TW202330026A - Combination of an anti-btn3a activating antibody and an il-2 agonist for use in therapy - Google Patents

Abstract

The present application relates to a therapeutic combination of an anti-BTN3A activating antibody and of an IL-2 agonist that is particularly useful for the treatment of cancer. The present disclosure more particularly related to the combined use of a BTN3A activating antibody that specifically binds to BTN3A and activates the cytolytic function of Vγ9Vδ2 T cells and of an IL-2 agonist which binds to the IL-2 receptor βγ _cheterodimer but is α-independent. The present application further relates to a method of treatment of cancer comprising the administration of an anti-BTN3A activating antibody in combination with an IL-2 agonist which binds to the IL-2 receptor βγ _cheterodimer but is α-independent.

Description

Combination of anti-BTN3A activating antibodies and IL2 agonists for therapy

The following discloses therapeutic combinations of anti-BTN3A activating antibodies and IL-2 agonists, which are particularly useful in the treatment of cancer. More specifically, the present invention relates to the combined use of a BTN3A activating antibody and a non-naturally occurring IL-2 agonist. The BTN3A activating antibody specifically binds to BTN3A and activates the lytic function of Vγ9Vδ2 T cells. The IL-2 agonist Binds to the IL-2 receptor βƔ _c heterodimer but is α-independent.

White blood cells are cells of the immune system that participate in defending the body against pathogens. Among these cells, lymphocytes, monocytes and dendritic cells can be cited. Monocytes can migrate from the bloodstream to other tissues and differentiate into tissue-resident macrophages or dendritic cells. Dendritic cells function as antigen presenting cells (APCs) of activated lymphocytes. Among lymphocytes, T cells can be divided into αβ T cells and γδ T cells. Vγ9Vδ2 (the major subset of γδ T cells in peripheral blood) is an important effector of the immune defense system. It directly lyses pathogen-infected or abnormal cells. In addition, it regulates immune responses by inducing dendritic cell (DC) maturation and isotype switching and immunoglobulin production. This important subset of cells of the immune system is tightly regulated by surface receptors, chemokines, and cytokines.

Recent discoveries in the field of immunology have pointed to the emerging role of butyrophilin/butyrophilin-like molecules (BTN/BTNL in humans, Btn/Btnl in mice) in the regulation of γδ T cells. Specifically, the BTN3A protein, which belongs to the B7 costimulatory family of molecules, has been identified as a key mediator of phosphoantigen sensing by human Vγ9Vδ2 T cells. BTN3A, and especially BTN3A1, can trigger Vγ9Vδ2 T cell activation in the presence of phosphoantigen (Harly C, et al. Blood 2012 Vol. 120, Issue 11, pp. 2269-79; Blazquez, J. L., et al. Front Immunol 2018, pp. 9 Vol. 1601).

IL-2 was originally identified as a key component for maintaining T cell homeostasis and proper immune regulation (Nelson B et al. J Immunol 2004 Vol. 172 No. 7 pp. 3983-8). IL-2 (Proleukin) has a long history as a cancer therapeutic, being able to induce complete and durable responses in a small subset of patients with metastatic renal cell carcinoma and metastatic melanoma (Amin A et al. Oncology 2013 Vol. 27 Issue 7 pp. 680-91). However, high-dose IL-2 therapy is associated with severe toxic side effects, including hypotension, vascular leak syndrome (VLS), liver dysfunction, and neurological disorders (Schwartz R et al. Oncology 2002 Volume 16 Issue 11 Supplement 13 pp. 11-20). Therefore, high-dose IL-2 therapy is limited to carefully selected patients with good cardiopulmonary function and is performed only in a few centers with experience in immunotherapy.

Interleukin-2 receptor (IL-2R) is a heterotrimeric protein expressed on the surface of certain immune cells (such as lymphocytes) that binds and responds to IL-2 . IL-2R is made up of different combinations of three different proteins, often called "chains": alpha (alpha) (also known as IL-2Rα, CD25), beta (beta) (also known as IL-2Rβ, or CD122), and Ɣ _c (gamma) (also known as IL-2RƔ _c , CD132). The three receptor chains are expressed separately and in different ways on different cell types and can be assembled in different combinations and orders to generate medium- and high-affinity IL-2 receptors. The combination of β and Ɣ chains forms a complex that binds IL-2 with moderate affinity and is known to manifest primarily on memory αβ T cells and NK cells, whereas the combination of all three receptor chains (α, β, and γ) forms Complexes that bind IL-2 with high affinity (Kd about 10-11 M) are generally expressed on activated T cells and regulatory T cells (Treg).

Early studies indicated that T cells are key cellular mediators of IL-2-mediated toxicity (Rosenstein M et al. J Immunol 1986 Vol. 137 No. 5 pp. 1735-42). Subsequently, mouse studies implicated NK cells (Peace D et al. J Exp Med 1989 Volume 169 Issue 1 Pages 161-73) and Treg homeostasis and dysfunction (Li Y et al. Nat Commun 2017 Volume 8 Issue 1 issue page 1762). Lung endothelial cells also display functional IL-2 receptors, suggesting their role in the initiation of VLS (Krieg C et al. Proc Natl Acad Sci U S A 2010 Vol. 107, Issue 26, pp. 11906-11). Taken together, these studies indicate that the cause of VLS is complex, in which both hematopoietic and non-hematopoietic cell targets may be involved. In addition, as mentioned above, IL2 induces the preferential proliferation of immunosuppressive Tregs by binding to IL-2R-α, which is preferentially expressed on this T cell subset at steady state (Ahmadzadeh M et al. Blood 2006 Volume 107 Issue 6 Pages 2409-14). Depletion of Treg cells has been shown to enhance IL-2-induced anti-tumor immunity, suggesting that Tregs may be a major obstacle to IL-2-mediated CTL expansion (Imai H et al. Cancer Sci 2007 Vol. 98 Issue 3 No. 416 -page 23).

In summary, IL-2's short in vivo half-life, severe toxicity, and tendency to expand Treg cells have limited its widespread use in cancer treatment.

Previous studies have shown that IL-2 (Proleukin®) promotes Vγ9Vδ2 T cell expansion following stimulation with anti-BTN3A antibodies, including humanized anti-BTN3A therapeutic antibodies as described in WO2020025703. This may be clinically useful, provided that Vγ9Vδ2 T cells typically constitute less than 5% of total T cells in the peripheral blood of healthy adult humans and in cancer patients. In contrast to αβ T cells, activated Vγ9Vδ2 T cells do not produce IL-2 themselves and therefore require an external source to promote survival and expansion. Furthermore, agonist anti-BNT3A antibodies triggered increased surface expression of the α chain of IL-2R (CD25) on Vγ9Vδ2 T cells. Therefore, it was hypothesized that combination with IL-2 would promote Vγ9Vδ2 T cell expansion via high-affinity IL-2 binding on IL-2Rα.

Surprisingly, the inventors have now demonstrated the use of an IL-2Rα-independent agonist (more specifically IL-2 with eliminated affinity or no binding site for IL-2Rα) in combination with an anti-BTN3A agonist antibody. Agonist) not only promotes synergistic and specific Vγ9Vδ2 T cell expansion in human PBMC but is also significantly more robust than clinical IL-2 (Proleukin®). Furthermore, although Treg expansion is maintained with higher doses of clinical IL-2, the combination of the anti-BTN3A agonist antibodies of the invention with IL-2Rα-independent agonist treatment as disclosed herein disrupts the expansion of immunosuppressive Tregs. .

The present invention thus relates to a therapeutic combination of an anti-BTN3A activating antibody and an IL-2 receptor agonist that binds to the IL-2 receptor βƔ _c heterodimer for the treatment of cancer in an individual in need thereof; wherein the IL-2 2. An agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) X1 is a peptide comprising the amino acid sequence described in SEQ ID NO: 30; (b) A peptide of an amino acid; (c) X3 is a peptide comprising the amino acid sequence described in SEQ ID NO: 31; (d) X4 is a peptide comprising the amino acid sequence described in SEQ ID NO: 32 ; wherein X1, X2, X3, and Consistent amino acid sequence.

or, IL-2 agonists are polypeptides comprising domains X1, X2, X3, and X4, wherein: (a) X1 is a peptide comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the sequence KIQLHAEHALYDALMILNI (SEQ ID NO: 33); (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 34); (d) X4 is a peptide comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the sequence EDEQEEMANAIITILQSWIFS (SEQ ID NO: 35); Among them, X1, X2, X3 and X4 can be in any order in the polypeptide; The amino acid linker can exist between any one of the domains.

or, IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the amino acid sequence described in any one of SEQ ID NO: 37-49 , an amino acid sequence that is at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

In some embodiments, the anti-BTN3A antibody binds to human BTN3A with a _KD of 10 nM or less, preferably with a _KD of 5 nM or less, as measured by surface plasmon resonance.

Typically, anti-BTN3A antibodies induce activation of γδ T cells (typically Vγ9Vδ2 T cells) in co-culture with BTN3-expressing cells, preferably with an _EC50 below 5 µg/ml as measured in a degranulation assay. 1 µg/ml or less.

In some embodiments, the anti-BTN3A antibody: - Comprises (a) a variable heavy chain (VH) polypeptide, which contains an amine group that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1 acid sequence, and (b) a variable light chain (VL) polypeptide comprising at least about 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 2 or SEQ ID NO: 3 The amino acid sequence; - Contains HCDR 1-3 of SEQ ID NO: 12-14 and LCDR 1-3 of SEQ ID NO: 15-17; - Contains HCDR 1-3 of SEQ ID NO: 18-20 and LCDR 1-3 of SEQ ID NO: 21-23, or - Compete for binding with an antibody selected from the following: mAb 20.1 produced by the fusion tumor deposited with CNCM under deposit number I-4401, mAb 7.2 produced by the fusion tumor deposited at CNCM under deposit number I-4402, and Antibodies having the heavy chain of SEQ ID NO: 4 and the light chain of SEQ ID NO: 6.

In some embodiments, an anti-BTN3A antibody comprises a mutated or chemically modified IgG1 constant region, wherein the mutated or chemically modified IgG1 constant region is such that it does not interact with Fcγ when compared to a corresponding antibody with a wild-type IgG1 isotype constant region. The body binds or reduces binding to Fcγ receptors. In some embodiments, the mutant IgG1 constant region is an IgG1 triple mutation L247F, L248E, and P350S.

In some embodiments, anti-BTN3A is a mAb comprising the heavy chain of SEQ ID NO: 4 and the light chain of SEQ ID NO: 6.

In some embodiments, the IL-2 agonist is a polypeptide comprising domains X1, X2, X3, and X4, wherein: (a) X1 is a peptide comprising the amino acid sequence EHALYDAL (SEQ ID NO: 30); (b) ) : 32) peptide; wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can exist between any one of the domains; and wherein the polypeptide and IL-2 receptor βƔ _cHeterodimer (IL-2RβƔ _c ) binding.

In some embodiments, the IL-2 agonist is a polypeptide comprising domains X1, X2, X3, and X4, wherein: (a) % or 100% identical amino acid sequence; (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide containing at least 90%, A peptide with an amino acid sequence that is at least 95% or 100% identical; and (d) ; and wherein X1 _, X2, X3, and body (IL-2RβƔ _c ) binding.

In some embodiments, the IL-2 agonist is a polypeptide comprising domains X1, ) 35 (EDEQEEMANAIITILQSWIFS) peptide; wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can exist between any one of the domains; and wherein the polypeptide and the IL-2 receptor βƔ _c heterodimer (IL-2R βƔ _c ) binding.

In some embodiments, the X2 domain of the IL-2 agonist is a peptide comprising an amino acid sequence that is at least 90%, at least 94%, or 100% identical to SEQ ID NO: 36 (KDEAEKAKRMKEWMKRIKT).

In some embodiments, the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94% an amino acid sequence selected from any one of SEQ ID NO: 38-49. %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical polypeptides.

In some embodiments, the IL-2 agonist polypeptide is linked to a moiety containing polyethylene glycol (“PEG (polyethylene glycol)”), optionally wherein the PEG-containing moiety is at a cysteine residue in the polypeptide. Linking, optionally where the PEG-containing moiety is linked to a cysteine residue via a maleimide group; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 43 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 62 is present and linked to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 46 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 73 is present and linked to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 45 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 69 is present and connected to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 44 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 66 is present and linked to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 48 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 82 is present and linked to the PEG-containing moiety.

In some embodiments of therapeutic combinations as disclosed herein, the anti-BTN3A antibody is administered at a dose between 0.1 and 10 mg/kg body weight.

In some embodiments of therapeutic combinations as disclosed herein, the anti-BTN3A antibody is administered as an intravenous infusion.

In some embodiments of therapeutic combinations as disclosed herein, the IL-2 agonist is administered at a dose between 0.1 μg/kg and 100 mg/kg body weight.

In some embodiments of therapeutic combinations as disclosed herein, the IL-2 agonist is administered before or after administration of the anti-BTN3A antibody.

In some embodiments of therapeutic combinations as disclosed herein, the cancer is selected from hematological cancer or solid tumor cancer, preferably selected from the group consisting of: B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma Non-Hodgkin lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma ( mantle cell lymphoma (MCL), NK cell lymphoma, myeloid cell line colon cancer, breast cancer, lung cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone cancer, cervical cancer, ovarian cancer cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, testicular cancer and skin cancer.

The present invention also encompasses a method of treating cancer in a patient in need thereof, comprising administering to the patient simultaneously, sequentially, or separately, a therapeutically effective amount of an anti-BTN3A activating antibody, in combination with a therapeutically effective amount of an IL2 agonist that binds to the IL-2 receptor βγc heterodimer; Wherein the IL-2 agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) X1 is a peptide containing the amino acid sequence EHALYDAL (SEQ ID NO: 30); (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide containing the amino acid sequence YAFNFELI (SEQ ID NO: 31); (d) X4 is a peptide containing the amino acid sequence ITILQSWIF (SEQ ID NO: 32); Among them, X1, X2, X3 and X4 can be in any order in the polypeptide; The amino acid linker can exist between any one of the domains; Or wherein the IL-2 agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) X1 is a peptide comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the sequence KIQLHAEHALYDALMILNI (SEQ ID NO: 33); (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 34); (d) X4 is a peptide comprising an amino acid sequence that is at least 90%, at least 95% or 100% identical to the sequence EDEQEEMANAIITILQSWIFS (SEQ ID NO: 35); Among them, X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker may be present between any of the domains; or Or wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, the amino acid sequence described in any one of SEQ ID NO: 37-49. An amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In some embodiments of methods as disclosed herein, the anti-BTN3A antibody has a _KD of 10 nM or less, preferably with a KD of 5 nM or less, as measured by surface plasmon resonance _. Binds to human BTN3A.

In some embodiments of methods as disclosed herein, an anti-BTN3A antibody induces activation of γδ T cells (typically Vγ9Vδ2 T cells) in co-culture with BTN3-expressing cells, wherein the EC is measured in a degranulation assay. ₅₀ is less than 5 μg/ml, preferably 1 μg/ml or less.

In some embodiments of methods as disclosed herein, the anti-BTN3A antibody: - Comprises (a) a variable heavy chain (VH) polypeptide, which contains an amine group that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1 acid sequence, and (b) a variable light chain (VL) polypeptide comprising at least about 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or SEQ ID NO: 3 The amino acid sequence; - Contains HCDR 1-3 of SEQ ID NO: 12-14 and LCDR 1-3 of SEQ ID NO: 15-17; - Contains HCDR 1-3 of SEQ ID NO: 18-20 and LCDR 1-3 of SEQ ID NO: 21-23, or - Compete for binding with an antibody selected from the following: mAb 20.1 produced by the fusion tumor deposited with CNCM under deposit number I-4401, mAb 7.2 produced by the fusion tumor deposited at CNCM under deposit number I-4402, or Antibodies having the heavy chain of SEQ ID NO: 4 and the light chain of SEQ ID NO: 6.

In some embodiments of the methods as disclosed herein, the anti-BTN3A antibody comprises a mutated or chemically modified IgGl constant region, wherein the mutated or chemically modified IgGl constant region is The region prevents binding to Fcγ receptors or reduces binding to Fcγ receptors. In a specific embodiment, the mutated IgG1 constant region is the IgG1 triple mutations L247F, L248E and P350S.

In some embodiments of the methods as disclosed herein, the anti-BTN3A antibody is a monoclonal antibody comprising the heavy chain of SEQ ID NO: 4 and the light chain of SEQ ID NO: 6.

In some embodiments of the methods as disclosed herein, the IL-2 agonist is a polypeptide comprising domains X1, X2, X3, and X4, wherein: (a) A peptide having an amino acid sequence that is at least 90%, at least 95% or 100% identical; (b) X2 is a peptide with a length of at least 8 amino acids; (c) ) is a peptide with an amino acid sequence that is at least 90%, at least 95% or 100% identical to the peptide; and (d) a peptide with an amino acid sequence; and wherein X1, X2, X3, and Receptor βƔ _c heterodimer (IL-2R βƔ _c ) binds.

In some embodiments of the methods as disclosed herein, the IL-2 agonist is a polypeptide comprising domains X1, X2, X3, and X4, wherein: (a) X1 is a polypeptide comprising the amino acid sequence SEQ ID NO: 33 ( KIQLHAEHALYDALMILNI); (b) X2 is a peptide of at least 8 amino acids in length; (c) X3 is a peptide comprising the amino acid sequence SEQ ID NO: 34 (LEDYAFNFELILEEIARLFESG); and (d) X4 is a peptide comprising an amine A peptide with the amino acid sequence SEQ ID NO: 35 (EDEQEEMANAIITILQSWIFS); wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can exist between any one of the domains; and wherein The peptide binds to the IL-2 receptor βƔ _c heterodimer (IL-2R βƔ _c ).

In some embodiments of the methods as disclosed herein, the X2 domain of the IL-2 agonist is one that includes an amino acid sequence that is at least 90%, at least 94%, or 100% identical to SEQ ID NO: 36 (KDEAEKAKRMKEWMKRIKT) Peptides.

In some embodiments of the methods as disclosed herein, the IL-2 agonist is at least 90%, at least 91%, at least 92% identical to an amino acid sequence selected from any one of SEQ ID NOs: 38-49. %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence.

In some embodiments of methods as disclosed herein, an IL-2 agonist polypeptide is linked to a polyethylene glycol ("PEG")-containing moiety, optionally wherein the PEG-containing moiety is a cysteine in the polypeptide. The residue is connected, and optionally the part containing PEG is connected to the cysteine residue via a maleimide group; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 43 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 62 is present and linked to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 46 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 73 is present and linked to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 45 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 69 is present and connected to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 44 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 66 is present and linked to the moiety containing PEG; Optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% of the amino acid sequence described in SEQ ID NO: 48 %, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 82 is present and linked to the PEG-containing moiety.

In some embodiments of methods as disclosed herein, the anti-BTN3A antibody is administered at a dose between 0.1 and 10 mg/kg body weight.

In some embodiments of methods as disclosed herein, the anti-BTN3A antibody is administered as an intravenous infusion.

In some embodiments of methods as disclosed herein, the IL2 agonist is administered at a dose between 0.1 μg/kg and 100 mg/kg body weight.

In some embodiments of methods as disclosed herein, an IL2 agonist is administered before or after administration of the anti-BTN3A antibody.

In some embodiments of methods of treating cancer as disclosed herein, the cancer is selected from hematological cancer or solid tumor cancer, preferably selected from the group consisting of: B-cell lymphoma, T-cell lymphoma, non-Hodgkin lymphoma NHL, B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK cell lymphoma, myeloid cell line Colon cancer, breast cancer, lung cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone cancer, cervical cancer, ovarian cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer , stomach cancer, testicular cancer and skin cancer.

In some embodiments of methods of treating cancer as disclosed herein, the combination of an IL-2 agonist and an anti-BTN3A antibody provides a synergistic effect in the treatment of cancer or for inhibiting tumor cell proliferation.

