Combination of BTN3A activating antibody and immune checkpoint inhibitor

Abstract

This application relates to therapeutic combinations of BTN3A-activating antibodies with immune checkpoint inhibitors selected from LAG-3 inhibitors (anti-LAG-3 antibodies and LAG-3 fusion proteins), anti-TIGIT antibodies, or anti-TIM-3 antibodies, which are particularly useful for treating cancer. More specifically, this disclosure relates to the combination of BTN3A-activating antibodies that specifically bind to BTN3A and activate the cytolytic function of Vy9V52 T cells with LAG-3 inhibitors that can directly bind to LAG-3 molecules or their ligands and block the interaction of LAG-3 with these ligands to promote a cooperative anti-tumor effect.

Description

[Technical Field]

[0001] Disclosed below are therapeutic combinations of BTN3A-activating antibodies with immune checkpoint inhibitors selected from LAG-3 inhibitors (e.g., anti-LAG-3 antibodies or LAG-3 fusion proteins), anti-TIGIT antibodies, or anti-TIM-3 antibodies that are particularly useful for treating cancer. More specifically, the disclosure relates to the combination of a BTN3A-activating antibody that specifically binds to BTN3A and activates the cytolytic function of Vy9V52 T cells with a LAG-3 inhibitor that can directly bind to LAG-3 molecules or their ligands and block the interaction of LAG-3 with these ligands, to promote a cooperative anti-tumor effect. [Background technology]

[0002] [background] Tumors evade immune-mediated recognition by several mechanisms. Among these, immune checkpoint inhibitor pathways have been well studied (Thommen et al., 2018 Cancer Cell. Apr 9;33(4):547-562). In particular, the immune checkpoint receptors PD-1 and CTLA-4, which are highly expressed on T cells, render them unresponsive or exhausted. Immune checkpoint inhibitor therapy targeting these receptors has shown promising clinical responses across a wide range of cancers. However, many patients still do not respond to them, and there is a need to identify alternative targets and approaches to improve therapeutic benefit to patients (Ribas and Wolchok 2018, Science. Mar 23;359(6382):1350-1355; Postow et al. 2015. J Clin Oncol. 2015 Jun 10;33(17):1974-82; Kong X. 2020. Adv Exp Med Biol. 1248:61-82). Besides PD-1 / PD-L1 and CTLA-4, novel immune checkpoint receptors such as LAG-3, TIM-3, and TIGIT have been well studied in cancer immunology, and antibody-based therapies targeting these inhibitor receptors are being developed and investigated in clinical trials as monotherapy or in combination with other drugs (Chauvin et al., 2020. J Immunother Cancer. Sep;8(2):e000957; He et al., 2018 Oct 16;11:7005-7009; Huo et al., 2022. Front Immunol. Jul 26;13:956090).In 2022, Opdualag, a combination of leratolimab, an anti-LAG-3 mAb developed by Bristol Myers Squibb (BMS), and nivolumab, an anti-PD-1 monoclonal antibody, was approved by the FDA as a single formulation for the treatment of unresectable or metastatic melanoma, demonstrating significant therapeutic advantages over nivolumab alone (Chocarro et al., 2022. Cutting-Edge: Preclinical and Clinical Development of the First Approved Lag-3 Inhibitor. Cells. Jul 30;11(15):2351). This approval indicates that combining several immune checkpoint inhibitors may offer therapeutic benefits to patients. Furthermore, with the exception of MHC-restricted αβ T cells, which have been primarily documented during immune checkpoint inhibitor therapy, the role of immune checkpoint receptors on nonconventional, non-MHC-restricted T cells has not been fully characterized, especially given that immune checkpoint receptors are also expressed by these cells.

[0003] Although these drugs represent a major advance in cancer therapy, there remains an unmet medical need for the large cancer patient population that does not respond to currently available treatments.

[0004] Among intriguing non-conventional T cells, Vγ9Vδ2 T cells are emerging as promising effector cells with potent cytolytic and proinflammatory activities and are associated with a favorable prognosis in cancer patients (Holtmeier et al., 2005. Chem Immunol Allergy. 86:151-183; Gentles et al., 2015. Nat Med. Aug;21(8):938-945; Tosolini et al., 2017. Oncoimmunology. Feb 6;6(3):e1284723).

[0005] Therefore, in recent years, a number of Vγ9Vδ2 T cell-based immuno-oncological therapeutic approaches have been investigated in a variety of tumors. These approaches have either involved in vivo activation of Vγ9Vδ2 T cells using aminobisphosphonates (ABPs), such as zoledronate, or synthetic pAgs (i.e., BrHPP) in combination with IL-2, or adoptive transfer of autologous or allogeneic Vγ9Vδ2 T cells into patients after in vitro / ex vivo expansion (Kabelitz et al., 2020. Cell Mol Immunol. Sep;17(9):925-939). Although these Vγ9Vδ2 T cell-based therapies appear to be safe, the clinical responses achieved have been variable among patients (Kabelitz et al., 2020. Cell Mol Immunol. Sep;17(9):925-939), suggesting the need for a better understanding of the underlying mechanisms regulating Vγ9Vδ2 T cells in the tumor microenvironment in order to develop more effective immunotherapies. The antitumor activity of Vγ9Vδ2 T cells in tumor immunosurveillance is triggered by TCR-mediated recognition of pAg, which is molecularly dependent on the transmembrane butyrophilin (BTN) molecules BTN3A1 and BTN2A1 (Kabelitz et al., 2020. Cell Mol Immunol. Sep;17(9):925-939).

[0006] BTN3A-activating antibodies have been described as potent activators of Vγ9Vδ2 T cells (Harly et al., 2012. Blood, Sep 13;120(11):2269-79; De Gassart et al., 2021. Sci Transl Med, 2021 Oct 20;13(616):eabj0835). Among these activating antibodies, ICT01, a first-in-class anti-BTN3A mAb that activates Vγ9Vδ2 T cells, is being evaluated as a single agent and in combination with a PD-1 blockade (pembrolizumab) in a phase 1 / 2a clinical study in solid tumors and hematological malignancies (EVICTION, NCT04243499).

[0007] The present disclosure relies on the use of a BTN3A activating antibody, such as ICT01, in combination with a LAG-3 or TIM-3 or TIGIT blocker, and optionally in combination with a PD-1 inhibitor, to synergistically enhance BTN3A-mediated tumor killing and / or activation of Vy9V52 T cells, and more generally to enhance anti-tumor immunity mediated by immune effector cells. Summary of the Invention

[0008] [overview] The present disclosure relates to a BTN3A activating antibody for use in treating cancer in a subject in need thereof, wherein a therapeutically effective amount of the BTN3A activating antibody is administered to the subject in combination with a therapeutically effective amount of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is selected from the group consisting of a LAG-3 inhibitor, a TIGIT inhibitor, and a TIM-3 inhibitor, and optionally the BTN3A activating antibody is further administered in combination with a PD-1 inhibitor.

[0009] In a preferred embodiment, the BTN3A activating antibody is ICT01.

[0010] In certain embodiments, the immune checkpoint inhibitor is a LAG-3 inhibitor. In more specific embodiments, the LAG-3 inhibitor is (i) a LAG-3 Fc fusion protein, e.g., eftiragimod alfa, (ii) an anti-LAG-3 antibody molecule, e.g., favezelimab, (iii) an anti-LAG-3 / anti-PD-1 bispecific antibody, e.g., tebotelimab, or (iv) a fixed-dose combination of an anti-LAG-3 antibody and an anti-PD-1 antibody, e.g., opdualag (a combination of leratolimab and nivolumab).

[0011] In certain embodiments, the immune checkpoint inhibitor is a TIM-3 inhibitor. In more specific embodiments, the TIM-3 inhibitor is (i) an anti-TIM-3 antibody molecule, e.g., sabatolimab, or (ii) an anti-LAG-3 / anti-PD-1 bispecific antibody, e.g., RO7121661.

[0012] In certain embodiments, the immune checkpoint inhibitor is a TIGIT inhibitor. In more specific embodiments, the TIGIT inhibitor is (i) an anti-TIGIT antibody molecule, such as tiragolumab, or (ii) an anti-TIGIT / anti-PD-1 bispecific antibody, such as AZD2936. [Brief explanation of the drawings]

[0013] [Figure 1A] Figure 1: Expression of LAG-3, TIM-3 and TIGIT on circulating Vy9V52 T cells as determined by flow cytometry. A. LAG-3, TIM-3 and TIGIT surface expression on circulating Vy9V52 T cells in ICT-1 stimulated healthy donor (HD) hu-PBMCs in vitro. B. LAG-3, TIM-3 and TIGIT surface expression on circulating Vy9V52 T cells from solid tumor patients treated with ICT01 + pembrolizumab. Two-way ANOVA and Holm-Sidak multiple comparison test were used: *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001). [Figure 1B] Figure 1: Expression of LAG-3, TIM-3 and TIGIT on circulating Vy9V52 T cells as determined by flow cytometry. A. LAG-3, TIM-3 and TIGIT surface expression on circulating Vy9V52 T cells in ICT-1 stimulated healthy donor (HD) hu-PBMCs in vitro. B. LAG-3, TIM-3 and TIGIT surface expression on circulating Vy9V52 T cells from solid tumor patients treated with ICT01 + pembrolizumab. Two-way ANOVA and Holm-Sidak multiple comparison test were used: *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001). [Figure 2A]Figure 1: Expression of LAG-3, TIM-3, and TIGIT on tumor-infiltrating Vy9V52 T cells and CD8+ T cells as determined by flow cytometry. A. Representative flow cytometry profiles of PD-1, LAG-3, TIM-3, and TIGIT surface expression on tumor-infiltrating Vy9V52 T cells and CD8+ T cells from dissociated tumor cells from bladder cancer. B. Percentage of cells positive for PD-1, LAG-3, TIM-3, and TIGIT on tumor-infiltrating Vy9V52 T cells (left panel) and on tumor-infiltrating CD8+ T cells (right panel) from different solid tumors: bladder cancer (n=15-16), head and neck cancer (n=6-9), and melanoma (n=4). [Figure 2B] Figure 1: Expression of LAG-3, TIM-3, and TIGIT on tumor-infiltrating Vy9V52 T cells and CD8+ T cells as determined by flow cytometry. A. Representative flow cytometry profiles of PD-1, LAG-3, TIM-3, and TIGIT surface expression on tumor-infiltrating Vy9V52 T cells and CD8+ T cells from dissociated tumor cells from bladder cancer. B. Percentage of cells positive for PD-1, LAG-3, TIM-3, and TIGIT on tumor-infiltrating Vy9V52 T cells (left panel) and on tumor-infiltrating CD8+ T cells (right panel) from different solid tumors: bladder cancer (n=15-16), head and neck cancer (n=6-9), and melanoma (n=4). [Figure 3]Figure 1 shows the density of intratumoral CD8+ T cells co-expressing PD-1, TIM-3, and LAG-3, as assessed by multiplex IHC using formalin-fixed and paraffin-embedded (FFPE) biopsy samples from patients in the EVICTION trial. The number of intratumoral CD3+CD8+PD1+LAG3+TIM3+ cells per mm2 of tissue was quantified after multiplex IHC staining performed on FFPE biopsy samples taken before (Pre) and 28 days after the start of treatment in cancer patients from the EVICTION trial. Data are shown from patients treated with ICT01 monotherapy (Group A) or ICT01 + pembrolizumab (Group C). Symbols accompanying the lines correspond to pre- and post-treatment biopsy samples from the same patient. Quantification of CD3+CD8+PD1+LAG3+TIM3+ cells was performed in the entire tumor (left panel), within the tumor area (middle panel), and within the stroma (right panel). [Figure 4A] Figure 1 shows the effect of blocking anti-LAG-3, anti-TIM-3 and anti-TIGIT mAbs on ICT01-mediated activation and expansion of Vy9V52 T cells in Hu-PBMCs. A. Activation (MFI CD25) and expansion (absolute counts) of Vy9V52 T cells determined by flow cytometry on days 3 and 6, respectively, in the presence of 10 μg / mL of blocking mAb (anti-LAG-3 or anti-TIM-3 or anti-TIGIT mAb) or corresponding isotypes in non-activated (NA) and ICT01-stimulated HD Hu-PBMCs. B. IFN-γ and granzyme-B secretion in culture supernatants determined by Luminex in the presence of 10 μg / mL of anti-LAG-3 mAb or corresponding isotypes in NA and ICT01-stimulated HD Hu-PBMCs. Wilcoxon test was used to compare two paired groups *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001) [Figure 4B]Figure 1 shows the effect of blocking anti-LAG-3, anti-TIM-3 and anti-TIGIT mAbs on ICT01-mediated activation and expansion of Vy9V52 T cells in Hu-PBMCs. A. Activation (MFI CD25) and expansion (absolute counts) of Vy9V52 T cells determined by flow cytometry on days 3 and 6, respectively, in the presence of 10 μg / mL of blocking mAb (anti-LAG-3 or anti-TIM-3 or anti-TIGIT mAb) or corresponding isotypes in non-activated (NA) and ICT01-stimulated HD Hu-PBMCs. B. IFN-γ and granzyme-B secretion in culture supernatants determined by Luminex in the presence of 10 μg / mL of anti-LAG-3 mAb or corresponding isotypes in NA and ICT01-stimulated HD Hu-PBMCs. Wilcoxon test was used to compare two paired groups *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001) [Figure 5A] Figure 1 shows the effect of blocking anti-LAG-3, anti-TIM-3 and anti-TIGIT mAbs on ICT01-mediated killing of tumor cells (SKOV-3) by in vitro expanded Vy9V52 T cells. A. LAG-3, TIM-3 and TIGIT surface expression on expanded Vy9V52 T cells as determined by flow cytometry. B. Tumor cell killing by expanded Vy9V52 T cells as determined by the percentage of viable target cells by flow cytometry after overnight co-culture with unstimulated (NA) and ICT01-activated Vy9V52 T cells in the presence of 10 μg / mL of blocking mAb (anti-LAG-3 or anti-TIM-3 or anti-TIGIT mAb) or the corresponding isotype (mIgG1). Wilcoxon test was used to compare two paired groups. *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001). [Figure 5B]Figure 1 shows the effect of blocking anti-LAG-3, anti-TIM-3 and anti-TIGIT mAbs on ICT01-mediated killing of tumor cells (SKOV-3) by in vitro expanded Vy9V52 T cells. A. LAG-3, TIM-3 and TIGIT surface expression on expanded Vy9V52 T cells as determined by flow cytometry. B. Tumor cell killing by expanded Vy9V52 T cells as determined by the percentage of viable target cells by flow cytometry after overnight co-culture with unstimulated (NA) and ICT01-activated Vy9V52 T cells in the presence of 10 μg / mL of blocking mAb (anti-LAG-3 or anti-TIM-3 or anti-TIGIT mAb) or the corresponding isotype (mIgG1). Wilcoxon test was used to compare two paired groups. *(p<0.05), **(p<0.01), ***(p<0.001) and ****(p<0.0001). [Figure 6A] Figure 1 shows the effect of blocking anti-LAG-3, anti-TIM-3 and anti-TIGIT mAbs on ICT01-mediated killing of tumor cells (THP1) by in vitro expanded Vy9V52 T cells. A. Representative flow cytometry profiles of LAG-3, TIM-3 and TIGIT surface expression on expanded Vy9V52 T cells (upper panel). Representative flow cytometry profiles of BTN3A and the ligands for LAG-3 (MHC class II), TIM-3 (galectin 9) and TIGIT (PVR and nectin 2) on THP1. B. Tumor cell killing by in vitro expanded Vy9V52 T cells as determined by flow cytometry analysis of the percentage of viable target cells after 4 hours of co-culture with naive (NA) and ICT01-activated Vy9V52 T cells. Co-cultures were performed in the presence of 10 μg / mL of the indicated immune checkpoint blockade mAb (anti-LAG-3, anti-TIM-3, or anti-TIGIT) or the corresponding isotype control (mIgG1). Two paired groups were compared using the Wilcoxon test. *(p<0.05) [Figure 6B]Figure 1 shows the effect of blocking anti-LAG-3, anti-TIM-3 and anti-TIGIT mAbs on ICT01-mediated killing of tumor cells (THP1) by in vitro expanded Vy9V52 T cells. A. Representative flow cytometry profiles of LAG-3, TIM-3 and TIGIT surface expression on expanded Vy9V52 T cells (upper panel). Representative flow cytometry profiles of BTN3A and the ligands for LAG-3 (MHC class II), TIM-3 (galectin 9) and TIGIT (PVR and nectin 2) on THP1. B. Tumor cell killing by in vitro expanded Vy9V52 T cells as determined by flow cytometry analysis of the percentage of viable target cells after 4 hours of co-culture with naive (NA) and ICT01-activated Vy9V52 T cells. Co-cultures were performed in the presence of 10 μg / mL of the indicated immune checkpoint blockade mAb (anti-LAG-3, anti-TIM-3, or anti-TIGIT) or the corresponding isotype control (mIgG1). Two paired groups were compared using the Wilcoxon test. *(p<0.05) [Figure 7A] Figure 1 shows the effect of blocking anti-LAG-3 and anti-TIM-3 mAbs on ICT01-mediated killing of SKOV3 by HD hu-PBMCs. A. Tumor cell killing indicated by the mean number of SKOV3 monitored over time during co-culture with HD hu-PBMCs (n=8) in the presence of ICT01 used at 0.1 μg / mL, or humanized anti-LAG-3 (leratolimab; 10 μg / mL) versus its corresponding isotype (hIgG4), or the ICT01 + leratolimab combination. B. Tumor cell killing indicated by the mean number of SKOV3 monitored over time during co-culture with HD hu-PBMCs (n=8) in the presence of ICT01 used at 0.1 μg / mL, or humanized anti-TIM-3 (sabatolimab; 10 μg / mL) versus its corresponding isotype (hIgG4), or the ICT01 + sabatolimab combination. Two-way ANOVA and Holm-Sidak multiple comparison test were used: ****(p<0.0001) [Figure 7B]Figure 1 shows the effect of blocking anti-LAG-3 and anti-TIM-3 mAbs on ICT01-mediated killing of SKOV3 by HD hu-PBMCs. A. Tumor cell killing indicated by the mean number of SKOV3 monitored over time during co-culture with HD hu-PBMCs (n=8) in the presence of ICT01 used at 0.1 μg / mL, or humanized anti-LAG-3 (leratolimab; 10 μg / mL) versus its corresponding isotype (hIgG4), or the ICT01 + leratolimab combination. B. Tumor cell killing indicated by the mean number of SKOV3 monitored over time during co-culture with HD hu-PBMCs (n=8) in the presence of ICT01 used at 0.1 μg / mL, or humanized anti-TIM-3 (sabatolimab; 10 μg / mL) versus its corresponding isotype (hIgG4), or the ICT01 + sabatolimab combination. Two-way ANOVA and Holm-Sidak multiple comparison test were used: ****(p<0.0001) [Figure 8A]Figure 1 shows the effect of soluble LAG-3 Fc on ICT01-mediated activation and expansion of Vy9V52 T cells in HD hu-PBMCs. A. Vy9V52 T cell activation (MFI CD25, upper panels) and expansion (absolute counts, lower panels) determined by flow cytometry on days 3 and 6 in non-activated (NA) and ICT01 (0.1 μg / mL)-stimulated HD hu-PBMCs in the presence of 10 μg / mL LAG-3 Fc or the corresponding control (Ig Fc), respectively. Data are shown for individual donors (n=5). B. Vy9V52 T cell activation (MFI CD25) determined on day 2 in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Data were normalized to the untreated condition for each individual donor. Individual donor data and mean ± SEM are shown (left panel). The mean fold change in CD25 MFI relative to the untreated control is shown in the heat map in the right panel. C. IFN-γ (left panel) and TNF-α (right panel) measured in day 2 culture supernatants determined by Luminex in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Average values ​​for three donors are shown. D. Activation of NK cells, CD4+, and CD8+ T cells (MFI CD25) determined by flow cytometry on day 2 in non-activated (NA) and ICT01 (0.1 μg / mL) stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0–10 μg / mL) or its corresponding control (Ig Fc). [Figure 8B]Figure 1 shows the effect of soluble LAG-3 Fc on ICT01-mediated activation and expansion of Vy9V52 T cells in HD hu-PBMCs. A. Vy9V52 T cell activation (MFI CD25, upper panels) and expansion (absolute counts, lower panels) determined by flow cytometry on days 3 and 6 in non-activated (NA) and ICT01 (0.1 μg / mL)-stimulated HD hu-PBMCs in the presence of 10 μg / mL LAG-3 Fc or the corresponding control (Ig Fc), respectively. Data are shown for individual donors (n=5). B. Vy9V52 T cell activation (MFI CD25) determined on day 2 in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Data were normalized to the untreated condition for each individual donor. Individual donor data and mean ± SEM are shown (left panel). The mean fold change in CD25 MFI relative to the untreated control is shown in the heat map in the right panel. C. IFN-γ (left panel) and TNF-α (right panel) measured in day 2 culture supernatants determined by Luminex in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Average values ​​for three donors are shown. D. Activation of NK cells, CD4+, and CD8+ T cells (MFI CD25) determined by flow cytometry on day 2 in non-activated (NA) and ICT01 (0.1 μg / mL) stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0–10 μg / mL) or its corresponding control (Ig Fc). [Figure 8C]Figure 1 shows the effect of soluble LAG-3 Fc on ICT01-mediated activation and expansion of Vy9V52 T cells in HD hu-PBMCs. A. Vy9V52 T cell activation (MFI CD25, upper panels) and expansion (absolute counts, lower panels) determined by flow cytometry on days 3 and 6 in non-activated (NA) and ICT01 (0.1 μg / mL)-stimulated HD hu-PBMCs in the presence of 10 μg / mL LAG-3 Fc or the corresponding control (Ig Fc), respectively. Data are shown for individual donors (n=5). B. Vy9V52 T cell activation (MFI CD25) determined on day 2 in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Data were normalized to the untreated condition for each individual donor. Individual donor data and mean ± SEM are shown (left panel). The mean fold change in CD25 MFI relative to the untreated control is shown in the heat map in the right panel. C. IFN-γ (left panel) and TNF-α (right panel) measured in day 2 culture supernatants determined by Luminex in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Average values ​​for three donors are shown. D. Activation of NK cells, CD4+, and CD8+ T cells (MFI CD25) determined by flow cytometry on day 2 in non-activated (NA) and ICT01 (0.1 μg / mL) stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0–10 μg / mL) or its corresponding control (Ig Fc). [Figure 8D]Figure 1 shows the effect of soluble LAG-3 Fc on ICT01-mediated activation and expansion of Vy9V52 T cells in HD hu-PBMCs. A. Vy9V52 T cell activation (MFI CD25, upper panels) and expansion (absolute counts, lower panels) determined by flow cytometry on days 3 and 6 in non-activated (NA) and ICT01 (0.1 μg / mL)-stimulated HD hu-PBMCs in the presence of 10 μg / mL LAG-3 Fc or the corresponding control (Ig Fc), respectively. Data are shown for individual donors (n=5). B. Vy9V52 T cell activation (MFI CD25) determined on day 2 in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Data were normalized to the untreated condition for each individual donor. Individual donor data and mean ± SEM are shown (left panel). The mean fold change in CD25 MFI relative to the untreated control is shown in the heat map in the right panel. C. IFN-γ (left panel) and TNF-α (right panel) measured in day 2 culture supernatants determined by Luminex in non-activated (NA) and ICT01 (0.03 and 0.1 μg / mL)-stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0-10 μg / mL) or its corresponding control (Ig Fc). Average values ​​for three donors are shown. D. Activation of NK cells, CD4+, and CD8+ T cells (MFI CD25) determined by flow cytometry on day 2 in non-activated (NA) and ICT01 (0.1 μg / mL) stimulated HD hu-PBMCs (n=3) co-cultured with increasing concentrations of LAG-3 Fc (0–10 μg / mL) or its corresponding control (Ig Fc). [Figure 9A]Figure 1 shows the effect of soluble LAG-3 Fc on ICT01-mediated cytolysis of tumor cells by in vitro expanded Vy9V52 T cells. A. Representative flow cytometry profiles of MHC class II expression on tumor cell lines (THP1 and SKOV3) and in vitro expanded Vy9V52 T cells. B. Flow cytometry assessment of Vy9V52 T cell degranulation (CD107) after co-culture of THP1 with non-activated or ICT01 (1 μg / mL) stimulated Vy9V52 T cells in the presence of LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for individual donors (n=6). Two paired groups were compared using a Wilcoxon test. *(p<0.05). C. Flow cytometry assessment of Vγ9Vδ2 T cell degranulation (CD107) after co-culture of SKOV3 with non-activated or ICT01 (0.1 μg / mL)-stimulated Vγ9Vδ2 T cells in the presence of LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for individual donors (n=6). Two paired groups were compared using a Wilcoxon test. *(p<0.05). [Figure 9B]Figure 1 shows the effect of soluble LAG-3 Fc on ICT01-mediated cytolysis of tumor cells by in vitro expanded Vy9V52 T cells. A. Representative flow cytometry profiles of MHC class II expression on tumor cell lines (THP1 and SKOV3) and in vitro expanded Vy9V52 T cells. B. Flow cytometry assessment of Vy9V52 T cell degranulation (CD107) after co-culture of THP1 with non-activated or ICT01 (1 μg / mL) stimulated Vy9V52 T cells in the presence of LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for individual donors (n=6). Two paired groups were compared using a Wilcoxon test. *(p<0.05). C. Flow cytometry assessment of Vγ9Vδ2 T cell degranulation (CD107) after co-culture of SKOV3 with non-activated or ICT01 (0.1 μg / mL)-stimulated Vγ9Vδ2 T cells in the presence of LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for individual donors (n=6). Two paired groups were compared using a Wilcoxon test. *(p<0.05). [Figure 9C]Figure 1 shows the effect of soluble LAG-3 Fc on ICT01-mediated cytolysis of tumor cells by in vitro expanded Vy9V52 T cells. A. Representative flow cytometry profiles of MHC class II expression on tumor cell lines (THP1 and SKOV3) and in vitro expanded Vy9V52 T cells. B. Flow cytometry assessment of Vy9V52 T cell degranulation (CD107) after co-culture of THP1 with non-activated or ICT01 (1 μg / mL) stimulated Vy9V52 T cells in the presence of LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for individual donors (n=6). Two paired groups were compared using a Wilcoxon test. *(p<0.05). C. Flow cytometry assessment of Vγ9Vδ2 T cell degranulation (CD107) after co-culture of SKOV3 with non-activated or ICT01 (0.1 μg / mL)-stimulated Vγ9Vδ2 T cells in the presence of LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for individual donors (n=6). Two paired groups were compared using a Wilcoxon test. *(p<0.05). [Figure 10A]Figure 1: Assessment of the direct activity of soluble LAG-3 Fc on MHC class II-expressing Vy9V52 T cells. A. Activation (MFI CD25) and MHC class II expression determined by flow cytometry on Vy9V52 T cells from hu-PBMCs that were not pre-activated (NA) or pre-activated with ICT01 (0.1 μg / mL). Data are shown for each individual donor using different symbols. B. Activation of Vy9V52 T cells (MFI CD25) determined by flow cytometry after 24 h culture of total T cells sorted from not pre-activated (NA) and ICT01-pre-activated hu-PBMCs (n=3) cultured with LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for each individual donor. C. Representative flow cytometry profiles of MHC class II expression on in vitro expanded Vy9V52 T cells from three donors (left panel) and Vy9V52 T cell degranulation (%CD107) after incubation with LAG-3 Fc (1 μg / mL) or the corresponding control (Ig Fc). Data are shown for each individual donor. [Figure 10B]Figure 1: Assessment of the direct activity of soluble LAG-3 Fc on MHC class II-expressing Vy9V52 T cells. A. Activation (MFI CD25) and MHC class II expression determined by flow cytometry on Vy9V52 T cells from hu-PBMCs that were not pre-activated (NA) or pre-activated with ICT01 (0.1 μg / mL). Data are shown for each individual donor using different symbols. B. Activation of Vy9V52 T cells (MFI CD25) determined by flow cytometry after 24 h culture of total T cells sorted from not pre-activated (NA) and ICT01-pre-activated hu-PBMCs (n=3) cultured with LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for each individual donor. C. Representative flow cytometry profiles of MHC class II expression on in vitro expanded Vy9V52 T cells from three donors (left panel) and Vy9V52 T cell degranulation (%CD107) after incubation with LAG-3 Fc (1 μg / mL) or the corresponding control (Ig Fc). Data are shown for each individual donor. [Figure 10C]Figure 1: Assessment of the direct activity of soluble LAG-3 Fc on MHC class II-expressing Vy9V52 T cells. A. Activation (MFI CD25) and MHC class II expression determined by flow cytometry on Vy9V52 T cells from hu-PBMCs that were not pre-activated (NA) or pre-activated with ICT01 (0.1 μg / mL). Data are shown for each individual donor using different symbols. B. Activation of Vy9V52 T cells (MFI CD25) determined by flow cytometry after 24 h culture of total T cells sorted from not pre-activated (NA) and ICT01-pre-activated hu-PBMCs (n=3) cultured with LAG-3 Fc (1 μg / mL) or its corresponding control (Ig Fc). Data are shown for each individual donor. C. Representative flow cytometry profiles of MHC class II expression on in vitro expanded Vy9V52 T cells from three donors (left panel) and Vy9V52 T cell degranulation (%CD107) after incubation with LAG-3 Fc (1 μg / mL) or the corresponding control (Ig Fc). Data are shown for each individual donor. DETAILED DESCRIPTION OF THE INVENTION

