Anti-ICOS antibody

Abstract

To provide antibodies that bind ICOS (inducible T cell co-stimulator), compositions, and methods of producing the antibodies.SOLUTION: An isolated antibody binding to the extracellular domain of a human and / or mouse ICOS comprises: a VH domain comprising an amino acid sequence having at least 95% sequence identity to the STIM003 VH domain with specific sequence; and a VL domain comprising an amino acid sequence having at least 95% sequence identity to the STIM003 VL domain with another specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions for stimulating a mammalian immune response, in particular a T cell response. The invention also relates to immuno-oncology, which involves anti-tumor therapy through the promotion of anti-tumor T cell responses in patients. and the medical use of such compositions in, for example, stimulating effector T cells and / or alters the balance between effector and regulatory T cells through the depletion of regulatory T cells. Other diseases and conditions in which modulating effector T cells in favor of them has therapeutic benefit. The present invention relates to the use of the composition in the treatment of [Background technology]

[0002] ICOS (inducible T cell costimulator) is a protein involved in the regulation of immune responses, particularly humoral immune responses. It is a member of the CD28 gene family and was first identified in 1999 [1]. It is a disulfide-linked homodimer with two differently glycosylated subunits. ICOS is a 55 kDa transmembrane protein present as a single molecule on T lymphocytes. It is expressed exclusively in various T cell subsets, including naive T lymphocytes. Although present at low levels on TCR, its expression is rapidly induced by immune activation and It is upregulated in response to proinflammatory stimuli, such as inflammation and costimulation with CD28 [2, 3]. COS is a pathway that is involved in the late stages of T cell activation, the late stages of memory T cell formation, and importantly, the T cell It plays a role in regulating humoral responses via B cell-dependent responses [4, 5]. ICOS binds to PI3K and binds to the kinase phophoinositides. Activates tide-dependent kinase 1 (PDK1) and protein kinase B (PKB) Activation of ICOS prevents cell death and upregulates cellular metabolism. In the absence (ICOS knockout) or in the presence of anti-ICOS neutralizing antibodies, a proinflammatory response There is a suppression of

[0003] ICOS is expressed on B cells and antigen-presenting cells (APCs) by the ICOS ligand (IC It binds to the IL-1 receptor (OSL) [6, 7], which acts as a costimulatory molecule and mediates TCR-mediated responses to antigens. Helps regulate immune and antibody responses. This cell type plays a negative role in the immune surveillance of cancer cells. The expression of ICOS on T regulatory cells is important because it has been suggested that they play a role in the regulation of ovarian function. There is emerging evidence for this in follicular carcinoma [8]. Importantly, ICOS expression , compared with CD4+ and CD8+ effector cells present in the tumor microenvironment, It has been reported that Fc-mediated cell effector functions are higher on regulatory T cells (TReg). Depletion of TRegs using antibodies with Treg-specific T cell proliferation has shown strong antitumor effects in preclinical models. [9] Increasing evidence supports the finding that ICOS can be effectively treated with immune checkpoint inhibitors. ICOS or IFN-γ-phosphate dehydrogenase (ICOS) has been shown to be involved in antitumor effects in both animal models and patients. Although the antitumor effect of anti-CTLA4 therapy is reduced in ICOSL-deficient mice

[10] , In normal mice, ICOS ligand inhibits anti-CTLA4 treatment in melanoma and prostate cancer.

[11] Furthermore, in humans, a retrospective study of patients with advanced melanoma Furthermore, ICOS levels were shown to increase after ipilimumab (anti-CTLA4) treatment

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

[0013] . In cancer patients treated with anti-CTLA4 therapy, tumor-specific IFNγ-producing CD4 The majority of T cells were ICOS-positive, and at the same time, the persistent ICOS-positive CD4 T cells It has also been observed that elevation correlates with survival [ 12 , 13 , 14 ].

[0004] WO2016 / 120789 describes an anti-ICOS antibody that activates T cells and their use for treating cancer, infections, and / or sepsis has been proposed. A number of murine anti-ICOS antibodies have been generated, a subset of which are homologous to the human ICOS receptor. Antibody 422.2 was selected as the lead anti-ICOS antibody. The antibody was selected and humanized to produce a human IgG4PE antibody named H2L5. L5 has a 1.34 nM response to human ICOS and a 0.9 nM response to cynomolgus monkey ICOS. It has an affinity of 5 nM and induces cytokine production in T cells, and together with CD3 stimulation, It was reported that it up-regulates cell activation markers. However, transplanted human melanoma cells Mice bearing the H2L5 hIgG4PE treatment showed significantly higher IgG4 expression than control-treated mice. It was reported that this antibody showed only minimal tumor growth delay or increased survival. In combination experiments with ipilimumab (anti-CTLA-4) or pembrolizumab (anti-PD-1), demonstrated further inhibition of tumor growth compared with ipilimumab or pembrolizumab monotherapy Finally, in mice bearing transplanted colon cancer cells (CT26), In this study, low-dose mice were administered ipilimumab or pembrolizumab in combination with mouse surrogates. Cross-reactive H2L5 surrogates improved overall survival compared with anti-CTL4 and anti-PD1 therapy alone In mice bearing transplanted EMT6 cells, A similar lack of strong therapeutic effect was demonstrated.

[0005] Further examples of anti-ICOS antibodies are described in WO2016 / 154177. These antibodies target CD4+ T cells, including effector CD8+ T cells (TEff). It has been reported to be an agonist and to deplete T regulatory cells (TRegs). The selective effect of antibodies on TEFF cells versus TReg cells has been described, and these antibodies can preferentially deplete TRegs, but not TRegs expressing lower levels of ICOS. Anti-ICOS antibodies have minimal effect on TEff, which is involved in the treatment of cancer. It has been proposed as a treatment for PD-1 / PD-L1 in combination with other anti-PD-1 or anti-PD-L1 antibodies. Summary of the Invention

[0006] Antibodies against ICOS that act to increase effector T cell activity have been reported in various Diseases and conditions and immune responses in which a CD8+ T cell response is beneficial, including vaccination regimens Represents a therapeutic approach in immuno-oncology and other medical settings. Many of the therapeutic approaches involve an immune component. In diseases and conditions, effector T cells (TEs) mediate CD8+ T cell immune responses. ff), and regulatory T cells (T The present invention aims to utilize this TEff / TReg balance. The present invention relates to antibodies that favorably modulate effector T cell activity. Antibodies that cause depletion of regulatory T cells relieve the suppression of TEff, thereby suppressing the effector The addition of anti-ICOS antibodies or A complementary mechanism is through agonistic activity at the ICOS receptor level, which activates effector T cells. The goal is to stimulate a response.

[0007] Relative ICOS on effector T cells (TEff) compared to regulatory T cells (TReg) The differential expression and relative activity of these cell populations will determine the overall efficacy of anti-ICOS antibodies in vivo. The expected mode of action is agonism of effector T cells. and depletion of ICOS-positive regulatory T cells. The differential and even opposing effects on IL-1 and IL-2 are due to their different levels of ICOS expression. This may be achievable by double engineering of the variable and constant regions of the anti-ICOS antibody. This has a net positive effect on effector T cell responses by affecting the CD8 / TReg ratio. It is possible to provide a molecule that exerts the effect of activating the ICOS receptor. The antigen-binding domain of an antibody may be involved in down-regulation and / or clearance of highly expressing cells to which the antibody binds. The effector positive constant region may be combined with an antibody constant (Fc) region to enhance target activity. Recruiting cellular effector functions against target cells (TReg) to e.g., antibody-dependent cellular mediators It can be used to promote antibody-mediated cell cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). Therefore, antibodies play a key role in promoting effector T cell activation and suppressing immune-suppressive T regulatory cells. ICOS may act both to downregulate TRegs and to downregulate TEffs. Since TRef is highly expressed, a therapeutic balance is achieved, whereby TRef depletion At the same time, Teff function is promoted, enhancing T cell immune responses (e.g., anti-tumor responses or other therapeutic responses). This results in a net increase in beneficial T cell responses.

[0008] Several preclinical and clinical studies have demonstrated that it is a potent effector in the tumor microenvironment (TME). A strong positive correlation was shown between the ratio of T cells to T-reg cells and overall survival in ovarian cancer patients. It has been reported that the ratio of CD8:T-reg cells is an indicator of favorable clinical outcomes.

[15] Similar findings were observed in patients with metastatic melanoma after receiving ipilumab

[0016] . Preclinical studies have shown that a high effector cell:T-reg ratio in the TME is associated with anti-tumor activity. It has also been shown to be associated with response

[43] .

[0009] The present invention provides an antibody that binds to human ICOS. By targeting the IFNγ receptor, it binds to T cells expressing ICOS. Agonistic activity on ICOS as demonstrated by its ability to increase secretion Examples of antibodies designed to enhance effector T cell function are provided. Thus, anti-ICOS antibodies may be engineered to deplete the cells to which they bind. This may be due to preferential downregulation of regulatory T cells, resulting in their inhibition of effector T cell responses. These cells should enhance the suppressive effect of T cells, thereby promoting overall effector T cell responses. Regardless of their mechanism of action, the anti-ICOS antibodies according to the present invention are It has been experimentally demonstrated that it stimulates T cell responses and has antitumor effects in vivo. have the desired level of Fc effector function, or where appropriate, Select an appropriate antibody format, such as one containing a constant region lacking essential effector functions. By this, the anti-ICOS antibody is beneficial in inducing effector T cell responses and / or for use in a variety of medical settings, including the treatment of diseases and conditions in which suppression of regulatory T cells is desirable. can be adjusted to suit.

[0010] Exemplary antibodies include those having the sequences described herein: STIM001, STIM0 02, STIM002-B, STIM003, STIM004, STIM005, STI These include M006, STIM007, STIM008, and STIM009.

[0011] The antibodies according to the present invention are identified as STIM001, STIM002 for binding to human ICOS. , STIM002-B, STIM003, STIM004, STIM005, STIM0 Heavy and light chain complementarity determination of STIM006, STIM007, STIM008, or STIM009 An antibody (e.g., human IgG1, or scFv) comprising the CDRs, optionally STIM0 01, STIM002, STIM002-B, STIM003, STIM004, STI M005, STIM006, STIM007, STIM008, or STIM009 V It may be one that competes with an antibody comprising H and VL domains.

[0012] The antibodies according to the present invention include STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, STIM009, STIM1001, STIM102, STIM103, STIM104, STIM105, STIM106, STIM107, STIM108-B, STIM109, M003, STIM004, STIM005, STIM006, STIM007, STI M008, and one or more CDRs of STIM009 (e.g., any such all six CDRs of a suitable antibody, or a set of HCDRs and / or LCDRs), or It may also include variants thereof as described in the specification.

[0013] The antibody has an antibody VH domain containing CDRs HCDR1, HCDR2, and HCDR3. and an antibody VL domain containing CDRs LCDR1, LCDR2, and LCDR3. HCDR3 may comprise STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, HCDR3 of an antibody selected from STIM008 and STIM009, or , and its HCDR3 with 2, 3, 4 or 5 amino acid modifications. , which may be the HCDR2 of the selected antibody, or may be 1, 2, 3, 4 or 5 amino acids. HCDR1 may comprise its HCDR2 having an amino acid modification. DR1 or its H with 1, 2, 3, 4 or 5 amino acid modifications. It may also include CDR1.

[0014] The antibody has an antibody VL domain containing CDRs HCDR1, HCDR2, and LCDR3. and an antibody VL domain containing CDRs LCDR1, LCDR2, and LCDR3. and LCDR3 may include STIM001, STIM002, STIM002-B , STIM003, STIM004, STIM005, STIM006, STIM007 , STIM008, and STIM009; or LCDR3 having 1, 2, 3, 4 or 5 amino acid alterations. LCDR2 may be the LCDR2 of the selected antibody, or may be 1, 2, 3, 4 or 5 LCDR1 may comprise its LCDR2 having amino acid modifications. It may be CDR1 or its variants with 1, 2, 3, 4 or 5 amino acid modifications. It may also include LCDR1.

[0015] The antibody is an antibody VH domain comprising complementarity determining regions HCDR1, HCDR2 and HCDR3; an antibody VL domain comprising complementarity determining regions LCDR1, LCDR2 and LCDR3; It may also include The antibody heavy chain CDRs are: STIM001, STIM002, STIM002-B, STIM 003, STIM004, STIM005, STIM006, STIM007, STIM 008 or STIM009, or 1, 2, 3, 4 or 5 meshes STIM001, STIM002, STIM002-B, and STIM0 have the acid modification 03, STIM004 or STIM005, STIM006, STIM007, ST IM008 or STIM009 heavy chain complementarity determining regions; and / or The antibody light chain CDRs are those of antibodies STIM001, STIM002, STIM002-B, ST IM003, STIM004, STIM005, STIM006, STIM007, ST IM008 or STIM009, or 1, 2, 3, 4 or 5 STIM001, STIM002, STIM002-B, and STI, which have amino acid modifications M003, STIM004, STIM005, STIM006, STIM007, STI Contains the M008 or STIM009 light chain complementarity determining region.

[0016] The antibody comprises one set of heavy chain complementarity determining regions (HCDRs): HCDR1, HCDR2 and HCDR3: HCDR1 is HCDR1 of STIM003; HCDR2 is HCDR2 of STIM003, HCDR3 is that of STIM003 or said HCDR sequence having 1, 2, 3, 4, 5 or 6 amino acid modifications. The VH domain may comprise a VH domain containing a VH fragment.

[0017] The antibody contains one set of light chain complementarity determining regions (LCDRs): LCDR1, LCDR2 and LCDR3: LCDR1 is LCDR1 of STIM003, LCDR2 is the LCDR2 of STIM003, LCDR3 is the LCDR3 of STIM003. or a set of LCDRs having 1, 2, 3 or 4 amino acid alterations. It may also comprise a VL domain containing

[0018] The amino acid modification (e.g., substitution) can be at any residue position in the CDR. Examples are shown in Figures 35, 36, and 37, which show alignments of variant sequences of anti-ICOS antibodies. Therefore, the amino acid modifications in the STIM003 CDRs are shown in Figure 36 As shown in Figure 1, the corresponding position in antibody CL-74570 or antibody CL-71642 The substitution may be for a residue that is

[0019] Examples of amino acid modifications in the STIM003 CDRs are defined according to IMGT as follows: The substitution at residue position is:

[0020] In HCDR1, a substitution at position 28 of IMGT, optionally a conservative substitution, e.g., V2 8F.

[0021] In HCDR2, substitutions at positions 59, 63, and / or 64 of IMGT. , the substitution at position 59 is N59I, the substitution at position 63 is G63D, and / or The substitutions at positions are D64N and / or D64S.

[0022] In HCDR3, substitutions at positions 106, 108, 109, and / or 112 of IMGT Optionally, the substitution at position 106 is R106A and the substitution at position 108 is F108Y and the substitution at position 109 is Y109F, and / or the substitution at position 112 is H112 It's N.

[0023] A substitution at position 36 in LCDR1, for example R36S.

[0024] Substitutions at positions 105, 108, and / or 109 in LCDR3. the substitution at position 05 is H105Q and the substitution at position 108 is D108G, and / or The substitution at position 109 is M109N or M109S.

[0025] The antibodies of the invention may comprise VH and / or VL sequences corresponding to human germline gene segment sequences. It may include domain framework regions, for example, STIM001, STIM0 02, STIM002-B, STIM003, STIM004, STIM005, STI One or more frames of M006, STIM007, STIM008, or STIM009 The framework region(s) may include FR1, FR2, F It may be R3, and / or FR4.

[0026] As described in Example 12, Table E12-1 shows the V Table E12 shows the human germline V, D, and J gene segments that generated the H domain. -2 is derived from the human germline V and J genes that recombined to generate the VL domains of these antibodies. The antibody VH and VL domains of the present invention are composed of these V(D)J segments. The criteria may be based on the

[0027] The antibody of the present invention (i) a human heavy chain V gene segment, a human heavy chain D gene segment, and a human heavy chain J gene a VH domain derived from recombination of segments, The V segment is IGHV1-18 (e.g., V1-18 * 01), IGVH3-20( For example, 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), IGH D3-10 (e.g., IGHD3-10 * 01) or IGHD3-9 (e.g., IGH D3-9 * 01) and / or The J gene segment is IGHJ6 (e.g., IGHJ6 * 02), IGHJ4 (e.g. , IGHJ4 * 02) or IGHJ3 (e.g., IGHJ3 * 02) VH Dome In, or (ii) a VH domain comprising framework regions FR1, FR2, FR3, and FR4; So, FR1 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGHV1-18 (e.g., V1-18 * 01), IGVH3-2 0 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) Young IGVH2-5 (e.g., V2-5 * 10) and aligned, FR2 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGHV1-18 (e.g., V1-18 * 01), IGVH3-2 0 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) Young IGVH2-5 (e.g., V2-5 * 10) and aligned, FR3 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGHV1-18 (e.g., V1-18 * 01), IGVH3-2 0 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) Young IGVH2-5 (e.g., V2-5 * 10) and / or FR4 may have 1, 2, 3, 4 or 5 amino acid modifications. Cytoplasmic V gene segment IJH6 (e.g., JH6 * 02), IGJH4 (e.g., JH 4 * 02) or IGJH3 (e.g., JH3 * 02), containing a VH domain That's fine too.

[0028] FR1, FR2, and FR3 of a VH domain are typically from the same germline V gene. Thus, for example, the present antibody is a human heavy chain V gene segment I GHV3-20 (e.g., VH3-20 * d01), human heavy chain D gene segment and human Heavy chain J gene segment IJH4 (e.g., JH4 * VH domain derived from recombination of The antibody may comprise VH domain framework regions FR1, FR2, FR3, , and FR4, and FR1, FR2, and FR3 are 1, 2, 3, 4, or a human germline V gene segment IGHV3 with up to five amino acid modifications. -20 (e.g., IGVH3-20 * d01), and FR4 is 1, 2, 3, 4, or Human germline J gene segment IGHJ4 (e.g., For example, IGHJ4 * 02). The alignment may be exact, but in some cases Depending on the amino acid sequence, one or more residues can be mutated from the germline. Acid substitutions or, more rarely, deletions or insertions may be present.

[0029] The antibody of the present invention (i) an antibody derived from the recombination of a human light chain V gene segment and a human light chain J gene segment; a VL domain, 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 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) an antibody VL domain comprising framework regions FR1, FR2, FR3, and FR4; And, FR1 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGKV2-28 (e.g., IGKV2-28 * 01), IGKV 3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1 D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned , FR2 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGKV2-28 (e.g., IGKV2-28 * 01), IGKV 3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1 D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned , FR3 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGKV2-28 (e.g., IGKV2-28 * 01), IGKV 3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1 D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned and / or FR4 may have 1, 2, 3, 4 or 5 amino acid modifications. Cytoplasmic V gene segment IGKJ4 (e.g., IGKJ4 *01), IGKJ2 (e.g., IGKJ2 * 04), IGKJ3 (e.g., IGKJ3 * 01) or IGKJ1 (e.g. , IGKJ1 * 01).

[0030] FR1, FR2, and FR3 of a VL domain are typically from the same germline V gene. Thus, for example, the present antibody is a human light chain V gene segment I GKV3-20 (e.g., IGKV3-20 * 01), human light chain J gene segment IGK J3 (e.g., IGKJ3 * 01). may include VL domain framework regions FR1, FR2, FR3, and FR4. FR1, FR2, and FR3 may each contain up to 1, 2, 3, 4, or 5 amino acid modifications. a human germline V gene segment IGKV3-20 (e.g., IGKV 3-20 * 01), and FR4 may contain up to 1, 2, 3, 4, or 5 amino acid modifications. having a human germline J gene segment IGKJ3 (e.g., IGKJ3 * 01) and The alignment can be accurate, but in some cases, the One or more residues can be mutated, resulting in amino acid substitutions, or, more rarely, deletions or deletions. There may be an insertion or

[0031] The antibodies according to the present invention include STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, STIM009, STIM1001, STIM102, STIM103, STIM104, STIM105, STIM106, STIM107, STIM108-B, STIM109, M003, STIM004 or STIM005, STIM006, STIM007, the VH domain of STIM008 or STIM009, or STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM00 5. Antibody V of STIM006, STIM007, STIM008 or STIM009 an antibody VH domain having an amino acid sequence that is at least 90% identical to the H domain sequence The amino acid sequence identity may be at least 95%.

[0032] The antibodies are STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM0 08 or STIM009, or the VL domain of STIM001, STIM0 02, STIM002-B, STIM003, STIM004, STIM005, STI The antibody VL domain of M006, STIM007, STIM008 or STIM009 It may also comprise an antibody VL domain having an amino acid sequence that is at least 90% identical to the sequence The amino acid sequence identity may be at least 95%.

[0033] STIM001, STIM002, STIM002-B, STIM003, STIM0 04, STIM005, STIM006, STIM007, STIM008 or ST An antibody VH domain having the HCDRs of IM009 or a variant of these CDRs The antibody VL domains have the same antibody LCDRs or variants of those CDRs. Similarly, STIM001, STIM002, STIM002-B , STIM003, STIM004, STIM005, STIM006, STIM007 the VH domain of any of STIM008 and STIM009, or the VH domain thereof A variant of the antibody is paired with a VL domain of the same antibody or a VL domain variant of the same antibody. obtain.

[0034] For example, the antibody may comprise the antibody STIM001 VH domain and the STIM001 VL domain. In another example, the antibody may comprise a VH domain of antibody STIM002 and a S domain. In another example, the antibody may comprise the antibody STIM002 VL domain. 3 VH domains and the STIM003 VL domain.

[0035] The antibody may comprise a constant region, optionally a human heavy and / or light chain constant region. A typical isotype is IgG, for example, human IgG1.

[0036] A further aspect of the invention is a nucleic acid molecule encoding the antibody sequences described herein, and culturing the host cells to express the antibody and optionally produce the antibody. The present invention also includes a method for producing an antibody by isolating or purifying the antibody expressed thereby. The VH and VL domains of the antibodies described herein can be similarly produced: Also an aspect of the invention is a pharmaceutical composition comprising the antibody.

[0037] Another aspect of the present invention is an ICOS knockout non-human animal and an antibody against human ICOS. In ICOS knockout animals, for example, I The gene encoding COS is inactivated or deleted from the animal's genome, resulting in ICO Such animals do not express both human ICOS and ICOS from non-human species. It is useful for generating species-cross-reactive antibodies that recognize the normal process of immune tolerance. Lymphocytes that recognize "self" antigens are deleted or inactivated to prevent autoimmune reactions in the body. On the other hand, the absence of endogenous ICOS antigen in non-human knockout animals When injected as a recombinant protein, or with cell lines or vesicles expressing ICOS If used, the animal's immune system should not be tolerized to the antigen, so I This means that the immune response to COS can be generated in knockout animals. The immune repertoire is a set of proteins capable of recognizing ICOS proteins from that species. Non-human test animals (e.g., mice) immunized with human ICOS should contain In this way, both human ICOS and test animal ICOS (e.g., mouse ICOS) Antibodies that bind to the

[0038] This has at least two advantages: first, species-cross-reactive antibodies can be generated in non-human test animals; These compounds can be used in preclinical studies in humans before being developed in human clinical trials. Second, the immune system of knockout animals exhibits a distinct immune response compared to that recognized by ICOS-expressing animals. This allows the recognition of a larger number of potential epitopes on the human ICOS molecule, As a result, the immune repertoire of knockout animals contains greater functional diversity of antibodies. Because of the similarity between the sequences of homologous ICOS molecules from different species, non-human The immune system of animals typically produces human ICOS proteins that match the proteins in non-human animals. Although animals can be tolerized to regions of the protein, this tolerization does not occur in knockout animals.

[0039] The ability to use ICOS knockout animals and their use to generate cross-reactive antibodies The advantages are demonstrated in the examples, where ICOS knockout animals were successfully immunized. It is particularly surprising that ICOS itself can generate antibody responses in germinal centers. It is involved in immune system biology, such as formation and maintenance, and is expressed in T follicular helper cells, which are ICOS-positive cells. This is because it contributes to the generation of immune responses through its role against inflammatory cytokines

[37] . Therefore, ICOS knockout animals are predicted to produce poor antibody responses at best. Surprisingly, strong titers were obtained in ICOS knockout mice, and the desired Highly functional antibodies, including cross-reactive antibodies, have been isolated from the antibody repertoire.

[0040] Exemplary embodiments of the invention are set forth in the accompanying claims. [Brief explanation of the drawings]

[0041] Certain aspects and embodiments of the present invention will now be described in more detail with reference to the accompanying drawings. explain.