In some embodiments, the IL-2 agonist is Neo-2/15. Neo-2/15 has been assigned CAS registration number 2407798-79-0.

In some embodiments, the IL-2 agonist is NL-201. NL-201 was generated from Neo-2/15 by introducing a cysteine residue at position 62 for site-specific binding of an unbranched 40 kDa polyethylene glycol (PEG) molecule. NL-201 has been assigned CAS registration number 2738533-90-7.

Definitions In order that the present invention may be more easily understood, certain terms are first defined. Other definitions are set forth throughout the description.

The term "agonist" is used herein as a molecule (eg, a smaller organic molecule or a polypeptide, such as an antibody) that binds to a receptor and activates the receptor to produce a biological response. A selective agonist is selective for a particular type of receptor. Binding to the receptor can be specific binding at a biologically relevant concentration, for example, as determined by surface plasmon resonance.

As used herein, the term "affinity" means the strength of a ligand's binding to its binding site on its receptor. Agonists that bind to a receptor can be targeted at how much of a physiological response can be triggered (i.e., efficacy or potency) and at the concentration of agonist required to produce a physiological response (usually measured as _EC50 , i.e., to produce a half concentration required for maximal reaction). For example, high-affinity ligand binding means that relatively low ligand concentrations are required to maximize ligand binding site occupancy and trigger physiological responses. The binding affinity (K _D ) of a ligand for its receptor (see also definition below) can be determined by surface plasmon resonance (SPR).

The term "Kon" or "Kass (Ka)" as used herein is intended to refer to the rate of association of a specific ligand-receptor interaction, and the term "Kdis (Kd)" or "Koff" as used herein is intended to refer to a specific ligand-receptor interaction. Dissociation rate of body-receptor interactions.

As used herein, the term “K _D ” is intended to refer to the equilibrium dissociation constant, which is derived from the ratio of k _off to _kon (i.e., k _off / _kon ) and is expressed as molar concentration (M). The _KD value is related to the concentration of the ligand (the amount of ligand required for a particular experiment), and therefore, the lower the _KD value (lower concentration) and thus the higher the affinity of the ligand for its receptor.

As used herein, the terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of their length or post-translational modifications (such as glycosylation).

In the context of the present invention, the term "IL-2" refers to any source of IL-2, including mammalian sources and which may be natural or obtained by recombinant or synthetic techniques, including recombinant IL-2 polypeptides produced by microbial hosts . As used herein, the term "IL-2" has its ordinary meaning in the art and generally refers to native IL-2 polypeptide. In some exemplary aspects, the IL-2 polypeptide is derived from a human source and includes recombinant human IL-2, particularly recombinant human IL-2 produced by a microbial host. In specific embodiments, the term "IL-2" refers to human IL-2 (interleukin 2) of SEQ ID NO: 50 and is disclosed, for example, in Genbank ref P60568.

As used herein, the term IL-2 (IL-2) agonist refers to a polypeptide capable of activating IL-2 receptor-mediated signaling.

The terms "peptide mimetic" and "peptidomimetic" have the same meaning herein and refer to a polypeptide or a modified polypeptide that biologically mimics an active ligand of a biomolecule.

As used herein, the term "IL-2 mimetic" refers to a polypeptide that has less than 60%, particularly less than 50%, sequence similarity to native IL-2, particularly human IL-2 of SEQ ID NO: 50. ; less than 40%; less than 30%; or less than 20% identical amino acid sequences. In some embodiments, the IL-2 mimetic is between 10% and 60% identical to human IL-2 of SEQ ID NO: 50, specifically between 10% and 40%, or between 10% and 30% sex. The IL-2 peptide mimetic of the present invention binds to the IL- _2βƔc receptor and can activate IL-2 receptor-mediated signaling. Exemplary IL-2 mimetics used in the methods of the invention induce heterodimerization of IL- _2RβƔc , resulting in phosphorylation of STAT5.

The term "IL-15" has its ordinary meaning and refers to human interleukin-15. Similar to IL-2, IL-15 is composed of the IL-2/IL-15 receptor beta chain (IL-2Rβ, also known as CD122) and a shared gamma chain (IL- _2RƔc , also known as CD132) The complex binds and communicates through it. In the context of the present invention, the term "IL-15" refers to any source of IL-15, including mammalian sources and which may be natural or obtained by recombinant or synthetic techniques, including recombinant IL-15 polypeptides produced by microbial hosts . In some exemplary aspects, IL-15 polypeptides are derived from human sources and include recombinant human IL-15, particularly recombinant human IL-15 produced by a microbial host. In a specific embodiment, it refers to human IL-15 (interleukin 15) of SEQ ID NO: 51.

As used herein, the term "BTN3A" has its ordinary meaning in the art. In specific embodiments, it refers to a human BTN3A polypeptide, including BTN3A1 of SEQ ID NO: 24, BTN3A2 of SEQ ID NO: 25, or BTN3A3 of SEQ ID NO: 26.

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen-binding site that immunospecifically binds an antigen. The terms "antibody" or "immunoglobulin" have the same meaning and will be used equally in the present invention. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies. As used herein, the term "antibody" also includes bispecific or multispecific molecules. The antibody can be derivatized or linked to another functional molecule, such as another peptide or protein (eg, another antibody or ligand for the receptor) to create a bispecific binding to at least two different binding sites or target molecules. molecular. Antibodies may indeed be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be formed as described herein The term "bispecific molecules" is used to encompass this. To form a bispecific molecule, an antibody of the invention can be functionally linked (eg, by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules (such as another antibody, Antibody fragments, peptides or binding mimetics), resulting in bispecific molecules. In addition, for embodiments where the bispecific molecule is multispecific, in addition to the first and second target epitopes, the molecule may further include a third binding specificity.

In natural antibodies from rodents and primates, 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 main classes (or isotypes) of heavy chains that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. In a typical IgG antibody, the light chain consists of two domains: the variable domain (VL) and the constant domain (CL). The heavy chain consists of four domains: one variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity for the antigen. The constant regions of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement fixation and binding to Fc receptors (FcR).

The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of a light chain and a variable part of a heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. Antibody binding sites are composed of residues primarily from hypervariable regions or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FR) may participate in the antibody binding site or affect the overall domain structure, and thus the binding site. Complementarity determining regions or CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light chain and heavy chain of immunoglobulins each have three CDRs, called L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3 respectively. Thus, an antigen binding site typically includes six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequence inserted between CDRs. Accordingly, the variable regions of the light and heavy chains typically include 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

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, hereafter "Kabat et al.") middle. This numbering system is used in this manual. Kabat residue names do not always correspond directly to the linear numbering of amino acid residues in the SEQ ID sequence. Corresponding to the shortening or insertion of structural components of the basic variable domain structure, whether framework or complementarity determining regions (CDRs), the actual linear amino acid sequence may contain fewer or additional amino acids than strict Kabat numbering . For a given antibody, the correct Kabat numbering of residues in the antibody sequence can be determined by aligning the homologous residues with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain according to the Kabat numbering system are located at residues 31 to 35 (H-CDR1), residues 50 to 65 (H-CDR2) and residues 95 to 102 (H-CDR3). The CDRs of the light chain variable domain according to the Kabat numbering system are located at residues 24 to 34 (L-CDR1), residues 50 to 56 (L-CDR2) and residues 89 to 97 (L-CDR3).

It is currently recognized in the art that non-CDR regions of mammalian antibodies can be replaced by similar regions of homospecific or heterospecific antibodies while retaining the epitope specificity of the original antibody. This is most clearly demonstrated in the development and use of "humanized" antibodies, in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to generate functional antibodies.

As used herein, "humanized" describes an antibody in which some, most, or all of the amino acids outside the CDR region are 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 incorporated herein by reference. middle. The above US Patent Nos. 5,585,089 and 5,693,761 and WO 90/07861 also propose four possible criteria that can be used to design humanized antibodies. The first recommendation is for the recipient to use a framework from a specific human immunoglobulin that is rare homologous to the donor immunoglobulin to be humanized, or to use a shared framework from multiple human antibodies. The second recommendation is that if the amino acid in the human immunoglobulin framework is unusual and the donor amino acid at that position is typical for the human sequence, the donor amino acid may be selected instead. receptor. The third suggestion is to select the donor amino acid rather than the acceptor amino acid at the positions immediately adjacent to the three CDRs in the humanized immunoglobulin chain. The fourth recommendation is to use donor amino acids present at framework positions where the amino acid is predicted to have side chain atoms within 3A of the CDR in the three-dimensional model of the antibody and is predicted to be able to interact with the CDR interaction. The above methods are only illustrative of some of the methods that can be used by those skilled in the art to make humanized antibodies. Those skilled in the art will be familiar with other methods for humanizing antibodies. In some humanized forms of antibodies, some, most, or all of the amino acids outside the CDR regions can be replaced with amino acids from human immunoglobulin molecules, but one or more of the amino acids within the CDR regions Some, most or all remain unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not eliminate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules will include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. "Humanized" antibodies retain similar antigen specificity to the original antibody. However, using certain humanization methods, the binding affinity and/or specificity of antibodies can be improved using "directed evolution" methods, as described by Wu et al., Mol. Biol. 294:151, 1999, the contents of which are incorporated by reference. method is incorporated into this article.

Fully human monoclonal antibodies can also be produced by immunizing transgenic mice for most human immunoglobulin heavy and light chain loci. See, for example, U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584 and the references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the product of endogenous (eg, murine) antibodies. The animals are further modified to contain all or a portion of the human germline immunoglobulin locus such that immunization of such animals will result in the production of fully human antibodies to the relevant antigen. After immunization of such mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard fusion tumor techniques. These monoclonal antibodies will have human immunoglobulin amino acids sequence and therefore does not elicit a human anti-mouse antibody (KAMA) response when administered to humans.

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

As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to the full length of the antibody or to one or more fragments of the antibody that retain specificity for the antigen (e.g., the BTN3A protein as defined above) The capacity for sexual union. In specific embodiments, the antibodies provided herein are antibody fragments, and more specifically, include any protein that is the antigen-binding domain of an antibody as disclosed herein. Well-known antibody fragments include: Fab fragment, a monovalent fragment composed of VL, VH, CL and CH1 domains; F(ab)2 fragment, a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge in the hinge region; VH and CH1 domain; Fv fragment consisting of the VL and VH domains of one arm of the antibody; dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of the VH domain or contains such antigen-binding fragments Composed of any fusion protein; bifunctional antibodies, which refer to smaller antibody fragments with two antigen-binding sites, these fragments contain a heavy chain that is linked to a light chain variable domain (VL) in the same polypeptide chain. Variable domain (VH) (VH-VL). By using a linker that is too short to allow pairing between two domains on the same chain, the domain is forced to pair with a complementary domain on the other chain and form two antigen-binding sites. Additionally, although the two domains of the Fv fragment (VL and VH) are encoded by separate genes, they can be joined using recombinant methods via a synthetic linker that allows them to be made such that the VL and VH regions pair to form a monovalent molecule single-chain proteins (called single-chain Fv (scFv); see, for example, 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 fragments" of antibodies (also referred to herein simply as antibody fragments). More generally, antibody fragments as herein are also intended to encompass single domain antibodies, which are antibody fragments that comprise all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, U.S. Patent No. 6,248,516 B1). Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for use in the same manner as intact antibodies. Well-suited 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 as described herein, including, but not limited to, proteolytic breakdown of intact antibodies and production by recombinant host cells.

As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules with a single specificity. Monoclonal antibodies display a single binding specificity and affinity for a specific epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing monoclonal antibodies is independent of binding specificity.

"Recombinant antibody" is an antibody produced, expressed, generated or isolated by recombinant means, such as an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; an antibody derived from human immune cells Antibodies isolated from transgenic animals (such as mice) whose protein genes are transgenic; or antibodies produced, expressed, generated or isolated in any other manner in which specific immunoglobulin gene sequences (such as human immunoglobulin gene sequence) are assembled together with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies. In some embodiments, a recombinant human antibody of the invention has the same amino acid sequence as a corresponding naturally occurring human antibody but is structurally different from the naturally occurring human antibody. For example, in some embodiments the glycosylation pattern differs depending on the recombinant product of the recombinant human antibody. In some embodiments, a recombinant human antibody is chemically modified by adding or subtracting at least one covalent chemical bond relative to the structure of a human antibody found naturally in humans.

As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to BTN3A is substantially free of specific binding to other antigens other than BTN3A of antibodies). However, isolated antibodies that specifically bind BTN3A may cross-react with other antigens, such as related BTN3A molecules from other species. Additionally, isolated antibodies can be substantially free of other cellular material and/or chemicals.

The phrases "antibody that recognizes an antigen" and "antibody that is specific for the antigen" are used interchangeably herein with the term "antibody that specifically binds to the antigen." The term "anti-BTN3A antibody" or "BTN3A antibody" is also used briefly herein to have the meaning of "antibody that recognizes BTN3A".

As used herein, the term "activating antibody" refers to an antibody capable of inducing, directly or indirectly, the immune function of effector cells. Specifically, as used herein, an activating anti-BTN3A antibody has at least the ability to induce activation of γδ T cells (typically Vγ9Vδ2 T cells) in co-culture with BTN3 expressing cells, where the EC ₅₀ is measured in a degranulation assay. Less than 5 μg/ml, preferably 1 μg/ml or less (see detailed examination WO/2020/025703).

As used herein, the term "binding" in the context of binding of an antibody to a predetermined antigen or epitope (particularly BTN3) typically means binding with a certain affinity, as determined by, for example, surface plasmon resonance (SPR) technology. This affinity corresponds to about ^10-7 M or less, such as about ^10-8 M or less, such as about ^10- K _D of ⁹ M or less, about 10 ^-10 M or less, or about 10 ^-11 M or even less. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of various surface plasmon resonance assays routinely used for epitope mapping of monoclonal antibodies. Typically, the antibody binds to the predetermined antigen with an affinity that corresponds to a _KD that is at least ten times lower, such as at least 100 times lower, such as at least 1,000 times lower, than the K of the antibody binding to a non-specific antigen (e.g., BSA, casein). ; such as a K _D that is at least 10,000-fold lower, for example, at least 100,000 times lower, which is not consistent with or closely related to the predetermined antigen. If the _K of the antibody is very low (ie, the antibody has high affinity), the antibody's _K for binding to the antigen is typically at least 10,000 times lower than its _K for binding to a non-specific antigen.

As used herein, the term "affinity" in the context of an antibody means the strength of the binding of the antibody to an epitope.

The term "Kon" or "Kass (Ka)" as used herein is intended to refer to the rate of association of a specific antibody-antigen interaction, and the term "Kdis (Kd)" or "Koff" as used herein is intended to refer to a specific antibody-antigen Dissociation rate of interaction.

As used herein, the term “K _D ” is intended to refer to the equilibrium dissociation constant, which is derived from the ratio of k _off to _kon (i.e., k _off / _kon ) and is expressed as molar concentration (M). The _KD value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), and therefore, the lower the _KD value (lower concentration) and therefore the higher the affinity of the antibody. The _KD value of an antibody can be determined using methods recognized in the art. Preferred methods for determining the _K value of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988) and 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 references are incorporated herein by reference in their entirety. One way to determine the K _D of an antibody is by using surface plasmon resonance or by using an instrument such as Biacore® (see also details on affinity assessment 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 biosensor system of the Octet® system. The Octet® platform is based on bio-layer interferometry (BLI) technology. The principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces: the immobilized protein layer and the internal reference layer. Binding between the ligand immobilized on the biosensor tip surface and the analyte in solution produces an increase in the optical thickness at the biosensor tip, which results in a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in the optical thickness of a biological layer. When this shift is measured over time and its magnitude is plotted as a function of time, a classical association/dissociation curve is obtained. This interaction is measured in real time, allowing monitoring of binding specificity, association and dissociation rates, and concentration. (See Abdiche et al. 2008 and Results for details). Affinity measurements are typically performed at 25°C.

As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen, such as BTN3A. In some embodiments, it is intended to refer to antibodies that bind to human BTN3A as expressed on peripheral blood marrow cells (PBMC), preferably as described in Examples (tests and protocols typically disclosed in WO/2020/ 025703, with particular reference to the EC ₅₀ determined in Table 4) below 50 μg/ml and preferably below 10 μg/ml. In other embodiments, it is 100 nM or less, 10 nM or less, 1 nM or Binds to the antigenic recombinant polypeptide with a _KD of lower, 100 pM or lower, or 10 pM or lower.

"Cross-reactive with an antigen other than BTN3A" antibody is intended to mean an antibody that binds an antigen other than BTN3A with a _KD of 10 nM or less, 1 nM or less, or 100 pM or less. An antibody that "does not cross-react with a specific antigen" is intended to mean an antibody that binds to that antigen with a _KD of 1 μM or greater or a _KD of 10 μM or greater. In certain embodiments, such antibodies do not cross-react with the antigen in standard binding assays, exhibiting essentially undetectable binding to such proteins. In a specific embodiment, the humanized antibody of the invention (eg mAbl) is identical to SEQ ID NO: 27, for example, as measured in a Biacore assay (see in particular the relevant assay exemplified in WO/2020/025703 with reference to Table 26). , SEQ ID NO: 28 and SEQ ID NO: 29 cross-react with cynomolgus monkey BTN3A1, BTN3A2 and BTN3A3.

Specificity can further be determined by, for example, about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1, or greater binding to a specific antigen versus nonspecific binding to other unrelated molecules ( In this case the specific antigen is the BTN3A polypeptide) affinity/affinity ratio.

As used herein, the term "affinity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It is controlled by three main factors: antibody epitope affinity; the valence of both antigen and antibody; and the structural arrangement of the interacting parts. Ultimately, these factors define the specificity of an antibody, that is, the likelihood that a particular antibody binds to the epitope of the precise antigen.

As used herein, the term "individual" includes any human or non-human animal. The term "non-human animals" includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.

As used herein, the term "optimized" means that the better codon changes in the production cell or organism (generally eukaryotic cells, such as Chinese Hamster Ovary (CHO) or human cells) have been used Nucleotide sequences encode amino acid sequences. The optimized nucleotide sequence is engineered to retain all or as much as possible of the amino acid sequence originally encoded by the starting nucleotide sequence. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as optimized.

As used herein, the term "identity" with respect to a polypeptide sequence refers to the amino acid sequence identity between two molecules. If an amino acid position in two molecules is occupied by the same amino acid, the molecules are identical at that position. The identity between two polypeptides is a direct function of the number of identical positions. In general, sequences are aligned to obtain the highest order match (including gaps if necessary). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. (That is, consistency % = number of consistent positions/total number of positions × 100). Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms, as described below.

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

When determining the consistency of the present invention, especially the consistency of IL-2 agonists, it is also important to consider the positioning of the binding interface with the IL-2 receptor. If amino acids are added or deleted, this should be done in a manner that does not substantially interfere with presentation of the protein to its binding partner and does not interfere with secondary structure.

Generally, but not necessarily, it is preferred that the amino acid substitutions relative to the reference peptide domain be conservative amino acid substitutions.

As used herein, "conservative amino acid substitution" means that the amino acid contained therein can be replaced by a residue with similar physiochemical characteristics, for example, one aliphatic residue is substituted for another aliphatic residue (such as with Ile, Val , Leu or Ala for each other), or one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions are known, for example substitution of entire regions with similar hydrophobic characteristics. Polypeptides containing conservative amino acid substitutions can be tested in any of the assays described herein to demonstrate retention of the desired activity of the native or reference polypeptide, such as antigen-binding activity and specificity. Amino acids can be grouped according to similarities in their side chain properties (in A. L. Lehninger, in Biochemistry, 2nd ed., pp. 73-75, Worth Publishers, New York (1975)): (1) Nonpolar: Gly ( G), Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) No polarity : Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidity: Asp (D), Glu (E); (4) Base Properties: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be grouped based on common side chain properties: (1) Hydrophobicity: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr ,Phe. A non-conservative substitution replaces a member of one of these categories necessarily with another category. Particularly preferred conservative substitutions include, for example: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Glu is replaced by Asn; Glu is replaced by Asp; Gly is replaced is Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu Or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp; and/or Phe is replaced by Val, Ile or Leu.