[0014] [Detailed explanation] definition In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0015] The terms "polypeptide," "protein," or "peptide," as used herein, refer to any chain of amino acid residues, regardless of chain length or post-translational modification (e.g., glycosylation).

[0016] As used herein, the term "BTN3A" has its general meaning in the art. In certain embodiments, the term refers to a human BTN3A polypeptide, including any of BTN3A1 of SEQ ID NO:24, BTN3A2 of SEQ ID NO:25, or BTN3A3 of SEQ ID NO:26.

[0017] As used herein, the term "PD-1" has its common meaning in the art and refers to the programmed death-1 receptor. The term "PD-1" also refers to a type I transmembrane protein that belongs to the CD28-B7 signaling family of receptors, which includes CD28, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), inducible costimulatory factor (ICOS), and B- and T-lymphocyte attenuator (BTLA). In a specific embodiment, the term refers to human PD-1 of SEQ ID NO: 130 (also found at UniprotKB accession number Q15116).

[0018] As used herein, PD-L1 (programmed death-ligand-1) is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. In a specific embodiment, PD-L1 refers to human PD-L1 of SEQ ID NO: 131 (also found in UniprotKB accession number Q9NZQ7-1).

[0019] As used herein, the term "LAG-3" (also referred to as CD223 or FDC protein) is an immune checkpoint inhibitor. In a specific embodiment, the term refers to the receptor for human LAG-3 of SEQ ID NO: 127 (also found in UniprotKB Accession No. P18627-1).

[0020] As used herein, the term "TIM-3" (also referred to as HAVCR2) is an immune checkpoint inhibitor. In certain embodiments, the term refers to the receptor for human TIM-3 of SEQ ID NO: 128 (also found at UniprotKB accession number Q8TDQ0-1).

[0021] As used herein, the term "TIGIT" (also called T cell immunoreceptor having an Ig domain and an ITIM domain) is an immune checkpoint inhibitor. In a specific embodiment, the term refers to the human TIGIT receptor of SEQ ID NO: 129 (also found in UniprotKB Accession No. Q495A1-1).

[0022] The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses whole antibody molecules as well as antibody fragments and antibody variants (including derivatives). The term "antibody," as used herein, also encompasses bispecific or multispecific molecules. Antibodies can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or receptor ligand), to create bispecific molecules that bind to at least two different binding sites or target molecules. Indeed, antibodies can be derivatized or linked to more than one other functional molecule to create multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the disclosure can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, etc.) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, to yield a bispecific molecule. Moreover, in embodiments where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity in addition to the first and second target epitopes.

[0023] In natural rodent and primate antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains domains with different sequences. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The constant region domains of the light chain (CL) and the constant region domains of the heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, placental mobility, complement fixation, and Fc receptor (FcR) binding.

[0024] An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) may also participate in the antibody binding site or influence the overall domain structure and ultimately the binding site. Complementarity-determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively.

[0025] Thus, an antigen-binding site typically comprises six CDRs, each comprising a set of CDRs from a heavy-chain and a light-chain V region. Framework regions (FRs) refer to the amino acid sequences interposed between the CDRs. Thus, the variable regions of light and heavy chains typically comprise four framework regions and three CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0026] Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al. This system is described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in the sequence of SEQ ID NO: 1. The actual linear amino acid sequence, whether in the framework of the basic variable domain structure or in the complementarity-determining regions (CDRs), may contain fewer or additional amino acids relative to the strict Kabat-numbered sequence, corresponding to truncations of or insertions into structural elements. The correct Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in the antibody sequence against the "standard" Kabat-numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31 to 35 (H-CDR1), 50 to 65 (H-CDR2), and 95 to 102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24 to 34 (L-CDR1), 50 to 56 (L-CDR2), and 89 to 97 (L-CDR3) according to the Kabat numbering system.

[0027] As used herein, "humanized" refers to an antibody in which some, most, or all amino acids outside the CDR regions have been replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Patent Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are hereby incorporated by reference. U.S. Patent Nos. 5,585,089 and 5,693,761, as well as WO 90 / 07861, also propose four possible criteria that can be used to design humanized antibodies. The first proposal was to use as the acceptor the framework of a specific human immunoglobulin that is exceptionally similar to the donor immunoglobulin being humanized, or to use the consensus framework of many human antibodies. The second proposal was that if an amino acid in the human immunoglobulin framework is unusual and the donor amino acid at this position is typical in human sequences, the donor amino acid can be selected rather than the acceptor amino acid. The third proposal was that the donor amino acid can be selected rather than the acceptor amino acid for positions immediately adjacent to the three CDRs in the humanized immunoglobulin chain. The fourth proposal was to use a donor amino acid located in a framework position where, in a three-dimensional model of the antibody, the amino acid is predicted to have a side chain atom within 3 A of the CDR and is predicted to be able to interact with the CDR. The above methods are merely illustrative of some of the approaches that those skilled in the art can employ to create humanized antibodies. Those skilled in the art will be familiar with other methods for antibody humanization. In some humanized forms of antibodies, some, most, or all of the amino acids outside the CDR regions may be replaced with amino acids from a human immunoglobulin molecule, but some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not destroy the desired properties of the parent antibody.

[0028] Suitable human immunoglobulin molecules can include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. A "humanized" antibody preferably retains antigen specificity, e.g., binding affinity, similar to that of the original antibody. However, when using certain humanization methods, the binding affinity and / or specificity of the antibody can be increased using the method of "directed evolution," as described in Wu et al., Mol. Biol. 294:151, 1999, the contents of which are incorporated herein by reference.

[0029] Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, and 6,150,584, the contents of which are incorporated herein by reference, and the references cited therein. These animals have been genetically modified to lack endogenous (e.g., murine) antibody production function. The animals have been further modified to contain all or part of human germline immunoglobulin gene loci, such that immunization of these animals results in the production of fully human antibodies against the antigen of interest. After immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke a human anti-mouse antibody (HAMA) response when administered to humans.

[0030] In vitro methods for generating human antibodies also exist. These methods include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0031] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to the full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the BTN3A protein, LAG-3 receptor, TIM-3 receptor, or TIGIT receptor, as defined above). In certain embodiments, the antibodies provided herein are antibody fragments, more particularly, any protein that comprises the antigen-binding domain of an antibody disclosed herein. Well-known antibody fragments include: Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; dAb fragments consisting of the VH domain (Ward et al., 1989 Nature 341:544-546), or any fusion protein containing such an antigen-binding fragment; and diabodies (small antibody fragments having two antigen-binding sites; these fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) (VH-VL) on the same polypeptide chain). The two domains on the same chain are forced to pair with complementary domains on another chain, creating two antigen-binding sites, using a linker that is too short to allow inter-domain pairing. Furthermore, although the two domains of the Fv fragment, i.e., VL and VH, are encoded by separate genes, they can be joined using recombinant techniques with a synthetic linker that allows the VL and VH domains to pair into a single-chain protein to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody (also referred to herein for short as antibody fragment).More generally, antibody fragments contemplated herein also encompass single-domain antibodies, which are antibody fragments comprising all or a portion of an antibody heavy chain variable domain or all or a portion of an antibody light chain variable domain. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 (B1)). These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Suitable antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells as described herein.

[0032] The term "monoclonal antibody," as used herein, refers to a single-specificity preparation of antibody molecules. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity that has variable and constant regions derived from or based on human germline immunoglobulin sequences, or from entirely synthetic sequences. The method of preparation of the monoclonal antibody is not related to the binding specificity.

[0033] A "recombinant antibody" is an antibody that is produced, expressed, created, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; an antibody isolated from an animal (e.g., a mouse) that is transgenic with human immunoglobulin genes; or an antibody that is produced, expressed, created, or isolated by any other method that combines particular immunoglobulin gene sequences (e.g., human immunoglobulin gene sequences) with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies. In some embodiments, recombinant human antibodies of the present disclosure have the same amino acid sequence as a corresponding naturally occurring human antibody, but are structurally different from said naturally occurring human antibody. For example, in some embodiments, recombinant human antibodies have different glycosylation patterns as a result of recombinant production. In some embodiments, recombinant human antibodies are chemically modified by adding or subtracting at least one covalent chemical bond from the structure of a human antibody naturally occurring in humans.

[0034] An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to BTN3A is substantially free of antibodies that specifically bind to antigens other than BTN3A). However, an isolated antibody that specifically binds to BTN3A may have cross-reactivity with other antigens, for example, related BTN3A molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0035] The phrases "antibody that recognizes an antigen" and "antibody having specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen." Additionally, the term "anti-LAG-3 antibody" is used herein simply to mean "antibody that recognizes LAG-3." Additionally, the term "anti-BTN3A antibody" is used herein simply to mean "antibody that recognizes BTN3A."

[0036] As used herein, the term "activating antibody" refers to an antibody that can directly or indirectly induce immune function of effector cells. In particular, as used herein, a BTN3A-activating antibody has at least the ability to induce activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3A-expressing cells, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay (see WO2020 / 025703 for detailed assays).

[0037] As used herein, the term "binding" in the context of antibody binding to a given antigen or epitope, particularly BTN3A, LAG-3, or TIGHT, typically refers to a binding activity of approximately 10 -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K to M and below D where affinity is as determined, for example, by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument, typically using a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats commonly used for epitope bin panels of monoclonal antibodies. Typically, an antibody binds to a given antigen with an affinity corresponding to a K of 0.05 for binding to a nonspecific antigen (e.g., BSA, casein) that is neither identical nor closely related to the given antigen. D K of 1 / 10 or less, for example, 1 / 100 or less, for example, 1 / 1,000 or less, for example, 1 / 10,000 or less, for example, 1 / 100,000 or less D The antibody binds with an affinity corresponding to K D If the K is very low (i.e., the antibody has high affinity), the K D However, typically, K DIt is less than 1 / 10,000 of that.

[0038] The term "affinity," as used herein in the context of antibodies, refers to the strength of the binding of an antibody to an epitope.

[0039] "K on " or "Kass" (K a The term "K"), as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, while "K dis " (Kd) or "K off The term " ", as used herein, is intended to refer to the off-rate of a particular antibody-antigen interaction.

[0040] The term “K D ", as used herein, refers to k off k on to (i.e., k off / k on ) is intended to refer to the equilibrium dissociation constant, expressed as a molar concentration (M), obtained from D The value is related to the antibody concentration (the amount of antibody needed for a particular experiment) and therefore the K D The lower the value (the lower the concentration), the higher the affinity of the antibody. D The K value can be determined using methods well established in the art. D Preferred methods for determining K values ​​can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Coligan et al. (eds.), Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. DMethods for determining affinity are those using surface plasmon resonance or those using biosensor systems, examples of which are the Biacore® system (for detailed information on affinity evaluation, see also Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug / human serum albumin interactions using BIACORE®. Anal Biochem. 2001 Sep. 15;296(2):197-207) or the Octet® system. The Octet® platform is based on biolayer interferometry (BLI) technology. The principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces: a layer of immobilized protein and an internal reference layer. Binding between a ligand immobilized on the surface of the biosensor tip and an analyte in solution increases the optical thickness at the biosensor tip, resulting in a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in optical thickness of the biological layer; measuring this shift over a period of time and plotting its magnitude as a function of time yields a classical association / dissociation curve. This interaction is measured in real time, allowing the monitoring of binding specificity, association and dissociation rates, and concentration (see Abdiche et al., 2008, but also see results for details). Affinity measurements are typically performed at 25°C.

[0041] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope present on an antigen, eg, BTN3A, LAG-3, or TIGHT.

[0042] In some embodiments, antibodies with specificity for BTN3A preferably have an EC of less than 50 μg / ml, more preferably less than 10 μg / ml, as determined in the assays disclosed in WO 2020 / 025703, the contents of which are incorporated by reference herein, particularly with respect to Table 4. 50 In another embodiment, the antibody binds to an antigenic recombinant polypeptide with a K of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less, as measured by SPR measurements as described above. D (However, see also Table 4 of WO 2020 / 025703 for details).

[0043] An antibody that "cross-reacts with an antigen other than BTN3A" has a K of 10 nM or less, 1 nM or less, or 100 pM or less for this antigen other than BTN3A. D An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen with a K of 1 μM or greater. D , or a K of 10 μM or more D The term "antibody" is intended to refer to an antibody that binds to the antigen. In certain embodiments, such antibodies that do not cross-react with the antigen have essentially undetectable binding to these proteins in standard binding assays. In certain embodiments, a humanized antibody of the present disclosure, e.g., mAb1, cross-reacts with cynomolgus monkey BTN3A1, BTN3A2, and BTN3A3 of SEQ ID NOs: 27, 28, and 29, respectively, as measured, for example, by Biacore assay (see, in particular, the relevant assays exemplified in WO 2020 / 025703, with reference to Table 26).

[0044] The terms "inhibition," "inhibitor," or "antagonist" include a decrease in a particular parameter, e.g., activity, of a given molecule, e.g., an immune checkpoint inhibitor. For example, inhibition of activity, e.g., the activity of a given molecule, e.g., an inhibitor molecule, by at least or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more is encompassed by this term. Thus, inhibition need not be 100%.

[0045] "Fusion protein" and "fusion polypeptide" refer to a protein or polypeptide having at least two covalently linked portions, each of which is a polypeptide with a distinct property. The property can be a biological property, e.g., in vitro or in vivo activity. The property can also be a simple chemical or physical property, e.g., binding to a target molecule, catalysis of a reaction, etc. The two portions can be directly linked by a single peptide bond or through a peptide linker, but are in reading frame with each other. Examples of fusion proteins include, among others, receptor binding domains fused to immunoglobulin constant regions (e.g., Fc or Ig fusion proteins).

[0046] Specificity may be further indicated by an affinity / avidity ratio for binding to a specific antigen versus non-specific binding to other unrelated molecules, e.g., about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or greater (where the specific antigen is a BTN3A polypeptide, LAG-3, TIM-3 or TIGIT receptor, respectively, or alternatively, MHC class II in the case of a soluble hLAG-3 Ig molecule).

[0047] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It depends on three major factors: antibody epitope affinity; the valency of both the antigen and the antibody; and the structural arrangement of the interacting moieties. Ultimately, these factors define antibody specificity, i.e., the likelihood that a particular antibody will bind to a precise antigen epitope.