[0042] [Figure 1] Measurement of serum titers of ICOS KO and wild-type Kymice against human and mouse ICOS expressed on CHO cells by flow cytometry. Data show the ability of immunoglobulins in the serum of (a) ICOS KO mice (KO) or (b) wild-type non-ICOS KO mice (HK or HL) immunized with human ICOS-expressing MEF cells and human ICOS protein, respectively, to bind to human ICOS (human ICOS binding) or mouse ICOS (mouse ICOS binding) expressed on CHO cells. The geometric mean is a measure of the fluorescence intensity of immunoglobulin binding to cells as determined by flow cytometry. [Figure 2]Human ICOS-ligand neutralization HTRF using the human ICOS receptor. Neutralization profiles of STIM001-STIM009 anti-ICOS mAbs in human IgG1 format compared to C398.4A and their respective isotype controls. Data are representative of four experiments. [Figure 3] Mouse ICOS-ligand neutralization HTRF using the mouse ICOS receptor. Neutralization profiles of STIM001-STIM009 anti-ICOS mAbs in human IgG1 format compared to C398.4A and their respective isotype controls. Data are representative of three experiments. [Figure 4] Direct neutralization HTRF of human ICOS-ligand using the human ICOS receptor. Neutralization profiles of STIM001-STIM009 anti-ICOS mAbs in human IgG4.PE format compared to C398.4A and their respective isotype controls. Data are representative of four experiments. [Figure 5] Mouse ICOS-ligand neutralization HTRF using the mouse ICOS receptor. Neutralization profiles of STIM001-STIM009 anti-ICOS mAbs in human IgG4.PE format compared to C398.4A and their respective isotype controls. Data are representative of four experiments. [Figure 6]a. Concentration-dependent study of STIM001-mediated ADCC on MJ cells using freshly isolated NK cells as effector cells. Effector and target cells (effector:target ratio 5:1) were incubated with antibody for 2 hours. BATDA released from lysed target cells was measured as described in the manufacturer's kit instructions. HC is a hybrid isotype control. b, c, d. Concentration-dependent study of STIM001- and STIM003-mediated ADCC on MJ cells using freshly isolated NK cells as effector cells. Effector and target cells (effector:target ratio 5:1) were incubated with antibody for 2 hours. BATDA released from lysed target cells was measured as described in the manufacturer's kit instructions. HC is a hybrid isotype control. e, f, g. Concentration-dependent study of STIM001 (hIgG1)- and STIM003 (hIgG1)-mediated ADCC in ICOS-transfected CCRF-CEM cells with freshly isolated NK cells as effector cells. Effector and target cells (effector:target ratio 5:1) were incubated with antibody for 4 hours. BATDA released from lysed target cells was measured as described in the manufacturer's kit instructions. HC is a hybrid isotype control. [Figure 7] Anti-ICOS antibodies inhibited CT26 tumor growth and improved survival when administered as monotherapy or in combination with anti-PDL1. STIM001 mIgG2a is more potent than the mIgG1 format. The number of animals cured or stable is shown in each graph. [Figure 8] Anti-ICOS antibodies inhibited CT26 tumor growth and improved survival when administered as monotherapy or in combination with anti-PDL1. STIM001 mIgG2a is more potent than the mIgG1 format. The number of animals cured or stable is shown in each graph. [Figure 9]Anti-ICOS antibodies inhibited CT26 tumor growth and improved survival when administered as monotherapy or in combination with anti-PDL1. STIM001 mIgG2a is more potent than the mIgG1 format. The number of animals cured or stable is shown in each graph. [Figure 10] CT26 in vivo efficacy study of 2x2 combinations. Each treatment group is represented by a "spider plot" showing tumor size for individual animals (n=10 per group). When combined with an anti-PDL1 antibody, STIM001 slows tumor growth and improves survival of treated animals. The efficacy observed in the presence of STIM001 mIgG2a is superior to that of STIM001 mIgG1. Finally, STIM001 mIgG2a in combination with anti-PDL1 mIgG2a was the most potent combination to elicit an anti-tumor response, with 60% of animals cured of the disease. For each group, the number of animals cured of the disease is shown in the upper right corner of each graph. Dosing occurred on days 6, 8, 10, 13, 15, and 17. [Figure 11] Graphs showing CT26 tumor volume over time for animals treated with anti-ICOS or anti-PDL1 monotherapy or combination therapy. Each treatment group is represented by a "spider plot" showing tumor size for individual animals (n=10 per group). For each group, the number of animals with tumor size less than 100 mm^3 (stable / cured disease) is indicated in the upper right corner of each graph. Dosing occurred on days 6, 8, 10, 13, 15, and 17. Administration times are indicated by shaded areas. (a) Isotype control; (b) anti-PDL1 mIgG2a AbW; (c) anti-ICOS STIM003 mIgG1; (d) anti-ICOS STIM003 mIgG2a; (e) anti-PDL1 mIgG2a AbW + STIM003 mIgG1; (f) anti-PDL1 mIgG2a AbW + STIM003 mIgG2a. STIM003 mIgG2 significantly inhibits CT26 tumor growth when combined with anti-PDL1(AbW) mIgG2a. [Figure 12]MJ cell in vitro activation assay—bead binding. Stimulation profiles of bead-bound STIM001, STIM002, and STIM003 anti-ICOS mAbs compared to anti-ICOS C398.4A and their respective isotype controls. Data represent the average of two experiments (n=1 for C398.4A isotype control beads). [Figure 13] MJ cell in vitro activation assay—plate binding. Stimulation profiles of plate-bound STIM001, STIM002, STIM003, and STIM004 anti-ICOS mAbs compared to anti-ICOS C398.4A and their respective isotype controls. Data represent the average of two experiments. [Figure 14] FACS analysis of STIM001 and STIM003 hIgG1 binding to activated T cells. (a) shows a representative experiment of dose response of pre-labeled antibody binding to activated T cells, (b) shows binding after dose response of naked antibody followed by detection with a secondary labeled antibody. The table shows the associated EC50 (M) determined using GraphPad Prism. [Figure 15]STIM001 and STIM003 showed isotype-dependent effects on the T cell compartment at the tumor site. A total of 1x10E5 CT-26 tumor cells were subcutaneously implanted into Balb / c female mice. Animals were intraperitoneally administered antibody or saline on days 13 and 15 post-implantation (n=10 per group). On day 16 post-implantation, spleens and tumors were harvested from tumor-bearing animals (n=8 per group), dissociated, and stained for FACS analysis. A, Percentage of CD3 cells positive for CD4 cells. B, Percentage of CD3 cells positive for CD8 cells. C, Percentage of CD4 cells positive for Foxp3+ and CD25+. D, Percentage of CD4 cells in the spleen positive for Foxp3+ and CD25+. E, Percentage of CD4 effector cells among total CD4 cells. F, Ratio of CD8 effector to T-Reg cells. G, Ratio of CD4 effector to T-Reg cells. Statistical analysis was performed using GraphPad Prism to compare all antibody-treated groups with the saline-treated group, and P values ​​were recorded when significant (p<0.05). Values ​​represent mean + SD (n=8 mice / group). For F, values ​​represent mean + SEM. [Figure 16] Example data from a concentration-dependent study of the agonistic effects of STIM001 (hIgG1) and STIM003 (hIgG1) on isolated human T cells costimulated with anti-CD3 / anti-CD28 Dynabeads for 3 days in T cell activation assay 1 (see Example 9b). IFN-γ production was used as an indicator of agonist effect. STIM001 (hIgG1) and STIM003 (hIgG1) were tested in plate-bound, soluble, or cross-linked soluble (Fc-binding Ab) formats and compared to a hybrid isotype control (HC hIgG1). Hamster antibody C398.4A and its isotype control (hamster IgG) were included for comparison in the plate-binding assay. The top panel shows data from the plate-bound antibody. The bottom panel shows data from the IgG1 antibody in soluble and cross-linked formats. The left and right panels each use T cells from two independent human donors. [Figure 17]Example dataset for STIM001 in T cell activation assay 1 (see Example 9). Data show the levels of IFN-γ induced by STIM001 (hIgG1) or its hybrid isotype control (HC IgG1) at a given dose for T cells from eight independent human donors. Plate-bound antibody (Figure 17a) was used at 5 μg / ml. Soluble antibody (Figure 17b) was used at 15 μg / ml. Each point represents one donor, identified by number (e.g., D214). Significance was assessed using the Wilcoxon statistical test. *p<0.05, **p<0.01. [Figure 18] Example dataset for STIM003 in T cell activation assay 1 (see Example 9). Data show the levels of IFN-γ induced by STIM003 (hIgG1) or its hybrid isotype control (HC hIgG1) at a given dose in T cells from eight independent healthy human donors. Soluble antibody (Figure 18a) was used at 15 μg / ml. Plate-bound antibody (Figure 18b) was used at 5 μg / ml. Each point represents one donor, identified by number (e.g., D214). Significance was assessed using the Wilcoxon statistical test. *p<0.05, **p<0.01. [Figure 19]Example data from T cell activation assay 2 (see Example 9c). Study of STIM001 (hIgG1) and STIM003 (hIgG1) agonist effects on isolated human T cells stimulated with anti-CD3 / anti-CD28 Dynabeads for 3 days, then rested in culture for 3 days, and finally restimulated with plate-bound STIM001, STIM003, or C398.4A Ab + / - CD3 Ab. Data comparing the levels of IFN-γ (A, B), TNF-α (C, D), and IL-2 (E, F) induced by STIM001, STIM003 at a given dose and in combination with CD3 Ab (TCR engagement) with their hybrid control IgG1 (A, C, E) or C398.4A and their hamster IgG control (B, D, F). Each point represents an independent donor, identifiable by its number (e.g., D190). Statistical significance between Abs and their isotype controls was assessed using the Wilcoxon statistical test and p-values ​​shown. Note that the concentration of STIM003 was slightly different from that of HC IgG1 (5.4 vs. 5 μg / ml). [Figure 20] Graph showing the percentage of immune cells (CD8 T-effector, CD4 T-effector, and CD4 / FoxP3 TReg cells) in CT26 tumors and the spleens of tumor-bearing animals expressing ICOS on their surface. Values ​​represent mean ± SD (n = 8). P values ​​were calculated using the nonparametric Dunn's multiple comparison test. NS = not significant, *** = p < 0.001, **** = p < 0.0001. [Figure 21] Relative expression of ICOS on the surface of immune cells CD8 T effector, CD4 T effector, and CD4 / FoxP3 TReg, as determined by mean fluorescence intensity (MFI). Values ​​represent mean ± SD (n = 8). P values ​​were calculated using the non-parametric Dunn's multiple comparison test. **** = p < 0.0001, ** = p < 0.01. Note the difference in fluorescence intensity between the spleen (low) and tumor (high). [Figure 22]Effect of STIM001 and STIM003 on the percentage of different immune cells in the CT26 tumor microenvironment. *=p<0.05. [Figure 23] Effect of antibodies STIM001 and STIM003 on the percentage of regulatory T cells (CD4+ / FoxP3+ cells) in the CT26 tumor microenvironment. **=p<0.05, ****=p<0.0001. Values ​​represent mean ± SD (n=8). P values ​​were calculated using the non-parametric Dunn's multiple comparison test. [Figure 24] STIM001 and STIM003 mIgG2 significantly increase the ratio of CD8 effector T cells to TRegs and CD4 effector T cells to TRegs in CT26 tumors, as determined by dividing the percentage of effector cells in the tumor by the percentage of regulatory T cells in the tumor. [Figure 25] Effect of antibodies on the percentage of immune cells in the spleens of CT26 tumor-bearing animals. [Figure 26] Effect of antibodies on the percentage of regulatory T cells (CD4+ / FoxP3+ cells) in the spleens of CT26 tumor-bearing animals. [Figure 27] (A) CD8 effector:Treg ratio and (B) CD4:Treg ratio in the spleens of CT26 tumor-bearing animals. [Figure 28] Surface staining of AF647-conjugated STIM001, STIM003, and hIgG1 hybrid control (HC IgG1) on activated Mauritius cynomolgus pan T cells. Data from assays using different donor sources of T cells are shown in A and B, respectively. EC50 values ​​are shown in the table. [Figure 29] Kaplan-Meier curves for the CT26 Balb / C model. The shaded area indicates the dosing window. Log-rank p<0.0001. [Figure 30]Figure 32 is a graph showing A20 tumor volume over time in mice for the study described in Example 20. Each treatment group is represented by a spider plot showing tumor size in individual animals (n=8 per group). For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing occurred on days 8, 11, 15, 18, 22, 25, and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 30) and the anti-PD-L1 treatment group (Figure 31), the STIM001 mIgG2a (Figure 32) and STIM003 mIgG2a (Figure 33) treatment groups showed significant inhibition of A20 tumor growth. [Figure 31] Figure 32 is a graph showing A20 tumor volume over time in mice for the study described in Example 20. Each treatment group is represented by a spider plot showing tumor size in individual animals (n=8 per group). For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing occurred on days 8, 11, 15, 18, 22, 25, and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 30) and the anti-PD-L1 treatment group (Figure 31), the STIM001 mIgG2a (Figure 32) and STIM003 mIgG2a (Figure 33) treatment groups showed significant inhibition of A20 tumor growth. [Figure 32] Figure 32 is a graph showing A20 tumor volume over time in mice for the study described in Example 20. Each treatment group is represented by a spider plot showing tumor size in individual animals (n=8 per group). For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing occurred on days 8, 11, 15, 18, 22, 25, and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 30) and the anti-PD-L1 treatment group (Figure 31), the STIM001 mIgG2a (Figure 32) and STIM003 mIgG2a (Figure 33) treatment groups showed significant inhibition of A20 tumor growth. [Figure 33]Figure 32 is a graph showing A20 tumor volume over time in mice for the study described in Example 20. Each treatment group is represented by a spider plot showing tumor size in individual animals (n=8 per group). For each group, the number of animals with no signs of tumor (indicating cure of disease) is shown at the bottom left of the graph. Dosing occurred on days 8, 11, 15, 18, 22, 25, and 29 after tumor cell implantation, with dosing times indicated by grey shaded areas. Compared to the control group (Figure 30) and the anti-PD-L1 treatment group (Figure 31), the STIM001 mIgG2a (Figure 32) and STIM003 mIgG2a (Figure 33) treatment groups showed significant inhibition of A20 tumor growth. [Figure 34] Data from the CT26 in vivo efficacy study described in Example 11c, using a combination of an anti-PD-L1 mIgG2a antibody and single or multiple doses of STIM003 mIgG2a. Each treatment group is represented by a "spider plot" showing tumor size for individual animals (n=8 per group). For each group, the number of animals cured of disease is indicated at the bottom right of each graph. The number of days of dosing for each antibody is indicated by the arrow below each graph. [Figure 35] Amino acid sequences of the STIM002 VH (top) and VL (bottom) domains, showing residues that differ from the corresponding sequences of STIM001, STIM002B, and related antibodies CL-61091, CL-64536, CL-64837, CL-64841, and CL-64841, and / or human germline. SEQ ID NOs are according to IMGT. [Figure 36] Amino acid sequences of the STIM003 VH (top) and VL (bottom) domains, showing residues that differ from the corresponding sequences of related antibodies CL-71642 and CL-74570 and / or human germline sequences. SEQ ID NOs are according to IMGT. The VL domain of antibody CL-71642 obtained from sequencing is shown here without the N-terminal residue. This alignment shows that the complete VH domain sequence includes an N-terminal glutamic acid. [Figure 37]Amino acid sequences of the STIM007 VH (top) and VL (bottom) domains, showing residues that differ from the corresponding sequence of STIM007 and / or human germline. SEQ ID NOs are according to IMGT. [Figure 38] Effect of STIM003 (anti-ICOS) and AbW (anti-PD-L1) mIgG2a antibodies in the J558 syngeneic model. Each treatment group is represented by a "spider plot" showing tumor size for individual animals (n=10 or n=8 per group). STIM003 monotherapy showed some efficacy, with 3 of 8 animals cured of disease. Similarly, anti-PDL1 was effective in this model, with 6 of 8 animals cured of disease by day 37. When combined with anti-PDL1 antibody, STIM003 mIgG2 completely inhibited tumor growth and improved survival of treated animals. For each group, the number of animals cured of disease is indicated at the bottom right of the respective graph. Days of dosing are indicated by dotted lines (days 11, 15, 18, 22, 25, and 29). [Figure 39] Quantification of ICOS expression (percentage of positive cells and relative expression / dMFI) on different TILS cell subtypes in tumor tissue. (A) % of immune cell subtypes positive for ICOS expression, (B) ICOS dMFI (relative ICOS expression on ICOS-positive cells) of immune cell subtypes from animals treated with saline, anti-PD-L1, or anti-PD-1 surrogate antibodies. Mice were implanted with 100 μl of 1×10 viable cells / ml on day 0 (n=7 or n=8). Animals were intraperitoneally administered 130 μg of antibody on days 13 and 15. Tissue samples were isolated and analyzed on day 16. CD4+ / FOXP3+ cells were exclusively present in the TReg population (far right graph) and excluded from "effector" CD4 cells (far left graph), which were all Foxp3-negative. See Example 22. [Figure 40]A20 Data from in vivo efficacy study. Each treatment group is represented by a "spider plot" showing tumor size for individual animals (n=10 per group). For each group, the number of animals cured of disease is shown on the respective graph. For multiple doses, dosing occurred on days 8, 11, 15, 18, 22, and 25, indicated by dotted lines. For single doses, animals received an intraperitoneal injection only on day 8. (A) Saline, (B) STIM003 mIgG2a multiple doses, (C) STIM003 mIgG2a single dose. See Example 23. [Figure 41] Kaplan-Meier curves for the study reported in Example 23 with a fixed dose of 60 μg of STIM003 mIgG2a. SD = single dose, day 8. MD = multiple dose BIW from day 8. [Figure 42] ICOS expression on major T cell subsets (T-reg [CD4+ / FoxP3+], CD4 Eff [CD4+ / FoxP3-] cells, and CD8+) from saline-treated CT26 tumor-bearing animals (n=4 per time point). Immune cell phenotyping was performed on days 1, 2, 3, 4, and 8 post-treatment, and all tissues were stained for ICOS expression at all time points. A-D show the percentage of ICOS-positive cells at all time points in four different tissues. E-H show ICOS dMFI (relative expression) at all time points in four different tissues. See Example 24. [Figure 43] FACS analysis demonstrating T-reg depletion of the TME in response to the STIM003 mIgG2a antibody. CT-26 tumor-bearing animals were treated with a single dose of STIM003 (6, 60, or 200 μg) on ​​day 12 after tumor cell implantation. Tissues (n=4 per time point) were harvested for FACS analysis on days 1, 2, 3, 4, and 8 after treatment. The percentage of T-reg cells (CD4+ CD25+ Foxp3+ ) in the total tumor (A) and the percentage of T-reg cells in the blood (B) are shown at different time points. See Example 24. [Figure 44]Increased CD8:Treg and CD4eff:Treg ratios in response to STIM003 mIgG2a. CT-26 tumor-bearing animals received a single dose of STIM003 mIgG2a (6, 60, or 200 μg) on ​​day 12 after tumor cell implantation. Tissues (n=4 per time point) were collected for FACS analysis on days 1, 2, 3, 4, and 8 after treatment, and the Teff to Treg ratios were calculated. (A) and (B) are CD8:Treg ratios in tumor and blood, and (C) and (D) are CD4eff:Treg ratios in tumor and blood. See Example 24. [Figure 45] STIM003 treatment correlates with increased degranulation and Th1 cytokine production by TILs. Eight days after treatment, TILs were isolated and FACS analysis was performed to detect CD107a expression on CD4 and CD8 T cells (A-B). In parallel, dissociated tumor-derived cells were incubated for 4 hours in the presence of Brefeldin-A, and the cells were stained for T cell markers and permeabilized for intracellular staining to detect IFN-γ and TNF-α (C-H). See Example 24. DETAILED DESCRIPTION OF THE INVENTION

[0043] ICOS The antibody according to the present invention binds to the extracellular domain of human ICOS. , binds to ICOS-expressing T lymphocytes. The "S receptor" may be human ICOS unless the context dictates otherwise. The sequences of macaque and mouse ICOS are shown in the attached sequence listing and the human NCBI ID :NP_036224.1, mouse NCBI ID:NP_059508.2, and crab It is available from NCBI as Macaca mollusc GenBank ID: EHH55098.1. be.

[0044] Cross-reactivity The antibodies according to the invention are preferably cross-reactive, e.g., with murine ICOS and human The antibody can bind to the extracellular domain of ICOS. Anti-ICOS antibodies intended for therapeutic use in humans can bind to other non-human ICOS, including The antibody must bind to human ICOS, but binding to ICOS from other species is not human. It has no direct therapeutic relevance in clinical situations. Nevertheless, the data herein The data showed that antibodies that bind to both human and mouse ICOS agonize them. It has been shown that the ATP-binding domains have properties that make them particularly suitable as target and depletion molecules. It may arise from one or more specific epitopes targeted by specific antibodies. Regardless of the theory, cross-reactive antibodies are highly valuable and are a promising therapeutic candidate for preclinical and clinical trials. It is an excellent candidate for therapeutic molecules.

[0045] As described in the experimental examples, mouse ICOS (ICOS knockout) was deficient in expression. The STIM gene was developed using Kymouse™ technology, which uses mice engineered as described herein. Antibodies were generated in ICOS knockout transgenic animals, and cross-reactive antibodies were obtained. Its use for producing is a further aspect of the invention.

[0046] One method for quantifying the degree of species cross-reactivity of antibodies is to compare the activity of one species against an antigen of another species. The fold difference in its affinity compared to the antigen, e.g., parental affinity for human ICOS versus mouse ICOS. Affinity is measured as a fold difference in affinity. K refers to the equilibrium dissociation constant of the antibody-antigen reaction determined by SPR using the same antibody. D and Species cross-reactive anti-ICOS antibodies may be quantified using the antibody against human ICOS and mouse ICOS. The fold difference in affinity for binding to S is 30-fold or less, 25-fold or less, 20-fold or less, 15-fold or less, or 1 In other words, the extracellular domain of human ICOS K binding to ketone D K binds to the extracellular domain of mouse ICOS D 30 times, 25 times The difference may be within 20, 15, 10, or 5 times. Antibodies may also bind to both antigens. RuK D If K satisfies the threshold, for example, K binding to human ICOS D and mouse ICOS K to bind D However, both of them are 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. If the K is less than or equal to M, it can be considered cross-reactive. D is 10nM or less, 5 It may be nM or less, 2 nM or less, or 1 nM or less. D is less than 0.9nM, 0. 8nM or less, 0.7nM or less, 0.6nM or less, 0.5nM or less, 0.4nM or less, 0. It may be 3 nM or less, 0.2 nM or less, or 0.1 nM or less.

[0047] Another measure of cross-reactivity for binding to human and mouse ICOS is HTRF. The ICOS receptor is a target of ATP synthesis. (See Example 8.) Examples of species cross-reactive antibodies are provided, each of which has the ability to bind to HT Binding of human B7-H2 (ICOS ligand) to human ICOS in RF assay and was confirmed to neutralize the binding of mouse B7-H2 to mouse ICOS. STIM001, STIM002, STIM002-B, STIM003, STI These include M005 and STIM006. Cross-reactive with human and mouse ICOS. If a specific antibody is desired, any of these antibodies or variants thereof can be selected. Species-cross-reactive anti-ICOS antibodies inhibit the binding of human ICOS to the human ICOS receptor The IC50 of mouse ICOS was determined in an HTRF assay. 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold greater than the IC50 for inhibiting binding to the OS receptor The antibody also inhibits the binding of human ICOS to the human ICOS receptor. IC50 for inhibiting the binding of mouse ICOS to the mouse ICOS receptor and both are 1 mM or less, preferably 0.5 mM or less, for example, 30 mM or less, 20 If the concentration is less than 10 nM, it can be considered cross-reactive. The C50 may be 5 nM or less, 4 nM or less, 3 nM or less, or 2 nM or less. In some cases, the IC50 is at least 0.1 nM, at least 0.5 nM, or less At most 1 nM.

[0048] specificity The antibodies according to the invention are preferably specific for ICOS, i.e., they bind to the target The protein ICOS (human ICOS, and preferably mouse and / or rabbit ICOS as described above) It binds to its epitope on the ferret (ICOS) but is a member of the CD28 gene family. It does not exhibit significant binding to molecules that do not display its epitope, including other molecules. The antibody in question preferably does not bind to human CD28. It also does not bind to cynomolgus monkey CD28.

[0049] CD28 is a key regulator of TCR-mediated antigen recognition in the context of its binding to professional antigen-presenting cells. Costimulates T cell responses when engaged by ligands CD80 and CD86 For various in vivo uses of the antibodies described herein, the cycle of binding to CD28 may be varied. Avoidance of CD28 is considered advantageous. 8 interacts with its natural ligand to generate the appropriate costimulatory signals for T cell activation. In addition, the non-binding of anti-ICOS antibodies to CD28 This avoids the risk of superagonism. Overstimulation of CD28 inhibits TCR activation. Proliferation is induced in resting T cells without the normal requirement for recognition of cognate antigen via This may result in runaway activation of T cells and the resulting cytokines, particularly in human subjects. Therefore, CD40 release syndrome may be caused by the antibody according to the present invention. The non-recognition of 28 represents an advantage in terms of their safe clinical use in humans.

[0050] As discussed elsewhere herein, the present invention provides multispecific antibodies (e.g., bispecific antibodies). Multispecific (e.g., bispecific) antibodies include: (i) antibody-antigen binding for ICOS; and (ii) a further antigen-binding site (optionally) that recognizes another antigen (e.g., PD-L1). Optionally, the antibody may comprise an antigen-binding site (as described herein). Therefore, antibodies that specifically bind to ICOS may be an antibody comprising an antigen-binding site that specifically binds to S, e.g., ICOS and PD-L1; and bispecific antibodies that bind to the antigen-binding domain of ICOS. The site is contained within an antigen-binding site that further comprises one or more additional binding sites for one or more other antigens. It can be enjoyed.

[0051] affinity The binding affinity of the antibody to ICOS can be determined. The affinity of the antibody for its antigen is , equilibrium dissociation constant K D , the association or on-rate (Ka) and dissociation or off-rate of the antibody-antigen interaction The Kd, Ka, for antibody-antigen binding can be quantified in terms of the ratio Ka / Kd of the kd. and Kd can be measured using surface plasmon resonance (SPR).

[0052] The antibody according to the present invention is at a concentration of 10 mM or less, preferably 5 mM or less, more preferably 1 mM or less. K below D It can bind to the EC domain of human ICOS at K D is 50nM or less, 10nM It may be 5 nM or less, 2 nM or less, or 1 nM or less. D is 0.9nM or more Bottom, 0.8nM or less, 0.7nM or less, 0.6nM or less, 0.5nM or less, 0.4nM or less The K may be 0.3 nM or less, 0.2 nM or less, or 0.1 nM or less. D Is a little at least 0.001 nM, for example, at least 0.01 nM, or at least 0.1 nM may be.

[0053] Affinity quantification can be performed using SPR with antibodies in Fab format. A suitable protocol is as follows: 1. Anti-human (or other species-matched antibody constant region) IgG is bound via primary amine coupling or other methods. The sample is then attached to a biosensor chip (e.g., a GLM chip). 2. Anti-human IgG (or other matched species antibody) is added to the test IgG, e.g., in Fab format. The test antibody is captured on the chip by exposure to antibody G. 3. For example, 5000nM, 1000nM, 200nM, 40nM, 8nM, and 2nM Test antigens were applied to the capture surface of the chip at concentrations ranging from 0 nM to 10 nM (i.e., buffer only). Let it pass. 4. Determine the binding affinity of the test antibody to the test antigen using surface plasmon resonance at 25°C. The buffer solution is pH 7.6, 150 mM NaCl, 0.05% surfactant (e.g., For example, P20), and 3 mM EDTA. HBS-EP may contain EPES. HBS-EP can be used as the running buffer. HBS-EP is manufactured by Teknova Inc. (California), catalog number H80 22).

[0054] Regeneration of the capture surface can be performed with 10 mM glycine at pH 1.7. The captured antibody is then removed, making the surface available for another interaction. The data is analyzed using standard techniques, for example, with ProteOn XPR36™ analysis software. A software-specific model can be used to match the specific model 1:1.

[0055] Biacore (trademark), ProteOn XPR36 (trademark) (Bio-Rad (registered trademark) )), and KinExA® (Sapidyne Instruments, Inc. Various SPR devices are known, such as those described in Example 7. A working example of SPR is found in Example 7.

[0056] As described, affinity chromatography was performed using antibodies in Fab format to attach antigen to the chip surface. The test antibody is passed over the chip in solution in Fab format to form a monomeric antibody. The affinity of the antibody-antigen interaction can also be determined by determining the affinity of the antibody-antigen interaction. Any desired pH, e.g., pH 5.5 or pH 7.6, and any desired temperature, e.g., It can be determined at 25° C. or 37° C. As reported in Example 7, The antibody has a specificity of 2n as determined by SPR using the antibody in monovalent (Fab) format. It bound to human ICOS with an apparent affinity of less than M

[0057] Other methods for measuring antibody binding to ICOS include, for example, binding to the exogenous surface of ICOS. Cells with expression (e.g., CHO cells) or activated cells expressing endogenous levels of ICOS This includes fluorescence-activated cell sorting (FACS) using primary T cells. Antibody binding to ICOS-expressing cells results in the antibody binding to the extracellular (EC) domain of ICOS. It is shown that they can be combined.

[0058] ICOS receptor agonism ICOS ligand (ICOSL, also known as B7-H2) binds to the ICOS receptor This intercellular ligand-receptor interaction is a cell surface-expressed molecule that mediates T cell proliferation and proliferation

[17] . Promotes ICOS multimerization on the surface, activates receptors, and induces downstream signaling in T cells In effector T cells, this receptor activation stimulates effector T cell responses are stimulated.

[0059] Anti-ICOS antibodies act as agonists of ICOS, inhibiting the natural ICO receptor. This stimulatory effect of S ligands can be mimicked and even surpassed. This may be due to the ability of antibodies to promote the multimerization of ICOS on T cells. The mechanism of this is that antibodies bind to ICOS on the surface of T cells and to adjacent cells (e.g., B cells, antigen-presenting cells, or other immune cells), forming an intercellular bridge between the receptors, e.g., Fc receptors. Another mechanism is the formation of multiple (e.g., two) antigen-binding sites (e.g., two VH-VL domains). Antibodies bearing α-pairs cross-link multiple ICOS receptor molecules, thereby promoting multimerization A combination of these mechanisms may also occur.

[0060] Agonism can be achieved by using soluble forms (e.g., immunoglobulins) with or without cross-linking agents. a tandem format or two spatially separated antigen binding sites, e.g., two VH-VL pairs )) antibodies can be used or attached to a solid surface to provide an array of tethered antigen binding sites. The combined antibodies can be used to test in an in vitro T cell activation assay. The rhesus assay uses human ICOS-positive T lymphocytes such as MJ cells (ATCC CRL-8294). A cytoplasmic cell line may be used as the target T cell for activation in such an assay. One or more measures of T cell activation can be determined for the subject, and a reference molecule or negative control The T cell activation mediated by the test antibody compared to a reference molecule or control is It can be determined whether there is a statistically significant (p<0.05) difference. One suitable measure of cell activation is the expression of cytokines, such as IFNγ, TNFα, or I Those skilled in the art will be able to include suitable controls, where appropriate, and to verify the association between the test antibody and the control. A suitable negative control would be a mixture of the same format that does not bind to ICOS, which would standardize the assay conditions. a control antibody (e.g., an isotype control), e.g., for an antigen not present in the assay system Antibodies that are specific for the antibody. Cognate isotype controls within the dynamic range of the assay. A significant difference observed for a test subject compared to an IC It has been shown to act as an agonist of the OS receptor.

[0061] Agonist antibodies, when tested in T cell activation assays, had a significantly lower EC50 for inducing IFNγ production compared to the control antibody; Induces significantly higher maximum IFNγ production compared to the control antibody, It has a significantly lower EC50 for inducing IFNγ production compared to ICOSL-Fc. , Induces significantly higher maximum IFNγ production compared to ICOSL-Fc, Significantly lower EC50 for induction of IFNγ production compared to reference antibody C398.4A and / or It induces significantly higher maximum IFNγ production compared to the reference antibody C398.4A. can be defined as

[0062] In vitro T cell assays include the bead binding assay described in Example 13, the pre-binding assay described in Example 14, and the These assays include the phosphate binding assay, and the soluble form assay of Example 15.

[0063] A significantly lower or significantly higher value is, for example, at most 0.5-fold higher than the reference or control value. Different, Different by 0.75 times, Different by 2 times, Different by 3 times, Different by 4 times or may differ by up to 5-fold.

[0064] Thus, in one example, an MJ cell activation assay using antibodies in a bead-bound format is In this study, the antibodies according to the present invention showed a significantly lower induction of IFNγ compared to the control. For example, it has an EC50 that is at least 2-fold lower.

[0065] Bead binding assays involve the binding of antibodies (and antibodies) to the surface of beads (and a control or reference experiment). Use a control antibody, reference antibody, or ICOSL-Fc. Various types of beads, such as tosyl-activated DYNABEADS M-450 (DYNAL Inc,5 Delaware Drive,Lake Success,NY1 1042 Prod No. 140.03, 140.04) are commercially available. , as described in Example 13, or generally in carbonate buffer (pH 9.6, 0.2M). The coating may be applied by dissolving the coating material or by other methods known in the art. The use of beads allows for a good determination of the amount of protein bound to the bead surface. Standard Fc protein quantification methods advantageously allow for determination with a degree of accuracy. This can be used to quantify the proteins bound to the beads. Any suitable method for determining the validity of the standard within the range may be used. As exemplified in Example 13, ELISA or other methods can be used.

[0066] The agonistic activity of antibodies can also be measured ex vivo in primary human T lymphocytes. The ability of antibodies to induce IFNγ expression in such T cells is also Two T cell activation assays using primary cells are described herein. (See Example 2, T Cell Activation Assay 1 and T Cell Activation Assay 2.) Preferably, the antibody is selected from T cell activation assay 1 and / or T cell activation assay 2. In comparison with the control antibody, a significant (p<0.05) induction of IFNγ was observed at 5 μg / mL. As mentioned above, anti-ICOS antibodies can inhibit the activity of ICOS-L or ICOS-C in such assays. 398.4. Thus, the antibody can stimulate T cell activation to a greater extent than 398.4. Significant increase in cytotoxicity at 5 μg / mL compared to control or reference antibody in cell activation assay 1 or 2 The induction of IFNγ was significantly greater than that of TNFα or IL-2 (p<0.05). The ATP concentration can be measured as a typical assay readout.

[0067] The agonism of anti-ICOS antibodies is thought to be due to the suppression of T cell proliferation in pathological sites, such as the tumor microenvironment. Ef f cells, and their ability to alter the balance of TReg and TEff cell populations in vivo As discussed elsewhere herein, activated ICOS-positive enzymes may contribute to The ability of the antibody to enhance tumor cell killing by effector T cells can be determined.

[0068] T cell dependent killing Effector T cell function is biologically relevant using in vitro co-culture assays This assay involves the in vivo analysis of tumor cells together with relevant immune cells. Incubation to induce immune cell-dependent killing and inhibit tumor cell killing by TEff The effect of anti-ICOS antibody will be observed.

[0069] Ability of antibodies to enhance tumor cell killing by activated ICOS-positive effector T cells The anti-ICOS antibody showed a significantly greater potency than the control antibody (p<0.05). Anti-ICOS antibodies can stimulate tumor cell killing. Similar or greater tumor cell killing in such assays compared to a reference molecule such as A similar degree of tumor cell killing may stimulate the assay readout for the test antibody. and the readout of the reference molecule can be expressed as less than a two-fold difference.

[0070] ICOS Ligand-Receptor Neutralization The antibody according to the present invention is an antibody that inhibits the binding of ICOS to its ligand ICOSL. It is also possible.

[0071] The extent to which an antibody inhibits binding between the ICOS receptor and its ligand depends on the ligand-receptor Neutralizing potency is usually expressed as an IC50 value in pM unless otherwise stated. In ligand binding studies, IC50 is the 50% of the maximum specific binding level. The IC50 is the concentration that reduces receptor binding by a specific The % specific receptor binding was plotted and analyzed using a software program such as Prism (GraphPad). The calculated values ​​were calculated using a sigmoid function to fit the data and generate IC50 values. Neutralizing capacity may be determined in an HTRF assay. A detailed working example of an HTRF assay for neutralizing capacity is provided in Example 8.

