ISOLATED ANTIBODY, COMPOSITION, METHOD FOR PRODUCING AN ANTIBODY AND METHOD FOR SELECTING AN ANTIBODY THAT BINDS TO ICOS

Abstract

This refers to antibodies that bind to icos (inducible co-stimulatory molecules of T cells). Therapeutic use of anti-icos antibodies to modulate the ratio between regulatory T cells and effector T cells in order to stimulate the immune system of patients, including use in the treatment of cancers. Methods for producing anti-icos antibodies, including cross-species reacting antibodies, using transgenic knockout mice.

Description

ISOLATED ANTIBODY, COMPOSITION, METHOD FOR PRODUCING AN ANTIBODY AND METHOD FOR SELECTING AN ANTIBODY THAT BINDS TO ICOS FIELD OF INVENTION

[001] The present invention relates to compositions for stimulating the immune response of mammals, especially the T cell response. The invention also relates to the medical use of such compositions in immuno-oncology, including antitumor therapy by promoting the antitumor T cell response in a patient, as well as to the use of the compositions in other diseases and conditions where there is therapeutic benefit by modulating the balance between effector T cells and regulatory T cells in favor of effector T cell activity, for example, by stimulating effector T cells and / or by depleting regulatory T cells. BACKGROUND

[002] ICOS (Inducible T-cell co-stimulator) is a member of the CD28 gene family involved in the regulation of immune responses, particularly humoral immune responses, first identified in 1999 (1). It is a 55 kDa transmembrane protein that exists as a disulfide-linked homodimer with two differentially glycosylated subunits. ICOS is expressed exclusively on T lymphocytes and is found in several T cell subsets. It is present at low levels in naive T lymphocytes, but its expression is rapidly induced by immune activation, being upregulated in response to pro-inflammatory stimuli, such as TCR involvement. Petition 870260039875, dated 04 / 29 / 2026, page 8 / 86 2 / 456 and co-stimulation with CD28 [2, 3]. ICOS plays a role in the late phase of T cell activation, in the formation of memory T cells and, mainly, in the regulation of humoral responses through T cell-dependent B cell responses [4, 5]. Intracellularly, ICOS binds to PI3K and activates phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B (PKB) kinases. ICOS activation prevents cell death and increases cellular metabolism. In the absence of ICOS (ICOS knockout) or in the presence of anti-ICOS neutralizing antibodies, there would be a suppression of pro-inflammatory responses.

[003] ICOS binds to the ICOS ligand (ICOSL) expressed on B cells and antigen-presenting cells (APCs) [6, 7]. As a co-stimulatory molecule, it serves to regulate TCR-mediated immune responses and antibody responses to antigen. ICOS expression on regulatory T cells may be important, as it has been suggested that this cell type exerts a negative function in the immunosurveillance of cancer cells – there is growing evidence of this in ovarian cancer [8]. It is important to note that ICOS expression has been reported to be higher in intratumoral regulatory T cells (TRegs) compared to CD4+ and CD8+ effector cells present in the tumor microenvironment. Depletion of TRegs using antibodies with Fc-mediated cell effector function has demonstrated strong antitumor efficacy in a preclinical model [9].Evidence suggests ICOS has an antitumor effect in both animal models, as well as in patients treated with immune checkpoint inhibitors. In mice deficient in ICOS or ICOSL, the antitumor effect of the therapy was observed. Petition 870230019896, dated 08 / 03 / 2023, page 22 / 576 3 / 456 anti-CTLA4 is decreased

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

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

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

[13] . It has also been observed that in cancer patients treated with anti-CTLA-4 therapy the mass of tumor-specific proIFNy T cells that produce cells is ICOS-positive while the prolonged elevation of ICOS-positive CD4 T cells correlates with survival [12, 13, 14].

[004] Document no. WO2016 / 120789 described anti-ICOS antibodies and proposed their use to activate T cells and to treat cancer, infectious diseases and / or sepsis. Several murine anti-ICOS antibodies were generated, a subset of which was described as being agonists of the human ICOS receptor. The antibody “422.2” was selected as the main anti-ICOS antibody and was humanized to produce a human “IgG4PE” antibody called “H2L5”. H2L5 was reported to have an affinity of 1.34 nM for human ICOS and 0.95 nM for cynomolgus ICOS, induces cytokine production in T cells and positively regulates T cell activation markers in combination with CD3 stimulation. However, mice carrying implanted human melanoma cells have been reported to show only a minimal delay in tumor growth or an increase in survival when treated with H2L5 hIgG4PE, compared to the control group.The antibody also failed to produce inhibition. Petition 870230019896, dated 08 / 03 / 2023, page 23 / 576 4 / 456 showed a significant increase in tumor growth in combination experiments with ipilimumab (anti-CTLA-4) or pembrolizumab (anti-PD-1), compared to ipilimumab or pembrolizumab monotherapy. Finally, in mice bearing implanted colon cancer cells (CT26), low doses of a cross-reactive mouse H2L5 surrogate in combination with a mouse ipilimumab or pembrolizumab surrogate only moderately improved overall survival compared to anti-CTL4 and anti-PD1 therapy alone. A similar lack of significant therapeutic benefit appeared in mice bearing implanted EMT6 cells.

[005] Document no. WO2016 / 154177 described other examples of anti-ICOS antibodies. These antibodies were reported to be agonists of CD4+ T cells, including effector CD8+ T cells (EffT cells), and deplete regulatory T cells (RegT cells). The selective effects of the antibodies on EffT cells versus RegT cells were described, whereby the antibodies could preferentially deplete RegT cells, although they have a minimal effect on EffT cells that express a lower level of ICOS. Anti-ICOS antibodies have been proposed for use in cancer treatment, and combination therapy with anti-PD-1 or anti-PD-L1 antibodies has been described. SUMMARY OF THE INVENTION

[006] An antibody to ICOS that acts to increase the activity of effector T cells represents a therapeutic approach in immuno-oncology and other medical contexts where a CD8+ T cell response is beneficial, including various diseases and conditions and in vaccination regimens. In many diseases and conditions involving a component Petition 870230019896, dated 08 / 03 / 2023, p. 24 / 576 5 / 456 In the immune system, there is a balance between effector T cells (TEff) that exert the CD8+ T cell immune response and regulatory T cells (TReg) that suppress this immune response by downregulating TEffs. The present invention relates to antibodies that modulate this TEff / TReg balance in favor of effector T cell activity. Antibodies that trigger the depletion of highly ICOS-positive regulatory T cells alleviate TEff suppression and therefore have the ultimate effect of promoting the effector T cell response. An additional or complementary mechanism for an anti-ICOS antibody is through agonistic activity at the ICOS receptor level in order to stimulate the effector T cell response.

[007] The relative expression of ICOS in effector T cells (TEff) compared to regulatory T cells (TReg) and the relative activities of these cell populations will influence the overall effect of an anti-ICOS antibody in vivo. One predicted mode of action combines agonism of effector T cells with depletion of ICOS-positive regulatory T cells. Differential and even opposing effects on these two different T cell populations can be obtained due to their different levels of ICOS expression. Dual engineering of the variable and constant regions, respectively, of an anti-ICOS antibody can provide a molecule that exerts a positive net effect on the effector T cell response by affecting the CD8 / TReg ratio. An antigen-binding domain of an agonist antibody that activates the ICOS receptor can be combined with a constant antibody region (Fc) that promotes downregulation and / or clearance of highly expressive cells to which the antibody is directed. Petition 870230019896, dated 08 / 03 / 2023, page 25 / 576 6 / 456 is switched on. A constant positive effector region can be used to recruit effector cellular functions against target cells (TRegs), for example, to promote antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP). In this way, the antibody can act both to promote the activation of effector T cells and to downregulate immunosuppressive Regulatory T cells. Since ICOS is expressed more highly on TRegs than on TEffs, a therapeutic balance can be achieved whereby Teff function is promoted while TRegs are depleted, resulting in a net increase in the T cell immune response (e.g., antitumor response or other beneficial therapeutic T cell response).

[008] Several preclinical and clinical studies have shown a strong positive correlation between a high effector T cell to T-reg ratio in the tumor microenvironment and overall survival. In ovarian cancer patients, the CD8:T-reg ratio has been reported as an indicator of good clinical outcome

[15] . A similar observation was made in patients with metastatic melanoma after receiving ipilimumab

[16] . In preclinical studies, it has also been demonstrated that a high effector cell:T-reg ratio in the TME is associated with an antitumor response

[43] .

[009] The present invention provides antibodies that bind to human ICOS. The antibodies target the extracellular domain of ICOS and thereby bind to T cells expressing ICOS. Examples are provided of antibodies that have been designed to have an agonist effect on ICOS, thereby enhancing the function of effector T cells, as indicated by Petition 870230019896, dated 08 / 03 / 2023, page 26 / 576 7 / 456 a capacity to increase the expression and secretion of IFNγ. As noted, anti-ICOS antibodies can also be modified to deplete the cells to which they bind, which should have the effect of downregulating regulatory T cells, increasing the suppressive effect of these cells on the effector T cell response and thus generally promoting the effector T cell response. Regardless of their mechanism of action, it is empirically demonstrated that anti-ICOS antibodies according to the present invention stimulate the T cell response and have antitumor effects in vivo, as shown in the Examples.Through the selection of appropriate antibody formats, such as those that include constant regions with a desired level of Fc effector function or absence of such effector function when appropriate, anti-ICO antibodies can be tailored for use in a variety of medical contexts including the treatment of diseases and conditions where a beneficial effector T cell response and / or where suppression of regulatory T cells is desired.

[010] Exemplary antibodies include STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009, whose sequences are described in this document.

[011] An antibody, according to the invention, may be one that competes for binding to human ICOS with an antibody (e.g., human IgG1, or an scFv) comprising the heavy and light chain complementarity-determining regions (CDRs) of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, optionally an antibody comprising the VH and VL domains of STIM001, Petition 870230019896, dated 08 / 03 / 2023, page 27 / 576 8 / 456 STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.

[012] An antibody according to the present invention may comprise one or more CDRs of any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 (for example, all 6 CDRs of any of these antibodies, or a set of HCDRs and / or LCDRs) or variants thereof as described herein.

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

[014] The antibody may comprise an antibody VL domain comprising CDRs HCDR1, HCDR2 and HCDR3 and an antibody VL domain comprising CDRs LCDR1, LCDR2 and LCDR3, wherein LCDR3 is an LCDR3 of an antibody selected from STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or comprises such LCDR3 with 1, 2, 3, 4 or 5 amino acid changes. LCDR2 may be the LCDR2 of the selected antibody or may comprise such LCDR2 with 1, 2, 3, 4 or 5 changes Petition 870230019896, dated 08 / 03 / 2023, page 28 / 576 9 / 456 of amino acids. LCDR1 may be the LCDR1 of the selected antibody or may comprise that LCDR1 with 1, 2, 3, 4, or 5 amino acid changes.

[015] An antibody may comprise:

[016] an antibody VH domain comprising the complementarity-determining regions HCDR1, HCDR2 and HCDR3, and

[017] an antibody VL domain comprising the complementarity-determining regions LCDR1, LCDR2 and LCDR3,

[018] wherein the heavy chain complementarity-determining regions are those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprise the heavy chain complementarity-determining regions of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 with 1, 2, 3, 4 or 5 amino acid changes; and / or

[019] wherein the light chain complementarity determinant regions are those of antibody STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprise the light chain complementarity determinant regions of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 with 1, 2, 3, 4 or 5 amino acid changes.

[020] An antibody may comprise a VH domain comprising a set of heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, wherein Petition 870230019896, dated 08 / 03 / 2023, page 29 / 576 10 / 456

[021]

[022]

[023]

[024] HCDR1 is the HCDR1 of STIM003, HCDR2 is the HCDR2 of STIM003, HCDR3 is the HCDR3 of STIM003, or comprises such a set of HCDRs with 1, 2, 3, 4, 5 or 6 amino acid changes.

[025] An antibody may comprise a VL domain comprising a set of complementarity-determining regions in which light chain (LCDRs) LCDR1, LCDR2 and LCDR3,

[026] LCDR1 is the LCDR1 of STIM003,

[027] LCDR2 is the LCDR2 of STIM003,

[028] LCDR3 is the LCDR3 of STIM003,

[029] or comprising this set of LCDRs with 1, 2, 3 or 4 amino acid changes.

[030] Amino acid changes (e.g., substitutions) can be at any residual position in CDRs. Examples of amino acid alterations are those illustrated in Figure 35, Figure 36, and Figure 37, which show alignments of variant sequences of anti-ICOS antibodies. Thus, an amino acid alteration in a CDR of STIM003 could be a substitution of the residue present at the corresponding position in antibody CL-74570 or antibody CL-71642, as indicated in Figure 36.

[031] Examples of amino acid changes in the CDRs of STIM003 are substitutions at the following residual positions, defined according to IMGT:

[032] In HCDR1, the replacement at position IMGT 28, optionally a conservative substitution, for example, V28F.

[033] In HCDR2, replacement at position IMGT 59, Petition 870230019896, dated 08 / 03 / 2023, page 30 / 576 11 / 456 and / or 64. Optionally, the replacement in position 59 is N59I, the replacement in position 63 is G63D and / or the replacement in position 64 is D64N and / or D64S.

[034] In HCDR3, replacement at position 106, 108, 109 and / or 112 of the IMGT. Optionally, the replacement in position 106 is R106A, the replacement in position 108 is F108Y, the replacement in position 109 is Y109F and / or the replacement in position 112 is H112N.

[035] In LCDR1, replacement in position 36, for example, R36S.

[036] In LCDR3, replacement in position 105, 108 and / or 109. Optionally, the replacement in position 105 is H105Q, the replacement in position 108 is D108G and / or the replacement in position 109 is M109N or M109S.

[037] The antibodies of the invention may comprise VH and / or VL domain framework regions corresponding to human germline gene segment sequences. For example, it may include one or more framework regions of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009. The framework region or regions can be FR1, FR2, FR3, and / or FR4.

[038] As described in Example 12, the Table Table E12-1 shows the human germline gene segments V, D, and J that generated the VH domains of these antibodies through recombination, and Table E12-2 shows the germline gene segments V and J that generated the VL domains of these antibodies through recombination. The VH and VL antibody domains of the present invention may be based on these V(D)J segments. Petition 870230019896, dated 08 / 03 / 2023, page 31 / 576 12 / 456

[039] An antibody of the invention may comprise an antibody VH domain that

[040] (i) is derived from the recombination of a human heavy chain V gene segment, a human heavy chain D gene segment and a human heavy chain J gene segment, wherein

[041] segment V is IGHV1-18 (e.g., V118*01), IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V3-11*01) or IGVH2-5 (e.g., V2-5*10);

[042] gene segment D is IGHD6-19 (e.g., IGHD6-19*01), IGHD3-10 (e.g., IGHD3-10*01) or IGHD3-9 (e.g., IGHD3-9*01); and / or

[043] the J gene segment is IGHJ6 (e.g., IGHJ6*02), IGHJ4 (e.g., IGHJ4*02) or IGHJ3 (e.g., IGHJ3*02) or

[044] (ii) comprises the FR1, FR2, FR3 and FR4 framework regions, where

[045] FR1 aligns with the human germline gene segment V IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V311*01), or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4, or 5 amino acid changes,

[046] FR2 aligns with the human germline gene segment V IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V311*01), or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4, or 5 amino acid changes,

[047] FR3 aligns with the human germline gene segment V IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V3 Petition 870230019896, dated 08 / 03 / 2023, page 32 / 576 13 / 456 11*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4, or 5 amino acid changes, and / or

[048] FR4 aligns with the germline gene segment J IGJH6 (e.g., JH6*02), IGJH4 (e.g., JH4*02), or IGJH3 (e.g., JH3*02), optionally with 1, 2, 3, 4, or 5 amino acid changes.

[049] FR1, FR2 and FR3 of the VH domain normally align with the same germline V gene segment. Thus, for example, the antibody may comprise a VH domain derived from recombination of the human heavy chain V gene segment IGHV3-20 (e.g., VH3-20*d01), a human heavy chain D gene segment and a human heavy chain J gene segment IGJH4 (e.g., JH4*02). An antibody may comprise VH domain framework regions FR1, FR2, FR3, and FR4, wherein FR1, FR2, and FR3 each align to the human germline gene segment V IGHV320 (e.g., IGVH3-20*d01) with up to 1, 2, 3, 4, or 5 amino acid changes, and an FR4 that aligns to the human germline gene segment J IGHJ4 (e.g., IGHJ4*02) with up to 1, 2, 3, 4, or 5 amino acid changes. The alignment may be exact, but in some cases one or more residues may undergo mutation from the germline, therefore, there may be amino acid substitutions present or, in rarer cases, deletions or insertions.

[050] An antibody of the invention may comprise a VL antibody domain that

[051] (i) is derived from the recombination of the human light chain V gene segment and a human light chain J gene segment, wherein

[052] segment V is IGKV2-28 (for example, Petition 870230019896, dated 08 / 03 / 2023, page 33 / 576 14 / 456 IGKV2-28*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01) or IGKV3-11 (e.g., IGKV311*01) and / or

[053] 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

[054] (ii) comprises the FR1, FR2, FR3 and FR4 framework regions, in which

[055] FR1 aligns with human germline gene segment V IGKV2-28 (e.g., IGKV228*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01), or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4, or 5 amino acid changes,

[056] FR2 aligns with human germline gene segment V IGKV2-28 (e.g., IGKV228*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01), or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4, or 5 amino acid changes,

[057] FR3 aligns with human germline gene segment V IGKV2-28 (e.g., IGKV228*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01), or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4, or 5 amino acid changes, and / or

[058] FR4 aligns with the germline gene segment J IGKJ4 (e.g., IGKJ4*01), IGKJ2 (e.g., IGKJ2*04), IGKJ3 (e.g., IGKJ3*01), or IGKJ1 (e.g., IGKJ1*01), optionally with 1, 2, 3, 4, or 5 amino acid changes.

[059] FR1, FR2 and FR3 of the VL domain normally Petition 870230019896, dated 08 / 03 / 2023, page 34 / 576 15 / 456 align with the same germline V gene segment. Thus, for example, the antibody may comprise a VL domain derived from the recombination of the human light chain V gene segment IGKV3-20 (e.g., IGKV3-20*01) and the human light chain J gene segment IGKJ3 (e.g., IGKJ3*01). An antibody may comprise the VL domain framework regions FR1, FR2, FR3, and FR4, wherein FR1, FR2, and FR3 each align to the human germline gene segment V IGKV3-20 (e.g., IGKV3-20*01) with up to 1, 2, 3, 4, or 5 amino acid changes, and FR4 aligns to the human germline gene segment J IGKJ3 (e.g., IGKJ3*01) with up to 1, 2, 3, 4, or 5 amino acid changes. The alignment may be exact, but in some cases one or more residues may be mutated from the germline, therefore amino acid substitutions may be present or, in rarer cases, deletions or insertions.

[060] An antibody according to the invention may comprise an antibody VH domain that is the VH domain of STIM001, STIM002, STIM002-B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 or that has an amino acid sequence at least 90% identical to the antibody VH domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009. The amino acid sequence identity may be at least 95%.

[061] The antibody may comprise an antibody VL domain that is the VL domain of STIM001, STIM002, STIM002B, STIM003, STIM004 or STIM005, STIM006, STIM007, STIM008 or STIM009 or that has an amino acid sequence of at least 90% Petition 870230019896, dated 08 / 03 / 2023, page 35 / 576 16 / 456 identical to the antibody VL domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009. The amino acid sequence identity must be at least 95%.

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

[063] For example, the antibody may comprise the VH domain of antibody STIM001 and the VL domain of STIM001. In another example, the antibody may comprise the VH domain of antibody STIM002 and the VL domain of STIM002. In yet another example, the antibody may comprise the VH domain of antibody STIM003 and the VL domain of STIM003.

[064] Antibodies may include constant regions, optionally constant regions of human heavy and / or light chains. An example isotype is IgG, for example, human IgG1.

[065] Other aspects of the invention include molecules comprising nucleic acid molecules encoding antibody sequences described herein, host cells containing such nucleic acids, and methods for producing the antibodies by culturing the host cells and expressing, and optionally isolating or Petition 870230019896, dated 08 / 03 / 2023, page 36 / 576 17 / 456 by purifying the antibodies. The expressed antibody is obtained in this way. The VH and VL domains of the antibodies described herein can be similarly produced and are aspects of the present invention. Pharmaceutical compositions comprising the antibodies are also provided.

[066] Other aspects of the invention relate to non-human ICOS knockout animals and their use to generate antibodies to human ICOS. In an ICOS knockout animal, ICOS is not expressed, for example, because the gene encoding ICOS has been inactivated or deleted from the animal's genome. These animals are useful for generating cross-reacting antibodies from species that recognize ICOS and human ICOS from non-human species.The normal process of immune tolerance means that lymphocytes that recognize “self” antigens are deleted or inactivated to prevent autoimmune reactions in the body, whereas the absence of the endogenous ICOS antigen in the non-human knockout animal means that the animal's immune system should not tolerate this antigen and therefore may mount an immune response against ICOS when injected as a recombinant protein or using cell lines or vesicles that express ICOS. The immune repertoire of the knockout animal must contain lymphocytes capable of recognizing the ICOS protein of that animal species. A non-human test animal (e.g., a mouse) immunized with human ICOS can thus generate antibodies that bind to both human ICOS and test animal ICOS (e.g., mouse ICOS).

[067] This has at least two advantages. First, a cross-reacting antibody of the species can be Petition 870230019896, dated 08 / 03 / 2023, page 37 / 576 18 / 456 is used for preclinical testing in non-human test animals before being taken on to development in human clinical trials. Secondly, the immune system of a knockout animal can recognize a greater number of possible epitopes in a human ICOS molecule compared to those recognized by an animal expressing ICOS, so the knockout animal's immune repertoire may contain a greater diversity of antibodies. Since there is similarity between the sequences of homologous ICOS molecules from different species, the immune system of a non-human animal may normally tolerate regions of the human ICOS protein that correspond to those of the non-human animal ICOS, but this tolerance does not occur in a knockout animal.

[068] The ability to use an ICOS knockout animal and its advantage in generating cross-reactive antibodies are shown in the Examples. It is particularly surprising that an ICOS knockout animal can be successfully immunized to produce an antibody response, given that ICOS itself is involved in the biology of the immune system, such as the formation and maintenance of germinal centers, and contributes to the generation of an immune response through its function in follicular helper T cells that are ICOS-positive [Error! Bookmark not defined.]. In this sense, it can be predicted that an ICOS knockout animal generates an insufficient antibody response, at a minimum. Surprisingly, intense titers were obtained in ICOS knockout mice, and highly functional antibodies were isolated from the antibody repertoire, including desirable cross-reactive antibodies. Petition 870230019896, dated 08 / 03 / 2023, p. 38 / 576 19 / 456

[069] Exemplary embodiments of the invention are presented in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

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

[071] Figure 1: Determination of serum titers of wild-type KO mice with ICOS and Kymouse against both human ICOS and mouse ICOS expressed on CHO cells by flow cytometry. The data illustrate the ability of immunoglobulin in sera from (a) wild-type KO mice with ICOS (KO) or (b) wild-type KO mice without ICOS (HK or HL), each immunized with MEF cells expressing human ICOS and human ICOS protein, 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.

[072] Figure 2: Ligand neutralization HTRF Human ICOS receptor neutralization profiles of STIM001 to STIM009 anti-ICOS mAbs in human IgG1 format compared to C398.4A and respective isotype controls. Representative data from four experiments.

[073] Figure 3: Ligand neutralization HTRF Mouse ICOS receptor neutralization profiles of STIM001 to STIM009 anti-ICOS mAbs in human IgG1 format compared to C398.4A and respective isotype controls. Representative data from three experiments. Petition 870230019896, dated 08 / 03 / 2023, page 39 / 576 20 / 456

[074] Figure 4: Direct neutralization HTRF of human ICOS ligand with human ICOS receptor. Neutralization profiles of STIM001 to STIM009 anti-ICOS mAbs in human IgG4.PE format compared to C398.4A and respective isotype controls. Representative data from four experiments.

[075] Figure 5: Neutralization of the mouse HTRF ICOS ligand with the mouse ICOS receptor. Neutralization profiles of STIM001 to STIM009 anti-ICOS mAbs in human IgG4.PE format compared to C398.4A and respective isotype controls. Representative data from four experiments.

[076] Figure 6a: Concentration-dependent study of ADCC mediated by STIM001 in MJ cells using newly isolated NK cells as effector cells. Effector cells and target cells (effector:target ratio of 5:1) were incubated together with the antibody for 2 hours. BATDA release from lysed target cells was measured as described in the manufacturer's kit instructions. HC is the hybrid isotype control.

[077] Figure 6b, c, d: Concentration-dependent study of ADCC mediated by STIM001 and STIM003 in MJ cells with newly isolated NK cells as effector cells. Effector cells and target cells (effector:target ratio of 5:1) were incubated together with the antibody for 2 hours. BATDA release from lysed target cells was measured as described in the manufacturer's kit instructions. HC is the hybrid isotype control.

[078] Figure 6e, f, g: Concentration-dependent study of ADCC mediated by STIM001 (hIgG1) and STIM003 (hIgG1) in CCRF-CEM cells transfected with ICOS with newly isolated NK cells as effector cells. As Petition 870230019896, dated 08 / 03 / 2023, page 40 / 576 21 / 456 effector and target cells (effector:target ratio 5:1) were incubated together with the antibody for 4 hours. BATDA release from lysed target cells was measured as described in the manufacturer's kit instructions. HC is the hybrid isotype control.

[079] Figure 7, Figure 8, Figure 9: The anti-ICOS antibody inhibits CT26 tumor growth and improves survival when administered as monotherapy or in combination with anti-PDL1. The mIgG2a STIM001 is more potent than the mIgG1 format. The number of animals cured or with stable disease is indicated in each graph.

[080] Figure 10: 2x2 combinations from the in vivo efficacy study of CT26. Each treatment group is represented by a “radar chart” showing the tumor size of each animal (n=10 per group). When combined with anti-PDL1 antibodies, STIM001 slows tumor growth and improves the survival of treated animals. The efficacy observed in the presence of mIgG2a STIM001 is superior to that of mIgG1 STIM001. Finally, mIgG2a STIM001 in combination with mIgG2a anti-PDL1 was the most potent combination to trigger the antitumor response, resulting in 60% of animals being cured of the disease. For each group, the number of animals cured of their disease is indicated in the upper right corner of the respective graphs. Dosing was performed on days 6, 8, 10, 13, 15, and 17.

[081] Figure 11: Graphs showing CT26 tumor volumes over time in animals treated with anti-ICOS or anti-PDL1 monotherapies or combination therapies. Each treatment group is represented by a radar chart showing the tumor size of each animal (n=10 per group). For each group, the number of animals with a size of Petition 870230019896, dated 08 / 03 / 2023, page 41 / 576 22 / 456 tumor below 100 mmA3 (stable / cured of their disease) is indicated in the upper right corner of the respective graphs. Dosing was performed on days 6, 8, 10, 13, 15, and 17. Dosing time is indicated by the shaded area. (a) Isotype control; (b) AbW mIgG2a Anti-PDL1; (c) mIgG1 STIM003 Anti-ICOS; (d) mIgG2a STIM003 Anti-ICOS; (e) AbW mIgG2a Anti-PDL1 + mIgG1 STIM003; (f) mIgG2a AbW Anti-PDL1 + mIgG2a STIM003. mIgG2 STIM003 significantly inhibits CT26 tumor growth when combined with mIgG2a (AbW) anti-PDL1.

[082] Figure 12: In vitro activation assay of microsphere-bound MJ cells. Stimulation profiles of microsphere-bound anti-ICOS mAbs STIM001, STIM002 and STIM003 compared to C398.4A anti-ICOS and respective isotype controls. Data represent the average of two experiments (n = 1 in the case of C398.4A isotype control spheres).

[083] Figure 13: In vitro activation assay of MJ cells - plate-bound. Stimulation profiles of plate-bound anti-ICOS mAbs STIM001, STIM002, STIM003 and STIM004 compared to C398.4A anti-ICOS and respective isotype controls. Data represent the average of two experiments.

[084] Figure 14: FACS analysis of hIgG1 STIM001 and STIM003 binding to activated T cells. (a) shows a representative experiment of the dose response of pre-labeled antibodies binding to activated T cells, while (b) shows the binding-after-dose response of bare antibodies followed by detection with a secondary labeled antibody. Tables indicate relevant EC50 (M), as determined using GraphPad Prism.

[085] Figure 15: STIM001 and STIM003 showed Petition 870230019896, dated 08 / 03 / 2023, p. 42 / 576 23 / 456 isotype-dependent effects on the T-cell compartment at the tumor site. A total of 1x10E5 CT-26 tumor cells were subcutaneously implanted into female Balb / c mice. On days 13 and 15 post-implantation, animals were dosed with antibodies or saline solution intraperitoneally (n=10 / each group). On day 16 post-implantation, spleen and tumors were harvested from tumor-bearing animals (n=8 / each group), dissociated, and labeled for FACS analysis. A, percentage of CD3 cells that are positive for CD4 cells. B, percentage of CD3 cells positive for CD8 cells. C, percentage of CD4 cells that are Foxp3+ & CD25+. D, percentage of CD4 cells in the spleen that are positive for Foxp3+ & CD25+. E, percentage of CD4 effector cells in the total number of CD4 cells. F, ratio between CD8 effector cells and T-Reg cells. G, ratio between CD4 effector cells and T-Reg cells.Statistical analysis was performed using GraphPad Prism. All antibody-treated groups were compared to the saline-treated group. P-values ​​were observed when significant (p<0.05). Values ​​denote means + SD (n=8 mice / group). For F: Values ​​denote mean + SEM.

[086] Figure 16: Exemplary data from the concentration-dependent study of the agonist effect of STIM001 (hIgG1) and STIM003 (hIgG1) on isolated human T cells co-stimulated 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 the agonist effect. STIM001 (hIgG1) and STIM003 (hIgG1) were tested in plate-bound, soluble, or cross-linked soluble (Ab-linked by Fc) formats and compared to an isotype control. Petition 870230019896, dated 08 / 03 / 2023, page 43 / 576 24 / 456 hybrid (HC hIgGl). Hamster antibody C398.4A and the isotype control of the same (hamster IgG) were included for comparison in the plate-bound assay. The top panel shows plate-bound antibody data. The bottom panel shows IgGl antibody data in soluble and cross-linked forms. The left and right panels use T cells from two independent human donors, respectively.

[087] Figure 17: Exemplary dataset for STIM00l in cell activation assay l T (see Example 9). The data indicate IFN-γ levels induced by STIM00l (hIgGl) or the hybrid isotype control (HC IgGl) of the same at a given dose for independent human donor T cells. Plate-bound antibody (Figure 17a) was used at 5 μg / ml. A soluble antibody (Figure 17b) was used at 15 μg / ml. Each point represents a donor, identified by number (D2l4 for example). Significance was assessed using the Wilcoxon statistical test: *, p<0.05 and **, p<0.01.

