1 having 1, 2, 3, 4, or 5 amino acid changes thereof, HCDR2 is the HCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises an HCDR2 having 1, 2, 3, 4, or 5 amino acid changes thereof, and / or HCDR3 is the HCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises an HCDR3 having 1, 2, 3, 4, or 5 amino acid changes thereof; and / or LCDR1 is the LCDR1 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises an LCDR1 having 1, 2, 3, 4, or 5 amino acid changes thereof, LCDR2 is the LCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises an LCDR2 having 1, 2, 3, 4, or 5 amino acid changes thereof, and / or LCDR3 is the LCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009, or comprises an LCDR3 having 1, 2, 3, 4, or 5 amino acid changes thereof, optionally, i) The heavy-chain CDRs of the antibody are those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or include the heavy-chain CDRs of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 having 1, 2, 3, 4 or 5 amino acid changes; and / or the light-chain CDRs of the antibody are those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or include the light-chain CDRs of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 having 1, 2, 3, 4 or 5 amino acid changes, optionally, the VH domain of the antibody has the heavy-chain CDRs of STIM00 3 and / or the VL domain of the antibody has the light-chain CDRs of STIM003; ii) The antibody includes the framework regions of the VH and / or VL domains of human germline gene segment sequences; and / or iii) The antibody comprises a VH domain that is the VH domain of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or has an amino acid sequence that is at least 90% identical to the VH domain sequence of an antibody of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009; and / or the antibody comprises a VL domain that is the VL domain of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or has an amino acid sequence that is at least 90% identical to the VL domain sequence of an antibody of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, optionally the antibody comprises the VH domain of STIM003 and the VL domain of STIM003, optionally the anti-ICOS antibody is STIM0003, An ICOS modulator for use according to claim 9.

11. a) The antibody comprises an antibody constant region, optionally the constant region comprises a human heavy chain and / or light chain constant region, optionally the constant region is Fc effector positive; and / or b) The antibody is a multispecific antibody. An ICOS modulator for use according to claim 9 or 10.

12. The anti-ICOS antibody is an antibody that binds to the extracellular domain of human and mouse ICOS with an affinity (KD) of less than 50 nM as determined by surface plasmon resonance, or less than 5 nM as determined by surface plasmon resonance, an ICOS modulator for use according to claim 9.