[0072] The IC50 value may represent the average of multiple determinations. Thus, for example, the results of three experiments The results may give IC50 values, after which an average IC50 value may be calculated.

[0073] The antibody may have an IC50 of 1 mM or less in a ligand-receptor neutralization assay, e.g., 0 The IC50 may be 0.5 mM or less. The IC50 may be 30 nM or less, 20 nM or less, or 10 nM or less. , 5 nM or less, 4 nM or less, 3 nM or less, or 2 nM or less. It may be at least 0.1 nM, at least 0.5 nM, or at least 1 nM.

[0074] antibody As described in more detail in the Examples, STIM001, STIM002, STIM00 2-B, STIM003, STIM004, STIM005, STIM006, STIM Antibodies of particular interest, designated STIM007, STIM008, and STIM009, were isolated. In various aspects of the present invention, unless otherwise indicated by context, the antibody Any of these antibodies, or STIM001, STIM002, STIM003, STI The sequences of each of these antibodies can be selected from the subset of: M004, STIM005, and STIM006. The columns are provided in the accompanying sequence listing, in which for each antibody the following sequences are shown: The nucleotide sequence encoding the VH domain, the amino acid sequence of the VH domain, and the V H CDR1 amino acid sequence, VH CDR2 amino acid sequence, VH CDR3 amino acid sequence , the nucleotide sequence encoding the VL domain, the amino acid sequence of the VL domain; CDR1 amino acid sequence, VL CDR2 amino acid sequence, and VL CDR3 amino acid sequence The present invention relates to the VH and / or VL domains of all antibodies shown in the accompanying sequence listing and / or figures. Anti-ICOS antibodies having the HCDRs and / or LCDRs of these antibodies and optionally includes antibodies having full-length heavy chain and / or full-length light chain amino acid sequences.

[0075] STIM001 contains the CDRH1 amino acid sequence of SEQ ID NO: 363, the CDRH2 amino acid sequence of SEQ ID NO: 364, SEQ ID NO: 366, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 365 The heavy chain variable region (V H ) amino acid sequence.H The heavy chain nucleic acid sequence of the domain is shown in SEQ ID NO: 367. STIM001 has the CDRL1 amino acid sequence of SEQ ID NO: 370, SEQ ID NO: 371. 371, and the CDRL3 amino acid sequence of SEQ ID NO: 372. , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 374. H The domains are the heavy chain constant region sequences described herein. Sequences, e.g., SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, No. 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: 524, SEQ ID NO: No. 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or SEQ ID NO: 534 May be combined with any of the following: V L The domains are the light chain constants described herein. Common region sequences, e.g., SEQ ID NOs: 207, 209, 211, 213, 215, 217, 21 9, 221, 223, 225, 227, 229, 231, 233, 235, 237, 53 The full-length heavy chain amino acid sequence may be combined with any of the sequences: 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).

[0076] STIM002 contains the CDRH1 amino acid sequence of SEQ ID NO: 377, the CDRH2 amino acid sequence of SEQ ID NO: 378, SEQ ID NO: 3, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 379 80 heavy chain variable region (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is STIM002 is the CDRL1 amino acid sequence of SEQ ID NO: 384, sequence The CDRL2 amino acid sequence of SEQ ID NO: 385 and the CDRL3 amino acid sequence of SEQ ID NO: 386 a light chain variable region (V) of SEQ ID NO: 387 L ) amino acid sequence. L Domain Light Chain The nucleic acid sequence is SEQ ID NO: 388 or SEQ ID NO: 519. H The domains are as described herein. The heavy chain constant region sequences described herein include, 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 domain is Honmei Light chain constant region sequences described in the specification, e.g., SEQ ID NOs: 207, 209, 211, 213 , 215, 217, 219, 221, 223, 225, 227, 229, 231, 233 , 235, 237, 536, and 538. The heavy chain amino acid sequence is SEQ ID NO: 382 (heavy chain nucleic acid sequence SEQ ID NO: 383). The amino acid sequence is SEQ ID NO: 389 (light chain nucleic acid sequence SEQ ID NO: 390 or SEQ ID NO: 520). .

[0077] STIM002-B contains the CDRH1 amino acid sequence of SEQ ID NO: 391, the CDRH1 amino acid sequence of SEQ ID NO: 392, SEQ ID NO: 393, including the CDRH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 394 The heavy chain variable region (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is SEQ ID NO: 395. STIM002-B has the CDRL1 amino acid sequence of SEQ ID NO: 398. the CDRL2 amino acid sequence of SEQ ID NO: 399, and the CDRL3 amino acid sequence of SEQ ID NO: 400 The light chain variable region (V) of SEQ ID NO: 401, comprising the amino acid sequence L ) amino acid sequence. L Domain The light chain nucleic acid sequence of V is SEQ ID NO: 402. H The domains are the overlapping domains described herein. Chain constant region sequences, e.g., 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: May be combined with any of the numbers 534. L The domains are described herein. light chain constant region sequences, e.g., 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: 396 (heavy chain nucleic acid sequence SEQ ID NO: 397). is SEQ ID NO: 403 (light chain nucleic acid sequence SEQ ID NO: 404).

[0078] STIM003 has the CDRH1 amino acid sequence of SEQ ID NO: 405, the CDRH2 amino acid sequence of SEQ ID NO: 406 SEQ ID NO: 4, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 407 The heavy chain variable region (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is No. 409 or SEQ ID NO: 521. STIM003 is CDRL1 of SEQ ID NO: 412 The amino acid sequence, the CDRL2 amino acid sequence of SEQ ID NO: 413, and the CDR of SEQ ID NO: 414 The light chain variable region (V) of SEQ ID NO: 415, comprising the L3 amino acid sequence L ) amino acid sequence. V L The light chain nucleic acid sequence of domain V is SEQ ID NO: 4416. H A domain is defined herein as The heavy chain constant region sequences described, e.g., SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 19 7, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 34 0, SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 53 2, or SEQ ID NO: 534. L The domain is The light chain constant region sequences described herein, e.g., SEQ ID NOs: 207, 209, 211, 21 3, 215, 217, 219, 221, 223, 225, 227, 229, 231, 23 3, 235, 237, 536, and 538. The long heavy chain amino acid sequence is SEQ ID NO: 410 (heavy chain nucleic acid sequence SEQ ID NO: 411 or SEQ ID NO: 52 2). The full-length light chain amino acid sequence is SEQ ID NO: 417 (light chain nucleic acid sequence SEQ ID NO: 418). be.

[0079] STIM004 contains the CDRH1 amino acid sequence of SEQ ID NO: 419, the CDRH2 amino acid sequence of SEQ ID NO: 420, SEQ ID NO: 4, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 421 22 heavy chain variable regions (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is STIM004 is the CDRL1 amino acid sequence of SEQ ID NO: 426, sequence The CDRL2 amino acid sequence of SEQ ID NO: 427 and the CDRL3 amino acid sequence of SEQ ID NO: 428 a light chain variable region (V) of SEQ ID NO: 429 L ) amino acid sequence. L Domain Light Chain The nucleic acid sequence is SEQ ID NO: 430 or SEQ ID NO: 431. H The domains are as described herein. The heavy chain constant region sequences described herein include, 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 domain is Honmei Light chain constant region sequences described in the specification, e.g., SEQ ID NOs: 207, 209, 211, 213 , 215, 217, 219, 221, 223, 225, 227, 229, 231, 233 , 235, 237, 536, and 538. The heavy chain amino acid sequence is SEQ ID NO: 424 (heavy chain nucleic acid sequence SEQ ID NO: 425). The amino acid sequence is SEQ ID NO: 432 (light chain nucleic acid sequence SEQ ID NO: 433 or SEQ ID NO: 434). .

[0080] STIM005 contains the CDRH1 amino acid sequence of SEQ ID NO: 435, the CDRH2 amino acid sequence of SEQ ID NO: 436, SEQ ID NO: 4, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 437 38 heavy chain variable regions (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is STIM005 is the CDRL1 amino acid sequence of SEQ ID NO: 442, sequence The CDRL2 amino acid sequence of SEQ ID NO: 443 and the CDRL3 amino acid sequence of SEQ ID NO: 444 a light chain variable region (V) of SEQ ID NO: 445 L ) amino acid sequence. L Domain Light Chain The nucleic acid sequence is SEQ ID NO: 446. H The domains are the heavy chain constant region domains described herein. Region sequences, e.g., 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: 53 4. V L The domains are light proteins as described herein. Chain constant region sequences, e.g., 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 No. 447 (light chain nucleic acid sequence SEQ ID NO: 448).

[0081] STIM006 contains the CDRH1 amino acid sequence of SEQ ID NO: 449, the CDRH2 amino acid sequence of SEQ ID NO: 450, SEQ ID NO: 4, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 451 52 heavy chain variable region (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is STIM006 is the CDRL1 amino acid sequence of SEQ ID NO: 456, sequence The CDRL2 amino acid sequence of SEQ ID NO: 457 and the CDRL3 amino acid sequence of SEQ ID NO: 458 a light chain variable region (V) of SEQ ID NO: 459 L ) amino acid sequence. L Domain Light Chain The nucleic acid sequence is SEQ ID NO: 460. H The domains are the heavy chain constant region domains described herein. Region sequences, e.g., 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: 53 4. V L The domains are light proteins as described herein. Chain constant region sequences, e.g., 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: 454 (heavy chain nucleic acid sequence SEQ ID NO: 455). The full length light chain amino acid sequence is No. 461 (light chain nucleic acid sequence SEQ ID NO: 462).

[0082] STIM007 has the CDRH1 amino acid sequence of SEQ ID NO: 463, the CDRH2 amino acid sequence of SEQ ID NO: 464, SEQ ID NO: 4, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 465 66 heavy chain variable region (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is STIM007 is the CDRL1 amino acid sequence of SEQ ID NO: 470, sequence The CDRL2 amino acid sequence of SEQ ID NO: 471 and the CDRL3 amino acid sequence of SEQ ID NO: 472 a light chain variable region (V) of SEQ ID NO: 473 L ) amino acid sequence. L Domain Light Chain The nucleic acid sequence is SEQ ID NO: 474. H The domains are the heavy chain constant region domains described herein. Region sequences, e.g., 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: 53 4. V L The domains are light proteins as described herein. Chain constant region sequences, e.g., 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 No. 475 (light chain nucleic acid sequence SEQ ID NO: 476).

[0083] STIM008 contains the CDRH1 amino acid sequence of SEQ ID NO: 477, the CDRH2 amino acid sequence of SEQ ID NO: 478, SEQ ID NO: 4, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 479 80 heavy chain variable region (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is STIM008 is the CDRL1 amino acid sequence of SEQ ID NO: 484, sequence The CDRL2 amino acid sequence of SEQ ID NO: 485 and the CDRL3 amino acid sequence of SEQ ID NO: 486 a light chain variable region (V) of SEQ ID NO: 487 L ) amino acid sequence. L Domain Light Chain The nucleic acid sequence is SEQ ID NO: 488. H The domains are the heavy chain constant region domains described herein. Region sequences, e.g., 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: 53 4. V L The domains are light proteins as described herein. Chain constant region sequences, e.g., 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: 482 (heavy chain nucleic acid sequence SEQ ID NO: 483). The full length light chain amino acid sequence is No. 489 (light chain nucleic acid sequence SEQ ID NO: 490).

[0084] STIM009 contains the CDRH1 amino acid sequence of SEQ ID NO: 491, the CDRH2 amino acid sequence of SEQ ID NO: 492, SEQ ID NO: 4, which contains the RH2 amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 493 94 heavy chain variable region (V H ) amino acid sequence. H The heavy chain nucleic acid sequence of the domain is STIM009 is the CDRL1 amino acid sequence of SEQ ID NO: 498, sequence The CDRL2 amino acid sequence of SEQ ID NO: 499 and the CDRL3 amino acid sequence of SEQ ID NO: 500 a light chain variable region (V) of SEQ ID NO: 501 L ) amino acid sequence. L Domain Light Chain The nucleic acid sequence is SEQ ID NO: 502. H The domains are the heavy chain constant region domains described herein. Region sequences, e.g., 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: 53 4. V L The domains are light proteins as described herein. Chain constant region sequences, e.g., 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 No. 503 (light chain nucleic acid sequence SEQ ID NO: 504).

[0085] Antibodies according to the present invention may be natural or partially or wholly synthetically produced immunoglobulins. Antibodies are molecules that contain a globulin domain or an immunoglobulin domain. whether derived from any species native to that country or created by recombinant DNA technology. and from serum, B cells, hybridomas, transfectomas, yeast, or bacteria. IgG, IgM, IgA, IgD or IgE molecules, whether isolated or not; Its antigen-specific antibody fragments (Fab, F(ab')2, Fv, disulfide-linked Fv, scF v, single domain antibody, closed structure multispecific antibody, disulfide bond scfv, diabody Antibodies can be prepared by conventional methods. The term antibody refers to any polypeptide that contains an antibody antigen-binding site. The antigen-binding site (paratope) is the site of the target antigen (I). The portion of an antibody that binds to and is complementary to an epitope of COS.

[0086] The term "epitope" refers to the region of an antigen to which an antibody binds. A functional epitope may be defined as a structural epitope. An epitope is a subset of a given protein that contains residues that directly contribute to the affinity of the interaction. It may also be composed of conformational, i.e., non-linear, amino acids. An epitope is a chemical entity of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group. In certain embodiments, certain triglycerides may be present in the form of determinants that are surface groups that are chemically active. It may have dimensional structural characteristics and / or particular charge characteristics.

[0087] An antigen-binding site is a polypeptide that contains one or more CDRs of an antibody and is capable of binding to an antigen. For example, a polypeptide may comprise a CDR3 (e.g., an HCDR3) For example, the polypeptide may comprise CDR1 and CDR2 (e.g., HCD1 and HCD2) of a variable domain of an antibody. R1 and R2) or CDR1 to 3 (e.g., HCDR1 to 3).

[0088] An antibody antigen-binding site may be provided by one or more antibody variable domains. Generally, an antibody binding site comprises a single variable domain, e.g., a heavy chain variable domain (VH domain) or is provided by the light chain variable domain (VL domain). Thus, an antibody antigen-binding site comprises a VH and VL pair or two or more such pairs. and VL.

[0089] The antibody may be a whole immunoglobulin including the constant region, or may be an antibody fragment An antibody fragment may contain a portion of an intact antibody, e.g., the antigen-binding and -binding functions 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) It is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region. F(ab')2 fragments containing (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of one arm of an antibody; (v) a dAb fragment consisting of a VH or VL domain (which is incorporated herein by reference in its entirety). Ward et al., (1989) Nature 341:544-546), and to (vi) An isolated complementarity-determining region (CDR) that retains specific antigen-binding function.

[0090] Further examples of antibodies include heavy chain dimers (5'-VH-(optional hinge)-CH 2-C H3-3') and lacks light chains.

[0091] Single chain antibodies (e.g., scFv) are commonly used fragments. Multispecific antibodies are Antibodies of the present invention may be formed from any such format, as appropriate. It is possible to adopt

[0092] Optionally, the antibody immunoglobulin domain may comprise additional polypeptide sequences, and / or target The antibody may be fused or conjugated to a recognition, tag, toxin, or other molecule. The ICOS may be fused or conjugated to one or more different antigen-binding domains to produce a second The antibody of the present invention can provide a molecule capable of binding to an antigen. an antibody antigen-binding site and (ii) a further antigen binding site that recognizes another antigen (e.g., PD-L1); and a multispecific antibody, e.g., a nucleotide sequence comprising a nucleotide sequence of the ... It may also be a bispecific antibody.

[0093] An antibody typically comprises an antibody VH and / or VL domain. The L domain is also part of the present invention. An antibody variable domain is a region that contains complementarity determining regions (CDRs, i.e. i.e., CDR1, CDR2, and CDR3) and the amino acid sequence of the framework region (FR) Thus, the VH domain and the VL domain are portions of the light and heavy chains of an antibody. Within each domain are CDRs and FRs. A VH domain contains one set of HCDRs, and a V The L domain contains one set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to Each VH and VL typically comprises FR1, CDR1, FR2, , CDR2, FR3, CDR3, FR4 are arranged in this order from the amino terminus to the carboxy terminus. It also consists of three CDRs and four FRs. The amino acid positions assigned to DR and FR are based on the Kabat (Sequences of Proteins of Immunological Interest(Nati onal Institutes of Health,Bethesda,Md.,1 987 and 1991) or according to the IMGT nomenclature. The antibody comprises an antibody VH domain comprising VH CDR1, CDR2 and CDR3, and a framework. Alternatively or additionally, the antibody may comprise a VL CDR1, CDR2 and The antibody VL domain may comprise a CDR3, as well as a framework. Examples of antibody VH and VL domains and CDRs are listed in the accompanying sequence listing, which forms part of this disclosure. The CDRs shown in the sequence listing are defined according to the IMGT system.

[18] . All VH and VL sequences, CDR sequences, and a set of C The DR and one set of HCDRs and one set of LCDRs are used to realize aspects and embodiments of the present invention. As used herein, a "set of CDRs" refers to CDR1, CDR2 and Therefore, one set of HCDRs, HCDR1, HCDR2 and CDR3 R3 refers to one set of LCDR, LCDR1, LCDR2 and LCDR3. Unless otherwise specified, a "set of CDRs" includes HCDRs and LCDRs.

[0094] The antibodies of the present invention may comprise one or more CDRs as described herein to form a set of CDRs. DR, for example, CDR3, and optionally CDR1 and CDR2. R or CDR set is STIM001, STIM002, STIM002-B, STI M003, STIM004, STIM005, STIM006, STIM007, STI The CDR or set of CDRs may be any of M008 and STIM009. It may also be any of the variants described in the specification.

[0095] The present invention relates to antibodies STIM001, STIM002, STIM002-B, STIM00 3, STIM004, STIM005, STIM006, STIM007, STIM00 8, and HCDR1, HCDR2, and / or HCDR1 of any of STIM009 3, and / or the LCDR1, LCDR2, and / or LCDR3 of any of these antibodies. The present invention provides antibodies that contain a set of CDRs, such as a DR3. The antibody may optionally comprise a VH CDR set of one of these antibodies. and the VL CDRs may comprise one VL CDR set of the same antibody as the VH CDRs. It may be derived from the same antibody or from a different antibody.

[0096] a VH domain comprising a disclosed set of HCDRs and / or a disclosed set of LCDRs Also provided by the present invention are VL domains comprising

[0097] Typically, a VH domain pairs with a VL domain to provide an antibody antigen-binding site. As discussed further below, only the VH domain or the VL domain can be used to bind to the antigen. The STIM003 VH domain may be paired with the STIM003 VL domain. The resulting antibody may contain both the STIM003 VH and VL domains. Similar embodiments can be used with other VH and In another embodiment, the STIM003 VH is Light chain promiscuity is not known in the art. Again, similar embodiments are well established in other areas disclosed herein. The VH and VL domains of the above are provided by the present invention.

[0098] Therefore, antibodies STIM001, STIM002, STIM003, STIM004 , and STIM005, the VH of either of the antibodies STIM001, STIM002, ST May be paired with any of the VLs of IM003, STIM004, and STIM005 In addition, antibodies STIM001, STIM002, STIM002-B, and STIM002 , STIM003, STIM005, STIM006, STIM007, STIM008 , and STIM009 are used in combination with antibodies STIM001, STIM002, ST IM002-B, STIM003, STIM004, STIM005, STIM006, Even when paired with the VL of STIM007, STIM008, or STIM009 good.

[0099] An antibody comprises one or more CDRs, e.g., a set of CDRs, within an antibody framework. The framework regions may also be human germline gene segment sequences. Therefore, the antibody contains one set of HCDRs in a human germline framework. The antibody may be a human antibody having a VH domain comprising It also has a VL domain containing a set of LCDRs in a cell lineage framework. "gene segment," e.g., a VH gene segment, a D gene segment, or a JH gene segment A subsegment refers to an oligonucleotide having a nucleic acid sequence from which that portion of an antibody is derived. For example, a VH gene segment is a polypeptide VH from FR1 to CDR3. These are oligonucleotides containing nucleic acid sequences corresponding to the human V, D, and J gene domains. The daughter segments are recombined to generate the VH domain, and the human V and J segments are recombined. The D domain or region is the diversity domain or region of the antibody chain. J domain or region refers to the joining domain or region of an antibody chain. Variants have framework regions that do not exactly match or align with the corresponding gene segment. Although a sequence alignment can be used to identify the most closely related antibody VH or VL domains, and identifying gene segments that contain specific VH or VL domains. It is possible to identify which specific combination of genes the antibody sequence originates from. When aligned with the gene segments, the antibody amino acid sequence is identified as the amino acid sequence encoded by the gene segments. The antibody nucleotide sequence may be aligned with the amino acid sequence of the gene segment. It may also be directly aligned with the octide sequence.

[0100] STIM antibody VH and VL domain sequences relative to related antibodies and human germline sequences The alignment is shown in Figures 35, 36 and 37.

[0101] The antibodies of the present invention may be human antibodies comprising human variable regions and non-human (e.g., murine) constant regions. The antibody of the present invention may be, for example, a chimeric antibody having a human variable region, and optionally It also has a human constant region.

[0102] Thus, the antibody optionally comprises a constant region or portion thereof, e.g., a human antibody constant region or For example, a VL domain may contain at its C-terminus an antibody light chain kappa or lambda constant. Similarly, an antibody VH domain may be linked at its C-terminus to any antibody VH domain. IgA, IgE, and IgM, as well as IgG1 or Ig2. Immunoglobulin heavy chain constants from any of the isotype subclasses, such as gG4 It may be attached to all or part of the region (eg, the CH1 domain or Fc region).

[0103] Examples of human heavy chain constant regions are shown in Table S1.

[0104] The constant region of the antibody of the present invention may alternatively be a non-human constant region. For example, the antibody are produced in transgenic animals (examples of which are described elsewhere herein). When this is done, chimeric antibodies containing human variable regions and non-human (host animal) constant regions are produced. Some transgenic animals produce fully human antibodies. Others It has been engineered to generate antibodies containing chimeric heavy chains and fully human light chains. If the antibody contains the above non-human constant regions, replacing them with human constant regions will improve its immunogenicity. Therefore, it is possible to provide an antibody that is more suitable for administration to humans as a therapeutic composition. can.

[0105] Digestion of antibodies with the enzyme papain produces two identical fragments, also known as "Fab" fragments. an antigen-binding fragment, and an "Fc" fragment that does not have antigen-binding activity but has the ability to be crystallized "Fab" as used herein refers to one constant fragment of each of the heavy and light chains. The term "Fc region" as used herein refers to a fragment of an antibody that contains one or more variable domains. The term "antibody" refers to an antibody or antibody fragment of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Used to define the C-terminal region. "Fc fragment" is a fragment held together by disulfides. The effector functions of antibodies are determined by certain The Fc region is also recognized by Fc receptors (FcRs) found on cells. Digestion of antibodies with the enzyme pepsin links the two arms of the antibody molecule. The F(ab')2 fragment remains intact and contains two antigen-binding sites. The fragments have the ability to cross-link antigens.

[0106] "Fv" as used herein refers to an antibody that retains both an antigen-recognition site and an antigen-binding site. This refers to the smallest fragment of an antibody that binds to the target molecule. This region is associated with strong non-covalent or covalent bonds. It consists of a dimer of one heavy chain variable domain and one light chain variable domain. The three CDRs of each variable domain interact to form an antigen-binding domain on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or Fv) containing only three CDRs specific for an antigen Even half of the total binding site has the ability to recognize and bind to the antigen, but this is less than the entire binding site. Low affinity.

[0107] The antibodies disclosed herein may be modified to increase or decrease their serum half-life. In one embodiment, one or more of the mutations T252L, T254S, or T256F The introduction of the above increases the biological half-life of the antibody. As described in US Pat. Nos. 869,046 and 6,121,022 (as described therein) (These modifications are incorporated herein by reference), heavy chain constant region CH1 domain or CL domain The region was modified to salvage two loops from the CH2 domain of the Fc region of IgG. It can also be increased by including receptor binding epitopes. The Fc hinge region of the antibody or antigen-binding fragment of the invention may be mutated to improve the production of the antibody or fragment. One or more amino acid mutations in the CH2-C of the Fc hinge fragment decrease the biological half-life. When introduced into the H3 domain interface region, the antibody or fragment binds to Staphylococcus aureus protein A. (SpA) binding is impaired compared to native Fc hinge domain SpA binding. Serum half-life Other methods for increasing the antibody activity are known to those skilled in the art. In another embodiment, the antibody or fragment is PEGylated. In another embodiment, the antibody is fused to an albumin-binding single domain antibody (dAb). In this case, the antibody or fragment is PASed (i.e., an uncharged antibody having a large hydrodynamic volume). It consists of PAS (XL-Protein GmbH) which forms a random coil structure In another embodiment, the antibody or fragment is a gene fusion of an XTENyl polypeptide sequence. ated® / rPEGylated (i.e., non-exact repeat peptide sequences (Amun ix, Versartis) with a therapeutic peptide). The fragment or fragment is ELP-conjugated (i.e., the gene is converted to an ELP repeat sequence (PhaseBio) These various half-life extending fusions are described by Strohl, BioDrugs (20 15) 29:215-239, e.g., in Tables 2 and 6. The fusions of these are incorporated herein by reference.

[0108] The antibody may have a modified constant region to increase stability. In one embodiment, the heavy chain constant region comprises a Ser228Pro mutation. The antibodies and fragments disclosed herein may be modified to alter the number of cysteine ​​residues. The heavy chain hinge region is modified to facilitate assembly of the light and heavy chains and to can be used to increase or decrease the stability of the antibody.

[0109] Fc effector functions, ADCC, ADCP, and CDC As discussed above, anti-ICOS antibodies are available in various isotypes and in different constant Examples of human IgG antibody heavy chain constant region sequences are shown in Table S1. The Fc region of the body is responsible for Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and complement dependence. The cytotoxicity (CDC) activity and antibody-dependent cellular phagocytosis (ADCP) activity were mainly investigated. These "cellular effector functions" determine the effector T cell mechanisms. Unlike the antibody-binding activity, it involves the recruitment of Fc receptor-bearing cells to the target cell site, and In addition to ADCC and CDC, the ADCP mechanism

[19] also targets antibody-bound T cells, resulting in their death. deplete Tregs, thereby representing a means to target high ICOS-expressing TRegs for deletion. .

[0110] The cell effector functions ADCC, ADCP, and / or CDC also lack an Fc region. It can be displayed by an antibody. An antibody can contain multiple different antigen binding sites, one One is directed at ICOS, and the other is directed at a target molecule, which, when engaged, prevents AD. CC, ADCP, and / or CDC are derived, e.g., joined by a linker. It contains two scFv regions, one of which can engage effector cells. It is an antibody that

[0111] The antibodies according to the present invention may exhibit ADCC, ADCP, and / or CDC. Alternatively, the antibodies according to the present invention lack ADCC, ADCP, and / or CDC activity. In either case, the antibody according to the invention may bind to one or more Fc receptors. They may contain an Fc region, or optionally lack one. Different antibody formats The use of FcR-binding and cellular effector functions allows the present invention to be As discussed elsewhere in this document, antibodies may be tailored for specific therapeutic uses. This makes it possible to:

[0112] Some suitable antibody formats for therapeutic applications use wild-type human IgG1 constant regions. The constant region optionally comprises an enzyme having ADCC and / or CDC and / or ADCP activity. A suitable wild-type human IgG1 constant region sequence may be an effector-compatible IgG1 constant region. The column is SEQ ID NO: 340 (IGHG1 * 01). Further examples of human IgG1 constant regions are Shown in Table S1.

[0113] Effector-positive human antibodies for testing candidate therapeutic antibodies in mouse models of human disease Instead of the mouse constant region, effector-positive mouse IgG2a (mIgG2a) A constant region may also be included.

[0114] The constant region may be engineered for enhanced ADCC and / or CDC and / or ADCP. good.

[0115] The efficacy of Fc-mediated effects can be determined by manipulating the Fc domain through a variety of established techniques. By such methods, the affinity for a particular Fc receptor can be increased. This increases the activity of the ATP-dependent ATPases, thereby creating a diverse profile of potential enhanced activation. This can be achieved by modifying one or more amino acid residues

[20] . n297 specific mutations or altered glycosylation (e.g., EU index numbering) The human IgG1 constant region containing N297Q of the IgG1 domain enhances binding to Fc receptors. Exemplary mutations are those in the human IgG1 constant region (or other IgG isotypes). For the equivalent positions in the nucleotide sequence, a nucleotide sequence of residues selected from 239, 332, and 330 is used. Thus, the antibody may be one or more of N297Q, S239D, I332E, and One or more mutations independently selected from A330L (EU index numbering) It may contain a human IgG1 constant region. Triple mutation to enhance binding to FcRn. (M252Y / S254T / T256E) may also be used, as well as other mutations that affect FcRn binding. Mutations of the above are discussed in Table 2 of

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

[0116] Increased affinity for Fc receptors can be achieved, for example, by fucosylation or defucosylation of the variants. By altering the native glycosylation profile of the Fc domain through production in vitro This can be achieved by using F that does not have fucose residues

[22] . The trimannosyl core structure of the complex N-glycan of Fc. These glycoengineered antibodies lacking core fucose residues may result in enhanced FcγRIIIa binding ability. Therefore, it may exhibit stronger ADCC than its fucosylated counterpart. For example, it may increase ADCC. To increase binding to Fc-gamma RIII, residues in the hinge region are modified.