[088] Figure 18: Exemplary dataset for STIM003 in T-cell activation assay 1 (see Example 9). The data indicate IFN-γ levels induced by STIM003 (hIgG1) or the hybrid isotype control (HC hIgG1) at a given dose for T cells from 8 independent healthy donors. A soluble antibody (Figure 18a) was used at 15 μg / ml. The plate-bound antibody (Figure 18b) was used at 5 μg / ml. Each point represents a donor, identified by number (D214 for example). Significance was assessed using the Wilcoxon statistical test: *, p<0.05 and **, p<0.01.

[089] Figure 19: Illustrative data of Petition 870230019896, dated 08 / 03 / 2023, page 44 / 576 25 / 456 T cell activation assay 2 (see Example 9c). Study of the agonist effect of STIM001 (hIgG1) and STIM003 (hIgG1) on isolated human T cells stimulated with anti-CD3 / anti-CD28 dynabeads for 3 days, then placed in resting medium for 3 days and finally stimulated again with Ab STIM001, STIM003 or C398.4A + / - Ab CD3 bound to the plate. Data comparing the levels of IFN-γ (A, B), TNF-α (C, D) and IL-2 (E, F) induced by STIM001, STIM003 vs. the IgG1 of the hybrid control of the same (A, C, E) or C398.4A vs. hamster IgG control of the same (B, D, F) at a given dose and in combination with Ab CD3 (TCR coupling). Each point represents an independent donor identifiable by its number (D190 for example). The statistical significance between the Abs and the isotype control was evaluated using the Wilcoxon statistical test, and the p-value was indicated.It is observed that the concentration of STIM003 was slightly different from that of HC IgG1 (5.4 vs. 5 μg / ml).

[090] Figure 20: Graph showing the percentage of immune cells (CD8 T-Efferent cells, CD4 T-Efferent cells and CD4 / FoxP3 TReg) in CT26 tumors and in the spleen of tumor-bearing animals expressing ICOS on their surface. Values ​​denote mean ±SD (n=8). P-values ​​were calculated using Dunn's multiple comparisons test. NS = not significant; *** = p<0.001; ****=p<0.0001.

[091] Figure 21: Relative expression of ICOS on the surface of immune cells - CD8 T effector, CD4 T effector and CD4 / FoxP3 TRegs - as determined by mean fluorescence intensity (MFI). Values ​​denote mean ±SD (n=8). P values ​​were calculated using the test of Petition 870230019896, dated 08 / 03 / 2023, page 45 / 576 26 / 456 Dunn's multiple comparisons. ****=p<0.0001, **=p<0.01. A difference in fluorescence intensity is observed between the spleen (high) and the tumors (high).

[092] Figure 22: Effect of STIM001 and STIM003 on the percentage of different immune cells in the microenvironment of CT26 tumors. *=p<0.05.

[093] Figure 23: Effect of STIM001 and STIM003 antibodies on the percentage of regulatory T cells (CD4+ / FoxP3+ cells) in the microenvironment of CT26 tumors. **=p<0.05, ****=p<0.0001. Values ​​denote mean ± SD (n=8). P-values ​​were calculated using Dunn's multiple comparisons test.

[094] Figure 24: mIgG2 STIM001 and STIM003 significantly increase the ratio between effector CD8 T cells and TReg and the ratio between effector CD4 T cells and TReg in CT26 tumors. The ratio was determined by dividing the percentage of effector cells in the tumor by the percentage of regulatory T cells in the tumor.

[095] Figure 25: Effect of antibodies on the percentage of immune cells in the spleen of animals bearing CT26 tumor.

[096] Figure 26: Effect of antibodies on the percentage of regulatory T cells (CD4+ / FoxP3+ cells) in the spleen of animals bearing CT26 tumor.

[097] Figure 27: (A) CD8:Treg effector ratio and (B) CD4:TReg ratio in the spleen of animals bearing the CT26 tumor.

[098] Figure 28: Surface labeling of STIM001, STIM003 and hybrid control of hIgG1 (HC IgG1) conjugated to AF647 on pan T cells of Cynomolgus mauridianus Petition 870230019896, dated 08 / 03 / 2023, page 46 / 576 27 / 456 activated. Data from assays using different T-cell donor sources are shown in A and B respectively. EC50 values ​​are indicated in the table.

[099] Figure 29: Kaplan-Meier curves for the CT26 Balb / C model. Shading shows the dosage window. LogRank p<0.0001.

[0100] Figure 30, Figure 31, Figure 32, Figure 33: Graphs showing A20 tumor volumes over time in mice for the study described in Example 20. Each treatment group is represented by a radar chart showing tumor size in individual animals, n=8 per group. For each group, the number of animals with no tumor signal (indicating disease cure) is shown in the lower left of the graph. Dosing was performed on days 8, 11, 15, 18, 22, 25, and 29 after tumor cell implantation, and the dosing time is indicated by the gray shaded area. Compared to the control group (Figure 30) and the antiPD-L1 treatment group (Figure 31), the mIgG2a STIM001 (Figure 32) and mIgG2a STIM003 (Figure 33) treatment groups show significant inhibition of A20 tumor growth.

[0101] Figure 34: Data from the CT2 6 in vivo efficacy study described in Example 11c using the combination of the mIgG2a anti-PD-L1 antibody with single vs. multiple doses of mIgG2a STIM003. Each treatment group is represented by a “radar chart” showing the tumor size of individual animals (n=8 per group). For each group, the number of animals cured of this disease is indicated in the lower right of the respective chart. Dosing days for each antibody are indicated by the arrows below the respective charts.

[0102] Figure 35: Amino acid sequences of Petition 870230019896, dated 08 / 03 / 2023, page 47 / 576 28 / 456 domain VH (upper) and VL (lower) of STIM002 showing residues that are different from the corresponding sequences of STIM001, STIM002B and related antibodies CL-61091, CL64536, CL-64837, CL-64841 and CL-64912 and / or the human germline. Sequence numbering is according to IMGT.

[0103] Figure 36: Amino acid sequences of the VH (top) and VL (bottom) domains of STIM003 showing residues that are different from the corresponding sequences of the related antibodies CL-71642 and CL-74570 and / or the human germline. Sequence numbering is according to IMGT. The VL domain of the CL-71642 antibody obtained from sequencing is shown in this document without the N-terminal residue. From the alignment, it can be seen that the complete VH domain sequence comprises an N-terminal glutamic acid.

[0104] Figure 37: Amino acid sequences of the VH (upper) and VL (lower) domains of STIM007 showing different residues in the corresponding sequences of STIM008 and / or in the human germline. Sequence numbering is according to IMGT.

[0105] Figure 38: Effect of mIgG2a STIM003 (anti-ICOS) and AbW (anti-PD-L1) antibodies in the J558 syngeneic model. Each treatment group is represented by a “radar graph” showing the tumor size of individual animals (n=10 or n=8 per group). Monotherapy with STIM003 demonstrated some efficacy, with 3 out of 8 animals cured of their disease. Similarly, anti-PDL1 was effective in this model, with 6 out of 8 animals cured of their disease at approximately day 37. When combined with anti-PDL1 antibodies, the mIgG2 STIM003 Petition 870230019896, dated 08 / 03 / 2023, page 48 / 576 29 / 456 completely inhibited tumor growth and improved the survival of treated animals. For each group, the number of animals cured of this disease is indicated in the lower right of the respective graph. Dosage days are indicated by dashed lines (days 11, 15, 18, 22, 25 and 29).

[0106] Figure 39: The quantification of the expression ICOS (percentage of cells and relative expression / dMFI) in different TILS cell subtypes in tumor tissue. (A) The % of immune cell subtypes that are positive for ICOS expression and (B) the dMFI of ICOS (relative ICOS expression in ICOS-positive cells) of immune cell subtypes from animals treated with saline or anti-PD-L1 or anti-PD-1 surrogate antibodies. Mice were implanted with 100 pl of 1x10⁶ viable cells / ml np day 0 (n=7 or n=8). The animals were dosed intraperitoneally with 130 pg of antibody on day 13 and day 15. Tissue samples were isolated and analyzed on day 16. CD4+ / FOXP3+ cells were included only for the TReg population (right-hand side graphs) and were excluded from effector CD4 cells (left-hand side graphs) which are all Foxp3-negative. See Example 22.

[0107] Figure 40: Data from the in vivo efficacy study A20. Each treatment group is represented by a radar chart showing the tumor size of each animal (n=10 per group). For each group, the number of animals cured of their disease is indicated on the respective chart. For the multiple dose, dosing occurred on days 8, 11, 15, 18, 22, and 25, indicated by the dashed lines. For the single dose, animals received an IP injection only on day 8. (A) Solution Petition 870230019896, dated 08 / 03 / 2023, page 49 / 576 30 / 456 saline; (B) multiple dose of mIgG2a STIM003; (C) single dose of mIgG2a STIM003. See Example 23.

[0108] Figure 41: Kaplan-Meier curves for the study reported in Example 23 with a dose of 60 pg of mIgG2a STIM003. SD = single dose, day 8. MD = multiple doses twice a week starting on day 8.

[0109] Figure 42: ICOS expression in subsets of larger T cells (T-reg [CD4+ / FoxP3+], CD4 Eff [CD4+ / FoxP3- cells] and CD8+) from animals bearing CT26 tumors (n=4 per time point) dosed with saline. Immune cell phenotyping was conducted on days 1, 2, 3, 4, and 8 post-treatment and labeled for ICOS expression in all tissues at all time points. A to D show the percentage of ICOS-positive cells at all time points in four different tissues. E to H shows the dMFI of ICOS (relative expression) at all time points in all four different tissues. See Example 24.

[0110] Figure 43: FACS analysis demonstrating T-reg depletion in the TME in response to the mIgG2a antibody STIM003. Animals bearing the CT-26 tumor were treated with a single dose (6, 60, or 200 pg) of STIM003 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 after 8 treatment. The percentage of T-reg cells (CD4+CD25+Foxp3+) in total tumor (A) and the percentage of T-reg cells in blood (B) are shown at different time points. See Example 24.

[0111] Figure 44: Increase in the CD8:T-reg ratio and in the CD4 eff:T-reg ratio in response to mIgG2a STIM003. The animals Petition 870230019896, dated 08 / 03 / 2023, p. 50 / 576 31 / 456 CT-26 tumor carriers received a single dose (6, 60, or 200 μg) of mIgG2a STIM003 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 after 8 treatment, and the ratios between T eff T-reg were calculated. (A) & (B), CD8:T-reg ratio in tumor and blood, (C) & (D) CD4-eff:T-reg ratio in tumor and blood. See Example 24.

[0112] Figure 45: STIM003 treatment correlates with increased dreganulation and Th1 cytokine production by TILs. On day 8 post-treatment, TILs were isolated and FACS analysis was performed to detect CD107a expression on CD4 and CD8 T cells (AB). In parallel, dissociated tumor cells rested for 4 hours in the presence of Brefeldin-A, the cells were labeled for T cell markers and permeabilized for intracellular labeling to detect IFN-γ and TNF-α (CH). See Example 24. DETAILED DESCRIPTION ICOS

[0113] The antibodies according to the present invention bind to the extracellular domain of human ICOS. In this way, the antibodies bind to T lymphocytes expressing ICOS. “ICOS” or “the ICOS receptor referred to herein may be human ICOS, unless the context indicates otherwise. The sequences of human, cynomolgus and mouse ICOS are shown in the attached sequence listing and are available from NCBI as NCBI ID: NP_036224.1, NCBI ID: NP_059508.2 and GenBank ID for cynomolgus: EHH55098.1. CROSS-REACTIVITY

[0114] Antibodies according to the present Petition 870230019896, dated 08 / 03 / 2023, p. 51 / 576 The 32 / 456 inventions are preferably cross-reacting and can, for example, bind to the extracellular domain of mouse ICOS as well as human ICOS. The antibodies can bind to other non-human ICOS, including primate ICOS such as cynomolgus. An anti-ICOS antibody intended for therapeutic use in humans must bind to human ICOS, since binding to ICOS from other species has no direct therapeutic relevance in the human clinical context. However, the data in the present document indicate that antibodies that bind to both human and mouse ICOS have properties that make them particularly suitable as agonist and deletion molecules. This may result from one or more particular epitopes being targeted by the cross-reacting antibodies. However, regardless of the underlying theory, cross-reacting antibodies are of high value and are excellent candidates as therapeutic molecules for preclinical and clinical studies.

[0115] As explained in the Experimental Examples, the STIM antibodies described herein were generated using Kymouse™ technology, in which the mouse had been modified to have no expression of mouse ICOS (an ICOS knockout). ICOS knockout transgenic animals and their use to generate cross-reacting antibodies are further aspects of the present invention.

[0116] One way to quantify the extent of cross-reactivity of an antibody species is as the difference in times in its affinity for an antigen or species compared to the antigen of another species, for example, the difference in times in affinity with human ICOS versus ICOS of Petition 870230019896, dated 08 / 03 / 2023, page 52 / 576 33 / 456 mouse. Affinity can be quantified as Kd, referring to the equilibrium dissociation constant of the antibody-antigen reaction, as determined by SPR with the antibody in Fab format as described elsewhere in this document. A cross-reacting anti-ICOS antibody of the species may have a difference in times in binding affinity to human and mouse ICOS that is 30 times or less, 25 times or less, 20 times or less, 15 times or less, times or less, or 5 times or less. Otherwise, the binding Kd of the extracellular domain of human ICOS may be within 30 times, 25 times, 20 times, 15 times, 10 times, or 5 times the binding Kd of the extracellular domain of mouse ICOS.Antibodies can also be considered cross-reactive if the Kd for antigen binding of both species reaches a threshold value, for example, if both the Kd for human ICOS binding and the Kd for mouse ICOS are 10 nM or less, preferably 5 nM or less, more preferably 1 nm or less. Kd can be 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. Kd can be 0.9 nM or less, 0.8 nM or less, 0.7 nm or less, 0.6 nm or less, 0.5 nm or less, 0.4 nm or less, 0.3 nm or less, 0.2 nM or less, or 0.1 nM or less.

[0117] An alternative measure of cross-reactivity for human ICOS and mouse ICOS is the ability of an antibody to neutralize the ICOS ligand that binds to the ICOS receptor, such as in an HTRF assay (see Example 8). Examples of cross-reacting antibody species are provided herein, including STIM001, STIM002, STIM002-B, STIM003, STIM005, and STIM006, each of which has been confirmed to neutralize the Petition 870230019896, dated 08 / 03 / 2023, page 53 / 576 34 / 456 binding of human B7-H2 (ICOS ligand) to human ICOS and neutralizing the binding of mouse B7-H2 to mouse ICOS in an HTRF assay. Any of these antibodies or variants thereof may be selected when a cross-reacting antibody to mouse and human ICOS is desired. A cross-reacting anti-ICOS antibody of the species may have an IC50 to inhibit the binding of human ICOS to the human ICOS receptor that is within 25-fold, 20-fold, 15-fold, 10-fold, or 5-fold the IC50 to inhibit mouse ICOS to the mouse ICOS receptor, as determined in an HTRF assay. Antibodies can also be considered cross-reactive if both the IC50 to inhibit the binding of human ICOS to the human ICOS receptor and the IC50 to inhibit the binding of mouse ICOS to the mouse ICOS receptor are 1 mM or less, preferably 0.5 mM or less, for example, 30 nM or less, 20 nM or less, 10 nM or less.The IC50 values ​​can be 5 nM or less, 4 nM or less, 3 nM or less, or 2 nM or less. In some cases, the IC50 values ​​will be at least 0.1 nM, at least 0.5 nM, or at least 1 nM. SPECIFICITY

[0118] The antibodies according to the present invention are preferably ICOS-specific. That is, the antibody binds to its epitope on the target protein, the ICOS (human ICOS and, preferably, mouse and / or cynomolgus ICOS, as noted above), but does not show significant binding to molecules that do not have this epitope, including other molecules in the CD28 gene family. Preferably, an antibody according to the present invention does not bind to human CD28. Preferably, the antibody also Petition 870230019896, dated 08 / 03 / 2023, p. 54 / 576 35 / 456 does not bind to mouse CD28 or cynomolgus.

[0119] CD28 co-stimulates cell responses T cells, when bound by the CD80 and CD86 ligands of the same antibodies, enter professional antigen-presenting cells in the context of antigen recognition via the TCR. For several in vivo uses of the antibodies described in this document, avoiding binding to CD28 is considered advantageous. Non-binding of the anti-ICOS antibody to CD28 should allow CD28 to interact with its native ligands and generate an appropriate co-stimulatory signal for T cell activation. Additionally, non-binding of the anti-ICOS antibody to CD28 avoids the risk of superagonism. Excessive CD28 stimulation can induce proliferation in resting T cells without normally requiring recognition of a cognate antigen via the TCR, potentially leading to uncontrolled T cell activation and consequent cytokine release syndrome, especially in humans.Therefore, the non-recognition of CD28 by antibodies according to the present invention represents an advantage in terms of the safe clinical use of these antibodies in humans.

[0120] As discussed in another section of this document, the present invention extends to multispecific (e.g., bispecific) antibodies. A multispecific (e.g., bispecific) antibody may comprise (i) an antibody antigen-binding site to ICOS and (ii) an additional antigen-binding site (optionally an antibody antigen-binding site, as described herein) that recognizes another antigen (e.g., PD-L1). The specific binding of binding sites to individual antigens can be determined. Thus, antibodies Petition 870230019896, dated 08 / 03 / 2023, p. 55 / 576 36 / 456 that specifically bind to ICOS include antibodies comprising an antigen-binding site that specifically binds to ICOS, optionally, the antigen-binding site for ICOS is comprised in an antigen-binding molecule that additionally includes one or more additional binding sites for one or more other antigens, for example, a bispecific antibody that binds to ICOS and PD-L1. AFFINITY

[0121] The binding affinity of an antibody to ICOS can be determined. The affinity of an antibody for its antigen can be quantified in terms of the dissociation equilibrium constant Kd, the association ratio Ka / Kd (Ka), and the dissociation rate (kd) of the antibody-antigen interaction. The Kd, Ka, and Kd for antibody-antigen binding can be measured using surface plasmon resonance (SPR).

[0122] An antibody according to the present invention may bind the EC domain of human ICOS with a Kd of 10 mM or less, preferably 5 mM or less, surface plasmon resonance of 1 mM or less. Kd may be 50 nM or less, 10 nM or less, 5 nM or less, 2 nM or less, or 1 nM or less. Kd may be 0.9 nM or less, 0.8 nM or less, 0.7 nm or less, 0.6 nm or less, 0.5 nm or less, 0.4 nm or less, 0.3 nm or less, 0.2 nM or less, or 0.1 nM or less. Kd may be at least 0.001 nM, for example, at least 0.01 nM or at least 0.1 nM.

[0123] Affinity quantification can be performed using SPR with the antibody in Fab format. A suitable protocol is as follows: Petition 870230019896, dated 08 / 03 / 2023, p. 56 / 576 37 / 456

[0124] 1. Attaching anti-human IgG (or another constant region of the antibody species) to a biosensor chip (e.g., GLM chip), such as by primary amine coupling;

[0125] 2. Expose anti-human IgG (or other species-matched antibody) to a test antibody, for example, in Fab fragment form, to capture the test antibody on the chip;

[0126] 3. Pass the test antigen over the surface of the capture chip at a range of concentrations, for example, at 5,000 nM, 1,000 nM, 200 nM, 40 nM, 8 nM and 2 nM, and at 0 nM (i.e., buffer only); and

[0127] 4. Determine the binding affinity of the test antibody to the test antigen using SPR at 25 °C. The buffer may be at pH 7.6, 150 mM NaCl, 0.05% detergent (e.g., P20), and 3 mM EDTA. The buffer may optionally contain 10 mM HEPES. HBS-EP can be used as a routine buffer. HBS-EP is available from Teknova Inc. (California; catalog number H8022).

[0128] Regeneration of the capture surface can be performed with 10 mM glycine at pH 1.7. This removes the captured antibody and allows the surface to be used for another interaction. Binding data can be fitted to the inherent 1:1 model using standard techniques, for example, using a model inherent to the ProteOn XPR36TM analysis software.

[0129] A variety of SPR instruments are known, such as Biacore™, ProteOn XPR36™ (Bio-Rad®) and KinExA® (Sapidyne Instruments, Inc). Worked examples of SPR are found in Example 7. Petition 870230019896, dated 08 / 03 / 2023, page 57 / 576 38 / 456

[0130] As described, affinity can be determined by SPR with the antibody in Fab format, with the antigen attached to the chip surface and the test antibody passed over the Fab-format chip in solution, to determine the affinity of the monomeric antibody-antigen interaction. Affinity can be determined at any desired pH, for example, pH 5.5 or pH 7.6, and any desired temperature, for example, 25 °C or 37 °C. As reported in Example 7, the antibodies according to the present invention bound to human ICOS with an apparent affinity of less than 2 nM, as determined by SPR using the antibody in monovalent format (Fab).

[0131] Other ways of measuring antibody binding to ICOS include fluorescence-activated cell separation (FACS), for example, using cells (e.g., CHO cells) with exogenous surface expression of ICOS or activated primary T cells expressing endogenous levels of ICOS. Antibody binding to cells expressing ICOS, as measured by FACS, indicates that the antibody has the capacity to bind to the extracellular (EC) domain of ICOS. ICOS RECEPTOR AGONISM

[0132] The ICOS ligand (ICOSL, also known as B7-H2) is a molecule expressed on the cell surface that binds to the ICOS receptor

[17] . This intercellular ligand-receptor interaction promotes ICOS multimerization on the surface of T cells, activating the receptor and stimulating downstream signaling in the T cell. In effector T cells, this receptor activation stimulates the effector T cell response. Petition 870230019896, dated 08 / 03 / 2023, page 58 / 576 39 / 456

[0133] Anti-ICOS antibodies can act as ICOS agonists, mimicking and even overcoming this stimulatory effect of the native ICOS ligand on the receptor. Such agonism may result from the antibody's ability to promote ICOS multimerization on the T cell. One mechanism for this is when antibodies form intercellular bridges between ICOS on the surface of T cells and receptors on an adjacent cell (e.g., B cell, antigen-presenting cell, or other immune cell), such as Fc receptors. Another mechanism is when antibodies that have multiple (e.g., two) antigen-binding sites (e.g., two pairs of VH-VL domains) bind multiple ICOS receptor molecules and thus promote multimerization. A combination of these mechanisms may occur.

[0134] Agonism can be tested in in vitro T-cell activation assays using antibody in soluble form (e.g., in immunoglobulin format or other antibody format comprising two spatially separated antigen-binding sites, e.g., two VHVL pairs), including or excluding a cross-linking agent, or using antibody bound to a solid surface to provide a linked antigen-binding site arrangement. Agonism assays can use a human ICOS-positive T lymphocyte cell line, such as MJ cells (ATCC CRL8294), as the target T cell for activation in such assays. One or more T-cell activation measures can be determined for a test antibody and compared to a reference molecule or a negative control to determine if there is a statistically significant difference (p < 0.05) in T-cell activation effected by the test antibody in Petition 870230019896, dated 08 / 03 / 2023, page 59 / 576 40 / 456 comparison to the reference molecule or control. An appropriate measure of T cell activation is the production of cytokines, for example, IFNγ, TNFα, or IL-2. A person skilled in the art will include appropriate controls, as needed, which standardize the assay conditions between the test antibody and control. An appropriate negative control is an antibody in the same format (e.g., isotype control) that does not bind to ICOS, for example, an antibody specific for an antigen that is not present in the assay system. A significant difference observed for the test antibody compared to a cognate isotype control within the assay's dynamic range indicates that the antibody acts as an ICOS receptor agonist in this assay.

[0135] An agonist antibody can be defined as one that, when tested in a T-cell activation assay:

[0136] has a significantly lower EC50 for inducing IFNγ production compared to the control antibody;

[0137] induces a significantly higher maximum production of IFNγ compared to the control antibody;

[0138] has a significantly lower EC50 for inducing IFNy production compared to ICOSL-Fc;

[0139] induces a significantly higher maximum production of IFNy compared to ICOSL-Fc;

[0140] has a significantly lower EC50 for inducing IFNγ production compared to the reference antibody C398.4A; and / or

[0141] induces maximum IFNy production Petition 870230019896, dated 08 / 03 / 2023, page 60 / 576 41 / 456 significantly higher compared to the reference antibody C398.4A.

[0142] In vitro T-cell assays include the microsphere-bound assay of Example 13, the microsphere-bound assay of Example 14, and the soluble assay of Example 15.

[0143] A significantly lower or significantly higher value may be, for example, up to 0.5 times different, up to 0.75 times different, up to 2 times different, up to 3 times different, up to 4 times different, or up to 5 times double different, compared to the reference or control value.

[0144] Thus, in one example, an antibody according to the present invention has a significantly lower EC50, for example, at least 2 times lower, for IFNγ induction in an MJ cell activation assay using the antibody in microsphere-bound form, compared to the control.

[0145] The bead-bound assay uses antibody (and, for control or reference experiments, control antibody, reference antibody, or ICOSL-Fc) bound to the surface of microspheres. Magnetic microspheres can be used, and several types are commercially available, for example, Tosyl-activated DYNABEADS M-450 (DYNAL Inc, 5 Delaware Drive, Lake Success, NY 11042 Prod No. 140.03, 140.04). The microspheres can be coated as described in Example 13, or more generally by dissolving the coating material in carbonate buffer (pH 9.6, 0.2 M) or another method known in the art. The use of beads allows the amount of protein bound to the surface of the Petition 870230019896, dated 08 / 03 / 2023, page 61 / 576 42 / 456 granule size can be determined with a good degree of precision. Standard Fc protein quantification methods can be used for the quantification of proteins coupled to microspheres. Any suitable method can be used, with reference to a relevant standard within the assay's dynamic range. DELFIA is exemplified in Example 13, but ELISA or other methods can be used.

[0146] The agonism activity of an antibody can also be measured in primary human T lymphocytes ex vivo. The ability of an antibody to induce IFNγ expression in such T cells is indicative of ICOS agonism. In this document, two T cell activation assays are described using primary cells – see Example 2. T1 cell activation assay and T2 cell activation assay. Preferably, an antibody will show significant (p <0.05) IFNγ induction at 5 pg / ml compared to the control antibody in the T1 cell activation assay and / or T2 cell activation assay. As noted above, an anti-ICOS antibody may stimulate T cell activation to a greater degree than ICOS-L or C398.4 in this assay. Thus, the antibody may show significantly (p <0.05) greater IFNγ induction at 5 pg / ml compared to the control or reference antibody in the T1 or 2 cell activation assay. TNFα or IL-2 induction may be measured as an alternative assay readout.

[0147] The agonism of an anti-ICOS antibody may contribute to its ability to alter the balance between populations of TReg and TEff cells in vivo, for example, in a pathological site, such as a tumor microenvironment, in favor of TEff cells. The ability of an antibody to Petition 870230019896, dated 08 / 03 / 2023, page 62 / 576 43 / 456 Intensifying the killing of tumor cells by activated ICOS-positive effector T cells can be determined, as described in another section of this document. T-cell dependent death

[0148] The function of effector T cells can be determined in a biologically relevant context using an in vitro co-culture assay in which tumor cells are incubated with relevant immune cells to trigger immune cell-dependent killing, in which the effect of an anti-ICOS antibody on tumor cell killing by TEffs is observed.

[0149] The ability of an antibody to increase tumor cell death by activated ICOS-positive effector T cells can be determined. An anti-ICOS antibody can stimulate significantly greater cell death (p <0.05) compared to a control antibody. An anti-ICOS antibody can stimulate similar or superior tumor cell death in such an assay compared to a reference molecule, such as the ICOS ligand or the C398.4 antibody. A similar degree of tumor cell death can be represented by the assay reading for the antibody being less than twice that of the reference molecule. ICOS Ligand Receptor Neutralizing Power

[0150] An antibody according to the present invention may be one that inhibits the binding of ICOS to its ICOSL ligand.

[0151] The degree to which an antibody inhibits the binding of the ICOS receptor to its ligand is termed potency. Petition 870230019896, dated 08 / 03 / 2023, p. 63 / 576 44 / 456 ligand receptor neutralizing agent. Potency is normally expressed as an IC50 value, in pM, unless otherwise indicated. In ligand binding studies, IC50 is the concentration that reduces receptor binding by 50% of the maximum specific binding level. IC50 can be calculated by plotting the % specific receptor binding as a function of the log of the antibody concentration and using a software program such as Prism (GraphPad) to fit a sigmoidal function to the data in order to generate the IC50 values. Neutralizing potency can be determined in an HTRF assay. A detailed working example of an HTRF assay for ligand receptor neutralizing potency is presented in Example 8.

[0152] An IC50 value can represent the average of a plurality of measurements. Thus, for example, IC50 values ​​can be obtained from the results of triplicate experiments, and an average IC50 value can then be calculated.

[0153] An antibody may have an IC50 of 1 mM or less in a receptor-ligand neutralization assay, for example, 0.5 mM or less. The IC50 may be 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, or 2 nM or less. The IC50 may be at least 0.1 nM, at least 0.5 nM, or at least 1 nM. ANTIBODIES

[0154] As described in more detail in the Examples, antibodies of particular interest have been isolated and characterized, designated STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009. In several aspects of the invention, except where the Petition 870230019896, dated 08 / 03 / 2023, page 64 / 57645 / 456 context determine otherwise, antibodies may be selected from any of these antibodies or from the subset of STIM001, STIM002, STIM003, STIM004 and STIM005. The sequences of each of these antibodies are provided in the attached sequence listing, where for each antibody the following sequences are shown: nucleotide sequence encoding the VH domain; amino acid sequence of the VH domain; amino acid sequence of CDR1 VH; amino acid sequence of CDR2 VH; amino acid sequence of CDR3 VH; nucleotide sequence encoding the VL domain; amino acid sequence of the VL domain; amino acid sequence of CDR1 VL; amino acid sequence of CDR2 VL; and amino acid sequence of CDR3 VL, respectively.The present invention encompasses anti-ICO antibodies that have the VH and / or VL domain sequences of all antibodies shown in the attached sequence listing and / or drawings, as well as antibodies comprising the HCDRs and / or LCDRs of these antibodies and that optionally have the complete amino acid sequence of the heavy chain and / or full-length light chain.

[0155] STIM001 has an amino acid sequence of the variable heavy chain (VH) region of Seq ID No:366 comprising the amino acid sequence of CDRH1 of Seq ID No:363, the amino acid sequence of CDRH2 of Seq ID No:364 and the amino acid sequence of CDRH3 of Seq ID No:365. The nucleic acid sequence of the heavy chain of the VH domain is Seq ID No:367. STIM001 has an amino acid sequence of the variable light chain (VL) region of Seq ID No:373 comprising the amino acid sequence of CDRL1 of Seq ID No:370, the amino acid sequence of CDRL2 of Seq ID No:371 Petition 870230019896, dated 08 / 03 / 2023, p. 65 / 576 46 / 456 and the amino acid sequence of CDRL3 of Seq ID No:372. The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:374. The Vh domain can be combined with any of the sequences of the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:368 (heavy chain nucleic acid sequence Seq ID No:369).A full-length light chain amino acid sequence is Seq ID No: 375 (light chain nucleic acid sequence Seq ID No: 376).