13. a) The PD-L1 inhibitor is i. An anti-PD-L1 antibody selected from the group consisting of atezolizumab (Roche), avelumab (Merck), durvalumab / MedImmune 4736 (MedImmune), KN035, CK-301, AUNP12, CA-170, BMS-936559 / MDX-1105 (BMS), FAZ-053 M7824, AbbV-368, LY-3300054, GNS-1480, YW243.55.S70, REGN3504, and those disclosed in WO2017 / 220990, WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801, WO2016 / 111645, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 160792, WO2016 / 022630, WO2016 / 007235, WO2015 / 179654, WO2015 / 173267, WO2015 / 181342, WO2015 / 109124, WO2015 / 112805, WO2015 / 061668, WO2014 / 159562, WO2014 / 165082, WO2014 / 100079, WO2014 / 055897, WO2013 / 181634, WO2013 / 173223, WO2013 / 079174, WO2012 / 145493, WO2011 / 066389, WO2010 / 077634, WO2010 / 036959, WO2010 / 089411 or WO2007 / 005874; or an anti-PD-L1 antibody selected from the group consisting of any of the foregoing; or ii. Pembrolizumab, nivolumab, semiprimab, JTX-401, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224 and AMP-514, MEDI-0680 / AMP514, PDR001, ranburizumab, BMS-936558, REGN2810, BGB-A317, BGB-108, PDR-001, SHR-1210, JS-001, JNJ-63723283, AGEN-2034, PF-06801591, genolimzumab, MGA-012 (INCMGA00012), IBI-308, BCD-100, TSR-042 ANA011, AUNP-12, KD033, MCLA-134, mDX400, muDX400, STI-A1110, AB011, 244C8, 388D4, XCE853, or pidilizumab / CT-011, or from the group consisting of WO2015 / 112800 and US2015 / 0203579 (including the antibodies in Tables 1-3), US9,394,365, US5,897,862 and US7,488,No. 802, WO2017 / 087599 (including antibodies SSI-361 and SHB-617), WO2017 / 079112, WO2017 / 071625 (deposit C2015132, hybridoma LT004, and including antibodies 6F5 / 6F5(Re), 6F5H1L1 and 6F5H2L2), WO2017 / 058859 (including PD1AB-1 to PD1AB-6), WO2017 / 058115 (including 67D9, c67D9 and hu67D9), WO2017 / 055547 (including 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375 and 13112.15380), WO2017 / 040790 (including AGEN2033w, AGEN2034w, AGEN2046w, AGEN2047w, AGEN2001w and AGEN2002w), WO2017 / 025051 and WO2017 / 024515 (1.7.3 hAb, 1.49.9 hAb, 1.103.11, including hAb, 1.103.11-v2 hAb, 1.139.15 hAb and 1.153.7 hAb), WO2017 / 025016 and WO2017 / 024465 (including antibodies A to I), WO2017 / 020858 and WO2017 / 020291 (including 1.4.1, 1.14.4, 1.20.15 and 1.46.11), WO2017 / 019896 and WO2015 / 112900 and US2015 / 0210769 (including BAP049-hum01 to BAP049-hum16 and BAP049-clone-A to BAP049-clone-E), WO2017 / 019846 (including PD-1 mAb 1 to PD-1 mAb 15), WO2017 / 016497 (including MHC723, MHC724, MHC725, MHC728, MHC729, m136-M13, m136-M19, m245-M3, m245-M5 and m136-M14), WO2016 / 201051 (including antibody EH12.2H7, antibody hPD-1 mAb2, antibody hPD-1 mAb7, antibody hPD-1 mAb9, antibody hPD-1 mAb15, or an anti-PD-1 antibody selected from Table 1), WO2016 / 197497 (including DFP D1-1 to DFP D1-13), WO2016 / 197367 (including 2.74.15 and 2.74.15.hAb4 to 2.74.15.hAb8), WO2016 / 196173 (including Table 5 and the antibodies in Figures 1 to 5), WO2016 / 127179 (including R3A1, R3A2, R4B3 and R3D6), WO2016 / 077397 (including the antibodies described in Table 1 of Example 9), WO2016 / 106159 (including the mouse antibodies in Table 3 of Example 2 and the humanized antibodies in Tables 7, 8 and 9 of Example 3), WO2016 / 092419 (including C1, C2, C3, EH12.1, mAb7-G4, mAb15-G4, mAb-AAA, mAb15-AAA), WO2016 / 068801 (including clone A3 and its variants, and the other antibodies described in Figures 1 to 4), WO2016 / 014688 (including 10D1, 4C10, 7D3, 13F1, 15H5, 14A6, 22A5, 6E1, 5A8, 7A4, and 7A4D, and the humanized antibodies of Examples 9 / 10), WO2016 / 015685 (including 10F8, BA08-1, BA-08-2 and 15H6), WO2015 / 091911 and WO2015 / 091910 (including the anti-canine PD-1 antibodies in Examples 2, 3 and 4), WO2015 / 091914 (including the anti-canine PD-1 antibodies in Table 3), WO2015 / 085847 (including mAb005, H005-1 to H005-4), WO2015 / 058573 (including cAB7), WO2015 / 036394 (including LOPD180), WO2015 / 035606 (including the antibodies in Table 1 of Example 2, Tables 14, 15 and 16 of Example 7, and Tables 20, 21 and 22 of Example 11), WO2014 / 194302 (including GA2, RG1B3, RG1H10, RG2A7, RG2H10, SH-A4, RG4A6, GA1, GB1, GB6, GH1, A2, C7, H7, SH-A4, SH-A9, RG1H11 and RG6B), WO2014 / 179664 (including 9A2, 10B11, 6E9, APE1922, APE1923, APE1924, APE1950, APE1963 and APE2058), WO2014 / 206107 (including clones 1, 10, 11, 55, 64, 38, 39, 41 and 48), WO2012 / 135408 (including h409A11, h409A16 and h409A17), WO2012 / 145493 (antibodies 1E3, 1E8, 1H3 and h1H3 Var 1 to h1H3 Varan anti-PD-1 antibody selected from any one of the anti-PD-1 antibodies described in WO2011 / 110621 (including antibody 949 and modified versions disclosed in Figures 1 to 11), WO2011 / 110604 (including antibody 948 and modified versions disclosed in Figures 3 to 11), WO2010 / 089411 (including CNCM deposit numbers 1-4122, 1-4080 or 1-4081), WO2010 / 036959 (including the antibodies in Table 1 of Example 1), WO2010 / 029435 and WO2010 / 029434 (including clones 2, 10 and 19), WO2008 / 156712 (including hPD-1.08A, hPD-1.09A, h409A11, h409A16 and h409A17, and the antibodies described in Example 2, Table H, Example 4 and Table IV), WO2006 / 121168 (including clones 17D8, 4H1, 5C4, 4A11, 7D3, 5F4 and 2D3), WO2004 / 004771 and WO2004 / 056875 (including PD1-17, PD1-28, PD1-33, PD1-35, PD1-F2 and the Abs described in Table 1) is; b) The ICOS modulator is an IgG1 anti-ICOS antibody, and / or the PD-L1 inhibitor is an IgG1 anti-PD-L1 antibody or an IgG1 anti-PD-1 antibody. Optionally, the IgG1 anti-ICOS antibody and / or the IgG1 anti-PD-L1 antibody or anti-PD-1 antibody comprises a human IgG1 constant region comprising the amino acid sequence of SEQ ID NO: 340; and / or c) The cancer is liver cancer (e.g., hepatocellular carcinoma), renal cell carcinoma, head and neck cancer (e.g., metastatic squamous cell carcinoma), melanoma, non-small cell lung cancer, diffuse large B-cell lymphoma, breast cancer (e.g., triple-negative breast cancer), penile cancer, pancreatic cancer or esophageal cancer. An ICOS modulator for use according to any one of claims 1 to 12.

14. An ICOS modulator for use in a method of treating cancer in a patient, a) the patient has a PD-L1 negative tumor or a tumor with low PD-L1 expression; or b) the patient has previously received treatment for cancer and the patient has not responded to the previous treatment or the response to the previous treatment has ceased, and the previous treatment for cancer was a PD-L1 inhibitor. An ICOS modulator.

15. a) The ICOS modulator is for use in combination with a PD-L1 inhibitor. Optionally, the ICOS modulator is an agonist anti-ICOS antibody. Optionally, the ICOS modulator is a bispecific antibody that is an anti-ICOS agonist and an anti-PD-L1 antagonist, or a bispecific antibody that is an anti-ICOS agonist and an anti-PD-1 antagonist; and / or b) The method is the method according to any one of claims 1 to 13. An ICOS modulator for use according to claim 14.

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