[23] Therefore, antibodies can be produced using mutant versions of the wild-type human IgG heavy chain constant region. The variant human IgG heavy chain constant region may comprise a human IgG heavy chain constant region, and the variant human IgG heavy chain constant region may comprise a human IgG heavy chain constant region. a human Fcγ receptor selected from the group consisting of FcyRIIB and FcyRIIA, Human IgG heavy chain constant regions bind to human Fcγ receptors with higher affinity than human IgG heavy chain constant regions bind to human Fcγ receptors. This antibody contains a human IgG heavy chain constant region that is a mutant of the wild-type human IgG heavy chain constant region. The mutant human IgG heavy chain constant region may comprise a variant of human FcγRIIB, which variant may comprise a variant of wild-type human IgG heavy chain constant region. The IgG heavy chain constant region binds to human FcγRIIB with higher affinity than the IgG heavy chain constant region binds to human FcγRIIB. The mutant human IgG heavy chain constant region may be a mutant human IgG1, a mutant human IgG2, or In one embodiment, the mutated human IgG4 heavy chain constant region may be a mutated human IgG4 heavy chain constant region. The common region is G236D, P238D, S239D, S267E, L328F, and L32 Contains one or more amino acid mutations selected from 8E (EU index numbering system) In another embodiment, the mutant human IgG heavy chain constant region comprises S267E and L328F; P2 38D and L328E; P238D, E233D, G237D, H268D, P271G and one or more substitutions selected from the group consisting of A330R; P238D and E233D , G237D, H268D, P271G, A330R; G236D and S267E ;S239D and S267E;V262E, S267E, and L328F;and V264 E, S267E, and L328F (EU index numbering system). The enhanced CDC activity is due to the activation of the classical complement activation cascade. This can be achieved by amino acid changes that increase affinity for C1q, the first component of the

[24] Another approach is to use human immunoglobulin G (IgG3) to exploit the higher affinity of IgG3 for C1q. By creating a chimeric Fc domain made from human IgG1 and human IgG3 segments,

[25] The antibodies of the present invention may be mutated at residues 329, 331, and / or 322. The inclusion of such amino acids may result in a reduction or abolishment of C1q binding and / or CDC activity. In another embodiment, the antibody or antibody fragment disclosed herein has a nucleotide sequence between residues 231 and 23 9, which alters the ability of the antibody to fix complement. In one embodiment, the antibody or fragment has an amino acid substitution such that the amino acid is E345K. , E430G, R344D, and D356R, especially R344D and D356R (EU In A set containing one or more mutations selected from a set containing double mutations (including the Index numbering system) It has a normal region.

[0117] WO2008 / 137915 describes modified Fc with enhanced effector function. describes anti-ICOS antibodies with VH and VK domains and Enhanced ADCC activity compared to the level mediated by a parent antibody containing a wild-type Fc region The antibodies according to the present invention are also capable of mediating ADCC activity as described therein. Such variant Fc regions may be used that have effector functions as described above.

[0118] The ADCC activity of an antibody can be determined by the assays described herein. ADCC activity was measured in vitro using ICOS-positive T cell lines as described in Example 10. The ADCC activity of anti-PD-L1 antibodies may be determined using PD-L1-expressing cells. This may be determined in vitro in an ADCC assay.

[0119] In certain applications (e.g., in the context of vaccination), Fc effector functions It may be preferable to use an antibody without a constant region, or an Fc Examples of such antibody formats are provided elsewhere herein. Alternatively, the antibody may have a constant region that is effector null. The antibody may have a heavy chain constant region that does not bind to an Fcγ receptor, e.g., the constant region The eu235Glu mutation (i.e., a wild-type leucine residue is changed to a glutamic acid residue) Another optional mutation in the heavy chain constant region may be to enhance stability. The heavy chain constant region contains the Leu235Glu mutation and Ser The "IgG4-PE" heavy chain may be an IgG4 containing both the 228Pro mutation and the 228Pro mutation. The chain constant region is effector null.

[0120] An alternative effector-null human constant region is a disabled IgG1. The IgG1 heavy chain constant region has amino acids at positions 235 and / or 237 (EU index numbering). lanine, for example, L235A and / or G237A mutations ("LAGA" ) containing IgG1 * It may also be a 01 sequence.

[0121] The variant human IgG heavy chain constant region is human FcγRIIIA, human FcγRIIA, or Contains one or more amino acid mutations that reduce the affinity of IgG for human FcγRI. In one embodiment, FcγRIIB is expressed by macrophages, monocytes, B cells, dendritic cells, and the like. The antibody is expressed on cells selected from the group consisting of cytoplasmic reticulocytes, endothelial cells, and activated T cells. In this embodiment, the mutant human IgG heavy chain constant region has the amino acid mutations G236A, S23 9D, F243L, T256A, K290A, R292P, S298A, Y300L, V 305I, A330L, I332E, E333A, K334A, A339T, and P39 6L (EU index numbering system). The human IgG heavy chain constant region is S239D; T256A; K290A; S298A; I33 2E;E333A;K334A;A339T;S239D and I332E;S239D, A330L, I332E;S298A, E333A, K334A;G236A, S239D and I332E; and F243L, R292P, Y300L, and V305 I and P396L (EU index numbering system) In one embodiment, the variant human IgG heavy chain constant region comprises a set of mutations, including S239D , A330L, or I332E amino acid mutations (EU index numbering system) In one embodiment, the variant human IgG heavy chain constant region comprises the S239D and I332E amino acids. In one embodiment, the mutant human IgG The heavy chain constant region contains S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the antibody or fragment is a variant human IgG1 heavy chain constant region comprising a mutated human IgG1 heavy chain constant region (a mutated human IgG1 heavy chain constant region). In another embodiment, the antibody or fragment thereof comprises an afucosylated Fc region. In another embodiment, the antibody or fragment is fucosylated.

[0122] Antibodies bind to one or more Fc receptors but do not induce cellular effector functions; That is, it may have a heavy chain constant region that does not mediate ADCC, CDC, or ADCP activity. Such constant regions are responsible for inducing ADCC, CDC, or ADCP activity. May fail to bind to a specific Fc receptor(s).

[0123] Antibody generation and modification Methods for identifying and preparing antibodies are well known. Antibodies may be directed against ICOS or fragments thereof. or immunization with a synthetic peptide containing the desired ICOS sequence motif, followed optionally by and humanized antibodies, in which the constant and / or variable regions have been humanized to produce human or humanized antibodies. transgenic mice (e.g., Kymouse™, Velocimouse™) Omnimouse®, Xenomouse®, HuMab M ouse®, or MeMo Mouse®), rats (e.g., Omni rat, camelid, shark, rabbit, chicken, or any other non-human animal In one example, the vectors can be produced using yeast, phage, or other microorganisms, as will be apparent to those skilled in the art. Alternatively, display techniques such as ribosome display can be used. Standard affinity maturation using spray technology has been demonstrated using transgenic animals, phage display Further steps after isolation of antibodies derived from a leukocyte library or other library Representative examples of suitable techniques are incorporated herein by reference in their entirety. This is described in US20120093818 (Amgen, Inc.), for example, in the method shown in paragraphs

[0309] to

[0346] .

[0124] Immunization of ICOS knockout non-human animals with human ICOS antigen revealed that human The production of antibodies that recognize both human and non-human ICOS is promoted. As described in the literature and in the Examples, ICOS knockout mice express human ICOS. Immunization with cells expressing ICOS was performed to detect the effects of ICOS on mouse and human ICOS in mice. The production of antibodies against human ICOS and mouse ICOS can be stimulated. Antibodies can be recovered and tested for binding. In this way, cross-reactive antibodies can be selected. which can be screened for other desirable properties as described herein. Expression of an endogenous antigen (e.g., an endogenous mouse antigen) has been knocked out in the animal. Producing antibodies against an antigen (e.g., a human antigen) by immunizing an animal with the antigen The method may be performed in an animal capable of producing antibodies comprising human variable domains. The genome of such animals contains human variable region gene segments and, optionally, endogenous constant region genes. to include human or humanized immunoglobulin loci encoding human constant regions. The recombinant human variable region gene segments can be used to engineer non-human or human The human antibody can then be generated with any of the non-human constant regions. When intended for in vivo use in humans, the constant regions are replaced by human constant regions. Such a method and a knockout transgenic animal may be used. / 061078.

[0125] Generally, Kymouse™, VELOCIMMUNE®, or other mice or rats (optionally ICOS knockout mice or rats, as described) The mice are challenged with the target antigen, and lymphocytes (B cells, etc.) are collected from the mice that express antibodies. Lymphocytes can be fused with myeloma cell lines to prepare immortal hybridoma cell lines. Such hybridoma cell lines are then screened to identify antibodies specific to the antigen of interest. The hybridoma cell line encoding the heavy and light chain variable regions may be identified. The DNA may be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, antigen-specific DNA encoding the chimeric antibody or the light and heavy chain variable domains is then transferred to antigen-specific lymphocytes. It may also be isolated directly from

[0126] First, a high affinity chimeric antibody having human variable regions and murine constant regions is isolated. These antibodies have been characterized based on affinity, selectivity, agonism, T cell-dependent killing, neutralizing capacity, and epitope identity. The murine constant regions are optionally characterized and selected for desirable properties, including and the desired human constant region is substituted to produce a fully human antibody of the present invention, e.g., a wild-type or modified antibody. IgG1 or IgG4 (see, e.g., US2005 / 0129994, incorporated herein by reference in its entirety) 11 / 0065902). The regions may vary depending on the particular application, but the high affinity antigen binding properties and target specificity properties are variable. Belongs to the area.

[0127] Thus, in a further aspect, the present invention provides a method for producing a human or humanized immunoglobulin locus. and a transgenic non-human mammal having a genome comprising an ICO. The mammal does not express S. The mammal may be, for example, a knockout mouse or rat, or other living Transgenic mice, such as Kymouse™, can be used to express the human The heavy and light chain immunoglobulin loci are derived from the corresponding endogenous mouse immunoglobulin loci. The transgenic mammals according to the present invention are It may contain insertions of human overexpression fragments or may contain human overexpression fragments that are randomly inserted into the genome. The high and light chain immunoglobulin loci or immunoglobulin genes are located at the endogenous Ig loci or It may be inserted at an exogenous location or provided on an additional chromosome or chromosomal fragment.

[0128] A further aspect of the present invention is the use of such non-human IgG4-associated ... Use of mammals and the production of antibodies or antibody heavy and / or light chains in such mammals. A method for producing a chain variable domain.

[0129] Methods for producing antibodies that bind to the extracellular domain of human and non-human ICOS include human or non-human ICOS. is a transgenic mouse that does not express ICOS and has a genome containing a humanized immunoglobulin locus. providing a transgenic non-human mammal, (a) Immunizing a mammal with a human ICOS antigen (e.g., cells expressing human ICOS) or 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 to (d) selecting one or more antibodies that bind to both human ICOS and non-human ICOS; It may include.

[0130] Tests for the ability to bind to human ICOS and non-human ICOS were performed using surface plasmon resonance imaging (SPIR). This may be done using ultrasound, HTRF, FACS, or any other method described herein. Optionally, the binding affinity to human and mouse ICOS is determined. The affinity or fold difference in affinity of binding to mouse ICOS may be determined, but Thus, antibodies showing species cross-reactivity can be selected (an affinity threshold can be used as a selection criterion). (and fold differences are exemplified elsewhere herein). Neutralizing Ability or Neutralization of Antibodies to Block Binding of Iodides to Human and Mouse ICOS Receptors The fold difference in activity is used to screen for cross-reactive antibodies, for example, in an HTRF assay. Additionally or alternatively, it may be determined as a method. Again, it may be used as a selection criterion. Possible thresholds and fold differences are exemplified elsewhere herein.

[0131] The method involves binding to non-human ICOS from the same or a different species as the immunized mammal. This may include testing the antibody for its ability to inhibit the transgenic mammalian cell line. If the animal is a mouse (e.g., Kymouse™), the antibody binds to mouse ICOS. When the transgenic mammal is a rat, the antibody can be tested for their ability to bind to rat ICOS. It may be equally useful to determine the cross-reactivity of isolated antibodies to ICOS. Therefore, antibodies produced in goats were shown to be effective for binding to rat or mouse ICOS. Optionally, binding to goat ICOS may be determined instead or in addition. This may be done.

[0132] In another embodiment, the transgenic non-human mammal contains, in place of human ICOS, The recipient may be immunized with non-human ICOS, optionally with ICOS of the same mammalian species (e.g. Alternatively, ICOS knockout mice may be immunized with mouse ICOS. The affinity of isolated antibodies for binding to human ICOS and non-human ICOS is the same. and antibodies that bind to both human and non-human ICOS are selected.

[0133] Encoding the antibody heavy chain variable domain and / or the antibody light chain variable domain of the selected antibody The nucleic acid may be isolated. Such nucleic acid may comprise a complete antibody heavy and / or light chain, or It may encode a variable domain(s) without an associated constant region(s). Thus, the coding nucleotide sequence may be obtained directly from mouse antibody-producing cells, or from B cells. Immortalizing or fusing the cells to generate hybridomas that express antibodies, and Optionally, the nucleic acid encoding the variable domain(s) may then be , joined to a nucleotide sequence encoding a human heavy chain constant region and / or a human light chain constant region. and encoding a human antibody heavy chain and / or a human antibody light chain, e.g., both the heavy chain and the light chain. As described elsewhere herein, a nucleic acid encoding an antibody comprising The step of administering the immunized mammal to a human as a pharmaceutical is preferably Non-human constant regions are substituted with human constant regions to produce antibodies that are less immunogenic when This is particularly useful when generating chimeric antibodies with common regions. The presence of constant regions is also important for determining the effector functions of an antibody, and many preferred Suitable heavy chain constant regions are discussed herein.

[0134] As described herein, nucleic acids encoding the heavy and / or light chain variable domains of an antibody Other modifications to the sequence, such as mutations and alterations of residues, can be made.

[0135] The isolated (optionally mutated) nucleic acid can be cultured in a host cell, e.g., CHO, as described. The antibody or antibody heavy and / or light chain variable domains can then be expressed. The host cells are cultured under conditions for expression in any desired antibody format. Possible antibody formats, e.g., whole immunoglobulins, antigen-binding fragments, and other designs are discussed herein. This will be stated in the specification.

[0136] any of the VH and VL domains or CDRs whose sequences are specifically disclosed herein Variable domain amino acid sequence variants of the, as described, may be used in accordance with the present invention.

[0137] Optimizing antibody sequences for large-scale manufacturing, facilitating purification, and enhancing stability or to improve suitability for inclusion in a desired pharmaceutical formulation. There are a number of reasons why it may be desirable to do so. Protein engineering can be done to isolate specific fragments in an antibody sequence. The substitution can be performed at more than one target residue, e.g., by substituting one amino acid with an alternative amino acid. (Optionally, all naturally occurring amino acids at this position, excluding Cys and Met if possible) Mutants containing ribonucleotides were generated and their effects on function and expression were assessed to determine the best substitutions. For example, through the formation of new intramolecular or intermolecular cysteine-cysteine ​​bonds. Therefore, residues may be substituted with Cys or Met, or the residues may be substituted with Cys or Met, which may cause manufacturing difficulties. Introducing these residues into the sequence is undesirable in some cases. When a compound is selected and optimized for manufacturing and clinical development, its antigen-binding properties are maximized. It is generally desirable to have a smaller affinity and potency than the parent molecule. However, variants can be engineered to modulate important antibody properties such as affinity, cross-reactivity, or neutralization potency. can also be generated.

[0138] The antibody may have a structure similar to that of any one set of H CDRs and / or L CDRs of any of the disclosed antibodies. and one or more amino acid mutations within the disclosed H CDR and / or L CDR sets. The mutations may be amino acid substitutions, deletions, or insertions. For example, one or more amino acid substitutions within the disclosed set of H CDRs and / or L CDRs. For example, there may be up to 12, 1 There may be 1, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations, e.g., substitutions. For example, there can be up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions, in HCDR3, and / or LCDR3, there may be up to 6, 5, 4, 3, or 2 mutations, e.g., substitutions. The antibody may comprise any of the HCDR sets, LCDs, and HCDRs shown for any STIM antibody herein. It may contain a set of R, or a set of six (H and L) CDRs, or one or two conserved It may comprise a set of CDRs with substitutions.

[0139] One or more amino acid mutations may be made in the framework of the antibody VH or VL domain disclosed herein. For example, the corresponding human germline segment sequences can be optionally generated in a germline region. One or more residues that differ from the sequence can be reverted to germline. The human germline gene segment sequences corresponding to the VH and VL domains of the OS antibodies are shown in Table 1. E12-1, Table E12-2, and Table E12-3, and the corresponding germline sequences. The alignment of antibody VH and VL domains is shown in the image.

[0140] The antibody may be a hybrid of at least 60, 7 or 8 VH domains of any of the antibodies shown in the attached sequence listing. VH domains with 0, 80, 85, 90, 95, 98, or 99% amino acid sequence identity and / or a combination of at least 60, 70, 80, 90, 100, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 34 VL domains with 0, 80, 85, 90, 95, 98, or 99% amino acid sequence identity Algorithms that can be used to calculate the percent identity of two amino acid sequences include Algorithms include, for example, BLAST, FASTA, or Smith-Waterman algorithms. A particular variant may involve one or more The above amino acid sequence modifications (addition, deletion, substitution, and / or insertion of amino acid residues) may be included.

[0141] Modifications may occur in one or more framework regions and / or one or more CDRs. Variants are optionally provided by CDR mutagenesis. Alterations usually do not result in loss of function. Therefore, antibodies containing such modified amino acid sequences do not bind ICOS. For example, the modification may retain the ability to The modified amino acid sequence can retain the same quantitative binding ability as an antibody without the modified amino acid sequence. Antibodies comprising the following may have an improved ability to bind ICOS.

[0142] The modification may be a substitution of one or more amino acid residues with a non-natural or non-standard amino acid; or more non-naturally occurring or non-standard forms of the amino acid residues, or or the insertion of non-standard amino acids into the sequence. Examples of amino acids and positions are described elsewhere herein. Naturally occurring amino acids are represented by the standard single letter Depending on the chord, G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R The 20 "standard" L-amino acids are identified as A, H, D, and E. Non-standard amino acids are Any amino acid residue that can be incorporated into the polypeptide backbone or that can result from the modification of an existing amino acid residue. Non-standard amino acids may be naturally occurring or non-naturally occurring.

[0143] As used herein, the term "mutant" refers to a mutant that has one or more amino acid or nucleic acid deletions, substitutions, or deletions. A polypeptide or nucleic acid that differs from the parent polypeptide or nucleic acid by substitution or addition, but retains one or more specific functions of the parent molecule. It refers to a peptide or nucleic acid that retains its function or biological activity. Modifications in which a different naturally occurring amino acid residue is substituted are included. Such substitutions are referred to as "conservative" substitutions. In this case, the amino acid residues contained in the polypeptide may be classified as polar, side chain Substitute another naturally occurring amino acid with similar characteristics, either in terms of functionality or size. Such conservative substitutions are well known in the art. Substitutions encompassed by the present invention include: They may also be "non-conserved", where the amino acid residues present in the peptide are: Substitution with amino acids with different properties (e.g., charge or (a hydrophobic amino acid is replaced with alanine) or a natural amino acid is replaced with a non-conventional amino acid. In some embodiments, the amino acid substitutions are conservative. When used in reference to a oligonucleotide or polypeptide, variants are encompassed within the term and compared to a reference polynucleotide or polypeptide, respectively (e.g., wild-type polynucleotide or polypeptide). Changes in primary, secondary, or tertiary structure (compared to a oligonucleotide or polypeptide) It refers to a polynucleotide or polypeptide that can

[0144] In some embodiments, "synthetic variants" are used, which are isolated or produced using methods well known in the art. "variant," "recombinant variant," or "chemically modified" polynucleotide variant or A "modified variant" can be used in any of the following polypeptide variants: The polynucleotide changes may include conservative or non-conservative amino acid changes, such as Amino acid substitutions, additions, deletions, fusions, and deletions in the polypeptide encoded by the reference sequence. Some embodiments of the use can result in truncation of, for example, human proteins. Mutant forms of ornithine insertions, including but not limited to, ornithine insertions that do not normally occur in proteins. Insertions and substitutions of amino acids and other molecules that do not normally occur in the underlying peptide sequence (including insertion mutants, deletion mutants, and substitution mutants with amino acid substitutions.) The term "substantially substituted," when describing a polypeptide, refers to a compound that substantially alters the activity of the polypeptide. For example, a conservative substitution refers to a change in the amino acid composition of a polypeptide that does not alter the amino acid sequence. Residues can be grouped by similar chemical properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar). Conservative amino acid substitution refers to the substitution of a different amino acid residue with a different amino acid residue that has a similar function (e.g., a specificity). Substitution of isine by isoleucine or valine, aspartate by glutamate or threonine with serine. Conservative substitution tables for these purposes are well known in the art. For example, the following six groups are Each of the amino acids contains an amino acid that is a conservative substitution in the following: 1) alanine (A), serine (S), threonine (T). 1) aspartic acid (D), glutamic acid (E); 2) asparagine (N), glutamine 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (H) 6) phenylalanine (F), tyrosine (Y), tryptophan ( W). (Creighton, Proteins, WH Freeman and Co. (See company (1984), incorporated by reference in its entirety.) In an embodiment, individual amino acids are modified, added, or deleted by one or a small number of amino acids. Substitutions, deletions, or additions are also considered "conservative substitutions" if the changes do not reduce the activity of the peptide. The insertions or deletions are typically in the range of about 1 to 5 amino acids. The selection of a suitable amino acid depends on the position of the amino acid to be substituted in the peptide, e.g., based on whether it is on the outside of the peptide and exposed to the solvent, or on the inside and not exposed to the solvent. You may also select

[0145] The amino acid that will replace the existing amino acid is subjected to a series of reactions, including exposure to solvent, to the existing amino acid. The position of the amino acid (i.e., whether the amino acid is solvent-exposed or in a non-solvent-exposed internal position) (whether present on the exterior of the peptide or polypeptide compared to the amino acids surrounding it) Such conservative amino acid substitutions can be selected, for example, by the method described in Dordo et al., J. .Mol Biol,1999,217,721-739 and Taylor et al., J. Theor. Biol. 119(1986);205-218 and S. Fr ench and B. Robson, J. Mol. Evol. 19 (1983) 171 As disclosed herein, it is well known in the art. Selecting conservative amino acid substitutions favoring exterior amino acids (i.e., solvent-exposed amino acids) For example, but not limited to, the following substitutions can be used: F for Y Substitution of T by S or K, substitution of P by A, substitution of E by D or Q, N Substitution of D or G for R, substitution of K for G, substitution of N or A for T, substitution of S or K for substitution of N or E for D, substitution of L or V for I, substitution of Y for F, substitution of T for S or substitution by A, substitution of R by K, substitution of G by N or A, substitution of K by R, A by S, K, or P.

[0146] In an alternative embodiment, amino acids suitable for use in the interior of proteins or peptides are Conservative amino acid substitutions can also be selected, for example, to make substitutions within the protein or peptide. Conservative substitutions can be used for certain amino acids (i.e., amino acids that are not solvent-exposed). For example, the following conservative substitutions can be used: Y is replaced with F, T is replaced with A or is replaced by S, I is replaced by L or V, W is replaced by Y, M is replaced by L, and N is D is substituted, G is substituted with A, T is substituted with A or S, D is substituted with N, and I is L or V, F is replaced by Y or L, S is replaced by A or T, and A is replaced by S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also included within the scope of the variations. Included.

[0147] The present invention relates to the VH and / or VL domains of antibodies shown in the attached sequence listing. The present invention also includes a method for producing an antibody containing a L domain variant, which comprises: (i) Addition, deletion, substitution, or deletion of one or more amino acids in the amino acid sequence of the parent antibody VH domain The insertion provides an antibody VH domain that is an amino acid sequence variant of the parent antibody VH domain. To provide The parent antibody VH domains are those of antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, The VH domain of either STIM008 or STIM009 or any of their antibodies providing a VH domain comprising a heavy chain complementarity-determining region of any one of the heavy chains; (ii) optionally combining the VH domain so provided with a VL domain, providing a VH / VL combination; (iii) testing the VH domain or VH / VL domain combination so provided; and identifying an antibody having one or more desired properties. do.

[0148] Desired properties include binding to human ICOS, binding to mouse ICOS, and binding to crab ICOS. This includes binding to other non-human ICOS, such as monkey ICOS. Antibodies with affinity comparable to or higher than mouse ICOS can be identified Other desirable characteristics include indirectly via depletion of immunosuppressive TRegs or T effectors. - Directly via activation of ICOS signaling on T cells to regulate effector T cell function Identifying antibodies with desired properties involves either The affinity, cross-reactivity, specificity, The present invention relates to the use of agonists, antagonists, and / or antagonists of the present invention, such as ICOS receptor agonism, neutralizing efficacy, and / or promotion of T cell-dependent killing. The method may include identifying antibodies having functional attributes as described in

[0149] When a VL domain is included in the method, the VL domain may be selected from the group consisting of STIM001, STIM0 02, STIM002-B, STIM003, STIM004, STIM005, STI M006, STIM007, STIM008 or STIM009 VL driver The parent VL domain may be STIM001, STIM002, S TIM002-B, STIM003, STIM004, STIM005, STIM006 , STIM007, STIM008, and STIM009 VL domains. or a parent VL domain containing the light chain complementarity-determining region of any of these antibodies. Addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of the L domain The variant may be provided by insertion.

[0150] The method for generating a variant antibody optionally includes the steps of: The method may further include expressing the resulting antibody. Nucleotide sequences corresponding to the desired antibody VH and / or VL domains are optionally It can be produced in one or more expression vectors. Suitable expression methods, including those described in detail herein.

[0151] Nucleic acid encoding and expression methods An isolated nucleic acid encoding an antibody according to the invention can be provided. It may be genomic DNA, cDNA, mRNA or RNA of synthetic origin. Other RNAs, or any combination of these, may encode antibodies.

[0152] The present invention also provides a plasmid, vector, transfection vector, or transfection vector comprising at least one of the above polynucleotides. Constructs are provided in the form of transcription or expression cassettes. Exemplary nucleotide sequences are provided in the Sequence Listing. Reference to a nucleotide sequence described herein refers to a DNA sequence having the specified sequence. A molecule having the specified sequence in which U is replaced with T unless the context requires otherwise. It includes RNA molecules.

[0153] The invention also provides recombinant host cells containing one or more nucleic acids encoding the antibodies. Methods for producing encoded antibodies include, for example, culturing recombinant host cells containing the nucleic acid. In this way, antibodies can be obtained. can be isolated and / or purified using any suitable technique and then used as appropriate. The production method may include adding the composition to a composition containing at least one additional component, such as a pharmaceutically acceptable excipient. The preparation can include formulating the preparation.

[0154] 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 bacteria. These include roviral systems, and transgenic plants and animals.

[0155] 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 cells in culture is also possible, depending on the production choice. are available to those skilled in the art as tools for the expression of heterologous polypeptides. Possible mammalian cell lines include Chinese hamster ovary (CHO) cells, HeLa cells, Baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 rat myeloma cells, human These include human fetal kidney cells, human fetal retinal cells, and many others.

[0156] The vector contains a promoter sequence, a terminator sequence, a polyadenylation sequence, an enhancer sequence, and a The antibody may contain appropriate regulatory sequences, including a sequence for a marker gene, and other sequences as appropriate. The nucleic acid encoding the calcium phosphate tranferase can be introduced into a host cell. Infection, DEAE-dextran, electroporation, liposome-mediated Sexual transfection and the use of retroviruses or other viruses, e.g., vaccinia by various methods, including transduction with baculovirus for insect cells. The introduction of nucleic acids into host cells, particularly eukaryotic cells, is a Virus or plasmid-based systems can be used. Plasmid systems are episomal The vector may be maintained in the host cell or may be integrated into an artificial chromosome. may be due to random integration of one or more copies at single or multiple loci, or In bacterial cells, the expression can be by targeted integration or by suitable techniques. Using calcium chloride transformation, electroporation, and bacteriophage After introduction, conditions for expression of the gene, e.g., The nucleic acid is expressed by culturing the host cells and then optionally isolating or purifying the antibody. It can be done.

[0157] The nucleic acids of the invention can be integrated into the genome (e.g., chromosome) of the host cell. This can be facilitated by including sequences that facilitate recombination with the genome, according to standard techniques. do.

[0158] The invention also relates to the use of the nucleic acids described herein in expression systems to express antibodies. The present invention provides a method comprising:

[0159] therapeutic use The antibodies described herein may be used in methods of treatment of the human or animal body by therapy. Antibodies may be used to treat disease, including the treatment of cancer or solid tumors, and in the context of vaccination. These compounds find use in increasing effector T cell responses, which are beneficial in a range of diseases or pathological conditions. The increased Teff response is due to the imbalance between Teff and Treg that favors Teff activity. This can be achieved using antibodies that modulate the ratio or intensity.

[0160] Anti-ICOS antibodies may deplete regulatory T cells and / or inhibit effector T cell responses in patients. can be used to increase the response to a given antibody, administered to a patient to deplete regulatory T cells, and and / or can be therapeutic by increasing the patient's effector T cell response. The present invention may treat a disease or pathological condition.

[0161] The antibodies of the present invention, or compositions comprising such antibody molecules or their encoding nucleic acids, may be administered in any may be used or provided for use in any such method. The use of an antibody or a composition comprising the antibody or its encoding nucleic acid for the manufacture of a medicament for The method typically includes administering the antibody or composition to a mammal. Suitable formulations and methods of administration are described elsewhere herein.

[0162] One envisioned therapeutic use of the antibodies is in the treatment of cancer. Cancer can be a solid tumor, such as a renal tumor. cell carcinoma (optionally renal cell carcinoma, e.g., renal clear cell carcinoma), head and neck cancer, melanoma (optionally malignant melanoma), non-small cell lung cancer (e.g., adenocarcinoma), bladder cancer, ovarian cancer, cervical cancer, stomach cancer, liver cancer, pancreatic cancer Could it be pancreatic cancer, breast cancer, testicular germ cell carcinoma, or a metastasis of a solid tumor such as those listed? or liquid hematologic tumors, such as lymphomas (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma) lymphoma (e.g., diffuse large B-cell lymphoma, DLBCL) or leukemia (e.g., Anti-ICOS antibodies can be used to treat melanoma, head and neck cancer, and non-small cell lung cancer. Anti-ICOS antibodies can enhance tumor clearance in cancers with moderate to high mutational burden, as well as other cancers with moderate to high mutational burden.

[0026] By enhancing the patient's immune response to these neoplastic lesions, Body-based immunotherapy offers the possibility of durable cures or long-term remissions, potentially in late-stage disease. It is also provided in the setting of illness.

[0163] Cancer is a heterogeneous group of diseases, but anti-ICOS antibodies can identify mutant or overexpressing cancer cells that distinguish cancer from normal tissue. Patients with the potential to kill any cancer cells by recognizing overexpressed epitopes Harnessing one's own immune system offers the potential to treat a range of different cancers. By modulating the f / Treg balance, anti-ICOS antibodies enhance the immune recognition and Therefore, anti-ICOS antibodies have broad spectrum Although these are useful therapeutic agents for cancers of the genus I, anti-ICOS therapy is particularly suitable and / or anti-I There are certain categories of cancers for which COS therapy may be effective when other treatments are not. .