[0156] STIM002 has an amino acid sequence of the variable heavy chain (VH) region of Seq ID No:380 comprising the amino acid sequence of CDRH1 of Seq ID No:377, the amino acid sequence of CDRH2 of Seq ID No:378 and the amino acid sequences of CDRH3 of Seq ID No:379. The nucleic acid sequence of the heavy chain of the VH domain is Seq ID No:381. STIM002 has an amino acid sequence of the variable light chain (VL) region of Seq ID No:387 comprising the amino acid sequence of CDRL1 of Seq ID No:384, the amino acid sequence of CDRL2 of Seq ID No:385 and the amino acid sequence of CDRL3 of Seq ID No:386. The nucleic acid sequence of the light chain of the VL domain is the Petition 870230019896, dated 08 / 03 / 2023, p. 66 / 576 47 / 456 Seq ID No:388 or Seq ID No:519. The Vh domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The Vl domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No: 382 (heavy chain nucleic acid sequence Seq ID No: 383). A full-length light chain amino acid sequence is Seq ID No: 389 (light chain nucleic acid sequence Seq ID No: 390 or Seq ID No: 520).

[0157] STIM002-B has an amino acid sequence of the variable region of the heavy chain (Vh) of Seq ID No:394 comprising the amino acid sequence of CDRH1 of Seq ID No:391, the amino acid sequence of CDRH2 of Seq ID No:392 and the amino acid sequence of CDRH3 of Seq ID No:393. The nucleic acid sequence of the light chain of the Vh domain is Seq ID No:395. STIM002-B has an amino acid sequence of the variable region of the light chain (Vl) of Seq ID No:401 comprising the amino acid sequence of CDRL1 of Seq ID No:398, the amino acid sequence of CDRL2 of Seq ID No:399 and the amino acid sequence of CDRL3 of Seq ID No:400. The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:402. The Vh domain can be combined with any of the sequences in the constant region of the heavy chain. Petition 870230019896, dated 08 / 03 / 2023, p. 67 / 576 48 / 456 described in this document, 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. The Vl domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No: 396 (heavy chain nucleic acid sequence Seq ID No: 397). A full-length light chain amino acid sequence is Seq ID No: 403 (light chain nucleic acid sequence Seq ID No: 404).

[0158] STIM003 has an amino acid sequence of the variable heavy chain (VH) region of Seq ID No:408 comprising the amino acid sequence of CDRH1 of Seq ID No:405, the amino acid sequence of CDRH2 of Seq ID No:406 and the amino acid sequences of CDRH3 of Seq ID No:407. The nucleic acid sequence of the heavy chain of the VH domain is Seq ID No:409 or Seq ID No:521. STIM003 has an amino acid sequence of the variable light chain (VL) region of Seq ID No:415 comprising the amino acid sequence of CDRL1 of Seq ID No:412, the amino acid sequence of CDRL2 of Seq ID No:413 and the amino acid sequence of CDRL3 of Seq ID No:414. The nucleic acid sequence of the light chain of the VL domain is Seq ID No:4416. The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, for example, Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID Petition 870230019896, dated 08 / 03 / 2023, p. 68 / 576 49 / 456 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. The Vl domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No: 410 (heavy chain nucleic acid sequence Seq ID No: 411 or Seq ID No: 522). A full-length light chain amino acid sequence is Seq ID No: 417 (light chain nucleic acid sequence Seq ID No: 418).

[0159] STIM004 has an amino acid sequence of the variable region of the heavy chain (Vh) of Seq ID No:422 comprising the amino acid sequence of CDRH1 of Seq ID No:419, the amino acid sequence of CDRH2 of Seq ID No:420 and the amino acid sequences of CDRH3 of Seq ID No:421. The nucleic acid sequence of the light chain of the Vh domain is Seq ID No:423. STIM004 has an amino acid sequence of the variable region of the light chain (Vl) of Seq ID No:429 comprising the amino acid sequence of CDRL1 of Seq ID No:426, the amino acid sequence of CDRL2 of Seq ID No:427 and the amino acid sequence of CDRL3 of Seq ID No:428. The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:430 or Seq ID No:431.The Vh domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. Petition 870230019896, dated 08 / 03 / 2023, page 69 / 576 50 / 456 Seq ID No: 532 or Seq ID No: 534. The Vl domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:424 (heavy chain nucleic acid sequence Seq ID No:425). A full-length light chain amino acid sequence is Seq ID No:432 (light chain nucleic acid sequence Seq ID No:433 or Seq ID No:434).

[0160] STIM005 has an amino acid sequence of the variable region of the heavy chain (Vh) of Seq ID No:438 comprising the amino acid sequence of CDRH1 of Seq ID No:435, the amino acid sequence of CDRH2 of Seq ID No:436 and the amino acid sequences of CDRH3 of Seq ID No:437. The nucleic acid sequence of the light chain of the Vh domain is the Seq ID No:439. STIM005 has an amino acid sequence of the variable region of the light chain (Vl) of Seq ID No:445 comprising the amino acid sequence of CDRL1 of Seq ID No:442, the amino acid sequence of CDRL2 of Seq ID No:443 and the amino acid sequence of CDRL3 of Seq ID No:444. The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:446. The Vh domain can be combined with any of the sequences of the constant region of the heavy chain described in this document, 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. The Vl domain can be combined with any of the sequences from the constant region of the light chain described. Petition 870230019896, dated 08 / 03 / 2023, page 70 / 576 51 / 456 in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536, and 538. A full-length heavy chain amino acid sequence is Seq ID No:440 (heavy chain nucleic acid sequence Seq ID No:441). A full-length light chain amino acid sequence is Seq ID No:447 (light chain nucleic acid sequence Seq ID No:448).

[0161] STIM006 has an amino acid sequence of the variable region of the heavy chain (Vh) of Seq ID No:452 comprising the amino acid sequence of CDRH1 of Seq ID No:449, the amino acid sequence of CDRH2 of Seq ID No:450 and the amino acid sequence of CDRH3 of Seq ID No:451. The nucleic acid sequence of the light chain of the VH domain is the Seq ID No:453. STIM006 has an amino acid sequence of the variable region of the light chain (Vl) of Seq ID No:459 comprising the amino acid sequence of CDRL1 of Seq ID No:456, the amino acid sequence of CDRL2 of Seq ID No:457 and the amino acid sequence of CDRL3 of Seq ID No:458. The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:460. The Vh domain can be combined with any of the sequences of the constant region of the heavy chain described in this document, 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. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, Petition 870230019896, dated 08 / 03 / 2023, p. 71 / 576 52 / 456 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:454 (heavy chain nucleic acid sequence Seq ID No:455). A full-length light chain amino acid sequence is Seq ID No: 461 (light chain nucleic acid sequence Seq ID No: 462).

[0162] STIM007 has an amino acid sequence of the variable heavy chain (VH) region of Seq ID No:466 comprising the amino acid sequence of CDRH1 of Seq ID No:463, the amino acid sequence of CDRH2 of Seq ID No:464 and the amino acid sequences of CDRH3 of Seq ID No:465. The nucleic acid sequence of the light chain of the VH domain is Seq ID No:467. STIM007 has an amino acid sequence of the variable light chain (VL) region of Seq ID No:473 comprising the amino acid sequence of CDRL1 of Seq ID No:470, the amino acid sequence of CDRL2 of Seq ID No:471 and the amino acid sequence of CDRL3 of Seq ID No:472. The nucleic acid sequence of the light chain of the VL domain is Seq ID No:474.The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No: 468 (acid sequence). Petition 870230019896, dated 08 / 03 / 2023, p. 72 / 576 53 / 456 heavy chain nucleic acids Seq ID No:469). A full-length light chain amino acid sequence is Seq ID No: 475 (light chain nucleic acid sequence Seq ID No: 476).

[0163] STIM008 has an amino acid sequence of the variable region of the heavy chain (Vh) of Seq ID No:480 comprising the amino acid sequence of CDRH1 of Seq ID No:477, the amino acid sequence of CDRH2 of Seq ID No:478 and the amino acid sequence of CDRH3 of Seq ID No:479. The nucleic acid sequence of the light chain of the VH domain is the Seq ID No:481. STIM008 has an amino acid sequence of the variable light chain (VL) region of Seq ID No:487 comprising the amino acid sequence CDRL1 of Seq ID No:484, the amino acid sequence CDRL2 of Seq ID No:485 and the amino acid sequence CDRL3 of Seq ID No:486. The nucleic acid sequence of the VL domain light chain is Seq ID No:488. The VH domain can be combined with any of the constant region sequences of the heavy chain described in this document, 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. The VL domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536, and 538. A full-length heavy chain amino acid sequence is Seq ID No:482 (heavy chain nucleic acid sequence Seq ID No:483). A full-length light chain amino acid sequence is Seq ID Petition 870230019896, dated 08 / 03 / 2023, p. 73 / 576 54 / 456 No:489 (light chain nucleic acid sequence Seq ID No:490)

[0164] STIM009 has an amino acid sequence of the variable region of the heavy chain (Vh) of Seq ID No:494 comprising the amino acid sequence of CDRH1 of Seq ID No:491, the amino acid sequence of CDRH2 of Seq ID No:492 and the amino acid sequence of CDRH3 of Seq ID No:493. The nucleic acid sequence of the light chain of the VH domain is the Seq ID No:495. STIM009 has an amino acid sequence of the variable region of the light chain (Vl) of Seq ID No:501 comprising the amino acid sequence of CDRL1 of Seq ID No:498, the amino acid sequence of CDRL2 of Seq ID No:499 and the amino acid sequence of CDRL3 of Seq ID No:500. The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:502. The Vh domain can be combined with any of the sequences of the constant region of the heavy chain described in this document, 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. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:496 (heavy chain nucleic acid sequence Seq ID No:497). A full-length light chain amino acid sequence is Seq ID No: 503 (light chain nucleic acid sequence Seq ID No: 504). Petition 870230019896, dated 08 / 03 / 2023, p. 74 / 576 55 / 456

[0165] Antibodies according to the present invention are immunoglobulins or molecules comprising immunoglobulin domains, either naturally occurring or partially or wholly synthetically produced. Antibodies may be IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments (including, but not limited to, Fb, F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibody, closed-conformation multispecific antibody, disulfide-linked scfv, diabody), or derived from any species that naturally produces an antibody, or created by recombinant DNA technology; or isolated from serum, B cells, hybridomas, transfectomas, yeast or bacteria. Antibodies may be humanized using routine technology. The term antibody encompasses any polypeptide or protein comprising an antibody antigen-binding site.An antigen-binding site (paratope) is the part of an antibody that binds to and is complementary to its target epitope (ICOS).

[0166] The term epitope refers to a region of an antigen that is bound by any antibody. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes can also be conformational, i.e., composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface clusters of molecules, such as amino acids, sugar side chains, phosphoryl groups, or groups Petition 870230019896, dated 08 / 03 / 2023, p. 75 / 576 56 / 456 sulfonyl and, in certain embodiments, may have specific three-dimensional structural features and / or specific charge characteristics.

[0167] The antigen-binding site is a polypeptide or domain comprising one or more CDRs of an antibody and having the capacity to bind to the antigen. For example, the polypeptide comprises a CDR3 (e.g., HCDR3). For example, the polypeptide comprises CDRs 1 and 2 (e.g., HCDR1 and 2) or CDRs 1 to 3 of a variable domain of an antibody (e.g., HCDRs 1 to 3).

[0168] An antibody antigen-binding site may be provided by one or more antibody variable domains. In one example, the antibody binding site is provided by a single variable domain, for example, a heavy chain variable domain (VH domain) or a light chain variable domain (VL domain). In another example, the binding site comprises a VH / VL pair or two or more such pairs. Thus, an antibody antigen-binding site may comprise a VH and a VL.

[0169] The antibody may be a complete immunoglobulin, including constant regions, or it may be an antibody fragment. An antibody fragment is a portion of an intact antibody, for example, comprising the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include:

[0170] (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment that includes two Fab fragments linked by a disulfide bridge in the hinge region; Petition 870230019896, dated 08 / 03 / 2023, page 76 / 576 57 / 456

[0171] (iii) an Fd fragment consisting of the VH and CH1 domains;

[0172] (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody,

[0173] (v) a dAb fragment (Ward et al., (1989) Nature 341: 544 to 546; which is incorporated herein by reference in its entirety), which consists of a VH or VL domain; and

[0174] (vi) an isolated complementarity-determining region (CDR) that retains specific antigen-binding functionality.

[0175] Other examples of antibodies are H2 antibodies which comprise a dimer of a heavy chain (5'-VH-(optional hinge)-CH2-CH3-3') and are devoid of a light chain.

[0176] Single-chain antibodies (e.g., scFv) are a commonly used fragment. Multispecific antibodies can be formed from antibody fragments. An antibody of the invention may employ any such format, as appropriate.

[0177] Optionally, the antibody immunoglobulin domains may be fused or conjugated to additional polypeptide sequences and / or to markers, tags, toxins, or other molecules. The antibody immunoglobulin domains may be fused or conjugated to one or more different antigen-binding regions, providing a molecule that has the ability to bind to a second antigen in addition to ICOS. An antibody of the present invention may be a multispecific antibody, for example, a bispecific antibody comprising (i) a site of Petition 870230019896, dated 08 / 03 / 2023, p. 77 / 576 58 / 456 antibody antigen binding to ICOS and (ii) an additional antigen binding site (optionally an antibody antigen binding site, as described herein) that recognizes another antigen (e.g., PD-L1).

[0178] An antibody typically comprises a VH antibody and / or VL domain. Isolated VH and VL domains of antibodies are also part of the invention. Variable antibody domains are the portions of the light and heavy chains of antibodies that include amino acid sequences of complementarity-determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). Thus, within the VH and VL domains are the CDRs and FRs. A VH domain comprises a set of CDRs and a VL domain comprises a set of LCDRs. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to the methods used in the present invention, the amino acid positions assigned to CDRs and FRs can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)) or according to IMGT nomenclature. An antibody may comprise an antibody VH domain comprising a VH CDR1, CDR2, and CDR3 and a framework. The same may additionally or alternatively comprise an antibody VL domain comprising a VL CDR1, CDR2, and CDR3 of a framework.Examples of antibody VH and VL domains and CDRs according to the present invention are listed in the attached sequence listing which forms part of the present disclosure. The CDRs shown in the listing... Petition 870230019896, dated 08 / 03 / 2023, p. 78 / 576 59 / 456 sequences are defined according to the IMGT system

[18] . All VH and VL sequences, CDR sequences, CDR sets and HCDR sets and LCDR sets disclosed in this document represent aspects and embodiments of the invention. As described in this document, a “CDR set” comprises CDR1, CDR2 and CDR3. Thus, an HCDR set refers to HCDR1, HCDR2 and HCDR3, and an LCDR set refers to LCDR1, LCDR2 and LCDR3. Unless otherwise indicated, a “CDR set” includes HCDRs and LCDRs.

[0179] An antibody of the invention may comprise one or more CDRs as described herein, for example, a CDR3, and optionally also a CDR1 and CDR2 to form a set of CDRs. The CDR or set of CDRs may be a CDR or a set of CDRs from any of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or may be a variant thereof, as described herein.

[0180] The invention provides antibodies comprising an HCDR1, HCDR2 and / or HCDR3 of any of the antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 and / or an LCDR1, LCDR2 and / or LCDR3 of any of these antibodies, for example, a set of CDRs. The antibody may comprise a set of HV CDRs from one of these antibodies. Optionally, it may also comprise a set of VL CDRs from one of these antibodies, and the VL CDRs may be from the same antibody or from a different antibody than the HV CDRs.

[0181] A VH domain comprising a revealed set of HCDRs and / or a VL domain comprising a set Petition 870230019896, dated 08 / 03 / 2023, page 79 / 576 60 / 456 revealed LCDRs are also provided by the invention.

[0182] Typically, a VH domain is paired with a VL domain to provide an antibody antigen-binding site, although, as discussed further below, only one VH or VL domain can be used for antigen binding. The VH domain of STIM003 can be paired with the VL domain of STIM003, so that an antibody antigen-binding site comprising the VH and VL domains of STIM003 is formed. Analogous embodiments are provided for the other VH and VL domains disclosed herein. In other embodiments, the STIM003 VH is paired with a VL domain other than the STIM003 VL. Light chain promiscuity is well established in the art. Again, analogous embodiments are provided by the invention for the other VH and VL domains disclosed herein.

[0183] Thus, the HV of any of the antibodies STIM001, STIM002, STIM003, STIM004 and STIM005 can be paired with the LV of any of the antibodies STIM001, STIM002, STIM003, STIM004 and STIM005. In addition, the HV of any of the antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 can be paired with the LV of any of the antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.

[0184] An antibody may comprise one or more CDRs, for example, a set of CDRs, within an antibody framework. Framework regions may be sequences of human germline gene segments. Thus, the antibody may be a human antibody that has a VH domain comprising a set of CDRs in a framework. Petition 870230019896, dated 08 / 03 / 2023, page 80 / 576 61 / 456 of the human germline. Typically, the antibody also has a VL domain comprising a set of LCDRs, for example, in a human germline framework. An “antibody gene segment,” for example, a VH gene segment, D gene segment, or JH gene segment, refers to an oligonucleotide that has a nucleic acid sequence from which that part of an antibody is derived; for example, a VH gene segment is an oligonucleotide, a nucleic acid sequence that corresponds to a VH polypeptide domain from FR1 to part of CDR3. Human V, D, and J gene segments recombine to generate the VH domain, and human V and J segments recombine to generate the VL domain. The D domain or region refers to the diversity domain or region of an antibody chain. The J domain or region refers to the binding domain or region of an antibody chain.Somatic hypermutation can result in an antibody VH or VL domain with framework regions that do not exactly match or align with the corresponding gene segments; however, sequence alignment can be used to identify the closest gene segments and determine which particular combination of gene segments a given VH or VL domain is derived from. During the alignment of antibody sequences with gene segments, the antibody amino acid sequence can be aligned with the amino acid sequence encoded by the gene segment, or the antibody nucleotide sequence can be directly aligned with the nucleotide sequence of the gene segment.

[0185] The alignments of the VH and VL domain sequences of the STIM antibody against related antibodies and against human germline sequences are Petition 870230019896, dated 08 / 03 / 2023, page 81 / 576 62 / 456 shown in Figure 35, Figure 36 and Figure 37.

[0186] An antibody of the invention may be a human antibody or a chimeric antibody comprising human variable regions and non-human constant regions (e.g., mouse). The antibody of the invention, for example, has human variable regions and optionally also has human constant regions.

[0187] Thus, antibodies optionally include constant regions, or parts thereof, for example, constant regions of the human antibody or parts thereof. For example, a VL domain may be linked at its C-terminal end to the kappa or lambda constant domains of an antibody light chain. Similarly, an antibody VH domain may be linked at its C-terminal end to all or part (for example, a CH1 domain or Fc region) of a constant region of the immunoglobulin heavy chain derived from any antibody isotype, for example, IgG, IgA, IgE and IgM and any of the isotype subclasses, such as IgG1 or IgG4.

[0188] Examples of constant regions of the human heavy chain are shown in Table S1.

[0189] The antibody constant regions of the invention may alternatively be non-human constant regions. For example, when antibodies are generated in transgenic animals (examples of which are described in another section of this document), chimeric antibodies may be produced comprising human variable regions and non-human constant regions (host animal). Some transgenic animals generate fully human antibodies. Others have been engineered to generate antibodies that Petition 870230019896, dated 08 / 03 / 2023, page 82 / 576 63 / 456 comprises chimeric heavy chains and fully human light chains. When antibodies comprise one or more non-human constant regions, these may be replaced by human constant regions to provide antibodies more suitable for administration to humans as therapeutic compositions, since their immunogenicity is thereby reduced.

[0190] Antibody digestion with the enzyme papain results in two identical antigen-binding fragments, also known as Fab fragments, and an Fc fragment, which has no antigen-binding activity but has the ability to crystallize. Fab, as used in this document, refers to a fragment of an antibody that includes a constant domain and a variable domain from each of the heavy and light chains. The term Fc region is used in this document to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. The Fc fragment refers to the carboxy-terminal portions of both H chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, and this region is also recognized by Fc receptors (FcRs) found on certain cell types.Antibody digestion with the enzyme pepsin results in the F(ab')2 fragment, in which the two arms of the antibody molecule remain linked and comprise two antigen-binding sites. The F(ab')2 fragment has the ability to cross-link the antigen.

[0191] Fv when used in this document refers to the minimum antibody fragment that retains both the antigen recognition site and the binding sites. Petition 870230019896, dated 08 / 03 / 2023, p. 83 / 576 64 / 456 to antigen. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain and a tight covalent non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with a lower affinity than the full binding site.

[0192] The antibodies disclosed in this document can be modified to increase or decrease the serum half-life. In one embodiment, one or more of the following mutations: T252L, T254S, or T256F are introduced to increase the biological half-life of the antibody. The biological half-life can also be increased by altering the CH1 domain of the constant region of the heavy chain or the CL region to contain an epitope obtained from two loops of a CH2 domain of an Fc region of an IgG, as described in US Patents Nos. 5,869,046 and 6,121,022, the modifications described therein being incorporated herein by reference. In another embodiment, the Fc hinge region of an antibody or antigen-binding fragment of the invention is mutated to decrease the biological half-life of the antibody or fragment.One or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment so that the antibody or fragment has conferred staphylococcal protein A (SpA) binding relative to SpA binding to the native Fc-hinge domain. Other methods to increase... Petition 870230019896, dated 08 / 03 / 2023, page 84 / 576 The serum half-life of 65 / 456 is known to those skilled in the art. Thus, in one embodiment, the antibody or fragment is PEGylated. In another embodiment, the antibody or fragment is fused to an albumin-binding domain, for example, a single-domain albumin-binding antibody (dAb). In another embodiment, the antibody or fragment is PAsylated (i.e., genetic fusion of polypeptide sequences composed of PAS (XL-Protein GmbH) that forms uncharged random folded structures with large hydrodynamic volume). In another embodiment, the antibody or fragment is XTENylated® / rPEGylated (i.e., genetic fusion of the non-exact repeat peptide sequence (Amunix, Versartis) to the therapeutic peptide). In another embodiment, the antibody or fragment is ELPylated (i.e., genetic fusion to the ELP repeat sequence (PhaseBio)).These various fusions that extend into the half-life are described in more detail in Strohl, BioDrugs (2015) 29: 215 to 239, whose fusions, for example, in Tables 2 and 6, are incorporated into this document by way of reference.

[0193] The antibody may have a modified constant region that increases stability. Thus, in one embodiment, the constant region of the heavy chain comprises a Ser228Pro mutation. In another embodiment, the antibodies and fragments disclosed herein comprise a heavy chain hinge region that has been modified to alter the number of cysteine ​​residues. This modification may be used to facilitate the assembly of the light and heavy chains or to increase or decrease the stability of the antibody. Effector functions of FC, ADCC, ADCP and CDC Petition 870230019896, dated 08 / 03 / 2023, page 85 / 576 66 / 456

[0194] As discussed above, anti-ICOS antibodies can be supplied in various isotypes and with different constant regions. Examples of sequences of the constant region of the human IgG antibody heavy chain are shown in Table S1. The Fc region of the antibody primarily determines its effector function in terms of Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cellular phagocytosis (ADCP) activity. These “cellular effector functions,” distinct from the function of effector T cells, involve the recruitment of cells containing Fc receptors to the site of target cells, resulting in the death of the antibody-bound cell. In addition to ADCC and CDC, the ADCP mechanism

[19] represents a means of depleting antibody-bound T cells and thus directing TRegs expressing elevated ICOS towards deletion.

[0195] The cellular effector functions ADCC, ADCP and / or CDC can also be exhibited by antibodies without Fc regions. Antibodies may comprise multiple different antigen-binding sites, one directed to ICOS and another directed to a target molecule where the binding of that target molecule induces ADCC, ADCP and / or CDC, for example, an antibody comprising two scFv regions linked by a ligand, and an scFv may envelop an effector cell.

[0196] An antibody according to the present invention may be one that exhibits ADCC, ADCP and / or CDC. Alternatively, an antibody according to the present invention may not have ADCC, ADCP and / or CDC activity. In any case, an antibody according to the present invention may Petition 870230019896, dated 08 / 03 / 2023, p. 86 / 576 67 / 456 may or may not include, or may optionally lack, an Fc region that binds to one or more types of Fc receptors. The use of different antibody formats and the presence or absence of FcR binding and cellular effector functions allow the antibody to be tailored for use for particular therapeutic purposes, as discussed in another section of this document.

[0197] A suitable antibody format for some therapeutic applications employs a wild-type human IgG1 constant region. A constant region can be an enabled effector constant region of IgG1 that optionally has ADCC and / or CDC and / or ADCP activity. A suitable wild-type human IgG1 constant region sequence is SEQ ID NO:340 (IGHG1*01). Other examples of human IgG1 constant regions are shown in Table S1.

[0198] For testing therapeutic antibody candidates in mouse models of human disease, a mouse effector constant positive region, such as mouse IgG2a (mIgG2a), may be included instead of a human effector constant positive region.

[0199] A constant region can be designed for enhanced ADCC and / or CDC and / or ADCP.

[0200] The potency of Fc-mediated effects can be enhanced by genetically modifying the Fc domain using various established techniques. Such methods increase the affinity for certain Fc receptors, thus creating diverse potential activation enhancement profiles. This can be achieved by modifying one or more amino acid residues

[20] . Human IgG1 constant regions containing specific mutations or altered glycosylation at the Asn297 residue (e.g., N297Q, EU index numbering) Petition 870230019896, dated 08 / 03 / 2023, page 87 / 576 68 / 456 enhanced binding to Fc receptors. Examples of mutations are one or more of the residues selected from 239, 332, and 330 for human IgG1 constant regions (or the equivalent positions in other IgG isotypes). Thus, an antibody may comprise a human IgG1 constant region that has one or more mutations independently selected from N297Q, S239D, I332E, and A330L (EU index numbering). A triple mutation (M252Y / S254T / T256E) can be used to enhance FcRn binding, and other mutations affecting FcRn binding are discussed in Table 2 of

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

[0201] Increased affinity for Fc receptors can also be obtained by altering the natural glycosylation profile of the Fc domain, for example, by generating fucosylated or defucosylated variants

[22] . Non-fucosylated antibodies harbor a tri-aminoglycan core structure of Fc complex-type N-glycans without a fucose residue. These glycomodified antibodies lacking the fucose residue of the Fc N-glycan core can exhibit more intense ADCC than fucosylated equivalents due to enhanced FcyRIIIa binding capacity. For example, in order to increase ADCC, residues in the hinge region can be altered to increase binding to Fc-gamma RIII

[23] .Thus, an antibody may comprise a constant region of human IgG heavy chain, which is a variant of a constant region of human IgG heavy chain, wherein the variant constant region of human IgG heavy chain binds to selected human Fcy receptors from the group consisting of FcyRIIB and FcyRIIA with higher affinity than the variant. Petition 870230019896, dated 08 / 03 / 2023, page 88 / 576 69 / 456 The constant region of the wild-type human IgG heavy chain binds to human Fcy receptors. The antibody may comprise a constant region of human IgG heavy chain that is a variant of a constant region of wild-type human IgG heavy chain, wherein the variant constant region of human IgG heavy chain binds to human FcyRIIB with greater affinity than the wild-type human IgG heavy chain constant region binds to human FcyRIIB. The variant human IgG heavy chain constant region may be a variant human IgG1, a variant human IgG2, or a variant human IgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (EU index numbering system).In another embodiment, the constant region of the variant human IgG heavy chain comprises a set of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G and A330R; P238D, E233D, G237D, H268D, P271G and A330R; G236D and S267E; S239D and S267E; V262E, S267E and L328F; and V264E, S267E and L328F (EU index numbering system). The intensification of CDC enhancement can be achieved by amino acid changes that increase affinity for C1q, the first component of the classical complement activation cascade

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

[25] . The antibodies of the present invention may comprise amino acids that have undergone mutation at residues 329. Petition 870230019896, dated 08 / 03 / 2023, page 89 / 576 70 / 456 331 and / or 322, to alter C1q binding and / or reduced or abolished CDC activity. In another embodiment, the antibodies or antibody fragments described herein may contain Fc regions with modifications at residues 231 and 239, wherein the amino acids are substituted to alter the antibody's ability to bind to complement. In one embodiment, the antibody or fragment has a constant region comprising one or more mutations selected from E345K, E430G, R344D, and D356R, in particular, a double mutation comprising R344D and D356R (EU index numbering system).

[0202] Document WO2008 / 137915 described anti-ICOS antibodies with modified Fc regions that have enhanced effector function. It was reported that the antibodies mediated increased ADCC activity compared to the level of ADCC activity mediated by a parental antibody comprising VH and VK domains and a wild-type Fc region. The antibodies according to the present invention may employ such variant Fc regions that have the effector function as described in this document.

[0203] The ADCC activity of an antibody can be determined in an assay as described in this document. The ADCC activity of an anti-ICOS antibody can be determined in vitro using an ICOS-positive T cell line, as described in Example 10. The ADCC activity of an anti-PD-L1 antibody can be determined in vitro in an ADCC assay using cells expressing PD-L1.

[0204] For certain applications (such as in the context of vaccination) it may be preferable to use antibodies. Petition 870230019896, dated 08 / 03 / 2023, page 90 / 576 71 / 456 without the effector function of the Fc. Antibodies may be supplied without a constant region, or without an Fc region – examples of such antibody formats are described elsewhere in this document. Alternatively, an antibody may have a constant region that has no effector function. An antibody may have a heavy chain constant region that does not bind to Fcy receptors, for example, the constant region may comprise a Leu235Glu mutation (i.e., where the wild-type leucine residue is mutated to a glutamic acid residue). Another optional mutation for a heavy chain constant region is Ser228Pro, which increases stability. A heavy chain constant region may be an IgG4 comprising the Leu235Glu mutation and the Ser228Pro mutation. This IgG4-PE heavy chain constant region has no effector function.

[0205] An alternative human null effector constant region is a disabled IgG1. A disabled IgG1 heavy chain constant region may contain alanine at position 235 and / or 237 (EU index numbering), for example, it may be an IgG1*01 sequence comprising the L235A and / or G237A (LAGA) mutations.

[0206] A variant of the constant region of the human IgG heavy chain may comprise one or more amino acid mutations that reduce the affinity of IgG for human FcyRIIIA, human FcyRIIA, or human FcyRI. In one embodiment, FcyRIIB is expressed in a cell selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells. In one embodiment, the variant human IgG heavy chain constant region comprises one or more of the following mutations. Petition 870230019896, dated 08 / 03 / 2023, page 91 / 576 72 / 456 amino acids G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L (EU index numbering system). In one embodiment, the constant region of the variant human IgG heavy chain comprises a set of amino acid mutations selected from the group consisting of: S239D; T256A; K290A; S298A; I332E; E333A; K334A; A339T; S239D and I332E; S239D, A330L and I332E; S298A, E333A and K334A; G236A, S239D and I332E; and F243L, R292P, Y300L, V305I and P396L (index numbering system) In one embodiment, the constant region of the variant human IgG heavy chain comprises an S239D, A330L, or I332E amino acid mutation (EU index numbering system). In another embodiment, the constant region of the variant human IgG heavy chain comprises an S239D and I332E amino acid mutation (EU index numbering system). In another embodiment, the constant region of the variant human IgG heavy chain is a constant region of the variant human IgG1 heavy chain comprising S239D and I332E amino acid mutations (EU index numbering system). In one embodiment, the antibody or fragment comprises an afucosylated Fc region. In another embodiment, the antibody or fragment thereof is defucosylated. In yet another embodiment, the antibody or fragment is fucosylated.