The percent identity between nucleotide sequences can also be determined using, for example, an algorithm (such as the BLASTN program for nucleic acid sequences) using a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and The comparison between the two strands is determined as a default.

Additional antibodies can be identified based on their ability to cross-compete (eg, competitively inhibit binding in a statistically significant manner) with other antibodies of the invention in standard antigen binding assays, such as ELISA binding assays. The ability of a test antibody to inhibit the binding of an antibody of the invention to a target indicates that the test antibody can compete with that antibody for binding to the target; according to non-limiting theory, such antibodies can bind to a target that is identical or related (e.g., structurally) to the antibody it competes with. Similar or spatially close) epitopes. Accordingly, another aspect of the invention provides antibodies that bind to the same antigen as and compete with the antibodies disclosed herein. As used herein, a competitive antibody inhibits target binding of an antibody or antigen-binding fragment of the invention by more than 50%, 51%, 52%, 53%, 54%, 55%, 56% in the presence of an equimolar concentration of the competing antibody. ,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%, At 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, the antibodies "compete" for binding.

As used herein, the term synergy or synergistic effect, when used in connection with a description of the efficacy of a combination of agents, means that any measured effect of the combination is greater than the effect predicted from the sum of the effects of the individual agents (i.e., greater than the additive effect). 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., if the drug combination produces an additive effect, when the tumor growth rate is The drug combination is synergistic when the expected rate is slow). In some embodiments, survival time is used to determine whether a drug combination is synergistic (eg, a drug combination is synergistic when an individual or population of individuals survives longer than expected if the drug combination produces an additive effect). In some embodiments, T cell expansion can also be used to determine whether a drug combination is synergistic (e.g., if a drug combination produces an additive effect) when the rate of expansion of a specific T cell subset (measured as a population increase compared to baseline values) A drug combination is synergistic when the expansion rate is higher than the expected expansion rate (measured as a percentage increase or absolute cell number increase).

Activated BTN3A Antibodies Activated anti-BTN3A antibodies of the invention typically exhibit one or more of the following properties: (i) As measured by SPR (eg as described in the examples of patent application WO2020025703) at 10 nM or _lower , preferably binds to BTN3A with a _K of 1 nM or lower; (ii) as measured by SPR (for example, as described in the examples of patent application WO2020025703), which binds to BTN3A with a K of 100 nM or lower _KD , preferably cross-reacts with cynomolgus BTN3A with a _KD of 10 nM or lower; (iii) as assayed by flow cytometry (as described in the examples of patent application WO2020025703) It binds to human PBMC with an _EC50 of 50 μg/ml or less, preferably 10 μg/ml or less, as measured in Activation of cells (typically Vγ9Vδ2 T cells) with an _EC50 below 5 μg/ml, preferably 1 μg/ml or lower, as described in the examples of patent application WO2020025703. (v) It induces in vitro activation of Vγ9Vδ2 T cells in human PBMCs, with an EC ₅₀ of less than 0.1 μg/mL, preferably 0.01 μg/mL or less, as measured by surface expression of the activation marker CD69 , for example, between 100 pg/mL and 0.1 μg/mL.

Example anti-BTN3A activating antibodies are described in the following paragraphs. In some embodiments, the anti-BTN3A activating antibody is selected from the group consisting of anti-BTN3A antibodies such as those described in international patent applications WO2012080769; WO2012080351 and WO2020025703. In some specific embodiments, the BTN3 activating antibody is selected from the humanized antibodies described in WO2020025703 or is a humanized version of the BTN3A agonist antibody described in WO2012080769 and WO2012080351. In some embodiments, the anti-BTN3 antibody can be selected from mAb 20.1 and mAb 7.2, which can be obtained from one of the fusion tumors obtained under CNCM deposit numbers I-4401 and I-4402, such as those described in WO2012080769 and WO2012080351, or a humanized version thereof; and selected from humanized mAbs 1-6 described in WO2020025703.

In some embodiments, the anti-BTN3 antibody comprises six CDRs (CDR1 (also known as HCDR1), VH CDR2 (also known as HCDR1), VH CDR2 (also known as (HCDR2), VH CDR3 (also known as HCDR1), VL CDR1 (also known as LCDR1), VL CDR2 (also known as LCDR2), VL CDR3 (also known as HCDR3)). In specific embodiments, the anti-BTN3A activating antibodies comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and HCDR3 as shown in Table 1 below: Table 1 : mAb1, mAb2, mAb4 and mAb5 according to Kabat numbering as defined in WO2020025703, CDR regions of the parent murine mAb 7.2 and murine mAb 20.1 antibodies. original antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 mAb 7.2 mAb1 mAb2 mAb4 mAb5 SEQ ID NO: 12 SEQ ID NO: 13 SEQ ID NO: 14 SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 mAb 20.1 SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23

In some embodiments of the antibodies as disclosed herein, the 6 CDR regions are 100% identical to the 6 CDR regions of antibodies 20.1 or 7.2 described in WO2012080769 and WO2012080351 or mAbs 1-6 described in WO2020025703, in particular In some embodiments, the six CDR regions of an antibody as disclosed herein are 100% identical to the six CDR regions of Table 1 (specifically mAb 7.2; 1; 2; 4; and 5).

Other antibodies as disclosed herein include those that have been mutated by amino acid deletions, insertions or substitutions but are not identical to antibodies 20.1 or 7.2 described in WO2012080769 and WO2012080351 or mAbs 1-6 described in WO2020025703 Compared with the 6 CDR areas, especially compared with the 6 CDR areas defined in Table 1 , the CDR areas still have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% consistent amino acids. In some embodiments, according to the present invention, compared with the CDR sequences of antibodies 20.1 or 7.2 described in WO2012080769 and WO2012080351 or mAbs 1-6 described in WO2020025703, in particular compared with the CDR sequences of Table 1 , more Specifically, compared with the CDR sequences of mAb 7.2; 1; 2; 4; and 5, the antibody may have between 1, 2, 3 or 4 amino acid changes (including deletions) in one or more CDRs. , insert or replace).

Antibodies of the invention also include those having at least 90%, in particular at least 95%, 96%, 97%, 98%, 99% or 100% identity to the VH and VL regions as defined in Table 2 . More specifically, the antibodies of the invention include selected humanized recombinant antibodies mAb1, mAb2, mAb4, and mAb5, which are structurally characterized by their variable heavy and light chain amino acid sequences as described in Table 2 below. And human constant region (isotype): Table 2 : Variable heavy chain and light chain amino acid sequences of mAb1-mAb6 antibody VH amino acid sequence VL amino acid sequence homotypic constant region mAb1 SEQ ID NO:1 (VH2 7.2) SEQ ID NO:2 (Vk1 7.2) Silencing IgG1 L247F/L248E/P350S mAb2 SEQ ID NO:1 (VH2 7.2) SEQ ID NO:3 (Vk2 7.2) Silencing IgG1 L247F/L248E/P350S mAb3 Humanized variant from 20.1 Humanized variant from 20.1 Silencing IgG1 L247F/L248E/P350S mAb4 SEQ ID NO:1 (VH2 7.2) SEQ ID NO:2 (Vk1 7.2) IgG4 S241P/L248E mAb5 SEQ ID NO:1 (VH2 7.2) SEQ ID NO:3 (Vk2 7.2) IgG4 S241P/L248E mAb6 Same as mAb3 Same as mAb3 IgG4 S241P/L248E mAb3 and mAb6 are humanized antibodies of the parent murine anti-BTN3A antibody, termed mAb 20.1 as described in WO2012/080351.

The corresponding amino acid and nucleotide coding sequences of the constant isotype regions of IgG1, IgG4 and their mutant forms IgG1 L247F/L248E/P350S and IgG4 S241P/L248E used to generate mAb1 to mAb6 are well known in the art (Oganesyan et al. , 2008;Reddy et al., 2000). 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 additionally deleted in constructs from mAb1 to mAb6.

The full-length light and heavy chains of mAb1, mAb2, mAb4 and mAb5 and the corresponding coding sequences are shown in Table 3 below. Table 3 : Full-length heavy chain and light chain DNA coding sequences antibody amino acid sequence DNA coding sequence mAb1 Heavy chain: SEQ ID NO: 4 Light chain: SEQ ID NO: 6 Heavy chain: SEQ ID NO: 8 Light chain: SEQ ID NO: 10 mAb2 Heavy chain: SEQ ID NO: 4 Light chain: SEQ ID NO: 7 Heavy chain: SEQ ID NO: 8 Light chain: SEQ ID NO: 11 mAb4 Heavy chain: SEQ ID NO: 5 Light chain: SEQ ID NO: 6 Heavy chain: SEQ ID NO: 9 Light chain: SEQ ID NO: 10 mAb5 Heavy chain: SEQ ID NO: 5 Light chain: SEQ ID NO: 7 Heavy chain: SEQ ID NO: 9 Light chain: SEQ ID NO: 11

In certain embodiments that can be combined with the previous embodiments, the antibodies provided herein are antibody fragments of an antibody as defined above. Antibody fragments include, for example, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, monofunctional and scFv fragments, diabodies, single domain or nanobodies and other fragments. Preferably, it is a monovalent antibody, such as a Fab of scFv fragment.

In some embodiments, the antibody of the invention competes for binding to the BTN3 antibody described above, particularly the antibody of the invention is selected from the group consisting of mAb 20.1 and mAb 7.2 (which can be obtained from CNCM deposit numbers such as those described in WO2012080769 and WO2012080351 One of the available fusion tumors I-4401 and I-4402) competes for binding with an antibody selected from mAbs 1-6 described in WO2020025703. In more specific embodiments, an antibody of the invention competes for binding with an antibody selected from mAb 7.2 produced by a fusionoma deposited with CNCM under Accession No. 1-4402 and having SEQ ID NO: 4 chain and the light chain of SEQ ID NO: 6.

In some embodiments, the antibodies of the invention are chimeric, humanized or human antibodies. In a preferred embodiment of the invention, the BTN3 antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while having at least the same affinity (or superior affinity) of the parent non-human antibody. More specifically, the BTN3 antibody is a humanized form of antibody 20.1 or 7.2 disclosed in WO2012080351. In a preferred embodiment, the antibody of the invention is the parent antibody mAb 7.2 humanized antibody as disclosed in WO2012080351. Generally, humanized antibodies contain one or more variable domains, where the CDRs (or portions thereof) are derived from a non-human antibody (e.g., murine mAb 7.2) and the FRs (or portions thereof) are derived from a domain with mutations to reduce immunity. Original murine antibody sequences. Humanized antibodies will optionally also contain at least a portion of a human constant region. Preferably, the recombinant antibody of the present invention is a humanized silent antibody, typically a humanized silent IgG1 or IgG4 antibody. Well-suited humanized anti-BTN3A antibodies of the present invention are typically described in WO2020025703 and include mAbs with the VH/VL polypeptide sequences of Table 2 and mAbs with the light/heavy chains of Table 3 .

As used herein, the term "silent" antibody refers to an antibody that exhibits no or lower FcyR binding and/or Clq binding as measured in binding assays such as those described in WO2020025703. In one embodiment, the term "no binding or low FcγR and/or C1q binding" means that the silenced antibody exhibits FcγR and/or C1q binding that is at least lower than that observed with the corresponding antibody with wild-type human IgG1 or IgG4 isotype FcγR and/or C1q binding is 50%, for example less than 80%.

Framework or Fc Engineering Antibodies of the invention may include modifications to framework residues within VH and VL to reduce their immunogenicity.

In some specific embodiments, the antibody of the invention is a humanized monoclonal antibody of the parent murine antibody mAb 7.2, which includes 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.

In other specific embodiments, the antibody of the invention is a humanized monoclonal antibody of the parent murine antibody mAb 7.2, which includes at least the following amino acid mutations in the VH framework region compared to mAb 7.2: 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.

In addition to modifications made within the framework regions, antibodies of the invention can be engineered to include modifications within the Fc region, typically altering one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or Antigen-dependent cellular cytotoxicity.

In addition, the antibodies of the invention can be chemically modified (eg, one or more chemical moieties can be attached to the antibody), or modified to alter its glycosylation, thereby altering one or more functional properties of the antibody. Each of these embodiments are described in further detail below.

As used herein, the terms "isotypic 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 as comprising the amino acid residues from position C226 or from P230 of the IgG antibody to the carboxyl terminus, where the numbering is according to the EU numbering system. The C-terminal lysine of the Fc region (residue K447) can be removed, for example, during production or purification of the antibody or its corresponding codon is deleted in the recombinant construct. Thus, the composition of the antibodies of the invention may include a population of antibodies with all K447 residues removed, a population of antibodies with no K447 residues removed, and a population of antibodies with a mixture of antibodies with or without K447 residues.

In some embodiments, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is changed, eg, increased or decreased. This method is further described in US Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the hinge region of CH1 is changed to, for example, promote the assembly of light and heavy chains or to increase or decrease the stability of the antibody.

In other embodiments, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment so that the antibody binds to Staphylococcyl protein A (Staphylococcyl protein A; SpA) relative to the binding of the native Fc hinge domain SpA. weaken. This method is described in further detail in U.S. Patent No. 6,165,745 to Ward et al.

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, one or more amino acids can be replaced with different amino acid residues such that the antibody has altered affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The affinity-modified effector ligand may be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in US Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

In another embodiment, one or more amino acids selected from the group consisting of amino acid residues may be replaced with different amino acid residues such that the antibody has altered C1q binding and/or reduced or eliminated complement-dependent cellular Toxicity (complement dependent cytotoxicity, CDC). This method is described in further detail in U.S. Patent No. 6,194,551 to Idusogie et al.

In another example, one or more amino acid residues are altered thereby altering the ability of the antibody to fix complement. This method is further described in Bodmer et al., PCT Publication WO 94/29351.

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 sensitivity of the antibody to Fcγ receptors. affinity. Such antibodies with reduced effector function and especially reduced ADCC include silent antibodies.

In certain embodiments, the Fc domain of the IgGl isotype is used. In some embodiments, mutated variants of the IgG1 Fc fragment are used, such as 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.

In certain embodiments, the Fc domain of the IgG4 isotype is used. In some embodiments, mutant variants of IgG4 Fc fragments are used, such as 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.

Silencing effector functions can be obtained by mutations in the Fc constant portion of antibodies and have been described in this technology (Baudino et al., J. Immunol. 2008; Strohl, CO Biotechnology 20 2009). Examples of silent IgG1 antibodies include triple mutation variants IgG1 L247F, L248E, P350S. Examples of silent IgG4 antibodies include double mutation variants IgG4 S241P L248E.

In certain embodiments, the Fc domain is a silent Fc mutation that prevents glycosylation at position 314 of the Fc domain. For example, the Fc domain contains an amino acid substitution of asparagine at position 314. An example of such an amino acid substitution is the replacement of N314 by glycine or alanine.

In yet other embodiments, the glycosylation of the antibody is modified. For example, aglycosylated antibodies can be prepared (ie, antibodies lack glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such non-glycosylation increases the affinity of the antibody for the antigen. Such methods are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 to Co et al.

Another modification to the antibodies herein contemplated by the invention is pegylation or hydroxyethylation or related techniques. Antibodies can be pegylated, for example, to extend the biological (eg, serum) half-life of the antibody. To PEGylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG) (such as a reactive ester or aldehyde derivative of PEG) so that one or more PEG groups become associated with the antibody or antibody fragment. react under the conditions of connection. PEGylation can be performed by chelation or alkylation with reactive PEG molecules (or similar reactive water-soluble polymers). 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 pegylating proteins are known in the art and can be applied to the antibodies of the invention. See, for example, Nishimura et al. EP 0 154 316 and Ishikawa et al. EP 0 401 384.

Another possibility is that fusions of at least the antigen-binding region of the antibodies of the invention and proteins are able to bind to serum proteins, such that human serum albumin prolongs the half-life of the resulting molecule. Such methods are described, for example, in Nygren et al., EP 0 486 525.

In certain embodiments, the C-terminal lysine typically found on the constant domain of human IgG heavy chains is engineered to reduce heterogeneity due to the reduced cleavage typically observed during manufacturing or storage. . Such modifications do not imperceptibly alter the desired functions of the antibodies while conferring stability benefits to the molecules.

Nucleic Acid Molecules Encoding Antibodies of the Invention Nucleic acid molecules encoding anti-BTN3A antibodies of the invention are also disclosed herein. Examples of variable light and heavy chain nucleotide sequences are those encoding the variable light and heavy chain amino acid sequences of any of mAb1, mAb2, mAb4, and mAb5, variable light Chain and heavy chain amino acid sequences are readily derived from Tables 1 and 2 , using the genetic code and taking into account codon bias as appropriate , depending on the host cell species.

The present invention is also directed to nucleic acid molecules derived from variable light and heavy chain amino acid sequences that have been optimized for protein expression in mammalian cells (eg, CHO cell lines).

The nucleic acid may be present in whole cells, cell lysates, or may be present in partially purified or substantially pure form. When nucleic acids are separated from other cellular components or other contaminants (e.g., other cells) by standard techniques including alkaline/SDS treatment, CsCl conjugation, column chromatography, agarose gel electrophoresis, and other techniques well known in the art, When a nucleic acid or protein is isolated and purified, a nucleic acid is "isolated" or "renders substantially pure" (Ausubel et al., 1988, Current Protocols in Molecular Biology (John Wiley & Sons)). The nucleic acid of the invention may be, for example, DNA or RNA and may or may not contain intronic sequences. In one embodiment, the nucleic acid may be present in a vector, such as a phage display vector or a recombinant plasmid vector.

Nucleic acids of the invention can be obtained using standard molecular biology techniques. Once DNA fragments encoding, for example, VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In such manipulations, a DNA fragment encoding VL or VH (e.g., VL and VH as 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 an antibody constant region. Flexible connector. As used in this context, the term "operably linked" is intended to mean that two DNA fragments join in, for example, a functional manner such that the amino acid sequences encoded by the two DNA fragments remain in frame, or so that the desired promoter Expression of proteins under control.

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

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding VL 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 covering these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

To form a scFv gene, a DNA fragment encoding VH and VL is operably linked to another fragment encoding a flexible linker (e.g., encoding an amino acid sequence (Gly4-Ser) ₃ ) such that the VH and VL sequences can be expressed are linked single-chain proteins in which the VL and VH regions are joined by a 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).

Generation of Monoclonal Antibody-Producing Metastases Antibodies of the invention may be produced in host cell transfectomas using, for example, a combination of recombinant DNA techniques and gene transfection methods as are well known in the art (Morrison, 1985; Science 229 , 1202 -1207).

For example, to express antibodies or antibody fragments thereof, DNA encoding partial or full-length light and heavy chains can be synthesized by standard molecular biology or biochemical techniques such as DNA chemical synthesis, PCR amplification, or the use of antibodies expressing the antibody of interest. Fusionomas are subject to cDNA selection) and the DNA can be inserted into an expression vector to operably link the gene to transcription and translation control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is joined into the vector such that the transcription and translation control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. Select expression vectors and expression control sequences that are 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 (eg, ligation of the antibody gene fragment and complementary restriction sites on the vector or blunt-end ligation if no restriction sites are present). The light and heavy chain variable regions of the antibodies described herein can be used to form full-length antibody genes of any antibody isotype by insertion into an expression vector that already encodes the heavy and light chain constant regions of the desired isotype, such that the VH segment The VL segment is operably connected to the CH segment within the carrier and the VL segment is operably connected to the CL segment within the carrier. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be selected and cloned into the vector so that the signal peptide is connected 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 (ie, a signal peptide from a non-immunoglobulin).