[0048] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0049] As used herein, the term "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons preferred by the production cell or organism, which is generally a eukaryotic cell, such as a Chinese hamster ovary cell (CHO) or a human cell. An optimized nucleotide sequence is one that has been engineered to completely retain, or retain as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence. An amino acid sequence encoded by an optimized nucleotide sequence is also referred to as optimized.

[0050] The term "identity," as used herein with reference to polypeptide sequences, refers to the identity of amino acid sequences between two molecules. These molecules are identical at a given amino acid position in both molecules if that position is occupied by the same amino acid. Identity between two polypeptides is a direct function of the number of identical positions. Generally, sequences are aligned (including gaps, if necessary) to obtain the highest order match. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity (%) = number of identical positions / total number of positions × 100), taking into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using the mathematical algorithm described below.

[0051] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using published techniques and widely available computer programs, examples of which are BLASTP, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990), or the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970) incorporated into the GAP program in the GCG software package (Devereux et al., Nucleic Acids Res. 12:387, 1984, generally available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0052] The percent identity between two nucleotide or amino acid sequences can also be determined using an algorithm such as the BLASTN program for nucleic acid sequences, which uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0053] Additional antibodies can be identified based on their ability to cross-compete with other antibodies of the present disclosure (e.g., competitively inhibit the binding of other antibodies of the present disclosure in a statistically significant manner) in a standard antigen-binding assay, such as an ELISA binding assay. The ability of a test antibody to inhibit the binding of an antibody of the present disclosure to its target indicates that the antibody and the test antibody can compete for binding to the target; such an antibody may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximate) epitope on the target as the competing antibody. Thus, another aspect of the present disclosure provides antibodies that bind to the same antigen as the antibodies disclosed herein and compete with them. As used herein, an antibody "competes" for binding when the competing antibody inhibits binding to a target by an antibody or antigen-binding fragment of the disclosure by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% in the presence of an equimolar concentration of the competing antibody.

[0054] "Combination therapy," "co-administration," "concurrent administration," or "concurrent administration" refers to the combined administration of at least two therapeutic agents, wherein a first agent, typically a BTN3A-activating compound, is administered to the same subject in need thereof, simultaneously or separately with a second agent, e.g., an immune checkpoint inhibitor selected from a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor, or a combination thereof, and optionally with a third agent, e.g., a PD-1 / PD-L1 inhibitor, or an IL2 or IL15 cytokine or a derivative thereof, within a time interval that allows the combined partners to exert a cooperative or synergistic effect to treat a disorder, e.g., cancer.

[0055] Examples of cancers that can be treated using the methods of the present invention include skin cancer, cutaneous or intraocular malignant melanoma, head or neck cancer, breast cancer, lung cancer, kidney cancer, bladder cancer, liver cancer, bone cancer, pancreatic cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, kidney or ureter cancer, penile cancer, central nervous system (CNS) neoplasms, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, childhood solid tumors, hematological malignancies, and the like. and any combination of the foregoing cancers.

[0056] The present invention is also applicable to the treatment of metastatic, refractory, or recurrent malignancies. While these delivery methods are within the scope described herein, they are not intended to imply that therapeutic agents must be administered simultaneously and / or formulated for delivery together. The BTN3A-activating antibodies disclosed herein, e.g., ICT01, may be administered concurrently with, before, or after one or more other additional therapies or therapeutic agents. These terms are also intended to encompass therapeutic regimens in which agents are not necessarily administered by the same route of administration.

[0057] As used herein, the terms synergy or synergistic effect, when used in connection with describing the effectiveness of a drug combination, means that the measured effect of the combination is greater than that expected from the sum of the effects of the individual drugs (i.e., greater than additive). In some embodiments, tumor growth rate or tumor size (e.g., rate of change in tumor size (e.g., volume, mass)) is used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the tumor growth rate is slower than expected if the drug combination results in an additive effect). In some embodiments, median overall survival (e.g., less than 12 months) is used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the median overall survival of a subject or population of subjects is longer than expected if the drug combination results in an additive effect). In some embodiments, γδ T cell activation may also be used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the rate of activation (determined as an increase in expression of an activation marker compared to baseline values) of a particular γδ T cell subset (typically the Vγ9Vδ2 T cell subset) is higher than would be expected if the drug combination were to have an additive effect). In some embodiments, γδ T cell proliferation and expansion may also be used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the rate of proliferation or expansion (determined as an increase in percentage of the population or an increase in absolute cell numbers compared to baseline values) of a particular γδ T cell subset (typically the Vγ952 T cell subset) is higher than would be expected if the drug combination were to have an additive effect).In some embodiments, γδ T cell cytotoxicity may also be used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the cytotoxic potency (determined as an increase in the percentage of effector cell degranulation or an increase in the percentage of target cell apoptosis or a decrease in the percentage and / or absolute cell number of hepatocytes among the target cells compared to baseline values) of a particular T cell subset (typically the Vy9δ2 T cell subset) is higher than would be expected if the drug combination resulted in an additive effect).

[0058] BTN3A activation antibody BTN3A activating antibodies according to the present disclosure typically exhibit one or more of the following properties: (i) the antibody has a K of 10 nM or less, as measured, for example, by SPR as described in the Examples of Patent Application WO 2020 / 025703; D , preferably a K of 1 nM or less D binds to BTN3A at ; (ii) the antibody has an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by the flow cytometry assay described in the Examples of patent application WO 2020 / 025703; 50 binds to human PBMCs; (iii) The antibody has an EC of less than 5 μg / ml, preferably 1 μg / ml or less, in co-culture with BTN3A-expressing cells, as described in the Examples of Patent Application WO 2020 / 025703. 50 induce activation of γδ T cells, typically Vγ9Vδ2 T cells; and / or (iv) the antibody induces in vitro activation of Vy9V52 T cells in human peripheral blood mononuclear cells (PBMCs) with an EC50 of less than 0.1 μg / mL, preferably 0.01 μg / mL or less, e.g., between 100 pg / mL and 0.1 μg / mL, as measured by surface expression of the activation marker CD69; and (v) Optionally, the antibody has a K of 100 nM or less, e.g., as measured by SPR as described in the Examples of Patent Application WO 2020 / 025703. D and preferably a K of 10 nM or less D It cross-reacts with cynomolgus monkey BTN3A.

[0059] Preferably, as used herein, a BTN3A activating antibody for use according to the present disclosure has at least properties (i) and (iv) above.

[0060] Specific examples of BTN3A activating antibodies are described in the following paragraphs. In some embodiments, the BTN3A activating antibody is selected from the group consisting of the BTN3A antibodies described in International Patent Application Publication Nos. WO 2012080769, WO 2012080351, and WO 2020025703. In some specific embodiments, the BTN3A activating antibody is selected from the humanized antibodies described in WO 2020025703, or is a humanized version of the BTN3A agonist antibodies described in WO 2012080769 and WO 2012080351. In some embodiments, the BTN3A antibody is selected from mAb 20.1 and mAb 7.2, or humanized versions thereof, obtainable from one of the hybridomas accessible under CNCM deposit numbers I-4401 and I-4402, such as those described in WO2012080769 and WO2012080351, and from humanized mAbs 1-6 described in WO2020 / 025703.

[0061] In some embodiments, a BTN3A activating antibody comprises the six CDRs (CDR1 (also referred to as HCDR1), VH CDR2 (also referred to as HCDR2), VH CDR3 (also referred to as HCDR3), VL CDR1 (also referred to as LCDR1), VL CDR2 (also referred to as LCDR2), VL CDR3 (also referred to as LCDR3)) of antibody 20.1 or 7.2 described in WO 2012 / 080769 and WO 2012080351, or mAb 1-6 described in WO 2020 / 025703. In specific embodiments, a BTN3A activating antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in Table 1 below:

[0062] [Table 1]

[0063] In some embodiments of the antibodies for use disclosed herein, the six CDR regions are 100% identical to the six CDR regions of antibody 20.1 or 7.2 described in WO 2012 / 080769 and WO 2012 / 080351, or mAbs 1-6 described in WO 2020 / 025703; particularly, in some embodiments, the six CDR regions of the antibodies disclosed herein are 100% identical to the six CDR regions of Table 1, particularly mAb 7.2, mAb 1, mAb 2, mAb 4, and mAb 5.

[0064] Other antibodies disclosed herein include antibodies with mutated amino acids by amino acid deletion, insertion, or substitution, but which still have at least 60, 70, 80, 90, 95, 96, 97, 98, 99, or 100 percent identity in the CDR regions to the six CDR regions of antibodies 20.1 or 7.2 described in WO2012 / 080769 and WO2012 / 080351, or to the six CDR regions of mAbs 1-6 described in WO2020 / 025703, particularly to the six CDR regions defined in Table 1.

[0065] In some embodiments, according to the present disclosure, an antibody may have 1, 2, 3, or 4 amino acid differences (including deletions, insertions, or substitutions) in one or more CDRs relative to the CDR sequences of antibodies 20.1 or 7.2 described in WO2012 / 080769 and WO2012 / 080351, or relative to the CDR sequences of mAbs 1-6 described in WO2020 / 025703, particularly relative to the CDR sequences in Table 1, and more particularly relative to the CDR sequences of mAb 7.2, mAb 1, mAb 2, mAb 4, and mAb 5.

[0066] Antibodies of the present disclosure also include antibodies having at least 90%, particularly at least 95, 96, 97, 98, 99 or 100% identity to the VH and VL regions defined in Table 2. More particularly, antibodies of the present disclosure include selected humanized recombinant antibodies mAb1, mAb2, mAb4 and mAb5, which are structurally characterized by variable heavy and light chain amino acid sequences and human constant regions (isotypes) as set forth in Table 2 below:

[0067] [Table 2]

[0068] mAb3 and mAb6 are humanized versions of the parent murine BTN3A activating antibody called mAb 20.1, which is described in WO 2012 / 080351.

[0069] The corresponding amino acid and nucleotide coding sequences of the constant isotype regions of IgG1, IgG4, and their mutant versions, IgG1 L247F / L248E / P350S and IgG4 S241P / L248E, used to generate mAb1 to mAb6, are well known in the art (Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 2008 June; 64(Pt 6):700-4; Reddy et al., J Immunol. 2000 February 15; 164(4):1925-33). The C-terminal lysine found in IgG can be naturally cleaved, and this modification does not affect the properties of the antibody; therefore, this residue can be further deleted in the constructs of mAb1 to mAb6.

[0070] The full-length light and heavy chains and corresponding coding sequences of mAb1, mAb2, mAb4 and mAb5 are shown in Table 3 below.

[0071] [Table 3]

[0072] Other BTN3A activating antibodies, in particular variants of the mAb20.1 activating antibody, are disclosed in WO 2023 / 161457 (Evobright GmbH), the contents of which are incorporated herein in their entirety.

[0073] A preferred exemplary BTN3A activating antibody for use according to the present disclosure is ICT01, defined by its heavy chain of SEQ ID NO:4 and its light chain of SEQ ID NO:6.

[0074] In certain embodiments that can be combined with the preceding embodiments, the antibody provided herein is an antibody fragment of the antibody defined above. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab'), Fv, unibody, and scFv fragments, diabodies, single domains, or nanobodies, and other fragments. Preferably, the antibody fragment is a monovalent antibody, e.g., a Fab of an scFv fragment.

[0075] In some embodiments, antibodies of the present disclosure compete for binding with the BTN3A antibody described above, and in particular, antibodies of the present disclosure compete for binding with an antibody selected from mAb 20.1 and mAb 7.2, obtainable from one of the hybridomas accessible under CNCM deposit numbers I-4401 and I-4402, such as those described in WO 2012080769 and WO 2012080351, and also compete for binding with an antibody selected from mAbs 1-6 described in WO 2020025703. In a more specific embodiment, antibodies of the present disclosure compete for binding with an antibody selected from mAb 7.2 produced by the hybridoma deposited at the CNCM under deposit number I-4402 and the ICT01 antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0076] In some embodiments, antibodies for use according to the present disclosure are chimeric, humanized, or human antibodies. In preferred embodiments of the present disclosure, the BTN3A antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans, while retaining at least the same affinity (or better affinity) to the parent non-human antibody. More specifically, the BTN3A antibody is a humanized form of the murine antibody mAb 20.1 or 7.2 disclosed in WO 2012 / 080351. In a preferred embodiment, the BTN3A antibody for use according to the present disclosure is a humanized version of the parent antibody mAb 7.2 disclosed in WO 2012 / 080351.

[0077] Generally, a humanized antibody comprises one or more variable domains, with the CDRs (or portions thereof) derived from a non-human antibody, such as mouse mAb 7.2, and the FRs (or portions thereof) derived from a mouse antibody sequence with mutations that reduce immunogenicity. The humanized antibody also optionally comprises at least a portion of a human constant region. Preferably, recombinant antibodies for use according to the present disclosure are humanized silenced antibodies, typically humanized silenced IgG1 or IgG4 antibodies. Suitable humanized anti-BTN3A antibodies for use according to the present disclosure are typically described in WO 2020 / 025703 and include mAbs having the VH / VL polypeptide sequences in Table 2 and the light chain / heavy chain sequences in Table 3.

[0078] As used herein, the term "silent" antibody refers to an antibody that has no or low FcγR binding and / or C1q binding, as measured in binding assays such as those described in WO 2020 / 025703. In one embodiment, the term "no or low FcγR binding and / or C1q binding" means that the silent antibody exhibits at least 50% less, e.g., less than 80%, of the FcγR binding and / or C1q binding observed with a corresponding antibody having a wild-type human IgG1 or IgG4 isotype.

[0079] LAG-3 inhibitors In certain embodiments, the combination therapy comprises administering a therapeutically effective amount of a BTN3A activating antibody described above, such as ICT01, in combination with a therapeutically effective amount of a LAG-3 inhibitor.

[0080] LAG-3 inhibitors that may be used in the combination therapies disclosed herein include, but are not limited to, anti-LAG-3 antibodies or recombinant LAG-3 proteins. In some embodiments, an anti-LAG-3 antibody molecule is intended to refer to a monospecific or bispecific antibody molecule that exhibits one or more of the following properties: (i) The molecule specifically binds to LAG-3, e.g., human LAG-3. The binding specificity of the Ab can be determined by monitoring binding of the antibody to cells expressing LAG-3 protein (e.g., transfected cells or primary immune cells) in a flow cytometry assay, preferably with an EC50 of less than 100 nM, more preferably less than 10 nM. Additionally or alternatively, the antibody binds to recombinant human LAG-3 protein with high affinity, with a KD of 100 nM or less, 10 nM or less, 1 nM or less, or 100 pM or less, as measured by SPR, BLI, or other suitable binding assay; (ii) the molecule inhibits the binding of LAG-3 to its ligand, e.g., a major histocompatibility class (MHC) II molecule, with an IC50 of less than 100 nM, more preferably less than 10 nM; (iii) The molecule modulates (e.g., stimulates, enhances, or revives) an immune response. The ability of an antibody to modulate an immune response may be demonstrated by one or more of the following: enhancing antibody-specific T cell responses; enhancing T cell activation and / or proliferation; enhancing cytokine (e.g., IL-2, IFN-g) and / or chemokine secretion, enhancing T cell homeostasis, enhancing tumor-infiltrating lymphocytes; reducing the suppressive activity of Tregs, or slowing tumor growth (e.g., in in vivo models).

[0081] (iv) Optionally, the molecule may also bind to non-human primate LAG-3, such as cynomolgus monkey LAG-3.

[0082] In one embodiment, the LAG-3 inhibitor for use in combination therapy is an anti-LAG-3 antibody molecule. In some embodiments, the LAG-3 inhibitor is selected from leratolimab. In some embodiments, the LAG-3 inhibitor is selected from favezelimab (also known as MK-4280) (Merck Sharp & Dohme). In some embodiments, the anti-LAG-3 antibody comprises a VH of SEQ ID NO: 52 and a VL of SEQ ID NO: 53, or a functional variant having a VH and VL with at least 90%, 95%, or at least 98% identity to SEQ ID NO: 52 and SEQ ID NO: 53, respectively. In some embodiments, the anti-LAG-3 antibody comprises a heavy chain of SEQ ID NO: 50 and a light chain of SEQ ID NO: 51, or a functional variant having a heavy chain and a light chain with at least 90%, 95%, or at least 98% identity to SEQ ID NO: 50 and SEQ ID NO: 51, respectively. Other anti-LAG-3 antibody molecules are disclosed in WO 2016 / 028672, the contents of which are incorporated herein in their entirety. In some embodiments, the anti-LAG-3 antibody comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, HCDR2, LCDR3) set forth in SEQ ID NOs: 54-59, respectively.

[0083] Other anti-LAG-3 antibody molecules are disclosed in WO 2016 / 028672, the contents of which are incorporated herein in their entirety.

[0084] In one embodiment, the LAG-3 inhibitor for use in combination therapy with a BTN3A-activating antibody, e.g., ICT01, is a fixed-dose combination of an anti-LAG-3 antibody and an anti-PD-1 antibody. In some embodiments, the LAG-3 inhibitor is selected from nivolumab / relatolimab (sold under the trade name Opdualag; BMS), a fixed-dose combination of nivolumab (an anti-PD-1 antibody) and relatolimab (an anti-LAG-3 antibody). Other examples of fixed-dose combinations are disclosed in WO 2020 / 081928. In some embodiments, the LAG-3 inhibitor is a fixed-dose combination of an anti-LAG-3 antibody having heavy and light chains of SEQ ID NOs: 33 and 34, or variants of the heavy and light chains having at least 90%, 95%, or at least 98% identity to SEQ ID NOs: 33 and 34, respectively, and an anti-PD-1 antibody having heavy and light chains of SEQ ID NOs: 31 and 32, or variants of the heavy and light chains having at least 90%, 95%, or at least 98% identity to SEQ ID NOs: 31 and 32, respectively. In some embodiments, the anti-LAG-3 antibody comprises a VH and VL of SEQ ID NO: 37 and SEQ ID NO: 38, respectively, or a related anti-LAG-3 antibody having a VH and VL having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 37 and SEQ ID NO: 38, respectively.

[0085] In some embodiments, the LAG-3 inhibitor is an anti-LAG-3 antibody. In certain embodiments, the anti-LAG-3 antibody is a full-length antibody. In certain embodiments, the antibody is a multispecific antibody, such as a dual affinity retargeting antibody (DART), DVD-Ig, or bispecific antibody. In some embodiments, the anti-LAG-3 antibody is BMS-986016, IMP731 (H5L7BW), MK-4280 (fabezelimab), REGN3767, GSK2831781, humanized BAP050, IMP-701 (LAG-525), aLAG3(0414), aLAG3(0416), Sym022, TSR-033, TSR-075, XmAb22841, MGD013, B1754111, FS118, P 13B02-30, or AVA-017.

[0086] In one embodiment, the LAG-3 inhibitor for use in combination therapy with a BTN3A-activating antibody, e.g., ICT01, is tebotelimab (Macrogenics), a bispecific DART molecule designed to block PD-1 and LAG-3 checkpoint molecules independently or cooperatively. In some embodiments, related bispecific DART molecules are described in WO 2016 / 200782, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-LAG-3 antibody is a bispecific DART molecule comprising a first polypeptide of SEQ ID NO: 60 and a second polypeptide of SEQ ID NO: 61, or variant sequences thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 60 and SEQ ID NO: 61, respectively. In some embodiments, the anti-LAG-3 antibody is a bispecific DART molecule comprising a first VH sequence of SEQ ID NO: 62 and a second VH sequence of SEQ ID NO: 64, and a first VL sequence of SEQ ID NO: 63 and a second VL sequence of SEQ ID NO: 132, or variant sequences thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 132, respectively. In some embodiments, the anti-LAG-3 antibody is a bispecific DART molecule comprising a first VH sequence having three CDRs of SEQ ID NOs: 65-67, respectively, a first VL sequence having three CDRs of SEQ ID NOs: 68-70, respectively, a second VH sequence having three CDRs of SEQ ID NOs: 71-73, and a second VL sequence having three CDRs of SEQ ID NOs: 74-76.

[0087] In some embodiments, recombinant LAG-3 protein for use in the combination therapy disclosed herein refers to the whole LAG-3 protein or a derivative including a mutant, variant, or fragment of LAG-3, or a recombinant soluble human LAG-3Ig fusion protein (also referred to as a LAG-3 Fc fusion protein), provided that said recombinant LAG-3 protein exhibits one or more of the following properties: (i) The recombinant LAG-3 protein specifically binds to human and mouse MHC class II. Binding specificity can be determined by monitoring binding of the recombinant LAG-3 protein or derivative to MHC class II-expressing antigen-presenting cells (e.g., transfected cells or primary immune cells) of human or mouse origin in a flow cytometry assay, preferably with an EC50 of less than 100 nM, more preferably less than 10 nM. Additionally or alternatively, soluble LAG-3-Ig fusion proteins, e.g., hLAG-3 Ig molecules, bind to MHC class II-positive cells, e.g., DAUDI B cells, at 37°C with a KD of less than 100 nM, more preferably less than 10 nM, as defined by Scatchard analysis; (ii) The recombinant LAG-3 protein induces phenotypic maturation of human monocytes or dendritic cells. Maturation can be monitored by increased expression of cell surface markers, such as CD40, CD80, and CD86, and induction of CD83 expression. Alternatively, activity on myeloid cells can be measured by increased production of cytokines, such as TNFα or CCL4.

[0088] (iii) The recombinant LAG-3 protein modulates (e.g., stimulates, enhances, or revives) an immune response. The ability of a soluble hLAG-3Ig molecule to modulate an immune response may be demonstrated by one or more of the following: enhancing the activation and / or proliferation and / or survival of T and / or NK cells; enhancing cytokine (e.g., IL-2, IFN-γ, TNF-α) secretion by T and / or NK cells; or enhancing the cytotoxic activity of T and / or NK cells against target cells (e.g., in an in vitro assay).