[0207] An antibody may have a constant region of heavy chain that binds to one or more types of Fc receptors, but does not induce cellular effector functions, that is, it does not mediate ADCC, CDC, or ADCP activity. Such a constant region may be unable to bind to the particular Fc receptor (or particular Fc receptors) responsible for triggering ADCC, CDC, or ADCP activity. Petition 870230019896, dated 08 / 03 / 2023, p. 92 / 576 73 / 456 Generation and Modification of Antibodies

[0208] The methods for identifying and preparing antibodies are well known. Antibodies can be generated using transgenic mice (e.g., Kymouse™, Velocimouse®, Omnimouse®, Xenomouse®, HuMab Mouse®, or MeMo Mouse®), rats (e.g., Omnirat®), camelids, sharks, rabbits, chickens, or other non-human animals immunized with ICOS or a fragment thereof or a synthetic peptide comprising an ICOS sequence motif of interest, optionally followed by humanization of the constant and / or variable regions to produce human or humanized antibodies. In one example, display technologies can be used, such as yeast, phage, or ribosome display, as will be evident to the skilled person.Standard affinity maturation, for example, using a display technology, can be performed in an additional step after isolating a lead antibody from a transgenic animal, phage display library, or other library. Representative examples of suitable technologies are described in document no. US20120093818 (Amgen, Inc), which is incorporated herein by reference in its entirety, for example, the methods set forth in paragraphs

[0309] to

[0346] .

[0209] Immunization of a non-human ICOS knockout animal with human ICOS antigen facilitates the generation of antibodies that recognize both human and non-human ICOS. As described herein and illustrated in the Examples, an ICOS knockout mouse can be immunized with cells expressing human ICOS to stimulate the production of antibodies to both human and mouse ICOS in the mouse that Petition 870230019896, dated 08 / 03 / 2023, page 93 / 57674 / 456 can be recovered and tested for binding to human ICOS and mouse ICOS. Cross-reactive antibodies can thus be selected, which can be screened for other desirable properties as described in this document. Methods for generating antibodies to an antigen (e.g., a human antigen) by immunizing animals with the antigen in which the expression of the endogenous antigen (e.g., endogenous mouse antigen) has been knocked out in the animal can be performed in animals capable of generating antibodies comprising human variable domains. The genomes of such animals can be genetically modified to comprise a human or humanized immunoglobulin locus encoding gene segments of the human variable region and, optionally, an endogenous constant region or a human constant region.Recombination of gene segments from the human variable region generates human antibodies, which may have either a non-human or human constant region. Non-human constant regions can be subsequently replaced by human constant regions where the antibody is intended for in vivo use in humans. Such methods and transgenic knockout animals are described in document no. WO2013 / 061078.

[0210] In general, a Kymouse™, VELOCIMMUNE®, or other mouse or rat (optionally, an ICOS knockout mouse or rat, as noted) can be challenged with the antigen of interest, and lymphatic cells (such as B cells) are recovered from the antibody-expressing mice. The lymphatic cells can be fused with a myeloma cell line to prepare immortal hybridoma cell lines, and these cell lines of Petition 870230019896, dated 08 / 03 / 2023, page 94 / 576 75 / 456 hybridoma cells are screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. The DNA encoding the variable regions of the heavy and light chains can be isolated and ligated to desirable isotypic constant regions of the heavy and light chains. Such an antibody protein can be produced in a cell, such as a CHO cell. Alternatively, the DNA encoding the antigen-specific chemical antibodies or the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0211] Initially, high-affinity chimeric antibodies are isolated that have a human variable region and a mouse constant region. The antibodies are characterized and selected for desirable characteristics, including affinity, selectivity, agonism, T-cell-dependent killing, neutralizing potency, epitope, etc. The mouse constant regions are optionally replaced by a desired human constant region to generate the fully human antibody of the invention, for example, wild-type or modified IgG1 or IgG4 (e.g., SEQ ID NO:751, 752, 753 in document no. US2011 / 0065902 (which is incorporated by reference herein in its entirety)). Although the selected constant region may vary according to the specific use, the high-affinity antigen-binding and target-specificity characteristics reside in the variable region.

[0212] Thus, in a further aspect, the present invention provides a non-human transgenic mammal having a genome comprising an immunoglobulin locus Petition 870230019896, dated 08 / 03 / 2023, page 95 / 576 76 / 456 human or humanized, wherein the mammal does not express ICOS. The mammal may, for example, be a knockout mouse or rat or other species of laboratory animals. Transgenic mice, such as Kymouse™, contain human heavy and light chain immunoglobulin loci inserted into the corresponding endogenous mouse immunoglobulin loci. A transgenic mammal according to the present invention may be one that contains such targeted insertions or may contain human heavy and light chain immunoglobulin loci or immunoglobulin genes that are randomly inserted into its genome, inserted into a locus other than the endogenous Ig locus, or provided on an additional chromosome or chromosomal fragment.

[0213] Other aspects of the invention are the use of such non-human mammals for the production of antibodies to ICOS, and methods for producing antibodies or variable domains of the heavy and / or light chain of antibody in such mammals.

[0214] One way to produce an antibody that binds to the extracellular domain of human and non-human ICOS may involve providing a transgenic non-human mammal that has a genome comprising a human or humanized immunoglobulin locus, wherein the mammal does not express ICOS, and

[0215] (a) immunize the mammal with human ICOS antigen (e.g., with cells expressing human ICOS or with purified recombinant ICOS protein);

[0216] (b) isolate the antibodies generated by the mammal;

[0217] (c) test antibodies for their ability to bind to human ICOS and non-human ICOS; and Petition 870230019896, dated 08 / 03 / 2023, page 96 / 576 77 / 456

[0218] (d) select one or more antibodies that bind to both human and non-human ICOS.

[0219] The binding capacity of human and non-human ICOS can be tested using surface plasmon resonance, HTRF, FACS, or any other method described in this document. Optionally, the binding affinities for mouse and human ICOS are determined. The affinity, or difference in times of affinity, of binding to human ICOS and mouse ICOS can be determined, and antibodies exhibiting cross-species reactivity (affinity thresholds and folding differences that can be used as selection criteria are exemplified throughout this document) can then be selected. The neutralizing potency, or the difference in times of neutralizing potency, of the antibody to inhibit the binding of human and mouse ICOS ligands to the human and mouse ICOS receptor, respectively, can be further or alternatively determined as a way to screen cross-reacting antibodies, for example, HTRF assay. Again, possible thresholds and differences that can be used as selection criteria are exemplified throughout this document.

[0220] The method may involve testing antibodies for their ability to bind to non-human ICOS of the same species or a different species from the immunized mammal. Thus, when the transgenic mammal is a mouse (e.g., a Kymouse™), antibodies can be tested for their ability to bind to mouse ICOS. When the transgenic mammal is a rat, antibodies can be tested for their ability to bind to rat ICOS. In Petition 870230019896, dated 08 / 03 / 2023, page 97 / 576 78 / 456 However, it may be equally useful to determine the cross-reactivity of an isolated antibody to non-human ICOS from another species. Thus, antibodies generated in goats can be tested for binding to rat or mouse ICOS. Optionally, binding to goat ICOS can be determined instead or additionally.

[0221] In other embodiments, the transgenic non-human mammal may be immunized with non-human ICOS, optionally ICOS from the same mammalian species (e.g., a knockout mouse with ICOS may be immunized with mouse ICOS) instead of human ICOS. The affinity of isolated antibodies for binding to human ICOS and non-human ICOS is then determined in the same manner, and antibodies that bind to both human ICOS and non-human ICOS are selected.

[0222] The nucleic acid encoding a variable domain of an antibody heavy chain and / or a variable domain of an antibody light chain from a selected antibody can be isolated. Such nucleic acid may encode the complete antibody heavy chain and / or light chain or the variable domain (or variable domains) without an associated constant region (or associated constant regions). As verified, the coding nucleotide sequences can be obtained directly from antibody-producing cells of a mouse, or B cells can be immortalized or fused to generate hybridomas expressing the antibody and encoding the nucleic acid obtained from such cells. Optionally, the nucleic acid encoding the variable domain (or variable domains) is then conjugated with a nucleotide sequence encoding a constant region of the heavy chain. Petition 870230019896, dated 08 / 03 / 2023, page 98 / 576 79 / 456 human and / or human light chain constant region, to provide nucleic acid encoding a human antibody heavy chain and / or human antibody light chain, for example, encoding an antibody comprising both the heavy chain and the light chain. As described throughout this document, this step is particularly useful when the immunized mammal produces chimeric antibodies with non-human constant regions that are preferably replaced by human constant regions to generate an antibody that will be less immunogenic when administered to humans as a drug. The provision of constant regions from particular human isotypes is also significant in determining the effector function of the antibody, and several suitable heavy chain constant regions are discussed in this document.

[0223] Other alterations in the nucleic acid that encodes the variable domain of the antibody heavy and / or light chain, such as residue mutation and variant generation, as described in this document.

[0224] The isolated nucleic acid (optionally, mutated) can be introduced into host cells, for example, CHO cells as discussed. The host cells are then cultured under conditions of expression of the antibody or the variable domain of the antibody heavy and / or light chain, in any desired antibody format. Some possible antibody formats are described in this document, for example, complete immunoglobulins, antigen-binding fragments, and other designs.

[0225] Variants of the variable domain amino acid sequence of either of the VH and VL domains or CDRs whose sequences are specifically disclosed herein. Petition 870230019896, dated 08 / 03 / 2023, page 99 / 576 80 / 456 document can be employed in accordance with the present invention, as discussed.

[0226] There are many reasons why it may be desirable to create variants, including optimizing the antibody sequence for large-scale production, which facilitates purification, enhances stability, or improves suitability for inclusion in a desired pharmaceutical formulation. Protein engineering work can be performed on one or more target residues in the antibody sequence, for example, to replace an amino acid with an alternative amino acid (optionally generating variants containing all naturally occurring amino acids at that position, with the possible exception of Cys and Met) and to monitor the impact on function and expression in order to determine the best substitution. In some examples, it is desirable to replace a residue with Cys or Met or to introduce these residues into a sequence, as this may generate manufacturing difficulties – for example, through the formation of new intramolecular or intermolecular cysteine-cysteine ​​linkages.When a prime candidate antibody is selected and optimized for manufacturing and clinical development, it is generally desirable to alter its antigen-binding properties as little as possible, or at least maintain the affinity and potency of the parent molecule. However, variants can also be generated in order to modulate key antibody characteristics such as affinity, cross-reactivity, or neutralizing potency.

[0227] An antibody may comprise a set of H and / or L CDRs of any of the antibodies described with one or more amino acid mutations within the disclosed set. Petition 870230019896, dated 08 / 03 / 2023, page 100 / 576 81 / 456 of CDR H and / or L. The mutation may be a substitution, deletion, or insertion of amino acids. Thus, for example, there may be one or more amino acid substitutions within the disclosed set of CDR H and / or L. For example, there may be up to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations, for example, substitutions, within the set of CDR H and / or L. For example, there may be up to 6, 5, 4, 3, or 2 mutations, for example, substitutions in HCDR3 and / or there may be up to 6, 5, 4, 3, or 2 mutations, for example, substitutions, in LCDR3. An antibody may comprise the set of HCDRs, LCDRs, or a set of 6 CDRs (H and L) shown for any STIM antibody of the present invention, or it may comprise that set of CDRs with one or two conservative substitutions.

[0228] One or more amino acid mutations may optionally be produced in framework regions of an antibody VH or VL domain disclosed herein. For example, one or more residues that are different from the sequence of corresponding human germline segments may be reverted to the germline. The human germline gene segment sequences corresponding to the VH and VL domains of example anti-ICOS antibodies are indicated in Table E121, Table E12-2, and Table E12-3, and the alignments of the antibody VH and VL domains to the corresponding germline sequences are shown in the drawings.

[0229] An antibody may comprise a VH domain that has at least 60, 70, 80, 85, 90, 95, 98, or 99% amino acid sequence identity to a VH domain of any of the antibodies shown in the attached sequence listing and / or comprise a VL domain that has at least Petition 870230019896, dated 08 / 03 / 2023, p. 101 / 576 82 / 456 60, 70, 80, 85, 90, 95, 98, or 99% amino acid sequence identity with a VL domain of any of these antibodies. Algorithms that can be used to calculate the % identity of two amino acid sequences include, for example, BLAST, FASTA, or the Smith-Waterman algorithm, which employs standard parameters. Particular variants may include one or more changes in the amino acid sequence (addition, deletion, substitution, and / or insertion of an amino acid residue).

[0230] Changes can be made to one or more structural regions and / or one or more CDRs. Variants are optionally provided by CDR mutagenesis. Changes typically do not result in loss of function, so an antibody comprising a subsequently altered amino acid sequence may retain an ability to bind to ICOS. It may retain the same quantitative binding capacity as an antibody in which the change is not made, for example, as measured in an assay described in this document. The antibody comprising a subsequently altered amino acid sequence may have an enhanced ability to bind to ICOS.

[0231] The alteration may comprise replacing one or more amino acid residues with a non-standard, unnaturally occurring amino acid, modifying one or more amino acid residues in a non-standard or unnaturally occurring form, or inserting one or more non-standard, unnaturally occurring amino acids into the sequence. Examples of the number and locations of alterations in the sequences of the invention are described throughout this document. Naturally occurring amino acids include the 20 “standard” L amino acids. Petition 870230019896, dated 08 / 03 / 2023, page 102 / 576 83 / 456 identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, D, E by their standard single-letter codes. Non-standard amino acids include any other residue that can be incorporated into a polypeptide backbone or that can result from the modification of an existing amino acid residue. Non-standard amino acids can occur naturally or occur unnaturally.

[0232] The term variant, as used in this document, refers to a peptide or nucleic acid that differs from a parent polypeptide or nucleic acid by one or more deletions, substitutions, or additions of amino acids or nucleic acids, but retains one or more specific functions or biological activities of the parent molecule. Amino acid substitutions include alterations in which an amino acid is replaced by a different naturally occurring amino acid residue. Such substitutions can be classified as conservative, in which case an amino acid residue contained in a polypeptide is replaced by another naturally occurring amino acid of similar character or with respect to polarity, side chain functionality, or size. Such conservative substitutions are well known in the art.The substitutions covered by the present invention may also be non-conservative, wherein an amino acid residue present in a peptide is replaced by an amino acid with different properties, such as the naturally occurring amino acid of a different group (for example, replacing a charged or hydrophobic amino acid with alanine), or alternatively, wherein a naturally occurring amino acid is replaced by an unconventional amino acid. In some embodiments, the substitutions of... Petition 870230019896, dated 08 / 03 / 2023, p. 103 / 576 84 / 456 amino acids are conservative. Also encompassed in the term variant when used with reference to a polynucleotide or polypeptide, it refers to a polynucleotide or polypeptide that may vary in primary, secondary, or tertiary structure, compared to a reference polynucleotide or polypeptide, respectively (e.g., compared to a wild-type polynucleotide or polypeptide).

[0233] In some respects, synthetic variants, recombinant variants, or polynucleotide variants or chemically modified polypeptide variants isolated or generated using methods well known in the art may be used. “Modified variants” may include conservative or non-conservative amino acid alterations, as described below. Polynucleotide alterations may result in substitutions, additions, deletions, fusions, and truncations of amino acids in the polypeptide encoded by the reference sequence. Some aspects used include insertion variants, deletion variants, or variants substituted with amino acid substitutions, including insertions and substitutions of amino acids and other molecules that do not normally occur in the peptide sequence that is the basis of the variant, for example, but not limited to, insertion of ornithine, which does not normally occur in human proteins.The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of the polypeptide that does not substantially alter the polypeptide's activity. For example, a conservative substitution refers to the replacement of one amino acid residue with a different amino acid residue that has similar chemical properties (e.g., acidic, basic, etc.). Petition 870230019896, dated 08 / 03 / 2023, p. 104 / 576 85 / 456 positively or negatively charged, polar or nonpolar, etc.). Conservative amino acid substitutions include the substitution of leucine for isoleucine or valine, aspartate for glutamate, or threonine for serine. Conservative substitution tables that provide functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Acid Aspartic (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See also Creighton, Proteins, W.H. Freeman and Company (1984), incorporated by reference in its entirety.) In some embodiments, individual substitutions, deletions, or additions that alter, add, or eliminate a single amino acid or a small percentage of amino acids may also be considered “conservative substitutions” if the alteration does not reduce the activity of the peptide. Insertions or deletions are typically in the range of about 1 to 5 amino acids. The choice of conservative amino acids may be selected based on the location of the amino acid to be substituted in the peptide, for example, if the amino acid is outside the peptide and exposed to solvents or inside and not exposed to solvents.

[0234] The amino acid that will replace an existing amino acid based on the location of the existing amino acid, including its exposure to solvents (i.e., whether the amino acid is exposed to solvents or is present on the surface). Petition 870230019896, dated 08 / 03 / 2023, p. 105 / 576 86 / 456 external peptide or polypeptide compared to internally located amino acids not exposed to solvents) can be selected. The selection of these conservative amino acid substitutions is well known in the art, for example, as described in Dordo et al. J. Mol. Biol., 1999, 217, 721 to 739 and Taylor et al. J. Theor. Biol. 119(1986);205 to 218 and S. French and B. Robson, J. Mol. Evol. 19(1983)171. Therefore, suitable conservative amino acid substitutions for amino acids on the exterior of a protein or peptide (e.g., amino acids exposed to a solvent) can be selected, for example, but without limitation, the following substitutions can be used: substitution of Y for F, T with S or K, P with A, E with D or Q, N with D or G, R with K, G with N or A, T with S or K, D with N or E, I with L or V, F with Y, S with T or A, R with K, G with N or A, K with R, A with S, K or P.

[0235] In alternative embodiments, suitable conservative amino acid substitutions for amino acids within a protein or peptide may be selected, for example, suitable conservative substitutions for amino acids within a protein or peptide may be used (i.e., amino acids are not exposed to a solvent), for example, but without limitation, the following conservative substitutions may be used: where Y is substituted for F, T with A or S, I with L or V, W with Y, M with L, N with D, G with A, T with A or S, D with N, I with L or V, F with Y or L, S with A or T and A with S, G, T or V. In some embodiments, non-conservative amino acid substitutions are also covered within the term variants.

[0236] The invention includes methods for producing Petition 870230019896, dated 08 / 03 / 2023, p. 106 / 576 87 / 456 antibodies containing VH and / or VL domain variants of the antibody VH and / or VL domains shown in the attached sequence listing. Such antibodies can be produced by a method comprising

[0237] (i) provide, by means of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent antibody VH domain, a VH antibody domain that is an amino acid sequence variant of the parent antibody VH domain,

[0238] wherein the VH domain of the parent antibody is the VH domain of any of the antibodies STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or a VH domain comprising the heavy chain complementarity-determining regions of any of these antibodies,

[0239] (ii) optionally combine the endowed VH domain with a VL domain to provide a VH / VL combination, and

[0240] (iii) test the VH / VL domain combination or VH domain combination then provided in order to identify an antibody with one or more desired characteristics.

[0241] Desired features include binding to human ICOS, binding to mouse ICOS, and binding to other non-human ICOS, such as cynomolgus ICOS. Antibodies with comparable or superior affinity to mouse and / or human ICOS may be identified. Other desired features include enhancing effector T cell function indirectly, through depletion of immunosuppressive TRegs, or directly through activation of ICOS signaling in effector T cells. Identifying an antibody with a desired feature may involve Petition 870230019896, dated 08 / 03 / 2023, page 107 / 576 88 / 456 the identification of an antibody with a functional attribute described in this document, such as affinity, cross-reactivity, specificity, ICOS receptor agonism, neutralization potency and / or promotion of T cell-dependent death, any of which may be determined in the assays as described in this document.

[0242] When VL domains are included in the method, the VL domain may be a VL domain of any one of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or may be a variant provided by adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence of a parental VL domain, wherein the primary VL domain is the VL domain of any one of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 and STIM009 or a VL domain comprising the complementarity-determining regions of the light chain of any one of these antibodies.

[0243] Methods for generating variant antibodies may optionally comprise producing copies of the antibody or of the VH / VL domain combination. The methods may further comprise expressing the resulting antibody. It is possible to produce nucleotide sequences corresponding to an antibody VH and / or VL domain, optionally, in one or more expression vectors. Suitable expression methods, including recombinant expression in host cells, are detailed in this document. Nucleic acid coding and expression methods Petition 870230019896, dated 08 / 03 / 2023, page 108 / 576 89 / 456

[0244] Isolated nucleic acid may be provided, encoding antibodies according to the present invention. The nucleic acid may be DNA and / or RNA. DNA, cDNA, mRNA or other genomic RNA, of synthetic origin, or any combination thereof, may encode an antibody.

[0245] The present invention provides constructs in the form of plasmids, vectors, transcripts or expression cassettes comprising at least one polynucleotide as above. Exemplary nucleotide sequences are included in the sequence listing. Reference to a nucleotide sequence as presented herein covers a DNA molecule with the specified sequence and covers an RNA molecule with the specified sequence wherein U is replaced by T, unless the context otherwise requires.

[0246] The present invention also provides a recombinant host cell comprising one or more nucleic acids encoding the antibody. Methods for producing the encoded antibody may comprise nucleic acid expression, for example, by culturing recombinant host cells containing the nucleic acid. The antibody may then be obtained and may be isolated and / or purified using any suitable technique, then used as appropriate. A production method may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

[0247] Systems for cloning and expressing a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, cells Petition 870230019896, dated 08 / 03 / 2023, page 109 / 576 90 / 456 vegetables, filamentous fungi, yeast and bacillovirus systems, and transgenic plants and animals.

[0248] The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is E. coli. Expression in cultured eukaryotic cells is also available to those skilled in the art as an option for production. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese Hamster Ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2 / 0 mouse myeloma cells, human embryonic kidney cells, human embryonic retinal cells, and many others.

[0249] Vectors may contain appropriate regulatory sequences, including promoter sequences, termination sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences, as appropriate. The nucleic acid encoding an antibody may be introduced into a host cell. The nucleic acid may be introduced into eukaryotic cells by various methods, including transfection with calcium phosphate, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, for example, vaccinia or, for insect cells, baculoviruses. The introduction of nucleic acid into the host cell, in particular a eukaryotic cell, may use a viral or plasmid-based system. The plasmid system may be maintained episomally or may be incorporated into the host cell or an artificial chromosome. Incorporation may Petition 870230019896, dated 08 / 03 / 2023, page 110 / 576 91 / 456 occur either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques include transformation with calcium chloride, electroporation, and transfection using a bacteriograph. Introduction can be followed by nucleic acid expression, for example, by culturing host cells under conditions for gene expression, then optionally isolating or purifying the antibody.

[0250] The nucleic acid of the invention can be integrated into the genome (e.g., chromosome) of the host cell. Integration can be promoted by the inclusion of sequences that promote recombination with the genome, according to standard techniques.

[0251] The present invention also provides a method comprising using the nucleic acid described herein in an expression system in order to express an antibody. THERAPEUTIC USE

[0252] An antibody described in this document can be used in a method of treating the human or animal body by therapy. Antibodies are useful in increasing the response of effector T cells, which is beneficial for a variety of diseases or conditions, including treatment against cancers or solid tumors and in the context of vaccination. The increased Teff response can be obtained with the use of an antibody that modulates the balance or ratio between Teffs and Tregs in favor of Teff activity.

[0253] Anti-ICOS antibodies can be used to deplete regulatory T cells and / or enhance the effector T cell response in a patient and can be administered Petition 870230019896, dated 08 / 03 / 2023, page 111 / 576 92 / 456 to a patient to treat a disease or condition treatable with therapy by depleting regulatory T cells and / or increasing the response of effector T cells.

[0254] An antibody of the present invention, or a composition comprising such an antibody molecule or the coding nucleic acid thereof, may be used or provided for use in any of these methods. The use of the antibody, or of a composition comprising the same or the coding nucleic acid thereof, for the manufacture of a medicament for use in such a method is also envisioned. The method typically comprises administering the antibody or composition to a mammal. Suitable formulations and methods of administration are described throughout this document.

[0255] An ideal therapeutic use of antibodies is the treatment of cancer. Cancer may be a solid tumor, for example, renal cell carcinoma (optionally renal cell carcinoma, e.g., clear cell renal cell carcinoma), head and neck cancer, melanoma (optionally, malignant melanoma), non-small cell lung cancer (e.g., adenocarcinoma), bladder cancer, ovarian cancer, cervical cancer, gastric cancer, liver cancer, pancreatic cancer, breast cancer, testicular germ cell carcinoma, or metastases from a solid tumor, such as those in striped cells, or it may be a liquid hematologic tumor, for example, lymphoma (such as Hodgkin lymphoma or non-Hodgkin lymphoma, e.g., diffuse large B-cell lymphoma, DLBCL) or leukemia (e.g., acute myeloid leukemia). An anti-ICOS antibody may increase clearance. Petition 870230019896, dated 08 / 03 / 2023, page 112 / 576 93 / 456 tumoral in melanoma, head and neck cancer and non-small cell lung cancer and other cancers with a moderate to high mutational burden

[26] . By enhancing the patients' immune response to their neoplastic lesions, immunotherapy using an anti-ICOS antibody offers the expectation of durable cures or long-term remissions, potentially even in the context of advanced-stage disease.

[0256] Cancers are a diverse group of diseases, but anti-ICO antibodies offer the possibility of treating a variety of different cancers by exploiting the patient's own immune system, which has the potential to kill any cancer cell through the recognition of mutant or overexpressed epitopes that distinguish cancer cells from normal tissue. By modulating the Teff / Treg balance, anti-ICO antibodies can activate and / or promote immune recognition and death of cancer cells. Therefore, while anti-ICO antibodies are useful therapeutic agents for a wide variety of cancers, there are particular categories of cancers for which anti-ICO therapy is especially suitable and / or in which anti-ICO therapy may be effective when other therapeutic agents are not.

[0257] One of these groups is cancer that is positive for ICOS ligand expression. Cancer cells can acquire ICOS ligand expression, as described for melanoma

[27] . ICOS ligand expression can provide cells with a selective advantage since the ligand expressed on the surface binds to ICOS on Tregs, promoting Treg expansion and activation and thus suppressing the immune response against cancer. Cancer cells Petition 870230019896, dated 08 / 03 / 2023, page 113 / 576 94 / 456 cells expressing the ICOS ligand may depend on its survival in this immune system suppression by Tregs and thus would be vulnerable to treatment with anti-ICOS antibodies targeting Tregs. This also applies to cancers derived from cells that naturally express the ICOS ligand. Continued expression of the ICOS ligand by these cells again provides a survival advantage through immunosuppression. A cancer expressing the ICOS ligand may be derived from antigen-presenting cells such as B cells, dendritic cells, and monocytes, and may be a liquid hematologic tumor, such as those mentioned in this document. Interestingly, these cancer types have also been shown to be rich in ICOS and FOXP3 expression (TCGA data) – see Example 25.Example 20 here demonstrates the effectiveness of exemplary anti-ICOS antibodies in the treatment of cancerous B-cell-derived tumors (A20 syngeneic cells) expressing the ICOS ligand.

[0258] Therefore, anti-ICOS antibodies can be used in treatment methods against cancers that are positive for ICOS ligand expression. Furthermore, a cancer to be treated with anti-ICOS antibody, according to the present invention, can be one that is positive for ICOS and / or FOXP3 expression and also optionally expresses the ICOS ligand.

[0259] Patients may undergo testing to determine if the cancer is positive for the expression of the protein of interest (e.g., ICOS ligand, ICOS and / or FOXP3), for example, by taking a test sample (e.g., tumor biopsy) from the patient and determining the expression of the protein of interest. Patients whose cancer Petition 870230019896, dated 08 / 03 / 2023, page 114 / 576 95 / 456 cells characterized as positive for the expression of one, two, or all of these proteins of interest are selected for treatment with anti-ICOS antibody. As discussed in another section, anti-ICOS antibody can be used as monotherapy or in combination with one or more other therapeutic agents.

[0260] Anti-ICO antibodies also offer hope to patients whose cancers are refractory to treatment with antibodies or other drugs targeting immune checkpoint molecules, such as CTLA-4, PD-1, PD-L1, CD137, GITR, or CD73. These immunotherapies are effective against some cancers, but in some cases a cancer may not respond or may become unresponsive to continued antibody treatment. In common with antibodies to immune checkpoint inhibitors, anti-ICO antibodies modulate the patient's immune system—however, an anti-ICO antibody may succeed when these other antibodies fail. This paper shows that animals carrying A20 B-cell lymphomas can be treated with anti-ICO antibodies to reduce tumor growth, shrink the tumor, and indeed remove the tumor from the body, whereas treatment with an anti-PD-L1 antibody was no better than the control.The A20 cell line has also been reported to be resistant to anti-CTLA-4

[28] .

[0261] Consequently, anti-ICO antibodies can be used in treatment methods against cancers that are refractory to treatment with one or more immunotherapies, such as (any or all) an anti-CTLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody, anti-GITR antibody, or anti-CD73 antibody. A cancer Petition 870230019896, dated 08 / 03 / 2023, page 115 / 576 96 / 456 can be characterized as refractory to treatment with an antibody or other drug if treatment with that antibody or drug does not significantly reduce cancer growth, for example, if a tumor continues to grow or does not decrease in size, or if after a period of response the tumor resumes its growth. Non-response to a therapeutic agent can be determined ex vivo by testing a sample (e.g., tumor biopsy sample) for cancer cell killing or growth inhibition and / or in the clinical setting by observing (e.g., using imaging technology, including MRI) that a patient treated with the therapy does not respond to treatment. Patients whose cancer has been characterized as refractory to treatment with such immunotherapy are selected for treatment with anti-ICOS antibodies.

[0262] In addition, anti-ICOS antibodies can be used to treat B-cell-derived cancers that are resistant to treatment with an anti-CD20 antibody. Anti-ICOS antibodies represent a treatment for cancers that do not respond to or become resistant to anti-CD20 antibody therapy such as rituximab. The anti-ICOS antibody can be used as a second-line (or complementary or additional) treatment for these cancers. Cancer resistant to anti-CD20 antibodies can be a B-cell cancer, for example, B-cell lymphoma, such as diffuse large B-cell lymphoma. The resistance of a cancer to anti-CD20 can be determined ex vivo by testing a sample (e.g., tumor biopsy sample) for the killing of cancer cells or inhibition of growth by the anti-CD20 antibody, and / or in the clinical setting by observing that a Petition 870230019896, dated 08 / 03 / 2023, page 116 / 576 97 / 456 patients treated with the anti-CD20 antibody do not respond to treatment. Alternatively or additionally, the cancer (e.g., a tumor biopsy sample) can be tested to assess CD20 expression, where an absence or low level of CD20 expression indicates loss of sensitivity to the anti-CD20 antibody.