In addition to the antibody chain genes, the recombinant expression vectors disclosed herein also carry regulatory sequences that control the expression of the antibody chain genes in host cells. The term "regulatory sequences" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody chain genes. Such control sequences are described, for example, in Goeddel's publication (Goeddel, DV (1990). [1] Systems for heterologous gene expression. In Methods in Enzymology, (Academic Press), pp. 3-7). Those familiar with this technology should understand that the design of expression vectors includes the selection of regulatory sequences, which may depend on factors such as the selection of host cells to be transformed and the amount of expression of the desired protein. Regulatory sequences for mammalian host cell expression include viral elements that direct high protein expression in mammalian cells, such as those derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (For example, adenovirus major late promoter (AdMLP)) and polyomavirus promoters and/or enhancers. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the P-hemoglobulin promoter. Furthermore, the regulatory elements are composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeats of human T-cell leukemia virus type 1 (Takebe et al., 1988, Mol. Cell. Biol. 8 , 466-472).

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vector of the present invention can also carry other sequences, such as sequences that regulate the replication of the vector in host cells (eg, origin of replication) and selectable marker genes. Selectable marker genes facilitate selection of host cells into which the vector has been introduced (see, eg, U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017, all issued to Axel et al.). For example, a selectable marker gene typically 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 dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of light and heavy chains, expression vectors encoding heavy and light chains are transfected into host cells by standard techniques. Various forms of the term "transfection" are intended to encompass various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-polydextrose transfection, and similar techniques. It is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells. The expression of antibodies in eukaryotic cells (eg, mammalian host cells, yeast, or filamentous fungi) is discussed because such eukaryotic cells, and especially mammalian cells, are more likely than prokaryotic cells to assemble and secrete appropriately folded and immunologically active antibodies.

In a specific embodiment, the selection or expression vector of the invention comprises one of the coding sequences of the heavy chain and the light chain of any of mAb1, mAb2, mAb4 and mAb5 operably linked to a suitable promoter sequence By.

Mammalian host cells for expressing recombinant antibodies of the invention include Chinese hamster ovary (CHO cells), including dhfr-CHO cells (described in Urlaub and Chasin, 1980), CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells, such as the GS CHO cell line in conjunction with the GS Xceed ^™ Gene Expression System (Lonza). When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody system is generated by culturing the host cell for a time sufficient to express the antibody in the host cell and, optionally, secrete the antibody into the medium in which the host cell is grown. produce. Standard protein purification methods such as recovery and purification of the antibody from the culture medium can be used after antibody secretion (Shukla et al., 2007, J. Chromatogr. B 848 , 28-39).

In a specific embodiment, the host cell of the invention is a host cell transfected with an expression vector having an expression vector suitable for the expression of mAb1, mAb2, mAb4 and mAb5, respectively, operably linked to a suitable promoter sequence. coding sequence.

For example, the present invention relates to a host cell comprising at least the nucleic acids of SEQ ID NO: 8 and 10 encoding the heavy chain and light chain of mAbl, respectively.

The subsequent host cells can then be further cultured under appropriate conditions to express and produce antibodies of the invention selected from the group consisting of mAb1, mAb2, mAb4 and mAb5 respectively.

Alternatively, a cell-free expression system can be used to generate any of mAb1, mAb2, mAb4, and mAb5. Typically, methods for cell-free expression of proteins or antibodies have been described (Stech et al., 2017, Sci. Rep. 7 , 12030).

Anti -BTN3A Immunoconjugates In another aspect, the invention features an anti-BTN3A antibody or fragment thereof as disclosed herein bound to a therapeutic moiety. Such conjugates are referred to herein as "immunoconjugates". Immunoconjugates that include one or more cytotoxins are called "immunotoxins." Cytotoxic or cytotoxic agents include any agent that is harmful to cells (eg, kills cells).

Cytotoxins can be conjugated to the antibodies of the invention using linker technology available in the art. Examples of linker types used to bind cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers, such as valine-citrulline linkers. The selected linker may, for example, be readily cleaved by low pH within the lysosomal compartment or be readily cleaved by proteases, such as proteases that are preferentially expressed in tumor tissue, such as cathepsins (eg cathepsins B, C, D).

For further discussion of cytotoxin types, linkers, and methods used to conjugate therapeutics to antibodies, see also Panowski et al., 2013 for a review of antibody drug conjugates.

The antibodies of the present invention can also be combined with radioactive isotopes to generate cytotoxic radiopharmaceuticals, also known as radioactive immunoconjugates. Examples of radioactive isotopes that can be combined with antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ^-131 , indium ^-111 , yttrium ^-90 , and gallium ^-177 . Methods for preparing radioimmunoconjugates are established in the art.

Bispecific or Multispecific Anti -BTN3A Antibodies In another aspect, further disclosed herein are bispecific or multispecific molecules comprising the anti-BTN3A antibodies of the invention. The antibody can be derivatized or linked to another functional molecule, such as another peptide or protein (eg, another antibody or ligand for the receptor) to create a bispecific binding to at least two different binding sites or target molecules. molecular. Antibodies may indeed be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also intended to be formed as described herein The term "bispecific molecules" is used to encompass this. To form a bispecific molecule, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other binding molecules (such as another antibody, an antibody fragments, peptides or binding mimetics), thereby creating bispecific molecules.

Accordingly, the present invention includes bispecific molecules that comprise at least a first binding specific for BTN3A, such as an antigen-binding portion of any one of mAb1, mAb2, mAb4, and mAb5, and an antigen-binding moiety directed against a first binding specific for BTN3A. A second binding specific for the two target epitopes.

In addition, for embodiments where the bispecific molecule is multispecific, in addition to the first and second target epitopes, the molecule may further include a third binding specificity.

In one embodiment, the bispecific molecules disclosed herein comprise as binding specificity at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab') ₂ , Fv, monofunctional antibody or single chain Fv. As described in US Patent No. 4,946,778 to Ladner et al., the antibody may also be a light chain or heavy chain dimer or any minimal fragment thereof, such as an Fv or single chain construct.

Other antibodies useful in the bispecific molecules disclosed herein are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules of the invention can be prepared by combining constitutive binding specifics using methods known in the art. For example, each binding specificity of a bispecific molecule can be generated separately and subsequently bound to each other. When the binding specific substance is a protein or peptide, various coupling agents or cross-linking agents can be used for covalent binding. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2- Nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP ) and 4-(N-maleimidomethyl)cyclohexane-l-carboxylic acid sulfosuccinimidyl ester (sulfo-SMCC) (Karpovsky et al., 1984 J. Exp. Med. 160 , 1686-1701; Liu et al., 1985 Proc. Natl. Acad. Sci. 82 , 8648-8652). Other methods include those described in Brennan et al., 1985, Science 229 , 81-83; Glennie et al., 1987. J. Immunol. 139 , 2367-2375; and Paulus, 1985 Behring Inst. Mitt. 118-132. .

Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly suitable when the bispecific molecule is mAb×mAb, mAb×Fab, Fab×F(ab') ₂ or ligand×Fab fusion protein. The bispecific molecule of the invention can be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants.

The binding of bispecific molecules to their specific targets can be accomplished by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, biological assays (such as growth inhibition and apoptosis). death) or Western ink spot test to confirm. Each of these assays typically detects the presence of the protein-antibody complex of interest by employing a labeled reagent (eg, an antibody) specific for the complex of interest.

IL-2 Agonist The IL-2 agonist of the present invention includes an IL2 mimetic polypeptide, which includes domains X1, X2, X3 and X4, wherein: (a) ); (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide containing the amino acid sequence YAFNFELI (SEQ ID NO: 31); (d) X4 is a peptide containing an amino acid A peptide of the sequence ITILQSWIF (SEQ ID NO: 32); wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can be present between any one of the domains.

The terms "peptide mimetic" and "peptidomimetic" have the same meaning herein and refer to polypeptides, modified polypeptides that biologically mimic the active ligands of biomolecules. IL-2 peptide mimetics bind to IL-2 βƔ _c receptors and can activate IL-2 βƔ _c receptor-mediated signaling. IL-2 mimetics are described in Silva et al., Nature 2019 Jan;565(7738):186-191. Exemplary IL-2 mimetics for use in the methods of the invention induce heterodimerization of the beta and gamma chains of the IL-2 receptor, resulting in phosphorylation of STAT5 (which may be defined previously with particular reference to Silva et al., to be evaluated in 2019). Briefly cells were stimulated with IL-2 agonists and subsequently fixed, permeabilized and incubated with anti-STAT5p antibodies such as anti-STAT5 pY694 (BD Biosciences) conjugated to Alexa Fluor® 647. MFI can be determined on a flow cytometer (Beckman-Coulter). Dose-response curves can be fit to a logistic model and the half-maximum effective concentration ( _EC50 value) and corresponding 95% confidence interval can be calculated. In some embodiments, IL-2 agonists of the invention induce phosphorylation of STAT5, as measured by the STAT5 phosphorylation assay detailed above, with an EC ₅₀ of 10 nM or less, specifically 1 nM. or less, 0.5 nM or less, 0.1 nM or less or 0.05 nM or less. In some embodiments, IL-2 agonists of the invention induce phosphorylation of STAT5, as measured by the STAT5 phosphorylation assay detailed above, with an EC ₅₀ between 0.001 nM and 100 nM, specifically Between 0.01 and 100 nM, between 0.01 and 50 nM, between 0.05 and 50 nM, between 0.1 and 50 nM.

More specifically, the IL-2 mimetics of the invention (i) bind to the IL-2 receptor βƔ _c heterodimer and (ii) are alpha-independent. By "alpha-independent" herein is meant that the IL-2R agonist is unable to detectably bind IL-2Rα and therefore has abrogated affinity for IL-2Rα (CD25). Typically, IL-2 mimetics of the invention are designed not to have an IL-2Rα binding interface.

In some preferred embodiments, the affinity of the IL-2 mimetics of the invention for IL- _2RβƔc is increased by at least 5-fold, 10-fold, 20-fold, 30-fold or 50-fold compared to native IL-2. In some embodiments, the IL-2 agonist has a binding affinity (i.e., K _D as typically determined by surface plasmon resonance SPR) for IL-2RβƔ _c of 200 nM or less, 100 nM or more. Low, 50 nM or less or 25 nM or less. Typically, the binding affinities of the IL-2 agonist combinations disclosed herein are between 0.1 and 100 nM, specifically between 1 and 100 nM, 1 and 50 nM, 5 and 100 nM, 5 and 50 nM, or Between 10 and 100 nM.

In some embodiments of the invention, the IL-2 mimetic is a long-acting IL-2 receptor agonist. "Long-acting" means that the IL-2 mimetic has a plasma or serum half-life of 3 hours or more, preferably 4 hours or more. In some aspects, the serum or plasma half-life of the IL-2 mimetic will be 9 or 10 hours or longer or 12 hours or longer. The half-life of a polypeptide is the time required for a 50% decrease in the concentration of the polypeptide as measured by an appropriate assay. The reduction may be caused by in vivo breakdown, clearance or chelation of the polypeptide. Whereas the half-life of the IL-2 mimetic of the invention can be determined by any means known in the art, such as by measuring the concentration of the IL-2 mimetic in the blood. For example, to measure the half-life of a polypeptide in vivo, a warm-blooded animal (i.e., a human or another suitable mammal, such as a mouse, rabbit, rat, pig, dog, or primate) is administered A suitable dose of the polypeptide; collecting blood samples or other samples from animals; measuring the content or concentration of the polypeptide in the sample; and calculating the time until the content or concentration of the polypeptide has been reduced by 50% based on the measured data. See, for example, Kenneth, A et al., Chemical Half-life of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic analysis: A Practical Approach (1996). As used herein, "half-life extension" or "longer half-life" refers to an increase compared to a control in any one or more of the parameters used to describe the half-life of a protein, such as tl/2-alpha, 11/2-beta, and Area under the curve (AUC). The long-acting properties of the IL-2 mimetic may be attributed to the moiety that binds or fuses with the IL-2 mimetic.

IL-2 mimetics can thus be further linked to other compounds to promote extended half-life in vivo (also known as half-life extension technology). Any such compound may be used in the present invention, provided that it is sufficiently safe for administration to human subjects.

Representative techniques for increasing protein half-life are typically described in Tan H, Su W, Zhang W, Wang P, Sattler M, Zou P. " Recent Advances in Half-life Extension Strategies for Therapeutic Peptides and Proteins ". Curr Pharm Des. 2018;24(41):4932-4946. Typical examples of compound linkages include albumin coupling, including chemical conjugation or genetic fusion to albumin (e.g., human serum albumin, HAS) or a derivative thereof (e.g., albumin binding domain, ABD); Chemical coupling of synthetic polymer chains to produce pegylated IL-2 mimetics) or polypropylene glycol (for more details see, for example, WO 87/00056 and Harris, J., Chess, R. Nat Rev Drug Discov 2, 214- 221 (2003)); chemically coupled with biodegradable hydroxyethyl starch (HES, to typically produce HESylated IL-2 mimetics) (for more details see Liebner R, Mathaes R, Meyer M, Hey T, Winter G, Besheer A. Eur J Pharm Biopharm. 2014;87(2):378-385); glycosylation (typically by introducing additional N-glycosylation or modules containing glycosylation sites (such as carboxyl terminal peptide) to achieve) and IgG Fc fusion (including dimeric or monomeric Fc regions and/or Fc variants).

"PEG" is a poly(ethylene glycol) molecule, which is a water-soluble polymer of ethylene glycol. PEG is available in different sizes and is also commercially available in chemically activated forms derivatized with chemically reactive groups capable of covalently binding to proteins. Linear PEGs are produced in different molecular weights, such as PEG polymers with weight average molecular weights of 5,000 Daltons, 10,000 Daltons, 20,000 Daltons, 30,000 Daltons and 40,000 Daltons. Branched chain PEG polymers have also been developed. Commonly used activated PEG polymers are N-hydroxysuccinimide groups (for binding to primary amines such as lysine residues and the N-terminus of proteins), aldehyde groups (for binding to the N-terminus) and These activated PEG polymers are derivatized with maleimide or iodoacetamide groups (for coupling with thiol groups such as cysteine residues). Methods of designing IL-2 mimetic moieties for binding to PEG are known in the art. For example, adding a polyethylene glycol ("PEG")-containing moiety may include linking a PEG group linked to a maleimide group (eg, PEG-MAL) to a cysteine residue of the polypeptide. Suitable examples of PEG-MAL include, but are not limited to, methoxyPEG-MAL 5 kD; methoxyPEG-MAL 20 kD; methoxy(PEG)2-MAL 40 kD; methoxyPEG(MAL)2 5 kD; MethoxyPEG(MAL)2 20 kD; MethoxyPEG(MAL)2 40 kD; or any combination thereof. See also US Patent No. 8,148,109.

Such linkages may be covalent or non-covalent.

Typically, an IL-2 agonist of the invention is less than 60% identical to a native IL-15 polypeptide (such as human IL-15 of SEQ ID 51), specifically less than 50%, 40%, 35%, 30 %, 25%, 20%, 15% or 10% consistency.

In some embodiments, the IL-2 agonists of the invention do not bind IL-15Rα (CD215), particularly human IL-15Rα (CD215) of SEQ ID 52.

In some embodiments, the IL-2 agonist of the invention does not comprise greater than 60%, specifically greater than 65%; 70%; 75 with IL-15Rα (CD215), especially human 15Rα (CD215) of SEQ ID 52 %; 80%; 85%; 90%; 91%; 92%; 93%; 94%; 95%; 96%; 97%; 98%; or 99% identical amino acid sequence.

Exemplary IL2 mimetic amino acid linkers can be of any length deemed appropriate for the intended use. Exemplary lengths of amino acids include those of 1-200, 1-100, 1-50, 1-20, 1-15, 1-10, 2-20, 2-15, or 2-10 amino acids in length. connector between.

In some embodiments, X1 is a peptide comprising the amino acid sequence EHALYDAL (SEQ ID NO:30); X2 is a peptide having a length of at least 8 amino acids; 31); and X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO: 32).

X1, X3 and Typically, each of X1, X3 and X4 contains at least 8 amino acids. In some aspects, each of X1, X3, and X4 includes at least 19 amino acids. In some such aspects, each of X1, X3, and X4 is no more than 200 or 100 or 50 amino acids in length.

In some embodiments, Peptide; 35) At least 90%, at least 95% or 100% identical amino acid sequences.

For all such embodiments, X1, X2, X3, and X4 may be in any order in the polypeptide. An exemplary domain order is X1-X3-X2-X4.

In some embodiments, (i) X1 includes 1, 2, 3, 4, or all 5 of the following: L at residue 4, H at residue 5, H at residue 8, residue 11 Y, M at residue 15; and/or (ii) X3 includes 1, 2, 3, 4, 5, 6, 7 or all 8 of the following: D at residue 3, D at residue 4 Y, F at residue 6, N at residue 7, L at residue 10, I at residue 11, E at residue 13 or E at residue 14. In yet another embodiment, (iii) X4 includes I at residue 19. Refer to SEQ ID NO: 33 for numbering the mentioned position of X1; refer to SEQ ID NO: 34 for numbering the mentioned position of X3; and refer to SEQ ID NO: 35 for the mentioned position of X4 number.

In some embodiments, amino acid substitutions relative to SEQ ID NO: 33 do not occur at positions 7E, 10L, 11Y, 12D, and 14L; amino acid substitutions relative to SEQ ID NO: 34 do not occur at position 1L , 4Y, 7N, 10L, 11I and 15I; relative to SEQ ID NO: 35, amino acid substitution does not occur at positions 12I, 16Q and 18W.

In some embodiments, X1 is a peptide comprising an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence KIQLHAEHALYDALMILNI (SEQ ID NO: 33); A peptide of amino acid; The amino acid sequence EDEQEEMANAIITILQSWIFS (SEQ ID NO: 35) is at least 90%, at least 95% or 100% identical to the amino acid sequence; wherein the amino acid sequence identical to EHALYDAL (SEQ ID NO: 30) is included in SEQ ID NO: 33; the amino acid sequence consistent with YAFNFELI (SEQ ID NO:31) is included in SEQ ID NO: 34; and the amino acid sequence consistent with ITILQSWIF (SEQ ID NO:32) is included in SEQ ID NO: 35 within.

As mentioned herein, domain X2 is a structural domain, and therefore any amino acid sequence that connects relevant other domains (depending on domain order) and allows its folding can be used. The required length will depend on the structure of the protein produced and can be 8 amino acids or longer, and in some aspects, 19 amino acids or longer. In some such aspects, X2 is no more than 200 or 100 or 50 amino acids in length. In any embodiment of the IL-2 mimetic provided herein, X2 can be a peptide comprising an amino acid sequence that is at least 90%, at least 94%, or 100% identical to the sequence KDEAEKAKRMKEWMKRIKT (SEQ ID NO: 36). In some aspects, the amino acid of SEQ ID NO: 36 is mutated to a cysteine residue. In some aspects, the amino acid is at one of positions 1, 2, 5, 9, 12, or 16 of SEQ ID NO: 36.

Exemplary IL-2 receptor agonists of the present invention comprise at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% of the amino acid sequence described in SEQ ID NO: 37 %, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequences. SEQ ID NO: 37 (NEO 2-15)： PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARL FESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS

In some such embodiments, at least 10, 11, 12, 13, or all 14 of the following are not true: position 7 is I, position 8 is T or M, position 11 is E, position 14 is K, position 18 is S, position 33 is Q, position 36 is R, position 37 is F, position 39 is K, position 40 is R, position 43 is R, position 44 is N, position 46 is W and position 47 is G. In yet another embodiment, one or both of the following are not true: position 68 is I and position 98 is F.

In some aspects, the IL-2 mimetic polypeptides of any embodiment disclosed herein bind to the IL-2 receptor _βƔc heterodimer (IL- _2RβƔc ) with a binding affinity (as determined by, e.g., Silva et al., The K _D ) measured by SPR as detailed in Nature 2019 is 200 nM or less, 100 nM or less, 50 nM or less, or 25 nM or less. In some aspects, the binding affinity is between 1 and 200 nM, specifically between 5 and 100 nM, more specifically between 10 and 100 nM, between 10 and 50 nM or between 5 and 50 nM. between 50 nM.