[0089] In one embodiment, the LAG-3 inhibitor for use in combination therapy is a recombinant LAG-3 protein or related Ig fusion protein. In some embodiments, the LAG-3 inhibitor is selected from eftiragimodo alfa / IMP321 (Immutep). Eftiragimod alfa is a recombinant soluble version of LAG-3 (LAG-3-Ig) consisting of the four extracellular domains of LAG-3 fused to the Fc region of IgG1. In some embodiments, the recombinant LAG-3 protein or related Ig fusion protein comprises the polypeptide sequence of SEQ ID NO: 30, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 30. Other recombinant LAG-3 proteins or related Ig fusion proteins are disclosed in WO 2009 / 044273, which discloses recombinant LAG-3 proteins and corresponding fusion proteins (the contents of which are incorporated herein by reference in their entirety).

[0090] Exemplary LAG-3 Inhibitors Table 4 below discloses certain exemplary LAG-3 inhibitors that can be used in combination therapy with a BTN3A activating antibody, such as ICT01.

[0091] [Table 4]

[0092] TIM-3 inhibitors In certain embodiments, the combination therapy comprises administering a therapeutically effective amount of a BTN3A activating antibody described above, such as ICT01, in combination with a therapeutically effective amount of a TIM-3 inhibitor.

[0093] TIM-3 inhibitors that may be used in the combination therapies disclosed herein include, but are not limited to, anti-TIM-3 antibodies.

[0094] In some embodiments, the anti-TIM-3 antibody molecule is a monospecific or bispecific antibody molecule that exhibits one or more of the following properties: (i) The molecule specifically binds to TIM-3, e.g., human TIM-3. The binding specificity of the Ab can be determined by monitoring binding of the antibody to cells expressing TIM-3 protein (e.g., transfected cells or primary immune cells) in a flow cytometry assay, preferably with an EC50 of less than 100 nM, more preferably less than 10 nM. Additionally or alternatively, the antibody binds to recombinant human TIM-3 protein with high affinity, with a KD of 100 nM or less, 10 nM or less, 1 nM or less, or 100 pM or less, as measured by SPR, BLI, or other suitable binding assay; (ii) the molecule inhibits binding of TIM-3 to a TIM-3 ligand, e.g., phosphatidylserine (PtdSer), HMGB1, or CEACAM-1 or galectin-9, with an IC50 of less than 100 nM, more preferably less than 10 nM; and / or (iii) The molecule modulates (e.g., stimulates, enhances, or revives) an immune response. The ability of the antibody to modulate an immune response may be demonstrated by one or more of the following: enhancing the activation and / or proliferation and / or survival of T cells; enhancing cytokine (e.g., IL-2, IFN-γ) secretion by T cells; enhancing the cytotoxic activity of T and / or NK cells against target cells (e.g., in in vitro assays); enhancing the ability of macrophages or antigen-presenting cells to stimulate T cell responses or slowing tumor growth (e.g., in in vivo models); and (iv) Optionally, the molecule binds to non-human primate TIM-3, e.g., cynomolgus monkey TIM-3.

[0095] In some embodiments, the TIM-3 inhibitor comprises an anti-TIM-3 antibody molecule. In some embodiments, the TIM-3 inhibitor comprises sabatolimab (MBG453), TSR-022, LY3321367, Sym023, BGB-A425, INCAGN02390, BMS-986258, RO7121661, BC-3402, SHR-1702, or LY-3415244. In some embodiments, the TIM-3 inhibitor comprises sabatolimab.

[0096] In some embodiments, the anti-TIM-3 antibody molecule comprises a variable heavy chain VH of SEQ ID NO: 79 and a variable light chain VL of SEQ ID NO: 80, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 79 and SEQ ID NO: 80, respectively. In some embodiments, the anti-TIM-3 antibody molecule comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, 1CDR2, LCDR3) of SEQ ID NOs: 81-86, respectively. In some embodiments, the anti-TIM-3 antibody molecule comprises a heavy chain of SEQ ID NO: 77 and a light chain of SEQ ID NO: 78, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 77 and SEQ ID NO: 78, respectively. Other TIM-3 antibody molecules are disclosed in WO2021123902, the contents of which are incorporated herein by reference in their entirety.

[0097] In other embodiments, the anti-TIM-3 antibody molecule is a bispecific molecule having binding specificities for TIM-3 and PD-1. In more specific embodiments, the TIM-3 / PD-1 bispecific antibody comprises the VH polypeptides of SEQ ID NOs: 91 and 93, respectively, and the VL polypeptides of SEQ ID NOs: 92 and 94, respectively, or functional variants thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NOs: 91-94, respectively.

[0098] In more specific embodiments, the TIM-3 / PD-1 bispecific antibody comprises the polypeptide of SEQ ID NOs: 87-90, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NOs: 87-90, respectively. Typically, the TIM-3 / PD-1 bispecific antibody for use in combination therapy is RO7121661 or other related bispecific antibodies disclosed in WO2017055404, the contents of which are incorporated herein by reference in their entirety.

[0099] Exemplary TIM-3 Inhibitors Table 5 below discloses certain exemplary TIM-3 inhibitors that can be used in combination therapy with a BTN3A activating antibody, such as ICT01.

[0100] [Table 5]

[0101] TIGIT inhibitors In certain embodiments, the combination therapy comprises administering a therapeutically effective amount of a BTN3A activating antibody described above, e.g., ICT01, in combination with a therapeutically effective amount of a TIGIT inhibitor.

[0102] TIGIT inhibitors that may be used in the combination therapies disclosed herein include, but are not limited to, anti-TIGIT antibodies.

[0103] In some embodiments, the anti-TIGIT antibody molecule is a monospecific or bispecific antibody molecule that exhibits one or more of the following properties: (i) The molecule specifically binds to TIGIT, e.g., human TIGIT. The binding specificity of the Ab can be determined by monitoring binding of the antibody to cells expressing TIGIT protein (e.g., transfected cells or primary immune cells) in a flow cytometry assay, preferably with an EC50 of less than 100 nM, more preferably less than 10 nM. Additionally or alternatively, the antibody binds to recombinant human TIGIT protein with high affinity, with a KD of 100 nM or less, 10 nM or less, 1 nM or less, or 100 pM or less as measured by SPR, BLI, or other suitable binding assay; and / or (ii) the molecule blocks the interaction of TIGIT with a TIGIT ligand, e.g., PVR (CD155) or CD112 / PVRL2 or CD113 / PVRL3, or inhibits signaling mediated by TIGIT binding to PVR, with an IC50 of less than 100 nM, more preferably less than 10 nM; (iii) modulates (e.g., stimulates, enhances, or revives) an immune response. The ability of an antibody to modulate an immune response may be demonstrated by one or more of the following: enhancing T cell activation and / or proliferation; enhancing cytokine (e.g., IL-2, IFN-γ) secretion by T cells; enhancing the cytotoxic activity of T and / or NK cells against target cells (e.g., in in vitro assays); reducing the suppressor activity of Tregs or slowing tumor growth (e.g., in in vivo models); and (iv) Optionally, the molecule binds to a non-human primate TIGIT, such as a cynomolgus monkey TIGIT.

[0104] In some embodiments, the TIGIT inhibitor comprises an anti-TIGIT antibody molecule. In some embodiments, the TIGIT inhibitor comprises BMS-986207, tiragolumab, MK-7684, AB154, COM902, IBI939, BGB-A1217, ASP8374, M6223, HLX301 (TIGITxPDL1 bispecific), AZD2936 (anti-TIGIT / anti-PD-1 bispecific).

[0105] In some embodiments, the TIGIT inhibitor comprises tiragolumab.

[0106] In some embodiments, the anti-TIGIT antibody molecule comprises a variable heavy chain VH of SEQ ID NO: 97 and a variable light chain VL of SEQ ID NO: 98, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 97 and SEQ ID NO: 98, respectively. In some embodiments, the anti-TIGIT antibody molecule comprises six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, 1CDR2, LCDR3) of SEQ ID NOs: 99-104, respectively. In some embodiments, the anti-TIGIT antibody molecule comprises a heavy chain of SEQ ID NO: 95 and a light chain of SEQ ID NO: 96, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 95 and SEQ ID NO: 96, respectively. Other TIGIT antibody molecules are disclosed in WO2017053748, the contents of which are incorporated herein by reference in their entirety.

[0107] In other embodiments, the anti-TIGIT antibody molecule is a bispecific molecule having TIGIT and PD-1 binding specificities. In more specific embodiments, the TIGIT / PD-1 bispecific antibody comprises the VH polypeptides of SEQ ID NOs: 109 and 111, respectively, and the VL polypeptides of SEQ ID NOs: 110 and 112, respectively, or functional variants thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NOs: 109-112, respectively. In some embodiments, the anti-TIGIT antibody is a bispecific TIGIT / PD-1 molecule comprising a first VH sequence having three CDRs of SEQ ID NOs: 113-115, respectively, a first VL sequence having three CDRs of SEQ ID NOs: 116-118, respectively, a second VH sequence having three CDRs of SEQ ID NOs: 119-121, and a second VL sequence having three CDRs of SEQ ID NOs: 122-124.

[0108] In more specific embodiments, the TIM-3 / PD-1 bispecific antibody comprises the polypeptide of SEQ ID NOs: 105-108, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NOs: 105-108, respectively. Typically, the TIM-3 / PD-1 bispecific antibody for use in combination therapy is AZD2936, or other related bispecific antibodies disclosed in WO 2022 / 229919, the contents of which are incorporated herein by reference in their entirety.

[0109] Exemplary TIGIT Inhibitors Table 6 below discloses certain exemplary TIGIT inhibitors that can be used in combination therapy with a BTN3A-activating antibody, such as ICT01.

[0110] [Table 6]

[0111] PD-1 inhibitors The disclosed combination therapies can also be further used in combination with PD-1 inhibitors, for example, the combined administration of ICT01 with an anti-LAG-3 inhibitor and a PD-1 inhibitor.

[0112] As used herein, the term "PD-1 inhibitor" includes, but is not limited to, PD-1-binding agents, PD-L1-binding agents, and PD-L2-binding agents. PD-1-binding agents include antibodies that specifically bind to PD-1. PD-L1 and PD-L2-binding agents include antibodies that specifically bind to PD-L1 and / or PD-L2, and soluble PD-1 polypeptides that bind to PD-L1 and / or PD-L2. In some embodiments, the PD-1 pathway inhibitor is a PD-1-binding agent, e.g., an anti-PD-1 antibody. In some embodiments, the PD-1 inhibitor is a PD-L1-binding agent, e.g., an anti-PD-L1 antibody. In a further embodiment, the PD-L1-binding agent is a soluble PD-1 polypeptide, e.g., a PD-1-Fc fusion polypeptide capable of binding to PD-L1. In a further embodiment, the PD-L2-binding agent is a soluble PD-1 polypeptide, e.g., a PD-1-Fc fusion polypeptide capable of binding to PD-L2.

[0113] In certain embodiments, art-recognized anti-PD-1 or anti-PD-L1 antibodies may be used.

[0114] In some embodiments, the anti-PD-1 antibody molecule is a monospecific or bispecific antibody molecule that exhibits one or more of the following properties: (i) This molecule binds to human PD-1 or human PD-L1 at 10 -7 K below M D Combine with (ii) the molecule inhibits the binding of PD-L1 and / or PD-L2 to PD-1; (iii) the molecule does not substantially bind to human CD28, CTLA-4, or ICOS; (iv) the molecule increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; and / or (v) The molecule increases interferon-gamma production in an MLR assay.

[0115] In some embodiments, the anti-PD-L1 antibody molecule is a monospecific or bispecific antibody molecule that exhibits one or more of the following properties: (i) This molecule binds to human PD-L1 at 10 -7 K below M D Combine with (ii) This molecule inhibits the binding of PD-L1 to PD-1. (iii) the molecule increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; and / or (iv) This molecule increases interferon-gamma production in an MLR assay.

[0116] In certain embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO 2008 / 156712), PDR001 (No vartis; also known as spartalizumab; see WO 2015 / 112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012 / 145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 70:136 (2017)), BGB-A317 ("tislelizumab" Beigene; see WO 2015 / 35606 and U.S. Patent Application Publication No. 2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; WO 2015 / 085847; see Si-Yang Liu et al., J. Hematol. Oncol. 70:136 (2017)), TSR-042 (Tesaro Biopharmaceuticals; also known as ANB011; see WO 2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu et al., J. Hematol. Oncol.70:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014 / 194302), AGEN2034 (Agenus; see WO 2017 / 040790), MGA012 (Macrogenics; see WO 2017 / 19846), IBI308 (Innovent; see WO 2017 / 024465, WO 2017 / 025016, WO 2017 / 132825, and WO 2017 / 133540), and BCD-100 (Biocad).

[0117] In certain embodiments, anti-PD-1 antibodies for use in combination therapy, particularly with an anti-LAG-3 inhibitor and ICT01, include, but are not limited to, nivolumab (BMS), pembrolizumab (Merck & Co), avelumab (EMD Serono), durvalumab (AstraZeneca), cemiplimab (Regeneron), pidilizumab (Pfizer), dostallimab (GlaxoSmithKline), or atezolizumab (Roche).

[0118] In certain embodiments, an anti-PD-1 antibody for use in combination therapy, particularly with an anti-LAG-3 inhibitor and ICT01, is an antibody having a VH of SEQ ID NO: 35 and a VL of SEQ ID NO: 36, or a functional variant thereof having at least 90%, 95%, or at least 98% identity to SEQ ID NO: 35 and SEQ ID NO: 36, respectively. In certain embodiments, an anti-PD-1 antibody for use in combination therapy, particularly with an anti-LAG-3 inhibitor and ICT01, is an antibody having an HCDR1 of SEQ ID NO: 39, an HCDR2 of SEQ ID NO: 40, an HDCR3 of SEQ ID NO: 41, an LCDR1 of SEQ ID NO: 42, an LCDR2 of SEQ ID NO: 43, and an LCDR3 of SEQ ID NO: 44.

[0119] In other specific embodiments, anti-PD-1 antibodies for use in combination therapy, particularly in bispecific molecules with additional binding specificity for LAG-3, TIM-3, or TIGIT, comprise the six CDRs of nivolumab (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively) of SEQ ID NOs: 39-44.

[0120] Framework or Fc manipulation BTN3A activating antibodies for use in the combination therapy of the present disclosure may include modifications made to framework residues in VH and VL to reduce immunogenicity.

[0121] In some specific embodiments, the antibodies of the disclosure are humanized monoclonal antibodies of the parent murine antibody mAb 7.2, which contain at least the following amino acid mutations in the VH framework region: V5Q, V11L, K12V, R66K, S74F, I75S, E81Q, S82AR, R82BS, R83T, D85E, T87S, L108S, and at least the following amino acid mutations in the Vκ framework region: T5N, V15L, R18T, V19I, K42N, A43I, D70G, ​​F73L, Q100G.

[0122] In other specific embodiments, an antibody of the disclosure is a humanized monoclonal antibody of the parent murine antibody mAb 7.2, which contains at least the following amino acid mutations relative to mAb 7.2 in the VH framework region: V5Q, V11L, K12V, R66K, S74F, I75S, E81Q, S82AR, R82BS, R83T, D85E, T87S, L108S, and at least the following amino acid mutations in the Vκ framework region: T5N, V15L, R18T, V19I, K42N, A43I, S63T, D70G, ​​F73L, Q100G.

[0123] In addition to modifications made to the framework regions, the antibodies of the disclosure typically may be engineered to contain modifications in the Fc region to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0124] Furthermore, antibodies for use in the combination therapies of the present disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter glycosylation to alter one or more functional properties of the antibody, each of these embodiments being described in further detail below.

[0125] As used herein, the terms "isotype constant region" or "Fc region" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined to include amino acid residues from position C226 or P230 to the carboxyl terminus of an IgG antibody, where numbering is according to the EU numbering system. The C-terminal lysine (K447 residue) of the Fc region can be removed, for example, during antibody production or purification, or the corresponding codon can be deleted in a recombinant construct. Thus, antibody compositions of the present disclosure can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0126] In certain embodiments, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0127] In other embodiments, the Fc-hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, the antibody has one or more amino acid mutations introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment to reduce Staphylococcus aureus protein A (SpA) binding compared to the SpA binding of the native Fc-hinge domain. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0128] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, an antibody can have its affinity for an effector ligand altered by replacing one or more amino acids with a different amino acid residue, but retain the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0129] In another embodiment, the antibody can have one or more selected amino acid residues replaced with different amino acid residues to alter C1q binding and / or reduce or eliminate complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0130] In another embodiment, one or more amino acid residues are altered to alter the ability of the antibody to fix complement. This approach is further described in PCT Publication No. WO 94 / 29351 by Bodmer et al.

[0131] In other embodiments, the Fc region is modified by modifying one or more amino acids to reduce the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to reduce the affinity of the antibody for Fcγ receptors. Such antibodies with reduced effector function, particularly reduced ADCC, include silenced antibodies.

[0132] In certain embodiments, an Fc domain of the IgG1 isotype is used, in some particular embodiments, a mutant variant of the IgG1 Fc fragment is used, for example, a silenced IgG1 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0133] In certain embodiments, an Fc domain of the IgG4 isotype is used, in some specific embodiments, a mutant variant of an IgG4 Fc fragment is used, for example, a silenced IgG4 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0134] Silenced effector functions can be obtained by mutations in the Fc constant portion of antibodies, and have been described in the art (Baudino L et al., J. Immunol. 2008 Sep. 15; 181(6):4107-12; Strohl, WR. Curr Opin Biotechnol. 2009 Dec. 20(6):685-91). An example of a silent IgG1 antibody is the triple mutation variant IgG1 L247F L248E P350S. An example of a silent IgG4 antibody is the double mutation variant IgG4 S241P L248E.

[0135] In certain embodiments, the Fc domain is a silent Fc mutant that prevents glycosylation of the Fc domain at position 314. For example, the Fc domain comprises an amino acid substitution of asparagine at position 314. Examples of such amino acid substitutions are substitutions of N314 with glycine or alanine.

[0136] In yet other embodiments, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more variable region framework glycosylation sites can be eliminated by making one or more amino acid substitutions, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for antigen. Such approaches are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0137] Another modification of the antibodies herein contemplated by the present disclosure is pegylation or hesylation or related techniques. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in attachment of one or more PEG groups to the antibody or antibody fragment. Pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, European Patent No. 0154316 to Nishimura et al. and European Patent No. 0401384 to Ishikawa et al.

[0138] Another possibility is to fuse at least the antigen-binding region of an antibody of the present disclosure to a protein capable of binding to a serum protein, such as human serum albumin, to increase the half-life of the resulting molecule. Such an approach is described, for example, in Nygren et al., EP 0486525.

[0139] In certain embodiments, the C-terminal lysine commonly present in human IgG heavy chain constant domains is engineered and removed to reduce heterogeneity due to cleavage of this lysine commonly observed during manufacturing or storage. Such modifications do not appreciably alter the desired function of these antibodies, and confer stability advantages to these molecules.

[0140] Nucleic acid molecules encoding antibodies of the present disclosure Also disclosed herein are nucleic acid molecules encoding BTN3A-activating antibodies for use in the present disclosure. Exemplary variable light and heavy chain nucleotide sequences are those encoding the variable light and heavy chain amino acid sequences of any one of mAb1, (ICT01), mAb2, mAb4, and mAb5, the latter sequences being readily derived from Tables 1 and 2 using the genetic code and, optionally, taking into account codon bias depending on the host cell type.

[0141] Nucleic acid molecules derived from the latter sequence are optimized for protein expression in mammalian cells, for example CHO cell lines.

[0142] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially purified" when they have been purified by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (Ausubel et al., 1988; Current Protocols in Molecular Biology (John Wiley & Sons)), to separate them from other cellular components or contaminants, such as other cellular nucleic acids or cellular proteins. Nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, the nucleic acids can be present in a vector, such as a phage display vector or a recombinant plasmid vector.

[0143] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For example, once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a VL- or VH-encoding DNA fragment (e.g., the VL and VH segments defined in Table 1) is operably linked to another DNA molecule or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that the two DNA fragments are operably joined, for example, so that the amino acid sequences encoded by the two DNA fragments are in frame or so that a protein is expressed under the control of a desired promoter.

[0144] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (Kabat et al., KS (1992). Sequences of Proteins of Immunological Interest (DIANE Publishing)), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In some embodiments, the heavy chain constant region is selected from an IgG1 isotype, e.g., a human IgG1 isotype. In other embodiments, the heavy chain constant region is selected from an IgG4 isotype, e.g., a human IgG4 isotype. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain constant region CH1.

[0145] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (e.g., into a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (Kabat et al., 1992, supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0146] To generate an scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible linker, for example, another fragment encoding the amino acid sequence (Gly4-Ser)3, thereby enabling expression of the VH and VL sequences as a contiguous single-chain protein in which the VL and VH domains are connected by the flexible linker (Bird et al., 1988, supra; Huston et al., 1988, supra; McCafferty et al., 1990; McCafferty, J. et al., 1990. Nature 348, 552-554).

[0147] Generation of transfectomas producing monoclonal antibodies The antibodies of the present disclosure can be produced in host cell transfectomas, for example, using a combination of recombinant DNA techniques and gene transfection methods, as are well known in the art (Morrison, 1985; Science 229, 1202-1207).

[0148] For example, to express an antibody, or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biological or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using a hybridoma expressing the antibody of interest), and this DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of controlling the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or, more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or, if no restriction sites are present, blunt-end ligation). The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting them into an expression vector already encoding heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to a CH segment(s) in the vector and the VL segment is operably linked to a CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0149] In addition to the antibody chain genes, the recombinant expression vectors disclosed herein carry control sequences that regulate the expression of the antibody chain genes in a host cell. The term "control sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such control sequences are described, for example, in Goeddel (Goeddel, DV (1990). [1] Systems for heterologous gene expression., in Methods in Enzymology (Academic Press), pp. 3-7). Those skilled in the art will understand that the design of the expression vector, including the selection of control sequences, can depend on factors such as the choice of host cell to be transformed and the desired expression level of protein. Control sequences for expression in mammalian host cells include promoters and / or enhancers derived from viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral control sequences, such as the ubiquitin promoter or P-globin promoter, can be used. Still further, control elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., 1988, Mol. Cell. Biol. 8, 466-472).