[0263] Samples obtained from patients can then be tested to determine the surface expression of a protein of interest, for example, ICOS ligand, ICOS, FOXP3 and / or a target receptor to which another therapeutic agent (e.g., anti-receptor antibody) is directed. The target receptor may be CD20 (to which anti-CD20 antibody therapy such as rituximab is directed), or another receptor such as PD1, EGFR, HER2 or HER3. Surface expression of ICOS ligand, ICOS, FOXP3 and / or lack or loss of surface expression of the target receptor is an indication that the cancer is amenable to anti-ICOS antibody therapy.Anti-ICOS antibodies may be provided for administration to a patient whose cancer is characterized by surface expression of the ICOS ligand, ICOS, FOXP3 and / or by the lack or loss of surface expression of a target receptor, optionally when the patient has been previously treated with anti-CTLA4, anti-PD1, anti-PD-L1 or with an antibody to the target receptor and has not responded or has ceased to respond to treatment with that antibody, as measured, for example, by continued or renewed growth of cancer cells, e.g., increased tumor size.

[0264] Any suitable method may be employed to determine whether cancer cells test positive for expression on the surface of a protein, Petition 870230019896, dated 08 / 03 / 2023, page 117 / 576 98 / 456 such as the ICOS ligand, CD20, or other target receptors mentioned in this document. A typical method is immunohistochemistry, in which a sample of cells (e.g., a tumor biopsy sample) comes into contact with an antibody for the protein of interest, and antibody binding is detected using a labeled reagent—typically a second antibody that recognizes the Fc region of the first antibody and carries a detectable label, such as a fluorescent marker. A sample can be declared positive when at least 5% of the cells are labeled, as visualized by cell staining or other label detection. Optionally, an upper limit, such as 10% or 25%, can be used. Antibody will generally be overused. Reagent antibodies for the molecules of interest are readily available or can be generated by direct methods.In order to test for the ICOS ligand, the MAB1651 antibody is currently available in 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 target ligand or ICOS receptor of interest is an alternative technique

[27] .

[0265] An additional indication that a tumor will respond to anti-ICOS antibody treatment is the presence of Tregs in the tumor microenvironment. Activated Tregs are characterized by surface expression with high ICOS and high Foxp3 content. The presence of Tregs in a tumor, especially in high numbers, provides an additional basis on which a patient can be selected for anti-ICOS antibody treatment. Tregs can be detected in an ex vivo tumor biopsy sample, for example, by Petition 870230019896, dated 08 / 03 / 2023, p. 118 / 576 99 / 456 immunohistochemistry (assay for co-expression of Foxp3 and ICOS, using antibodies to the target protein followed by marker detection, as described above) or by single-cell scattering of a sample for use in FACS with antibodies labeled for ICOS and Foxp3. FACS methods are exemplified in Example 17 and Example 18.

[0266] Anti-ICO antibodies can be used to treat cancers associated with infectious agents, such as virus-induced cancers. This category includes squamous cell carcinoma of the head and neck, cervical cancer, Merkel cell carcinoma, and many others. Cancer viruses include HBV, HCV, HPV (cervical cancer, oropharyngeal cancer), and EBV (Burkitts lymphoma, gastric cancer, Hodgkin lymphoma, other EBV-positive B-cell lymphomas, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease). The International Agency for Research on Cancer (Monograph 100B) has identified the following major cancer sites associated with infectious agents:

[0267] · Stomach / Gastric: Helicobacter pylori

[0268] • Liver: Hepatitis B virus, virus Hepatitis C (HCV), Opisthorchis viverrini, Clonorchis sinensis

[0269] • Cervix: human papillomavirus (HPV) with or without HIV

[0270] • Anogenital (penis, vulva, vagina, anus): HPV with or without HIV

[0271] • Nasopharynx: Epstein-Barr virus (EBV)

[0272] • Oropharynx: HPV with or without tobacco or alcohol consumption

[0273] • Kaposi's sarcoma: Human herpesvirus Petition 870230019896, dated 08 / 03 / 2023, page 119 / 576 100 / 456 type 8 with or without HIV

[0274] · Non-Hodgkin lymphoma: H. pylori, EBV with or without HIV, HCV, Human T-cell lymphotropic virus type 1

[0275] · Hodgkin's Lymphoma: EBV with or without HIV

[0276] · Bladder: Schistosoma haematobium.

[0277] The antibodies according to the present invention can be used for the treatment of cancer associated with or induced by any of these infectious agents, such as the cancers specified above.

[0278] Stimulation of the effector T cell response may also contribute to immunity against infectious disease and / or to recovery from infectious disease in a patient. Thus, an anti-ICOS antibody may be used to treat infectious diseases by administering the antibody to a patient.

[0279] Infectious diseases include those caused by pathogens, for example, bacterial, fungal, viral, or protozoan pathogens, and treatment may involve promoting an immune response in a patient against the pathogen infection. An example of a bacterial pathogen is tuberculosis. Examples of viral pathogens are hepatitis B and HIV. Examples of protozoan pathogens are the Plasmodium species that cause malaria, such as P. falciparum.

[0280] The antibody can be used to treat infections, for example, infection by any pathogen mentioned in this document. The infection may be persistent or chronic. The infection may be localized or systemic. Prolonged contact between a pathogen and the immune system may lead to exhaustion of the system. Petition 870230019896, dated 08 / 03 / 2023, page 120 / 576 101 / 456 immunological or to the development of tolerance (manifested, for example, by increased levels of Tregs and a shift in the Treg:Teff balance in favor of Tregs) and / or to immune evasion by the pathogen, through the evolution and modification of the pathogenic antigens displayed. These features reflect similar processes that occur in cancer. Anti-ICOS antibodies present a therapeutic approach for the treatment of infection by a pathogenic agent, for example, chronic infection, through the modulation of the Treg:Teff ratio in favor of Teff and / or other effects described in this document.

[0281] Treatment may be for patients who have been diagnosed as having an infectious disease or an infection. Alternatively, treatment may be preventative and administered to a patient in order to prevent the contraction of a disease, for example, as a vaccine, as described throughout this document.

[0282] Furthermore, it has been proposed that an immune response, particularly a systemic immune response dependent on IFNγ, could be beneficial for the treatment of Alzheimer's disease and other CNS pathologies that share a neuroinflammatory component

[29] . Document no. WO2015 / 136541 proposed the treatment of Alzheimer's disease with the use of an anti-PD-1 antibody. Anti-ICOS antibodies can be used in the treatment of Alzheimer's disease or other neurodegenerative diseases, optionally in combination with one or more other immunomodulators (e.g., antibody to PD-1). COMBINATION THERAPY

[0283] Treatment with an immunomodulatory antibody, such as anti-CTLA4, anti-PD1 or anti-PDL1, Petition 870230019896, dated 08 / 03 / 2023, page 121 / 576 102 / 456, especially one with Fc effector function, can create an environment in which further depletion of highly immunosuppressive ICOS cells is beneficial. Combining an anti-ICOS antibody with such an immunomodulator may be advantageous to enhance its therapeutic effects.

[0284] A patient who has been treated with an immunomodulatory antibody (e.g., anti-PDL-1, anti-PD-1, anti-CTLA-4) may particularly benefit from treatment with an anti-ICOS antibody. One reason for this is that an immunomodulatory antibody can increase the number of ICOS-positive Tregs (e.g., intratumoral Tregs) in the patient. This effect is also observed with certain other therapeutic agents, such as recombinant IL-2. The anti-ICOS antibody may reduce and / or reverse a surge or increase in ICOS+ Tregs (e.g., intratumoral Tregs) resulting from the patient's treatment with another therapeutic agent. A patient selected for treatment with an anti-ICOS antibody may thus be one who has already received treatment with a first therapeutic agent, the first therapeutic agent being an antibody (e.g., immunomodulatory antibody) or another agent (e.g., IL-2) that increases the number of ICOS+ Tregs in the patient.

[0285] Immunomodulators with which an anti-ICOS antibody may be combined include antibodies to any of the following: PDL1 (e.g., avelumab), PD-1 (e.g., pembrolizumab or nivolumab), or CTLA-4 (e.g., ipilimumab or tremelimumab). An anti-ICOS antibody may be combined with pidilizumab. In other embodiments, an anti-ICOS antibody is not administered in combination with the anti-CTLA-4 antibody and / or is optionally administered in combination with a therapeutic antibody that is not an antibody. Petition 870230019896, dated 08 / 03 / 2023, p. 122 / 576 103 / 456 anti-CTLA-4.

[0286] For example, an anti-ICOS antibody can be used in combination therapy with an anti-PDL1 antibody. Preferably, the anti-ICOS antibody is one that mediates ADCC, ADCP, and / or CDC. Preferably, the anti-PDL1 antibody is one that mediates ADCC, ADCP, and / or CDC. An example of such combination therapy is the administration of an anti-ICOS antibody with an anti-PDL1 antibody where both antibodies have positive effector constant regions. In this way, both the anti-ICOS antibody and the anti-PDL1 antibody can be capable of mediating ADCC, CDC, and / or ADCP. The effector function of Fc and the selection of constant regions are described in detail in other sections of this document, but as an example, a human anti-ICOS IgG1 can be combined with a human anti-PD-L1 IgG1. The anti-ICOS antibody and / or the anti-PD-L1 antibody may comprise a wild-type human IgG1 constant region.Alternatively, the positive effector constant region of an antibody may be one designed to enhance effector function, for example, enhanced CDC, ADCC, and / or ADCP. Examples of antibody constant regions, including wild-type human IgG1 sequences and mutations that alter effector function, are discussed in detail throughout this document.

[0287] Anti-PDL1 antibodies with which an anti-ICOS antibody can be combined include:

[0288] · The anti-PDL1 antibody that inhibits the binding of PD-1 to PDL1 and / or inhibits PDL1, optionally, as a positive effector human IgG1;

[0289] · The anti-PD-1 antibody that inhibits the binding of PD-1 to PDL1 and / or PDL2; Petition 870230019896, dated 08 / 03 / 2023, p. 123 / 576 104 / 456

[0290] · Avelumab, a human IgG1 antibody that inhibits the binding of PD-1 to PDL-1. See document no. WO2013 / 079174;

[0291] · Durvalumab (or “MEDI4736”), a human IgG1 variant antibody that has the L234A, L235A and 331 mutations. See document no. WO2011 / 066389;

[0292] · Atezolizumab, a variant human IgG1 antibody with N297A, D356E and L358M mutations. See document no. US2010 / 0203056;

[0293] · BMS-936559, a human IgG4 antibody comprising S228P with mutation. See document no. WO2007 / 005874.

[0294] Numerous additional examples of anti-PD-L1 antibodies are disclosed in this document and others are known in the art. Characterization data for many of the anti-PD-L1 antibodies mentioned in this document were published in documents no. US9,567,399 and no. US9,617,338, which are incorporated herein by reference. Examples of anti-PD-L1 antibodies have VH and / or VL domains that comprise the HCDRs and / or LCDRs of any of 1D05, 84G09, 1D05 HC mutant 1, 1D05 HC mutant 2, 1D05 HC mutant 3, 1D05 HC mutant 4, 1D05 LC mutant 1, 1D05 LC mutant 2, 1D05 LC mutant 3, 411B08, 411C04, 411D07, 385F01, 386H03, 389A03, 413D08, 413G05, 413F09, 414B06, or 416E01, as indicated in documents US9,567,399 or US9,617,338.The antibody may comprise the VH and VL domains of either of these antibodies and may optionally comprise a heavy and / or light chain having the amino acid sequence of the heavy and / or light chain of either of these antibodies. The VH and VL domains of these anti-PD-L1 antibodies are described. Petition 870230019896, dated 08 / 03 / 2023, p. 124 / 576 105 / 456 additionally throughout this document.

[0295] Other examples of anti-PD-L1 antibodies have VH and / or VL domains comprising the HCDRs and / or LCDRs of KN-035, CA-170, FAZ-053, M7824, ABBV-368, LY-3300054, GNS1480, YW243.55.S70, REGN3504 or an anti-PD-L1 antibody disclosed in any of the documents no. WO2017 / 034916, WO2017 / 020291, WO2017 / 020858, WO2017 / 020801, WO2016 / 111645, WO2016 / 197367, WO2016 / 061142, WO2016 / 149201, WO2016 / 000619, WO2016 / 160792, WO2016 / 022630, WO2016 / 007235, WO2015 / 179654, WO2015 / 173267, WO2015 / 181342, WO2015 / 109124, WO2015 / 112805, WO2015 / 061668, WO2014 / 159562, WO2014 / 165082, WO2014 / 100079, WO2014 / 055897, WO2013 / 181634, WO2013 / 173223, WO2013 / 079174, WO2012 / 145493, WO2011 / 066389, WO2010 / 077634, WO2010 / 036959, WO2010 / 089411 WO2007 / 005874. The antibody may comprise the VH domain and VL of any of these antibodies and may optionally comprise a heavy and / or light chain having the amino acid sequence of the heavy and / or light chain of any of these antibodies. The anti-ICOS antibody used in combination therapy with anti-PD-Ll may be an antibody of the present invention as disclosed herein. Alternatively, the anti-ICOS antibody may comprise the CDRs or a VH and / or VL domain of an anti-ICOS antibody disclosed in any of the following publications:

[0296] WO2016154177, US2016304610 - for example, any of the antibodies 7F12, 37A10, 35A9, 36E10, 16G10, 37A10S713, 37A10S714, 37A10S715, 37A10S716, 37A10S717, 37A10S718, 16G10S71, 16G10S72, 16G10S73, 16G10S83, 35A9S79, 35A9S710 or 35A9S89;

[0297] WO16120789, US2016215059 - for example, the antibody known as 422.2 and / or H2L5; Petition 870230019896, dated 08 / 03 / 2023, page 125 / 576 106 / 456

[0298] WO14033327, EP2892928, US2015239978 - for example, the antibody known as 314-8 and / or produced from the CNCM I-4180 hybridoma;

[0299] WO12131004, EP2691419, US9376493, US20160264666 - for example, the Icos145-1 antibody and / or antibody produced by the CNCM I-4179 hybridoma;

[0300] WO10056804 - for example, the JMAb antibody 136 or 136;

[0301] WO9915553, EP1017723B1, US7259247, US7132099, US7125551, US7306800, US7722872, WO05103086, EP1740617, US8318905, US8916155 - for example, the antibody MIC-944 or 9F3;

[0302] WO983821, US7932358B2, US2002156242, EP0984023, EP1502920, US7030225, US7045615, US7279560, US7226909, US7196175, US7932358, US8389690, WO02070010, EP1286668, EP1374901, US7438905, US7438905, WO0187981, EP1158004, US6803039, US7166283, US7988965, WO0115732, EP1125585, US7465445, US7998478 - for example, any JMAb antibody, for example, any one of JMAb-124, JMAb126, JMAb-127, JMAb-128, JMAb-135, JMAb-136, JMAb-137, JMAb138, JMAb-139, JMAb-140, JMAb-141, for example, JMAb136;

[0303] WO2014 / 089113 - for example, antibody 17G9;

[0304] WO12174338;

[0305] US2016145344;

[0306] WO11020024, EP2464661, US2016002336, US2016024211, US8840889;

[0307] US8497244.

[0308] The anti-ICOS antibody optionally comprises the CDRs of 37A10S713, as disclosed in Petition 870230019896, dated 08 / 03 / 2023, p. 126 / 576 107 / 456 document no. WO2016154177. It may comprise the VL and VH domains of 37A10S713 and may optionally have the antibody heavy and light chains of 37A10S713.

[0309] The combination of an anti-ICOS antibody with an immunomodulator may provide an increased therapeutic effect compared to monotherapy and may allow therapeutic benefit to be achieved with a lower dose of the immunomodulator(s). Thus, for example, an antibody (e.g., anti-PD-L1 antibody, optionally ipilimumab) that is used in combination with an anti-ICOS antibody may be dosed at 3 mg / kg instead of a more usual dose of 10 mg / kg. The administration regimen of anti-PD-L1 or other antibody may involve intravenous administration over a period of 90 minutes every 3 weeks for a total of 4 doses.

[0310] An anti-ICOS antibody can be used to increase the sensitivity of a tumor to treatment with an anti-PD-L1 antibody, which can be recognized as a dose reduction at which the anti-PD-L1 antibody exerts a therapeutic benefit. Thus, the anti-ICOS antibody can be administered to a patient to reduce the dose of anti-PD-L1 antibody effective in treating cancer or a tumor in the patient. Administration of anti-ICOS antibody can reduce the recommended or required dosage of anti-PD-L1 antibody to that patient by, for example, 75%, 50%, 25%, 20%, 10% or less compared to a dosage when the anti-PD-L1 antibody is administered without anti-ICOS. The patient can be treated by administering anti-ICOS antibody and anti-PD-L1 antibody in a combination therapy, as described in this document. Petition 870230019896, dated 08 / 03 / 2023, page 127 / 576 108 / 456

[0311] The benefit of combining anti-PD-L1 with anti-ICOS may extend to a reduction in the dosage of each agent when compared to use as monotherapy. The anti-PD-L1 antibody can be used to reduce the dose at which the anti-ICOS antibody exerts a therapeutic benefit and thus can be administered to a patient to reduce the dose of anti-ICOS antibody effective in treating cancer or a tumor in the patient. In this way, an anti-PD-L1 antibody can reduce the recommended or required dosage of anti-ICOS antibody administration to that patient by, for example, 75%, 50%, 25%, 20%, 10% or less compared to a dosage when the anti-ICOS antibody is administered without anti-PD-L1. The patient can be treated by administering anti-ICOS antibody and anti-PD-L1 antibody in a combination therapy, as described in this document.

[0312] As discussed in Example 22, treatment with anti-PD-L1 antibody, especially effector Fc-positive antibody, does not appear to increase ICOS expression in Teff cells. This is advantageous when administering such antibodies in combination with effector-positive anti-ICOS antibodies, where an increase in ICOS expression in Teffs makes these cells undesirably more susceptible to deletion by the anti-ICOS antibody. In combination with anti-PD-L1, anti-ICOS therapy can therefore exploit differential ICOS expression in Teffs compared to Tregs, preferably targeting high-ICOS concentration Tregs for depletion. This, in turn, alleviates Teff suppression and has the ultimate effect of promoting effector T cell response in a patient. The effect of targeting immune checkpoint molecules on expression Petition 870230019896, dated 08 / 03 / 2023, page 128 / 576 109 / 456 of ICOS in T cells was also previously studied (see Figure S6C in reference

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

[0313] As described in this document, a single dose of anti-ICOS antibody may be sufficient to provide a therapeutic effect, especially in combination with other therapeutic agents such as anti-PD-L1 antibody. In tumor therapy, the rationale for this single-dose benefit may be that the anti-ICOS antibody mediates the effect of the tumor, at least in part, by resetting or altering the tumor microenvironment sufficiently to make the tumor more sensitive to immune attack and / or the effects of other immunomodulators, such as those mentioned. The resetting of the tumor microenvironment is triggered, for example, by the depletion of ICOS-positive tumor infiltration T-regs. Thus, for example, a patient may be treated with a single dose of an anti-ICOS antibody followed by one or multiple doses of anti-PD-L1 antibody.During a treatment period, for example, six months or a year, the anti-ICOS antibody may be administered in a single dose, whereas other agents, for example, anti-PD-L1 antibody, are optionally administered multiple times during that period. Petition 870230019896, dated 08 / 03 / 2023, page 129 / 576 110 / 456 treatment, preferably with at least one of these doses being administered subsequent to treatment with the anti-ICOS antibody.

[0314] Other examples of combination therapy include combining anti-ICOS antibodies with:

[0315] - an antagonist of an adenosine A2A receptor (“A2AR inhibitor”);

[0316] - a CD137 agonist (e.g., antibody agonist);

[0317] - an antagonist of the enzyme indoleamine-2,3 dioxygenase that catalyzes the breakdown of tryptophan (“IDO inhibitor”). IDO is an immune checkpoint, activated in dendritic cells and macrophages, that contributes to immune suppression / tolerance.

[0318] Anti-ICOS antibodies can be used in combination therapy with IL-2 (e.g., recombinant IL-2 such as aldesleukin). IL-2 can be administered in high doses (HD). Typical HD IL-2 therapy involves bolus infusion of more than 500,000 IU / kg, e.g., 600,000 or 720,000 IU / kg bolus infusion, per therapy cycle, where 10 to 15 of these bolus infusions are administered at 5- to 10-hour intervals, e.g., up to 15 bolus infusions every 8 hours, and repeating the therapy cycle approximately every 14 to 21 days for up to 6 to 8 cycles. HD IL-2 therapy has been successful in treating tumors, especially melanoma (e.g., metastatic melanoma) and renal cell carcinoma; however, its use is limited by the high toxicity of IL-2, which can cause serious adverse effects.

[0319] Treatment with high doses of IL-2 Petition 870230019896, dated 08 / 03 / 2023, page 130 / 576 111 / 456 has been shown to increase the population of ICOS-positive Tregs in cancer patients

[31] . This increase in ICOS+ TRegs after the first cycle of HD IL-2 therapy has been reported to correlate with worse clinical outcome – the higher the number of ICOS+ TRegs, the worse the prognosis. An IL2 F42K variant has been proposed as an alternative therapy to avoid this undesirable increase in ICOS+ Tregs

[32] . However, another approach would be to exploit the increase in ICOS+ TRegs using an antibody according to the present invention as a second-line therapeutic agent.

[0320] It may be beneficial to combine IL-2 therapy with anti-ICOS antibodies, taking advantage of the ability of anti-ICOS antibodies to target ICOS-expressing TRegs, inhibiting these cells and improving the prognosis for patients undergoing IL-2 therapy. Concomitant administration of IL-2 and anti-ICOS antibodies may increase the response rate, avoiding or reducing adverse events in the treated patient population. The combination may allow IL-2 to be used at lower doses compared to IL-2 monotherapy, which reduces the risk or level of adverse events from IL-2 therapy, while maintaining or increasing clinical benefit (e.g., reduction in tumor growth, solid tumor clearance, and / or reduction in metastasis). In this way, the addition of anti-ICOS may improve the treatment of patients receiving IL-2, high-dose (HD) or low-dose (LD) IL-2.

[0321] Consequently, one aspect of the invention provides a method for treating a patient by administering an anti-ICOS antibody to the patient, wherein the patient is also treated with IL-2, for example, HD IL-2. Petition 870230019896, dated 08 / 03 / 2023, page 131 / 576 112 / 456 Another aspect of the invention is an anti-ICOS antibody for use in the treatment of a patient who is also being treated with IL-2, for example, HD IL-2. The anti-ICOS antibody can be used as second-line therapy. Thus, the patient could be one who has been treated with IL-2, for example, who has received at least one cycle of HD IL-2 therapy and who has an increased level of ICOS+ TRegs. Assays can be performed on cancer cell samples, for example, tumor biopsy samples, using immunohistochemistry or FACS, as described herein, to detect ICOS-positive cells, Foxp3, ICOSL, and optionally one or more additional markers of interest. The methods may comprise determining that the patient has an increased level of ICOS+ TRegs (for example, in peripheral blood or in a tumor biopsy) after IL-2 treatment, where an increased level indicates that the patient would benefit from ICOS antibody treatment.The increase in Tregs may be relative to control (untreated) individuals or to the patient before IL-2 therapy. These patients with elevated Tregs represent a group that may not benefit from continued IL-2 treatment, but for whom a combination of anti-ICOS antibody and IL-2 therapy, or treatment with anti-ICOS antibody alone, offers therapeutic benefit. Thus, after a positive determination that the patient has an increased level of ICOS+ TRegs, additional anti-ICOS antibody and / or IL-2 therapy may be administered. Treatment with anti-ICOS antibody can selectively target and deplete ICOS+ TRegs relative to other T cell populations in these patients. This provides a therapeutic effect by alleviating immunosuppression mediated by these cells and thus... Petition 870230019896, dated 08 / 03 / 2023, page 132 / 576 113 / 456 increase the activity of Teffs against target cells, for example, tumor cells or infected cells.

[0322] Combination therapy with anti-ICOS antibodies and IL-2 can be used for any therapeutic indication described in this document and particularly to treat a tumor, for example, melanoma such as metastatic melanoma or renal cell carcinoma. Thus, in one example, the patient treated with an anti-ICOS antibody is one who has metastatic melanoma and has been treated with IL-2, for example, IL-2 HD therapy or IL-2 LD therapy.

[0323] In general, when an anti-ICOS antibody is administered to a patient who has received treatment with a first therapeutic agent (e.g., immunomodulatory antibody) or another agent (e.g., IL-2), the anti-ICOS antibody may be administered after a minimum period of, for example, 24 hours, 48 ​​hours, 72 hours, 1 week, or 2 weeks after administration of the first therapeutic agent. The anti-ICOS antibody may be administered within 2, 3, 4, or 5 weeks after administration of the first therapeutic agent. This does not preclude further administrations of any agent at any time, although it may be desirable to minimize the number of treatments administered to facilitate patient adherence and to reduce costs.Instead, the relative timing of administrations will be selected to optimize their combined effect, the first therapeutic agent creating an immunological environment (e.g., elevated ICOS+ TRegs, or antigen release as discussed below) in which the anti-ICOS antibody effect is especially advantageous. Thus, sequential administration of the first therapeutic agent followed by the second... Petition 870230019896, dated 08 / 03 / 2023, page 133 / 576 114 / 456 anti-ICOS antibody may give the first agent time to act, creating in vivo conditions in which the anti-ICOS antibody can exhibit its enhanced effect. Several administration regimens, including simultaneous or sequential combination treatments, are described herein and may be used as appropriate. When the first therapeutic agent is one that increases the number of ICOS+ Tregs in the patient, the treatment regimen for the patient may comprise determining that the patient has an increased number of ICOS+ Tregs and then administering the anti-ICOS antibody.

[0324] As noted, the use of anti-ICOS antibodies in combination therapy may provide advantages in reducing the effective dose of therapeutic agents and / or counteracting the adverse effects of therapeutic agents that increase ICOS+ TRegs in patients. Still other therapeutic benefits can be achieved by selecting a first therapeutic agent that causes the release of antigens from target cells through the death of immune cells and administering the first therapeutic agent in combination with an anti-ICOS antibody. As noted, the administration of the anti-ICOS antibody may sequentially follow the administration of the first therapeutic agent, with the administration of the two agents separated by a certain time interval, as discussed above.

[0325] Immune cell death is a recognized mode of cell death, as opposed to apoptosis. It is characterized by the release of ATP and HMGB1 from the cell and exposure of calreticulin on the plasma membrane [33, 34].

[0326] The death of immune cells in a Petition 870230019896, dated 08 / 03 / 2023, page 134 / 576 115 / 456 Target tissue or target cells promote cell involvement by an antigen-presenting cell, resulting in the display of target cell antigens which, in turn, induces antigen-specific Teff cells. Anti-ICOS antibody can increase the magnitude and / or duration of the Teff response by acting as an ICOS agonist on Teff cells. Furthermore, when the anti-ICOS antibody has Fc effector function enabled (e.g., a human IgG1 antibody), the anti-ICOS antibody can cause depletion of antigen-specific Tregs. Thus, through a combination of one or both of these effects, the balance between Teff and Treg cells is modulated in favor of increased Teff activity.The combination of an anti-ICOS antibody with a treatment that induces the death of immune cells in a target tissue or cell type, such as in a tumor or cancer cells, thus promotes an immune response in the patient against the target tissue or cells, representing a form of vaccination in which the vaccine antigen is generated in vivo.

[0327] Consequently, one aspect of the invention is a method for treating cancer in a patient by in vivo vaccination of the patient against their cancer cells. Another aspect of the invention is an anti-ICOS antibody for use in such a method. The anti-ICOS antibodies can be used in a manner comprising:

[0328] treat the patient with a therapy that causes the death of immune cells of cancer cells, resulting in the presentation of the antigen to antigen-specific effector T cells, and

[0329] administer an anti-ICOS antibody to the patient, where the anti-ICOS antibody improves the response of Petition 870230019896, dated 08 / 03 / 2023, page 135 / 576 116 / 456 antigen-specific T cell against cancer cells.

[0330] Treatments that induce the death of immune cells include radiation (e.g., ionizing cell irradiation using UVS light or γ rays), chemotherapeutic agents (e.g., oxaliplatin, anthracyclines such as doxorubicin, idarubicin, or mitoxantrone, BK channel agonists such as phloretin or primaric acid, bortezomib, cardiac glycosides, cyclophosphamide, GADD34 / PP1 inhibitors with mitomycin, PDT with hypericin, polynosinic-polycyticidyl acid, 5-fluorouracil, gemcitabine, gefitnib, erlotinib, or thapsigargin with cisplatin), and antibodies to tumor-associated antigens. A tumor-associated antigen can be any antigen that is overexpressed by tumor cells relative to non-tumor cells of the same tissue, e.g., HER2, CD20, EGFR. Suitable antibodies include herceptin (anti-HER2), rituximab (anti-CD20), or cetuximab (anti-EGFR).

[0331] Thus, it is advantageous to combine an anti-ICOS antibody with one or more of these treatments. Optionally, the anti-ICOS antibody is administered to a patient who has already received such treatment. The anti-ICOS antibody can be administered after a period of, for example, 24 hours, 48 ​​hours, 72 hours, 1 week or 2 weeks after the treatment that induces the death of immune cells, for example, between 24 and 72 hours after treatment. The anti-ICOS antibody can be administered within 2, 3, 4 or 5 weeks after treatment. Other regimens for combination therapy are discussed throughout this document.

[0332] Although “in vivo vaccination” has been described above, it is also possible to treat tumor cells to Petition 870230019896, dated 08 / 03 / 2023, page 136 / 576 117 / 456 induce the death of immune cells ex vivo, after which the cells can be reintroduced into the patient. Instead of administering the agent or treatment that induces the death of immune cells directly to the patient, the treated tumor cells are administered to the patient. The patient's treatment may be in accordance with the administration regimens described above.

[0333] As previously noted, a single dose of an anti-ICOS antibody may be sufficient to provide therapeutic benefit. Thus, in the treatment methods described in this document, the anti-ICOS antibody is optionally administered as a single dose. A single dose of anti-ICOS antibody can deplete Tregs in a patient, with consequent beneficial effects in diseases such as cancer. Transient ablation of Tregs has been reported to have antitumor effects, including reducing tumor progression, treating established tumors and metastases, and prolonging survival, and may enhance the therapeutic effect of tumor irradiation

[35] . Administering a single dose of anti-ICOS can provide this Treg depletion and can be used to enhance the effects of other therapeutic approaches used in combination, such as radiotherapy.