[0164] One such group is cancers that are positive for expression of ICOS ligands. As described in

[2004] , cancer cells can acquire expression of ICOS ligand

[0027] . The expression of ICOS ligand occurs when the surface-expressed ligand binds ICOS on Tregs. , promotes the expansion and activation of Tregs, thereby suppressing the immune response to cancer, Cancer cells expressing ICOS ligands may provide a selective advantage to Tregs. Survival against this suppression of the immune system may depend on anti-ICOS targeting Tregs. These cells naturally express ICOS ligands and may be vulnerable to treatment with antibodies. This also applies to cancers of immune origin. Cancers that express ICOS ligands are known to suppress B cells, dendritic cells, and other immune cells, again providing a survival advantage through suppression. and antigen-presenting cells, such as cytoplasmic cells, and monocytes, and may be derived from fluid hematomas as described herein. Interestingly, these types of cancers show a marked decrease in ICOS and FOXP3 expression ( TCGA data) has also been shown to be high (see Example 25). Example 20 of the present specification in the treatment of tumors derived from cancerous B cells (A20 syngeneic cells) expressing ICOS ligands The efficacy of exemplary anti-ICOS antibodies is demonstrated.

[0165] Therefore, anti-ICOS antibodies are useful for treating cancers that are positive for ICOS ligand expression. Furthermore, cancers treated with the anti-ICOS antibodies of the present invention can be used in are positive for ICOS and / or FOXP3 expression, and optionally, an ICOS ligand It may also be a cancer that manifests.

[0166] For example, a test sample (e.g., a tumor biopsy) is obtained from the patient and the protein of interest is analyzed. By determining expression, it is possible to determine whether these cancers express a protein of interest (e.g., ICOS ligand). To determine whether the test is positive for expression of IFN-γ, ICOS, and / or FOXP3 The expression of one, two, or all such proteins of interest can be tested. Patients whose cancer is characterized as positive for ICOS are selected for treatment with anti-ICOS antibodies. As described elsewhere herein, anti-ICOS antibodies can be used as monotherapy or in combination with other anti-ICOS antibodies. can be used in combination with one or more other therapeutic agents.

[0167] Anti-ICOS antibodies also inhibit CTLA-4, PD-1, PD-L1, CD137, and GITR or treatment with antibodies or other drugs against immune checkpoint molecules such as CD73 These immunotherapies offer hope for patients with refractory cancers. Although effective against cancers, in some cases the cancer does not respond or does not tolerate continued treatment with the antibody. As with antibodies to immune checkpoint inhibitors, Anti-ICOS antibodies regulate the patient's immune system, but they do so where other such antibodies fail. Herein, animals bearing A20 B-cell lymphoma were treated with anti-ICOS Treatment with antibodies can reduce tumor growth, shrink tumors, and actually eliminate tumors from the body. However, treatment with anti-PD-L1 antibodies was not superior to the control. 20 Cell lines have also been reported to be resistant to anti-CTLA-4

[28] .

[0168] Therefore, anti-ICOS antibodies are similar to anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-PD-L1 antibodies, Anti-CD137 antibody, anti-GITR antibody, or anti-CD73 antibody (any or all of these), etc. The present invention can be used in methods of treating cancer that is refractory to treatment with one or more of the following immunotherapies: Cancers may be considered to be cancerous if treatment with the antibody or drug does not significantly reduce cancer growth, e.g. If the tumor continues to grow or does not decrease in size, or if the tumor continues to grow or does not decrease in size after the response period, If the growth recurs, the tumor is said to be refractory to antibody or other drug therapy. Non-response to a therapeutic agent can be characterized by the ability to kill or inhibit the growth of cancer cells. (e.g., tumor biopsy samples) and / or in a clinical setting. by observing that patients treated with the method do not respond to treatment (e.g., M Refractory to such immunotherapy treatments can be determined using imaging techniques, including radioimmunotherapy. Patients whose cancer has been characterized as a genital tumour are selected for treatment with anti-ICOS antibodies.

[0169] Furthermore, anti-ICOS antibodies have been shown to treat B-cell derived cancers that are resistant to treatment with anti-CD20 antibodies. Anti-ICOS antibodies can be used to treat rheumatoid arthritis. Anti-ICOS antibodies are used to treat cancers that do not respond to or become resistant to treatment with anti-ICOS antibodies. , can be used as a second-line (or further or additional) treatment for such cancers. Anti-CD20 antibody resistant cancers include B cell cancers, e.g., B cell lymphomas, e.g., diffuse large B cell lymphomas. Cancer resistance to anti-CD20 may be due to the killing of cancer cells by anti-CD20 antibodies. or by testing samples (e.g., tumor biopsies) for growth inhibition. Patients treated with anti-CD20 antibodies in the NIH and / or clinical setting have not responded to treatment Alternatively or additionally, cancer (e.g., tumor growth) may be detected by observing the presence or absence of a tumor. The test sample can be tested to assess CD20 expression, and the absence or presence of CD20 expression can be assessed. Low levels indicate a loss of sensitivity to anti-CD20 antibodies.

[0170] Thus, a sample obtained from a patient can be tested to detect the protein of interest, e.g., ICOS ligand. targeting of the receptor, ICOS, FOXP3, and / or another therapeutic agent (e.g., an anti-receptor antibody) Surface expression of the receptor can be determined. The target receptor can be an anti-CD2 (For antibody therapy) CD20, or PD1, EGFR, HER2, or HER3, etc. The surface expression and / or targeting of ICOS ligand, ICOS, FOXP3 may be a receptor for ICOS. Absence or loss of surface expression of target receptors may contribute to the susceptibility of cancers to anti-ICOS antibody therapy. Anti-ICOS antibodies are an indicator of ICOS ligand, ICOS, and FOXP3 expression. Cancers characterized by surface expression and / or lack or loss of surface expression of target receptors. and optionally, the patient has previously received anti-CTLA4 , anti-PD1, anti-PD-L1 or antibodies against target receptors, e.g. For example, continued or recurrent cancer cell growth, as measured by growth in tumor size. As previously reported, they have not responded or have stopped responding to antibody treatment.

[0171] Any suitable method may be used to detect whether the cancer cells express ICOS ligand, CD20, or the antibodies described herein. Determine whether the antibody tests positive for surface expression of other target receptors or other proteins listed A typical method is immunohistochemistry, in which a sample of cells (e.g., tumor tissue) is The test sample is contacted with an antibody to the protein of interest, and antibody binding is mediated by a labeled reagent, typically a first a second antibody that recognizes the Fc region of the antibody and has a detectable label such as a fluorescent marker; At least one of the following is determined: If even 5% of cells are labeled, the sample can be declared to test positive. Optionally, a higher cutoff such as 10% or 25% can be used. is generally used in excess. Reagent antibodies to the molecule of interest are available or To test ICOS ligands, the antibody MAB1 651 is currently available from R&D Systems as a mouse IgG that recognizes the human ICOS ligand. Rituximab can be used to test for CD20 expression. Detection of mRNA levels of the ICOS ligand or target receptor of interest is an alternative technique.

[0172] Further indications of tumor response to anti-ICOS antibody therapy include the tumor microenvironment. Activated Tregs have high surface expression of ICOS and Fo The presence of Tregs in tumors is characterized by high surface expression of xp3. Increasing numbers of patients can be selected for treatment with anti-ICOS antibodies. Tregs can be identified in ex vivo tumor biopsies, e.g., by immunohistochemistry. (Assay for co-expression of both Foxp3 and ICOS as above, detection of labeling followed by labeling by using antibodies against target proteins) or by labeling against ICOS and Foxp3 The antibodies can be detected by single cell dispersion of the sample for use in FACS. The ACS method is exemplified in Examples 17 and 18.

[0173] Anti-ICOS antibodies can be used to treat cancers associated with infectious agents, such as virus-induced cancers. This category includes head and neck squamous cell carcinoma, cervical cancer, Merkel cell carcinoma, and many other cancers. Other viruses associated with cancer include HBV, HCV, and HPV (cervical cancer, pharyngeal cancer), and EBV (Burkitt's lymphoma, gastric cancer, Hodgkin's lymphoma, other EBV-positive B These include lymphoma, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disorder. National Agency for Research on Cancer( Monograph 100B) identified the following major cancer sites associated with infectious agents: : ●Stomach / Gastric: Heliobacter pylori Liver: Hepatitis B virus, Hepatitis C virus (HCV), Opisthorchis viverrini, Clonorchis sinensis ●Cervix: HIV positive or negative human papillomavirus (HPV) Anogenital (penis, vulva, vagina, anus): HIV positive or negative HPV Nasopharynx: Epstein-Barr virus (EBV) Oropharyngeal: HPV regardless of tobacco or alcohol consumption Kaposi's sarcoma: Human herpesvirus type 8 in HIV-positive or HIV-negative individuals Non-Hodgkin's lymphoma: H. pylori, HIV positive or negative EBV, HCV, Human T-cell lymphotropic virus type 1 Hodgkin's lymphoma: HIV positive or negative EBV ●Bladder: Schistosoma haematobium

[0174] The antibodies according to the invention can be used to treat any of these infectious agents, e.g., the cancers identified above. It may be used to treat cancers associated with or induced by it.

[0175] Stimulation of effector T cell responses may also contribute to immunity to infectious diseases and / or to the development of immune responses in patients. Therefore, anti-ICOS antibodies may contribute to recovery from infectious diseases. The compounds can be used to treat infectious diseases by administering the compounds to patients.

[0176] Infectious diseases include those caused by pathogens, such as bacterial, fungal, viral, or protozoan pathogens. The treatment includes those caused by pathogen infections, and involves enhancing the patient's immune response to the pathogen infection. An example of a bacterial pathogen is tuberculosis. Examples of viral pathogens are hepatitis B and Examples of protozoan pathogens include HIV, P. falciparum, which causes malaria, and It is a Plasmodium species.

[0177] The antibodies can be used to treat infections, such as infections by any of the pathogens described herein. The infection may be persistent or chronic. The infection may be localized or systemic. Extensive contact between pathogens and the immune system can result in increased levels of Tregs, Tre, and Immune system exhaustion or tolerance (manifested by modulation of the Treg:Teff balance in favor of g) The immune response by pathogens through the development of immune responses and / or the evolution and modification of presented pathogen antigens. These features reflect similar processes thought to occur in cancer. Anti-ICOS antibodies may be used to induce a Treg:Teff ratio favorable for Teff and / or to induce Treg:Teff phenotypes as described herein. for treating infections by pathogens, e.g., chronic infections, through modulation of other effects described in Therapeutic approaches are presented.

[0178] The treatment may be of a patient diagnosed with an infectious disease or infection; or As described elsewhere herein, treatment may be prophylactic and may involve administering to a patient The virus can be administered to prevent disease, for example as a vaccine.

[0179] Immune responses, particularly IFNγ-dependent systemic immune responses, are part of the neuroinflammatory component. It is believed that this could be beneficial for Alzheimer's disease and other CNS pathologies that share common principles. It has also been proposed

[29] . WO2015 / 136541 uses anti-PD-1 antibodies Anti-ICOS antibodies are a promising treatment for Alzheimer's disease or other neurodegenerative diseases. In the treatment of degenerative diseases, optionally one or more other immunomodulatory agents (e.g., PD-1 inhibitors) It can be used in combination with antibodies against the

[0180] Combination therapy Immunomodulatory antibodies, such as anti-CTLA4, anti-PD1, or anti-PDL1, particularly those that inhibit Fc effector mechanisms Treatment with immunomodulatory antibodies with ICOS-high expression is effective in further depleting immunosuppressive cells. To enhance the therapeutic effect, anti-ICOS antibodies and other It may be advantageous to combine it with an immunomodulatory agent such as

[0181] Patients treated with immunomodulatory antibodies (e.g., anti-PDL-1, anti-PD-1, anti-CTLA-4) may particularly benefit from treatment with anti-ICOS antibodies. One reason for this is that immunological Immunomodulatory antibodies may 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 are used to treat patients with other therapeutic agents that result in ICOS+ Tregs (e.g. For example, the rapid rise or rise of intratumoral Tregs can be reduced and / or reversed. Therefore, patients selected for treatment with an anti-ICOS antibody should be treated with a first therapeutic agent. The patient may already be receiving the first therapeutic agent, and the first therapeutic agent increases the number of ICOS+ Tregs in the patient. The antibody may be an immunomodulatory antibody or other agent (e.g., IL-2).

[0182] Immunomodulatory agents that may be combined with anti-ICOS antibodies include PDL1 (e.g., avelumab), PD-1 (e.g., pembrolizumab or nivolumab), or CTLA-4 (e.g., ipilimumab) Anti-ICOS antibodies include antibodies against either PI3K / PI4K / C16 / C16 / C26 ... In another embodiment, the anti-ICOS antibody is an anti-CTLA-4 antibody. and / or optionally a therapeutic antibody that is not an anti-CTLA-4 antibody. It is administered in combination with the body.

[0183] For example, an anti-ICOS antibody can be used in combination therapy with an anti-PDL1 antibody. Alternatively, 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 a combination therapy is one in which both antibodies have effector-positive constant regions. Therefore, the administration of anti-ICOS antibodies and anti-PDL1 antibodies is Both anti-PDL1 and anti-PDL1 antibodies can mediate ADCC, CDC, and / or ADCP. Fc effector functions and constant region selection are described in detail elsewhere herein. For example, anti-ICOS human IgG1 is combined with anti-PD-L1 human IgG1. The anti-ICOS antibody and / or anti-PD-L1 antibody may be a wild-type human IgG1 Alternatively, the effector positive constant region of the antibody may comprise a constant region having an enhanced effect. engineered for target function, e.g., enhanced CDC, ADCC, and / or ADCP Examples include wild-type human IgG1 sequences and mutations that alter effector function. Exemplary antibody constant regions are described in detail elsewhere herein.

[0184] Anti-PDL1 antibodies that can be combined with anti-ICOS antibodies include: Optionally, as an effector-positive human IgG1, it blocks PD-1 binding to PDL1. anti-PDL1 antibodies that harm and / or inhibit PDL1; • anti-PD-1 antibodies that inhibit the binding of PD-1 to PDL1 and / or PDL2; Avelumab, a human IgG1 antibody that inhibits PD-1 binding to PDL-1, and WO2 See 013 / 079174; Dextromethorphan is a mutant human IgG1 antibody with mutations L234A, L235A, and 331. Urvalumab (or "MEDI4736"), see WO2011 / 066389 ; A mutant human IgG1 antibody with mutations N297A, D356E, and L358M. atezolizumab, see US2010 / 0203056; BMS-936559, a human IgG4 antibody containing the S228P mutation, WO2007 See also: / 005874.

[0185] Numerous additional examples of anti-PD-L1 antibodies are disclosed herein, and others are incorporated herein by reference. Characterization data for many of the anti-PD-L1 antibodies mentioned herein is known in the art. are incorporated herein by reference in their entirety. 17,338. Exemplary anti-PD-L1 antibodies are disclosed in U.S. Pat. No. 9,567,399. or 1D05, 84G09, 1D05 as described in US 9,617,338 HC mutant 1, 1D05HC mutant 2, 1D05HC mutant 3, 1D05HC mutant 4, 1D05LC mutant 1, 1D05LC mutant 2, 1D05LC mutant 3, 411B08, 411C04, 411D07, 385F01, 386H03, 389A03, 413D0 8, 413G05, 413F09, 414B06, or 416E01 HCD The antibodies have VH and / or VL domains containing R and / or LCDR. and the heavy chain and / or VH and VL domains of any of these antibodies. These anti-PD-1 antibodies may optionally contain a heavy chain and / or a light chain having a light chain amino acid sequence. The VH and VL domains of the L1 antibody are further described elsewhere herein.

[0186] Further exemplary anti-PD-L1 antibodies include KN-035, CA-170, and FAZ-053. , M7824, ABBV-368, LY-3300054, GNS-1480, YW24 3.55.S70, including the HCDR and / or LCDR of REGN3504, or W O2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801, WO2016 / 111645, WO2016 / 197367 , WO2016 / 061142, WO2016 / 149201, WO2016 / 00061 9, WO2016 / 160792, WO2016 / 022630, WO2016 / 0072 35, WO2015 / 179654, WO2015 / 173267, WO2015 / 181 342, WO2015 / 109124, WO2015 / 112805, WO2015 / 06 1668, WO2014 / 159562, WO2014 / 165082, WO2014 / 1 00079, WO2014 / 055897, WO2013 / 181634, WO2013 / 173223, WO2013 / 079174, WO2012 / 145493, WO011 / 066389, WO2010 / 077634, WO2010 / 036959, WO010 / 089411, and the anti-PD-L1 antibody disclosed in WO2007 / 005874 The antibody may have the VH and / or VL domains of any of these antibodies. VL domain and having the heavy and / or light chain amino acid sequence of any of these antibodies. The antibody may optionally contain heavy and / or light chains that are capable of binding to PD-L1. The anti-ICOS antibody may be an antibody of the invention disclosed herein. The S antibody may be selected from the CDRs of the anti-ICOS antibodies disclosed in any of the following publications, or the VH thereof: and / or VL domains: WO2016154177, US20163046 10 - e.g. 7F12, 37A10, 35A9, 36E10, 16G10, 37A10S 713, 37A10S714, 37A10S715, 37A10S716, 37A10S 717, 37A10S718, 16G10S71, 16G10S72, 16G10S73 , 16G10S83, 35A9S79, 35A9S710, or 35A9S89 antibody WO16120789, US2016215059 - e.g. 422.2 and / or is an antibody known as H2L5; WO14033327, EP2892928, US 2015239978 - known as e.g. 314-8 and / or hybrid Antibody produced from CNCM I-4180; WO12131004, EP26914 19, US9376493, US20160264666 - e.g. antibody Icos145 - 1 and / or antibodies produced by hybridoma CNCM I-4179; WO10 056804 - e.g., antibody JMAb136 or "136"; WO9915553, EP1 017723B1, US7259247, US7132099, US7125551, U S7306800, US7722872, WO05103086, EP1740617, US8318905, US8916155 - e.g. antibodies MIC-944 or 9F3; WO 983821, US7932358B2, US2002156242, EP098402 3, EP1502920, US7030225, US7045615, US727956 0, US7226909, US7196175, US7932358, US838969 0, WO02070010, EP1286668, EP1374901, US74389 05, US7438905, WO0187981, EP1158004, US68030 39, US7166283, US7988965, WO0115732, EP11255 85, US7465445, US7998478 - e.g., any JMAb antibody, e.g., J MAb-124, JMAb-126, JMAb-127, JMAb-128, JMAb- 135, JMAb-136, JMAb-137, JMAb-138, JMAb-139, JMAb-140, JMAb-141, for example, JMAb136; WO201 4 / 089113 - e.g., antibody 17G9; WO12174338; US20161453 44;WO11020024, EP2464661, US2016002336, US2 016024211, US8840889;US8497244.

[0187] The anti-ICOS antibody is optionally 37A1 as disclosed in WO2016154177. It contains the CDRs of 37A10S713. It contains the VH and VL domains of 37A10S713. and optionally having the antibody heavy and light chains of 37A10S713.

[0188] The combination of anti-ICOS antibodies with immunomodulatory agents provides increased therapeutic efficacy compared with monotherapy. and can achieve therapeutic benefit with lower doses of the immunomodulatory agent(s). Thus, for example, an antibody (e.g., an anti-P D-L1 antibody, optionally with ipilimumab) was administered at a dose of 3 mg / kg instead of the more usual 10 mg / kg. The dosing regimen for anti-PD-L1 or other antibodies may be a total of 4 doses. It may involve intravenous administration over a 90 minute period every three weeks.

[0189] Anti-PD-L1 antibodies may be recognized as a reduction in the dose at which they exert therapeutic benefit. Use of anti-ICOS antibodies to increase tumor sensitivity to treatment with L1 antibodies Therefore, anti-ICOS antibodies can be effective in treating cancer or tumors in patients. It may be administered to patients to reduce the dose of an anti-PD-L1 antibody. The dose is higher than that when the anti-PD-L1 antibody is administered without anti-ICOS, e.g. , 75%, 50%, 25%, 20%, or less than 10% of patients receiving anti-PD-L1 antibodies. The recommended or required dosage of the drug may be reduced. It can be treated by administering an ICOS antibody and an anti-PD-L1 antibody.

[0190] The benefits of combining anti-PD-L1 and anti-ICOS compared with their use as monotherapy In this case, the dose of each drug may be reduced. Anti-PD-L1 antibodies may be used in combination with anti-ICOS antibodies. It can be used to reduce the beneficial dose, thus curing the cancer or tumor in the patient. The dose of anti-ICOS antibody effective to treat the patient may be reduced. Therefore, anti-PD-L1 antibodies may be more effective than anti-ICOS antibodies when administered without anti-PD-L1. For example, to less than 75%, 50%, 25%, 20%, or 10% of the administered dose. The recommended or required dose of anti-ICOS antibody to the patient may be reduced. This can be treated by administering an anti-ICOS antibody and an anti-PD-L1 antibody in combination therapy.

[0191] As described in Example 22 herein, anti-PD-L1 antibodies, particularly effector-positive Treatment with Fc-containing antibodies does not appear to increase ICOS expression on Teff cells. This is because such antibodies are administered in combination with an effector-positive anti-ICOS antibody. This is advantageous in cases where increased ICOS expression on Teffs allows these cells to be differentiated by anti-ICOS antibodies. This makes them undesirably more susceptible to depletion by the body. In this study, anti-ICOS therapy suppressed the differential expression of ICOS on Teffs compared with Tregs. This could be used to preferentially target ICOS-high Tregs for depletion. , which has the net effect of relieving TEff suppression and promoting effector T cell responses in patients. The effect of targeting immune checkpoint molecules on ICOS expression on T cells has also been previously reported. Treatment with anti-CTLA-4 and / or anti-PD-1 antibodies has been investigated It has been reported that CD4+ Tregs increase the proportion of CD4+ Tregs that are involved in immune responses to inflammatory cytokines. See Figure S6C in the figure (sample). The efficacy of therapeutic agents in combination with anti-ICOS antibodies will determine the selection of appropriate agents. The effect of anti-ICOS antibodies may be due to the high level of ICOS on Tregs versus Teffs. Note that differential expression may be enhanced under certain conditions.

[0192] As described herein, a single dose of an anti-ICOS antibody may be administered in a manner that is consistent with the efficacy and safety of an anti-PD-L1 antibody. In combination with other therapeutic agents such as The rationale underlying this single-dose benefit is, at least in part, due to tumor microenvironmental changes. The microenvironment can be sufficiently reset or altered to allow immune attack on the tumor and / or other immune responses as mentioned. By making them more sensitive to the effects of immunomodulators, anti-ICOS antibodies mediate their effects. The resetting of the tumor microenvironment may be mediated by, for example, the activation of ICOS-positive tumor-infiltrating T cells. Thus, for example, a patient may receive a single dose of anti-IC The patient may be treated with an OS antibody followed by one or more doses of an anti-PD-L1 antibody. For example, the anti-ICOS antibody may be administered in a single dose over a period of six months or one year. On the other hand, other agents, such as anti-PD-L1 antibodies, may optionally be administered multiple times over the course of treatment. at least one such dose administered following treatment with an anti-ICOS antibody, preferably and then administered.

[0193] 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); -Indoleamine-2,3-diamine, which catalyzes the breakdown of tryptophan ("IDO inhibitor") IDO is an immune checkpoint inhibitor that inhibits dendritic cells. It is activated in alveoli and macrophages and contributes to immunosuppression / tolerance.

[0194] Anti-ICOS antibodies interact with IL-2 (e.g., recombinant IL-2 such as aldesleukin). It can be used in combination therapy. IL-2 can be administered in high doses (HD). Standard HD IL-2 therapy is recommended at doses exceeding 500,000 IU / kg per treatment cycle. with a bolus injection, e.g., 600,000 or 720,000 IU / kg. , 10-15 bolus injections at intervals of 5-10 hours, e.g., every 8 hours maximum 15 bolus injections, up to 6-8 treatment cycles approximately every 14-21 days Repeat. HD IL-2 therapy is effective in treating tumors, especially melanoma (e.g., metastatic melanoma), and kidney cancer. Although successful in treating cell carcinomas, its use is associated with IL-2, which can cause serious adverse effects. Limited to high toxicity.

[0195] Treatment with high doses of IL-2 increases the population of ICOS-positive Tregs in cancer patients. It has been shown that ICOS following the first cycle of HD IL-2 therapy can improve survival

[31] . This increase in +TReg has been reported to correlate with worse clinical outcomes - ICOS+T The higher the number of reg cells, the worse the prognosis. IL-2 mutant F42K is a key regulator of ICOS+ Treg cells. It has been proposed as an alternative therapy to avoid this unwanted increase in cells

[32] . However, another approach is to use the antibodies according to the invention as second-line therapeutic agents. This will likely take advantage of the increase in ICOS+ Tregs.

[0196] Targeting Tregs with high ICOS expression, inhibiting these cells and preventing IL-2 therapy Taking advantage of the ability of anti-ICOS antibodies to improve the prognosis of patients with IL-1, we have developed a combination of anti-ICOS antibodies and IL-1. Combining IL-2 therapy with anti-ICOS antibodies may be beneficial. may increase response rates while avoiding or reducing adverse events in the treated patient population. This combination allows for the use of lower doses of IL-2 compared to IL-2 monotherapy. and reduce the risk or level of adverse events resulting from IL-2 therapy, while clinical benefit (e.g., reduced tumor growth, clearance of solid tumors and / or reduced metastasis) In this way, the addition of anti-ICOS can be used in high dose (HD) or low dose (HD) Improve the treatment of patients receiving IL-2, regardless of the dose (LD) of IL-2. can be done.

[0197] Thus, one aspect of the present invention is to treat a patient with HIV by administering an anti-ICOS antibody to the patient. The present invention provides a method of treating IL-2, e.g., HD IL-2, in which the patient is also treated with IL-2. Another aspect is an anti-ICOS antibody for use in treating a patient, wherein the patient is receiving IL-2, e.g. For example, HD is also treated with IL-2. Anti-ICOS antibodies are used as second-line therapy. Thus, patients may receive, for example, at least one cycle of HD IL-2 therapy. and IL-2 treated patients with increased ICOS+ Treg levels The assay can be performed to identify cells positive for ICOS, Foxp3, ICOSL, and Optionally, to detect one or more additional markers of interest, the methods described elsewhere herein may be used. Immunohistochemistry or FACS as described can be used to detect the presence of cancer cells in a sample, e.g., a tumor biopsy sample. The method can be performed on a patient after IL-2 treatment (e.g., in peripheral blood or and determining that the patient has elevated ICOS+ Treg levels (in a tumor biopsy). and elevated levels indicate that the patient will benefit from treatment with anti-ICOS antibodies. The increase in Tregs is relative to control (untreated) individuals or to patients prior to IL-2 treatment. Such patients with elevated Tregs may benefit from continuous IL-2 treatment alone. Although this represents a group in which it is not possible to obtain the above-mentioned results, the combination of anti-ICOS antibody and IL-2 therapy, or anti-ICOS Treatment with S antibodies alone provides therapeutic benefit. Following a positive determination that the patient has elevated levels of ICOS antibodies and / or Treatment with anti-ICOS antibodies is effective in treating such patients. Selectively target and deplete ICOS+ Tregs compared with other T cell populations in This may alleviate the immune suppression mediated by these cells, thereby By enhancing the activity of Teff against target cells, such as tumor cells or infected cells, provides a therapeutic effect.

[0198] Combination therapy with anti-ICOS antibodies and IL-2 may be used for any of the therapeutic indications described herein. , in particular for the treatment of tumors, for example melanoma, such as metastatic melanoma, or renal cell carcinoma. Thus, in one example, a patient treated with anti-ICOS antibodies presented with metastatic melanoma and I and patients treated with HD IL-2 therapy or LD IL-2 therapy.

[0199] Generally, the anti-ICOS antibody is administered in combination with a first therapeutic agent (e.g., an immunomodulatory antibody) or other agent (e.g., When administered to patients treated with IL-2, anti-ICOS antibodies may be effective in preventing the first A minimum period of time, e.g., 24 hours, 48 ​​hours, 72 hours, 1 week, or 2 weeks, after administration of the therapeutic agent. The anti-ICOS antibody may be administered 2, 3, 4, or 5 days after the administration of the first therapeutic agent. This is to facilitate patient compliance and reduce costs. Although it is desirable to minimize the number of treatments administered, it is important to This does not preclude additional dosing. Rather, the relative timing of the doses may affect the effectiveness of the combination. The first therapeutic agent is selected to optimize the effect of the anti-ICOS antibody. Create an immune environment that stimulates immune responses (e.g., elevated ICOS+ Tregs or antigen release as described below) Therefore, the sequential administration of the first therapeutic agent and then the anti-ICOS antibody is This allows time for the anti-ICOS antibody to act and demonstrate its enhanced efficacy. Various administration regimens, including simultaneous or sequential combination therapy, are described herein. The first therapeutic agent can be used to increase the number of ICOS+ Tregs in a patient. If the patient's treatment regimen is designed to promote the development of increased numbers of ICOS+ Tregs, the patient's treatment regimen should be designed to promote the development of increased numbers of ICOS+ Tregs. The method may include determining whether or not a patient has an ICOS antibody, and then administering an anti-ICOS antibody.

[0200] As noted above, the use of anti-ICOS antibodies in combination therapy reduces the effective dose of the therapeutic agent. The benefits of countering the adverse effects of therapeutic agents that increase the number of ICOS+ Tregs in patients Furthermore, it is possible to provide a first method for releasing antigens from target cells by "immunological cell death". and administering the first therapeutic agent in combination with the anti-ICOS antibody. As mentioned above, anti-ICOS antibodies can be used to achieve further therapeutic benefits. The administration of the first therapeutic agent may be sequentially followed by the administration of the second therapeutic agent, and the administration of the two agents may be sequentially followed by the administration of the first therapeutic agent, as described above. Separated by a specific time window.

[0201] Immunological cell death, as opposed to apoptosis, is a recognized mode of cell death. release of ATP and HMGB1 from cells and exposure of calreticulin on the plasma membrane It is characterized by [33, 34].

[0202] Immunological cell death in target tissues or cells promotes phagocytosis of the cells by antigen-presenting cells. promotes antigen presentation from target cells, which then induces antigen-specific Teff cells Anti-ICOS antibodies act as agonists of ICOS on Teff cells. Furthermore, anti-I can increase the magnitude and / or duration of the Teff response. If the COS antibody is capable of Fc effector function (e.g., human IgG1 antibody), anti-ICO S antibodies can cause the depletion of antigen-specific Tregs. By either or a combination of both, the balance between Teff and Treg cells can be improved to enhance Teff activity. The anti-ICOS antibody is preferably administered to, for example, tumor or cancer cells. Combination with a therapy that induces immunological cell death in the target tissue or cell type thereby The vaccine stimulates a patient's immune response against target tissues or cells, and the vaccine antigen is produced in vivo. It is expressed in the form of vaccination.