[0150] In addition to the antibody chain genes and control sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0151] For expression of the light and heavy chains, expression vector(s) encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The antibodies of the present disclosure can theoretically be expressed in either prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi, is contemplated because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0152] In one specific embodiment, a cloning or expression vector according to the present disclosure comprises one of the coding sequences for the heavy and light chains of any of mAb1, mAb2, mAb4, and mAb5 operably linked to a suitable promoter sequence.

[0153] Mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker (described in Kaufman and Sharp, 1982), the CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells, and SP2 cells, e.g., the GS CHO cell line used with the GS Xceed™ Gene Expression System (Lonza). When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient for expression of the antibody within the host cells and, optionally, for secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered and purified from the culture medium, for example, after secretion, using standard protein purification methods (Shukla et al., 2007, J. Chromatogr. B 848, 28-39).

[0154] In one particular embodiment, the host cell of the present disclosure is a host cell transfected with an expression vector having coding sequences suitable for expressing the antibody heavy and light chains or recombinant proteins for use as disclosed herein, operably linked to a suitable promoter sequence.

[0155] For example, the present disclosure relates to host cells comprising at least the nucleic acids of SEQ ID NO:8 and SEQ ID NO:10 encoding the heavy and light chain ICT01, respectively.

[0156] The latter host cells can then be further cultured under conditions suitable for the expression and production of antibodies for use as disclosed herein.

[0157] Alternatively, cell-free expression systems can be used for the production of either antibodies or recombinant proteins. Typically, cell-free expression methods for proteins or antibodies have been previously described (Stech et al., 2017, Sci. Rep. 7, 12030).

[0158] Combination Kits and Compositions Combination Kit The combinations of the present disclosure, comprising a BTN3A antibody as defined above and an immune checkpoint inhibitor, typically an anti-LAG-3 antibody, an anti-TIM-3 antibody and / or an anti-TIGIT antibody, may be presented as a combination kit.

[0159] The term "combination kit" or "kit of parts," as used herein, refers to a pharmaceutical composition or compositions used to administer a BTN3A activating antibody according to the present disclosure and an immune checkpoint inhibitor.

[0160] When both compounds are administered simultaneously, the combination kit may, for example, comprise the components, suitably a BTN3A activating antibody and an immune checkpoint inhibitor, in separate pharmaceutical compositions. When the components, suitably a BTN3A activating antibody and an immune checkpoint inhibitor, are not administered simultaneously, the combination kit comprises the active ingredients in separate pharmaceutical compositions in a single package, or alternatively, in separate pharmaceutical compositions in separate packages.

[0161] In one aspect, a composition comprising a BTN3A activating antibody, typically a BTN3A activating antibody as defined above, together with a pharmaceutically acceptable excipient, diluent, or carrier; and A composition comprising an immune checkpoint inhibitor, typically an immune checkpoint inhibitor as defined above, typically an anti-LAG-3 antibody, an anti-TIM-3 antibody and / or an anti-TIGIT antibody, together with a pharmaceutically acceptable excipient, diluent or carrier. A kit of parts is provided comprising:

[0162] In one embodiment of the present disclosure, the kit of parts comprises: a composition comprising a BTN3A activating antibody, typically a BTN3A activating antibody as defined above, together with a pharmaceutically acceptable excipient, diluent, or carrier; and A composition comprising an immune checkpoint inhibitor, typically an immune checkpoint inhibitor as defined above, typically an anti-LAG-3 antibody, an anti-TIM-3 antibody and / or an anti-TIGIT antibody, together with a pharmaceutically acceptable excipient, diluent or carrier. wherein the components are provided in a form suitable for sequential, separate and / or simultaneous administration.

[0163] In one embodiment, the kit of parts comprises: a first container containing a composition comprising a BTN3A activating antibody, typically a BTN3A activating antibody as defined above, together with a pharmaceutically acceptable excipient, diluent, or carrier; and a second container containing a composition comprising an immune checkpoint inhibitor, typically an immune checkpoint inhibitor as defined above, typically an anti-LAG-3 antibody, an anti-TIM-3 antibody and / or an anti-TIGIT antibody, together with a pharmaceutically acceptable excipient, diluent or carrier; and a container for containing said first and second containers. Equipped with.

[0164] The combination kit may also be provided with instructions, e.g., dosage and administration instructions, which may be of the type provided to a physician or by a physician, such as instructions to a patient.

[0165] Pharmaceutical Composition Pharmaceutical compositions suitable for administration to human patients are typically formulated for parenteral administration, e.g., in a liquid carrier or suitable for reconstitution into a solution or suspension for intravenous administration.

[0166] In general, such compositions typically include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a government regulatory agency or listed in the United States Pharmacopoeia or another generally recognized pharmacopeia for use in animals, and particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, glycerol polyethylene glycol ricinoleate, and the like. Water or Aqueous Solutions: Saline and aqueous dextrose and glycerin solutions can be employed as carriers, particularly for injectable solutions (e.g., containing a BTN3A-activating antibody, anti-LAG-3, and / or anti-PD-1 antibody).

[0167] Antibodies for use in combination therapy can be formulated into the compositions defined above in neutral or salt forms. Liquid compositions for parenteral administration can be formulated for administration by injection or continuous infusion. Routes of administration by injection or infusion include intravenous, intraperitoneal, intramuscular, intrathecal, and subcutaneous routes. In one embodiment, the BTN3A-activating antibody, anti-LAG-3 antibody, and / or anti-PD-1 antibody are administered intravenously (e.g., in separate formulations, or together (in the same formulation or in separate formulations)).

[0168] Suitable formulations for infusion or subcutaneous injection solutions of antibodies have been described in the art and are reviewed, for example, in Cui et al. (Drug Dev Ind Pharm 2017, 43(4):519-530), the contents of which are incorporated herein in their entirety.

[0169] Pharmaceutical compositions and dosing regimens comprising BTN3A activating antibodies, anti-LAG-3 and / or anti-PD-1 antibodies Thus, in some embodiments, the BTN3A activating antibodies, anti-LAG-3 and / or anti-PD-1 antibodies defined herein can be formulated into a composition, e.g., a pharmaceutical composition as defined above, comprising any of the following: one or a combination of the BTN3A-activating antibodies disclosed herein, for example, an antibody selected from the group consisting of ICT01 (mAb1), mAb2, mAb3, mAb4, mAb5, and mAb6, or antigen-binding portions thereof, formulated together with a pharmaceutically acceptable carrier; one or a combination of LAG-3 inhibitors as defined herein, for example a LAG-3 inhibitor selected from the group consisting of eftiragimoda alfa, opdualag (nivolumab / leratlimab), tebotelimab; and Optionally, one of the PD-1 inhibitors as defined herein or a combination thereof, for example an anti-PD-1 antibody selected from the group consisting of nivolumab (BMS), pembrolizumab (Merck&Co), avelumab (EMD Serono), durvalumab (AstraZeneca), cemiplimab (Regeneron), pidilizumab (Pfizer), dostallimab (GlaxoSmithKline), or atezolizumab (Roche).

[0170] In a preferred embodiment, the pharmaceutical compositions for use in combination therapy comprise a first pharmaceutical composition having ICT01 formulated for intravenous infusion as defined above, a second pharmaceutical composition comprising a LAG-3 inhibitor preferably selected from eftiragimoda alfa, opdualag (nivolumab / relatolimab), tebotelimab, also formulated for intravenous administration, and optionally a third pharmaceutical composition comprising a PD-1 inhibitor preferably selected from the group consisting of nivolumab (BMS), pembrolizumab (Merck&Co), avelumab (EMD Serono), durvalumab (AstraZeneca), cemiplimab (Regeneron), pidilizumab (Pfizer), dostallimab (GlaxoSmithKline), or atezolizumab (Roche), also in formulations for intravenous administration.

[0171] Pharmaceutical compositions comprising a BTN3A activating antibody (e.g., ICT01), a LAG-3 inhibitor, and optionally a PD-1 inhibitor, respectively, can be formulated at various concentrations.

[0172] For example, a formulation for use in combination therapy may contain an activating BTN3A-activating antibody (e.g., ICT01) at a concentration of 0.1 μM to 1 mM, more preferably 1 μM to 500 μM, 500 μM to 1 mM, 300 μM to 700 μM, 1 μM to 200 μM, 100 μM to 200 μM, 200 μM to 300 μM, 300 μM to 400 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 800 μM to 900 μM, or 900 μM to 1 mM. Typically, the formulation contains a BTN3A-activating antibody at a concentration of 300 μM to 700 μM.

[0173] Combination Uses and Methods of the Present Disclosure The present disclosure provides therapeutic combinations (also referred to as "combination therapies") for use in the treatment of cancer, which therapeutic combinations include a BTN3A activating antibody, e.g., ICT01, and an immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, an anti-TIM-3 antibody, or an anti-TIGIT antibody (and related bispecific molecules involving anti-PD-1 molecules), as defined above, optionally in combination with a PD-1 inhibitor, e.g., an anti-PD-1 or anti-PD-L1 antibody, as described in the previous section.

[0174] The present disclosure also provides a method of treating cancer in a subject in need thereof, comprising administering to the patient a therapeutically effective amount of a BTN3A activating antibody, e.g., ICT01, in combination with a therapeutically effective amount of an immune checkpoint inhibitor, either simultaneously, sequentially, or separately, wherein the immune checkpoint inhibitor is selected from a LAG-3 inhibitor, a TIM-3 inhibitor, and a TIGIT inhibitor, e.g., an anti-LAG-3 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody.

[0175] As used herein, the terms "treat," "treating," or "treatment" refer to one or more of the following: (1) inhibiting the disease; e.g., inhibiting the disease, condition, or disorder (i.e., arresting further progression of the symptoms and / or symptomology) in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder; and (2) ameliorating the disease; e.g., ameliorating the disease, condition, or disorder (i.e., ameliorating the symptoms and / or symptomology) in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder, e.g., reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. In particular, with respect to the treatment of tumors, the term "treatment" can refer to inhibiting tumor growth or reducing the size of the tumor.

[0176] BTN3A activating antibodies can activate the cytolytic function, cytokine production and / or proliferation of Vy9V52 T cells, and thereby be used to overcome immune suppressive mechanisms observed in cancer patients (see, inter alia, WO2012080769, WO2012080351, and WO2020025703). The results of the present disclosure now show that this combination therapy with an additional immune checkpoint inhibitor, such as a LAG-3, TIM-3, or TIGIT inhibitor, further promotes synergistic and specific Vy9V52 T cell activation and cytotoxicity in human PBMCs, highlighting the therapeutic importance of this combination, particularly for the treatment of cancer.

[0177] As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells capable of autonomous growth, i.e., an abnormal state or pathology characterized by rapidly proliferating cell proliferation. Hyperproliferative and neoplastic disease states may be classified as pathological, i.e., characterizing or constituting a disease state, or non-pathological, i.e., deviating from the norm but not associated with a disease state. The terms are meant to include all types of cancerous growths or tumorigenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0178] The term "cancer" or "neoplasm" includes malignant tumors of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic system, digestive system, and urinary tract, as well as adenocarcinoma, which includes malignant tumors such as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular cancer, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus.

[0179] Examples of cancer include, but are not limited to, hematological malignancies such as neoplasms of myeloid lineage, including acute myeloid leukemia (AML); B-cell lymphoid neoplasms, including Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma (B-NHL), diffuse large B-cell lymphoma (DLBCL), indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), multiple myeloma; T-cell lymphoid neoplasms and NK-cell lymphoid neoplasms.

[0180] Examples of non-hematological cancers (i.e., solid tumors) include, but are not limited to, advanced tumors, refractory or recurrent tumors, melanoma (e.g., metastatic malignant melanoma), lung cancer (e.g., small cell lung cancer), breast cancer, prostate cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), renal cancer, colon cancer, gastric cancer, esophageal cancer, liver cancer, prostate cancer, glioblastoma, ovarian cancer, cervical cancer, bile duct cancer, bladder cancer, and cancer of the kidney or ureter, and PD-L1 positive cancers.

[0181] In the combination therapies of the present disclosure, the BTN3A activating antibody and the immune checkpoint inhibitor, e.g., in the case of LAG-3 inhibition, the anti-TIM-3 antibody or anti-TIGIT antibody, and optionally the PD-1 inhibitor, can be independently administered orally or parenterally, where parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration.

[0182] Sequential administration (e.g., in separate pharmaceutical compositions) is particularly useful when the therapeutic agents of the combination therapy are in different dosage forms (e.g., one agent is a tablet or capsule and the other is a sterile liquid) and / or when the therapeutic agents of the combination therapy are administered on different dosing schedules, e.g., a chemotherapeutic agent that is administered at least daily and a biologic therapeutic agent that is administered less frequently, e.g., once per week, once every two weeks, or once every three weeks.

[0183] In some embodiments, a BTN3A activating antibody is administered in conjunction with an immune checkpoint inhibitor, such as a LAG-3 inhibitor, an anti-TIM-3 antibody, or an anti-TIGIT antibody.

[0184] In other embodiments, the BTN3A activating antibody is administered prior to administration of the immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, an anti-TIM-3 antibody, or an anti-TIGIT antibody, while in other embodiments, the BTN3A activating antibody is administered after administration of the immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, an anti-TIM-3 antibody, or an anti-TIGIT antibody.

[0185] The selection of a dosing regimen (also referred to herein as an administration regimen) for the combination therapy of the present disclosure depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs in the individual being treated. Preferably, the dosing regimen maximizes the amount of each therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Thus, the dosage and frequency of each biological and chemotherapeutic agent in the combination will depend, in part, on the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient. Guidance is available for selecting appropriate doses of antibodies, cytokines, and small molecules. See, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New See Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition); Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002).The determination of an appropriate dosing regimen can be made by the clinician using, for example, parameters or factors known or suspected in the art to affect treatment, or predicted to affect treatment, and will depend, for example, on the patient's clinical history (e.g., previous therapies), the type and stage of the cancer being treated, and response biomarkers to one or more of the therapeutic agents of the combination therapy.

[0186] Any suitable dosage range may be used as determined by the attending medical practitioner. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response).

[0187] In some embodiments, at least one of the therapeutic agents of the combination therapy is administered using the same dosing regimen (dose, frequency of treatment, and duration of treatment) that would typically be employed to treat the same cancer if the therapeutic agent were used as a monotherapy. In other embodiments, the patient is administered a lower total amount, e.g., a lower dose, less frequent administration, and / or shorter duration of treatment, of at least one of the therapeutic agents of the combination therapy than if the therapeutic agent were used as a monotherapy.

[0188] Regarding the dosing regimen of the BTN3A activating antibody, e.g., ICT01, and the immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, an anti-TIM-3 antibody, or an anti-TIGIT antibody, of the present combination therapy, any suitable dosing range can be used as determined by the attending medical professional.

[0189] Dosage regimens can be adjusted to obtain the optimum desired response (e.g., therapeutic or prophylactic response). The antibodies of the present disclosure can be formulated to contain about 0.0001 to 100.0 milligrams, or about 0.001 to 10 milligrams, or about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 milligrams, or even 1.0 to about 10 milligrams per dose in the therapeutic mixture. Multiple doses can also be administered. Typically, therapeutic doses of BTN3A-activating antibodies, e.g., ICT01, for human patients range from 100 pg to 700 mg per administration (based on a 70 kg body weight). For example, maximum therapeutic doses can range from 0.1 to 10 mg / kg per administration, e.g., 0.1 to 5 mg / kg, 1 to 5 mg / kg, or 0.1 to 2 mg / kg. It will be appreciated that such doses may be administered at different intervals as determined by the oncologist / physician; for example, doses may be administered daily, twice weekly, weekly, every other week, every three weeks or monthly.

[0190] Typically, therapeutic doses of a BTN3A-activating antibody (e.g., ICT01), a LAG-3 inhibitor (e.g., eftiragimoda alfa, opdualag (nivolumab / relatolimab), or tebotelimab) for use in combination therapy in human patients range from 100 pg to 700 mg per administration (based on a 70 kg body weight). For example, a maximum therapeutic dose may range from 0.1 to 10 mg / kg per administration, e.g., 0.1 to 5 mg / kg, or 1 to 5 mg / kg, or 0.1 to 2 mg / kg. It will be understood that such doses may be administered at different intervals as determined by the oncologist / physician; for example, doses may be administered daily, twice weekly, weekly, every other week, every three weeks, or monthly. One skilled in the art can also select a dosing regimen prescribed for an approved monotherapy of a LAG-3 inhibitor and / or a PD-1 inhibitor.

[0191] Typically, in certain embodiments, the activating BTN3A antibody (e.g., ICT01) is administered intravenously at doses containing 20 μg to 200 mg, particularly 1 mg to 200 mg or 7 to 200 mg, typically every 21 days.

[0192] In certain embodiments, the LAG-3 inhibitor, e.g., an anti-LAG-3 antibody, e.g., faverezimab, is administered intravenously at a unit dose ranging from 20 μg to 1000 mg, particularly about 100 to about 1000 mg, e.g., 800 mg, once every 21 days for 1 to 35 cycles, either concurrently or sequentially with a BTN3A-activating antibody (e.g., ICT01).

[0193] In another specific embodiment, the LAG-3 inhibitor, e.g., the fixed-dose combination OPDUALAG, is administered intravenously at a unit dose of 160 mg leratolimab and 480 mg nivolumab in a single intravenous infusion, e.g., every 4 weeks, either concurrently or sequentially with a BTN3A-activating antibody (e.g., ICT01).

[0194] In certain embodiments, a unit dose suitable for intravenous administration of an activating anti-BTN3A antibody can be selected from 1, 7, 10, 20, 50, 75, 100, 125, 150, 175, and 200 mg.

[0195] In certain embodiments, a suitable unit dose for subcutaneous administration of LAG-3 recombinant protein or Ig fusion may be selected from about 0.25 to 30 mg (eg, eftiragimdo alfa).

[0196] In certain embodiments, the unit dose suitable for intravenous administration of the PD-1 inhibitor can be selected from 100 to 2000 mg (e.g., preferably selected from the group consisting of nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, pidilizumab, dostallimab, or atezolizumab, which are also formulations for intravenous administration).

[0197] In certain embodiments, the BTN3A-activating antibody (preferably ICT01 (mAb1) described herein) is administered in this combination therapy (typically with a LAG-3 inhibitor and optionally an anti-PD-1 antibody) at a unit dose of about 7 to about 200 mg, e.g., 75 mg, e.g., once every 21 days for 1 to 22 cycles.

[0198] Advantageously, in certain embodiments, the combined effect of BTN3A activating antibody (e.g., ICT01) treatment with an immune checkpoint inhibitor (e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor, and optionally a PD-1 inhibitor) increases the overall survival of a subject by at least 10%, 20%, 30%, 40%, or at least 50% compared to monotherapy with either the BTN3A antibody or the immune checkpoint inhibitor (e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor).

[0199] "Overall survival" (OS) is defined herein as the time from the date of the first dose to the date of death from any cause in a clinical study participant.

[0200] In certain embodiments, the combined effect of BTN3A activating antibody (e.g., ICT01) treatment with an immune checkpoint inhibitor (e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor, and optionally a PD-1 inhibitor) increases progression-free survival by at least 10%, 20%, 30%, 40%, or at least 50% compared to monotherapy with either the BTN3A antibody or the immune checkpoint inhibitor (e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor).

[0201] The term "progression-free survival" (PFS), as used herein, is defined as the time from the date of the first dose to the date of confirmed progression.

[0202] In certain embodiments, the combination therapy may inhibit, delay, and / or reduce tumor growth in a subject. In certain embodiments, tumor growth is delayed by at least 50%, 60%, 70%, or 80% compared to an untreated control subject. In certain embodiments, tumor growth is delayed by at least 80% compared to an untreated control subject. In certain embodiments, tumor growth is delayed by at least 50%, 60%, 70%, or 80% compared to growth expected for an untreated tumor. In certain embodiments, tumor growth is delayed by at least 50%, 60%, 70%, or 80% compared to growth expected for a tumor untreated or treated with a corresponding monotherapy using a BTN3A-activating antibody or immune checkpoint inhibitor (e.g., a LAG-3, TIM-3, or TIGIT inhibitor). Tumor volume assessment can be determined using RECIST (Eisenhauer EA et al., Eur J Cancer. 2009 Jan;45(2):228-47) for patients with solid tumors and RECIL (Younes A et al., Ann Oncol. 2017 Jul 1;28(7):1436-1447) for patients with lymphoma.

[0203] In certain embodiments, administration of a combination therapy of the present disclosure can increase the length of survival of a subject. In certain embodiments, the increased survival is an increase compared to an untreated control subject or a control subject treated with standard therapy. In certain embodiments, the increased survival is an increase compared to the length of survival expected for a subject treated with standard therapy. In certain embodiments, the length of survival is increased by at least 3-fold, 4-fold, or 5-fold compared to an untreated control subject or a control subject treated with standard therapy. In certain embodiments, the length of survival is increased by at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, or 3 years compared to a control subject treated with standard therapy.

[0204] A BTN3A activating antibody as defined above and an immune checkpoint inhibitor, such as a LAG-3 inhibitor, a TIM-3 inhibitor or a TIGIT inhibitor, The combination therapy, whether combined in a single composition or in separate compositions, can further be administered with other drugs, for example, other drugs for the treatment or prevention of the diseases mentioned above.

[0205] In certain embodiments, the combination therapy disclosed herein (typically ICT01, as described above, and an immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor) can be administered in combination with an anti-neoplastic agent.

[0206] In other specific embodiments, the combination therapy disclosed herein (typically ICT01, as described above, and an immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor) can be administered in combination with a cell therapy (particularly a γδ T cell therapy).

[0207] In other specific embodiments, the combination therapy disclosed herein (typically ICT01, as described above, and an immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor) can be administered with other immune checkpoint inhibitors (particularly PD-1 inhibitors, e.g., anti-PD-1 or anti-PD-L1 antibodies, and anti-CTLA-4 antibodies).