[0334] ANTIBODIES FOR PD-L1

[0335] An antibody to PD-L1 for use in combination with an anti-ICOS antibody, or as a separate therapeutic agent or in a multispecific antibody, as described herein, may comprise the antigen-binding site of any anti-PD-L1 antibody. Numerous examples of anti-PD-L1 antibodies are disclosed in Petition 870230019896, dated 08 / 03 / 2023, p. 137 / 576 118 / 456 of this document and others are known in the art. Characterization data for many of the anti-PD-L1 antibodies mentioned in this document were published in documents No. US9,567,399 and No. US9,617,338, which are incorporated herein by reference.

[0336] 1D05 has a variable heavy chain (Vh) region amino acid sequence of Seq ID No:33 comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The light chain nucleic acid sequence of the Vh domain is Seq ID No:34. 1D05 has a variable light chain (Vl) region amino acid sequence of Seq ID No:43, which comprises the amino acid sequence of CDRL1 from Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the amino acid CDRL2 sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the amino acid sequence of CDRL3 from Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The nucleic acid sequence of the V1 domain light chain is Seq ID No:44. The Vh domain can be combined with any of the sequences from the constant region of the heavy chain described in this document, 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. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A sequence of Petition 870230019896, dated 08 / 03 / 2023, p. 138 / 576 119 / 456 full-length heavy chain amino acids is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full-length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

[0337] 84G09 has a variable heavy chain (Vh) region amino acid sequence of Seq ID No:13 comprising the CDRH1 amino acid sequence of Seq ID No:7 (IMGT) or Seq ID No:10 (Kabat), the CDRH2 amino acid sequence of Seq ID No:8 (IMGT) or Seq ID No:11 (Kabat) and the CDRH3 amino acid sequence of Seq ID No:9 (IMGT) or Seq ID No:12 (Kabat). The nucleic acid sequence of the Vh domain light chain is Seq ID No:14. 84G09 has an amino acid sequence of the variable light chain (VL) region of Seq ID No:23, which comprises the amino acid sequence CDRL1 of Seq ID No:17 (IMGT) or Seq ID No:20 (Kabat), the amino acid sequence CDRL2 of Seq ID No:18 (IMGT) or Seq ID No:21 (Kabat), and the amino acid sequence CDRL3 of Seq ID No:19 (IMGT) or Seq ID No:22 (Kabat). The nucleic acid sequence of the V1 domain light chain is Seq ID No:24. The Vh domain can be combined with any of the constant region sequences of the heavy chain described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A sequence of Petition 870230019896, dated 08 / 03 / 2023, p. 139 / 576 120 / 456 full-length heavy chain amino acids is Seq ID No:15 (heavy chain nucleic acid sequence Seq ID No:16). A full-length light chain amino acid sequence is Seq ID No:25 (light chain nucleic acid sequence Seq ID No:26).

[0338] 1D05 HC mutant 1 has a variable heavy chain (VH) region amino acid sequence of Seq ID No:47 which comprises the amino acid sequence of CDRH1 of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence from Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence from Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 1 has a variable light chain (VL) region amino acid sequence from Seq ID No:43, comprising the CDRL1 amino acid sequence from Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat). No:39 (IMGT) or Seq ID No:42 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No:44. The VH domain can be combined with any of the sequences of the heavy chain constant region described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The VL domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A sequence of light chain amino acids of length Petition 870230019896, dated 08 / 03 / 2023, p. 140 / 576 121 / 456 total is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

[0339] 1D05 HC mutant 2 has a variable heavy chain (VH) region amino acid sequence of Seq ID No:48 comprising the amino acid sequence of CDRH1da Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence from Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence from Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 2 has a variable light chain (VL) region amino acid sequence from Seq ID No:43, comprising the CDRL1 amino acid sequence from Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence from Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No:44. The VH domain can be combined with any of the sequences of the heavy chain constant region described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The VL domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

[0340] 1D05 HC mutant 3 has a sequence of Petition 870230019896, dated 08 / 03 / 2023, p. 141 / 576 122 / 456 amino acids from the variable heavy chain region (Vh) of the Seq ID No:49 which comprises the amino acid sequence of CDRH1 of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence from Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence from Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 3 has a variable light chain (V1) region amino acid sequence from Seq ID No:43, comprising the CDRL1 amino acid sequence from Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat). No:39 (IMGT) or Seq ID No:42 (Kabat). The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:44. The Vh domain can be combined with any of the sequences of the constant region of the heavy chain described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

[0341] 1D05 HC mutant 4 has a variable heavy chain (Vh) region amino acid sequence of Seq ID No:342 comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the sequence Petition 870230019896, dated 08 / 03 / 2023, p. 142 / 576 123 / 456 of the CDRH2 amino acid sequence from Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat) and the CDRH3 amino acid sequence from Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). 1D05 HC mutant 4 has an amino acid sequence of the variable light chain region (Vl) from Seq ID No:43, which comprises the CDRL1 amino acid sequence from Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the CDRL2 amino acid sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat), and the CDRL3 amino acid sequence from Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat). No:39 (IMGT) or Seq ID No:42 (Kabat). The nucleic acid sequence of the light chain of the V1 domain is Seq ID No:44. The VH domain can be combined with any of the sequences of the heavy chain constant region described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

[0342] 1D05 LC mutant 1 has a variable heavy chain (Vh) region amino acid sequence of Seq ID No:33 comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The nucleic acid sequence Petition 870230019896, dated 08 / 03 / 2023, p. 143 / 576 The 124 / 456 light chain of the Vh domain is Seq ID No:34. The 1D05 LC mutant has an amino acid sequence of the variable light chain (Vl) region of Seq ID No:50, which comprises the amino acid sequence of CDRL1 of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat) and the amino acid sequence of CDRL3 of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The CDRL2 sequence of the 1D05 LC Mutant 1 is as defined by the Kabat or systems. IMGT of the Vl sequence from Seq ID No:50. The Vh domain can be combined with any of the sequences from the constant region of the heavy chain described in this document, for example, Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No: 532 or Seq ID No: 534. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:5 (heavy chain nucleic acid sequence Seq ID No:36).

[0343] 1D05 LC mutant 2 has a variable heavy chain (Vh) region amino acid sequence of Seq ID No:33 comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The light chain nucleic acid sequence of the Vh domain is Seq ID No:34. 1D05 LC mutant 2 has a variable chain region amino acid sequence Petition 870230019896, dated 08 / 03 / 2023, p. 144 / 576 125 / 456 light (Vl) of Seq ID No:51, comprising the amino acid sequence of CDRL1 of Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat), the amino acid sequence of CDRL2 of Seq ID No:38 (IMGT) or Seq ID No:41 (Kabat) and the amino acid sequence of CDRL3 of Seq ID No:39 (IMGT) or Seq ID No:42 (Kabat). The Vh domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The Vl domain can be combined with any of the sequences from the light chain constant region described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36).

[0344] 1D05 LC mutant 3 has a variable heavy chain (Vh) region amino acid sequence of Seq ID No:33 comprising the CDRH1 amino acid sequence of Seq ID No:27 (IMGT) or Seq ID No:30 (Kabat), the CDRH2 amino acid sequence of Seq ID No:28 (IMGT) or Seq ID No:31 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:29 (IMGT) or Seq ID No:32 (Kabat). The light chain nucleic acid sequence of the Vh domain is Seq ID No:34. The 1D05 LC mutant 3 has an amino acid sequence of the variable light chain (Vl) region with Seq ID No:298, which comprises the amino acid sequence of CDRL1 with Seq ID No:37 (IMGT) or Seq ID No:40 (Kabat) and the amino acid sequence of CDRL3 with Seq ID No:39 Petition 870230019896, dated 08 / 03 / 2023, p. 145 / 576 126 / 456 (IMGT) or Seq ID No:42 (Kabat). The CDRL2 sequence of the 1D05 LC Mutant 3 is as defined by the Kabat or systems. IMGT of the V1 sequence of Seq ID No:298. The nucleic acid sequence of the light chain of the V1 domain is Seq ID No:44. The VH domain can be combined with any of the sequences of the heavy chain constant region described in this document, for example, Seq ID No:193, Seq ID No:195, Seq ID No:197, Seq ID No:199, Seq ID No:201, Seq ID No:203, Seq ID No:205 or Seq ID No:340, Seq ID No:524, Seq ID No:526, Seq ID No:528, Seq ID No:530, Seq ID No:532 or Seq ID No:534. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:35 (heavy chain nucleic acid sequence Seq ID No:36). A full-length light chain amino acid sequence is Seq ID No:45 (light chain nucleic acid sequence Seq ID No:46).

[0345] 411B08 has a variable heavy chain (VH) region amino acid sequence of Seq ID No:58 comprising the CDRH1 amino acid sequence 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), and the CDRH3 amino acid sequence of Seq ID No:54 (IMGT) or Seq ID No:57 (Kabat). The light chain nucleic acid sequence of the VH domain is Seq ID No:59. 411B08 has a variable light chain (VL) region amino acid sequence of Seq ID No: 68, which comprises the amino acid sequence of CDRL1 Petition 870230019896, dated 08 / 03 / 2023, p. 146 / 576 127 / 456 of Seq ID No:62 (IMGT) or Seq ID No:65 (Kabat), the amino acid CDRL2 sequence of Seq ID No:63 (IMGT) or Seq ID No:66 (Kabat), and the amino acid sequence of CDRL3 of Seq ID No:64 (IMGT) or Seq ID No:67 (Kabat). The nucleic acid sequence of the light chain of domain V1 is Seq ID No:69. The Vh domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538.A full-length heavy chain amino acid sequence is Seq ID No:60 (heavy chain nucleic acid sequence Seq ID No:61). A full-length light chain amino acid sequence is Seq ID No:70 (light chain nucleic acid sequence Seq ID No:71).

[0346] 411C04 has a variable heavy chain (VH) region amino acid sequence of Seq ID No:78 comprising the CDRH1 amino acid sequence of Seq ID No:72 (IMGT) or Seq ID No:75 (Kabat), the CDRH2 amino acid sequence of Seq ID No:73 (IMGT) or Seq ID No:76 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:74 (IMGT) or Seq ID No:77 (Kabat). The light chain nucleic acid sequence of the VH domain is Seq ID No:79. 411C04 has a variable light chain (VL) region amino acid sequence of Seq ID No:88, comprising the CDRL1 amino acid sequence. Petition 870230019896, dated 08 / 03 / 2023, p. 147 / 576 128 / 456 of Seq ID No:82 (IMGT) or Seq ID No:85 (Kabat), the amino acid CDRL2 sequence of Seq ID No:83 (IMGT) or Seq ID No:86 (Kabat), and the amino acid sequence of CDRL3 of Seq ID No:84 (IMGT) or Seq ID No:87 (Kabat). The nucleic acid sequence of the light chain of domain V1 is Seq ID No:89. The Vh domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538.A full-length heavy chain amino acid sequence is Seq ID No:80 (heavy chain nucleic acid sequence Seq ID No:81). A full-length light chain amino acid sequence is Seq ID No:90 (light chain nucleic acid sequence Seq ID No:91).

[0347] 411D07 has a variable heavy chain (VH) region amino acid sequence of Seq ID No:98 comprising the CDRH1 amino acid sequence 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), and the CDRH3 amino acid sequence of Seq ID No:94 (IMGT) or Seq ID No:97 (Kabat). The light chain nucleic acid sequence of the VH domain is Seq ID No:99. 411D07 has a variable light chain (VL) region amino acid sequence of Seq ID No:108, comprising the CDRL1 amino acid sequence. Petition 870230019896, dated 08 / 03 / 2023, p. 148 / 576 129 / 456 of Seq ID No:102 (IMGT) or Seq ID No:105 (Kabat), the amino acid CDRL2 sequence of Seq ID No:103 (IMGT) or Seq ID No:106 (Kabat), and the amino acid sequence of CDRL3 of Seq ID No:104 (IMGT) or Seq ID No:107 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No:109. The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538.A full-length heavy chain amino acid sequence is Seq ID No:100 (heavy chain nucleic acid sequence Seq ID No:101). A full-length light chain amino acid sequence is Seq ID No:110 (light chain nucleic acid sequence Seq ID No:111).

[0348] 385F01 has a variable heavy chain (VH) region amino acid sequence of Seq ID No:118 comprising the CDRH1 amino acid sequence of Seq ID No:112 (IMGT) or Seq ID No:115 (Kabat), the CDRH2 amino acid sequence of Seq ID No:113 (IMGT) or Seq ID No:116 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:114 (IMGT) or Seq ID No:117 (Kabat). The light chain nucleic acid sequence of the VH domain is Seq ID No:199. 385F01 has a variable light chain (VL) region amino acid sequence. Petition 870230019896, dated 08 / 03 / 2023, p. 149 / 576 130 / 456 of Seq ID No:128, which comprises the amino acid sequence of CDRL1 from Seq ID No:122 (IMGT) or Seq ID No:125 (Kabat), the amino acid sequence CDRL2 from Seq ID No:123 (IMGT) or Seq ID No:126 (Kabat), and the amino acid sequence of CDRL3 from Seq ID No:124 (IMGT) or Seq ID No:127 (Kabat). The nucleic acid sequence of the light chain of domain V1 is Seq ID No:129. The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538.A full-length heavy chain amino acid sequence is Seq ID No: 120 (heavy chain nucleic acid sequence Seq ID No: 121). A full-length light chain amino acid sequence is Seq ID No: 130 (light chain nucleic acid sequence Seq ID No: 131).

[0349] 386H03 has a variable heavy chain (VH) amino acid sequence from the Seq ID No:158 region comprising the CDRH1 amino acid sequence from Seq ID No:152 (IMGT) or Seq ID No:155 (Kabat), the CDRH2 amino acid sequence from Seq ID No:153 (IMGT) or Seq ID No:156 (Kabat), and the CDRH3 amino acid sequence from Seq ID No:154 (IMGT) or Seq ID No:157 (Kabat). The nucleic acid sequence of the VH domain light chain is Seq ID No:159. 386H03 has a Petition 870230019896, dated 08 / 03 / 2023, p. 150 / 576 131 / 456 amino acid sequence of the variable light chain (Vl) region of Seq ID No:168, comprising the amino acid sequence of CDRL1 of Seq ID No:162 (IMGT) or Seq ID No:165 (Kabat), the amino acid CDRL2 sequence of Seq ID No:163 (IMGT) or Seq ID No:166 (Kabat), and the amino acid sequence of CDRL3 of Seq ID No:164 (IMGT) or Seq ID No:167 (Kabat). The nucleic acid sequence of the light chain of the Vl domain is Seq ID No:169. The Vh domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:160 (heavy chain nucleic acid sequence Seq ID No:161). A full-length light chain amino acid sequence is Seq ID No: 170 (light chain nucleic acid sequence Seq ID No: 171).

[0350] 389A03 has a variable heavy chain (Vh) amino acid sequence from the Seq ID No:178 region comprising the CDRH1 amino acid sequence from Seq ID No:172 (IMGT) or Seq ID No:175 (Kabat), the CDRH2 amino acid sequence from Seq ID No:173 (IMGT) or Seq ID No:174 (Kabat), and the CDRH3 amino acid sequence from Seq ID No:173 (IMGT) or Seq ID No:177 (Kabat). The nucleic acid sequence of Petition 870230019896, dated 08 / 03 / 2023, p. 151 / 576 132 / 456 light chain of domain Vh is Seq ID No:179. 389A03 has an amino acid sequence of the variable region of the light chain (Vl) of Seq ID No:188, which comprises the amino acid sequence of CDRL1 of Seq ID No:182 (IMGT) or Seq ID No:185 (Kabat), the amino acid sequence of CDRL2 of Seq ID No:183 (IMGT) or Seq ID No:186 (Kabat), and the amino acid sequence of CDRL3 of Seq ID No:184 (IMGT) or Seq ID No:187 (Kabat). The nucleic acid sequence of the light chain of domain Vl is Seq ID No:189. The Vh domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The Vl domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:180 (heavy chain nucleic acid sequence Seq ID No:181). A full-length light chain amino acid sequence is Seq ID No:190 (light chain nucleic acid sequence Seq ID No:191).

[0351] 413D08 has a variable heavy chain (Vh) amino acid sequence from the Seq ID No:138 region comprising the CDRH1 amino acid sequence from Seq ID No:132 (IMGT) or Seq ID No:135 (Kabat), the CDRH2 amino acid sequence from Seq ID No:133 (IMGT) or Seq ID No:136 (Kabat), and the CDRH3 amino acid sequence from Seq ID No:134 (IMGT) or Seq ID No:135 (Kabat). Petition 870230019896, dated 08 / 03 / 2023, page 152 / 576 133 / 456 Seq ID No:137 (Kabat). The nucleic acid sequence of the Vh domain light chain is Seq ID No:139. 413D08 has an amino acid sequence of the variable light chain (VL) region of Seq ID No:148, which comprises the amino acid sequence of CDRL1 from Seq ID No:142 (IMGT) or Seq ID No:145 (Kabat), the amino acid sequence of CDRL2 from Seq ID No:143 (IMGT) or Seq ID No:146 (Kabat), and the amino acid sequence of CDRL3 from Seq ID No:144 (IMGT) or Seq ID No:147 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No:149. The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The VL domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:140 (heavy chain nucleic acid sequence Seq ID No:141). A full-length light chain amino acid sequence is Seq ID No:150 (light chain nucleic acid sequence Seq ID No:151).

[0352] 413G05 has a variable heavy chain (VH) amino acid sequence from the Seq ID No:244 region comprising the CDRH1 amino acid sequence from Seq ID No:238 (IMGT) or Seq ID No:241 (Kabat), the CDRH2 amino acid sequence from Seq ID No:239 (IMGT) or Seq ID No:242 (Kabat) and the Petition 870230019896, dated 08 / 03 / 2023, p. 153 / 576 134 / 456 amino acid sequence CDRH3 from Seq ID No:240 (IMGT) or Seq ID No:243 (Kabat). The nucleic acid sequence of the VH domain light chain is Seq ID No:245. 413G05 has an amino acid sequence of the variable light chain (VL) region from Seq ID No:254, which comprises the amino acid sequence CDRL1 from Seq ID No:248 (IMGT) or Seq ID No:251 (Kabat), the amino acid sequence CDRL2 from Seq ID No:252 (IMGT) or Seq ID No:249 (Kabat), and the amino acid sequence CDRL3 from Seq ID No:250 (IMGT) or Seq ID No:253 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No:255. The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The VL domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:246 (heavy chain nucleic acid sequence Seq ID No:247). A full-length light chain amino acid sequence is Seq ID No:256 (light chain nucleic acid sequence Seq ID No:257).

[0353] 413F09 has a variable heavy chain (VH) amino acid sequence from the Seq ID No:264 region comprising the CDRH1 amino acid sequence from Seq ID No:258 (IMGT) or Seq ID No:261 (Kabat), the amino acid sequence Petition 870230019896, dated 08 / 03 / 2023, p. 154 / 576 135 / 456 CDRH2 from Seq ID No:259 (IMGT) or Seq ID No:262 (Kabat) and the CDRH3 amino acid sequence from Seq ID No:260 (IMGT) or Seq ID No:263 (Kabat). The nucleic acid sequence of the Vh domain light chain is Seq ID No:265. 413F09 has a variable light chain (VL) region amino acid sequence from Seq ID No:274, which comprises the CDRL1 amino acid sequence from Seq ID No:268 (IMGT) or Seq ID No:271 (Kabat), the CDRL2 amino acid sequence from Seq ID No:269 (IMGT) or Seq ID No:270 (Kabat), and the CDRL3 amino acid sequence from Seq ID No:270 (IMGT) or Seq ID No:273 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No:275.The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No: 266 (heavy chain nucleic acid sequence Seq ID No: 267). A full-length light chain amino acid sequence is Seq ID No: 276 (light chain nucleic acid sequence Seq ID No: 277).

[0354] 414B06 has a variable heavy chain (VH) amino acid sequence from the Seq ID No:284 region comprising the CDRH1 amino acid sequence from Seq ID No:278 Petition 870230019896, dated 08 / 03 / 2023, p. 155 / 576 136 / 456 (IMGT) or Seq ID No:281 (Kabat), the CDRH2 amino acid sequence of Seq ID No:279 (IMGT) or Seq ID No:282 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:280 (IMGT) or Seq ID No:283 (Kabat). The nucleic acid sequence of the VH domain light chain is Seq ID No:285. 414B06 has an amino acid sequence of the variable light chain (VL) region with Seq ID No:294, comprising the amino acid sequence CDRL1 with Seq ID No:288 (IMGT) or Seq ID No:291 (Kabat), the amino acid sequence CDRL2 with Seq ID No:289 (IMGT) or Seq ID No:292 (Kabat), and the amino acid sequence CDRL3 with Seq ID No:290 (IMGT) or Seq ID No:293 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No:295.The VH domain can be combined with any of the sequences from the heavy chain constant region described in this document, 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. The VL domain can be combined with any of the constant region sequences of the light chain described in this document, for example, Seq ID Nos: 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No: 286 (heavy chain nucleic acid sequence Seq ID No: 287). A full-length light chain amino acid sequence is Seq ID No: 296 (light chain nucleic acid sequence Seq ID No: 297).

[0355] 416E01 has a variable heavy chain (VH) region amino acid sequence from Seq ID No:349 that Petition 870230019896, dated 08 / 03 / 2023, page 156 / 576 137 / 456 comprises the CDRH1 amino acid sequence of Seq ID No:343 (IMGT) or Seq ID No:346 (Kabat), the CDRH2 amino acid sequence of Seq ID No:344 (IMGT) or Seq ID No:347 (Kabat), and the CDRH3 amino acid sequence of Seq ID No:345 (IMGT) or Seq ID No:348 (Kabat). The nucleic acid sequence of the Vh domain light chain is Seq ID No:350. 416E01 has an amino acid sequence of the variable light chain (VL) region with Seq ID No: 359, comprising the amino acid sequence CDRL1 with Seq ID No: 353 (IMGT) or Seq ID No: 356 (Kabat), the amino acid sequence CDRL2 with Seq ID No: 354 (IMGT) or Seq ID No: 357 (Kabat), and the amino acid sequence CDRL3 with Seq ID No: 355 (IMGT) or Seq ID No: 358 (Kabat). The nucleic acid sequence of the VL domain light chain is Seq ID No: 360.The VH domain can be combined with any of the heavy chain constant region sequences described in this document, 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, SeqID No:528, Seq ID No:530, Seq ID No:532, or Seq ID No:534. The VL domain can be combined with any of the sequences from the constant region of the light chain described in this document, for example, Seq ID Nos:207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 536 and 538. A full-length heavy chain amino acid sequence is Seq ID No:351 (heavy chain nucleic acid sequence Seq ID No:352). A full-length light chain amino acid sequence is Seq ID No: 361 (light chain nucleic acid sequence Seq ID No: 362). Antibody-drug conjugates Petition 870230019896, dated 08 / 03 / 2023, page 157 / 576 138 / 456

[0356] Anti-ICOS antibodies can be used as carriers of cytotoxic agents to target Tregs. As reported in Example 18, Tregs located in the tumor microenvironment (TME) strongly express ICOS. ICOS is more strongly expressed in intratumoral Tregs than in intratumoral Teffs or peripheral Tregs. Thus, anti-ICOS antibodies labeled with a toxic drug or prodrug can preferentially target Tregs in the TME to deliver the toxic payload, selectively inhibiting these cells. Such targeting of cytotoxic agents provides an additional route to remove the immunosuppressive effect of Tregs, thereby altering the Treg:Teff balance in favor of Teff activity, and can be used as an alternative to, or in combination with, any one or more of the other therapeutic approaches discussed in this document (e.g., effector Fc-mediated inhibition of Tregs, effector T cell agonism).

[0357] Thus, the invention provides an anti-ICOS antibody that is conjugated to a cytotoxic drug or prodrug. In the case of a prodrug, the prodrug is acceptable at the EMT or other target site of therapeutic activity to generate the cytotoxic agent. Activation can be in response to a trigger, such as photoactivation, for example, using near-infrared light to activate a photoabsorbent conjugate

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

[0358] For use in an antibody conjugate Petition 870230019896, dated 08 / 03 / 2023, page 158 / 576 139 / 456 drug, the cytotoxic drug or prodrug is preferably non-immunogenic and non-toxic (dormant or inactive) during circulation of the antibody-drug conjugate in the blood. Preferably, the cytotoxic drug (or the prodrug, when activated) is potent – ​​for example, two to four molecules of the drug may be sufficient to kill the target cell. A photoactivatable prodrug is the silica-phallaocyanin dye (IRDye 700 DX), which induces lethal damage to the cell membrane after exposure to infrared light. Cytotoxic drugs include antimiotic agents, such as monomethyl-auristatin E inhibitors, and microtubule inhibitors, such as maytansine derivatives, for example, mertansine, DM1, emtansine.

[0359] Conjugation of the drug (or prodrug) to the antibody will generally be via a ligand. The ligand may be a cleavable ligand, for example, disulfide, hydrazone or peptide bond. Cathepsin-cleavable ligands may be used, so that the drug is released by cathepsin into tumor cells. Alternatively, non-cleavable ligands may be used, for example, thioether bond. Additional attachment groups and / or spacers may also be included.

[0360] The antibody in the antibody-drug conjugate may be an antibody fragment, such as Fab'2 or another antigen-binding fragment, as described herein, since the small size of such fragments may aid penetration into the tissue site (e.g., solid tumor).

[0361] An anti-ICOS antibody, according to the present invention, can be provided as an immunocytokine. Anti-ICOS antibodies can also be administered with Petition 870230019896, dated 08 / 03 / 2023, p. 159 / 576 140 / 456 Immunocytokines in combination therapy. Several examples of antibodies are described herein for use in combination therapy with anti-ICOS, and any one of them (e.g., an anti-PD-L1 antibody) can be provided as immunocytokines for use in the present invention. An immunocytokine comprises an antibody molecule conjugated to a cytokine, such as IL-2. Anti-ICOS:IL-2 conjugates and anti-PD-L1:IL-2 conjugates are therefore further aspects of the present invention.

[0362] An IL-2 cytokine may have activity at the high-affinity IL-2 receptor (αβγ) and / or the intermediate-affinity IL-2 receptor (αβ). IL-2, as used in an immunocytokine, may be wild-type human IL-2 or a variant IL-2 cytokine that has one or more amino acid deletions, substitutions, or additions, for example, IL-2 that has a deletion of 1 to 10 amino acids at the N-terminus. Other IL-2 variants include the R38A or R38Q mutations.

[0363] An example of an anti-PD-L1 immunocytokine comprises an immunoglobulin heavy chain and an immunoglobulin light chain, with the heavy chain comprising in the N- to C-terminal direction:

[0364] a) A VH domain comprising CDRH1, CDRH2 and CDRH3; and

[0365] b) A constant heavy chain region;

[0366] and wherein the light chain comprises, in the N- to C-terminal direction:

[0367] c) A Vl domain comprising CDRL1, CDRL2 and CDRL3;

[0368] d) A constant region of the light chain, (CL); Petition 870230019896, dated 08 / 03 / 2023, p. 160 / 576 141 / 456

[0369] e) Optionally, a binder, (L); and

[0370] f) An IL-2 cytokine;

[0371] wherein the Vh domain and the Vl domain consist of an antigen-binding site that binds specifically to human PD-L1; and

[0372] wherein the immunocytokine comprises a VH domain comprising a CDRH3 comprising motif X1GSGX2YGX3X4FD (SEQ ID NO:609), wherein X1, X2 and X3 are independently any amino acid and X4 is either present or absent and, if present, may be any amino acid.

[0373] The VH and VL domains may be the VH and VL domains of any anti-PD-L1 antibody mentioned in this document, for example, the VH and VL domains of 1D05.

[0374] IL-2 can be wild-type or variant human IL-2. VACCINATION

[0375] Anti-ICOS antibodies can be supplied in vaccine compositions or co-administered with vaccine preparations. ICOS is involved in the formation of follicular helper T cells and in the germinal center reaction

[37] . ICOS agonist antibodies therefore have potential clinical utility as molecular adjuvants to increase vaccine efficacy. Antibodies can be used to increase the protective efficacy of numerous vaccines, such as those against hepatitis B, malaria, and HIV.

[0376] In the context of vaccination, the anti-ICOS antibody will generally be one that lacks the effector function of the Fc region and therefore does not mediate ADCC, CDC, or ADCP. The antibody may be provided in a format without an Fc region or that has a null effector constant region. Optionally, Petition 870230019896, dated 08 / 03 / 2023, page 161 / 576 142 / 456 An anti-ICOS antibody may have a constant heavy chain region that binds to one or more types of Fc receptors, but which does not induce ADCC, CDC, or ADCP activity, or which exhibits lower ADCC, CDC, and ADCP activity compared to wild-type human IgG1. Such a constant region may have no binding capacity or may bind with lower affinity to the particular Fc receptor (or particular Fc receptors) responsible for triggering ADCC, CDC, or ADCP activity. Alternatively, when cellular effector functions are acceptable or desirable in the context of vaccination, the anti-ICOS antibody may comprise a constant heavy chain region that is positive for Fc effector function. Any of the IgG1, IgG4, and IgG4.PE formats may be used, for example, for anti-ICOS antibodies in vaccination regimens, and other examples of suitable isotypes and antibody constant regions are presented in more detail throughout this document. Formulations and Administration

[0377] Antibodies may be monoclonal or polyclonal, but are preferably supplied as monoclonal antibodies for therapeutic use. They may be supplied as part of a mixture of other antibodies, optionally including antibodies of different binding specificity.

[0378] Antibodies according to the invention that encode nucleic acid will generally be provided in isolated form. Thus, the antibodies, the VH and / or VL domains, and the nucleic acids can be provided purified from their natural environment or from their production environment. The isolated antibodies and the isolated nucleic acid will be free or Petition 870230019896, dated 08 / 03 / 2023, page 162 / 576 143 / 456 substantially free from the material to which they are naturally associated, such as other polypeptides or nucleic acids with which they are found in vivo, or the environment in which they are prepared (e.g., cell culture) when such preparation is done by recombinant DNA technology in vitro. Optionally, an isolated antibody or nucleic acid (1) is free from at least some other proteins with which it would normally be found, (2) is essentially free from other proteins from the same source, e.g., the same species, (3) is expressed by a cell of a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates or other materials to which it is associated in nature, (5) is operationally associated (by covalent or non-covalent linkage) to a polypeptide to which it is not associated in nature, or (6) does not occur in nature.

[0379] Antibodies or nucleic acids can be formulated with diluents or adjuvants and can also be isolated for practical purposes – for example, they can be mixed with carriers if used to coat microtiter plates for use in immunoassays and can be mixed with pharmaceutically acceptable carriers or diluents when used in therapy. As described throughout this document, other active ingredients can also be included in therapeutic preparations. Antibodies can be glycosylated, naturally in vivo or by heterologous eukaryotic cell systems such as CHO cells, or they can be (for example, if produced by expression in a prokaryotic cell) non-glycosylated. The invention encompasses antibodies that have a glycosylation pattern. Petition 870230019896, dated 08 / 03 / 2023, page 163 / 576 144 / 456 modified. In some applications, modification to remove undesirable glycosylation sites may be useful or, for example, removal of a fucose chemical moiety to increase ADCC function

[38] . In other applications, galactosylation modification may be done to modify CDC.

[0380] Typically, an isolated product constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. An antibody may be substantially free of proteins or polypeptides or other contaminants found in its natural or production environment that interfere with its therapeutic, diagnostic, prophylactic, or other use.