Cysteine residues present in the IL-2 mimetics described herein can be used to attach a moiety (eg, a stabilizing moiety, such as, for example, a water-stable moiety (such as a PEG-containing moiety)) to a polypeptide. The cysteine moiety can be in any of X1, X2, X3 or X4 or in the linker as appropriate. In some aspects, the cysteine moiety is in X2. For example, an exemplary IL-2 receptor agonist of the invention is a polypeptide in which the amino acid of Neo-2/15 is mutated to a cysteine residue such that a moiety (e.g., a stability moiety, such as (e.g., e.g., ) to which a water-stable moiety (such as a PEG-containing moiety) is attached. In some aspects, amino acid mutations at one or more of positions 50, 53, 56, 58, 59, 62, 66, 69, 73, 77, 82, or 85 of SEQ ID NO: 37 into a cysteine residue to allow a moiety (eg, a PEG-containing moiety) to be attached thereto.

Exemplary IL-2 receptor agonists of the present invention comprise at least 90%, at least 91%, and the amino acid sequence described in any one of SEQ ID NO: 38-49 as shown in Table 4 below. An amino acid sequence that is at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. Table 4 R50C SEQ ID NO: 38 PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS E53C SEQ ID NO: 39 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS D56C SEQ ID NO: 40 PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS K58C SEQ ID NO: 41 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS D59C SEQ ID NO: 42 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS E62C SEQ ID NO: 43 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS R66C SEQ ID NO: 44 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS E69C SEQ ID NO: 45 PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKCWMKRIKTTASEDEQEEMANAIITILQSWIFS R73C SEQ ID NO: 46 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS T77C SEQ ID NO: 47 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTCASEDEQEEMANAIITILQSWIFS E82C SEQ ID NO: 48 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS E85C SEQ ID NO: 49 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQECMANAIITILQSWIFS

IL-2 receptor agonists of the present invention include polypeptides that comprise at least 90%, at least 91%, or at least 92% of the amino acid sequence described in any one of SEQ ID NO. 38-49 , an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical. In illustrative of such embodiments, mutated cysteines (i.e., D56C; K58C; D59C; R66C; T77C; E85C; R50C; E53C; E62C; E69C; R73C; and/or E82C) are present and optionally mutated The cysteine is linked to a stabilizing moiety, such as, for example, a water-stable moiety (such as a PEG-containing moiety), as described herein. In some embodiments, at least 10, 11, 12, 13, or all 14 of the following are not true: position 7 is I, position 8 is T or M, position 11 is E, position 14 is K, position 18 is S, Position 33 is Q, position 36 is R, position 37 is F, position 39 is K, position 40 is R, position 43 is R, position 44 is N, position 46 is W and position 47 is G (numbering refers to SEQ ID NO : any of 38-49). In yet another embodiment, one or both of the following are not true: position 68 is I and position 98 is F (numbering refers to any of SEQ ID NO: 38-49).

Exemplary PEGylated IL-2 receptor agonists of the invention include polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 43 (NEO 2-15 E62C), wherein the cysteine at position 62 is PEGylated. The polyvinyl groups may be attached via any suitable attachment chemistry, including, for example, with maleimide (eg, maleimide-modified PEG (PEG-MAL) 5 kD; PEG- MAL 20 kD; or PEG-MAL 40 kD). In some embodiments, PEG-MAL 30 kD is used for PEGylation. In some embodiments, modified PEG-MAL 40 kD is used for PEGylation. In some embodiments, the repeating PEG units in the PEGylated peptide of SEQ ID NO: 43 range from about 800-1000. In some embodiments, the average number of repeating PEG units in the PEGylated peptide of SEQ ID NO: 43 is about 850-950. Those skilled in the art will understand that the PEG moiety may be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the invention include polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 48 (NEO 2-15 E82C), wherein the cysteine at position 82 is PEGylated. The polyethylene groups may be attached via any suitable attachment chemistry, including, for example, with maleimide-modified PEG (PEG-MAL) 5 kD; PEG-MAL 20 kD; or PEG-MAL 40 kD. In some embodiments, PEG-MAL 30 kD is used for PEGylation. In some embodiments, PEG-MAL 40 kD is used for PEGylation. In some embodiments, the repeating PEG units in the PEGylated peptide of SEQ ID NO: 48 range from about 800-1000. In some embodiments, the average number of repeating PEG units in the PEGylated peptide of SEQ ID NO: 48 is about 850-950. Those skilled in the art will understand that the PEG moiety may be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the invention include polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 45 (NEO 2-15 E69C), wherein the cysteine at position 69 is PEGylated. The polyvinyl groups may be attached via any suitable attachment chemistry, including, for example, using maleimide-modified PEG (e.g., PEG-MAL 5 kD; PEG-MAL 20 kD; or PEG-MAL 40 kD). In some embodiments, PEG-MAL 30 kD is used for PEGylation. In some embodiments, modified PEG-MAL 40 kD is used for PEGylation. In some embodiments, the repeating PEG units in the PEGylated peptide of SEQ ID NO: 45 range from about 800-1000. In some embodiments, the average number of repeating PEG units in the PEGylated peptide of SEQ ID NO: 45 is about 850-950. Those skilled in the art will understand that the PEG moiety may be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the invention include polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 46 (NEO 2-15 R73C), wherein the cysteine at position 73 PEGylated. The polyethylene groups may be attached via any suitable attachment chemistry, including, for example, with maleimide-modified PEG (PEG-MAL) 5 kD; PEG-MAL 20 kD; or PEG-MAL 40 kD. In some embodiments, PEG-MAL 30 kD is used for PEGylation. In some embodiments, modified PEG-MAL 40 kD is used for PEGylation. In some embodiments, the repeating PEG units in the PEGylated peptide of SEQ ID NO: 46 range from about 800-1000. In some embodiments, the average number of repeating PEG units in the PEGylated peptide of SEQ ID NO: 46 is about 850-950. Those skilled in the art will understand that the PEG moiety may be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the invention include polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 44 (NEO 2-15 R66C), wherein the cysteine at position 66 is PEGylated. The polyethylene groups may be attached via any suitable attachment chemistry, including, for example, with maleimide-modified PEG (PEG-MAL) 5 kD; PEG-MAL 20 kD; or PEG-MAL 40 kD. In some embodiments, PEG-MAL 30 kD is used for PEGylation. In some embodiments, modified PEG-MAL 40 kD is used for PEGylation. In some embodiments, the repeating PEG units in the PEGylated peptide of SEQ ID NO: 46 range from about 800-1000. In some embodiments, the average number of repeating PEG units in the PEGylated peptide of SEQ ID NO: 46 is about 850-950. Those skilled in the art will understand that the PEG moiety may be linear or branched.

The polypeptides and peptide domains described herein may include additional residues at the N-terminus, the C-terminus, or both; such additional residues are not included in determining the identity of the polypeptides or peptide domains of the invention relative to a reference polypeptide. in percentage. Such residues may be any residues suitable for the intended use, including (but not limited to) detection labels (i.e. fluorescent proteins, antibody epitope tags, etc.), adapters, ligands suitable for purification purposes (His tag, etc.), adding functional groups to other peptide domains of the polypeptide, etc. As disclosed in U.S. Patent Nos. 9,676,871 and 9,777,070, residues suitable for attachment of such groups may include cysteine, lysine, or p-acetylphenylalanine residues or may be labels, Such as amino acid markers suitable for reaction with transglutaminase.

Combination Kits and Compositions Combination Kits Combinations of the invention as previously defined comprising an anti-BTN3A antibody and an IL-2 agonist may be presented in the form of a combination kit. As used herein, the term "combination kit" or "kit of parts" means a pharmaceutical composition or composition for administration of an anti-BTN3A antibody and an IL-2 agonist of the invention. When the two compounds are administered simultaneously, the combination kit may contain, for example, the components (suitably an anti-BTN3A antibody and an IL-2 agonist) in separate pharmaceutical compositions. When the components (suitably anti-BTN3A antibody and IL-2 agonist) are not administered simultaneously, the combination set will be in separate pharmaceutical compositions (either in a single kit or in separate pharmaceutical compositions in separate kits). ) contains active agents.

In one aspect, a partial package is provided that contains: an anti-BTN3A antibody associated with a pharmaceutically acceptable excipient, diluent or carrier, typically a composition comprising an anti-BTN3A antibody as previously defined; and

The IL-2 agonist is associated with a pharmaceutically acceptable excipient, diluent and/or carrier, typically a composition comprising an IL-2 agonist as previously defined.

In one embodiment of the invention, the set of parts includes: an anti-BTN3A antibody associated with a pharmaceutically acceptable excipient, diluent and/or carrier, typically a composition comprising an anti-BTN3A antibody as previously defined; and IL-2 agonist in association with a pharmaceutically acceptable excipient, diluent and/or carrier, typically a composition comprising an IL-2 agonist as previously defined The components are provided in a form suitable for sequential, separate and/or simultaneous administration.

In one embodiment, the set of parts includes: a first container comprising an anti-BTN3A antibody associated with a pharmaceutically acceptable excipient, diluent and/or carrier, typically a composition comprising an anti-BTN3A antibody as previously defined; and A second container and container means for containing the first and second containers, the second container containing an IL-2 agonist associated with a pharmaceutically acceptable excipient, diluent and/or carrier agent, typically a composition comprising an IL-2R agonist as previously defined.

Combination kits may also be provided with instructions, such as dosage and administration instructions. Such dosage and administration instructions may be of the type provided to the physician, or they may be of the type provided by the physician, such as instructions provided to the patient.

Compositions Thus, according to the present invention, for example, an anti-BTN3A antibody as defined herein and an IL-2 agonist as defined herein may be formulated separately into separate compositions (eg pharmaceutical compositions), wherein the anti-BTN3A antibody and/or the IL-2 agonist is formulated together with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" means a diluent, adjuvant or excipient and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents that are physiologically compatible agents, isotonic and absorption delaying agents and the like. In addition to the active compound (ie, anti-BTN3A antibody and/or IL-2 agonist), the composition may further comprise one or more of the following compounds.

The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous route or intratumoral injection. Depending on the route of administration, the anti-BTN3A antibody as previously defined may be coated in a material to protect the compound from acids and other natural conditions that may inactivate the compound.

Sterile phosphate buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known in the art. (Remington and Gennaro, 1995) The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss from the vial surface, and the like.

The form, route of administration, dosage and regimen of the pharmaceutical composition naturally depend on the condition to be treated, the severity of the disease, the age, weight and gender of the patient, etc.

Pharmaceutical compositions of the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and similar modes of administration.

Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for injectable formulations. Such vehicles may in particular be isotonic sterile physiological saline solutions (monobasic or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride and the like or mixtures of such salts), or Drying, in particular freeze-drying, of the composition allows reconstitution into injectable solutions upon the addition of sterile water or physiological saline, as appropriate.

The dosage for administration may be adapted according to various parameters and, inter alia, according to the mode of administration used, the associated pathology or alternatively the desired duration of treatment.

To prepare a pharmaceutical composition, an effective amount of anti-BTN3A antibody and/or IL-2 agonist can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

Injectable compositions are preferably sterile. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders or lyophilisates for ready-to-use preparations of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that ease of injectability exists. The form must be stable under the conditions of manufacture and storage, and must be protected against the contaminating effects of microorganisms, such as bacteria and fungi.

Solutions of the active compounds in the form of the free base or pharmacologically acceptable salts can be prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols such as glycerol, propylene glycol and liquid polyethylene glycols and the like, suitable mixtures thereof, and vegetable oils. For example, proper flowability can be maintained by using coatings such as lecithin, by maintaining the desired particle size for dispersions, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In most cases it will be preferable to include an isotonic agent such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of an absorption-delaying agent in the composition, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent, together with various other ingredients enumerated above, as appropriate, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilizing active ingredients into a sterile vehicle that contains an alkaline dispersion medium and the required other ingredients from those enumerated above. In the case where sterile powders are used to prepare sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional required ingredients from a previously sterile filtered solution.

The preparation of more or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisaged to produce extremely fast penetration, delivering high concentrations of active agent into small tumor areas.

When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in various dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like may also be employed.

For example, for parenteral administration in the form of an aqueous solution, the solution should be appropriately buffered if necessary, and the liquid diluent first made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that may be employed in accordance with the present invention will be known to those skilled in the art. For example, a single dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the recommended infusion site (see, e.g., "Remington's Pharmaceutical Sciences", 15th ed., p. Pages 1035-1038 and 1570-1580). Depending on the condition of the individual being treated, some dosage variations will necessarily occur. In any case, the appropriate dose for the individual individual will be determined by the person responsible for administration. Parenteral compositions may be enclosed in ampoules, disposable syringes or multi-dose vials made of glass, plastic or other materials.

In addition to compounds formulated for parenteral administration (such as intravenous or intramuscular injection), other pharmaceutically acceptable forms include, for example, lozenges or other solids for oral administration; time-release capsules; and any other form currently in use. When the composition is in the form of a capsule (eg, a gelatin capsule), it may contain, in addition to the above types of materials, a liquid carrier such as polyethylene glycol, cyclodextrin or fatty oil. The compositions may be in liquid form, such as elixirs, syrups, solutions, emulsions or suspensions. Liquids can be used for oral administration or for delivery by injection. When intended for oral administration, the composition may include one or more of sweeteners, preservatives, dyes/colorants, and flavoring agents. Compositions for administration by injection may also include one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers and isotonic agents. Delayed release capsules are also covered, including those with enteric coating.

In certain embodiments, the use of liposomes and/or nanoparticles to introduce antibodies into host cells is contemplated. The forms and uses of liposomes and/or nanoparticles are known to those skilled in the art.

Nanocapsules can generally encapsulate compounds in a stable and reproducible manner. To avoid side effects caused by intracellular polymer overload, such ultrafine particles (approximately 0.1 µm in size) are generally designed using polymers that can degrade in vivo. Biodegradable polyalkyl cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be readily produced.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilamellar vesicles (also known as multilamellar vesicles (MLV)). MLVs generally have diameters from 25 nm to 4 µm. Sonic treatment of MLV results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å and containing aqueous solution at the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

The IL-2 agonist and/or anti-BTN3A antibody may be the only active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for the intended use.

Compositions and Dosing Regimen Comprising Anti -BTN3A Antibodies In some embodiments, an anti-BTN3A antibody as defined herein can thereby be formulated in the form of a composition (e.g., a pharmaceutical composition as defined above) containing One or a combination of the disclosed antibodies, for example, an antibody selected from the group consisting of mAb1, mAb2, mAb4 and mAb5 or the antigen-binding portion thereof, is formulated together with a pharmaceutically acceptable carrier.

The antibodies of the invention may be formulated in neutral or salt form into compositions as defined above. Pharmaceutically acceptable salts include acid addition salts (formed from free amine groups of proteins) and are prepared from inorganic acids such as, for example, hydrochloric acid or phosphoric acid or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. similar to acid) formation. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; and organic bases such as isopropylamine, trimethylamine, histamine Acids, procaine and similar bases. Suitable formulations of solutions for infusion or subcutaneous injection 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). In preferred embodiments, the anti-BTN3A antibody as defined above is formulated for intravenous infusion.

Pharmaceutical compositions containing activated anti-BTN3A antibodies can be formulated at different concentrations. For example, the formulation may comprise a concentration between 0.1 μM and 1 mM, more preferably between 1 μM and 500 μM, between 500 μM and 1 mM, between 300 μM and 700 μM, between 1 Between μM and 200 μM, between 100 μM and 200 μM, between 200 μM and 300 μM, between 300 μM and 400 μM, between 400 μM and 500 μM, between 500 μM and 600 μM between 600 μM and 700 μM, between 800 μM and 900 μM, or between 900 μM and 1 mM. Typically, formulations comprise activated anti-BTN3A antibody at a concentration between 300 μM and 700 μM.

Typically, therapeutic doses of activated anti-BTN3A antibodies in human patients will range from 100 pg to 700 mg per administration (based on 70 kg body weight). For example, the maximum therapeutic dose may range from 0.1 to 10 mg/kg per administration, such as between 0.1 and 5 mg/kg or between 1 and 5 mg/kg or between 0.1 and 2 mg/kg between. It is understood that such dosages may be administered at various intervals as determined by the oncologist/physician; for example, dosages may be daily, twice weekly, once weekly, every two weeks, every three weeks, or monthly. Invest.

Typically, in specific embodiments, the activating anti-BTN3A antibody is present in a dose of between 20 μg and 200 mg, specifically between 1 mg and 200 mg or at 7 mg per dose (typically every 21 days) Doses between 200 mg and 200 mg are administered intravenously.

In specific embodiments, suitable dosages for intravenous administration of activated anti-BTN3A antibodies may be selected from the group consisting of 1, 7, 10, 20, 50, 75, 100, 125, 150, 175 and 200 mg.

In specific embodiments, an activated anti-BTN3A antibody (preferably mAbl as described below) for use according to the methods of the invention is administered intravenously at a dose of between 20 μg and 200 mg per dose, less than Preferably the second dose is administered at least 15 days (typically after about 21 days) after the first dose.

Compositions and Dosage Regimes Comprising IL-2 Agonists Pharmaceutical compositions comprising the IL-2 agonists of the invention can be formulated such that the IL-2 agonist is a biological agent when the composition is administered to a patient. usable. IL-2 agonists are available as solutions, suspensions, emulsions, microgranules, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, lotions, aerosols, sprays , suspension form or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient in a pharmaceutical composition will depend on a variety of factors. Relevant factors include, but are not limited to, the type of animal (eg, human), the specific form of the IL-2 receptor agonist, the mode of administration, and the composition used.

An IL-2 agonist as defined herein may be administered by any convenient route, such as by infusion or bolus injection. Can be administered systemically or locally. Typical routes of administration include, but are not limited to, oral, topical, parenteral, sublingual, transrectal, transvaginal, ocular, intratumoral, and intranasal. Parenteral administration includes subcutaneous, intravenous, intramuscular, intrabrainstem injection or infusion techniques. In one aspect, the IL-2 agonist is administered parenterally. In another aspect, the IL-2 agonist is administered intravenously or subcutaneously. In specific embodiments, the IL-2 agonist is administered topically to the area in need of treatment. In one embodiment, administration may be by direct injection at the site of (or formation of) cancer, tumor, or neoplastic or pre-neoplastic tissue. In another embodiment, administration may be by direct injection at the site where the autoimmune disease manifests (or develops). An example of local administration is infusion via a catheter, such as intravesical infusion.

A suitable dosage range for IL-2R agonists and in particular for IL-2 mimetics as defined herein may, for example, be 0.1 ug/kg to 100 mg/kg body weight; alternatively, it may be 0.5 ug /kg to 50 mg/kg; 1 ug/kg to 25 mg/kg or 5 ug/kg to 10 mg/kg body weight. In some embodiments, an exemplary dosage is about 2 ug/mg to about 15 ug/kg. In some embodiments, the dosing regimen includes providing the IL-2 receptor agonist based on a 21-day cycle, with administration occurring once or twice during the 21-day cycle.

Uses and Methods of Combinations of the Invention The present invention provides a therapeutic combination comprising an anti-BTN3A antibody as previously defined and an IL-2 agonist for the treatment of cancer.

The invention also provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an anti-BTN3A activating antibody and a therapeutically effective amount of an IL2 agonist in combination, simultaneously, sequentially, or separately. Agonists bind to the IL-2 receptor βγc heterodimer and (i) have reduced affinity for IL-2Rα (CD25) or (ii) have no binding site for IL-2Rα.