[0208] In other specific embodiments, the combination therapy disclosed herein (typically ICT01, as previously described, and an immune checkpoint inhibitor, e.g., a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor) is further administered in combination with a cytokine (e.g., interleukin-2 (IL-2) (Choudhry H et al., 2018, Biomed Res Int. 2018 May 6), interleukin-15 (IL-15) (Patidar M et al., Cytokine Growth Factor Rev. 2016 Oct;31:49-59), interleukin-21 (IL-21) (Caccamo N. et al., PLoS One. 2012;7(7):e41940), or interleukin-33 (IL-33) (Duault C et al., J Immunol. 2016 Jan. 1;196(1):493-502), or their recombinant forms and derivatives thereof, or any cytokine or derivative capable of inducing lymphocyte activity and / or expanding lymphocytes (e.g., proliferation or cytokine production or metabolic changes). The term derivative is used in particular for any cytokine modification, which may rely on PEGylation (e.g., conjugation to polyethylene glycol (EPG) chains), mutations such as amino acid deletions, substitutions or insertions, or association with an enhancer (e.g., an IL15 / IL15Ra complex fused to IgG1 Fc, in which IL-15 is further mutated (asn72asp), which further increases the biological activity of this complex, making it an IL-2 and IL-15Rβγ superagonist (Rhode PR et al., Cancer Immunol Res. 2016;4(1):49-60)) (Barroso-Sousa R et al., Curr Oncol Rep. 2018 Nov 15;21(1):1).

[0209] An example of a suitable IL2 cytokine is Proleukin.

[0210] Suitable doses of IL-2 cytokines, e.g., Proleukin, that may be administered, e.g., subcutaneously, to a subject using the combination therapy of the present disclosure are m2 0.5.10 per 6 IU~18.10 6 IU. In a specific embodiment, the IL-2 cytokine is administered daily on days 1-5 of a 21-day administration cycle of a BTN3A activating antibody.

[0211] The term "IL-2" has its conventional meaning and refers to human interleukin-2. IL-2 is part of the body's innate immune response. IL-2 primarily regulates lymphocyte activity by binding to the IL-2 receptor.

[0212] The term "IL-15" has its conventional meaning and refers to human interleukin-15. Like IL-2, IL-15 binds to and signals through a complex composed of the IL-2 / IL-15 receptor beta chain (CD122) and the common gamma chain (gamma-C, CD132). IL-15 regulates the activation and proliferation of T cells and natural killer (NK) cells.

[0213] The term "IL-21" has its conventional meaning and refers to human interleukin-21. IL-21 is known to have pleiotropic properties, including, but not limited to, enhancing the cytotoxicity of NK cells and CD8+ T cells, modulating plasma cell differentiation, and inhibiting Treg cells.

[0214] The term "IL-33" has its general meaning and refers to human interleukin-33. IL-33, believed to be an alarmin released upon tissue stress or injury, is a member of the IL-1 family and binds to the ST2 receptor. IL-33 is known to be a potent stimulator of TH1 immune cells, natural killer (NK) cells, iNKT cells, and CD8 T lymphocytes.

[0215] In other specific embodiments, the combination therapy disclosed herein (typically ICT01, as described above, and an immune checkpoint inhibitor, e.g., an anti-LAG-3 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody) can be administered with an additional immunotherapeutic agent, e.g., an immune checkpoint inhibitor (particularly an anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibody).

[0216] As used herein, the term "cell therapy" refers to a therapy comprising the in vivo administration of at least a therapeutically effective amount of a cell composition to a subject in need thereof. The cells administered to a patient can be allogeneic or autologous. The term "γδ T cell therapy" refers to a cell therapy in which the cell composition comprises γδ T cells, in particular Vγ9V52 T cells, as an active ingredient. In a specific embodiment, said Vγ9V52 T cells have been expanded and / or activated ex vivo.

[0217] A cell therapy product refers to a cell composition administered to the patient for therapeutic purposes, comprising a therapeutically effective dose of cells and, optionally, additional excipients, adjuvants, or other pharmaceutically acceptable carriers.

[0218] Another therapeutic strategy is based on the use of the properties of the combination disclosed herein as an agent to selectively expand and / or activate Vy9V52 T cells isolated from a sample from a human subject. Accordingly, the present disclosure provides a method for treating a subject in need thereof, comprising: (a) isolating cells comprising Vy9V52 T cells, for example PBMCs from a blood sample or tumor infiltrating leukocytes (TILs) from tumor tissue of a subject; (b) culturing the isolated cells, optionally together with other tumor or accessory cells, in a suitable cell culture medium with effective amounts of a BTN3A activating antibody and an immune checkpoint inhibitor, such as a LAG-3 inhibitor, a TIM-3 inhibitor or a TIGIT inhibitor, which may be added simultaneously, in parallel or sequentially to the cell culture, thereby obtaining expanded Vy9V52 T cells; (c) harvesting expanded Vγ9Vδ2 T cells; (d) optionally formulating the expanded Vy9V52 T cells and administering a therapeutically effective amount of said Vy9V52 T cells to the subject. The present invention relates to a method comprising:

[0219] The present disclosure further relates to the use of the combinations disclosed herein for selectively expanding chimeric antigen receptor (CAR) Vy9V52 T cells. CAR γδ T cells and their use in adoptive T cell cancer immunotherapy are described, for example, in Mirzaei et al. (Cancer Lett 2016, 380(2):413-423).

[0220] The present disclosure also relates to a combination as defined herein for in vivo use to enhance tumor cell efficacy in γδ T cell therapy in a subject in need thereof, typically a subject suffering from cancer, wherein a BTN3A activating antibody and an immune checkpoint inhibitor, such as a LAG-3 inhibitor, a TIM-3 inhibitor or a TIGIT inhibitor, can be administered to the subject simultaneously, concurrently or sequentially.

[0221] As used herein, the term γδ T cell therapy refers to a therapy comprising administering at least an effective amount of γδ T cells to a subject in need thereof. Such γδ T cells can be allogeneic or autologous. In certain embodiments, γδ T cells can be genetically engineered by deletion or knockout or insertion or knockin of specific genes. In certain embodiments, the γδ T cells include γδ T cells expressing a chimeric antigen receptor. γδ T cells can be ex vivo expanded and / or purified. Alternatively, γδ T cells can also be included in a cell composition containing other blood cells, e.g., other cells of the immune system. For references regarding γδ T cell therapy, see Pauza CD. et al., Front Immunol. 2018 Jun. 8;9:1305.doi:10.3389 and Saudemont A. et al., Front Immunol. 2018 Feb. 5;9:153.doi:10.3389.

[0222] Accordingly, the present disclosure provides a method of treating a subject suffering from cancer, including a solid tumor or a hematological malignancy, particularly a leukemia, e.g., acute myeloid leukemia, and having tumor cells, e.g., hematological tumor cells, comprising: i. administering to the subject an effective amount of a BTN3A activating antibody disclosed herein, typically ICT01, mAb2, mAb3, mAb4, mAb5, or mAb6, in combination with an effective amount of an immune checkpoint inhibitor, such as a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor, wherein the BTN3A activating antibody and the immune checkpoint inhibitor, such as a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor, can be administered simultaneously, concurrently, or sequentially; and ii. administering to said subject an effective amount of a γδ T cell composition. wherein the combination of the effective amount of a BTN3A activating antibody with the effective amount of an immune checkpoint inhibitor, such as a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor, can enhance anti-tumor cytolysis of the tumor cells mediated by the γδ T cell composition.

[0223] The present disclosure provides a method for treating a subject suffering from cancers having solid tumor cells, e.g., advanced tumors, refractory or recurrent tumors, melanoma (e.g., metastatic malignant melanoma), lung cancer (e.g., non-small cell lung cancer), breast cancer, prostate cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), renal cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, glioblastoma, ovarian cancer, cervical cancer, bile duct cancer, bladder cancer, cancer of the kidney or ureter, and PD-L1 positive cancers, and hematological malignancies including, e.g., acute myeloid leukemia (AML), Hodgkin's lymphoma, multiple myeloma, diffuse large B-cell lymphoma (DLBCL), indolent non-Hodgkin's lymphoma (NHL), the method comprising: i. administering to the subject a therapeutically effective amount of a BTN3A activating antibody disclosed herein, typically ICT01, in combination with a therapeutically effective amount of an immune checkpoint inhibitor as defined herein, e.g., an anti-LAG-3 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody, wherein the BTN3A activating antibody and the immune checkpoint inhibitor, e.g., an anti-LAG-3 antibody, an anti-TIM-3 antibody, or an anti-TIGIT antibody, can be administered simultaneously, concurrently, or sequentially; and ii. administering to said subject an effective amount of a γδ T cell composition. Including, The present invention further relates to a method, wherein the combination of the therapeutically effective amount of the BTN3A activating antibody and the therapeutically effective amount of an immune checkpoint inhibitor, such as a LAG-3 inhibitor, a TIM-3 inhibitor, or a TIGIT inhibitor, can enhance anti-tumor cytolysis of the tumor cells mediated by the γδ T cell composition.

[0224] Specific Embodiments E1. A BTN3A activating antibody for use in treating cancer in a subject in need thereof, wherein said subject is administered a therapeutically effective amount of said BTN3A activating antibody in combination with a therapeutically effective amount of an immune checkpoint inhibitor, wherein said immune checkpoint inhibitor is selected from the group consisting of a LAG-3 inhibitor, a TIGIT inhibitor, and a TIM-3 inhibitor, and optionally said BTN3A activating antibody is further administered in combination with a PD-1 inhibitor.

[0225] E2. The BTN3A antibody binds to human BTN3A with a K of 10 nM or less as measured by surface plasmon resonance D , preferably a K of 5 nM or less D BTN3A activating antibody for use with E1 that binds at

[0226] E3. The BTN3A-activating antibody activates γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3A-expressing cells, as measured by a degranulation assay, with an EC of less than 5 μg / ml, preferably 1 μg / ml or less. 50BTN3A activating antibodies for use with either E1 or E2, induced by

[0227] E4. The BTN3A activating antibody: (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2 or SEQ ID NO:3; comprising HCDRs 1 to 3 of SEQ ID NOs: 12 to 14 and LCDRs 1 to 3 of SEQ ID NOs: 15 to 17; comprising HCDRs 1 to 3 of SEQ ID NOs: 18 to 20 and LCDRs 1 to 3 of SEQ ID NOs: 21 to 23; or competes for binding with an antibody selected from mAb 20.1 produced by the hybridoma deposited at the CNCM under deposit number I-4401, mAb 7.2 produced by the hybridoma deposited at the CNCM under deposit number I-4402, and / or an antibody having a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, A BTN3A activating antibody for use with any one of E1 to E3.

[0228] E5. A BTN3A activating antibody for use according to any one of E1 to E4, wherein the BTN3A activating antibody comprises HCDRs 1 to 3 of SEQ ID NOs: 12 to 14 and LCDRs 1 to 3 of SEQ ID NOs: 15 to 17.

[0229] E6. A BTN3A activating antibody for use with any one of E1-E5, wherein the BTN3A activating antibody comprises (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2.

[0230] E7. A BTN3A activating antibody for use with any one of E1-E6, wherein said BTN3A activating antibody is an antibody comprising or consisting essentially of a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0231] E8. A BTN3A activating antibody for use with any one of E1-E7, wherein the BTN3A activating antibody comprises a mutated IgG1 constant region or a chemically modified IgG1 constant region that abolishes or reduces binding to Fcγ receptors compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

[0232] E9. A BTN3A activating antibody for use with any one of E1-E8, wherein said mutated IgG1 constant region is the IgG1 triple mutant L247F, L248E and P350S. E10. The immune checkpoint inhibitor is a LAG-3 inhibitor, preferably (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38; comprising HCDRs 1 to 3 of SEQ ID NOs: 45 to 47 and LCDRs 1 to 3 of SEQ ID NOs: 48, 43 and 49; or Competes for binding with an anti-LAG-3 antibody selected from leratolimab and favezelimab a monospecific antibody, bispecific antibody, or fixed-dose combination comprising an anti-LAG-3 antibody; A BTN3A activating antibody for use with any one of E1 to E9.

[0233] E11. A BTN3A activating antibody for use with any one of E1 to E10, wherein the BTN3A activating antibody is administered in a unit dose ranging from about 1 to about 200 mg.

[0234] E12. A BTN3A activating antibody for use with any one of E1 to E11, wherein said immune checkpoint inhibitor is a LAG-3 inhibitor, for example an anti-LAG antibody administered in a unit dose ranging from 100 to 1000 mg.

[0235] E13. A BTN3A activating antibody for use with any one of E1-E11, wherein said immune checkpoint inhibitor is a recombinant LAG-3 protein, e.g., a LAG-3-Ig fusion protein.

[0236] E14. A BTN3A activating antibody for use with any one of E1-E13, wherein the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), lung cancer (e.g., non-small cell lung cancer), breast cancer, prostate cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), renal cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, glioblastoma, ovarian cancer, cervical cancer, bile duct cancer, bladder cancer, cancer of the kidney or ureter, PD-L1 positive cancer, and hematological malignancies, wherein the hematological malignancies include, for example, acute myeloid leukemia, Hodgkin's lymphoma, multiple myeloma, diffuse large B-cell lymphoma (DLBCL), and indolent non-Hodgkin's lymphoma (NHL).

[0237] E15. A BTN3A activating antibody for use according to any one of E1-E14, wherein the subject is refractory to or has relapsed from PD-1 inhibitor therapy, e.g., the subject is refractory to ipilimumab and / or nivolumab therapy.

[0238] E16. A BTN3A activating antibody for use with any one of E1 to E15, wherein the BTN3A activating antibody is administered once every three weeks or once every four weeks.

[0239] E17. A BTN3A activating antibody for use with any one of E1 to E16, wherein the BTN3A activating antibody is administered intravenously, for example, at a unit dose of about 1 to about 200 mg, e.g., 75 mg, for example, once every 21 days for 1 to 22 cycles.

[0240] E18. A BTN3A activating antibody for use with any one of E1 to E17, wherein the immune checkpoint inhibitor is an anti-LAG-3 / anti-PD-1 bispecific antibody administered intravenously at a unit dose of about 100 to about 1000 mg, e.g., 600 mg, e.g., once every 21 days for 1 to 35 cycles.

[0241] E19. A BTN3A activating antibody for use with any one of E1-E18, wherein the BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the immune checkpoint inhibitor is nivolumab / leratolimab.

[0242] E20. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a BTN3A activating antibody in combination with a therapeutically effective amount of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is selected from the group consisting of a LAG-3 inhibitor, a TIGIT inhibitor, and a TIM-3 inhibitor, and optionally, the method further comprises administering the BTN3A activating antibody in combination with a PD-1 inhibitor.

[0243] E21. The BTN3A antibody binds to human BTN3A with a K of 10 nM or less as measured by surface plasmon resonance. D , preferably a K of 5 nM or less D Combine using the E19 method.

[0244] E22. The BTN3A-activating antibody activates γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3A-expressing cells, as measured by a degranulation assay, with an EC of less than 5 μg / ml, preferably 1 μg / ml or less. 50 Induction by E20 or E21 method. E23. The BTN3A activating antibody (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2 or SEQ ID NO:3; comprising HCDRs 1 to 3 of SEQ ID NOs: 12 to 14 and LCDRs 1 to 3 of SEQ ID NOs: 15 to 17; comprising HCDRs 1 to 3 of SEQ ID NOs: 18 to 20 and LCDRs 1 to 3 of SEQ ID NOs: 21 to 23; or competes for binding with an antibody selected from mAb 20.1 produced by the hybridoma deposited at the CNCM under deposit number I-4401, mAb 7.2 produced by the hybridoma deposited at the CNCM under deposit number I-4402, and / or an antibody having a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, Methods for E20 to E22.

[0245] E24. The method of any one of E20 to E23, wherein the BTN3A activating antibody comprises HCDRs 1 to 3 of SEQ ID NOs: 12 to 14 and LCDRs 1 to 3 of SEQ ID NOs: 15 to 17.

[0246] E25. Any one of the methods of E20-E24, wherein the BTN3A activating antibody comprises (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2.

[0247] E26. The method of any one of E20 to E25, wherein said BTN3A activating antibody is an antibody comprising or consisting essentially of a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0248] E27. The method of any one of E20 to E26, wherein the BTN3A antibody comprises a mutated IgG1 constant region or a chemically modified IgG1 constant region, which abolishes or reduces binding to Fcγ receptors compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

[0249] E28. The method of any one of E20 to E27, wherein said mutated IgG1 constant region is of the IgG1 triple mutant L247F, L248E and P350S. E29. The immune checkpoint inhibitor is a LAG-3 inhibitor, for example: (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38; comprising HCDRs 1 to 3 of SEQ ID NOs: 45 to 47 and LCDRs 1 to 3 of SEQ ID NOs: 48, 43 and 49; or Competes for binding with an anti-LAG-3 antibody selected from leratolimab and favezelimab The method of any one of E20 to E28, wherein the anti-LAG-3 antibody is a monospecific antibody, a bispecific antibody, or a fixed-dose combination.

[0250] E30. The method of any one of E20 to E28, wherein said immune checkpoint inhibitor is a recombinant LAG-3 protein, for example, a LAG-3-Ig fusion protein.

[0251] E31. The method of any one of E20 to E30, wherein the BTN3A antibody is administered in a unit dose ranging from about 1 to about 200 mg.

[0252] E32. The method of any one of E20-E31, wherein said immune checkpoint inhibitor is a LAG-3 inhibitor, for example, an anti-LAG antibody administered at a dosage ranging from 100-1000 mg.

[0253] E33. The method of any one of E20-E31, wherein said immune checkpoint inhibitor is an anti-LAG-3 / anti-PD-1 bispecific antibody, e.g., tebotelimab.

[0254] E34. The method of any one of E20 to E28, wherein said immune checkpoint inhibitor is a TIM-3 inhibitor, for example, sabatolimab.

[0255] E35. The method of any one of E20 to E28, wherein the immune checkpoint inhibitor is an anti-TIM-3 / anti-PD-1 bispecific antibody, e.g., RO7121661.

[0256] E36. The method of any one of E20 to E28, wherein said immune checkpoint inhibitor is a TIGIT inhibitor, for example, tiragolumab.

[0257] E37. The method of any one of E20 to E28, wherein said immune checkpoint inhibitor is an anti-TIGIT / anti-PD-1 bispecific antibody, e.g., AZD2936.

[0258] E38. Any one of the methods of E20 to E37, wherein the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), lung cancer (e.g., non-small cell lung cancer), breast cancer, prostate cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), renal cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, glioblastoma, ovarian cancer, cervical cancer, bile duct cancer, bladder cancer, cancer of the kidney or ureter, PD-L1 positive cancer, and hematological malignancies, wherein the hematological malignancies include, for example, acute myeloid leukemia, Hodgkin's lymphoma, multiple myeloma, diffuse large B-cell lymphoma (DLBCL), and indolent non-Hodgkin's lymphoma (NHL).

[0259] E39. The method of any one of E20-E38, wherein said subject is refractory or has relapsed to PD-1 inhibitor therapy, e.g., a subject refractory to ipilimumab and / or nivolumab therapy.

[0260] E40. The method of any one of E20 to E39, wherein said BTN3A activating antibody is administered once every three weeks or once every four weeks.

[0261] E41. Any one of the methods of E20 to E40, wherein the BTN3A activating antibody is administered intravenously, e.g., at a unit dose of about 1 to about 200 mg, e.g., 75 mg, e.g., once every 21 days for 1 to 22 cycles.

[0262] E42. Any one of the methods of E20 to E28, wherein said immune checkpoint inhibitor is an anti-LAG-3 / anti-PD-1 bispecific antibody administered intravenously at a unit dose of about 100 to about 1000 mg, e.g., 600 mg, e.g., once every 21 days for 1 to 35 cycles.

[0263] E43. The method of any one of E20 to E28, wherein said BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and said immune checkpoint inhibitor is eftiragimode alfa.

[0264] E44. The method of any one of E20 to E28, wherein said BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and said immune checkpoint inhibitor is favezelimab.

[0265] E45. The method of any one of E20 to E28, wherein said BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and said immune checkpoint inhibitor is the fixed-dose combination nivolumab / leratolimab.

[0266] E46. The method of any one of E20 to E28, wherein the BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the immune checkpoint inhibitor is tebotelimab.

[0267] E47. The method of any one of E20 to E28, wherein said BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and said immune checkpoint inhibitor is sabatolimab.

[0268] E48. The method of any one of E20 to E28, wherein the BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the immune checkpoint inhibitor is RO7121661.

[0269] E49. The method of any one of E20 to E28, wherein said BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and said immune checkpoint inhibitor is tiragolumab.

[0270] E50. The method of any one of E20 to E28, wherein the BTN3A antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the immune checkpoint inhibitor is AZD2936.

[0271] The present disclosure having thus been fully described, will now be further described by the following examples, which are merely illustrative and are not meant to be further limiting.

[0272] [Table 7] JPEG2026506893000008.jpg180149 JPEG2026506893000009.jpg193149 JPEG2026506893000010.jpg193149 JPEG2026506893000011.jpg199149 JPEG2026506893000012.jpg193149 JPEG2026506893000013.jpg149149 JPEG2026506893000014.jpg175149 JPEG2026506893000015.jpg174149 JPEG2026506893000016.jpg187149 JPEG2026506893000017.jpg168149 JPEG2026506893000018.jpg180149 JPEG2026506893000019.jpg182149 TIFF2026506893000020.tif168149 JPEG2026506893000021.jpg175149 JPEG2026506893000022.jpg174149 JPEG2026506893000023.jpg188149 JPEG2026506893000024.jpg187149 JPEG2026506893000025.jpg175149 JPEG2026506893000026.jpg193149 JPEG2026506893000027.jpg187149 JPEG2026506893000028.jpg187149 JPEG2026506893000029.jpg187149 JPEG2026506893000030.jpg193149 JPEG2026506893000031.jpg194149 JPEG2026506893000032.jpg162149 JPEG2026506893000033.jpg162149 JPEG2026506893000034.jpg206149 JPEG2026506893000035.jpg168149 JPEG2026506893000036.jpg85149 [Example]

[0273] 1. Methods for Characterizing BTN3A Activating Antibodies for Use According to the Present Disclosure 1.1 Binding affinity assay: multi-cycle kinetic assay (SPR) Multi-cycle kinetic analysis can be performed with the BTN3A antibody using a Biacore T200 (serial number 1909913) instrument (Uppsala, Sweden) running Biacore T200 evaluation software V2.0.1.

