WO2023052541A1

WO2023052541A1 - Combination of an anti-btn3a activating antibody and an il-2 agonist for use in therapy

Info

Publication number: WO2023052541A1
Application number: PCT/EP2022/077184
Authority: WO
Inventors: Carl WALKEY; Aude DE GASSART; Paul Frohna
Original assignee: Imcheck Therapeutics; Neoleukin Therapeutics, Inc.
Priority date: 2021-09-30
Filing date: 2022-09-29
Publication date: 2023-04-06
Also published as: TW202330026A

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 βγc heterodimer 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 βγc heterodimer but is α-independent.

Description

COMBINATION OF AN ANTI-BTN3A ACTIVATING ANTIBODY AND AN IL-2 AGONIST FOR USE IN THERAPY

It is hereafter disclosed 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 BTN3 A activating antibody that specifically binds to BTN3A and activates the cytolytic function of Vy9V52 T cells and of a non-naturally occurring IL-2 agonist which binds to the IL-2 receptor py_c heterodimer but is a-independent.

BACKGROUND

White blood cells are cells of the immune system involved in defending the body against pathogens. Among these cells, lymphocytes, monocytes, and dendritic cells can be cited. Monocytes may migrate from the bloodstream to other tissues and differentiate into tissue resident macrophages or dendritic cells. Dendritic cells play a role as antigen presenting cells (APC) that activate lymphocytes. Among lymphocytes, T cells can be divided into aP T cells and y5 T cells. Vy9V52, a major subset of y5 T cells in the peripheral blood, are important effectors of the immune defense system. They directly lyse pathogen infected or abnormal cells. In addition, they regulate immune responses by inducing dendritic cell (DC) maturation as well as isotype switching and immunoglobulin production. This important cell subset of the immune system is tightly regulated by surface receptors, chemokines and cytokines.

Recent findings in the immunology field have pointed out the emergent role of butyrophilins/butyrophilin-like molecules (BTN/BTNL in human, Btn/Btnl in mouse) in the modulation of y5 T cells. In particular, BTN3 A proteins, which belongs to the B7 co-stimulatory family of molecules, have been identified as key mediators of phosphoantigen sensing by human Vy9V52 T cells. BTN3A, and in particular BTN3A1, can trigger the activation of Vy9V52 T cells in the context of phosphoantigen (Harly C, et al Blood 2012 Vol. 120 Issue 11 Pages 2269-79; Blazquez, J. L., et al. Front Immunol 2018 Vol. 9 Pages 1601).

IL-2 was originally identified as a critical component for the preservation of T cell homeostasis and proper immune regulation (Nelson B et al. J Immunol 2004 Vol. 172 Issue 7 Pages 3983- 8). IL-2 (Proleukin) has had a long history as a cancer therapeutic, being capable of eliciting complete and durable remissions in a small subset of patients with metastatic renal cell carcinoma and metastatic melanoma (Amin A et al. Oncology 2013 Vol. 27 Issue 7 Pages 680- 91). However, high-dose IL-2 therapy is associated with severe toxic side effects that include hypotension, vascular leak syndrome (VLS), liver dysfunction, and neurological disorders (Schwartz R et al. Oncology 2002 Vol. 16 Issue 11 Suppl 13 Pages 11-20). Accordingly, high- dose IL-2 treatment is limited to carefully selected patients with good cardiopulmonary functions and is only performed in a small number of centers with experience in immunotherapy.

The 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 composed of different combinations of three different proteins, often referred to as "chains": a (alpha) (also called IL-2Ra, CD25), p (beta) (also called IL-2RP, or CD122), and Ye (gamma) (also called IL-2Ry_c, CD 132). The three receptor chains are expressed separately and differently on various cell types and can assemble in different combinations and orders to generate intermediate and high affinity IL-2 receptors. The combination of P and Y chains forms a complex that binds IL- 2 with intermediate affinity and is known to be primarily expressed on memory aP T cells and NK cells, whereas the combination of all three receptor chains (a, P and y) forms a complex that binds IL-2 with high affinity (Kd ~ 10-11 M) generally expressed on activated T cells and regulatory T cells (Tregs).