As used herein, the term "treat" refers to one or more of the following: (1) Suppression of disease; e.g., suppression of disease in an individual who is experiencing or exhibiting lesions or symptoms of a disease, condition or disorder , a condition or disorder (i.e., arresting further progression of the disease, condition, or symptoms); and (2) alleviating the disease; e.g., alleviating the disease, condition, or disorder in an individual who is experiencing or exhibiting the lesions or symptoms of the disease, condition, or disorder (also i.e., reversing the disease and/or symptoms), such as reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. In particular, with reference to the treatment of tumors, the term "treatment" may refer to inhibiting tumor growth or reducing tumor size.

The antibodies of the invention are anti-BTN3A activating antibodies and can activate lytic function, interleukin production and/or proliferation of Vγ9Vδ2 T cells, and can thus be used to overcome cancer in patients (see in particular WO2012080769, WO2012080351 and WO2020025703) and in Immunosuppressive mechanisms observed during chronic infection. The results of the present invention now demonstrate that this combination further promotes synergistic and specific Vγ9Vδ2 T cell expansion in human PBMCs, highlighting their therapeutic importance, particularly for the treatment of cancer.

As used herein, the terms "cancer", "hyperproliferation" and "neoplastic" refer to an abnormal state or condition characterized by the ability of cells to grow autonomously, i.e., rapid proliferative cell growth. Hyperproliferative and neoplastic disease conditions may be classified as pathological, that is, characterizing or constituting the disease condition, or non-pathological, that is, as deviations from normal but not associated with the disease condition. The term is intended to include all types of cancerous growths or carcinogenic processes, metastatic tissue or malignantly transformed cells, tissues or organs regardless of histopathological type or stage of invasion.

The term "cancer" or "neoplasia" includes malignancies of various organ systems, such as those affecting the lungs, breasts, thyroid, lymphocytes, gastrointestinal tract, and urogenital tract; and adenocarcinoma, which includes malignancies such as most colon cancers, renal Cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, small intestine cancer and esophageal cancer.

Examples of cancers include, but are not limited to, hematological malignancies such as B-cell lymphoma, T-cell lymphoma, non-Hodgkin's lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL) ), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK cell lymphoma and myeloid cell line tumors (including acute myeloid leukemia).

Examples of non-blood cancers (i.e., solid tumors) include, but are not limited to, colon cancer, breast cancer, lung cancer, brain cancer, prostate cancer, head and neck cancer, pancreatic cancer, bladder cancer, colorectal cancer, bone cancer, cervical cancer, Ovarian cancer, liver cancer, oral cancer, esophageal cancer, thyroid cancer, kidney cancer, stomach cancer, testicular cancer and skin cancer.

Each therapeutic agent in the combination therapy of the present invention (i.e., anti-BTN3A antibody and IL-2 agonist as previously defined) can be used simultaneously (i.e., in the same drug), in parallel (i.e., in separate drugs), and One drug is administered immediately after another drug in any order) or sequentially in any order. Anti-BTN3A antibodies and IL-2 agonists are typically formulated into separate compositions as previously described. One or more compositions may be administered orally or parenterally, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration as previously detailed.

In the combination therapy of the present invention, the BTN3A antibody and IL-2 agonist can be administered orally or parenterally independently, including intravenous, intramuscular, intraperitoneal, subcutaneous, transrectal, topical and transdermal administration. way.

When the therapeutic agents in a combination therapy are in different dosage forms (one agent is a tablet or capsule and the other agent is a sterile liquid) and/or are administered according to different dosing schedules (e.g., a chemotherapeutic agent is administered at least daily and a biological agent is administered at least daily) Sequential administration (eg, in separate pharmaceutical compositions) is particularly useful when the therapeutic agent is administered less frequently, such as once a week, once every two weeks, or once every three weeks).

In some embodiments, the anti-BTN3A activating antibody is administered before the IL-2 agonist, while in other embodiments the anti-BTN3A activating antibody is administered after the IL-2 agonist is administered.

The dosage regimen selected for use in combination therapies of the invention (also referred to herein as a dosing regimen) depends on several factors, including the entity's serum or tissue turnover rate, the extent of symptoms, the immunogenicity of the entity, and the individual being treated. Accessibility of target cells, tissues or organs. Preferably, the dosing regimen maximizes the amount of each therapeutic agent delivered to the patient based on acceptable side effects. Accordingly, the dosage and frequency of administration of each biotherapeutic and chemotherapeutic agent in the combination will depend, in part, on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance is available on selecting appropriate doses of antibodies, interleukins, 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 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 ed.); Medical Economics Company; ISBN : 1563634457; Edition 57 (November 2002). Appropriate dosing regimens may be determined by the clinician, e.g., using 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., prior therapies), the conditions to be treated, It depends on the type and stage of the cancer and the biological markers of response to one or more therapeutic agents in the combination therapy.

Any appropriate dosage range may be used as judged by the attending medical provider. Dosage regimens can be adjusted to provide the optimal desired response (eg, therapeutic or prophylactic response). A suitable dosage range for IL-2 agonists and in particular for IL-2 mimetics as defined herein may, for example, be 0.1 ug/kg to 100 mg/kg body weight; alternatively, it may be 0.5 ug /kg to 50 mg/kg; 1 ug/kg to 25 mg/kg or 5 ug/kg to 10 mg/kg body weight. In some embodiments, an exemplary dosage is about 2 ug/mg to about 15 ug/kg. In some embodiments, the dosing regimen includes providing the IL-2 receptor agonist based on a 21-day cycle, with administration occurring once or twice during the 21-day cycle.

In some embodiments, at least one therapeutic agent in the combination therapy is administered using the same dosing regimen (dose, frequency, and duration of treatment) typically used when the agent is used as monotherapy to treat the same cancer. In other embodiments, the patient receives a lower total amount (e.g., smaller doses, less frequent dosing, and/or shorter duration of treatment) of at least one of the combination therapies than when the agent is used as monotherapy. Therapeutic agents.

Regarding the dosing regimen of the anti-BTN3A antibody and IL-2 agonist of this treatment combination, any suitable dosage range may be used as determined by the attending medical practitioner.

Dosage regimens can be adjusted to provide the optimal desired response (eg, therapeutic or prophylactic response). The antibodies of the invention may be formulated in a therapeutic mixture to contain about 0.0001 to 100.0 mg, or about 0.001 to 10 mg, or about 0.0001 to 1.0 mg, or about 0.001 to 0.1 mg, or about 0.1 to 1.0 mg, or Even 1.0 to about 10 mg. Multiple doses can also be administered. Typically, therapeutic doses of activated anti-BTN3A antibodies in human patients will range from 100 pg to 700 mg per administration (based on 70 kg body weight). For example, the maximum therapeutic dose may range from 0.1 to 10 mg/kg per administration, such as between 0.1 and 5 mg/kg or between 1 and 5 mg/kg or between 0.1 and 2 mg/kg between. It is understood that such dosages may be administered at various intervals as determined by the oncologist/physician; for example, dosages may be daily, twice weekly, once weekly, every two weeks, every three weeks, or monthly Invest.

Typically, in specific embodiments, the activated anti-BTN3A antibody is administered intravenously at a dose of between 20 μg and 200 mg per dose (typically every 21 days).

In specific embodiments, suitable dosages for intravenous administration of activated anti-BTN3A antibodies may be selected from the group consisting of 1, 7, 10, 20, 50, 75, 100, 125, 150, 175 and 200 mg.

In a specific embodiment, the activated anti-BTN3A antibody (preferably mAbl as described below) for use according to the methods of the invention is administered intravenously at a dose of between 20 μg and 200 mg per dose, Preferably the second dose is administered at least 15 days (typically about 21 days later) after the first dose.

For IL-2 agonists, a suitable dosage range for the polypeptide may, for example, be 0.1 ug/kg to 100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg or 5 ug/kg to 10 mg/kg body weight. In some embodiments, an exemplary dosage is about 2 ug/mg to about 15 ug/kg. In some embodiments, the dosing regimen includes providing the IL-2 receptor agonist based on a 21-day cycle, with administration occurring once or twice during the 21-day cycle.

Whether in the form of a mixed single composition or separate compositions, a combination therapy comprising an anti-BTN3A antibody and an IL-2 agonist as previously defined may be further administered in combination with other drugs, for example, for treatment or prophylaxis the above diseases.

In specific embodiments, combination therapies as disclosed herein (typically mAbl and an IL-2 agonist, particularly an IL-2 mimetic as previously described) may be administered in combination with an anti-tumor agent.

In other specific embodiments, combination therapies as disclosed herein (typically mAbl and IL-2 agonists as previously described, particularly IL-2 mimetics) can be combined with cell therapies, particularly γδ T cell therapies. Invest.

In other specific embodiments, combination therapies as disclosed herein (typically mAbl and an IL-2 agonist as previously described, particularly an IL-2 mimetic) may be administered with an immunotherapeutic agent, such as Checkpoint inhibitors (especially anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies).

As used herein, the term "cell therapy" refers to therapy involving the in vivo administration of at least a therapeutically effective amount of a cellular composition to an individual in need thereof. The cells administered to the patient may be allogeneic or autologous. The term "γδ T cell therapy" refers to cell therapy in which the cellular composition includes γδ T cells (especially Vγ9Vδ2 T cells) as an active ingredient. In specific embodiments, the Vγ9Vδ2 T cells have been expanded and/or activated ex vivo.

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

The term "PD-1" has its ordinary meaning in the art and refers to the programmed death-1 receptor. The term "PD-1" also refers to a type I transmembrane protein, which belongs to the CD28-B7 signaling receptor family, which includes CD28, cytotoxic T-lymphocyte-associated antigen 4 (CTLA- 4), inducible costimulator (ICOS) and B- and T-lymphocyte attenuator (BTLA) (Greenwald RJ et al., 2005, Annual Review of Immunology, Vol. 23, No. 515 -page 548).

The term "anti-PD-1 antibody" or "anti-PD-L1" has its ordinary meaning in the art and refers to an antibody that has binding affinity for PD-1 or PD-L1, respectively, and has antagonistic activity against PD-1 , that is, it inhibits the signal transduction cascade related to PD-1 and inhibits PD-1 ligand binding (PD-L1; PD-L2). Such anti-PD-1 antibodies or anti-PD-L1 antibodies are preferably compared with other subtypes or isomers of the CD28-B7 signaling receptor family (CD28; CTLA-4; ICOS; BTLA), respectively. The greater affinity and potency of the interaction between substances inactivates PD-1. Tests and assays for determining whether a compound is a PD-1 antagonist are well known to those skilled in the art, such as Shaabani S, et al. (2015-2018). Expert Opin Ther Pat. 2018 Sep; 28 (9):665-678; described in Seliger, B. J. Clin. Med. 2019, 8 , 2168.

Examples of such anti-PD1 or anti-PDL1 antibodies include, but are not limited to, nivolumab, pembrolizumab, avelumab, durvalumab , cemiplimab (cemiplimab) or atezolizumab (atezolizumab).

Examples of such anti-CTLA4 antibodies include, but are not limited to, ipilimumab.

Another therapeutic strategy is based on utilizing the properties of the combinations disclosed herein as agents to selectively expand and/or activate Vγ9Vδ2 T cells isolated from human individual samples.

The present invention thus relates to a method for treating an individual in need thereof, comprising: (a) Isolation of blood cells containing Vγ9Vδ2 T cells, such as PBMC from a blood sample of an individual, (b) Cultivate the isolated blood cells in an appropriate cell culture medium, and add effective amounts of anti-BTN3A antibodies and IL-2 agonists to the cell culture simultaneously, in parallel, or sequentially, and, as appropriate, add other tumors or accessories cells, and thereby obtain expanded Vγ9Vδ2 T cells, (c) Collect the expanded Vγ9Vδ2 T cells, (d) If appropriate, deploy the expanded Vγ9Vδ2 T cells and administer a therapeutically effective amount of such Vγ9Vδ2 T cells to the individual.

The present invention further relates to the use of the combination disclosed herein for selective expansion of Chimeric Antigen Receptor (CAR) Vγ9Vδ2 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).

The invention also relates to a combination as defined herein for in vivo use to enhance tumor cells in gamma delta T cell therapy in an individual in need thereof, typically suffering from cancer, wherein an anti-BTN3A antibody and an IL-2 agonist It can be administered to individuals simultaneously, in parallel or sequentially.

As used herein, the term γδ T cell therapy refers to therapy comprising the administration of at least an effective amount of γδ T cells to an individual in need thereof. Such γδ T cells can be allogeneic or autologous. In specific embodiments, γδ T cells can be genetically engineered by deletion or knockout or insertion or knock-in of specific genes. In specific embodiments, the γδ T cells include γδ T cells expressing chimeric antigen receptors. γδ T cells may have been expanded and/or purified ex vivo. Alternatively, γδ T cells may be included in a cell composition that includes other blood cells and, for example, other cells of the immune system. For references on gamma delta T cell therapy, see Pauza CD. et al., Front Immunol. 2018 Jun 8;9:1305. doi: 10.3389, Saudemont A. et al., Front Immunol. 2018 Feb 5 ;9:153. doi: 10.3389.

The present invention thus relates to a method of treating an individual suffering from cancer, including solid tumors or hematological malignancies, particularly leukemias (such as acute myeloid leukemia) and having tumor cells (e.g., hematological tumor cells), comprising: i. Administering to the subject an effective amount of an anti-BTN3A antibody as disclosed herein (typically mAb1, mAb2, mAb4, or mAb5) in combination with an effective amount of an IL-2 agonist, wherein the anti-BTN3A antibody and IL-2 2 Agonists may be administered simultaneously, concurrently, or sequentially, and, ii. Administer an effective amount of the gamma delta T cell composition to the individual, The combination of the effective amount of anti-BTN3A antibody and the effective amount of IL-2 agonist has the ability to enhance the anti-tumor cell lysis mediated by the γδ T cell composition against the tumor cells.

The invention further relates to a method for treating an individual suffering from cancer harboring solid tumor cells, such as ovarian cancer cells, the method comprising: i. Administer to the individual an effective amount of an anti-BTN3A antibody as disclosed herein (typically mAb1, mAb2, mAb4, or mAb5) in combination with an effective amount of an IL-2 agonist as defined herein, wherein the anti- The BTN3A antibody and IL-2 agonist can be administered simultaneously, concurrently, or sequentially, and, ii. Administer an effective amount of the gamma delta T cell composition to the individual, The combination of the effective amount of anti-BTN3A antibody and the effective amount of IL-2 agonist has the ability to enhance the anti-tumor cell lysis mediated by the γδ T cell composition against the tumor cells.

The invention having been fully described is now further illustrated by the following examples, which are illustrative only and are not intended to be further limiting. Table 5 : Sequences used in the present invention SEQ ID NO: Type Description of the sequence sequence 1 aa mAb 7.2 humanized heavy chain variable region VH2 QVQLVQSGAEVKKPGASVKLSCKASGYIFTRYYMYWVKQRPGQGLEWIGEINPNNGGTKFNEKFKNRATLTVDKSISTAYMELSRLRSDDTAVYYCSREDDYDGTPFAMDYWGQGTLVTVSS 2 aa mAb 7.2 humanized light chain variable region Vk1 DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASNLHTGVPSRFTGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK 3 aa mAb 7.2 humanized light chain variable region Vk2 DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIK 4 aa Full-length heavy chain of mAb 1 and mAb 2 (VH2 7.2 silenced IgG1) Question PSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 5 aa Full-length heavy chain of mAb 4 and mAb 5 (VH2 7.2 silenced IgG4) Question PSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 6 aa Full-length light chain (Vk1 7.2) of mAb 1 and mAb 4 DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASNLHTGVPSRFTGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC 7 aa Full-length light chain (Vk2 7.2) of mAb 2 and mAb 5 DIQMTQSPSSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC 8 nt Full-length heavy chain of mAb 1 and mAb 2 (VH2 7.2 silenced IgG1) CAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACATCTTCACCAGATACTATATGTATTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAATGGTGGTACTAAGTTCAATGAGAAGTTCAAGAACAGGGCCACACTGACTGTAGACAAATCCATCAGCACAGCATACATGGAGCTCAGCAGGCTGAGATCT GACGACACGGCGGTCTATTATTGTTCAAGAGGATGATTACGACGGGACCCCCTTTGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACCTTCCCGGCTG TCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGAACCACAGGTGTACACCCTGGCCCCATCCCCCC GAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC TCCGGGTTGA 9 nt Full-length heavy chain of mAb 4 and mAb 5 (VH2 7.2 silenced IgG4) CAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACATCTTCACCAGATACTATATGTATTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTAATCCTAACAATGGTGGTACTAAGTTCAATGAGAAGTTCAAGAACAGGGCCACACTGACTGTAGACAAATCCATCAGCACAGCATACATGGAGCTCAGCAGGCTGAGATCT GACGACACGGCGGCTATTATTGTTCAAGAGGATGATTACGACGGGACCCCCTTTGCTATGGACTACTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACCTTCCCGGCTG TCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAG GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGTCCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTC TCTGGGTTGA 10 nt Full-length light chain (Vk1 7.2) of mAb 1 and mAb 4 GACATCCAGATGACCCAGTCTCCATCCAGTCTGTCTGCATCCGTAGGAGACAGAGTCACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTATCTTGGTACCAGCAGAAACCAGGAAAAGCCCCTAAACTCTTGATCTATAAGGCTTCCAACTTGCACACAGGCGTCCCATCAAGATTTACTGGCAGTGGATCTGGAACAGATTTCACATTCACCATCAGCAGCCTGCAGCCTGAAGACATTGCCACTT ACTACTGTCAACAGGGTCAAACTTATCCATACACGTTCGGACAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 11 nt Full-length light chain (Vk2 7.2) of mAb 2 and mAb 5 GACATCCAGATGACCCAGTCTCCATCCAGTCTGTCTGCATCCGTAGGAGACAGAGTCACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTATCTTGGTACCAGCAGAAACCAGGAAAAGCCCCTAAACTCTTGATCTATAAGGCTTCCAACTTGCACACAGGCGTCCCATCAAGATTTAGTGGCAGTGGATCTGGAACAGATTTCACATTCACCATCAGCAGCCTGCAGCCTGAAGACATTGCCACTT ACTACTGTCAACAGGGTCAAACTTATCCATACACGTTCGGACAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC AGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG 12 aa HCDR1 of mAb 7.2, mAb 1, mAb 2, mAb 4 and mAb 5 RYYMY 13 aa HCDR2 of mAb 7.2, mAb 1, mAb 2, mAb 4 and mAb 5 EINPNNGGTKFNEKFKN 14 aa HCDR3 of mAb 7.2, mAb 1, mAb 2, mAb 4 and mAb 5 EDDYDGTPFAMDY 15 aa LCDR1 of mAb 7.2, mAb 1, mAb 2, mAb 4 and mAb 5 HASQNINVWLS 16 aa LCDR2 of mAb 7.2, mAb 1, mAb 2, mAb 4 and mAb 5 KASNLHT 17 aa LCDR3 of mAb 7.2, mAb 1, mAb 2, mAb 4 and mAb 5 QQGQTYPYT 18 aa mAb 20.1-HCDR1 RYYLY 19 aa mAb 20.1-HCDR2 EINPNNGGTKFNEKFKS 20 aa mAb 20.1-HCDR3 EDDYDGTPDAMDY twenty one aa mAb 20.1-LCDR1 HASQNINLWLS twenty two aa mAb 20.1-LCDR2 RASNLHT twenty three aa mAb 20.1-LCDR3 QQGHSYPYT twenty four aa Human BTN3A1 MKMASFLAFLLLNFRVCLLLLQLLMPHSAQFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELKWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHVDVKGYKDGGIHLECRSTGWYPQPQIQWSNNKGENIPTVEAPVVADGVGLYAVAAS VIMRGSSGEGVSCTIRSSLLGLEKTASISIADPFFRSAQRWIAALAGTLPVLLLLLGGAGYFLWQQQEEKKTQFRKKKREQELREMAWSTMKQEQSTRVKLLEELRWRSIQYASRGERHSAYNEWKKALFKPADVILDPKTANPILLVSEDQRSVQRAKEPQDLPDNPERFNWHYCVLGCESFISGRHYWEVEVGDRKEWHIGVCSKNVQRKGWVKMTPENGFWTM GLTDGNKYRTLTEPRTNLKLPKPPKKVGVFLDYETGDISFYNAVDGSHIHTFLDVSFSEALYPVFRILTLEPTALTICPA 25 aa Human BTN3A2 MKMASSLAFLLLNFHVSLLLVQLLTPCSAQFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELKWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSNLHVEVKGYEDGGIHLECRSTGWYPQPQIQWSNAKGENIPAVEAPVVADGVGLYEVAASVIMR GGSGEGVSCIIRNSLLGLEKTASISIADPFFRSAQPWIAALAGTLPILLLLLAGASYFLWRQQKEITALSSEIESEQEMKEMGYAATEREISLRESLQEELKRKKIQYLTRGEESSSDTNKSA 26 aa Human BTN3A3 MKMASSLAFLLLNFHVSLFLVQLLTPCSAQFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELRWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHIEVKGYEDGGIHLECRSTGWYPQPQIKWSDTKGENIPAVEAPVVADGVGLYAVAASVIMR GSSGGGVSCIIRNSLLGLEKTASISIADPFFRSAQPWIAALAGTLPISLLLLAGASYFLWRQQKEKIALSRETEREREMKEMGYAATEQEISLREKLQEELKWRKIQYMARGEKSLAYHEWKMALFKPADVILDPDTANAILLVSEDQRSVQRAEEPRDLPDNPERFEWRYCVLGCENFTSGRHYWEVEVGDRKEWHIGVCSKNVERKKGWVKMTPENGYWTMGLTD GNKYRALTEPRTNLKLPEPPRKVGIFLDYETGEISFYNATDGSHIYTFPHASFSEPLYPVFRILTLEPTALTICPIPKEVESSPDPDLVPDHSLETPLTPGLANESGEPQAEVTSLLLPAHPGAEVSPSATTNQNHKLQARTEALY 27 aa Cynomolgus macaque ( cynomolgus monkey) BTN3A1 extracellular domain for recombinant protein production MGSSLAFLLLSFHVCVLLLQLLMPHSAQFAVVGPPGPILAMVGEDADLPCHLFPTMSAETMELRWVSSNLRQVVNVYADGKEVEDRQSAAYRGRTSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHIDVKGYEDGGIHLECRSTGWYPQPQIRWSNDKGENIPAVEAPVFVDGVGLYAVAASVILR GSSGEGVSCTIRSSLLGLEKTTSISIAGHHHHHH 28 aa Cynomolgus macaque ( cynomolgus monkey) BTN3A2 extracellular domain for recombinant protein production MGSSLAFLLLNFHVSFFLVQLLTPCSAQFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELRWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRDDIAAGKAALRIHNVTASDSGKYLCYFQDADFYEKALVELKVAALGSNLHVEVKGYEDGGIHLECRSTGWYPQPKIQWSNAKGQNIPAVEAPVVADGVGLYAVAASVIMRGGS GESVSCIIRNSVLGLEKTASISIADHHHHHHH 29 aa Cynomolgus macaque ( cynomolgus monkey) BTN3A3 extracellular domain for recombinant protein production MANFLAFLLLNFRVCLLLVQLLTPCSAQFAVLGPHGPILAMVGEDVDLPCHLFPTMSAETMELRWVSSSLRQVVNVYSDGKEVEDRQSAPYRGRTSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHIEVKGYEDGGIHLECRSTGWYPQPQIQWSNTKGQHIPAVKAPVVADGVGLYAVAASVIMRGS SGEGVSCIIRNSLLGLEKTASISITDHHHHHH 30 aa peptide EHALYDAL 31 aa peptide YAFNFELI 32 aa polypeptide ITILQSWIF 33 aa polypeptide KIQLHAEHALYDALMILNI 34 aa polypeptide LEDYAFNFELILEEIARLFESG 35 aa polypeptide EDEQEEMANAIITILQSWIFS 36 aa polypeptide KDEAEKAKRMKEWMKRIKT 37 aa IL-2 agonist Neo-2/15 PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 38 aa IL-2 agonist R50C PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIACLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 39 aa IL-2 agonist E53C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFCSGDQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 40 aa IL-2 agonist D56C PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGCQKDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 41 aa IL-2 agonist K58C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQCDEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 42 aa IL-2 agonist D59C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKCEAEKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 43 aa IL-2 agonist E62C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEACKAKRMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 44 aa IL-2 agonist R66C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKCMKEWMKRIKTTASEDEQEEMANAIITILQSWIFS 45 aa IL-2 agonist E69C PKKKIQLHAEHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKCWMKRIKTTASEDEQEEMANAIITILQSWIFS 46 aa IL-2 agonist R73C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKCIKTTASEDEQEEMANAIITILQSWIFS 47 aa IL-2 agonist T77C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTCASEDEQEEMANAIITILQSWIFS 48 aa IL-2 agonist E82C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDCQEEMANAIITILQSWIFS 49 aa IL-2 agonist E85C PKKKIQLHAEHALYDALMILNIVKTNSPPAEEKLEDYAFNFELILEEIARLFESGDQKDEAEKAKRMKEWMKRIKTTASEDEQECMANAIITILQSWIFS 50 aa Human IL2 (P60568|21-153) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 51 aa Human IL15 (P40933|49-162) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS 52 aa Human IL15R subunit alpha (UniProtKB/Swiss-Prot: Q13261) MAPRRARGCRTLGLPALLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSR QTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