[0203] Thus, one aspect of the present invention is the in vivo vaccination of a patient against their cancer cells. Another aspect of the invention is a method for treating cancer in a patient using a method comprising administering to said patient a therapeutically effective amount of a compound selected from the group consisting of ribonucleotides, ...nucleotides, and nucleotides of formula (I) and (II). The anti-ICOS antibody may be used in methods including: : Treating patients with therapies that induce immunological cell death of cancer cells, thereby targeting antigen-specific and providing antigen presentation to target effector T cells. administering an anti-ICOS antibody to a patient, wherein the anti-ICOS antibody acts against cancer cells; administering a therapeutic agent that enhances antigen-specific effector T cell responses.

[0204] Treatments that induce immunological cell death include radiation (e.g., UVC light or gamma light). ionizing irradiation of cells), chemotherapeutic agents (e.g., oxaliplatin, doxorubicin, etc.) BK such as tracycline, idarubicin or mitoxantrone, phloretin or pimaric acid Channel agonists, bortezomib, cardiac glycosides, cyclophosphamide, mitomycin GADD34 / PP1 inhibitor with hypericin, PDT with polyinosinic acid-poly Cytidic acid, 5-fluorouracil, gemcitabine, gefitinib, erlotinib, or Thapsigargin with cisplatin), and antibodies against tumor-associated antigens. Related antigens bind to tumor cells in relation to non-tumor cells of the same tissue (e.g., HER2, CD20, EGFR). The antigen may be any antigen that is overexpressed by tumor cells. Suitable antibodies include Herceptin ( These include rituximab (anti-HER2), rituximab (anti-CD20), or cetuximab (anti-EGFR).

[0205] It may therefore be advantageous to combine an anti-ICOS antibody with one or more such treatments. Optionally, the anti-ICOS antibody is administered to a patient already undergoing such treatment. ICOS antibodies induce immunological cell death after treatment, e.g., 24 to 72 hours after treatment. for example, after a period of 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 treatment. Other regimens of combination therapy are described elsewhere herein.

[0206] "In vivo vaccination" is described above, but is not limited to the ex vivo immunological cell death. It is also possible to treat tumor cells to induce leukemia, and then reintroduce the cells into the patient. Rather than administering drugs or treatments that directly induce immunological cell death to the patient, The treated tumor cells are then administered to the patient. Treatment of the patient follows the administration regimen described above. can be done.

[0207] As already mentioned, a single dose of anti-ICOS antibody is sufficient to provide therapeutic benefit. Therefore, in the therapeutic methods described herein, the anti-ICOS antibody is administered as a single dose. A single dose of anti-ICOS antibody is optionally administered in a single dose to deplete Tregs in the patient. This may ultimately have beneficial effects on diseases such as cancer. These include antitumor efficacy, including reduction of tumor progression, treatment of established tumors and metastases, and prolongation of survival. It has been reported that a single dose of 1000 mg / kg of 1000 ribosomal iodine (RI) can enhance the therapeutic effect of tumor irradiation

[35] . Administration of anti-ICOS may provide such Treg depletion, and may be combined with other therapies such as radiation therapy. It may be used to enhance the effectiveness of other therapeutic approaches being used.

[0208] Antibodies against PD-L1 As described herein, anti-ICOS antibodies, whether as separate therapeutic agents or multispecific antibodies, Antibodies against PD-L1 used in combination with antibodies can be used to inhibit the antigen binding of any anti-PD-L1 antibody. Numerous examples of anti-PD-L1 antibodies are disclosed herein, including those Others are known in the art. Characterization of many of the anti-PD-L1 antibodies mentioned herein The value data are from US 9,567,399 and U Published in S9,617,338.

[0209] 1D05 is a CDRH1 amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat). CDRH2 amino acid sequence of SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) Sequence, and CDRH3 amino acid of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable region (V) of SEQ ID NO: 33, comprising the sequence H ) amino acid sequence. H Domain The heavy chain nucleic acid sequence is SEQ ID NO: 34. 1D05 is SEQ ID NO: 37 (IMGT) or SEQ ID NO: No. 40 (Kabat), SEQ ID NO: 38 (IMGT) or SEQ ID NO: 4 1 (Kabat), and the CDRL2 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 4 a light chain variable region (V) of SEQ ID NO: 43, comprising the CDRL3 amino acid sequence of SEQ ID NO: 2 (Kabat); L )a It has the amino acid sequence V L The light chain nucleic acid sequence of domain V is SEQ ID NO: 44. H Domain The sequence may be any of the heavy chain constant region sequences described herein, e.g., SEQ ID NO: 19. 3, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 20 3, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 52 8, SEQ ID NO: 530, SEQ ID NO: 532, or SEQ ID NO: 534. L Do The main sequence may be 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, Combined with 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 light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0210] 84G09 is a 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). The heavy chain variable (V) of SEQ ID NO: 13 H ) region amino acid sequence. H Domain Heavy Chain The nucleic acid sequence is SEQ ID NO: 14. 84G09 is SEQ ID NO: 17 (IMGT) or SEQ ID NO: 20 (Kabat), SEQ ID NO: 18 (IMGT) or SEQ ID NO: 21 (Kabat) CDRL2 amino acid sequence, and SEQ ID NO: 19 (IMGT) or SEQ ID NO: 22 A light chain variable region (V) of SEQ ID NO: 23, comprising the CDRL3 amino acid sequence of (Kabat) L )Ami It has the amino acid sequence VL The light chain nucleic acid sequence of domain V is SEQ ID NO: 24. H domain is any of the heavy chain constant region sequences described herein, e.g., 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 Domestic The amino acid sequence may be any of the light chain constant region sequences described herein, e.g., SEQ ID NO:2. 07, 209, 211, 213, 215, 217, 219, 221, 223, 225, 2 27, 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 light chain amino acid sequence is SEQ ID NO: 25 (light chain nucleic acid sequence SEQ ID NO: 26).

[0211] 1D05 HC Variant 1 is the amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat) CDRH1 amino acid sequence, SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) RH2 amino acid sequence and CD of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable (V) of SEQ ID NO: 47, comprising the RH3 amino acid sequence H ) region amino acid sequence. 1D05 HC Variant 1 is the C of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat) CDRs of DRL1 amino acid sequence, SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat) L2 amino acid sequence and CDRs of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat) The light chain variable region (V) of SEQ ID NO: 43, comprising the L3 amino acid sequenceL ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 44. H The domains are described herein. any of the heavy chain constant region sequences listed in SEQ ID NO: 193, SEQ ID NO: 195, Sequence number 197, sequence number 199, sequence number 201, sequence number 203, sequence number 205, sequence Sequence number 340, sequence number 524, sequence number 526, sequence number 528, sequence number 530, sequence May be combined with sequence number 532 or sequence number 534. L The domains are as described herein. Any of the light chain constant region sequences listed above, e.g., SEQ ID NOs: 207, 209, 211 , 213, 215, 217, 219, 221, 223, 225, 227, 229, 231 , 233, 235, 237, 536, and 538. The sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0212] 1D05 HC Variant 2 is SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat) CDRH1 amino acid sequence, SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) RH2 amino acid sequence and CD of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable (V) of SEQ ID NO: 48, comprising the RH3 amino acid sequence H ) region amino acid sequence. 1D05 HC variant 2 is C of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat) CDRs of DRL1 amino acid sequence, SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat) L2 amino acid sequence and CDRs of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat) The light chain variable region (V) of SEQ ID NO: 43, comprising the L3 amino acid sequence L) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 44. H The domains are described herein. any of the heavy chain constant region sequences listed in SEQ ID NO: 193, SEQ ID NO: 195, Sequence number 197, sequence number 199, sequence number 201, sequence number 203, sequence number 205, sequence Sequence number 340, sequence number 524, sequence number 526, sequence number 528, sequence number 530, sequence May be combined with sequence number 532 or sequence number 534. L The domains are as described herein. Any of the light chain constant region sequences listed above, e.g., SEQ ID NOs: 207, 209, 211 , 213, 215, 217, 219, 221, 223, 225, 227, 229, 231 , 233, 235, 237, 536, and 538. The sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0213] 1D05 HC variant 3 is SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat) CDRH1 amino acid sequence, SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) RH2 amino acid sequence and CD of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable (V) of SEQ ID NO: 49, comprising the RH3 amino acid sequence H ) region amino acid sequence. 1D05 HC variant 3 is C of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat) CDRs of DRL1 amino acid sequence, SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat) L2 amino acid sequence and CDRs of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat) The light chain variable region (V) of SEQ ID NO: 43, comprising the L3 amino acid sequence L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 44. H The domains are described herein. any of the heavy chain constant region sequences listed in SEQ ID NO: 193, SEQ ID NO: 195, Sequence number 197, sequence number 199, sequence number 201, sequence number 203, sequence number 205, sequence Sequence number 340, sequence number 524, sequence number 526, sequence number 528, sequence number 530, sequence May be combined with sequence number 532 or sequence number 534. L The domains are as described herein. Any of the light chain constant region sequences listed above, e.g., SEQ ID NOs: 207, 209, 211 , 213, 215, 217, 219, 221, 223, 225, 227, 229, 231 , 233, 235, 237, 536, and 538. The sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0214] 1D05 HC variant 4 is SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat) CDRH1 amino acid sequence, SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) RH2 amino acid sequence and CD of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable (V) of SEQ ID NO: 342, comprising the RH3 amino acid sequence H ) region amino acid sequence 1D05 HC variant 4 is SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat) CDRL1 amino acid sequence, SEQ ID NO: 38 (IMGT) or SEQ ID NO: 41 (Kabat) RL2 amino acid sequence and the CD of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat) a light chain variable region (V) of SEQ ID NO: 43, comprising the RL3 amino acid sequence L ) amino acid sequence. VL The light chain nucleic acid sequence of domain V is SEQ ID NO: 44. H The domains are described herein. any of the heavy chain constant region sequences listed in 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, It may be combined with SEQ ID NO: 532 or SEQ ID NO: 534. L A domain is defined herein as Any of the light chain constant region sequences described, e.g., SEQ ID NOs: 207, 209, 21 1, 213, 215, 217, 219, 221, 223, 225, 227, 229, 23 1, 233, 235, 237, 536, and 538. The nucleic acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0215] 1D05 LC variant 1 is SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat) CDRH1 amino acid sequence, SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) RH2 amino acid sequence and CD of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable (V) of SEQ ID NO: 33, comprising the RH3 amino acid sequence H ) region amino acid sequence. V H The heavy chain nucleic acid sequence of domain 1D05 is SEQ ID NO: 34. 1D05 LC variant 1 has the sequence The CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), and the sequence a sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat); The light chain variable region (V L ) amino acid sequence. The RL2 sequence is V of SEQ ID NO:50 L Determined from the sequence using the Kabat or IMGT system V H The domains are selected from the heavy chain constant region sequences described herein. Any of, 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: May be combined with number 534. L The domains are the light chain constant region sequences described herein. Any of the sequences, e.g., SEQ ID NOs: 207, 209, 211, 213, 215, 21 7, 219, 221, 223, 225, 227, 229, 231, 233, 235, 23 7, 536, and 538. The full-length heavy chain amino acid sequence is SEQ ID NO: 35 ( The heavy chain nucleic acid sequence is SEQ ID NO: 36).

[0216] 1D05 LC variant 2 is SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat) CDRH1 amino acid sequence, SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) RH2 amino acid sequence and CD of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable (V) of SEQ ID NO: 33, comprising the RH3 amino acid sequence H ) region amino acid sequence. V H The heavy chain nucleic acid sequence of domain 1D05 is SEQ ID NO: 34. 1D05 LC variant 2 has the sequence CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), SEQ ID NO: 38 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 41 (Kabat), and SEQ ID NO: SEQ ID NO: 39 (IMGT) or the CDRL3 amino acid sequence of SEQ ID NO: 42 (Kabat). The light chain variable region (V L ) amino acid sequence. H The domains are described herein. any of the heavy chain constant region sequences listed in SEQ ID NO: 193, SEQ ID NO: 195, Sequence number 197, sequence number 199, sequence number 201, sequence number 203, sequence number 205, sequence Sequence number 340, sequence number 524, sequence number 526, sequence number 528, sequence number 530, sequence May be combined with sequence number 532 or sequence number 534. L The domains are as described herein. Any of the light chain constant region sequences listed above, e.g., SEQ ID NOs: 207, 209, 211 , 213, 215, 217, 219, 221, 223, 225, 227, 229, 231 , 233, 235, 237, 536, and 538. The sequence is SEQ ID NO: 35 (heavy chain nucleic acid sequence SEQ ID NO: 36).

[0217] 1D05 LC variant 3 is the amino acid sequence of SEQ ID NO: 27 (IMGT) or SEQ ID NO: 30 (Kabat) CDRH1 amino acid sequence, SEQ ID NO: 28 (IMGT) or SEQ ID NO: 31 (Kabat) RH2 amino acid sequence and CD of SEQ ID NO: 29 (IMGT) or SEQ ID NO: 32 (Kabat) The heavy chain variable (V) of SEQ ID NO: 33, comprising the RH3 amino acid sequence H ) region amino acid sequence. V H The heavy chain nucleic acid sequence of domain 1D05 is SEQ ID NO: 34. 1D05 LC variant 3 has the sequence The CDRL1 amino acid sequence of SEQ ID NO: 37 (IMGT) or SEQ ID NO: 40 (Kabat), and the sequence a sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 39 (IMGT) or SEQ ID NO: 42 (Kabat); The light chain variable region (V L ) amino acid sequence of 1D05 LC mutant 3 The DRL2 sequence is V of SEQ ID NO: 298 L Sequences were analyzed using the Kabat or IMGT system. V L The light chain nucleic acid sequence of domain V is SEQ ID NO: 44. H Do The main sequence may be 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, Can be combined with sequence number 528, sequence number 530, sequence number 532, or sequence number 534 V L The domain may be any of the light chain constant region sequences described herein, e.g. , 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). The full-length light chain amino acid sequence is SEQ ID NO: 45 (light chain nucleic acid sequence SEQ ID NO: 46).

[0218] 411B08 is a CDRH1 of SEQ ID NO: 52 (IMGT) or SEQ ID NO: 55 (Kabat). The CDRH2 amino acid sequence of SEQ ID NO: 53 (IMGT) or SEQ ID NO: 56 (Kabat) The amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 54 (IMGT) or SEQ ID NO: 57 (Kabat) The heavy chain variable (V) of SEQ ID NO: 58, comprising the amino acid sequence H ) region amino acid sequence. H Domain The heavy chain nucleic acid sequence of 411B08 is SEQ ID NO: 62 (IMGT) or is the CDRL1 amino acid sequence of SEQ ID NO: 65 (Kabat), SEQ ID NO: 63 (IMGT) or The CDRL2 amino acid sequence of SEQ ID NO: 66 (Kabat) and SEQ ID NO: 64 (IMGT) or A light chain variable region of SEQ ID NO: 68, containing the CDRL3 amino acid sequence of SEQ ID NO: 67 (Kabat) (V L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 69. H The domain may be any of the heavy chain constant region sequences described herein, for example, the sequence No. 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: No. 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or SEQ ID NO: 534 .V L The domain may be any of the light chain constant region sequences described herein, e.g., SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, Paired with 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).

[0219] 411C04 is a CDRH1 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) The amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 74 (IMGT) or SEQ ID NO: 77 (Kabat) The heavy chain variable (V) of SEQ ID NO: 78, comprising the amino acid sequence H ) region amino acid sequence. H Domain The heavy chain nucleic acid sequence of 411C04 is SEQ ID NO: 79. is the CDRL1 amino acid sequence of SEQ ID NO: 85 (Kabat), SEQ ID NO: 83 (IMGT) or The CDRL2 amino acid sequence of SEQ ID NO: 86 (Kabat) and SEQ ID NO: 84 (IMGT) or A light chain variable region of SEQ ID NO: 88, containing the CDRL3 amino acid sequence of SEQ ID NO: 87 (Kabat) (V L ) amino acid sequence. L The light chain nucleic acid sequence of domain V is SEQ ID NO: 89. H The domain may be any of the heavy chain constant region sequences described herein, for example, the sequence No. 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: No. 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or SEQ ID NO: 534 .V L The domain may be any of the light chain constant region sequences described herein, e.g., SEQ ID NOs: 207, 209, 211, 213, 215, 217, 219, 221, 223, Paired with 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).

[0220] 411D07 is a CDRH1 of SEQ ID NO: 92 (IMGT) or SEQ ID NO: 95 (Kabat) The CDRH2 amino acid sequence of SEQ ID NO: 93 (IMGT) or SEQ ID NO: 96 (Kabat) The amino acid sequence and the CDRH3 amino acid sequence of SEQ ID NO: 94 (IMGT) or SEQ ID NO: 97 (Kabat) The heavy chain variable (V) of SEQ ID NO: 98, comprising the amino acid sequence H ) region amino acid sequence. H Domain The heavy chain nucleic acid sequence of 411D07 is SEQ ID NO: 102 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 105 (Kabat), SEQ ID NO: 103 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 106 (Kabat), and SEQ ID NO: 104 (IM SEQ ID NO: 10, which contains the CDRL3 amino acid sequence of SEQ ID NO: 107 (Kabat) 8 light chain variable region (V L ) amino acid sequence. L The light chain nucleic acid sequence of the domain is SEQ ID NO: No. 109. V H The domain may be any of the heavy chain constant region sequences described herein. or, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, Sequence number 201, sequence number 203, sequence number 205, sequence number 340, sequence number 524, sequence Sequence number 526, sequence number 528, sequence number 530, sequence number 532, or sequence number 534 Can be combined with V L The domains are selected from the light chain constant region sequences described herein. Any of, 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). The full-length light chain amino acid sequence is SEQ ID NO: 110 (light chain nucleic acid sequence SEQ ID NO: 111). Column number 111).

[0221] 385F01 is the CDR of SEQ ID NO: 112 (IMGT) or SEQ ID NO: 115 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 113 (IMGT) or SEQ ID NO: 116 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 114 (IMGT) or SEQ ID NO: 117 (Kabat) The heavy chain variable (V) of SEQ ID NO: 118, comprising the DRH3 amino acid sequence H ) region amino acid sequence V H The heavy chain nucleic acid sequence of domain 385F01 is SEQ ID NO: 119. 122 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 125 (Kabat), SEQ ID NO: 123 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 126 (Kabat), and the sequence containing the CDRL3 amino acid sequence of SEQ ID NO: 124 (IMGT) or SEQ ID NO: 127 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 129. H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534. L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 1 20 (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).

[0222] 386H03 is the CDR of SEQ ID NO: 152 (IMGT) or SEQ ID NO: 155 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 153 (IMGT) or SEQ ID NO: 156 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 154 (IMGT) or SEQ ID NO: 157 (Kabat) The heavy chain variable (V) of SEQ ID NO: 158, comprising the DRH3 amino acid sequence H ) region amino acid sequence V H The heavy chain nucleic acid sequence of domain 386H03 is SEQ ID NO: 159. 162 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 165 (Kabat), SEQ ID NO: 163 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 166 (Kabat), and the sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 164 (IMGT) or SEQ ID NO: 167 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 169. H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534. L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 1 60 (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).

[0223] 389A03 is the CDR of SEQ ID NO: 172 (IMGT) or SEQ ID NO: 175 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 173 (IMGT) or SEQ ID NO: 176 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 174 (IMGT) or SEQ ID NO: 177 (Kabat) The heavy chain variable (V) of SEQ ID NO: 178, comprising the DRH3 amino acid sequence H ) region amino acid sequence V H The heavy chain nucleic acid sequence of domain 389A03 is SEQ ID NO: 179. 182 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 185 (Kabat), SEQ ID NO: 183 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 186 (Kabat), and the sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 184 (IMGT) or SEQ ID NO: 187 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 189. H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534.L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 1 80 (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).

[0224] 413D08 is the CDR of SEQ ID NO: 132 (IMGT) or SEQ ID NO: 135 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 133 (IMGT) or SEQ ID NO: 136 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 134 (IMGT) or SEQ ID NO: 137 (Kabat) The heavy chain variable (V) of SEQ ID NO: 138, comprising the DRH3 amino acid sequence H ) region amino acid sequence V H The heavy chain nucleic acid sequence of domain 413D08 is SEQ ID NO: 139. 142 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 145 (Kabat), SEQ ID NO: 143 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 146 (Kabat), and the sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 144 (IMGT) or SEQ ID NO: 147 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 149. H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534. L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 1 40 (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).

[0225] 413G05 is the CDR of SEQ ID NO: 238 (IMGT) or SEQ ID NO: 241 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 239 (IMGT) or SEQ ID NO: 242 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 240 (IMGT) or SEQ ID NO: 243 (Kabat) The heavy chain variable (V) of SEQ ID NO: 244, comprising the DRH3 amino acid sequence H ) region amino acid sequence V H The heavy chain nucleic acid sequence of domain 413G05 is SEQ ID NO: 245. 248 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 251 (Kabat), SEQ ID NO: 249 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 252 (Kabat), and the sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 250 (IMGT) or SEQ ID NO: 253 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 255.H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534. L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 2 46 (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).

[0226] 413F09 is the CDR of SEQ ID NO: 258 (IMGT) or SEQ ID NO: 261 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 259 (IMGT) or SEQ ID NO: 262 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 260 (IMGT) or SEQ ID NO: 263 (Kabat) The heavy chain variable (V) of SEQ ID NO: 264, comprising the DRH3 amino acid sequence H ) region amino acid sequence V H The heavy chain nucleic acid sequence of domain 413F09 is SEQ ID NO: 265. 268 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 271 (Kabat), SEQ ID NO: 269 ​​(IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 272 (Kabat), and the sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 270 (IMGT) or SEQ ID NO: 273 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 275. H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534. L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 2 66 (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).

[0227] 414B06 is the CDR of SEQ ID NO: 278 (IMGT) or SEQ ID NO: 281 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 279 (IMGT) or SEQ ID NO: 282 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 280 (IMGT) or SEQ ID NO: 283 (Kabat) The heavy chain variable (V) of SEQ ID NO: 284 comprises the DRH3 amino acid sequence H ) region amino acid sequence V H The heavy chain nucleic acid sequence of domain 414B06 is SEQ ID NO: 285. 288 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 291 (Kabat), SEQ ID NO: 289 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 292 (Kabat), and the sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 290 (IMGT) or SEQ ID NO: 293 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 295. H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534. L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 2 86 (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).

[0228] 416E01 is the CDR of SEQ ID NO: 343 (IMGT) or SEQ ID NO: 346 (Kabat) CDRs of H1 amino acid sequence, SEQ ID NO: 344 (IMGT) or SEQ ID NO: 347 (Kabat) H2 amino acid sequence, and C of SEQ ID NO: 345 (IMGT) or SEQ ID NO: 348 (Kabat) The heavy chain variable region (V) of SEQ ID NO: 349, comprising the DRH3 amino acid sequence H ) having the amino acid sequence V HThe heavy chain nucleic acid sequence of domain 416E01 is SEQ ID NO: 350. 353 (IMGT) or the CDRL1 amino acid sequence of SEQ ID NO: 356 (Kabat), SEQ ID NO: 354 (IMGT) or the CDRL2 amino acid sequence of SEQ ID NO: 357 (Kabat), and the sequence comprising the CDRL3 amino acid sequence of SEQ ID NO: 355 (IMGT) or SEQ ID NO: 358 (Kabat) , the light chain variable region (V L ) amino acid sequence. L Light chain nucleic acid of the domain The sequence is SEQ ID NO: 360. H The domains are the heavy chain constant region sequences described herein. Any of the sequences, for example, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: No. 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 340, SEQ ID NO: SEQ ID NO: 524, SEQ ID NO: 526, SEQ ID NO: 528, SEQ ID NO: 530, SEQ ID NO: 532, or May be combined with SEQ ID NO: 534. L The domains are the light chain constant region domains described herein. Any of the sequences of the regions, e.g., 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: 3 51 (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).

[0229] Antibody-drug conjugates Anti-ICOS antibodies can be used as carriers of cytotoxic drugs to target Tregs. As reported in Example 18, Tr located in the tumor microenvironment (TME) can be ICOS is expressed more strongly in tumor Teffs than in peripheral Tregs. It is more strongly expressed in Tregs within tumors, and therefore is labeled with toxic drugs or prodrugs. The proposed anti-ICOS antibody preferentially targets Tregs in the TME, delivering its toxic payload. Such targeting of cytotoxic drugs can be achieved by delivering cytotoxic drugs to cells in the cytoplasm, selectively inhibiting those cells. provides an additional route to remove the immunosuppressive effects of e.g., thereby increasing Teff activity altering the Treg:Teff balance in favor of the other therapeutic approaches described herein. Any one or more of the following (e.g., Fc effector-mediated inhibition of Tregs, effector T These may be used as an alternative to or in combination with other methods (e.g., cellular agonism).

[0230] Thus, the present invention provides an anti-ICOS antibody conjugated to a cytotoxic drug or prodrug. In the case of prodrugs, the prodrug may be administered to the TME or other target site for therapeutic activity. The cytotoxic drug can be activated in a cytotoxic environment, for example, by near-infrared light. In response to a trigger such as photoactivation, e.g., activating a light absorber conjugate using

[36] Spatially selective activation of the prodrug is highly selective for these cells. In combination with high ICOS expression on intratumoral Tregs to provide a potent cytotoxic effect , further enhancing the cytotoxic effect of the antibody-drug conjugate.

[0231] For use in antibody-drug conjugates, the cytotoxic drug or prodrug may be , preferably non-immunogenic and non-toxic ( Preferably, the cytotoxic drug (or prodrug when activated) is Drugs are potent, for example, 2-4 molecules of the drug are sufficient to kill the target cell. Photoactivatable prodrugs induce lethal damage to cell membranes after near-infrared light exposure. It is a silacapthalocyanine dye (IRDye 700 DX) that inhibits cytotoxic drugs. antimitotic agents such as monomethyl auristatin E, and maytansine derivatives, e.g., metansine These include microtubule inhibitors such as rutansine, DM1, and emtansine.

[0232] Conjugation of a drug (or prodrug) to an antibody is usually via a linker. The linker may be a cleavable linker, such as a disulfide, hydrazone, or peptide bond. The drug may be released by cathepsin in tumor cells. Cleavable linkers may be used. Alternatively, non-cleavable linkers, such as thioethers, may be used. Additional attachment groups and / or spacers may be included. .

[0233] The antibody in the antibody-drug conjugate may be delivered to a tissue site (e.g., the small size of such fragments) where it is difficult to deliver the antibody to the tissue site (e.g., the small size of such fragments). Fab'2 or other antibodies described herein may aid in the penetration of target cells into the target organs (e.g., solid tumors). It may also be an antibody fragment, such as an antigen-binding fragment.

[0234] The anti-ICOS antibody according to the present invention may be provided as an immunocytokine. The antibody may also be administered with an immunocytokine in a combination therapy. Any of these are described herein for use in combination therapy with ICOS. or (e.g., anti-PD-L1 antibodies) are provided as immunocytokines for use in the present invention. Immunocytokines include antibody molecules conjugated to cytokines such as IL-2. Therefore, anti-ICOS:IL-2 conjugates and anti-PD-L1:IL-2 conjugates This is a further aspect of the present invention.

[0235] The IL-2 cytokine binds to the high (αβγ) affinity IL-2 receptor and / or the intermediate affinity (αβ IL-2 used in immunocytokines may have activity at one or more IL-2 receptors. Human wild-type IL-2 or mutant IL-2 sequences having the above amino acid deletions, substitutions, or additions. The protein may be an IL-2 having 1 to 10 amino acid deletions at the N-terminus. IL-2 mutants include the mutations R38A or R38Q.

[0236] Exemplary anti-PD-L1 immunocytokines include those containing immunoglobulin heavy chains and immunoglobulin light chains. The heavy chain comprises, from N- to C-terminus: a) V containing CDRH1, CDRH2, and CDRH3 H The domain and b) a heavy chain constant region; The light chain is arranged in the N-terminal to C-terminal direction as follows: c) a V comprising CDRL1, CDRL2, and CDRL3 L The domain and d) Light chain constant region (C L )and, e) optionally a linker (L); f) IL-2 cytokine, V H Domain and V L The domain is an antigen-binding site that specifically binds to human PD-L1. It is composed of The immunocytokine contains the motif X1GSGX2YGX3X4FD (SEQ ID NO: 609). V containing CDRH3 H domain, wherein X1, X2, and X3 are independently any amino acid, and X4 is either present or absent, and if present, It can be any amino acid.

[0237] The VH and VL domains of any of the anti-PD-L1 antibodies referred to herein may be L domains, for example, the 1D05 VH and VL domains.

[0238] The IL-2 can be human wild-type or mutant IL-2.

[0239] Vaccination The anti-ICOS antibodies may be provided in a vaccine composition or co-administered with a vaccine preparation. ICOS is involved in T follicular helper cell formation and germinal center reactions.

[0037] Therefore, agonistic ICOS antibodies may be used as molecular adjuvants to enhance vaccine efficacy. Antibodies have potential clinical utility as antibodies against hepatitis B, malaria, and HIV. It can be used to increase the protective efficacy of a number of vaccines, such as do.

[0240] In the context of vaccination, anti-ICOS antibodies generally lack Fc effector functions. and therefore do not mediate ADCC, CDC, or ADCP. The antibody may be provided in a format lacking a constant region or having an effector-null constant region. Optionally, anti-ICOS antibodies may bind to one or more types of Fc receptors, but may also inhibit ADCC, CD4+, or other Fc receptor-specific receptors. C, or does not induce ADCP activity or has lower ADCP activity compared to wild-type human IgG1 The antibody may have a heavy chain constant region that exhibits CC, CDC, and ADCP activity. Specific Fc receptor(s) responsible for DCC, CDC, or ADCP activity They may not be able to bind or may bind with lower affinity. Alternatively, cellular effector functions may be impaired. If acceptable or desirable in the context of immunosuppression, anti-ICOS antibodies may be used to inhibit Fc effector activity. For example, IgG1, IgG4, and IgG 4. Any of the PE formats may be used to target anti-ICOS antibodies in vaccination regimens. Other examples of suitable isotypes and antibody constant regions that may be used are described elsewhere herein. This is explained in more detail elsewhere.

[0241] Formulation and Administration Antibodies may be monoclonal or polyclonal, but are preferably of therapeutic use. These are optionally provided as monoclonal antibodies of different binding specificities. The antibody may be provided as part of a mixture of other antibodies.