Early studies suggested that T cells were critical cellular mediators of IL-2 mediated toxi cities (Rosenstein M et al. J Immunol 1986 Vol. 137 Issue 5 Pages 1735-42). Subsequently, mice studies implicated NK cells (Peace D et al. J Exp Med 1989 Vol. 169 Issue 1 Pages 161-73) and dysregulation of Tregs homeostasis and functions (Li Y et al. Nat Commun 2017 Vol. 8 Issue 1 Pages 1762). Lung endothelial cells were also shown to express a functional IL-2 receptor, suggesting their role in VLS initiation (Krieg C et al. Proc Natl Acad Sci U S A 2010 Vol. 107 Issue 26 Pages 11906-11). Altogether these studies suggest a complex etiology for VLS with the potential participation of both haematopoietic and non-haematopoietic cellular targets. In addition, IL2 induces preferential proliferation of the immunosuppressive Tregs by binding to IL-2R-a, which is preferentially expressed on this T cell subset at steady state as mentioned above (Ahmadzadeh M et al. Blood 2006 Vol. 107 Issue 6 Pages 2409-14). Depletion of Treg cells has been shown to enhance IL-2-induced antitumor immunity, suggesting that Treg may be a major barrier for IL-2 -mediated CTL expansion (Imai H et al. Cancer Sci 2007 Vol. 98 Issue 3 Pages 416-23).

Altogether, the short in vivo half-life of IL-2, severe toxicity, and propensity to amplify Treg cells have limited its widespread use for cancer therapy. Previous studies have shown that IL-2 (Proleukin®) promotes Vy9V52 T cell expansion following anti-BTN3A antibodies stimulation (including the humanized anti-BTN3A therapeutic antibody as described in W02020025703). This may be clinically useful given that Vy9V52 T cells are normally under 5% of total T cells in healthy human adult peripheral blood and in cancer patients. In contrast to a.p T cells, activated Vy9V52 T cells do not produce IL-2 themselves and therefore need an external source to promote survival and expansion. In addition, agonist anti-BNT3 A antibodies trigger the increase surface expression of the a-chain of the IL-2R (CD25) on Vy9V52 T cells. It was therefore hypothesized that combination with IL-2 would promote Vy9V52 T cell expansion through IL-2 binding on the high affinity IL- 2Ra.

Surprisingly, the inventors have now shown that the use of an IL-2Ra- independent agonist, more specifically an IL-2 agonist which has abolished affinity, or no binding site, for IL-2 Ra in combination with an anti-BTN3A agonist antibody not only promotes synergistic and specific Vy9V52 T cell expansion in human PBMCs but is also significantly more robust as compared to clinical IL-2 (Proleukin®). Furthermore, while Treg expansions persists with the higher doses of clinical IL-2, the present therapeutic combination of an anti-BTN3A agonist antibody with IL-2Ra- independent agonist as herein disclosed abolishes expansion of the immunosuppressives Tregs.

SUMMARY

The present disclosure thus relates to a therapeutic combination of an anti BTN3A activating antibody and an IL-2 receptor agonist which binds to the IL-2 receptor py_c heterodimer, for use in the treatment of cancer in a subject in need thereof; wherein the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(a) XI is a peptide comprising the amino acid sequence set forth in SEQ ID NO:30;

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence set forth in SEQ ID NO:31;

(d) X4 is a peptide comprising the amino acid sequence set forth in SEQ ID NO:32; wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide has an amino acid sequence having less than 60 % identity with an IL-2 native polypeptide.

Alternatively, the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(a) XI 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 of at least 8 amino acids in length;

(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); wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains.

Alternatively, the IL-2 agonist comprises an amino acid sequence that 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 to an amino acid sequence set forth in any one of SEQ ID NOs:37-49.

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, the anti-BTN3A antibody induces the activation of y5 T cells, typically Vy9V52 T cells, in co-culture with BTN3 expressing cells, with an EC50 below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay.

In some embodiments, the anti-BTN3A antibody: comprises (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to of SEQ ID NO: 2 or SEQ ID NO: 3;

- comprises HCDRs 1-3 of SEQ ID NO: 12- 14 and LCDRs 1-3 of SEQ ID NO: 15-17

- comprises HCDRsl-3 of SEQ ID NO: 18-20 and LCDRsl-3 of SEQ ID NO:21-23, or competes for binding with an antibody selected from mAb 20.1 as produced by the hybridoma deposited at the CNCM under deposit number 1-4401, mAb 7.2 as produced by the hybridoma deposited at the CNCM under deposit number 1-4402, and an antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

In some embodiments, the anti BTN3A antibody comprises a mutant or chemically modified IgGl constant region, wherein said mutant or chemically modified IgGl constant region confers no or decreased binding to Fey receptors when compared to a corresponding antibody with wild type IgGl isotype constant region. In some embodiments, said mutant IgGl constant region is the IgGl triple mutant L247F L248E and P350S.