EXAMPLES Methods and Assays of the Invention Methods for Characterizing Anti-BTN3A Activating Antibodies Used According to the Invention

1.1 Binding affinity test : Multi-cycle kinetic test (SPR) can use Biacore T200 (serial number 1909913) instrument to run Biacore T200 evaluation software V2.0.1 (Uppsala, Sweden) for multi-cycle kinetic analysis of anti-BTN3A antibodies.

Purified antibodies were diluted to a concentration of 2 μg/ml in 2% BSA/PBS. At the beginning of each cycle, each antibody was captured on Protein A with a density (RL) of approximately 146.5 RU (theoretical to obtain an RMax of approximately 50 RU). After capture, the surface was stabilized before injection of BTN3A1 antigen (Sino Biological catalog number 15973-H08H). BTN3A1 was titrated in 0.1% BSA/HBS-P+ (working buffer) at two-fold dilutions ranging from 25 to 0.78 nM. The association period was monitored for 400 seconds and the dissociation period was monitored for 35 minutes (2100 seconds). Kinetic data were obtained using a flow rate of 50 μl/min to minimize any possible mass transfer effects. Regeneration of the Protein A surface was performed at the end of each cycle using two injections of 10 mM glycine-HCl pH 1.5. Two blank (without BTN3A1) and one single concentration analyte replicates were performed for each test antibody to check surface and analyte stability during kinetic cycles. The signal from reference channel Fc1 was subtracted from the signals of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surface. Additionally, each Fc was subtracted from the blank run to correct for any antigen-independent signal variables, such as offset. Use a one-to-one combination mathematical model to fit the sensing map with the overall RMax parameter and no bulk signal (constant RI = 0 RU).

1.2 Binding assay to human PBMC by flow cytometry Anti-BTN3A antibodies used according to the present invention can also be characterized for their binding to human PBMC (isolated from the blood of healthy donors). PBMC were isolated from the leukocyte layer using density centrifugation with Lymphocyte Separator (Axis-shield, Dundee, UK). PBMC are then frozen and stored at -80°C or in liquid nitrogen until needed.

Transfer 100 μl of cells (1×10 ⁶ cells/ml) into each well of a new U-shaped bottom 96-well plate, then centrifuge the plate and discard the supernatant.

Prepare a series of antibody dilutions (0.001 μg/ml to 150 μg/ml) in PBS 2 mM EDTA. Human PBMC were resuspended in 50 μl of the prepared diluted test antibody titration series.

After incubation in the dark for 30 minutes at 4°C, the plate was centrifuged and washed twice with 150 μl/well of PBS 2 mM EDTA, after which the wells were resuspended in 50 μl of PBS 2 mM EDTA 1/100 In a mixture of diluted goat anti-human antibody (PE labeled) and Live/dead pure IR diluted 1/500.

After incubation in the dark at 4°C for 15 minutes, the plates were centrifuged and washed once with 150 μl/well PBS 2 mM EDTA, after which the wells were resuspended in 200 μl PBS 2 mM EDTA. Cells were analyzed on a BD LSR Fortessa cell counter. Data were analyzed using FlowJo software (version 10, FlowJo, LLC, Ashland, USA).

The same protocol can be performed on cynomolgus monkey PBMC and on the Daudi Burkitt's lymphoma cell line.

1.3 In vitro functional efficacy : γδ-T cell degranulation test The test consists of the following: measuring the activation or inhibition effect of anti-BTN3A antibody on γδ-T cell degranulation against Davoudi Burkitt's lymphoma cell line (Harly et al., 2012). γδ-T cells were expanded from PBMCs from healthy donors by culturing with zoledronic acid (1 μM) and IL2 (200 Ui/ml) for 11-13 days. IL2 was added on days 5, 8 and every 2 days thereafter. The percentage of γδ-T cells was determined at the beginning of culture and culture time assessed by flow cytometry until it reached at least 80%. Frozen or freshly prepared γδ-T cells were subsequently used in degranulation assays against the Daoudi cell line (E:T ratio 1:1), in which cells were incubated at 37°C at 10 μg/ml 7.2 and/or 20.1 human culture variants and/or chimeric versions thereof for 4 hours. Activation by PMA (20 ng/ml) plus ionomycin (1 µg/ml) served as a positive control for γδ-T cell degranulation, and medium alone served as a negative control. At the end of the 4-hour co-incubation, cells were analyzed by flow cytometry to evaluate the number of γδ-T cells positive for CD107a (LAMP-1, lysosome-associated membrane protein-1) + CD107b (LAMP-2). percentage. CD107 moves to the cell surface following activation-induced granule extracellular secretion, whereby measurement of surface CD107 is a sensitive marker for identifying recently degranulated lytic T cells.

The same protocol can be performed using AML blasts isolated from the patient as target cells instead of Daoudi cells.

1.4 In vitro functional efficacy : The Vγ9Vδ2 T cell activation assay in PBMC consists of the following: measuring the activation effect of anti-BTN3A antibodies on Vγ9Vδ2 T cells in PBMC. Human PBMCs were isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA-leukocyte layer or heparinized whole blood). When whole blood was used, RBCs were depleted using 1X RBC lysis buffer (eBioscience) for 10 minutes at room temperature and subsequently washed with PBS 1% FBS.

PBMC- or RBC-depleted cells were prepared in RPMI with increasing concentrations of anti-BTN3A antibody (dose range 0.00001 to 100 μg/mL) in _a round-bottom 96-well plate in a volume of 200 μL at 37 °C in 5% CO 1640 Cultured at 1.5 to 3 × 10 ⁶ cells/mL in 10% FBS, 1% P/S. Activation status was monitored after two days of culture by flow cytometric analysis of surface expression of activation markers. Wash cells in PBS 2% FBS 2 mM EDTA (FACS buffer). Cells were centrifuged at 1800 rpm for 5 min and subsequently incubated with 10 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at RT before adding 30 to 50 μL of the appropriate antibody mixture in FACS buffer. Prepared and containing at least fluorescently conjugated anti-CD3, anti-Vγ9 or Vδ2 TCR and anti-CD69 antibodies. A viability marker (LIVE/DEAD fixable dead cell dye) was added to all experiments to exclude dead cells from the analysis. Cells were incubated at 4°C for 30 minutes and washed twice in FACS buffer before being fixed in Cytofix fixation buffer (BD Bioscience) and analyzed by flow cytometry. Data were analyzed using flowjo V-10.6 software. Activated Vγ9Vδ2 T cells are defined as CD3+Vδ2+ (or Vγ9+ or Vδ2+Vγ9+) CD69+.

Method 2.1 for Characterizing IL-2 Agonists for Use According to the Invention Preparation of Exemplary PEGylated IL-2 Mimics : By introducing a cysteine residue that is subsequently combined with a single 40 kDa via maleic acid residue Amine-modified polyethylene glycol conjugation) produced NL-201 from Neo-2/15. Neo-2/15 stocks with a single mutation at position 62 were dialyzed into phosphate buffer (pH 7.0) and adjusted to 1.0-2.0 mg/ml. TCEP was added to the protein at a 10:1 molar ratio and incubated at RT for 10 minutes to reduce disulfide bonds. Add maleimide-modified PEG40k (PEG40k-MA) or PEG30k (PEG30k-MA) powder directly to the reduced protein solution at a molar ratio of 10:1 PEG cysteine and incubate under stirring. 2 hours. SDS-PAGE aliquots were taken directly from the reaction mixture. Formation of rapid, spontaneous, and nearly quantitative covalent bonds between PEG40k-MA or PEG30k-MA and Neo-2/15 cysteine mutations.

2.2 STAT5 phosphorylation study : In vitro study: Approximately 2 × 10 ⁵ YT-1, IL-2Rα ⁺ YT-1 or starved CTLL-2 cells were spread into each well of a 96-well plate and resuspended in cells containing hIL -2 or serial dilutions of engineered IL-2 mimics in RPMI complete medium. Cells were stimulated at 37°C for 15 min and then fixed by adding formaldehyde to 1.5% and incubating at room temperature for 10 min. Cells were permeabilized by resuspending in ice-cold 100% methanol for 30 min at 4°C. Fixed and permeabilized cells were washed twice with FACS buffer (phosphate-buffered saline [PBS] pH 7.2 containing 0.1% bovine serum albumin) and diluted 1:50 in FACS buffer at room temperature. Anti-STAT5 pY694 (BD Biosciences) conjugated to Alexa Fluor® 647 was incubated for 2 hours. Cells were then washed twice in FACS buffer and MFI determined on a CytoFLEX flow cytometer (Beckman-Coulter). The dose-response curve was fit to a logarithmic model and the half-maximum effective concentration ( _EC50 value) was calculated using GraphPad Prism data analysis software after subtracting the mean fluorescence intensity (MFI) of non-stimulatory cells and normalizing to the maximum signal intensity. The corresponding 95% confidence interval. The experiment was repeated three times and performed three times with similar results.

2.3 Binding Assay Surface Plasmon Resonance (SPR): For IL-2 receptor affinity titration studies, biotin-labeled human or mouse IL-2Rα, IL-2Rβ, and IL-2RƔ _c receptors were immobilized to the coating Streptavidin wafers were available for analysis on a Biacore T100 instrument (GE Healthcare). Irrelevant biotin-labeled proteins were immobilized in the reference channel to subtract non-specific binding. Less than 100 reaction units (RU) of each ligand were immobilized to minimize mass transfer effects. Three-fold serial dilutions of hIL-2 or engineered IL-2 mimetic were flowed through the immobilized ligand for 60 s and dissociation was measured for 240 s. For IL-2RβƔ _c binding studies, saturating concentrations of hIL-2Rβ (3 uM) were added to the indicated concentrations of hIL-2. Surface regeneration for all interactions was performed using 15 s exposure to 1 M MgCl ₂ /10 mM sodium acetate (pH 5.5). SPR experiments were performed at 25°C in HBS-P+ buffer (GE Healthcare) supplemented with 0.2% bovine serum albumin (BSA) and all binding studies were performed at a flow rate of 50 L/min to prevent Analytes rebind. Data were observed and processed using Biacore T100 evaluation software version 2.0 (GE Healthcare). Steady-state titration curve fitting and steady-state binding-dissociation (K _D ) value determination were performed using GraphPad Prism assuming that all binding interactions are first order. The SPR experiment was repeated three times with similar results. Biolayer interferometry: Binding data were collected in the Octet RED96 (ForteBio, Menlo Park, CA) and processed using the instrument's integrated software using a 1:1 binding model. Biotinylated target receptors human IL-2Rα, IL-2Rβ, or _Ɣc were dissolved in binding buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5 % skim milk powder) functionalized to a streptavidin-coated biosensor (SA ForteBio) at 1 μg/ml for 300 seconds. Analyte proteins were diluted from concentrated stocks into binding buffer. After baseline measurement in binding buffer alone, by dipping the biosensor into a well containing the indicated concentration of target protein (association step) and subsequently dipping the sensor back into baseline/buffer (dissociation) to monitor binding kinetics. For _{heterodimeric} receptor binding experiments with IL- _2RβƔc , allow _Ɣc to bind to the sensor while IL-2Rβ is in solution at a saturating concentration (i.e., at least approximately 2.5-fold molar excess compared to _Kd ).

Results 1. Resting V γ 9V δ 2 T cells express IL-2R β and γ chain interleukin-2 receptor (IL-2R) are heterotrimers expressed on the surface of certain immune cells (such as lymphocytes) Protein that binds and responds to IL-2. IL-2R is composed of different combinations of α (CD25), β (CD122), and γ (CD132) chains. The three receptor chains are expressed separately and in different ways on different cell types and can be assembled in different combinations and orders to generate medium- and high-affinity IL-2 receptors. The combination of β and γ chains forms a complex that binds IL-2 with moderate affinity and is primarily expressed on memory γδ T cells and NK cells, whereas the combination of all three receptor chains (α, β, and γ) forms a complex with The complex of IL-2 with high affinity (Kd about 10-11 M) is generally expressed on activated T cells and regulatory T cells (Treg).

Antibodies specific for CD25, CD122, and CD132 and flow cytometric analysis were used to monitor the expression of IL-2R chains on resting Vγ9Vδ2 T cells compared with αβ CD4 and CD8 T cells, Tregs, and NK cells.

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA-leukocyte layer) from healthy donors (HD; n=3). Cells were centrifuged at 1800 rpm for 5 min and then incubated with 10 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature before adding 50 μL in PBS 2% FBS 2 mM EDTA (FACS buffer) The following antibody mixtures were prepared: CD3-APC-A700, CD8-BV395, CD4-V660, CD56-V605, Vd2-FITC, FoxP3-PE for identifying immune subpopulations, and for separately assessing IL-2 receptor α , CD25-APC, CD122-PerCPCy5 and CD132-mCherry expressed on the surface of β and γ. CD69-PC7 was added to monitor activation status and a viability marker (LIVE/DEAD™ fixable dead cell dye) was added to exclude dead cells. Cells were incubated at 4°C for 30 minutes and washed twice in FACS buffer before being fixed in Cytofix fixation buffer (BD Bioscience) and analyzed by flow cytometry.

Figure 1 shows that IL-2R-γ (CD132) is expressed in similar amounts on most T cell subsets and NK cells. IL-2R-β (CD122) is expressed at low levels on T cell subsets and highly on NK cells, while high-affinity IL-2R-α (CD25) is expressed on resting CD8, CD4, Vγ9Vδ2 T cells and NK cells Undetectable but highly expressed on resting Tregs.

2. ICT01- mediated activation of Vγ9Vδ2 T cells induces an increase in the expression of IL-2-Rα chain (CD25). ICT01 (mAb1) is an anti - BTN3A therapeutic antibody that specifically activates Vγ9Vδ2 T cells. The effect of ICT01 on the expression of IL-2R chains α, β and γ on Vγ9Vδ2 T cells, αβ CD8 T cells, Tregs and NK cells was analyzed using antibodies specific for CD25, CD122 and CD132 and flow cytometry. to monitor.