[0274] Purified antibodies were diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, each antibody was captured onto Protein A at a density of approximately 146.5 RU (RL) (theoretical value for an RMax of approximately 50 RU). After capture, the surface was allowed to stabilize before injecting BTN3A1 antigen (Sino Biological cat. no. 15973-H08H). BTN3A1 was titrated over a 2-fold dilution range from 25 to 0.78 nM in 0.1% BSA / HBS-P+ (running buffer). The association phase was monitored for 400 s, and the dissociation phase was monitored for 35 min (2100 s). Kinetic data were obtained using a flow rate of 50 μl / min to minimize potential mass transfer effects. The Protein A surface was regenerated at the end of each cycle using two injections of 10 mM glycine-HCl pH 1.5. To confirm the stability of the surface and analyte throughout the kinetic cycle, two blank (no BTN3A1) and single-concentration analyte replicates are performed for each antibody tested. The signal from reference channel Fc1 is subtracted from the signals from Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. Additionally, blank runs are subtracted for each Fc to correct for antigen-independent signal fluctuations, e.g., drift. Sensorgrams are fitted using a one-to-one binding mathematical model with a global RMax parameter and no bulk signal (constant RI = 0 RU).

[0275] 1.2 Flow cytometric binding assay of human PBMCs BTN3A-activating antibodies for use according to the present disclosure can also be characterized for binding to human PBMCs isolated from the blood of healthy donors. PBMCs are isolated from buffy coats using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. PBMCs are then frozen and stored at -80°C or in liquid nitrogen until needed.

[0276] 1×10 6 100 μl of cells / ml were transferred to each well of a new U-bottom 96-well plate, the plate was then centrifuged, and the supernatant was discarded.

[0277] Serial dilutions of antibody from 0.001 μg / ml to 150 μg / ml are prepared in PBS 2 mM EDTA. Human PBMCs are resuspended in 50 μl of the prepared diluted test antibody dose titration series.

[0278] After 30 min of incubation at 4°C in the dark, the plates were centrifuged and washed twice with 150 μl / well of PBS 2 mM EDTA. The wells were then resuspended in 50 μl of a mix consisting of a 1 / 100 dilution of goat anti-human antibody (PE-labeled) and a 1 / 500 dilution of Live / Dead near-IR in PBS 2 mM EDTA.

[0279] After 15 minutes of incubation in the dark at 4°C, the plates are centrifuged and washed once with 150 μl / well of PBS 2mM EDTA, after which the cells are resuspended in 200 μl of PBS 2mM EDTA. Wells are analyzed on a BD LSR Fortessa Cytometer. Data are analyzed using FlowJo software (Version 10, FlowJo, LLC, Ashland, USA).

[0280] The same protocol can be performed for cynomolgus monkey PBMCs and Daudi-Burkitt lymphoma cell lines.

[0281] 1.3 In vitro functional efficacy: γδ-T cell degranulation assay The assay consisted of measuring the activating or inhibitory effect of the BTN3A antibody on γδ T cell degranulation in a Daudi-Burkitt lymphoma cell line (Harly et al., 2012). γδ T cells were expanded from PBMCs of healthy donors and cultured for 11–13 days with zoledronic acid (1 μM) and IL2 (200 IU / ml). IL2 was added on days 5 and 8, and every two days thereafter. The percentage of γδ T cells was determined at the beginning of culture and assessed throughout the culture period by flow cytometry until it reached at least 80%. Frozen or fresh γδ T cells were then used in a degranulation assay against the Daudi cell line (E:T ratio 1:1). In this assay, cells were co-cultured for 4 hours at 37°C in the presence of 10 μg / ml of 7.2 and / or 20.1 humanized variants and / or their chimeric versions. For γδ T cell degranulation, activation with PMA (20 ng / ml) plus ionomycin (1 μg / ml) served as a positive control, and medium alone served as a negative control. At the end of the 4-hour co-incubation, cells were analyzed by flow cytometry to assess the percentage of γδ T cells positive for CD107a (lysosomal-associated membrane protein-1 (LAMP-1)) and CD107b (LAMP-2). Because CD107 is mobilized to the cell surface following activation-induced granule exocytosis, measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytolytic T cells.

[0282] The same protocol can be performed using AML blasts isolated from patients as target cells instead of Daudi cells.

[0283] 1.4 In vitro functional efficacy: activation of Vγ9Vδ2 T cells in PBMCs The assay consisted of measuring the activating effect of BTN3A antibody on Vγ9Vδ2 T cells in PBMCs. Human PBMCs were isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA-buffy coat or heparinized whole blood). When using whole blood, RBCs were removed using 1× RBC lysis buffer (eBioscience) for 10 minutes at room temperature and then washed with PBS 1% FBS.

[0284] PBMCs or RBC-depleted cells were cultured in RPMI 1640 with 10% FBS, 1% P / S, at a concentration of 1.5–3 × 10 6 Cells were cultured at 200 μL in a round-bottom 96-well plate at 37°C and 5% CO2 with increasing concentrations of anti-BTN3A antibody (dose range: 0.00001–100 μg / mL). Activation status was monitored by flow cytometry analysis of activation marker surface expression after 2 days of culture. Cells were washed with PBS 2% FBS 2 mM EDTA (FACS buffer). Cells were centrifuged at 1800 rpm for 5 minutes and then incubated with 10 μL of FcR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature. Then, 30–50 μL of the appropriate antibody mix prepared in FACS buffer containing at least fluorescently conjugated anti-CD3, anti-Vg9 or Vd2 TCR, and anti-CD69 antibodies was added. A viability marker (LIVE / DEAD Fixable Dead Cell Stain) was added in all experiments to exclude dead cells from the analysis. Cells were incubated at 4°C for 30 minutes, washed twice with FACS buffer, and then fixed with Cytofix fixation buffer (BD Bioscience) for flow cytometry analysis. Data were analyzed using flowjo V-10.6 software. Activated Vy9V52 T cells were defined as CD3+Vd2+ (or Vg9+ or Vd2+Vg9+)CD69+.

[0285] 2. Experimental Section Materials and Methods 2.1 PBMC isolation and Vγ9Vδ2 T cell expansion Human ethylenediaminetetraacetic acid (EDTA)-buffy coats from healthy donors (HD) were obtained from the Etablissement Français du Sang (EFS) Provence Alpes Cote d'Azur (France). Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation of the EDTA-buffy coats.

[0286] To expand γδ T cells for killing assays, healthy donor human PBMCs (HD Hu-PBMCs) were resuspended in complete medium (RPMI 1640 medium supplemented with 10% FBS, 1% sodium pyruvate, and 1% P / S) in the presence of recombinant human interleukin-2 (rhIL-2 (Proleukin®, Novartis) (200 International Units (IU) / mL)) and aminobisphosphonate (zoledronate (Sigma-Aldrich #SML0223), 1 μM) for 12–13 days. Starting on day 5, rhIL-2 was replenished every 2 or 3 days by adding fresh complete culture medium supplemented with rhIL-2, and cells were cultured at a concentration of 1 × 10 6 The cells were maintained at a concentration of 1000 cells / mL. Expanded cells were harvested on days 12-13 for phenotypic and killing assessment.

[0287] 2.2 Flow cytometry assessment of surface expression of markers of interest on effector T cells and tumor cell lines For assessment of LAG-3, TIM-3, and TIGIT expression on unstimulated and ICT01-stimulated Vy9V52 T cells, freshly isolated Hu-PBMCs from healthy donors (HDs) were resuspended in complete medium and cultured for 5 days in the presence of 1 μg / mL ICT01 or its corresponding isotype control (hIgG1S), with or without 50 IU / ml rhIL-2 (Proleukin®). Before culture and at the indicated time points after incubation, cells were harvested for assessment of LAG-3, TIM-3, and TIGIT surface expression by flow cytometry performed on a Cytoflex LX instrument (Beckman Coulter). Briefly, cells were washed with PBS supplemented with 2% FBS and 2 mM EDTA (FACS buffer) and then incubated with FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature before staining with a mix of conjugated mAbs (anti-CD14 APC Vio770, anti-CD19 BV650, anti-CD3 Alexa fluor 700, anti-Vd2TCR FITC, anti-TIM-3 BV785, anti-TIGIT PE-cy7, and anti-LAG-3 APC) and a viability marker (LIVE / DEAD™ near-IR) for 20 min at 4 °C. Cells unstained for the markers of interest (TIM-3, LAG-3, and TIGIT) were used in parallel as controls. After incubation, stained cells were washed twice with FACS buffer and then collected.

[0288] Using the same staining procedure described above, in vitro expanded Vy9V52 T cells were analyzed for LAG-3, TIM-3, and TIGIT, as well as MHC class II surface expression by flow cytometry using conjugated mAbs (anti-CD3 Alexa fluor 700, anti-Vd2 TCR FITC, anti-LAG-3 APC, anti-TIGIT PE-cy7, anti-TIM-3 PE Dazzle594, and anti-MHC class II PE) and a viability marker (Live / Dead™ near-IR). To allow accurate gating, a second mix of mAbs containing isotype controls for each of anti-TIM-3, anti-LAG-3, anti-TIGIT, and anti-MHC class II was run in parallel.

[0289] Using the same staining procedure described above, we assessed the expression of BTN3A, the ligand for LAG-3 (MHC class II), the ligand for TIM-3 (galectin-9), and the ligand for TIGIT (PVR and nectin-2) on tumor target cell lines by flow cytometry using conjugated Abs (anti-MHC class II PE, anti-PVR PV421, anti-BTN3A AF546), purified mAbs (anti-nectin-2, anti-galectin-9), and a viability marker (Live / Dead™ near-IR). For staining with purified Abs, a second staining step with labeled secondary Abs was performed. To enable accurate gating, additional antibody cocktails containing isotype controls for each of the markers of interest were used in parallel.

[0290] Assessment of LAG-3, TIM-3, and TIGIT expression on circulating Vy9V52 T cells at baseline and after treatment with different doses of ICT01 + pembrolizumab in cancer patients enrolled in the EVICTION trial (listed in the table below) was performed by flow cytometry analysis using frozen PBMCs. After thawing, PBMCs were counted and 1 x 10 6Cells were mixed with an antibody cocktail containing anti-CD14 APC Vio770, anti-CD3 Alexa fluor 700, anti-Vd2TCR PE, anti-TIM-3 BB515, anti-TIGIT PE-Cy7, and anti-LAG-3 PE-Dazzle594, as well as a viability marker (Live / Dead™ near-IR). Additional antibody cocktails containing isotype controls for anti-TIM-3, anti-LAG-3, and anti-TIGIT were used in parallel.

[0291] [Table 8]

[0292] Assessment of PD-1, LAG-3, TIM-3 and TIGIT expression on tumor-infiltrating Vy9V52 T cells and CD8+ T cells from cancer patients (listed in the table below) was performed by flow cytometry using fresh and frozen dissociated tumor cells. Freshly dissociated tumor cells were isolated from fresh tumors by mechanical dissociation using a gentleMACS™ Octo Dissociator (Miltenyi Biotec). Briefly, fresh and frozen dissociated cells were washed with PBS supplemented with 2% FBS and 2 mM EDTA (FACS buffer) and then incubated with FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature, followed by staining with a mix of conjugated mAbs (anti-CD14 BUV805, anti-CD19 BUV496, anti-CD3 RedFluor710, anti-Vd2TCR PE, anti-CD8 BUV395, anti-CD4 BUV563, anti-PD-1 BV421, anti-LAG-3 PE-Dazzle594; anti-TIM-3 BB515, and anti-TIGIT PE-Cy7) and viability markers (Live / Dead™ near-IR) for 20 min at 4°C. To enable accurate gating, each sample was also stained with a second mix of mAbs containing anti-PD-1, anti-LAG-3, anti-TIM-3, and anti-TIGIT isotype controls. After incubation, stained cells were washed twice with FACS buffer and then collected on a Cytek® Aurora Spectral flow cytometer.

[0293] [Table 9]

[0294] 2.3 BrightPlex® IHC Staining of TIM-3, PD-1, LAG-3, CD8, and CD3 on FFPE Tissues from Cancer Patients Enrolled in the EVICTION Trial Quantification of intratumoral CD8+ T cells expressing PD-1, LAG-3, and TIM-3 was performed by immunohistochemistry (IHC) analysis using Veracyte BrightPlex® technology on FFPE biopsy samples (listed in the table below) from cancer patients enrolled in the EVICTION trial before and after (day 28) treatment with ICT01 as monotherapy or in combination with pembrolizumab. Briefly, sequential IHC staining was performed on a single 4 μm FFPE tissue slide using anti-PD-1 (Origene), anti-TIM-3 (Ozyme), anti-LAG-3 (Ozyme), anti-CD3 (Veracyte), and anti-CD8 (Veracyte). Sequential staining was performed on a Leica Bond RX using MACH 2 polymer detection (Biocare), AMEC chromogen (Vector Lab), followed by counterstaining with hematoxylin at the end of each staining. The stained sections are protected by manual coverslipping using Histodenz mounting medium. Slides are allowed to dry for a minimum of 15 minutes. Visual quality control is then performed to eliminate bubbles if necessary, and the slides are then cleaned to optimize the efficiency of the scanning step. Each slide is scanned using a Nanozoomer XR x20 / Nanozoomer S360 x20. The coverslip is removed from the slide using a warm water bath. The slides are then AMEC destained with ethanol, and the staining antibody is restriped with a denaturing solution and incubation in PT LINK. Digital pathology allows quantification of positive cells in the total tumor area, tumor cell area, and stromal area. Each sample is analyzed with Halo software using the Veracyte Digital Pathology Platform.

[0295] [Table 10]

[0296] 2.4 Activation and proliferation of Vγ9Vδ2 T cells in HD Hu-PBMCs by flow cytometry Fresh HD Hu-PBMCs from healthy donors were stained with Cell Trace Violet (CTV) reagent (Invitrogen) and cultured at 2.5 × 10 in RPMI 1640 medium supplemented with 10% FBS and 1% P / S. 6 Cells / mL were cultured in the presence or absence of 0.1 μg / mL ICT01 and 50 IU / mL rhIL-2 (Proleukin®) for 3 and 6 days at 37°C and 5% CO. 10 μg / mL of blocking anti-TIM-3 (F38-2E2 clone, Biolegend) or anti-TIGIT (MBSA43 clone, ThermoFisher Scientific) or anti-LAG-3 (17B4 clone, Adipogen) versus the corresponding control isotype (mIgG1, Miltenyi), or LAG-3 Fc (produced in CHO, Adipogen) versus the corresponding control (Ig Fc, produced in CHO, Adipogen) were added to ICT01-activated HD Hu-PBMCs at the initiation of culture. At the indicated time points, cells were harvested for assessment of Vy9V52 T cell frequency, activation, and proliferation using flow cytometry performed on a Cytoflex LX instrument (Beckman Coulter) and stained with conjugated Abs prepared in FACS buffer (anti-CD3 Alexa fluor 700, anti-Vg9 TCR FITC, anti-Vd2 TCR PE, anti-CD25 APC Live / Dead™ near IR). For γδ T cell expansion assessment, Count Bright Absolute Counting Beads (ThermoFisher Scientific) were added to the FACS buffer solution before collection, and relative cell numbers were calculated on day 6 according to the manufacturer's instructions.

[0297] To assess the potential synergistic interaction of LAG-3 and ICT01 on Vγ9Vδ2 T cell activation, fresh HD Hu-PBMCs were cultured at 2.5 × 10 in RPMI 1640 medium supplemented with 10% FBS, 1% P / S. 6Cells / mL were incubated with or without ICT01 used at 0.03 and 0.1 μg / mL, and rhIL-2 (Proleukin®) at 50 IU / mL for 2 days. LAG-3 Fc or Ig Fc (Adipogen) was added at the beginning of the culture at different concentrations ranging from 0.16 to 10 μg / mL. After 2 days of incubation, cell-free culture supernatants were collected and stored for further analysis, while cells were harvested for assessment of Vy9V52 T cell frequency and activation by flow cytometry performed on a Cytoflex LX instrument (Beckman Coulter) and stained with conjugated Abs prepared in FACS buffer (anti-CD3 BV786, anti-CD14 APC Vio770, anti-CD56 PE Vio770, anti-Vd2 TCR FITC, anti-CD8 Alexa Fluor 700, anti-CD11b BV650, anti-CD25 APC, anti-CD86 PE and Live / Dead™ aqua).

[0298] To assess the direct effect of LAG-3 Fc on the activation of Vγ9δ2 T cells, which highly express MHC class II, fresh HD Hu-PBMCs were cultured at 2.5 × 10 in RPMI 1640 medium supplemented with 10% FBS and 1% P / S. 6 The PBMCs were incubated at 0.1 μg / mL with or without ICT01 at 0.1 μg / mL for 2 days. After 2 days of incubation, total CD3+ T cells were selected by negative selection using the EasySep Human T cell enrichment Kit (STEMCELL technologies) from either untreated or ICT01-pretreated PBMCs. The selected T cells were then incubated at 1.10 μg / mL with conjugated Abs prepared in FACS buffer (anti-CD3 BV786, anti-CD11b BV650, anti-CD8 Alexa Fluor 700, anti-Vd2TCR FITC, anti-CD25 APC, anti-MHC II PE) for flow cytometry analysis on a Cytoflex LX instrument. 5CD25 and MHC class II expression on Vy952 T cells was first assessed by incubating 2.5 x 10 sorted T cells with 1000 ng / mL of IgG. 6 Cells / mL were incubated with LAG-3 Fc or Ig Fc control used at 1 μg / mL and 50 IU / mL rhIL-2 (Proleukin®) in RPMI 1640 medium supplemented with 10% FBS, 1% P / S for 1 day. After 24 h of incubation, cells were harvested for assessment of Vy9V52 T cell activation by flow cytometry performed on a Cytoflex LX instrument (Beckman Coulter) and stained with conjugated Abs prepared in FACS buffer (anti-CD3 Alexa fluor 700, anti-Vg9 TCR FITC, anti-Vd2 TCR PE, anti-CD25 APC, anti-CD69 PE Cy7, and Live / Dead™ near IR).

[0299] 2.5 Cytokine secretion evaluation by Elisa and Luminex Supernatants were collected from 3-day cultures of unstimulated and ICT01-stimulated HD Hu-PBMCs treated with blocking mAbs and analyzed for IFN-γ and Granzyme B levels by ThermoFisher Scientific using Luminex technology.

[0300] Supernatants were collected from 3-day cultures of unstimulated and ICT01-stimulated HD Hu-PBMCs treated with a single dose of LAG-3 Fc or Ig Fc protein and analyzed for IFN-γ and granzyme B using a human IFN-γ uncoated ELISA kit (ThermoFisher Scientific) and a human granzyme B Duoset ELISA (R&D Biotech), respectively, according to the manufacturer's instructions.

[0301] Quantification of IFN-γ and TNF-α in 2-day cultures of unstimulated and ICT01-stimulated HD Hu-PBMCs treated with different doses of LAG-3 Fc or Ig Fc proteins was performed by ThermoFisher Scientific using Luminex technology.

[0302] 2.6 Co-culture of expanded Vγ9Vδ2 T cells or HD hu-PBMCs with cancer cell lines (ovarian cancer-derived (SKOV-3) or leukemia cell line (THP1)) The SKOV-3 cell line (human ovarian adenocarcinoma) was purchased from the European Collection of Authenticated Cell Cultures (ECACC) and cultured in McCoy's Glutamax medium supplemented with 10% FBS and 1 nM sodium pyruvate at 37°C and 5% CO2. mKate-expressing SKOV3 cells were established by lentiviral transduction at a multiplicity of infection of 3 using IncuCyte® NucLight Red Lentivirus Reagent (Sartorius) in the presence of 8 μg / ml polybrene® (Sigma-Aldrich). Transduced cells were selected by adding 1 μg / ml puromycin (Invivogen) to the culture. Cells were tested for mycoplasma contamination by PCR using the MycoplasmaCheck platform (Eurofins).

[0303] The THP1 cell line (human acute monocytic leukemia) was purchased from ATCC and cultured in RPMI Glutamax 10% FBS 1 mM sodium pyruvate 2.5 g / L D-glucose 0.05 mM b-mercaptoethanol 10 mM HEPES at 37°C and 5% CO. Cells were tested for mycoplasma contamination by PCR using the MycoplasmaCheck platform (Eurofins).

[0304] For tumor cell degranulation assays, expanded Vy9V52 T cells were cultured with THP1 for 4 hours or with SKOV3 overnight at a 1:1 ratio in the presence of a mix of anti-CD107a and anti-CD107b PE mAbs, with or without ICT01, used at 1 or 0.1 μg / mL. LAG-3 Fc or the corresponding control (Ig Fc) was added at 1 μg / mL. After coculture, cells were washed and incubated in FcR blocking reagent for 10 minutes before staining with a mix of conjugated mAbs (anti-CD3 Alexa Fluor 700, anti-Vd2 TCR FITC) and a viability marker (Live / Dead™ near-IR) for 20 minutes at 4°C. Stained cells were washed extensively and then collected using a Cytoflex S instrument (Beckman Coulter).