[0242] Antibodies and encoding nucleic acids according to the invention are usually provided in isolated form. Thus, antibodies, VH and / or VL domains, and nucleic acids may be used in their natural environment or where they are produced. Isolated antibodies and isolated nucleic acids may be provided purified from their native environment. or, if such preparations are by in vitro recombinant DNA techniques, by which they are prepared. They may be present in the environment (e.g., cell culture) with which they are naturally associated, such as other polypeptides or nucleic acids found in the environment. Optionally, the isolated antibody or nucleic acid (1) is free or substantially free of associated substances. does not contain at least some other proteins in which it is normally found; (2) is of the same origin, e.g. (3) be expressed by cells from different species; (4) at least about 50 percent of the polynucleotides, lipids, carbohydrates, or (5) is separated from other substances with which it is naturally associated (covalently or non-covalently bound); (through bonded interactions) to enable it to function with polypeptides with which it is not naturally associated associated with, or (6) not occurring in nature.

[0243] The antibody or nucleic acid may be formulated with a diluent or adjuvant and, for practical purposes, may be isolated. may be used, for example, to coat microtiter plates for use in immunoassays. When used for treatment, it may be mixed with a carrier, and when used in therapy, it may be mixed with a pharmaceutically acceptable carrier. As described elsewhere herein, other carriers or diluents may be used. Active ingredients may also be included in therapeutic preparations. Antibodies may be produced naturally in vivo or in CHO cells. It may be glycosylated by a heterologous eukaryotic system such as a cell, or may be non-glycosylated. The present invention also provides modified genes. In some applications, antibodies with undesired glycosylation patterns may be used. Modifications that remove sylation sites may be useful, or, for example, increase ADCC function. For other applications, modifying CDCs may be useful

[38] . Galactosylation modifications can be performed to achieve this.

[0244] Typically, an isolated product comprises at least about 5%, at least about 10%, or a mixture of at least about 5% of a given sample. %, at least about 25%, or at least about 50%. or polypeptide, or its therapeutic, diagnostic, prophylactic, research, or other uses. and may be substantially free of other contaminants found in its natural or production environment. do.

[0245] Antibodies are identified and separated from components of their production environment (e.g., natural or recombinant). An isolated antibody can be, for example, an antibody that has been isolated and / or recovered by the FDA. isolated to an acceptable or recognized standard. It may be free from association with all other components. Contaminating components of the production environment, such as microbial contamination, may be present in the antibody for research, diagnostic, or therapeutic use. Substances that can interfere with the function of the enzymes, hormones, and other proteinaceous or non-proteinaceous substances. In some embodiments, the antibody may be (1) purified by, for example, the Lowry method; Typically, greater than 95% by weight of the antibody, and in some embodiments, greater than 99% by weight of the antibody, comprises (2) spinyltransferase. By using a sequenator, at least one amino acid sequence at the N-terminus or internal (3) Coomassie blue, preferably silver staining, or Purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using An isolated antibody will be free of at least one component of the antibody's natural environment. Generally, an isolated antibody or its derivatives are used. The nucleic acid encoding the will be prepared by at least one purification step.

[0246] The present invention provides therapeutic compositions comprising the antibodies described herein. Therapeutic compositions containing nucleic acids encoding the nucleic acids are also provided. The therapeutic methods described herein are described in more detail and include DNA and RNA, such as mRNA. In the method, the use of nucleic acid encoding the antibody and / or cells containing such nucleic acid can be used to produce the antibody. It can be used as an alternative to (or in addition to) a composition containing Thus, a cell containing an antibody-encoding nucleic acid, wherein the nucleic acid is stably integrated into the genome, is and represents a therapeutic agent for therapeutic use in a patient. Human B lymphocytes, optionally derived from the intended patient, are introduced ex vivo Optionally, memory B cells are used. Administration of cells containing the antibody may provide a longer-lasting effect compared to administration of isolated nucleic acids or isolated antibodies. A reservoir of cells capable of expressing anti-ICOS antibodies that may provide therapeutic benefit. Provide a bar.

[0247] The composition may be incorporated into a formulation to provide improved transport, delivery, tolerance, etc. Many suitable formulations are known to all pharmaceutical chemists. It can be found in the prescriptions listed at: Remington's Pharmacy ical Sciences,Mack Publishing Company,Ea These preparations include, for example, powders, pastes, ointments, jellies, waxes, lipids (cationic or is anionic), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, Emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and carbowax Semi-solid mixtures containing Bowax are included. Powell et al. endium of excipients for parenteral form PDA(1998)J Pharm Sci Technol 52: See also U.S. Pat. No. 2,238-311. The composition may be administered in combination with a medical injection buffer and / or adjuvant. The combination may include an antibody or a nucleic acid.

[0248] The antibody or its encoding nucleic acid may be administered by the desired route of administration to the patient, e.g., as a liquid for injection (optionally Various delivery systems are known and may be used to administer the pharmaceutical compositions of the present invention. It can be used to administer pharmaceutical compositions. Methods of introduction include intradermal, intramuscular, and intraperitoneal. These include, but are not limited to, intravenous, subcutaneous, intranasal, epidural, and oral routes. Formulation of antibodies for subcutaneous administration typically requires additional time for their administration compared to intravenous formulations. The high potency of the antibodies according to the invention is due to the fact that they are weak at doses low enough to make subcutaneous formulations practical, demonstrating advantages over anti-ICOS antibodies It may be possible to use

[0249] The compositions may be administered by any convenient route, for example, by infusion or bolus injection. It is administered by absorption through the intradermal or mucocutaneous lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration may be systemic or intravenous. It can be localized.

[0250] Pharmaceutical compositions can also be delivered in vesicles, in particular liposomes (Langer et al., 1999). 90)Science 249:1527-1533;Treat et al.(198 9)in Liposomes in the Therapy of Infecti ous Disease and Cancer,Lopez Berestein a nd Fidler (eds.), Liss, New York, pp. 353-365; See Lopez-Berestein, ibid., pp. 317-327; generally, ibid. (See the book).

[0251] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. Pumps can be used (Langer, supra; Sefton (1987) C (See RC Crit. Ref. Biomed. Eng. 14:201). In embodiments, polymeric materials may be used; tions of Controlled Release,Langer and W ise(eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled release system can be placed in proximity to the target of the composition. thus requiring only a fraction of the systemic dose (e.g., Goodson ,in Medical Applications of Controlled R See Elease, supra, vol. 2, pp. 115-138, 1984 stomach).

[0252] Injectable preparations include intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, and other dosage forms. These injection preparations can be prepared by known methods. The antibody or its salt is added to a sterile aqueous or oily medium conventionally used for injection, for example, An injectable preparation can be prepared by dissolving, suspending, or emulsifying the water. The solvent may include a suitable solvent, such as alcohol (e.g., ethanol), polyalcohol, or the like. Coal (e.g., propylene glycol, polyethylene glycol), non-ionic surfactants agents [e.g., polysorbate 80, HCO-50 (polyoxyethylene (5 0 mole) adduct)], etc., which may be used in combination with physiological saline, glucose-containing, etc. solubilizing agents, such as benzyl benzoate, benzyl alcohol, and other adjuvants. For example, sesame oil, soybean oil, etc. can be used in combination with cole, etc. The injection solution thus prepared can be filled into suitable ampoules. The substance can be delivered subcutaneously or intravenously using a standard needle and syringe. It is expected that the use of needle-free devices will not be limited to intravenous injection. For subcutaneous delivery, pen delivery devices facilitate the use of delivering the pharmaceutical compositions of the present invention. Such pen delivery devices may be reusable or disposable. Capable pen delivery devices generally use replaceable cartridges that contain the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge is The cartridge is easily discarded and easily replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. There are no replaceable cartridges in disposable pen delivery devices. The pharmaceutical composition is pre-filled in a reservoir within the syringe. Once empty, the entire device is discarded. Many reusable pen and autoinjector delivery devices have utility in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include, to name a few: , AUTOPEN(TM)(Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ Pen (Disetronic Medical Sy stems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ Pen, HUMALOG™ Pen, HUMALIN 70 / 30™ Penn (Eli Lilly and Co., Indianapolis, Ind.), N OVOPEN™ I, II, and III (Novo Nordisk, Copenhagen) gen,Denmark), NOVOPEN JUNIOR(trademark)(Novo Nordi sk, Copenhagen, Denmark), BD™ pen (Becton Digital Kinson, Franklin Lakes, NJ), OPTIPENT (trademark), OPTIPEN PRO™, OPTIPEN STARLET™, and OPTI CLIKT(TM)(Sanofi-Aventis, Frankfurt, Germany Examples of the subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, An example of a disposable pen delivery device having such uses is the SOLOSTAR™ pen. nofi-Aventis), FLEXPEN™ (Novo Nordisk), and These include, but are not limited to, KWIKPEN™ (Eli Lilly) .

[0253] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are adapted to the dose of the active ingredient. Such unit dose dosage forms are, for example, tablets, This includes pills, capsules, injections (ampoules), suppositories, etc. The content of the above-mentioned antibody is The dosage form is usually about 5 to about 500 mg, and in particular in the form of an injection, the above-mentioned antibody For other dosage forms, it can be contained in an amount of about 5 to about 100 mg, and for other dosage forms, it can be contained in an amount of about 10 to about 250 mg.

[0254] The antibody, nucleic acid, or composition comprising it may be placed in a medical container, such as a medicine vial, syringe, or intravenous fluid. In one example, the antibody, nucleic acid, or composition can be contained in an injection device. In one example, the method of treatment described herein may be in vitro and in a sterile container. Kits are provided that include the antibodies, packaging, and instructions for use.

[0255] One aspect of the present invention is a method for producing a compound comprising combining an antibody or nucleic acid with one or more pharmaceutically acceptable salts of the compounds listed above. "Pharmaceutically acceptable" means a composition that is pharmaceutically acceptable to animals, including humans. Approved by a U.S. federal or state regulatory agency for use in or approvable, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia. A pharmaceutically acceptable carrier, excipient, or adjuvant is a drug , for example, may be administered to a patient together with any of the antibodies or antibody chains described herein. When administered in a dose sufficient to deliver a therapeutic amount of the drug without impairing their pharmacological activity. It is non-toxic in most cases.

[0256] In some embodiments, the anti-ICOS antibody is the only active ingredient in a composition according to the invention. Thus, the composition may consist of the antibody, or may consist of the antibody and one or more pharmaceutically acceptable salts thereof. However, the compositions according to the present invention may optionally comprise one or more of: A detailed description of drugs with which anti-ICOS antibodies can be combined is provided in , as provided elsewhere herein. Optionally, the composition may comprise, for example, an anti-ICOS antibody and Multiple antibodies (e.g., IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG1, IgG1, IgG4, IgG1, IgG2, IgG4, IgG5, IgG6, IgG1, IgG1, IgG4, IgG1, IgG2, IgG4, IgG4, IgG5, IgG6, IgG1, IgG1, IgG4, IgG1, IgG4 or encoding nucleic acid). Other therapeutic agents that may be useful include pain relievers. Any such agent or combination of agents may be administered in combination. Whether combined or in separate preparations, the antibodies or nucleic acids according to the invention may be used in combination with the antibodies or nucleic acids according to the invention. The antibody or antibody according to the present invention may be administered in combination with the antibody or antibody according to the present invention, or may be provided in a composition together with the antibody or antibody according to the present invention. The nucleic acid may be administered separately, sequentially, or simultaneously with another therapeutic agent(s), such as those mentioned. , optionally administered as a combined preparation.

[0257] Anti-ICOS antibodies for use in specific therapeutic indications are considered to be accepted standards of care. Thus, in the case of anti-cancer therapy, antibody therapy may be used in combination with, for example, chemotherapy, It may be used in a treatment regimen that also includes surgery and / or radiation therapy. can be a single or fractionated dose delivered either directly to the affected tissue or systemically. do.

[0258] The multiple compositions may be administered separately or simultaneously. Separate administrations may be 10, 20, 30 or more. At intervals of 10 to 60 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 hours This refers to the administration of two compositions at an interval of 24 hours or longer. Alternatively, two or more compositions may be administered simultaneously, e.g., They may be administered less than 10 minutes apart or less than 5 minutes apart. In some embodiments, they are administered simultaneously. The composition may be a mixture with or without similar or different time release mechanisms for each of the components. The compounds may be administered as a combination.

[0259] The antibodies and their encoding nucleic acids can be used as therapeutic agents. The subject is generally a mammal, typically a human. The antibody or nucleic acid may be, for example, The compound may be administered to a mammal by any of the routes of administration mentioned above.

[0260] Administration is typically in a "therapeutically effective amount," which means that sufficient of the compound administered will show benefit to the patient. The precise amount will depend on the purpose of the treatment and can be determined by known techniques. The accuracy of the method will be confirmed by one skilled in the art using the method described in Lloyd (1999) The Art,Science and Technology of Pharmaceut (See ical Compounding). Prescribing treatment, e.g., determining dosage. The determination of the medical condition is within the responsibility of general practitioners and other medical professionals and depends on the severity and severity of the symptoms of the disease being treated. The therapeutically effective amount or suitable dose of the antibody or nucleic acid may depend on the progression of the disease. This can be determined by comparing its in vitro and in vivo activity in Methods for the extrapolation of effective dosages in mice and other experimental animals are known.

[0261] As demonstrated by the in vivo studies described in the Examples herein, anti-ICOS antibodies may be effective over a range of doses. Pharmacodynamic studies are reported in Example 24.

[0262] The anti-ICOS antibody may be administered in one of the following amounts per dose: Approximately 10 μg / kg body weight to approximately 100 mg / kg body weight, Approximately 50 μg / kg body weight to approximately 5 mg / kg body weight, Approximately 100 μg / kg body weight to approximately 10 mg / kg body weight, Approximately 100 μg / kg body weight to approximately 20 mg / kg body weight, Approximately 0.5 mg / kg to approximately 20 mg / kg of body weight, or Less than about 5 mg / kg of body weight, e.g., less than 4 mg / kg, less than 3 mg / kg, or 2 mg / kg or less than 1 mg / kg of antibody.

[0263] The optimal therapeutic dose in humans is 0.1 to 0.5 mg / kg, for example, about 0.1 mg / kg. kg, 0.15mg / kg, 0.2mg / kg, 0.25mg / kg, 0.3mg / kg, 0 may be 0.35mg / kg, 0.4mg / kg, 0.45mg / kg, or 0.5mg / kg For fixed doses in adult humans, the preferred doses are 8 to 50 mg or 8 to 25 mg. , for example, 15 mg or 20 mg.

[0264] In the treatment methods described herein, one or more doses may be administered. In some cases, a single dose may be effective in achieving long-term benefits. It may involve administering a single dose of the antibody, its encoding nucleic acid, or composition. Multiple doses may be administered, usually consecutively or separately at intervals of days, weeks, or months. Anti-ICOS antibodies should be administered at intervals of 4 to 6 weeks, for example, every 4 weeks, 5 weeks, or 6 weeks. The anti-ICOS antibody may be administered to the patient repeatedly at weekly intervals. Optionally, the anti-ICOS antibody may be administered once a month. The drug may be administered to a patient once a day, or less frequently, for example, every two or three months. Thus, a method of treating a patient may involve administering a single dose of an anti-ICOS antibody to the patient, and This administration should not be repeated for at least 1 month, at least 2 months, or at least 3 months. and may include not repeating the administration for at least 12 months.

[0265] As discussed in Example 11c, either single or multiple doses of anti-ICOS antibody Equivalent therapeutic efficacy may be achieved using either antibody or mAb, as a single dose of antibody may be sufficient to This may be the result of the effectiveness of resetting the environment. Physicians may be able to identify disease states as well as anti-ICO Any other therapeutic agent or procedure with which the S antibody is combined (e.g., surgery, radiation therapy) Taking into account the above, the administration regimen of anti-ICOS antibodies should be tailored to suit the disease and patient being treated. In some embodiments, an effective dose of an anti-ICOS antibody can be administered in combination with, for example, More frequently than once a month, such as once every three weeks, once every two weeks, or once a week Treatment with anti-ICOS antibodies should be continued for at least 1 month, at least 6 months, or may include multiple doses administered over a period of at least one year.

[0266] As used herein, "treat," "treatment," "treating," or "improvement" refers to The term "anticoagulant" refers to any agent that reverses, reduces, improves, or inhibits the progression or severity of a condition associated with a disease or disorder. Refers to a therapeutic procedure whose purpose is to harm, slow, or stop a disease. The term "treatment" refers to the reduction or amelioration of at least one side effect or symptom of a condition, disease, or disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. "Treatment" refers not only to the improvement of symptoms or markers, but also to the improvement of symptoms or markers that would be expected in the absence of treatment. This includes a cessation or at least a general decline in the progression or worsening of symptoms, compared to a beneficial or desired clinical outcome. Outcomes include relief of one or more symptoms, a reduction in the extent of disease, stable disease (i.e., (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state , remission (partial or complete), and / or detectable or undetectable The term "treatment" of a disease includes, but is not limited to, a reduction in mortality without treatment. It also includes providing relief from the symptoms or side effects of the disease (including palliative care). To be effective, a cure is not intended. The method may, in certain embodiments, include a cure. In the context of the present invention, treatment may also be prophylactic treatment.

[0267] T cell therapy WO2011 / 097477 describes a method for treating a T cell population with, optionally, IL-1β, IL-6, or IL-1β. In the presence of Th17 polarizing agents such as anti-IFNγ and / or anti-IL-4, primary activation signals are A first agent (e.g., an anti-CD3 antibody) that provides a signal and a second agent that activates ICOS. To generate and expand T cells by contacting them with an antibody (e.g., an anti-ICOS antibody) The use of anti-ICOS antibodies described herein has been described. In such a method, a cell population having therapeutic activity (e.g., anti-tumor activity) can be provided. A culture-expanded T cell population can be generated that is capable of expressing the T cells described in WO2011 / 097477. As such, such T cells may be used therapeutically in methods of treating patients by immunotherapy. It is possible.

[0268] Morphological assay for anti-ICOS antibodies as therapeutic candidates Candidate therapeutic anti-ICOS antibodies are bound to the solid surface and interact with ICOS-expressing T cells. It was found that when exposed to anti-I they were able to induce morphological changes in cells. Addition of ICOS+ T cells to wells internally coated with COS antibody resulted in the cells The cells changed from their original round shape to adopt a spindle shape and spread out to the antibody-coated surface. This morphological change was not observed with the control antibody. It was found to be dose dependent, with the increase in the concentration of antibody on the surface resulting in a faster and and / or a more pronounced shape change occurred. This shape change was due to T cell binding to ICOS and / or This assay provides a surrogate index of agonism by anti-ICOS antibodies. Antibodies that promote the multimerization of ICOS on the cell surface can be identified. , represent therapeutic candidate agonist antibodies. Advantageously, this assay provides Visual indication is a simple method, especially for screening large numbers of antibodies or cells. The assay may be automated to be performed in a high-throughput system.

[0269] Thus, one aspect of the present invention optionally provides a method for selecting ICOS agonist antibodies. an assay for selecting an antibody that binds to ICOS, comprising: providing an array of antibodies immobilized (bound or attached) to a substrate in a test well; Add ICOS-expressing cells (e.g., activated primary T cells or MJ cells) to test wells. And, Observing the morphology of cells; Detecting a shape change within the well from a round shape to a shape flattened against the substrate and wherein the shape change is due to the antibody binding to ICOS, optionally an ICOS agonist. detecting the antibody; and selecting the antibody from the test well.

[0270] The assay can be optionally performed in parallel, for example in a 96-well plate format. Multiple test wells may be performed, each containing a different antibody for testing. or the inner surface of a well. Thus, a two-dimensional surface is provided against which the cells Flattening may be observed, e.g., the bottom and / or walls of the wells may be coated with antibody. The linkage of the antibody to the substrate can be via the constant region of the antibody.

[0271] A negative control may be included, such that the antibody is known not to bind to ICOS. Preferably, the antibody does not bind to an antigen on the surface of the ICOS-expressing cells used. The assay quantifies the extent of morphological change and, if multiple antibodies are tested, and selecting an antibody that induces a greater morphological change than the other tested antibodies. .

[0272] Antibody selection involves expressing nucleic acid encoding the antibody present in the test well of interest. or expressing an antibody comprising the CDRs or antigen-binding domain of that antibody. The antibody is optionally an antibody that comprises, for example, the antigen-binding domain of a selected antibody, e.g., For example, antibody fragments or antibodies containing different constant regions can be reformatted to provide The antibody selected preferably contains a human IgG1 constant region or one of the constant regions described herein. The selected antibody may further comprise a composition comprising one or more additional components. Suitable pharmaceutical formulations are discussed elsewhere herein.

[0273] Terms Embodiments of the present invention are described in the following numbered clauses, which are a part of the specification:

[0274] Clause 1. An isolated antibody that binds to the extracellular domain of human and / or mouse ICOS. and has at least 95% sequence identity with the STIM003 VH domain (SEQ ID NO: 408). and a VH domain containing an amino acid sequence having the same nucleotide sequence as that of STIM003 VL domain (SEQ ID NO: 1). No. 415), and a VL domain comprising an amino acid sequence having at least 95% sequence identity with , including an antibody.

[0275] Clause 2. The VH domain comprises a set of heavy chain complementarity determining regions (HCDRs) HCDR1, H comprising CDR2 and HCDR3, HCDR1 is STIM003 HCDR1 having the amino acid sequence of SEQ ID NO: 405 can be, HCDR2 is STIM003 HCDR2 having the amino acid sequence of SEQ ID NO: 406 can be, HCDR3 is STIM003 HCDR3 having the amino acid sequence of SEQ ID NO: 407 The antibody of clause 1,

[0276] Clause 3. The VL domain comprises a set of light chain complementarity determining regions (LCDRs) LCDR1, L comprising CDR2 and LCDR3, LCDR1 is STIM003 LCDR1 having the amino acid sequence of SEQ ID NO: 412 can be, LCDR2 is STIM003 LCDR2 having the amino acid sequence of SEQ ID NO: 413 can be, LCDR3 is STIM003 LCDR3 having the amino acid sequence of SEQ ID NO: 414 The antibody of clause 1 or clause 2.

[0277] Clause 4. The amino acid sequence of the VH domain is SEQ ID NO: 408, and / or the VL domain is The antibody of clause 1, wherein the amino acid sequence of the antibody is SEQ ID NO: 415.

[0278] Clause 5. An isolated antibody that binds to the extracellular domain of human and / or mouse ICOS. There was, Antibody V, including complementarity determining regions (CDRs), HCDR1, HCDR2, and HCDR3 H Domestic In and, Antibody V comprising complementarity determining regions LCDR1, LCDR2 and LCDR3 L domain and Including, HCDR1 is STIM001, STIM002, STIM002-B, STIM00 3, STIM004, STIM005, STIM006, STIM007, STIM00 HCDR1 of STIM009 or HCDR1 of STIM009, or 1, 2, 3, 4 or 5 and its HCDR1 having an amino acid modification, HCDR2 is STIM001, STIM002, STIM002-B, STIM00 3, STIM004, STIM005, STIM006, STIM007, STIM00 HCDR2 of STIM009 or 1, 2, 3, 4 or 5 and / or comprising its HCDR2 having an amino acid modification HCDR3 is STIM001, STIM002, STIM002-B, STIM00 3, STIM004, STIM005, STIM006, STIM007, STIM00 HCDR3 of STIM009 or 1, 2, 3, 4 or 5 An antibody comprising an HCDR3 thereof having an amino acid modification.

[0279] Clause 6. The antibody heavy chain CDR is selected from the group consisting of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or 1, 2, 3, 4 or 5 STIM001, STIM002, STIM002-B, and STIM003-B each having an amino acid alteration TIM003, STIM004, STIM005, STIM006, STIM007, S The antibody of clause 5, comprising TIM008 or STIM009 heavy chain CDRs.

[0280] Clause 7. The antibody of Clause 6, wherein the antibody VH domain has the heavy chain CDRs of STIM003. antibody.

[0281] Clause 8. An isolated antibody that binds to the extracellular domain of human and / or mouse ICOS. There was, an antibody VH domain comprising complementarity determining regions HCDR1, HCDR2 and HCDR3; an antibody VL domain comprising complementarity determining regions LCDR1, LCDR2 and LCDR3; Including, LCDR1 is STIM001, STIM002, STIM002-B, STIM00 3, STIM004, STIM005, STIM006, STIM007, STIM00 8 or LCDR1 of STIM009, or 1, 2, 3, 4 or 5 and its LCDR1 having an amino acid modification, LCDR2, STIM001, STIM002, STIM002-B, STIM003 , STIM004, STIM005, STIM006, STIM007, STIM008 or LCDR2 of STIM009, or 1, 2, 3, 4 or 5 and / or comprising an LCDR2 thereof having an α-acid modification; LCDR3, STIM001, STIM002, STIM002-B, STIM003 , STIM004, STIM005, STIM006, STIM007, STIM008 or LCDR3 of STIM009, or 1, 2, 3, 4 or 5 An antibody comprising an LCDR3 thereof having an amino acid modification.

[0282] Clause 9. The antibody light chain CDR is selected from the group consisting of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, or 1, 2, 3, 4 or 5 STIM001, STIM002, STIM002-B, and STIM003-B each having an amino acid alteration TIM003, STIM004, STIM005, STIM006, STIM007, S 10. The method of claim 5, wherein the light chain CDR of the TIM008 or STIM009 molecule is a nucleotide sequence of the TIM008 or STIM009 light chain CDR. The antibodies listed.

[0283] Clause 10. The antibody according to Clause 9, wherein the antibody VL domain has the light chain CDRs of STIM003. antibodies.

[0284] Clause 11. VH and / or VL domain frames of human germline gene segment sequences 11. The antibody of any one of clauses 5 to 10, comprising a network region.

[0285] Article 12. (i) a human heavy chain V gene segment, a human heavy chain D gene segment, and a human heavy chain J gene a VH domain derived from recombination of segments, The V segment is IGHV1-18 (e.g., V1-18 * 01), IGVH3-20( For example, 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), IGH D3-10 (e.g., IGHD3-10 * 01) or IGHD3-9 (e.g., IGH D3-9 * 01) and / or The J gene segment is IGHJ6 (e.g., IGHJ6 * 02), IGHJ4 (e.g. , IGHJ4 * 02) or IGHJ3 (e.g., IGHJ3 * 02) VH Dome In, or (ii) a VH domain comprising framework regions FR1, FR2, FR3, and FR4; So, FR1 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGHV1-18 (e.g., V1-18 * 01), IGVH3-2 0 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) Young IGVH2-5 (e.g., V2-5 * 10) and aligned, FR2 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGHV1-18 (e.g., V1-18 * 01), IGVH3-2 0 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) Young IGVH2-5 (e.g., V2-5 * 10) and aligned, FR3 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGHV1-18 (e.g., V1-18 * 01), IGVH3-2 0 (e.g., V3-20 * d01), IGVH3-11 (e.g., V3-11 * 01) Young IGVH2-5 (e.g., V2-5 * 10) and / or FR4 may have 1, 2, 3, 4 or 5 amino acid modifications. Cytoplasmic V gene segment IJH6 (e.g., JH6 * 02), IGJH4 (e.g., JH 4 * 02) or IGJH3 (e.g., JH3 * 02), containing a VH domain that aligns with 12. The antibody according to any one of items 5 to 11.

[0286] Article 13. (i) an antibody derived from the recombination of a human light chain V gene segment and a human light chain J gene segment; a VL domain, 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 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) an antibody VL domain comprising framework regions FR1, FR2, FR3, and FR4; And, FR1 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGKV2-28 (e.g., IGKV2-28 * 01), IGKV 3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1 D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned , FR2 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGKV2-28 (e.g., IGKV2-28 * 01), IGKV 3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1 D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned , FR3 may have 1, 2, 3, 4 or 5 amino acid modifications. The cytoplasmic V gene segment IGKV2-28 (e.g., IGKV2-28 * 01), IGKV 3-20 (e.g., IGKV3-20 * 01), IGKV1D-39 (e.g., IGKV1 D-39 * 01) or IGKV3-11 (e.g., IGKV3-11 * 01) and aligned and / or FR4 may have 1, 2, 3, 4 or 5 amino acid modifications. Cytoplasmic V gene segment IGKJ4 (e.g., IGKJ4 * 01), IGKJ2 (e.g., IGKJ2 * 04), IGKJ3 (e.g., IGKJ3 * 01) or IGKJ1 (e.g. , IGKJ1 * 01), comprising an antibody VL domain aligned with any of clauses 5 to 12. The antibody described.

[0287] Article 14. STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 young or the VH domain of STIM009, or STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM00 6. The antibody VH domain sequence of STIM007, STIM008, or STIM009; Clause 5 to 7, comprising an antibody VH domain having an amino acid sequence that is at least 90% identical to the antibody VH domain of clause 5 to 7, 14. An antibody according to any one of 13.

[0288] Article 15. STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 young or the VL domain of STIM009, or STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM00 6. The antibody VL domain sequence of STIM007, STIM008 or STIM009 Clause 5 to 7, comprising an antibody VL domain having an amino acid sequence that is at least 90% identical to the antibody VL domain of clause 5 to 7, 14. An antibody according to any one of 13.

[0289] Article 16. STIM001, STIM002, STIM002-B, STIM003, STIM0 04, STIM005, STIM006, STIM007, STIM008 or ST or selected from the VH domains of STIM001, STIM002, S TIM002-B, STIM003, STIM004, STIM005, STIM006 , STIM007, STIM008, or STIM009 antibody VH domain sequence and an antibody VH domain having an amino acid sequence that is at least 90% identical to the VH domain; the VL domain of the selected antibody or the antibody VL domain sequence of the selected antibody and an antibody VL domain having an amino acid sequence at least 90% identical to 6. The antibody according to claim 5.

[0290] Clause 17. Comprising a STIM003 VH domain and a STIM003 VL domain, 17. The antibody of clause 16.

[0291] Clause 18. The antibody of any one of clauses 1 to 17, comprising an antibody constant region.

[0292] Clause 19. The antibody of clause 18, wherein the constant region comprises a human heavy chain and / or light chain constant region. body.

[0293] Clause 20. The antibody of clause 18 or clause 19, wherein the constant region is Fc effector positive. antibody.

[0294] Clause 21. Enhanced ADCC, ADCP, and / or C compared to native human Fc region The antibody of clause 20, comprising an Fc region with DC function.

[0295] Clause 22. The antibody of any one of clauses 18 to 21, wherein the antibody is an IgG1.