[0381] An antibody may have been identified, separated, and / or recovered from a component of its production environment (e.g., naturally or recombinantly). The isolated antibody may be free from association with all other components of its production environment, for example, such that the antibody has been isolated to an FDA-approved or approvable standard. Contaminating components of its production environment, such as those resulting from recombinant cells undergoing transfection, are materials that typically interfere with research, diagnostics, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody will be purified: (1) to more than 95% by weight of the antibody, as determined, for example, by the Lowry method and, in some embodiments, to more than 99% by weight; (2) to a sufficient degree Petition 870230019896, dated 08 / 03 / 2023, page 164 / 576 145 / 456 to obtain at least 15 N-terminal or internal amino acid sequence residues using a rotary cup sequencer or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. The isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. However, normally an isolated antibody or its encoding nucleic acid will be prepared by at least one purification step.

[0382] The invention provides therapeutic compositions comprising the antibodies described herein. Therapeutic compositions comprising antibody-encoding nucleic acids are also provided. The encoding nucleic acids are described in more detail throughout this document and include DNA and RNA, for example, mRNA. In the therapeutic methods described herein, the antibody-encoding nucleic acid and / or the cells containing that nucleic acid may be used alternatively (or additionally) to compositions comprising the antibody alone. Cells containing antibody-encoding nucleic acid, optionally wherein the nucleic acid is stably integrated into the genome, thus represent medicaments for therapeutic use in a patient. The anti-ICOS antibody-encoding nucleic acid may be introduced into human B lymphocytes, optionally, intended patient-derived B lymphocytes modified ex vivo.Optionally, memory B cells are used. Administering cells containing the coding nucleic acid to the patient provides a reservoir of cells. Petition 870230019896, dated 08 / 03 / 2023, page 165 / 576 146 / 456 capable of expressing the anti-ICOS antibody, which may provide long-term therapeutic benefit compared to administration of isolated nucleic acid or isolated antibody.

[0383] Compositions may contain carriers, excipients, and other suitable agents that are incorporated into formulations to provide improved transfer, delivery, tolerability, and the like. Multiple suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax (polyethylene glycols of various molecular weights) emulsions, semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al. “Compendium of excipients for parenteral formulations PDA (1998) J Pharm Sci Technol 52: 238 to 311.The compositions may comprise the antibody or nucleic acid in combination with medical injection buffer and / or adjuvant.

[0384] Antibodies or their encoding nucleic acids can be formulated for the desired route of administration for a patient, for example, in liquid (optionally aqueous solution) for injection. Several delivery systems are known and can be used to administer the pharmaceutical composition of the invention. Methods of delivery include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The formulation of Petition 870230019896, dated 08 / 03 / 2023, page 166 / 576 147 / 456 antibodies for subcutaneous administration typically require a smaller volume compared to intravenous preparations. The high potency of antibodies according to the present invention can make it possible to use them in sufficiently low doses to make subcutaneous formulation practical, which represents an advantage compared to less potent anti-ICO antibodies.

[0385] The composition can be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered in conjunction with other biologically active agents. Administration can be systemic or local.

[0386] The pharmaceutical composition can also be administered into a vesicle, in particular, a liposome (see Langer (1990) Science 249: 1527 to 1533; Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss, New York, pages 353 to 365; Lopez-Berestein, ibid., pages 317 to 327; see generally ibid.).

[0387] In certain situations, the pharmaceutical composition may be delivered in a controlled-release system. In one embodiment, a pump may be used (see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: 201). In another embodiment, polymeric materials may be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974). In yet another embodiment, a controlled-release system may be placed in close proximity to the target of the Petition 870230019896, dated 08 / 03 / 2023, p. 167 / 576 148 / 456 composition, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, above, volume 2, pages 115 to 138, 1984).

[0388] Injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous and intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared, for instance, by dissolving, suspending or emulsifying the antibody or salt thereof described above in a sterile aqueous medium or an oily medium conventionally used for injections. As an aqueous medium for injections, there is, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which can be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a non-ionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) hydrogenated castor oil adduct)], etc.As an oily medium, sesame oil, soybean oil, etc., are employed, which can be used in combination with a solubilizing agent, such as benzyl benzoate, benzyl alcohol, etc. The injection then prepared can be loaded into an appropriate ampoule. A pharmaceutically present composition of the invention can be administered subcutaneously or intravenously with a standard needle and syringe. It is envisioned that the treatment will not be restricted to clinical use. Therefore, subcutaneous injection using a needle-free device is also advantageous. Petition 870230019896, dated 08 / 03 / 2023, page 168 / 576 149 / 456 Regarding subcutaneous delivery, a pen-type delivery device has applications in delivering a pharmaceutical composition of the present invention. Such a pen-type delivery device can be reusable or disposable. A reusable pen-type delivery device generally uses a replaceable cartridge containing a pharmaceutical composition. Once all the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can be reused. In a disposable pen-type delivery device, there is no replaceable cartridge. Instead, the disposable pen-type delivery device comes pre-loaded with the pharmaceutical composition held in a reservoir within the device.Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded. Numerous reusable pen-type delivery devices and autoinjectors have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Inc., Inc., Woodstock, UK). Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPENT™, OPTIPEN PRO™, OPTIPEN STARLET™ and OPTICLIKT™ (Sanofi-Aventis, Frankfurt, Germany), for example. Examples of pen delivery devices. Petition 870230019896, dated 08 / 03 / 2023, page 169 / 576 Disposable devices that have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention include, but are not limited to, the SOLOSTAR™ pen (Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly).

[0389] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in a unit dose suitable for adjusting to a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforementioned antibody contained is generally from about 5 to about 500 mg in each dosage form in a unit dose; especially in the injection form, the aforementioned antibody may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms.

[0390] The antibody, nucleic acid, or composition comprising thereof may be contained in a medical container, such as a vial, syringe, IV container, or injection device. In one example, the antibody, nucleic acid, or composition is in vitro and may be in a sterile container. In one example, a kit is provided comprising the antibody, packaging, and instructions for use in a therapeutic method as described herein.

[0391] One aspect of the invention is a composition comprising an antibody or nucleic acid of the invention and one or more pharmaceutically acceptable excipients, examples of which are listed above. “Pharmaceutically acceptable” refers to approved or approvable by a U.S. federal government regulatory agency or a state government or listed Petition 870230019896, dated 08 / 03 / 2023, page 170 / 576 151 / 456 in the U.S. Pharmacopoeia or another generally recognized pharmacopoeia for use in animals, including humans. A pharmaceutically acceptable carrier, excipient, or adjuvant may be administered to a patient along with an agent, for example, any antibody or antibody chain described herein, and does not destroy the pharmacological activity of the agent and is non-toxic when administered in doses sufficient to deliver a therapeutic amount of the agent.

[0392] In some embodiments, an anti-ICOS antibody will be the only active ingredient in a composition according to the present invention. Thus, a composition may consist of the antibody or may consist of the antibody with one or more pharmaceutically acceptable excipients. However, compositions according to the present invention optionally include one or more additional active ingredients. A detailed description of the agents with which the anti-ICOS antibodies may be combined is provided throughout this document. Optionally, the compositions contain multiple antibodies (or encoding nucleic acids) in a combined preparation, for example, a single formulation comprising the anti-ICOS antibody and one or more other antibodies. Other therapeutic agents whose administration with antibodies or nucleic acids according to the present invention may be desirable include analgesic agents.Any agent or combination of such agents may be administered in combination with antibody or nucleic acid compositions according to the present invention, or provided therein, or as a combined or separate preparation. The antibody or nucleic acid according to the present invention may be administered separately, sequentially, or concomitantly. Petition 870230019896, dated 08 / 03 / 2023, page 171 / 576 152 / 456 and optionally as a combination preparation, with other agents or therapeutic agents, such as those mentioned.

[0393] Anti-ICOS antibodies for use in a given therapeutic indication can be combined with the accepted standard of care. Thus, for cancer treatment, antibody therapy can be employed in a treatment regimen that also includes chemotherapy, surgery and / or radiation therapy, for example. Radiation therapy can occur in a single dose or in fractionated doses, either directly to the affected tissues or throughout the body.

[0394] Several compositions may be administered separately or simultaneously. Separate administration refers to two compositions that are administered at different times, for example, at least 10, 20, 30, or 10 to 60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. In addition, compositions may also be administered at an interval of 24 hours or more. Alternatively, two or more compositions may be administered simultaneously, for example, with less than 10 or less than 5 minutes between doses. Compositions administered simultaneously may, in some respects, be administered as a mixture, with or without a similar or different time-release mechanism for each of the components.

[0395] Antibodies and their encoding nucleic acids can be used as therapeutic agents. The patients in this document are generally mammals, typically humans. An antibody or nucleic acid can be administered to a mammal, for example, by any of the routes of administration mentioned in Petition 870230019896, dated 08 / 03 / 2023, page 172 / 576 153 / 456 present document.

[0396] Administration is normally in a “therapeutically effective amount,” which is an amount that produces the desired effect for which it is administered, sufficient to show benefit to a patient. The exact amount will depend on the treatment objective and will be determinable by a specialist in the field using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding). Treatment prescription, for example, dosage decisions, etc., is within the responsibility of general practitioners and other physicians and may depend on the severity of symptoms and / or the progression of a disease being treated. A therapeutically effective amount or appropriate dose of antibody or nucleic acid can be determined by comparing its in vitro activity and in vivo activity in an animal model.The methods for extrapolating effective dosages from mice and other test animals to humans are known.

[0397] As indicated by the in vivo studies described in the Examples, the anti-ICOS antibody may be effective across a range of doses. Pharmacodynamic studies are reported in Example 24.

[0398] Anti-ICOS antibodies may be administered in an amount within one of the following ranges per dose:

[0399] about 10 μg / kg of body weight to about 100 mg / kg of body weight,

[0400] about 50 μg / kg of body weight to about 5 mg / kg of body weight, Petition 870230019896, dated 08 / 03 / 2023, page 173 / 576 154 / 456

[0401] about 100 μg / kg of body weight to about 10 mg / kg of body weight,

[0402] about 100 μg / kg of body weight to about 20 mg / kg of body weight,

[0403] about 0.5 μg / kg of body weight to about 20 mg / kg of body weight, or

[0404] approximately 5 mg / kg of body weight or less, for example, less than 4, less than 3, less than 2 or less than 1 mg / kg of antibody.

[0405] An ideal therapeutic dose may be between 0.1 and 0.5 mg / kg in a human being, for example, approximately 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg or 0.5 mg / kg. For fixed dosage in adult humans, an appropriate dose may be between 8 and 50 mg, or between 8 and 25 mg, for example, 15 mg or 20 mg.

[0406] In the treatment methods described in this document, one or more doses may be administered. In some cases, a single dose may be effective in achieving long-term benefit. Thus, the method may comprise the administration of a single dose of the antibody, its encoding nucleic acid, or the composition. Alternatively, multiple doses may be administered, usually sequentially over a period of days, weeks, or months. The anti-ICOS antibody may be administered repeatedly to a patient at intervals of 4 to 6 weeks, for example, every 4 weeks, every 5 weeks, or every 6 weeks. Optionally, the anti-ICOS antibody may be administered to a patient once a month or less frequently, for example, every two months or every three months. Consequently, a method for treating a patient Petition 870230019896, dated 08 / 03 / 2023, p. 174 / 576 155 / 456 may involve administering a single dose of the anti-ICOS antibody to the patient and not repeating the administration for at least one month, at least two months, at least three months, and optionally, not repeating the treatment for at least 12 months.

[0407] As discussed in Example 11c, comparable therapeutic effects can be obtained with the use of either one or multiple doses of anti-ICOS antibody, which may be a result of a single dose of the antibody being effective in resetting the tumor microenvironment. Physicians can adapt the anti-ICOS antibody administration regimen to the disease and the patient undergoing therapy, taking into account the disease status and any other therapeutic agents or therapeutic measures (e.g., surgery, radiotherapy, etc.) with which the anti-ICOS antibody is combined. In some modalities, an effective dose of an anti-ICOS antibody is administered more frequently than once a month, such as, for example, once every three weeks, once every two weeks, or once a week. Treatment with anti-ICOS antibody may include multiple doses administered over a period of at least one month, at least six months, or at least one year.

[0408] As used in this document, the terms treat, “treatment,” or “enhancement” refer to therapeutic treatments where the objective is to reverse, alleviate, improve, inhibit, slow, or halt the progression or severity of a condition associated with a disease or disorder. The term “treat” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is generally “effective” if one or more Petition 870230019896, dated 08 / 03 / 2023, p. 175 / 576 156 / 456 symptoms or clinical markers are reduced. Alternatively, treatment is effective if the progression of a disease is reduced or halted. That is, treatment includes not only the improvement of symptoms or markers, but also the cessation or at least the slowing of the progression or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction in disease extent, stabilized disease status (i.e., no worsening), delay or slowing of disease progression, improvement or palliation of disease status, remission (partial or total), and / or reduction in detectable or undetectable mortality. The term treatment of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment). For treatment to be effective, a complete cure is not contemplated. The method may also include, in certain aspects, a cure.In the context of the invention, the treatment can be a preventive treatment. T-cell therapy

[0409] Document WO2011 / 097477 described the use of anti-ICOS antibodies to generate and expand T cells by placing a population of T cells in contact with a first agent that provides a primary activation signal (e.g., an anti-CD3 antibody) and a second agent that activates ICOS (e.g., an anti-ICOS antibody), optionally in the presence of a Th17 polarizing agent, such as IL-β, IL-6, anti-IFNγ and / or neutralizing anti-IL-4. The anti-ICOS antibodies described in this document can be used in such methods to provide populations of T cells. They can be Petition 870230019896, dated 08 / 03 / 2023, page 176 / 576 157 / 456 generated populations of expanded cultured T cells with therapeutic activity (e.g., antitumor activity). As described in document no. WO2011 / 097477, such T cells can be used therapeutically in immunotherapy treatment methods for patients. Morphological Assay for Anti-Ic Antibodies as Therapeutic Candidates

[0410] It was observed that when candidate therapeutic anti-ICOS antibodies were coupled to a solid surface and placed in contact with ICOS-expressing T cells, they could induce morphological changes in the cells. Upon adding ICOS+ T cells to the cavities that were internally coated with anti-ICOS antibodies, it was observed that the cells changed their initial rounded shape, adopting a fusiform shape, spreading and adhering to the antibody-coated surface. This morphological alteration was not observed with the control antibody. Furthermore, it was found that the effect was dose-dependent, with more evident and / or faster shape change occurring as the antibody concentration on the surface increased. The shape change provides a surrogate indicator of T cell binding to ICOS and / or agonism by the anti-ICOS antibody.The assay can be used to identify an antibody that promotes ICOS multimerization on the T cell surface. Such antibodies represent candidate therapeutic agonist antibodies. Conveniently, the visual indicator provided by this assay is a simple method for selecting antibodies or cells, particularly in large numbers. The assay can be automated to operate in a high-throughput system. Petition 870230019896, dated 08 / 03 / 2023, page 177 / 576 158 / 456

[0411] Accordingly, an aspect of the invention is an assay for selecting an antibody that binds to ICOS, optionally, for selecting an ICOS agonist antibody, comprising the assay:

[0412] provide an array of antibodies immobilized (bound or attached) to a substrate in a test well;

[0413] add cells that express ICOS (e.g., activated primary T cells or MJ cells) to the test wells;

[0414] observe the morphology of the cells;

[0415] detect a change in the shape of the cells from rounded to flattened against the substrate within the cavities; the change in shape indicates that the antibody is an ICOS-binding antibody, optionally an ICOS agonist antibody, and

[0416] select antibody from test wells.

[0417] The assay can be performed with multiple test wells, each containing a different antibody for testing, optionally in parallel, for example, in a 96-well plate format. The substrate is preferably an internal surface of the wells. In this way, a two-dimensional surface is provided against which the flattening of the cells can be observed. For example, the bottom and / or wall of a well can be coated with antibody. The binding of the antibody to the substrate can occur through a constant region of the antibody.

[0418] A negative control may be included, such as an antibody that does not bind to ICOS, preferably, Petition 870230019896, dated 08 / 03 / 2023, p. 178 / 576 159 / 456 an antibody that does not bind to an antigen on the surface of cells expressing ICOS to be used. The assay may comprise the quantification of the degree of morphological change and, when multiple antibodies are tested, selecting an antibody that induces a greater morphological change than one or more other test antibodies.

[0419] Antibody selection may comprise expression of the nucleic acid encoding the antibody present in the test well of interest or expression of an antibody comprising the CDRs or antigen-binding domain of that antibody. The antibody may optionally be reformatted, for example, to provide an antibody comprising the antigen-binding domain of the selected antibody, for example, an antibody fragment or an antibody comprising a different constant region. A selected antibody is preferably endowed with a human IgG1 constant region or other constant region as described herein. A selected antibody may be further formulated in a composition comprising one or more additional ingredients – suitable pharmaceutical formulations are discussed herein throughout this document. CLAUSES

[0420] The embodiments of the invention are presented in the following numbered clauses, which are part of the description.

[0421] Clause 1. An isolated antibody that binds to the extracellular domain of human and / or mouse ICOS, wherein the antibody comprises a VH domain comprising an amino acid sequence that has at least 95% of Petition 870230019896, dated 08 / 03 / 2023, page 179 / 576 160 / 456 sequence identity with the VH domain of STIM003 SEQ ID NO:408 and a VL domain comprising an amino acid sequence that has at least 95% sequence identity with the VL domain of STIM003 SEQ ID NO:415.

[0422] Clause 2. An antibody, according to clause 1, wherein the VH domain comprises a set of heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein

[0423] HCDR1 is the HCDR1 of STIM003 with the amino acid sequence SEQ ID NO:405,

[0424] HCDR2 is HCDR2 from STIM003 with amino acid sequence SEQ ID NO:406,

[0425] HCDR3 is HCDR3 from STIM003 with amino acid sequence SEQ ID NO:407,

[0426] Clause 3. An antibody, according to clause 1 or 2, wherein the VL domain comprises a set of light chain complementarity-determining regions (LCDRs), LCDR1, LCDR2 and LCDR3, wherein

[0427] LCDR1 is the LCDR1 of STIM003 with the amino acid sequence SEQ ID NO:412,

[0428] LCDR2 is the LCDR2 of STIM003 with the amino acid sequence SEQ ID NO:413,

[0429] LCDR3 is the LCDR3 of STIM003 with the amino acid sequence SEQ ID NO:414.

[0430] Clause 4. An antibody, according to clause 1, in which the amino acid sequence of the VH domain is SEQ ID NO:408 and / or in which the amino acid sequence of the VL domain is SEQ ID NO:415.

[0431] Clause 5. An isolated antibody that binds to the extracellular domain of human and / or ICOS Petition 870230019896, dated 08 / 03 / 2023, page 180 / 576 161 / 456 mice comprising

[0432] an antibody VH domain comprising the complementarity-determining regions (CDRs) HCDR1, HCDR2 and HCDR3, and

[0433] an antibody VL domain comprising the complementarity-determining regions LCDR1, LCDR2 and LCDR3, where

[0434] HCDR1 is the HCDR1 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that HCDR1 with 1, 2, 3, 4 or 5 amino acid changes,

[0435] HCDR2 is the HCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that HCDR2 with 1, 2, 3, 4 and / or 5 amino acid changes,

[0436] HCDR3 is the HCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that HCDR3 with 1, 2, 3, 4 or 5 amino acid changes,

[0437] Clause 6. An antibody, according to clause 5, wherein the antibody heavy chain CDRs are those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprise the heavy chain CDRs of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 with 1, 2, 3, 4 or 5 amino acid changes.

[0438] Clause 7. An antibody, according to clause 6, in which the antibody VH domain has the heavy chain CDRs of STIM003.

[0439] Clause 8. An isolated antibody that Petition 870230019896, dated 08 / 03 / 2023, page 181 / 576 162 / 456 links to the extracellular domain of human and / or mouse ICOS comprising

[0440] an antibody VH domain comprising the complementarity-determining regions HCDR1, HCDR2 and HCDR3, and

[0441] an antibody VL domain comprising the complementarity-determining regions LCDR1, LCDR2 and LCDR3,

[0442] where LCDR1 is LCDR1 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises LCDR1 with 1, 2, 3, or 5 amino acid changes,

[0443] LCDR2 is LCDR2 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or includes that LCDR2 with 1, 2, 3, and / or 5 amino acid alterations,

[0444] LCDR3 is LCDR3 of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprises that LCDR3 with 1, 2, 3, or 5 amino acid changes,

[0445] Clause 9. An antibody, according to any of clauses 5 to 8, wherein the antibody light chains CDRs are those of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or comprise the light chain CDRs STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 with 1, 2, 3, 4 or 5 amino acid changes.

[0446] Clause 10. An antibody, according to clause 9, in which the antibody VL domain has the CDRs of Petition 870230019896, dated 08 / 03 / 2023, page 182 / 576 163 / 456 light chain of STIM003.

[0447] Clause 11. An antibody according to any of clauses 5 to 10 comprising structural regions of VH and / or VL domains of human germline gene segment sequences.

[0448] Clause 12. An antibody, according to any one of clauses 5 to 11, comprising a VH domain that

[0449] (i) is derived from the recombination of a human heavy chain V gene segment, a human heavy chain D gene segment and a human heavy chain J gene segment, wherein

[0450] segment V is IGHV1-18 (e.g., V118*01), IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V3-11*01) or IGVH2-5 (e.g., V2-5*10);

[0451] gene segment D is IGHD6-19 (e.g., IGHD6-19*01), IGHD3-10 (e.g., IGHD3-10*01) or IGHD3-9 (e.g., IGHD3-9*01); and / or

[0452] the J gene segment is IGHJ6 (e.g., IGHJ6*02), IGHJ4 (e.g., IGHJ4*02) or IGHJ3 (e.g., IGHJ3*02) or

[0453] (ii) comprises the FR1, FR2, FR3 and FR4 framework regions, where

[0454] FR1 aligns with the human germline gene segment V IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V311*01), or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4, or 5 amino acid changes,

[0455] FR2 aligns with the human germline gene segment V IGHV1-18 (e.g., V1-18*01), Petition 870230019896, dated 08 / 03 / 2023, p. 183 / 576 164 / 456 IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V311*01) or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4 or 5 amino acid changes,

[0456] FR3 aligns with the human germline gene segment V IGHV1-18 (e.g., V1-18*01), IGVH3-20 (e.g., V3-20*d01), IGVH3-11 (e.g., V311*01), or IGVH2-5 (e.g., V2-5*10), optionally with 1, 2, 3, 4, or 5 amino acid changes, and / or

[0457] FR4 aligns with the germline gene segment J IGJH6 (e.g., JH6*02), IGJH4 (e.g., JH4*02), or IGJH3 (e.g., JH3*02), optionally with 1, 2, 3, 4, or 5 amino acid changes.

[0458] Clause 13. An antibody according to any one of clauses 5 to 12, comprising a VL domain

[0459] (i) is derived from the recombination of the human light chain V gene segment and a human light chain J gene segment, wherein

[0460] segment V 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., IGKV311*01) and / or

[0461] 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

[0462] (ii) comprises the FR1, FR2, FR3 and FR4 framework regions, where

[0463] FR1 aligns with human germline gene segment V GKV2-28 (e.g., IGKV228*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., Petition 870230019896, dated 08 / 03 / 2023, p. 184 / 576 165 / 456 example, IGKV1D-39*01) or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4 or 5 amino acid changes,

[0464] FR2 aligns with human germline gene segment V IGKV2-28 (e.g., IGKV228*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01), or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4, or 5 amino acid changes,

[0465] FR3 aligns with human germline gene segment V IGKV2-28 (e.g., IGKV228*01), IGKV3-20 (e.g., IGKV3-20*01), IGKV1D-39 (e.g., IGKV1D-39*01), or IGKV3-11 (e.g., IGKV3-11*01), optionally with 1, 2, 3, 4, or 5 amino acid changes, and / or

[0466] FR4 aligns with the germline gene segment J IGKJ4 (e.g., IGKJ4*01), IGKJ2 (e.g., IGKJ2*04), IGKJ3 (e.g., IGKJ3*01), or IGKJ1 (e.g., IGKJ1*01), optionally with 1, 2, 3, 4, or 5 amino acid changes.

[0467] Clause 14. An antibody, according to any of clauses 5 to 13, comprising an antibody VH domain that is the Vh domain of STIM001, STIM002, STIM002B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or having an amino acid sequence at least 90% identical to the antibody VH domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009.

[0468] Clause 15. An antibody, according to any one of clauses 5 to 14, comprising an antibody VL domain that is the VL domain of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, Petition 870230019896, dated 08 / 03 / 2023, page 185 / 576 166 / 456 STIM008 or STIM009, or which has an amino acid sequence at least 90% identical to the antibody VL domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008, or STIM009.

[0469] Clause 16. An antibody, according to clause 15, comprising

[0470] an antibody VH domain that is selected from the VH domain of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009 or that has an amino acid sequence at least 90% identical to the antibody VH domain sequence of STIM001, STIM002, STIM002-B, STIM003, STIM004, STIM005, STIM006, STIM007, STIM008 or STIM009, and

[0471] an antibody VL domain that is the VL domain of said selected antibody or that has an amino acid sequence at least 90% identical to the antibody VL domain sequence of said selected antibody.

[0472] Clause 17. An antibody according to clause 16 comprising the VH domain of STIM003 and the VL domain of STIM003.

[0473] Clause 18. An antibody according to any of the preceding clauses comprising a constant region of antibody.

[0474] Clause 19. An antibody according to clause 18, wherein the constant region comprises a human heavy and / or light chain constant region.

[0475] Clause 20. An antibody according to clause 18 or clause 19, wherein the constant region is effector Fc positive.

[0476] Clause 21. An antibody, according to Petition 870230019896, dated 08 / 03 / 2023, page 186 / 576 167 / 456 clause 20, which comprises an Fc region that has enhanced ADCC, ADCP and / or CDC compared to a native human Fc region.

[0477] Clause 22. An antibody according to any of clauses 18 to 21, wherein the antibody is an IgG1.

[0478] Clause 23. An antibody according to clause 21 or 22, wherein the antibody is afucosylated.

[0479] Clause 24. An antibody according to any of the preceding clauses that is conjugated with a cytotoxic drug or prodrug.

[0480] Clause 25. An antibody according to any of the preceding clauses, which is a multispecific antibody.

[0481] Clause 26. An isolated antibody that binds to the extracellular domain of human and mouse ICOS with an affinity (Kd) of less than 50 nM, as determined by surface plasmon resonance.

[0482] Clause 27. An antibody according to clause 26, wherein the antibody binds to the extracellular domain of human and mouse ICOS with an affinity (Kd) of less than 5 nM as determined by surface plasmon resonance.

[0483] Clause 28. An antibody according to clause 26 or 27, wherein the Kd binding to the extracellular domain of human ICOS is less than 10 times the Kd binding to the extracellular domain of mouse ICOS.

[0484] Cell 29. A composition comprising an antibody isolated according to any of the preceding clauses and a pharmaceutically acceptable excipient.

[0485] Clause 30. A composition comprising isolated nucleic acid encoding an antibody according to Petition 870230019896, dated 08 / 03 / 2023, p. 187 / 576 168 / 456 any of clauses 1 to 28 and a pharmaceutically acceptable excipient.

[0486] Clause 31. A method for modulating the balance of regulatory T cells (Treg) to effector T cells (Teffs) in order to increase the Teff response in a patient comprising administering an antibody according to any of clauses 1 to 28 or a composition according to clause 29 to the patient.

[0487] Clause 32. A method for treating a disease or condition treatable by therapy depleting regulatory T cells (Tregs) and / or increasing the effector T cell (Teff) response in a patient, wherein the method comprises administering an antibody in accordance with any of clauses 1 to 28 or a composition in accordance with clause 29 to the patient.

[0488] Clause 33. An antibody, according to any of clauses 1 to 28, or a composition according to clause 29, for use in a method of treating the human body by therapy.

[0489] Cell 34. An antibody or composition for use in accordance with clause 33, for use in modulating the balance of regulatory T cells (Tregs) to effector T cells (Teffs) to enhance the effector T cell response in a patient.

[0490] Clause 35. An antibody or composition for use in accordance with clause 33 for use in the treatment of a disease or condition treatable with therapy depleting regulatory T cells (Tregs) and / or enhancing the effector response of T cells (Teff) in a patient.

[0491] Clause 36. A method, according to Petition 870230019896, dated 08 / 03 / 2023, page 188 / 576 169 / 456 clause 32, or an antibody or a composition for use in accordance with clause 35, wherein the disease is a cancer or a solid tumor.

[0492] Clause 37. An antibody, according to any one of clauses 1 to 28, or a composition according to clause 29, for use in a method for treating cancer in a human patient.

[0493] Clause 38. A method for treating cancer in a human patient comprising administering an antibody, in accordance with any of clauses 1 to 28, or a composition in accordance with clause 29, to the patient.

[0494] Clause 39. A method or an antibody or composition for use in accordance with any of clauses 36 to 38, wherein the cancer is renal cell carcinoma, head and neck cancer, melanoma, non-small cell lung cancer or diffuse large B-cell lymphoma.

[0495] Clause 40. A method or an antibody or composition for use in accordance with any of clauses 31 to 39, wherein the method comprises administering the antibody and another therapeutic agent and / or radiation therapy to the patient.

[0496] Clause 41. A method or an antibody or composition for use in accordance with clause 40, wherein the therapeutic agent is an anti-PD-L1 antibody.

[0497] Clause 42. A method or an antibody or composition for use, in accordance with clause 41, wherein the anti-PD-L1 antibody comprises a VH domain having an amino acid sequence SEQ ID NO:299 and a VL domain having an amino acid sequence SEQ ID NO:300.

[0498] Clause 43. A method or an antibody or Petition 870230019896, dated 08 / 03 / 2023, page 189 / 576 170 / 456 composition for use in accordance with clause 41 or clause 42, wherein the therapeutic agent is an anti-PD-L1-IL2 immunocytokine agent.

[0499] Clause 44. A method or an antibody or composition for use in accordance with clause 43, wherein the anti-PD-L1 antibody is an immunocytokine comprising wild-type or human variant IL-2.

[0500] Clause 45. A method or an antibody or composition for use in accordance with clause 44, wherein the anti-ICOS antibody and the anti-PDL1 antibody each have the capacity to mediate ADCC, ADCP and / or CDC.

[0501] Clause 46. A method or an antibody or composition for use, in accordance with any of clauses 41 to 45, wherein the anti-ICOS antibody is a human IgG1 antibody and the anti-PDL1 antibody is a human IgG1 antibody.

[0502] Clause 47. A method or an antibody or composition for use, in accordance with clause 40, wherein the therapeutic agent is an anti-DP-1 antibody.

[0503] Clause 48. A method or an antibody or composition for use in accordance with clause 40, wherein the other therapeutic agent is IL-2.

[0504] Clause 49. A method or an antibody or composition for use, in accordance with any of clauses 40 to 48, wherein the method comprises administering the anti-ICOS antibody after administration of the other therapeutic agent and / or radiation therapy.