[0305] For expanded Vy9V52 T cell-mediated killing of THP1 and SKOV3, in vitro expanded Vy9V52 T cells were pre-incubated with 10 μg / mL of a blocking mAb specific for TIM-3 (F38-2E2 clone, Biolegend), or TIGIT (MBSA43 clone, ThermoFisher Scientific), or LAG-3 (17B4 clone, Adipogen) or the corresponding control isotype (mIgG1; Miltenyi) for 30 min and then co-cultured at a 1:1 ratio with Cell trace violet (CTV)-targeted THP1 for 4 h or with CTV-labeled SKOV3 overnight. After incubation, cells were centrifuged and incubated in FcR blocking reagent for 10 minutes before staining with a mix of anti-CD3 Alexa Fluor 700, anti-Vd2 TCR FITC mAb, and a viability marker (Live / Dead™ Fixable) for 20 minutes at 4°C. After incubation, cells were washed twice with FACS buffer and Annexin V binding buffer and then stained with Annexin V-APC reagent prepared in the appropriate buffer for 20 minutes at room temperature. After incubation, cells were analyzed by flow cytometry within 4 hours of staining.

[0306] For HD Hu-PBMC-mediated killing of SKOV3, 2.5 x 10 3 mkate-expressing SKOV3 cells were seeded into 96-well plates and cultured overnight in the appropriate complete medium. On the day of the experiment, 12.5 x 10 3 Fresh HD hu-PBMCs were added and cultured for 120 hours in the presence of single agents (0.1 μg / mL ICT01, or 10 μg / mL anti-LAG-3 (Leratolimab, Selleckchem) or anti-TIM-3 (Sabatolimab, Selleckchem), versus the corresponding isotype (hIgG4)), or the combination of ICT01 + anti-LAG-3 or ICT01 + anti-TIM-3. Wells containing target cells alone served as controls for tumor cell growth and survival. The total number of tumor target cells was tracked by the fluorescent signal emitted by NucLight Red dye using an IncuCyte® Live Cell Imaging System. Tumor cell growth is presented over time as tumor cell counts normalized to baseline (tumor cell counts 3 hours after the start of co-culture).

[0307] 2.7 Statistics Two-way ANOVA and Holm-Sidak multiple comparison tests were used to determine the effects of two independent variables on continuous dependent variables. Two paired groups were compared using the Wilcoxon test. Statistics were calculated using GraphPad Prism 8.0 (GraphPad Software). Differences were considered significant when P values ​​were less than 0.05. * (p<0.05), ** (p<0.01) *** (p<0.001) and **** (p<0.0001)

[0308] 3. Experimental Results 3.1 ICT01 increases LAG-3, TIM-3, and TIGIT expression on circulating Vγ9δ2 T cells from cancer patients in vitro LAG-3, TIM-3 and TIGIT expression on Vy9V52 T cells was assessed at steady state and after ICT01 treatment by incubating hu-PBMCs from healthy donors (HD) with ICT01 or its corresponding isotype (hIgG1).

[0309] As shown in Figure 1A, LAG-3, TIM-3, and TIGIT were weakly detected on Vy9V52 T cells at steady state but were rapidly upregulated after ICT01 + rhIL-2 treatment compared to cells treated with hIgG1S + rhIL-2. Significant differences in LAG-3, TIM-3, and TIGIT expression on Vy9V52 T cells were observed between ICT01- and hIgG1S-treated samples over time, with LAG-3 and TIM-3 being particularly high on day 5 in the presence of ICT01. Similar results were observed in cell cultures without rhIL-2 (data not shown).

[0310] We then monitored LAG-3, TIM-3, and TIGIT expression on circulating Vγ9Vδ2 T cells in PBMCs of cancer patients from the EVICTION clinical trial (NCT04243499), a first-in-human, two-part, open-label clinical trial designed to evaluate the safety, tolerability, and activity of intravenous doses of ICT01 as monotherapy and in combination with pembrolizumab in patients with advanced-stage, relapsed / refractory cancer (samples detailed in the Materials and Methods section). Strong heterogeneity was observed in the expression of these molecules on circulating Vγ9Vδ2 T cells in cancer patients before treatment initiation. Importantly, the frequencies of TIM3+, LAG3+, and TIGIT+ Vγ9Vδ2 T cells increased in the majority of patients treated with ICT01 in combination with pembrolizumab, with a significant increase at day 7 post-dose (Figure 1B).

[0311] 3.2 LAG-3, TIM-3, and TIGIT are expressed on tumor-infiltrating Vγ9Vδ2 T cells Evaluation of immune checkpoint receptor expression, including LAG-3, TIM-3, TIGIT, and PD-1, was performed on tumor-infiltrating T cells obtained from dissected tumors of bladder cancer patients, head and neck cancer, and melanoma. Expression of these receptors on infiltrating Vγ9Vδ2 T cells varied widely across samples, with the overall frequency of PD1+Vγ9Vδ2 T cells ranging from 0 to 92% (median 29.2% SEM 3.6), TIM3+Vγ9Vδ2 T cells ranging from 0 to 68% (median 11.7% SEM 3.4), and TIGIT+Vγ9Vδ2 T cells ranging from 1.1 to 67.3% (median 18.9% SEM 3.4). LAG-3 was observed less frequently, with the overall frequency of LAG-3+Vγ9Vδ2 T cells ranging from 0 to 13.5% (median 2.4% SEM 0.76) (Figures 2A and B). In addition to Vγ9Vδ2 T cells, elevated levels of these immune checkpoint receptors were observed on infiltrating CD8+ T cells, particularly TIM-3 and PD-1 (Figures 2A and B).

[0312] 3.3 Intratumoral PD-1+TIM-3+LAG-3+CD8+ T cells are increased in patients treated with ICT01 The inventors investigated the effect of ICT01 treatment on the expression of PD-1, TIM-3, and LAG-3 on intratumoral CD8+ T cells in cancer patients enrolled in the EVICTION clinical trial (NCT04243499). Tumor biopsies were taken at baseline and on day 28 (7 days after the second dose of ICT01) and stained for PD-1, TIM-3, and LAG-3 using CD3- and CD8-specific antibodies together using a multiplex IHC method. Figure 3 depicts a significant increase (p=0.013) in CD8 T cells expressing the immune checkpoint receptors PD-1, LAG-3, and TIM-3 within the entire tumor after ICT01 treatment. Specific analysis of stromal and tumor areas within biopsy samples confirmed an increased density of PD1+LAG3+TIM3+CD8 T cells in tumors harvested after ICT01 treatment in both patients receiving ICT01 monotherapy (Group A) and patients receiving ICT01 + pembrolizumab (Group C) (Figure 3).

[0313] These findings highlight the rationale for combining ICT01 with other immune checkpoint therapies beyond anti-PD-1 to enhance effector immune responses.

[0314] 3.4 LAG-3 modulates ICT01-mediated Vγ9Vδ2 T cell activation and expansion in HD Hu-PBMCs The involvement of LAG-3, TIM-3, and TIGIT in the activation and expansion of Vy9V52 T cells suboptimally activated with ICT01 was assessed by incubating HD hu-PBMCs with low ICT01 doses in the presence of specific blocking mAbs against LAG-3, TIM-3, or TIGIT or their corresponding isotypes (mIgG1). As expected, ICT01 induced Vy9V52 T cell activation and expansion, as indicated by CD25 mean fluorescence intensity (MFI) on day 3 and absolute Vy9V52 T cell counts on day 6, respectively. Interestingly, exclusively LAG-3 blockade improved ICT01-mediated Vy9V52 T cell activation and expansion in vitro compared to isotype control, whereas TIM-3 and TIGIT blockade had less of an effect (Figure 4A). Additionally, LAG-3 blockade also enhanced the secretion of IFN-γ and granzyme B into the supernatants of ICT01-treated HD Hu-PBMCs (Figure 4B), suggesting a critical role for LAG-3 in regulating the ICT01-mediated ability of Vγ9Vδ2 T cells to produce IFN-γ and cytolytic granules.

[0315] 3.5 LAG-3, TIM-3, and TIGIT modulate ICT01-mediated killing of an ovarian cancer cell line (SKOV3) by Vγ9Vδ2 T cells Next, we tested the ability of anti-TIM-3, anti-LAG-3, and anti-TIGIT blocking mAbs to modulate tumor killing by ICT01-activated Vγ9V52 T cells. To this end, in vitro-expanded γδ T cells expressing TIM-3, LAG-3, and TIGIT on their plasma membrane (shown in Figure 5A) were cocultured with the SKOV3 cell line. ICT01 treatment induced the tumor-killing capacity of expanded Vγ9V52 T cells, as indicated by a decrease in the percentage of viable tumor cells (gated on live / dead and annexin V negative staining) at the end of the coculture, as determined by flow cytometry (Figure 5B). This killing capacity of ICT01-activated Vγ9V52 T cells was significantly enhanced by anti-TIM-3, anti-LAG-3, and anti-TIGIT blocking mAbs, demonstrating the role of these immune checkpoint receptors in negatively modulating Vγ9V52 T cell function (Figure 5B).

[0316] 3.6 LAG-3, TIM-3, and TIGIT modulate ICT01-mediated killing of leukemia cell lines by Vγ9Vδ2 T cells Given the effect of blocking LAG-3, TIM-3 or TIGIT on ICT01-mediated killing of SKOV3 cells by Vy9V52 T cells, we investigated the influence of anti-TIM-3, anti-LAG-3 and anti-TIGIT blocking mAbs on the cytotoxicity of ICT01-activated Vy9V52 T cells against the leukemia cell line THP-1. In vitro expanded Vy9V52 T cells expressing TIM-3, LAG-3 and TIGIT on the plasma membrane (Figure 6A, upper panel) were co-cultured with the leukemia cell line THP1, which expresses BTN3A, the ligand for LAG-3, the ligand for TIM-3 and the ligand for TIGIT (Figure 6A, lower panel).

[0317] The use of ICT01 during co-culture enhanced the tumor-killing capacity of expanded Vy9V52 T cells, as evidenced by a decrease in the percentage of viable tumor cells at the end of the co-culture (gating on live / dead and live cells negative for Annexin V staining), as determined by flow cytometry. Furthermore, the tumor-killing capacity of ICT01-activated Vy9V52 T cells was significantly enhanced by anti-TIM-3, anti-LAG-3, and anti-TIGIT blocking mAbs (Figure 6B). These results corroborate those obtained using solid tumor cell lines and clearly demonstrate the involvement of these immune checkpoint receptors in negatively modulating Vy9V52 T cell function.

[0318] 3.7 Combination of ICT01 with LAG-3 or TIM-3 blockade improves tumor cell killing by HD hu-PBMCs compared to single agents We then examined the effect of combining ICT01 with immune checkpoint blockade on HD Hu-PBMC-mediated cytolysis of tumor target cells by co-culturing the ovarian cancer cell line SKOV3 with HD Hu-PBMCs in the presence of therapeutic mAbs used as single agents or in combination. Cancer cell growth over time was monitored by live imaging using an IncuCyte device.

[0319] Treatment with ICT01 alone induced significant tumor cell killing by HD Hu-PBMCs compared to untreated conditions, as evidenced by a marked decrease in target cell counts over time (Figure 7). In addition, the humanized anti-LAG-3 blocking antibody (relatolimab) and the anti-TIM-3 blocking antibody (sabatolimab) demonstrated reduced target cell counts compared to their respective isotypes (hIgG4) (Figures 7A and 7B, respectively). Importantly, the combination of ICT01 with anti-LAG-3 (Figure 7A) or anti-TIM-3 (Figure 7B) both demonstrated significantly superior effects on HD Hu-PBMC-mediated killing of tumor cells compared to the single agents.

[0320] 3.8 Recombinant LAG-3 Fc fusion protein enhances ICT01-mediated activation / expansion of Vγ9Vδ2 T cells in HD Hu-PBMCs In addition to the use of anti-LAG-3 blocking mAbs, we evaluated the effect of a soluble LAG-3 immunoglobulin fusion protein (LAG-3 Fc), an MHC class II agonist currently undergoing clinical trials, on modulating Vy9V52 T cell responses.

[0321] First, we investigated the ability of LAG-3 Fc to affect ICT01-mediated activation and expansion of Vy9V52 T cells. HD Hu-PBMCs were incubated with a suboptimal concentration of ICT01 (0.1 μg / mL) for 3 or 6 days, with or without LAG-3 Fc or a corresponding control (Ig Fc). ICT01 stimulation induced Vy9V52 T cell activation and expansion, as evidenced by increased CD25 expression and absolute cell counts, respectively (Figure 8A). Importantly, LAG-3 Fc significantly enhanced these effects in the majority of donors tested, as evidenced by enhanced CD25 expression and elevated absolute cell counts (Figure 8A). Additionally, IFN-γ and granzyme B secretion, measured by ELISA in 3-day culture supernatants, was increased by treatment with LAG-3 Fc compared to the corresponding isotypes (data not shown).

[0322] Next, we investigated the possible synergistic effect of the combination of ICT01 and LAG-3 Fc on Vy9V52 T cell activation. HD Hu-PBMCs were incubated for 2 days with increasing concentrations of LAG-3 Fc or matched controls, with or without ICT01 used at suboptimal concentrations (0.03 and 0.1 μg / mL). LAG-3 Fc alone had a modest effect on resting Vy9V52 T cells, inducing a 1.3-fold increase in surface CD25 expression after 2 days of stimulation at 10 μg / mL. ICT01 used at suboptimal concentrations of 0.03 and 0.1 μg / mL induced a 1.9- and 3.9-fold increase in surface CD25 expression on Vy9V52 T cells, respectively. Importantly, when ICT01, used at the same suboptimal concentration, was combined with 10 μg / mL LAG-3 Fc, we observed a 9.3-fold and 31.5-fold increase in CD25 expression on Vy9V52 T cells, demonstrating the synergistic activity of these two compounds (Figure 8B). Additionally, IFN-γ and TNF-α secretion into the culture supernatant was increased by treatment with LAG-3 Fc compared to the corresponding control and was further enhanced in the presence of ICT01 (Figure 8C). Furthermore, consistent with its established biological activity described in the literature, LAG-3 Fc induced dose-dependent activation of NK cells and CD8+ T cells in HD Hu-PBMCs. These effects were unaltered by ICT01 treatment, consistent with its specific activity against Vy9V52 T cells (Figure 8D).

[0323] Taken together, our experimental results demonstrate that LAG-3 Fc can induce the activation of effector immune cells and synergize with ICT01 to trigger the activation of Vγ9Vδ2 T cells in HD Hu-PBMCs.

[0324] 3.9. LAG-3 Fc enhances ICT01-mediated cytotoxic activity of Vγ9Vδ2 T cells Given the synergistic enhancement of Vy9V52 T cell activation by the combination of ICT01 and LAG-3 Fc, we examined the effect of this combination on tumor cell cytolysis. The ovarian cancer cell line SKOV3 and the leukemia cell line THP1 were co-cultured with in vitro-expanded Vy9V52 T cells.

[0325] First, we characterized the expression of MHC class II, the primary target of LAG-3 Fc, on both tumor cells and expanded Vy9V52 T cells. Figure 9A illustrates that MHC class II was prominently expressed on the plasma membrane of the leukemia cell line THP1, but not on the ovarian cancer cell line SKOV3. High levels of MHC class II were also consistently detected on in vitro expanded Vy9V52 T cells from all tested donors (Figure 9A).

[0326] We then investigated the ability of the ICT01 + LAG3 Fc combination to modulate the cytotoxicity of in vitro expanded Vy9V52 T cells against THP1 (Figure 9B) and SKOV3 (Figure 9C) by monitoring Vy9V52 T cell degranulation using flow cytometry. ICT01 and LAG-3 Fc, when used individually, induced Vy9V52 T cell degranulation, as evidenced by increased CD107 expression on Vy9V52 T cells (Figure 9B and Figure 9C). This effect was significantly enhanced when both agents were used in combination, both when using THP1 (Figure 9B) and SKOV3 (Figure 9C) targets.

[0327] Taken together, our results demonstrate the remarkable synergy achieved by combining ICT01 with LAG-3 Fc, inducing potent activation of Vy9V52 T cells and robust cytolytic activity against cancer cells.

[0328] 3.10. LAG-3 Fc can act directly on Vγ9Vδ2 T cells Given the significant MHC class II expression on expanded Vy9V52 T cells and the ability of LAG-3 Fc to induce Vy9V52 T cell degranulation even in the absence of MHC class II on target cells such as SKOV3, we hypothesized that LAG-3 Fc may have direct activity on Vy952 T cells.

[0329] To address this issue, we performed a series of experiments, beginning with the evaluation of the activity of LAG-3 Fc on Vy952 T cells, which highly express MHC class II. Because MHC class II is strongly induced by ICT01 treatment, HD Hu-PBMCs were pretreated with ICT01 for 2 days to promote Vy952 T cell activation, as indicated by increased expression of the CD25 marker and high MHC class II expression on the surface of Vy952 T cells (Figure 10A). Total CD3+ T cells (including the Vy952 T cell subpopulation) were then sorted from either untreated or ICT01-pretreated PBMCs to eliminate all antigen-presenting, MHC class II-positive cells. Sorted T cells were then incubated with LAG-3 Fc or an Ig Fc control for 1 day. Figure 10B shows that LAG-3 Fc significantly induced activation of ICT01-preactivated Vy9δ2 T cells that highly express MHC class II, while it had no effect on non-preactivated cells that lowly express MHC class II, thus suggesting a direct effect of LAG-3 Fc on Vy9δ2 T cell activation (Figure 10B).

[0330] We confirmed these results using in vitro expanded Vy952 T cells, known to highly express surface MHC class II (Figure 10C, left panel). In vitro expanded Vy952 T cells were cultured in the presence of LAG-3 Fc or irrelevant Ig Fc in the absence of any additional cells and degranulation was assessed by flow cytometry. In these conditions, LAG-3 Fc induces Vy952 T cell degranulation, as depicted by an increase in CD107 compared to Ig Fc conditions.

[0331] Taken together, these findings suggest a direct effect of LAG-3 Fc on MHC class II-positive Vγ9δ2 T cells.

Claims

1. 1. A BTN3A activating antibody for use in treating cancer in a subject in need thereof, wherein a therapeutically effective amount of the BTN3A activating antibody is administered to the subject in combination with a therapeutically effective amount of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is selected from the group consisting of a LAG-3 inhibitor, a TIGIT inhibitor, and a TIM-3 inhibitor, and optionally the BTN3A activating antibody is further administered in combination with a PD-1 inhibitor.

2. The BTN3A-activating antibody activates γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3A-expressing cells, with an EC of less than 5 μg / ml, preferably 1 μg / ml or less, as measured by a degranulation assay. ５０ 2. The BTN3A activating antibody for use according to claim 1, which is induced by

3. The BTN3A activating antibody (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:2 or SEQ ID NO:3; comprising HCDR1-3 of SEQ ID NOs: 12-14 and LCDR1-3 of SEQ ID NOs: 15-17; comprising HCDR1-3 of SEQ ID NOs: 18-20 and LCDR1-3 of SEQ ID NOs: 21-23; or competes for binding with an antibody having a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6; A BTN3A activating antibody for use according to claim 1 or 2.

4. 4. A BTN3A activating antibody for use according to any one of claims 1 to 3, wherein the BTN3A activating antibody comprises HCDR1-3 of SEQ ID NOs: 12-14 and LCDR1-3 of SEQ ID NOs: 15-17.

5. 5. The BTN3A activating antibody for use according to any one of claims 1 to 4, wherein the BTN3A activating antibody is an antibody comprising or consisting essentially of a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:

6.

6. 6. A BTN3A activating antibody for use according to any one of claims 1 to 5, wherein said BTN3A activating antibody comprises an IgG1 constant region with the triple mutations L247F, L248E and P350S.

7. The immune checkpoint inhibitor is a LAG-3 inhibitor, preferably (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 38; comprising HCDR1-3 of SEQ ID NOs: 45-47 and LCDR1-3 of SEQ ID NOs: 48, 43 and 49; or Competes for binding with an anti-LAG-3 antibody selected from leratolimab and favezelimab The BTN3A activating antibody for use according to any one of claims 1 to 6, which is a monospecific antibody, a bispecific antibody or a fixed dose combination comprising an anti-LAG-3 antibody.

8. 8. The BTN3A activating antibody for use according to any one of claims 1 to 7, wherein the immune checkpoint inhibitor is an anti-LAG antibody administered at a dosage ranging from 100 to 1000 mg.

9. 9. The BTN3A activating antibody for use according to any one of claims 1 to 8, wherein the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), lung cancer (e.g., non-small cell lung cancer), breast cancer, prostate cancer, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), renal cancer, colorectal cancer, gastric cancer, esophageal cancer, liver cancer, pancreatic cancer, glioblastoma, ovarian cancer, cervical cancer, bile duct cancer, bladder cancer, cancer of the kidney or ureter, PD-L1 positive cancer and hematological malignancies, wherein the hematological malignancies include, for example, acute myeloid leukemia, Hodgkin's lymphoma, multiple myeloma, diffuse large B-cell lymphoma (DLBCL) and indolent non-Hodgkin's lymphoma (NHL).

10. 10. The BTN3A activating antibody for use according to any one of claims 1 to 9, wherein the subject is refractory or has relapsed to PD-1 inhibitor therapy, for example a subject refractory to ipilimumab and / or nivolumab therapy.

11. 11. The BTN3A activating antibody for use according to any one of claims 1 to 10, wherein the BTN3A activating antibody is administered once every three weeks or once every four weeks.

12. 12. The BTN3A activating antibody for use according to any one of claims 1 to 11, wherein the BTN3A activating antibody is administered intravenously, for example in a unit dose of about 1 to about 200 mg, for example 75 mg.

13. 13. The BTN3A activating antibody for use according to any one of claims 1 to 12, wherein the BTN3A activating antibody is administered intravenously at a unit dose of about 1 to about 200 mg, for example 75 mg, for 1 to 22 cycles.

14. 14. The BTN3A activating antibody for use according to any one of claims 1 to 13, wherein the immune checkpoint inhibitor is an anti-LAG-3 / anti-PD-1 bispecific antibody administered intravenously at a unit dose of about 100 to about 1000 mg, e.g., 600 mg, e.g., once every 21 days for 1 to 35 cycles.

15. 15. A BTN3A activating antibody for use according to any one of claims 1 to 14, wherein the BTN3A activating antibody comprises a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the immune checkpoint inhibitor is nivolumab / relatolimab.

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