Human PBMC from healthy donors (HD, n=3) were cultured in complete medium (RPMI 1640 medium supplemented with 10% fetal calf serum and 1% penicillin/streptomycin) and used at 1 μg/mL. ICT01 or its isotype control (hlgG1S) treatment. Resting cells (D0) or cells activated for 2 days were collected, centrifuged at 1800 rpm for 5 min and then incubated with 10 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at RT, after which 50 μL of PBS 2% was added. The following antibody mixture prepared in FBS 2 mM EDTA (FACS buffer): CD3-APC-A700, CD8-BV395, CD4-V660, CD56-V605, Vd2-FITC, FoxP3-PE for recognition of immune subpopulations, and CD25-APC, CD122-PerCPCy5 and CD132-mCherry are used to evaluate the surface expression of IL-2 receptor a, b and g respectively. CD69-PC7 was added to monitor activation status and a viability marker (LIVE/DEAD™ fixable dead cell dye) was added to exclude dead cells. Cells were incubated at 4°C for 30 minutes and washed twice in FACS buffer before being fixed in Cytofix fixation buffer (BD Bioscience) and analyzed by flow cytometry.

As expected, ICT01 induced specific activation of Vγ9Vδ2 T cells, as demonstrated by increased CD69 expression after 2 days of treatment. In addition, ICT01 triggering increases the expression of CD25 on Vγ9Vδ2 T cells but does not affect the expression of CD122 and CD132 on this immune cell subset. No constant activation or regulation of IL-2R subunits on CD8 T cells, Tregs and NK cells was noted. This result indicates that ICT01 can alter the response of Vγ9Vδ2 T cells to IL-2 by increasing the expression of high-affinity IL-2R-α subunit.

3. The IL-2 receptor α- independent agonist NL-201 induces pSTAT5 signaling on immune effector cell populations , including Vγ9Vδ2 T cells , in response to IL-2 (Proleukin ) or NL-201 (α-independent IL-2 agonist) IL-2R signaling is through tyrosine 694 (Y694) in Stat5 (signal transducer and activator of transcription-5) (well known to mediate IL-2 and its receptor Phosphorylation on biologically active transcription factors upon binding was assessed in Vγ9Vδ2 T cells, CD4 and CD8 T cells, Tregs and NK cells.

Human PBMC from healthy donors (HD, n=3) were incubated in PBS for 1 h and then treated with increasing concentrations of IL-2 (Proleukin) or NL-201 and ICT01 or its isotype control at 1 μg/mL. Culture in 100 μL complete medium (20 M cells/mL) in the presence of (hlgG1S). After 20 minutes, 100 μL of pre-warmed CytoFix solution (BD Bioscience) was added and cells were incubated at room temperature for 20 minutes. Cells were washed, resuspended in cold permeabilization solution (PermIII; BD Bioscience) and incubated on ice for 30 minutes. After washing in FACS buffer, cells were stained for 30 min with the following antibody mixture: Vd2-FITC, CD56-BV605, CD4-BV785, CD3-PeCy7, FoxP3-PE, Pstat5-APC, and FcR blocking reagent (Miltenyi Biotec) , and analyzed by flow cytometry. The response of Vγ9Vδ2 T cells, CD4 and CD8 T cells, Treg and NK cells to IL-2 or NL-201 was assessed by monitoring the P-Stat5 staining intensity (MFI) in each population.

Results demonstrate concentration-dependent increases in P-Stat5 signaling in response to IL-2 and the alpha-independent IL-2 agonist NL-201 in all immune subpopulations. However, NL-201 appears to be approximately 100X more potent than IL-2 in triggering IL-2R signaling in Vγ9Vδ2 T cells, approximately 50X more potent than IL-2 in triggering IL-2R signaling in CD8 T cells and NK cells, and approximately 50X more potent than IL-2 in triggering IL-2R signaling in CD8 T cells and NK cells. 2 triggers IL-2R signaling in Tregs about 50X weaker. NL-201 and IL-2 have similar activities on conventional CD4 T cells. ICT01 has no significant effect on IL-2R signaling under static conditions (Figure 3 and Table 6).

Similar experiments were performed on PBMC activated with ICT01 for 2 days and then incubated with increasing concentrations of IL-2 or NL-201. In these cases, as shown in Figure 2, Vγ9Vδ2 T cells exhibit increased surface expression of the high-affinity IL-2R subunit (CD25). The results show that (i) all cell subpopulations are equally sensitive to NL-201 at rest and after 2 days in culture w/o ICT01, (ii) culturing PBMC for 2 days renders all subpopulations susceptible to IL-2 (irrelevant ICT01) Increased sensitivity (iii) Tregs remain more than approximately 150X insensitive to NL-201 compared to IL-2, (iv) ICT01 tends to reduce sensitivity to IL-2 on CD4 T cells, CD8 T cells and NK cells sensitivity. This last effect is less pronounced with NL-201. Table 6 : EC ₅₀ of resting g9d2 T cells , CD4 T cells, CD8 T cells, Treg and NK cells in response to IL-2 and NL-201 EC ₅₀ (nM) g9d2 T cells Tregs CD4 Th cells CD8 T cells NK cells hIgG1S+IL-2 7.4390 0.0010 1.4930 8.9640 0.7489 ICT01+IL-2 6.9570 0.0006 1.5330 9.5960 0.9038 hIgG1S+NL-201 0.0558 0.0919 0.4973 0.1767 0.0143 ICT01+NL-201 0.0753 0.1035 0.7757 0.2373 0.0151 Table 7 : EC ₅₀ of g9d2 T cells, CD4 T cells, CD8 T cells, Treg and NK cells activated by ICT01 in response to IL-2 and NL-201 EC ₅₀ (nM) g9d2 T cells Tregs CD4 Th cells CD8 T cells NK cells hIgG1S+IL-2 0.0847 0.0008 0.0417 0.1181 0.0340 ICT01+IL-2 0.1157 0.0005 0.2748 0.5926 0.1801 hIgG1S+NL-201 0.0724 0.1307 0.2046 0.1102 0.0181 ICT01+NL-201 0.0676 0.1159 0.2389 0.1173 0.0205

4. The IL-2 receptor α -independent agonist NL-201 induces the expansion of Vγ9Vδ2 T cells in vitro . To further demonstrate the activity of the α-independent IL-2 agonist NL-201 on Vγ9Vδ2 T cells, we used Increasing concentrations of IL-2 or NL-201 (7 doses starting at 12 nM, serial dilution 3X) were cultured together for 8 days in the presence of hIgG1S (1 μg/mL) or ICT01 (0.01, 0.1, and 1 μg/mL). Human PBMC were used to monitor the expansion of Vγ9Vδ2 T cells. Cells were cultured in complete medium. ICT01 and IL-2 or NL-201 were added on day 0 and interleukins were updated on day 4. After 8 days, cells were stained with a mixture of antibodies to identify Vγ9Vδ2 T cells and Tregs prior to flow cytometric analysis. Counting beads were added to each well to deduce absolute cell numbers.

As shown in Figures 4A and 4B, the combination of ICT01 with IL-2 or NL-201 induced concentration-dependent synergistic expansion of Vγ9Vδ2 T cells, with this specific T cell subset reaching up to the total T cell compartment. 50% (compared to less than 5% when IL-2 or NL-201 was used as a single agent and approximately 20% when ICT01 alone) (Figure 4B). Furthermore, the combination of NL-201 and ICT01 demonstrated superior ability to trigger Vγ9Vδ2 T cell expansion compared to IL-2 at doses >0.2 nM, and was superior to ICT01 and NL when compared to IL-2 used at the same concentration. -201 combinations recovered 1.2 to 3.8 times more Vγ9Vδ2 T cells (absolute number) at the end of culture (Fig. 4A). Regarding the Treg compartment, IL-2 is approximately 100X more potent than NL-201 in inducing Treg expansion. ICT01 continuously inactivates IL-2 and NL-201-mediated Treg expansion and activation. Specifically, the combination of NL-201 and ICT01 appeared to completely inhibit Treg expansion even at high concentrations, whereas Treg expansion persisted when ICT01 was combined with IL-2 at concentrations >1 nM.

Therefore, the combination of ICT01 and NL201 may be a novel approach to enhance Vγ9Vδ2 T cell-mediated cancer immunotherapy by inducing robust expansion of cytotoxic Vγ9Vδ2 T cells and avoiding immunosuppressive Tregs.

5.IL-2 receptor α independent agonist NL-201 Induced in vivo in mouse models Vγ9Vδ2 T cells amplify

To confirm the pharmacology of ICT01+NL-201 combination on Vγ9Vδ2 T cells, human PBMC from two different healthy donors were transplanted into NOD CRISPR Prkdc Il2r Gamma (NCG) mice. One day later, mice were treated with ICT01 alone (1 mg/kg IV on day 1) or with IL-2 (0.3 M IU/kg IP on days 1, 2, 3, and 4) or NL-201 (IV on days 1, 2, 3, and 4). 1 day IV 1, 3 and 10 μg/kg) combined treatment (Figure 5). Repeat treatment after one week. Mice were monitored daily for signs of unexpected distress. Body weight and global clinical scores were monitored weekly. Flow cytometric analysis of blood cells was performed on day 7 to monitor Vγ9Vδ2 T cell numbers and occurrence.

Combination of ICT01 with IL-2 or NL-201 did not trigger apparent toxicity in PBMC-transplanted NCG mice (Fig. 6A). Immunophenotyping of mouse blood samples drawn on day 7 after PBMC transplantation demonstrated a small expansion of the Vγ9Vδ2 T cell compartment in the ICT01+IL-2 combination panel (compared to ICT01 alone in the ICT01+IL-2 panel about 1.5 times more cells). In contrast, the ICT01+NL-201 combination induced robust dose-dependent expansion of the Vγ9Vδ2 T cell compartment, with approximately 4.3 compared to ICT01 alone at 1, 3, and 10 μg/kg of ICT01+NL-201, respectively. , 20.8 and 23 times more cells, and the occurrence rates of Vγ9Vδ2 T cells in the ICT01+NL-201 groups at 1, 3 and 10 μg/kg reached an average of 22%, 34% and 42% of the total T cells respectively. (Figure 6B).

Figure 1 : Resting Vγ9Vδ2 T cells express IL - 2 - Rβγ . (A) Used to identify CD4 (CD3+, Vd2-, CD4+), CD8 (CD3+, Vd2-, CD8+) and Vγ9Vδ2 (CD3+, Vd2+) T cells, Treg (CD3+, Vd2-, CD4+, FoxP3+) and NK cells ( Examples of gating strategies for CD3-, CD56+). (B) Analysis of surface expression of CD122 (IL-2Rβ), CD132 (IL-2Rγ) and CD25 (IL-2Rα) on each cell subpopulation. Examples showing the FACS characteristic curve (B) of each cell subpopulation and the quantification of expression amount and occurrence rate (C). Figure 2 : Vγ9Vδ2 T cells activated by ICT01 exhibit high-affinity IL-2Rα chain. Immune subpopulations were identified as CD8 (CD3+, Vd2-, CD8+) and Vγ9Vδ2 (CD3+, Vd2+) T cells, Tregs (CD3+, Vd2-, CD4+, FoxP3+) and NK cells (CD3-, CD56+). Analysis of CD122 (IL-2Rβ), CD132 (IL-2Rγ), CD25 (IL-2Rα) and CD69 on each cell subset at baseline (D0) and after 2 days of culture with ICT01 or its isotype control (hIgG1S) surface expression. The graph shows the percentage of cells positive for CD25, CD122, CD132, and CD69 within each subpopulation (A) and the relative average fluorescence intensity value of each marker within the entire indicated population (B). Figure 3 : The alpha-independent IL-2 agonist NL-201 induces P-STAT5 signaling on a resting immune cell population. The mean fluorescence intensity (MFI) value of P-Stat5 staining was analyzed for each cell population. The graph represents the mean ± SEM of MFI of 3 PBMC donors. Data were tabulated and plotted using GraphPad Prism software. Curve fits were obtained using the S-shaped 4PL formula. Figure 4 : The combination of α -independent IL-2 agonist NL-201 and ICT01 induces the expansion of Vγ9Vδ2 T cells in vitro with increasing concentrations of IL - 2 or NL-201 alone or together with 0.01 The absolute number (A, C) and appearance rate (B, D) of Vγ9Vδ2 T cells (A, B) and Tregs (C, D) after culturing PBMC with ICT01 combinations of , 0.1 and 1 μg/mL for 8 days. The graph represents the mean ± SEM of values from 3 PBMC donors. Data were tabulated and plotted using GraphPad Prism software. Curve fits were obtained using the S-shaped 4PL formula. Figure 5 : In vivo pharmacology study design Figure 6 : ICT01+ NL -201 combination triggers Vγ9Vδ2 T cell expansion in the blood of PBMC - transplanted NCG mice . (A) Body weight of individual mice versus treatment time course. (B) Absolute numbers of Vγ9Vδ2 T cells (number of cells/mL blood) 7 days after PBMC transplantation into NCG mice treated with ICT01 or isotype control (hIgG1S) w/o IL-2 (Proleukin) or NL-201 and occurrence rate (% of total T cells). The graph represents individual values and average values for each group of animals. Data were tabulated and plotted using GraphPad Prism software.

TW202330026A_111137182_SEQL.xml

Claims (16)

Use of an anti-BTN3A activating antibody for the manufacture of a medicament for the treatment of cancer in an individual in need thereof, wherein the anti-BTN3A antibody binds IL-2 receptor βƔ _c heterodimer (IL-2RβƔ _c ) The agonists are administered simultaneously, sequentially or separately in combination; wherein the IL-2 agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) X1 is a polypeptide comprising the amino acid sequence EHALYDAL (SEQ ID NO: 30); (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide containing the amino acid sequence YAFNFELI (SEQ ID NO: 31); (d) X4 is a peptide containing A peptide with the amino acid sequence ITILQSWIF (SEQ ID NO: 32); wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can be present in any of these domains between; or wherein the IL-2 agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) X1 is at least 90%, at least 95% or A peptide with an amino acid sequence that is 100% identical; (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide that contains at least 90% and at least 95% of the sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO: 34) or a peptide with an amino acid sequence that is 100% identical; (d) X2, X3, and The amino acid sequence described in any one of -49 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, At least 99% or 100% identical amino acid sequence. The use of _claim 1 , wherein the anti-BTN3A antibody binds to human BTN3A with a KD of 10 nM or less, preferably with a KD of 5 nM or _less , as measured by surface plasmon resonance. . The use of any one of claims 1 or 2 , wherein the anti-BTN3A antibody induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with cells expressing BTN3A, wherein the anti-BTN3A antibody induces activation of γδ T cells, typically Vγ9Vδ2 T cells, as measured in a degranulation assay. _EC50 is less than 5 μg/ml, preferably 1 μg/ml or less. The use of any one of claims 1 to 3 , wherein the anti-BTN3A antibody: comprises (a) a variable heavy chain (VH) polypeptide comprising at least about 95% of the amino acid sequence of SEQ ID NO: 1, An amino acid sequence that is 96%, 97%, 98%, 99% or 100% identical, and (b) a variable light chain (VL) polypeptide comprising at least about 100% identity to SEQ ID NO: 2 or SEQ ID NO: 3 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence; including HCDR 1-3 of SEQ ID NO: 12-14 and LCDR 1-3 of SEQ ID NO: 15-17 ; Containing HCDR 1-3 of SEQ ID NO: 18-20 and LCDR 1-3 of SEQ ID NO: 21-23, or competing for binding with an antibody selected from the following: deposited in CNCM with the deposit number I-4401 mAb 20.1 produced by the fusion tumor, mAb 7.2 produced by the fusion tumor deposited at CNCM with deposit number I-4402, and an antibody having the heavy chain of SEQ ID NO: 4 and the light chain of SEQ ID NO: 6. The use of any one of claims 1 to 4 , wherein the anti-BTN3A antibody comprises a mutated or chemically modified IgG1 constant region, wherein the mutated or chemically modified IgG1 constant region when compared to a corresponding antibody having a wild-type IgG1 isotype constant region Modification of the IgG1 constant region confers no binding to Fcγ receptors or reduces binding to Fcγ receptors. The use of any one of claims 1 to 5 , wherein the mutant IgG1 constant region is IgG1 triple mutations L247F, L248E and P350S. The use of any one of claims 1 to 6 , wherein the anti-BTN3A antibody is a mAb comprising the heavy chain of SEQ ID NO: 4 and the light chain of SEQ ID NO: 6. The use of any one of claims 1 to 7 , wherein the IL-2 agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) X1 is a polypeptide comprising the amino acid sequence EHALYDAL (SEQ ID NO: 30); (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide containing the amino acid sequence YAFNFELI (SEQ ID NO: 31); (d) X4 is a peptide containing A peptide with the amino acid sequence ITILQSWIF (SEQ ID NO: 32); wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can be present in any of these domains between; and wherein the polypeptide binds to IL-2 receptor βƔ _c heterodimer (IL-2R βƔ _c ). The use of any one of claims 1 to 8 , wherein the IL-2 agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) ) is a peptide with an amino acid sequence that is at least 90%, at least 95% or 100% identical to the peptide; (b) X2 is a peptide with a length of at least 8 amino acids; (c) X3 is a peptide containing SEQ ID NO: 34 A peptide that has an amino acid sequence that is at least 90%, at least 95% or 100% identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO: 35); and (d) % identical amino acid sequence peptide; and wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can be present between any of these domains; and wherein This polypeptide binds to the IL-2 receptor βƔ _c heterodimer (IL-2R βƔ _c ). The use of any one of claims 1 to 8 , wherein the IL-2 agonist is a polypeptide comprising domains X1, X2, X3 and X4, wherein: (a) X1 is an amino acid sequence comprising SEQ ID NO: 33 (KIQLHAEHALYDALMILNI); (b) X2 is a peptide of at least 8 amino acids in length; (c) X3 is a peptide comprising the amino acid sequence SEQ ID NO: 34 (LEDYAFNFELILEEIARLFESG); and (d) X4 is A peptide comprising the amino acid sequence SEQ ID NO: 35 (EDEQEEMANAIITILQSWIFS); wherein X1, X2, X3 and X4 can be in any order in the polypeptide; wherein the amino acid linker can be present in any of these domains between; and wherein the polypeptide binds to IL-2 receptor βƔ _c heterodimer (IL-2R βƔ _c ). The use of any one of claims 1 to 10 , wherein the X2 domain of the IL-2 agonist contains an amine group that is at least 90%, at least 94% or 100% identical to SEQ ID NO: 36 (KDEAEKAKRMKEWMKRIKT) Acid sequence of peptides. The use of any one of claims 1 to 11 , wherein the IL-2 agonist comprises at least 90%, at least 91%, at least an amino acid sequence selected from any one of SEQ ID NO: 38-49. A polypeptide that is 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. The use of any one of claims 1 to 12 , wherein the IL-2 agonist polypeptide is linked to a polyethylene glycol ("PEG")-containing moiety, optionally wherein the PEG-containing moiety is in the polypeptide. linked at a cysteine residue, optionally wherein the PEG-containing moiety is linked to the cysteine residue via a maleimide group; optionally wherein the IL-2 agonist comprises SEQ ID The amino acid sequence described in NO: 43 is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or an amino acid sequence that is 100% identical, and the cysteine at position 62 is present and linked to the PEG-containing moiety; Optionally wherein the IL-2 agonist comprises an amine as described in SEQ ID NO: 46 Amino acids whose amino acid sequences are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical sequence, and the cysteine at position 73 is present and linked to the PEG-containing moiety; Optionally wherein the IL-2 agonist comprises at least 90% of the amino acid sequence described in SEQ ID NO: 45, An amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, and at position 69 Cysteine is present and linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92% of the amino acid sequence described in SEQ ID NO: 44 , an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical, and the cysteine at position 66 is present and consistent with the PEG-containing moiety is linked; optionally wherein the IL-2 agonist comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94% of the amino acid sequence described in SEQ ID NO: 48 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence, and the cysteine at position 82 is present and linked to the PEG-containing moiety. The use of any one of claims 1 to 13 , wherein the anti-BTN3A antibody is administered at a dose between 0.1 and 10 mg/kg body weight. The use of any one of claims 1 to 14 , wherein the IL-2 agonist is administered at a dose between 0.1 μg/kg and 100 mg/kg body weight. The use of any one of claims 1 to 15 , wherein the IL-2 agonist is NL-201.