[0505] Clause 50. A method or an antibody or composition for use in accordance with any of clauses 31 to 49, wherein

[0506] the anti-ICOS antibody is conjugated to a pro Petition 870230019896, dated 08 / 03 / 2023, p. 190 / 576 171 / 456 drug, and in which

[0507] the method or use comprises

[0508] administer the anti-ICOS antibody to a patient and

[0509] selectively activate the prodrug at a target tissue site.

[0510] Clause 51. A method or an antibody or composition for use, in accordance with clause 50, wherein the patient has a solid tumor and the method comprises selectively activating the prodrug in the tumor.

[0511] Clause 52. A method or an antibody or composition for use, in accordance with clause 50 or clause 51, comprising selectively activating the prodrug by photoactivation.

[0512] Clause 53. Combination of human anti-ICOS IgG1 antibody and human anti-PDL1 IgG1 antibody for use in a cancer treatment method in a patient.

[0513] Clause 54. A method of treating cancer in a patient comprising administering a human anti-ICOS IgG1 antibody and a human anti-PD-L1 IgG1 antibody to the patient.

[0514] Clause 55. Anti-ICOS antibody for use in a method for treating cancer in a patient, wherein the method comprises administering the anti-ICOS antibody and the anti-PD-L1 antibody to the patient, in which a single dose of the anti-ICOS antibody is administered.

[0515] Clause 56. Anti-ICOS antibody for use in accordance with clause 55, wherein the anti-ICOS antibody is a human IgG1 antibody and the anti-PD-L1 antibody is a human IgG1 antibody. Petition 870230019896, dated 08 / 03 / 2023, page 191 / 576 172 / 456

[0516] Clause 57. Combination, in accordance with clause 53, method in accordance with clause 54 or anti-ICOS antibody for use in accordance with clause 55 or clause 56, wherein the cancer is renal cell carcinoma, head and neck cancer, melanoma, non-small cell lung cancer or diffuse large B-cell lymphoma.

[0517] Clause 58. A method or an antibody, composition or combination for use in accordance with any of clauses 41 to 46 or 53 to 54, wherein the method comprises administering the anti-ICOS antibody and the anti-PD-L1 antibody to the patient, wherein a single dose of the anti-ICOS antibody is administered.

[0518] Clause 59. A method or an antibody, composition or combination for use in accordance with clause 58, wherein the method comprises administering a single dose of the anti-ICOS antibody followed by multiple doses of the anti-PD-L1 antibody.

[0519] Clause 60. A method or an antibody, composition or combination for use in accordance with any of clauses 41 to 46 or 53 to 54, wherein the anti-ICOS antibody and the anti-PDL1 antibody are provided in separate compositions for administration.

[0520] Clause 61. A method or an antibody, composition or combination for use in accordance with any of clauses 41 to 46 or 53 to 60, wherein the anti-ICOS antibody and / or the anti-PD-L1 antibody comprises a human IgG1 constant region comprising the amino acid sequence SEQ ID NO:340.

[0521] Clause 62. Anti-ICOS antibody for use in a method for treating a patient comprising the method Petition 870230019896, dated 08 / 03 / 2023, page 192 / 576 173 / 456 which comprises the administration of anti-ICOS antibody to a patient who has an increased level of ICOS-positive regulatory T cells after treatment with another therapeutic agent.

[0522] Clause 63. A method for treating a patient, wherein the method comprises administering an anti-ICOS antibody to a patient who has an increased level of ICOS-positive regulatory T cells following treatment with another therapeutic agent.

[0523] Clause 64. An anti-ICOS antibody for use in accordance with clause 62, or a method in accordance with clause 63, wherein the method comprises administering a therapeutic agent to the patient, to determine that the patient has an increased level of ICOS-positive regulatory T cells following treatment with said agent and administering an anti-ICOS antibody to the patient to reduce the level of regulatory T cells.

[0524] Clause 65. An anti-ICOS antibody for use or a method, in accordance with any of clauses 62 to 64, wherein the therapeutic agent is IL-2 or an immunomodulatory antibody (for example, anti-PDL-1, anti-DP-1 or anti-CTLA-4).

[0525] Clause 66. An anti-ICOS antibody for use or a method in accordance with any of clauses 62 to 65, wherein the method comprises treating a tumor, for example, melanoma, such as metastatic melanoma.

[0526] Clause 67. Anti-ICOS antibody for use in a method of treating cancer in a patient by means of in vivo vaccination of the patient against cancer cells, wherein the method comprises

[0527] treat the patient with a therapy that causes Petition 870230019896, dated 08 / 03 / 2023, page 193 / 576 174 / 456 the death of immune cells in cancerous cells, resulting in the presentation of the antigen to antigen-specific effector T cells, and

[0528] administer an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances the antigen-specific T cell response.

[0529] Clause 68. A method of treating cancer in a patient by means of in vivo vaccination of the patient against cancer cells, wherein the method comprises

[0530] treat the patient with a therapy that causes the death of immune cells of cancer cells, resulting in the presentation of the antigen to antigen-specific effector T cells, and

[0531] administer an anti-ICOS antibody to the patient, wherein the anti-ICOS antibody enhances the antigen-specific T cell response.

[0532] Clause 69. A method of treating cancer in a patient by means of in vivo vaccination of the patient against cancer cells, wherein the method comprises administering an anti-ICOS antibody to the patient in which

[0533] the patient is one who has been previously treated with a therapy that causes the death of immune cells of cancer cells, resulting in the presentation of the antigen to antigen-specific effector T cells, and in which

[0534] the anti-ICOS antibody enhances the antigen-specific T cell response.

[0535] Cell 70. Anti-ICOS antibody for use or a method in accordance with any of clauses 67 to 69, wherein the therapy causes death of immune cells is Petition 870230019896, dated 08 / 03 / 2023, page 194 / 576 175 / 456 radiation of cancer cells, administration of a chemotherapeutic agent and / or administration of an antibody directed against a tumor-associated antigen.

[0536] Clause 71. Anti-ICOS antibody for use or a method in accordance with clause 70, wherein the chemotherapeutic agent is oxaliplatin.

[0537] Clause 72. Anti-ICOS antibody for use or a method in accordance with clause 70, wherein the tumor-associated antigen is HER2 or CD20.

[0538] Clause 73. Anti-ICOS antibody for use in a method for treating cancer in a patient, wherein the cancer is or has been characterized as being positive for expression of the ICOS and / or FOXP3 ligand.

[0539] Clause 74. A method for treating cancer in a patient, wherein the cancer is or has been characterized as being positive for expression of ICOS and / or FOXP3 ligand, wherein the method comprises administering an anti-ICOS antibody to the patient.

[0540] Clause 75. Anti-ICOS antibody for use in accordance with clause 73, or a method in accordance with clause 74, wherein the method comprises:

[0541] test a patient sample to determine that the cancer expresses the ICOS and / or FOXP3 ligand;

[0542] select the patient for treatment with the anti-ICOS antibody; and

[0543] administer the anti-ICOS antibody to the patient.

[0544] Clause 76. Anti-ICOS antibody for use in accordance with clause 73 or a method in accordance with clause 74, wherein the method comprises administering a Petition 870230019896, dated 08 / 03 / 2023, page 195 / 576 176 / 456 anti-ICOS antibody for a patient in whom an assay sample indicated that the cancer is positive for expression of the ICOS and / or FOXP3 ligand.

[0545] Clause 77. Anti-ICOS antibody for use or a method in accordance with clause 75 or clause 76, wherein the sample is a biopsy sample of a solid tumor.

[0546] Clause 78. Anti-ICOS antibody for use in a method for treating cancer in a patient, wherein the cancer is or has been characterized as being refractory to treatment with an immuno-oncological drug, for example, anti-CTLA-4 antibody, anti-PD1 antibody, anti-PDL1 antibody, anti-CD137 antibody or anti-GITR antibody.

[0547] Clause 79. A method for treating cancer in a patient, wherein the cancer is or has been characterized as being refractory to treatment with an immuno-oncological drug, for example, anti-CTLA-4 antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-CD137 antibody or anti-GITR antibody, wherein the method comprises administering an anti-ICOS antibody to the patient.

[0548] Clause 80. Anti-ICOS antibody for use in accordance with clause 78, or a method in accordance with clause 79, wherein the method comprises:

[0549] treat the patient with immuno-oncological drug;

[0550] determine that the cancer does not respond to the drug;

[0551] select the patient for treatment with the anti-ICOS antibody; and

[0552] administer the anti-ICOS antibody to the patient. Petition 870230019896, dated 08 / 03 / 2023, page 196 / 576 177 / 456

[0553] Clause 81. Anti-ICOS antibody for use in accordance with clause 78, or a method in accordance with clause 79, wherein the method comprises: administering an anti-ICOS antibody to a patient whose cancer has not responded to previous treatment with the immuno-oncological drug.

[0554] Clause 82. Anti-ICOS antibody for use or a method in accordance with any of clauses 73 to 81, wherein cancer is a tumor derived from cells that have acquired the ability to express the ICOS ligand.

[0555] Clause 83. Anti-ICOS antibody for use or a method in accordance with clause 82, where the cancer is melanoma.

[0556] Cell 84. Anti-ICOS antibody for use or a method in accordance with any of clauses 73 to 81, wherein the cancer is derived from an antigen-presenting cell, such as a B lymphocyte (e.g., B-cell lymphoma, such as diffuse large B-cell lymphoma) or a T lymphocyte.

[0557] Cell 85. Anti-ICOS antibodies for use or a method in accordance with any of clauses 73 to 81, where the cancer is resistant to treatment with an anti-CD20 antibody.

[0558] Cell 86. Anti-ICOS antibody for use or a method in accordance with clause 85, wherein the cancer is B-cell lymphoma.

[0559] Cell 87. Anti-ICOS antibodies for use or a method in accordance with clause 86, wherein the anti-CD20 antibody is rituximab.

[0560] Cell 88. Anti-ICO antibodies for use Petition 870230019896, dated 08 / 03 / 2023, page 197 / 576 178 / 456 or a method in accordance with any of clauses 85 to 87, wherein the method comprises treating the patient with the anti-CD20 antibody;

[0561] determine that the cancer does not respond to anti-CD20 antibody;

[0562] test a patient sample to determine that the cancer expresses the ICOS ligand;

[0563] select the patient for treatment with the anti-ICOS antibody; and

[0564] administer the anti-ICOS antibody to the patient.

[0565] Clause 89. Anti-ICOS antibodies for use or a method in accordance with any of clauses 85 to 87, wherein the method comprises administering an anti-ICOS antibody to a patient whose cancer has not responded to previous treatment with anti-CD20 antibody.

[0566] Clause 90. Anti-ICOS antibody for use or a method in accordance with any of clauses 67 to 89, wherein cancer is a solid tumor.

[0567] Clause 91. Anti-ICOS antibody for use or a method in accordance with any of clauses 67 to 89, wherein cancer is a hematologic fluid tumor.

[0568] Clause 92. Anti-ICOS antibody for use or a method in accordance with clause 90 or 91, wherein the tumor has a high content of regulatory T cells.

[0569] Clause 93. Anti-ICOS antibody for use or a method, in accordance with any of clauses 53 to 92, wherein the anti-ICOS antibody is as defined in any of clauses 1 to 28, or is provided in a composition in accordance with clause 29. Petition 870230019896, dated 08 / 03 / 2023, page 198 / 576 179 / 456

[0570] Cell 94. A non-human transgenic mammal having a genome comprising a human or humanized immunoglobulin locus encoding gene segments of the human variable region, wherein the mammal does not express ICOS.

[0571] Clause 95. A method for producing an antibody that binds to the extracellular domain of human and non-human ICOS comprising

[0572] (a) immunize a mammal in accordance with clause 94 with human ICOS antigen;

[0573] (b) isolate the antibodies generated by the mammal;

[0574] (c) test antibodies for their ability to bind to human ICOS and non-human ICOS; and

[0575] (d) select one or more antibodies that bind to both human and non-human ICOS.

[0576] Clause 96. A method, according to clause 95, comprising immunizing mammals with cells expressing human ICOS.

[0577] Clause 97. A method in accordance with clause 95 or 96 comprising

[0578] (c) test antibodies for binding ability to human and non-human ICOS using surface plasmon resonance and determine binding affinities; and

[0579] (d) select one or more antibodies for which the human ICOS binding Kd is less than 50 nM and the non-human ICOS binding Kd is less than 500 nM.

[0580] Clause 98. A method in accordance with clause 97 comprising Petition 870230019896, dated 08 / 03 / 2023, p. 199 / 576 180 / 456

[0581] (d) select one or more antibodies for which the Kd binding to human ICOS is less than 10 nM, and the Kd binding to non-human ICOS is less than 100 nM.

[0582] Clause 99. A method in accordance with any of clauses 95 to 98 comprising

[0583] (c) test antibodies for binding capacity to human and non-human ICOS using surface plasmon resonance and determine binding affinities; and

[0584] (d) select one or more antibodies for which the Kd binding to human ICOS is less than 10 times the Kd binding to non-human ICOS.

[0585] Clause 100. A method in accordance with clause 99 comprising

[0586] (d) select one or more antibodies for which the Kd binding to human ICOS is less than 5 times the Kd binding to non-human ICOS.

[0587] Clause 101. A method in accordance with any of clauses 95 to 100 comprising testing antibodies for their ability to bind to non-human ICOS of the same species as the animal.

[0588] Clause 102. A method in accordance with any of clauses 95 to 101 comprising testing antibodies for their ability to bind to non-human ICOS of a species other than animal.

[0589] Clause 103. A method in accordance with any of clauses 95 to 102, wherein the mammal is a mouse or a rat.

[0590] Clause 104. A method in accordance with any of clauses 95 to 103, wherein the non-human ICOS Petition 870230019896, dated 08 / 03 / 2023, p. 200 / 576 181 / 456 is mouse ICOS or rat ICOS.

[0591] Clause 105. A method in accordance with any of clauses 95 to 104, wherein the human or non-human immunoglobulin locus comprises gene segments from a human variable region upstream of an endogenous constant region.

[0592] Clause 106. A method in accordance with clause 105 comprising

[0593] (a) immunizing a mammal in accordance with clause 94 with human ICOS antigen, wherein the mammal is a mouse;

[0594] (b) isolate the antibodies generated by the mouse;

[0595] (c) test antibodies for their ability to bind to human ICOS and mouse ICOS; and

[0596] (d) select one or more antibodies that bind to both human ICOS and mouse ICOS.

[0597] Clause 107. A method according to any of clauses 95 to 106 comprising the isolation of nucleic acid encoding a variable heavy chain antibody domain and / or a variable light chain antibody domain.

[0598] Clause 108. A method in accordance with any of clauses 95 to 107, wherein the mammal produces antibodies by recombination of gene segments from the human variable region and an endogenous constant region.

[0599] Clause 109. A method, according to clause 107 or 108, comprising conjugating nucleic acid encoding the variable heavy chain and / or light chain domain of a nucleotide sequence encoding a Petition 870230019896, dated 08 / 03 / 2023, page 201 / 576 182 / 456 constant region of the human heavy chain and / or the human light chain constant region respectively.

[0600] Clause 110. A method, according to any one of clauses 107 to 109, comprising introducing nucleic acid into a host cell.

[0601] Clause 111. A method, according to clause 110, comprising culturing the host cell under conditions for expression of the antibody or of the variable domain of the heavy and / or light chain of the antibody.

[0602] Clause 112. An antibody or variable domain of the heavy and / or light chain of the antibody produced by the method in accordance with any of clauses 95 to 111.

[0603] Clause 113. A method for selecting an antibody that binds to ICOS, optionally for selecting an ICOS agonist antibody, comprising the assay:

[0604] provide an array of antibodies immobilized (bound or attached) to a substrate in a test well;

[0605] add cells that express ICOS (e.g., activated primary T cells or MJ cells) to the test wells;

[0606] observe the morphology of the cells;

[0607] detect change in cell shape from rounded to flattened against the substrate within the cavities; the change in shape indicates that the antibody is an ICOS-binding antibody, optionally an ICOS agonist antibody;

[0608] select antibody from test wells;

[0609] express nucleic acid that codes for Petition 870230019896, dated 08 / 03 / 2023, page 202 / 576 183 / 456 CDRs of the selected antibody; and

[0610] to formulate the antibody in a composition comprising one or more additional components.

[0611] Several other aspects and embodiments of the present invention will become apparent to those skilled in the art by virtue of the present disclosure. All documents mentioned in this descriptive report, including the equivalents of any patents or patent applications cited, are incorporated herein by reference in their entirety. EXPERIMENTAL EXAMPLES

[0612] The following Examples describe the generation, characterization, and performance of anti-ICOS antibodies. The antibodies were generated using Kymouse™, a transgenic mouse platform capable of generating antibodies with human variable domains. Kymouse™ antibodies have human variable domains, generated from human V(D) and J segments, and mouse constant domains. Endogenous mouse variable genes have been silenced and constitute a very small portion of the repertoire (less than 0.5% of all heavy chain variable regions are of mouse origin). The Kymouse™ system is described in Lee et al 2014

[39] , WO2011 / 004192, WO2011 / 158009 and WO2013 / 061098. This project employed the HK Kymouse™ strain, in which the heavy chain locus and the light chain kappa are humanized.

[0613] Kymouse™kockout ICOS were immunized with ICOS protein or a combination of alternative protein enhancements and human and mouse ICOS-expressing cells.

[0614] The occurrences that were linked to human ICOS Petition 870230019896, dated 08 / 03 / 2023, page 203 / 576 184 / 456 were identified. The primary selection criteria for screening were binding to ICOS expressed in human cells (CHO cells) and binding to the ICOS protein (HTRF). Binding to mouse ICOS protein and to ICOS expressed in mouse cells (CHO cells) was also evaluated and considered during the selection of screening occurrences. Using these criteria, occurrences progressed to a second screening. In the secondary screening, the results were confirmed by determining binding to human and mouse ICOS expressed in CHO cells by flow cytometry.

[0615] From a large number of screened antibodies, a small panel was identified that binds to human / cynomolgus and mouse ICOS, as determined by surface plasmon resonance and flow cytometry. These antibodies included STIM001, STIM002 and variants STIM002-B, STIM003, STIM004 and STIM005 thereof. Four more antibodies, STIM006, STIM007, STIM008 and STIM009, were also added, showing less cross-reactivity with mouse ICOS but demonstrating agonism of the human ICOS receptor. The data presented in this document indicate the ability of anti-ICOS antibodies to act as ICOS receptor agonists in an ICOS-positive CD4+ cell line and also in a primary T cell-based assay, show the ability to kill cells in an ADCC assay and an ability to promote an antitumor immune response in vivo. EXAMPLE 1: KNOCKOUT MOUSE GENERATION WITH ICOS

[0616] A Kymouse™ knockout cell line with ICOS was generated by homologous recombination in Kymouse™ HK ES cells. Petition 870230019896, dated 08 / 03 / 2023, page 204 / 576 185 / 456 In summary, a 3.5 kb targeting vector encoding a puromycin selection was targeted to ES cells. Successful targeting resulted in the replacement of a small region (72 bp) of the mouse ICOS locus with a puromycin cassette, disrupting the signal peptide / start codon of the gene. The ES-positive clones were expanded and microinjected into mouse blastocysts, and the chimeras were cultured to ultimately generate homozygous animals for both the kappa heavy and humanized immunoglobulin heavy loci and the modified functionally null ICOS locus. EXAMPLE 2: ANTIGEN AND CELL LINE PREPARATION Generation of stably transfected MEF and CHO-S cells expressing human or mouse ICOs.

[0617] Full-length DNA sequences encoding human and mouse ICOS were encoded for mammalian expression, ordered as synthetic strand DNA, and cloned into an expression vector under the control of the CMV promoter and flanked by piggyBac-specific 3' and 5' terminal repeat sequences facilitating stable integration into the cell genome (see

[40] ). The expression vector contained a puromycin selection cassette to facilitate stable cell line generation. For the generation of cell lines expressing human ICOS and mouse ICOS, respectively, the human or mouse ICOS expression plasmid was transfected with a piggyBac transposase-encoding plasmid into an embryonic fibroblast cell line of Petition 870230019896, dated 08 / 03 / 2023, page 205 / 576 186 / 456 mouse (MEF) and CHO-S cells were transfected using FreeStyle Max transfection reagent (Invitrogen) according to the manufacturer's instructions. MEF cells were generated from embryos obtained from a 129S5 female mouse crossed with C57BL6. Twenty-four hours after transfection, the medium was supplemented with puromycin and cultured for at least two weeks to select stable cell lines. The cell culture medium was replaced every 3 to 4 days. The expression of human or mouse ICOS protein was evaluated by flow cytometry using anti-human or anti-mouse ICOS-PE conjugated antibodies (eBioscience), respectively. The complete MEF medium was formed from Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% v / v fetal bovine serum (Gibco). The complete CHO-S medium was composed of CD-CHO medium supplemented with 8 mM Glutamax (Gibco).CHO-S cells are the CHO-3E7 cell line included in the pTT5 system available from the National Research Council of Canada, but other CHO cell lines may be used. Preparation of MEF cells for mouse immunizations.

[0618] The cell culture medium was removed, and the cells were washed once with 1XPBS. The cells were treated for 5 minutes with trypsin to loosen the cells from the tissue culture surface. The cells were collected, and the trypsin was neutralized by the addition of complete medium containing 10% v / v fetal bovine serum (FCS). The cells were then centrifuged at 300 g for 10 minutes and washed with 25 ml of 1XPBS. The cells were counted and resuspended at the appropriate concentration in 1XPBS. Petition 870230019896, dated 08 / 03 / 2023, page 206 / 576 187 / 456 CLONING AND PROTEIN EXPRESSION RECOMBINANTS

[0619] Synthetic DNA encoding the extracellular domains of human ICOS (NCBI ID: NP_036224.1), mouse ICOS (NCBI ID: NP_059508.2), and cynomolgo ICOS (GenBank ID: EHH55098.1) were cloned into a pREP4 (Invitrogen) or a pTT5 expression plasmid (National Research Council of Canada) using standard molecular biology techniques. The constructs also contained a human Fc motif, a mouse Fc motif, or a peptide motif. FLAG His was used to aid in purification and detection. These were added to the DNA constructs by overlap extension. All constructs were sequenced prior to expression to ensure correct sequence composition. EXAMPLE 3: IMMUNIZATION

[0620] KymiceTM kockout with ICOS (see Example 1), the wild-type HK strain Kymouse™ and the wild-type HL strain Kymouse™ were immunized according to the regimens presented in Table E3. The wild-type HK and HL strains Kymouse™ express wild-type mouse ICOS. In the HK strain, the immunoglobulin chain locus and the kappa light chain locus are humanized, and in the HL strain, the immunoglobulin heavy chain locus and the lambda light chain locus are humanized. Mouse Regime Starter or Intensification 1 Intensification 2 Intensification 3 Final Intensification KM103 KO with ICOS micOS Fc hICOS MEF micOS Fc hICOS MEF micOS Fc KM103 KO with ICOS micOS Fc hICOS Fc micOS Fc hICOS Fc N / A Petition 870230019896, dated 08 / 03 / 2023, p. 207 / 576 188 / 456 KM111 KO with ICOS micOS Fc+ hICOS Fc micOS MEF + hICOS MEF micOS Fc + hICOS Fc micOS MEF + hICOS MEF micOS Fc + hICOS Fc KM111 KO with ICOS hICOS Fc hICOS MEF hICOS Fc hICOS MEF hICOS Fc KM111 KO with ICOS micOS Fc micOS MEF micOS Fc micOS MEF micOS Fc KM111 HK and HL hICOS Fc hICOS MEF hICOS Fc hICOS MEF hICOS Fc KM135 KO with ICOS Initiator micOS Fc 1 and 6 intensifications (RIMMS) KM135 KO with ICOS Initiator hICOS Fc 1 and 6 intensifications (RIMMS) TABLE E3 IMMUNIZATION REGIMES FOR STRAINS OF KYMOUSE™

[0621] Legend for the Table:

[0622] mICOS Fc = Mouse ICOS protein with human FC

[0623] hiCOS Fc = human ICOS protein with human Fc

[0624] mICOS MEF = Mouse ICOS expressed in MEF cells

[0625] hICOS MEF = human ICOS expressed in cells MEF

[0626] micOS Fc + hICOS Fc = mouse ICOS protein with human ICOS Fc + human Fc delivered simultaneously

[0627] micOS MEF + hICOS MEF = mouse ICOS expressed in MEF cells + human ICOS expressed in MEF cells administered simultaneously

[0628] KO with ICOS = Kymouse HK kocknout with ICOS

[0629] HK and HL = HK and HL genotype of Kymouse from Petition 870230019896, dated 08 / 03 / 2023, p. 208 / 576 189 / 456 wild type

[0630] RIMMS is a modified subcutaneous immunization procedure (rapid multi-site immunization); modified after Kilpatrick et al.

[41] ). The KM103 and KM111 immunization regimens were initiated-placed on rest intensified by intraperitoneal (ip) administration. The Sigma Adjuvant System was used for all immunizations and rest intervals were generally between 2 and 3 weeks. Final intensifications were administered intravenously in the absence of adjuvant.

[0631] Sera from serial or terminal blood samples were analyzed for the presence of specific antibodies by flow cytometry, and titer data were used (when possible) to select mice to be used for B cell separation. EXAMPLE 4: COMPARISON OF SERUM TITRATIONS BETWEEN KO MICE WITH ICOS AND WILD-TYPE MICE

[0632] Serum titers of immunized ICOS-KO and immunized wild-type Kymouse were determined using flow cytometry. In ICOS-KO mice, immunization with human ICOS antigen induced a serum immunoglobulin response with Ig binding to both mouse ICOS and human ICOS expressed in CHO cells (Figure 1a). In contrast, in wild-type Kymouse (which expresses mouse ICOS), immunization with the same human serum produced with human ICOS antigen showed markedly reduced binding to mouse ICOS compared to binding of the same serum to human ICOS (Figure 1b). METHOD Petition 870230019896, dated 08 / 03 / 2023, page 209 / 576 190 / 456

[0633] CHO-S cells expressing human ICOS or mouse ICOS (see Example 2) or non-transfected CHO cells (referred to as wild type (WT)), suspended in FACS buffer (PBS + 1% w / v BSA + 0.1% w / v sodium azide), were distribute...

Claims

CLAIMS 1. ISOLATED ANTIBODY, which binds to the extracellular domain of human and / or mouse ICOS, characterized by comprising: a VH domain comprising a set of heavy chain complementarity-determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, wherein HCDR1 is the HCDR1 of STIM003 with amino acid sequence SEQ ID NO: 405, HCDR2 is the HCDR2 of STIM003 with amino acid sequence SEQ ID NO: 406, HCDR3 is the HCDR3 of STIM003 with amino acid sequence SEQ ID NO: 407, and a VL domain comprising a set of light chain complementarity-determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, wherein LCDR1 is the LCDR1 of STIM003 with amino acid sequence SEQ ID NO: 412, LCDR2 is the LCDR2 of STIM003 with amino acid sequence SEQ ID NO: 413, LCDR3 is the LCDR3 of STIM003 with amino acid sequence SEQ ID NO:

414.

2. ANTIBODY, according to claim 1, characterized in that the amino acid sequence of the VH domain is SEQ ID NO: 408 and / or where the amino acid sequence of the VL domain is SEQ ID NO:

415.

3. ANTIBODY, according to claim 2, characterized in that 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. Petition 870260039875, dated 04 / 29 / 2026, p. 9 / 86 2 / 5 4. ANTIBODY, according to any one of claims 1 to 3, characterized by comprising a constant antibody region.

5. ANTIBODY, according to claim 4, characterized by the constant region comprising a constant region of human light and / or heavy chain.

6. ANTIBODY, according to any one of claims 4 or 5, characterized in that the constant region is Fc-positive effector.

7. ANTIBODY, according to any one of claims 4 to 6, characterized in that the antibody is an IgG1.

8. ANTIBODY, according to any one of claims 5 to 7, characterized by comprising a heavy chain amino acid sequence of SEQ ID NO:

410.

9. ANTIBODY, according to any one of claims 5 to 8, characterized by comprising a light chain amino acid sequence of SEQ ID NO:

417.

10. ANTIBODY, according to claim 9, characterized by comprising a heavy chain amino acid sequence of SEQ ID NO: 410 and a light chain amino acid sequence of SEQ ID NO:

417.

11. ANTIBODY, according to any one of claims 1 to 10, characterized by being a multispecific antibody.

12. COMPOSITION, characterized by comprising an isolated antibody, as defined in any one of claims 1 to 11, and a pharmaceutically acceptable excipient.

13. COMPOSITION, characterized by comprising isolated nucleic acid Petition 870260039875, dated 04 / 29 / 2026, p. 10 / 86 3 / 5 encoding an antibody, as defined in any of claims 1 to 12 and a pharmaceutically acceptable excipient, wherein the isolated nucleic acid sequence comprises the sequences SEQ ID NOs: 411 and 418.

14. METHOD FOR PRODUCING AN ANTIBODY that binds to the extracellular domain of human and non-human ICOS, as defined in claim 1, characterized by comprising (a) utilizing human ICOS antigen as an immunogen in a non-human mammal with human ICOS antigen, wherein the mammal has a genome comprising human or humanized immunoglobulin loci encoding gene segments of the human variable region; (b) isolating antibodies generated by the mammal; (c) testing the antibodies for binding capacity to human and non-human ICOS; and (d) selecting one or more antibodies that bind to both human and non-human ICOS.

15. METHOD, according to claim 14, characterized in that the mammal is a mouse or a rat.

16. METHOD, according to any one of claims 14 or 15, characterized in the non-human ICOS being a mouse ICOS or a rat ICOS.

17. METHOD, according to any one of claims 14 to 16, characterized by comprising isolating the nucleic acid encoding an antibody heavy chain variable domain and / or an antibody light chain variable domain.

18. METHOD, according to claim 17, characterized by comprising conjugating the nucleic acid that encodes the variable domain of the heavy chain and / or light chain into a nucleotide sequence that encodes a constant region of the human heavy chain and / or a constant region of the human light chain, respectively.

19. METHOD, according to any one of claims 17 or 18, characterized by comprising introducing nucleic acid into a host cell.

20. METHOD, according to claim 19, characterized by comprising cultivating the host cell under conditions for the expression of the antibody or of the antibody light and / or heavy chain variable domain, and isolating and purifying the antibody or of the antibody heavy and / or light chain variable domain.

21. METHOD FOR SELECTING AN ICOS-BINDING ANTIBODY, as defined in claim 1, optionally for selecting an ICOS agonist antibody, characterized by the assay comprising: providing an array of antibodies immobilized (bound or attached) to a substrate in a test well; adding ICOS-expressing cells (e.g., activated primary T cells or MJ cells) to the test well; observing cell morphology; detecting the shape change in the cells from rounded to flattened against the substrate within the well; wherein the shape change indicates that the antibody is an ICOS-binding antibody, optionally an ICOS agonist antibody; selecting the antibody from the test well; expressing the nucleic acid encoding the CDRs of the selected antibody; and formulating the antibody into a composition comprising one or more additional components.

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