[0296] Clause 23. The antibody of clause 21 or clause 22, wherein the antibody is non-fucosylated.

[0297] Article 24. A compound according to any one of Articles 1 to 23 conjugated to a cytotoxic drug or prodrug. The antibody described.

[0298] Clause 25. The antibody of any one of clauses 1 to 24, which is a multispecific antibody.

[0299] Clause 26. Affinity (K) of less than 50 nM as determined by surface plasmon resonance D )in An isolated antibody that binds to the extracellular domain of human and mouse ICOS.

[0300] Clause 27. The antibody has an affinity (as determined by surface plasmon resonance) of less than 5 nM K D 27. The antibody of clause 26, which binds to the extracellular domain of human and mouse ICOS at

[0301] Article 28. K binding to the extracellular domain of human ICOS D However, extracellular K binding domain D 28. The antibody of clause 26 or clause 27, wherein the antibody is within 10-fold of

[0302] Clause 29. An isolated antibody according to any one of clauses 1 to 28, and a pharmaceutically acceptable excipient. A composition comprising:

[0303] Clause 30. An isolated nucleic acid encoding the antibody of any one of clauses 1 to 29, and a pharmaceutical and a physiologically acceptable excipient.

[0304] Article 31. Maintaining a balance between regulatory T cells (Treg) and effector T cells (Teff) 28. A method for increasing a Teff response in a patient, comprising administering to a subject a therapeutically effective amount of a compound according to any one of clauses 1 to 28. or the composition of clause 29 to a patient.

[0305] Clause 32. Depleting regulatory T cells (Tregs) and / or effector T cells in a patient Diseases or pathologies that can be treated by increasing T cell (Teff) responses A method of treating a condition comprising administering to a subject an antibody according to any one of clauses 1 to 28 or to a subject according to clause 29. The method comprises administering the composition described above to a patient.

[0306] Article 33. A method of treating the human body by therapy according to any of Articles 1 to 28. An antibody according to any one of clauses 29 or a composition according to clause 29.

[0307] Article 34. Maintaining a balance between regulatory T cells (Treg) and effector T cells (Teff) 33 for use in increasing an effector T cell response in a patient. An antibody or composition for the use described.

[0308] Article 35. Depletion of regulatory T cells (Treg) and / or effector T cells ( in the treatment of diseases or pathological conditions amenable to therapy by increasing the Teff (Treg) response 34. An antibody or composition for use according to clause 33.

[0309] Clause 36. The method according to clause 32 or clause 35, wherein the disease is cancer or a solid tumor. The antibody or composition for the above use.

[0310] Clause 37. Any of clauses 1 to 28 for use in a method for treating cancer in a human patient. 29. An antibody according to any one of clauses 1 to 28 or a composition according to clause 29.

[0311] Clause 38. A method for treating cancer in a human patient, comprising administering to a subject an antibody according to any one of clauses 1 to 28. 30. A method comprising administering to a patient a composition according to claim 29.

[0312] Article 39. The cancer is renal cell carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, or diffuse large cell carcinoma. 39. The method for use or antibody-containing method according to any one of clauses 36 to 38, wherein the cancer is B-cell lymphoma. Or composition.

[0313] Clause 40. The method includes administering to the patient an antibody and another therapeutic agent and / or radiation therapy. 40. The method, antibody or composition according to any one of clauses 31 to 39.

[0314] Clause 41. The method or method for use according to clause 40, wherein the therapeutic agent is an anti-PD-L1 antibody. is an antibody or composition.

[0315] Clause 42. The anti-PD-L1 antibody comprises a VH domain having the amino acid sequence of SEQ ID NO: 299. and a VL domain having the amino acid sequence of SEQ ID NO: 300. Methods of use or antibodies or compositions.

[0316] Clause 43. Clause 41 or 42, wherein the therapeutic agent is an anti-PD-L1 IL-2 immunocytokine. 43. A method or antibody or composition for use according to clause 42.

[0317] Clause 44. The anti-PD-L1 antibody is an immunocytokinin containing human wild-type or mutant IL-2. 44. A method or antibody or composition for use according to clause 43, wherein the antibody or composition is

[0318] Clause 45. The anti-ICOS antibody and the anti-PDL1 antibody are capable of inhibiting ADCC, ADCP, and / or 45. A method or antibody for use according to clause 44, each of which is capable of mediating CDC Composition.

[0319] Article 46. The anti-ICOS antibody is a human IgG1 antibody, and the anti-PDL1 antibody is a human Ig 46. ​​The method for use or the antibody or method for use according to any one of clauses 41 to 45, wherein the antibody or composition.

[0320] Clause 47. The method for use according to clause 40, wherein the therapeutic agent is an anti-PD-1 antibody. Antibody or composition.

[0321] Clause 48. The method for use or the method for the treatment of an illness according to clause 40, wherein the other therapeutic agent is IL-2. Body or composition.

[0322] Clause 49. The method comprises administering an anti-ICOS antibody after administering other therapeutic agents and / or radiation therapy. 49. A method for use or antibody or method for use according to any one of clauses 40 to 48, comprising administering to a subject a is a composition.

[0323] Article 50. The anti-ICOS antibody is conjugated to a prodrug, The method or use administering an anti-ICOS antibody to the patient; and selectively activating the prodrug at the target tissue site. 9. A method, antibody, or composition according to any one of 9.

[0324] Clause 51. The patient has a solid tumor, and the method comprises selectively targeting the prodrug in the tumor. 51. A method for use or an antibody or composition according to clause 50, comprising activating .

[0325] Clause 52. Clause 5, including selectively activating a prodrug by photoactivation. 0 or a method or antibody or composition for use according to clause 51.

[0326] Clause 53. An anti-ICOS human IgG1 antibody for use in a method of treating cancer in a patient. and an anti-PDL1 human IgG1 antibody.

[0327] Article 54. A method of treating cancer in a patient, comprising administering an anti-ICOS human IgG1 antibody and an anti-PD-L1 antibody. 3. A method comprising administering to a patient a human IgG1 antibody, comprising administering to said patient a human IgG1 antibody,

[0328] Article 55. An anti-ICOS antibody for use in a method of treating cancer in a patient, comprising: The method comprises administering to a patient an anti-ICOS antibody and an anti-PD-L1 antibody, The anti-ICOS antibody is administered.

[0329] Article 56. The anti-ICOS antibody is a human IgG1 antibody and the anti-PD-L1 antibody is a human IgG1 antibody. 56. The anti-ICOS antibody for use according to clause 55, which is a gG1 antibody.

[0330] Article 57. The cancer is renal cell carcinoma, head and neck cancer, melanoma, non-small cell lung cancer, or diffuse large cell carcinoma. The combination of clause 53, the method of clause 54 or clause 55, wherein the tumor is a B-cell lymphoma. or an anti-ICOS antibody for use according to clause 56.

[0331] Clause 58. The method comprises administering to a patient an anti-ICOS antibody and an anti-PD-L1 antibody. 41-46 or 53-54, wherein a single dose of anti-ICOS antibody is administered. A method or antibody, composition or combination for the use as described.

[0332] Clause 59. The method comprises administering a single dose of an anti-ICOS antibody followed by multiple doses of an anti-PD-L1 antibody. 59. A method for use or antibody, composition or combination according to clause 58, comprising administering to a subject Match.

[0333] Article 60. The anti-ICOS antibody and the anti-PDL1 antibody are provided in separate compositions for administration. 54. A method for use or an antibody or composition according to any one of clauses 41 to 46 or 53 to 54. A composition or combination.

[0334] Clause 61. The anti-ICOS antibody and / or anti-PD-L1 antibody comprises the amino acid sequence of SEQ ID NO: 340 60. A method for producing a human IgG1 constant region comprising administering to a mammalian subject the present invention a ... The method or antibody, composition or combination for use as described above.

[0335] Article 62. An anti-ICOS antibody for use in a method of treating a patient, the method comprising: The method involves administering anti-ICOS antibodies to a patient with an increased level of ICOS positive regulatory activity after treatment with another therapeutic agent. administering to a patient having T cells.

[0336] 63. A method of treating a patient, said method comprising administering an anti-ICOS antibody in combination with another therapeutic agent It is intended to be administered to patients with increased levels of ICOS-positive regulatory T cells after treatment with Including, a method.

[0337] Clause 64. The method comprises administering a therapeutic agent to a patient, and determining whether the patient has an increased risk of developing a disease after treatment with the agent. The patient was determined to have a high level of ICOS-positive regulatory T cells and was administered anti-ICOS antibodies. and administering to said patient a therapeutically effective amount of said antibody ... or the method according to clause 63.

[0338] Clause 65. The therapeutic agent is IL-2 or an immunomodulatory antibody (e.g., anti-PDL-1, anti-PD-1 65. Anti-IC for use according to any of clauses 62 to 64, which is anti-CTLA-1 or anti-CTLA-1 OS antibodies or methods.

[0339] Clause 66. The method includes treating a tumor, e.g., melanoma, such as metastatic melanoma. 66. An anti-ICOS antibody for use or a method according to any one of Items 62 to 65.

[0340] Article 67. Detecting cancer in a patient by in vivo vaccination of the patient against their own cancer cells. 1. An anti-ICOS antibody for use in a method of treatment by performing but, Immunological cells of cancer cells that provide antigen presentation to antigen-specific effector T cells Treating a patient with a therapy that causes death; administering an anti-ICOS antibody to a patient, wherein the anti-ICOS antibody induces an antigen-specific effect and administering an anti-ICOS antibody to enhance a target T cell response.

[0341] Article 68. Detecting cancer in a patient by in vivo vaccination of the patient against their own cancer cells. 1. A method of treating a subject by performing the steps of: Immunological cells of cancer cells that provide antigen presentation to antigen-specific effector T cells Treating a patient with a therapy that causes death; administering an anti-ICOS antibody to a patient, wherein the anti-ICOS antibody induces an antigen-specific effect and administering a therapeutically effective amount of a compound to enhance a target T cell response.

[0342] Article 69. Detecting cancer in a patient by in vivo vaccination of the patient against their own cancer cells. a method of treating a patient by administering an anti-ICOS antibody to the patient, This includes: The patient undergoes immunization of cancer cells, resulting in presentation of antigen to antigen-specific effector T cells. have been previously treated with a therapy that causes biologic cell death, and A method in which the anti-ICOS antibody enhances antigen-specific effector T cell responses.

[0343] Article 70. Therapies that induce immunological cell death include irradiation of cancer cells, administration of chemotherapeutic agents, etc. 69. The method according to any one of clauses 67 to 69, wherein the administration of a tumor-associated antigen is a steroid therapy, and / or an antibody against a tumor-associated antigen is administered. The anti-ICOS antibody or method for use as described above.

[0344] Clause 71. The method for use according to clause 70, wherein the chemotherapeutic agent is oxaliplatin. ICOS antibodies or methods.

[0345] Article 72. The use according to Article 70, wherein the tumor-associated antigen is HER2 or CD20. Anti-ICOS antibodies or methods for

[0346] Clause 73. An anti-ICOS antibody for use in a method of treating cancer in a patient, the method comprising: are characterized as being positive for ICOS ligand and / or FOXP3 expression or characterized anti-ICOS antibody.

[0347] Article 74. A method of treating cancer in a patient, wherein the cancer is treated with an ICOS ligand and / or FO characterized or characterized as being positive for XP3 expression, The method comprises administering an anti-ICOS antibody to the patient.

[0348] Article 75. The method: Samples from patients are tested to determine whether the cancer expresses ICOS ligand and / or FOXP3. and determining that selecting the patient for treatment with an anti-ICOS antibody; 88. The method according to any one of clauses 85 to 87, comprising administering an anti-ICOS antibody to the patient. Anti-ICOS antibodies or methods for use.

[0349] 76. The method comprises administering an anti-ICOS antibody to a cancer targeting an ICOS ligand and / or FOXP Clause 7, including administering to a patient a test sample that has tested positive for expression of 3. 3. An anti-ICOS antibody for use according to clause 3 or the method according to clause 74.

[0350] Clause 77. Use according to clause 75 or clause 76, wherein the sample is a biopsy sample of a solid tumor. An anti-ICOS antibody or method for

[0351] Article 78. Cancer is treated with immuno-oncological drugs, such as anti-CTLA-4 antibodies, anti-PD1 antibodies, anti Refractory to treatment with PD-L1 antibody, anti-CD137 antibody, or anti-GITR antibody 2. The method of claim 1, wherein the compound is a compound selected from the group consisting of benzodiazepines, ... Anti-ICOS antibody for

[0352] Article 79. A method of treating cancer in a patient, wherein the cancer is treated with an immuno-oncological drug, e.g., an anti-C TLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody, or anti-GITR antibody characterized or characterized as refractory to treatment with antibodies, The method comprises administering an anti-ICOS antibody to the patient.

[0353] Article 80. The method: treating the patient with an immuno-oncology agent; determining that the cancer is unresponsive to the drug; selecting patients for treatment with anti-ICOS antibodies; and administering to the patient an anti-ICOS antibody. COS antibody or the method of clause 79.

[0354] Clause 81. The method comprises administering an anti-ICOS antibody to a patient whose cancer has responded to prior treatment with an immuno-oncological agent. 78. An anti-ICOS antibody or The method described in clause 79.

[0355] Article 82. The cancer is a tumor derived from cells that have acquired the ability to express ICOS ligand. 82. An anti-ICOS antibody or method for use according to any one of clauses 73 to 81.

[0356] Clause 83. The anti-ICOS antibody or method for use according to clause 82, wherein the cancer is melanoma. Law.

[0357] Article 84. The cancer is a B-lymphocyte (e.g., B-cell lymphoma, such as diffuse large B-cell lymphoma) 2. The antigen-presenting cells, such as lymphoma cells or T lymphocytes, described in any of Articles 73 to 81 2. An anti-ICOS antibody or method for use in

[0358] Article 85. Any of Articles 73 to 81, wherein the cancer is resistant to treatment with an anti-CD20 antibody. 2. An anti-ICOS antibody for use or a method according to claim 1.

[0359] Article 86. The anti-ICOS antibody for use according to Article 85, wherein the cancer is B-cell lymphoma. Body or method.

[0360] Clause 87. The anti-CD20 antibody for use according to clause 86, wherein the anti-CD20 antibody is rituximab. ICOS antibodies or methods.

[0361] Clause 88. The method comprises treating a patient with an anti-CD20 antibody; determining that the cancer is unresponsive to an anti-CD20 antibody; testing a sample from the patient to determine that the cancer expresses an ICOS ligand; , selecting the patient for treatment with an anti-ICOS antibody; 88. The method according to any one of clauses 85 to 87, comprising administering an anti-ICOS antibody to the patient. Anti-ICOS antibodies or methods for use.

[0362] Clause 89. The method comprises administering an anti-ICOS antibody to a patient whose cancer has responded to prior treatment with an anti-CD20 antibody. 88. The use according to any one of clauses 85 to 87, comprising administering to a patient who has not previously received The anti-ICOS antibody or method.

[0363] Article 90. For the use according to any one of Articles 67 to 89, wherein the cancer is a solid tumor. The anti-ICOS antibody or method.

[0364] Article 91. The use according to any one of Articles 67 to 89, wherein the cancer is a hematological liquid tumor. Anti-ICOS antibody or method for use.

[0365] Article 92. Anti-I for use according to Article 90 or 91, wherein the tumor is enriched in regulatory T cells COS antibodies or methods.

[0366] Article 93. The anti-ICOS antibody is as defined in any of Articles 1 to 28, or for use according to any of clauses 53 to 92, wherein the composition is provided in a composition according to clause 29. Anti-ICOS antibodies or methods for

[0367] Article 94. Human or humanized immunoglobulins encoding human variable region gene segments a transgenic non-human mammal having a genome comprising a gene locus, A mammal that does not express ICOS.

[0368] Article 95. Methods for producing antibodies that bind to the extracellular domain of human and non-human ICOS There was, (a) immunizing a mammal as described in clause 94 with a human ICOS antigen; (b) isolating the antibody produced by the mammal; and (c) testing the antibody for its ability to bind to human ICOS and non-human ICOS; (d) selecting one or more antibodies that bind to both human ICOS and non-human ICOS; , including , a method.

[0369] Clause 96. Immunizing a mammal with cells expressing human ICOS. 95. The method described in claim 95.

[0370] Article 97. (c) The ability to bind to human ICOS and non-human ICOS was investigated using surface plasmon resonance. testing the antibody against the antibody to determine binding affinity; (d) K of binding to human ICOS D The activity of the compound is less than 50 nM, and the effect on non-human ICOS is K of combination D and selecting one or more antibodies having a β-glucan value of less than 500 nM. is the method described in Article 96.

[0371] Article 98. (d) K of binding to human ICOS D The activity of the compound is less than 10 nM, and its effect on non-human ICOS is K of combination D Clause 97, comprising selecting one or more antibodies having a β-glucan value of less than 100 nM. How to do it.

[0372] Article 99. (c) The ability to bind to human ICOS and non-human ICOS was investigated using surface plasmon resonance. testing the antibody against the antibody to determine binding affinity; (d) K of binding to human ICOS D is the K value for binding to non-human ICOS. D Within 10 times and selecting one or more antibodies which are Law.

[0373] Article 100. (d) K of binding to human ICOS D is the K of binding to non-human ICO D Within 5 times of 99. The method of clause 99, comprising selecting one or more antibodies that

[0374] Article 101. Test antibodies for their ability to bind to non-human ICOS from the same species as the mammal. 101. The method of any one of clauses 95 to 100, comprising testing.

[0375] Article 102. Antibodies from species other than mammals for their ability to bind to non-human ICOS 102. The method of any of clauses 95 to 101, comprising testing:

[0376] Article 103. The method according to any one of Articles 95 to 102, wherein the mammal is a mouse or a rat. How to post.

[0377] Article 104. The non-human ICOS is mouse ICOS or rat ICOS. Article 95 A method according to any one of claims 1 to 103.

[0378] Article 105. Human or humanized immunoglobulin loci contain a human constant region upstream of the endogenous constant region. 105. The method of any of clauses 95 to 104, comprising variable region gene segments.

[0379] Article 106. (a) immunizing a mammal as described in clause 94 with a human ICOS antigen, immunizing the subject, wherein the subject is a mouse; and (b) isolating the antibodies produced by the mice; and (c) testing the antibody for its ability to bind to human ICOS and mouse ICOS; (d) selecting one or more antibodies that bind to both human and mouse ICOS. , the method described in clause 105.

[0380] Article 107. Nucleic acid encoding an antibody heavy chain variable domain and / or an antibody light chain variable domain 107. The method of any of clauses 95 to 106, comprising isolating

[0381] Article 108. A mammal is a recombinant human variable region gene segment and an endogenous constant region gene segment. 108. The method of any of clauses 95 to 107, wherein the antibody is produced by

[0382] Clause 109. The nucleic acid encoding the heavy and / or light chain variable domain is selected from the group consisting of a human heavy chain constant region and / or a human light chain constant region, respectively. The method described below.

[0383] Clause 110. Any of clauses 107 to 109, including introducing the nucleic acid into a host cell. The method described.

[0384] Clause 111. Conditions for expression of antibodies or antibody heavy and / or light chain variable domains 111. The method of clause 110, comprising culturing the host cells under

[0385] Article 112. An antibody or antibody produced by the method according to any one of Articles 95 to 111. is an antibody heavy and / or light chain variable domain.

[0386] Article 113. Optionally, to select ICOS agonist antibodies, binding to ICOS 1. A method for selecting an antibody that: providing an array of antibodies immobilized (bound or attached) to a substrate in a test well; Add ICOS-expressing cells (e.g., activated primary T cells or MJ cells) to test wells. And, Observing the morphology of cells; Detects cell shape changes within the well from round to flattened against the substrate wherein the shape change is due to an antibody that binds to ICOS, optionally ICOS detecting an agonist antibody; selecting antibodies from the test wells; expressing nucleic acids encoding the CDRs of the selected antibodies; and formulating the antibody in a composition comprising one or more additional components.

[0387] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure. This specification, including the published U.S. counterparts of any patents or patent applications mentioned, All documents mentioned in are incorporated herein by reference in their entirety.

[0388] Experimental Example The following examples describe the generation, characterization, and performance of anti-ICOS antibodies. Transgenic mouse platform capable of generating antibodies containing Antibodies were generated using Kymouse™, a mouse model of the genus Kymouse. The antibody contains human variable domains generated from human V(D) and J segments and mouse constant domains. The endogenous mouse variable genes are silenced and have a very small repertoire. (less than 0.5% of all heavy chain variable regions are of murine origin.) The se™ system is described in Lee et al. 2014

[39] , WO2011 / 004 192, WO2011 / 158009, and WO2013 / 061098 In this project, the heavy chain locus and the light chain kappa locus were humanized using Kymo The use™ HK strain was used.

[0389] The ICOS knockout Kymouse™ is injected with ICOS protein or human and mouse Immunization with either a combination of alternating boosts of protein and cells expressing mouse ICOS Granted.

[0390] Hits that bound to human ICOS were identified. The primary selection criteria for the screen were human cell-expressing The binding to ICOS (CHO cells) and ICOS protein (HTRF) were investigated. Binding to mouse ICOS protein and mouse cell-expressed ICOS (CHO cells) was also evaluated. These criteria were taken into consideration when selecting primary screen hits. were carried forward to the secondary screen. In the secondary screen, C By measuring binding to human and mouse ICOS expressed on HO cells, I checked the site.

[0391] From the large number of antibodies screened, surface plasmon resonance and flow cytometry were used. Identified a small panel of antibodies that bind to human / cynomolgus monkey and mouse ICOS as determined by These antibodies include STIM001, STIM002, and their mutant form STIM00. 2-B, STIM003, STIM004, and STIM005. Mouse I Four additional antibodies showed low cross-reactivity with ICOS but agonism of the human ICOS receptor. The antibodies STIM006, STIM007, STIM008, and STIM009 were also selected. The data presented here are based on ICOS-positive CD4+ cell lines and primary T cell assays. demonstrated the ability of anti-ICOS antibodies to act as agonists of the ICOS receptor in ADCs C assay and the ability to promote anti-tumor immune responses in vivo .

[0392] Example 1: Generation of ICOS knockout mice ICOS knockout by homologous recombination in Kymouse™ HK ES cells The Kymouse™ strain was generated using a plasmid encoding puromycin selection. The 3.5 kb targeting vector was targeted to ES cells. Successful targeting resulted in mouse IC A small region (72 bp) of the OS locus was replaced with a puromycin cassette, The signal peptide / start codon was disrupted. Positive ES clones were expanded and transfected into mouse embryonic discs. The resulting chimeras are then propagated to express humanized heavy chain and kappa immunoglobulin genes. Animals homozygous for both the locus and a modified functional null ICOS locus was finally generated.

[0393] Example 2: Preparation of antigens and cell lines Stably transfected MEFs and CHO cells expressing human or mouse ICOS Generation of -S cells The full-length DNA sequences encoding human and mouse ICOS were ordered as synthetic DNA sequences. Codon-optimized for mammalian expression, 3' and 5' under the control of a CMV promoter Cloned into an expression vector flanked by piggyBac-specific terminal repeat sequences and transfected into cells The expression vectors facilitate stable integration into the genome (see

[40] ). A puromycin selection cassette was included to facilitate generation of defined cell lines. For the generation of ICOS-expressing cell lines and mouse ICOS-expressing cell lines, the manufacturer's instructions were followed. FreeStyle Max transfection reagent (Invitrogen) was used. Human or mouse ICOS expression plasmids were transfected into mouse embryonic fibroblasts (MEFs) using the The cell line and CHO-S cells were transfected with a plasmid encoding the piggyBac transposase. The 129S5 mice were mated with C57BL6 female mice and co-transfected with the 129S5 mice. MEF cells were generated from the transfected embryos. 24 hours after transfection, the medium was filled with purified Supplement with bromocriptine and grow for at least 2 weeks to select for stable cell lines. The cell culture medium was changed every 4 days. Human or IgG was identified by flow cytometry using a purified antibody (eBioscience). Expression of mouse ICOS protein was evaluated. Complete MEF medium was supplemented with 10% v / v fetal bovine serum. The medium consisted of Dulbecco's modified Eagle's medium (Gibco) supplemented with fetal calf serum (Gibco). Complete CHO-S medium was prepared using CD-CHO supplemented with 8 mM Glutamax (Gibco). CHO-S cells were grown on the pTT5 line available from the National Research Council of Canada. Included is the CHO-3E7 cell line, although other CHO cell lines may be used.

[0394] Preparation of MEF cells for mouse immunization The cell culture medium was removed, and the cells were washed once with 1x PBS. The cells were then trypsinized for 5 min. The cells were treated to detach from the tissue culture surface. The cells were harvested and resuspended in 10% v / v fetal bovine serum (F Trypsin was neutralized by the addition of complete MEF medium containing CS. The cells were then cultured for 1 hour. The cells were centrifuged at 300 g for 10 minutes and washed with 25 ml of 1x PBS. The cells were resuspended in PBS at the appropriate concentration.

[0395] Cloning and Expression of Recombinant Proteins Using standard molecular biology techniques, human ICOS (NCBI ID: NP_036224 .1), mouse ICOS (NCBI ID: NP_059508.2), and cynomolgus monkey ( A synthetic DNA encoding the extracellular domain of the nucleotide sequence (GenBank ID: EHH55098.1) A was transformed into pREP4 (Invitrogen) expression plasmid or pTT5 (National Research Institute of Canada) The constructs were also cloned into either a genomic DNA (mechanism) or expression plasmid. To aid in this, either human Fc, mouse Fc, or FLAG His peptide motifs are used. These were added to the DNA construct by overlap extension. All constructs were sequenced before expression to ensure their correct sequence composition. .

[0396] Example 3: Immunization ICOS knockout HK Kymice™ according to the regimen shown in Table E3 (See Example 1), Kymouse™ wild-type HK strain, and Kymouse ( The Kymouse™ wild-type HL strain was immunized with the wild-type HK and HL strains. The HK strain expresses the immunoglobulin heavy chain locus and the light chain codon. The HL strain has a humanized IgE locus and a humanized IgE light chain locus. The rooster has been humanized. [Table 1]

[0397] RIMMS is a modified subcutaneous immunization procedure (rapid immunization at multiple sites); Kilp (Modified after Atrick et al.

[41] ). Immunization regimen KM103 and KM111 were prime-rest-boost administered intraperitoneally (ip). All immunizations were performed using the Sigma Adjuvant System. The interval was usually 2-3 weeks. The final boost was given intravenously in the absence of adjuvant. did.

[0398] Serial or terminal blood samples are examined for the presence of specific antibodies by flow cytometry. Serum from the sample is analyzed and titer data (if available) is used to determine the matrix used for B cell sorting. I selected Mouse.

[0399] Example 4: Comparison of serum titers between ICOS KO and wild-type mice Using flow cytometry, immunized ICOS KO and immunized wild-type mice were analyzed. The serum titers of the ICOS-KO mice were determined. Immunization with IgG inhibited Ig binding to both human and mouse ICOS expressed on CHO cells. Conversely, wild-type Kymouse (M. typhimurium) induced serum immunoglobulin responses against the IgG4-dependent IL-1 receptor (IL-1) (Fig. 1a). In mice (which express mouse ICOS), immunization with the same human ICOS antigen inhibits the expression of human ICOS. The serum showed significantly reduced binding to mouse ICOS compared to the binding of the same serum to mouse ICOS. The supernatant was produced (Fig. 1b).

[0400] method Suspension in FACS buffer (PBS + 1% w / v BSA + 0.1% w / v sodium azide) CHO-S cells expressing human ICOS or mouse ICOS (see Example 2) were suspended in water. (see below) or untransfected CHO-S cells (referred to as wild-type (WT) 10 per well 5 Cell density in 96-well, V-bottom plates (Greiner) The samples were diluted in FAC buffer to prepare titrations of mouse serum. 50 μL / well of this titration was added to the cell plates. Activity levels resulting from immunization were To determine changes in the IL-1 receptor, serum from each animal before immunization was diluted 1:1 in FACS buffer. 00 and added 50 μL / well to the cells. Incubate the cells at 4°C for 1 hour. The cells were washed twice with 150 μL of PBS and centrifuged after each washing step to remove the supernatant. The supernatant was aspirated (centrifuged at 300 × g for 3 minutes). Anti-mouse IgG (Jackson ImmunoResearch) was diluted in FACS buffer In some cases, AF647 goat cells were diluted 1 / 500 in PBS and 50 μL was added to the cells. Anti-mouse IgG (Jackson ImmunoResearch) was used. Incubate for 1 hour at 37°C in the dark, then wash twice with 150 μL of PBS as above. To fix the cells, 100 μL of 2% v / v paraformaldehyde was added. The cells were then incubated at 4°C for 30 minutes. The cells were then centrifuged at 300 x g. The cells were pelleted and resuspended in 50 μL of FACS buffer. Fluorescent signal intensity (several times) was measured by flow cytometry using the ACS Array instrument. The average value was measured.

[0401] Example 5: Sorting of antigen-specific B cells by FACS Using techniques substantially similar to those described in Example 1 of WO2015 / 040401 Then, B cells expressing anti-ICOS antibodies were collected from the immunized mice. Spleen cells and / or lymph node cells isolated from the imm...

Claims

1. (i) a set of heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3 A VH domain comprising R3, HCDR1 is STIM003 HCDR1 having the amino acid sequence of SEQ ID NO: 405; HCDR2 is STIM003 HCDR2 having the amino acid sequence of SEQ ID NO: 406; a VH domain, wherein HCDR3 is STIM003 HCDR3 having the amino acid sequence of SEQ ID NO: 407; A set of light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3 A VL domain comprising: LCDR1 is STIM003 LCDR1 having the amino acid sequence of SEQ ID NO: 412; LCDR2 is STIM003 LCDR2 having the amino acid sequence of SEQ ID NO: 413; a VL domain, wherein the LCDR3 is STIM003 LCDR3 having the amino acid sequence of SEQ ID NO:414; and Human IgG1 constant region Culturing a host cell containing a nucleic acid encoding (ii) expression of the antibody; Including, A method for producing an isolated antibody that binds to the extracellular domain of human and / or mouse ICOS.

2. the amino acid sequence of the VH domain is SEQ ID NO: 408 and the amino acid sequence of the VL domain is SEQ ID NO: 415; The method of claim 1.

3. moreover, (iii) isolating or purifying the antibody; Including, 3. The method according to claim 1 or 2.

4. The method of any one of claims 1 to 3, wherein the isolated antibody produced by said method comprises an Fc effector-positive constant region.

5. The method of any one of claims 1 to 4, wherein the isolated antibody produced by said method is a multispecific antibody.

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