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

In some embodiments, the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(a) XI is a peptide comprising the amino acid sequence EHALYDAL (SEQ ID NO: 30);

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:31);

(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO: 32); wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide binds to IL-2 receptor py_c heterodimer (IL-2Rpy_c).

(a) XI is a peptide comprising an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the peptide of SEQ ID NO:33 (KIQLHAEHALYDALMILNI);

(b) X2 is a peptide of at least 8 amino acids in length; (c) X3 is a peptide comprising an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the peptide of SEQ ID NO:34 (LEDYAFNFELILEEIARLFESG); and

(d) X4 is a peptide comprising an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO: 35); and wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide binds to IL-2 receptor PYc heterodimer (IL-2Rpy_c).

(a) XI is a peptide 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 the amino acid sequence SEQ ID NO 35

(EDEQEEMANAIITILQSWIFS) wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide binds to IL-2 receptor py_c heterodimer (IL-2Rpy_c).

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 a polypeptide that 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 to an amino acid sequence selected from any one of SEQ ID NOs: 38-49.

In some embodiments, the IL-2 agonist polypeptide is linked to a polyethylene glycol (“PEG”) containing moiety, optionally wherein the PEG containing moiety is linked at a cysteine residue in the polypeptide, optionally wherein the PEG containing moiety is linked to the cysteine reside via a maleimide group; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 43 and the cysteine at position 62 is present and is linked to a PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at leas 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 46 and the cysteine at position 73 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 45 and the cysteine at position 69 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 44 and the cysteine at position 66 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 48 and the cysteine at position 82 is present and is linked to the PEG-containing moiety.

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

In some embodiments of the therapeutic combination as herein disclosed, the anti-BTN3A antibody is administered as an intravenous infusion. In some embodiments of the therapeutic combination as herein disclosed, the IL-2 agonist is administered at a dosage comprised between 0.1 pg/kg tolOO mg/kg body weight.

In some embodiments of the therapeutic combination as herein disclosed, the IL-2 agonist is administered prior or after administration of said anti-BTN3 A antibody.

In some embodiments of the therapeutic combination as herein disclosed, the cancer is selected among haematological cancers or solid tumor cancers, preferably selected from the group consisting of B-cell lymphoid neoplasm, T-cell lymphoid neoplasm, non-Hodgkin lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK-cell lymphoid neoplasm, myeloid cell lineage neoplasmcolon 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.

The present disclosure also encompasses a method of treatment of cancer in a patient in need thereof comprising administering to said patient a therapeutically effective amount of an anti BTN3A activating antibody, in combination, simultaneously, sequentially, or separately with a therapeutically effective amount of an IL2 agonist which binds to the IL-2 receptor Pye heterodimer; wherein said IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:31);

(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO: 32); wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; or wherein said IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein: (a) XI 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 of at least 8 amino acids in length;

(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); wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; or or wherein said IL-2 agonist comprises an amino acid sequence that 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 to an amino acid sequence set forth in any one of SEQ ID NOs:37-49.

In some embodiments of the method as herein disclosed, 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.

In some embodiments of the method as herein disclosed, the anti-BTN3 A antibody induces the activation of y5 T cells, typically Vy9V52 T cells, in co-culture with BTN3 expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay.

In some embodiments of the method as herein disclosed, the anti-BTN3A antibody: comprises (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to of SEQ ID NO: 2 or SEQ ID NO: 3;

- comprises HCDRs 1-3 of SEQ ID NO: 12- 14 and LCDRs 1-3 of SEQ ID NO: 15-17 - comprises HCDRs 1-3 of SEQ ID NO: 18-20 and LCDRsl-3 of SEQ ID NO:21-23, or competes for binding with an antibody selected from mAb 20.1 as produced by the hybridoma deposited at the CNCM under deposit number 1-4401, mAb 7.2 as produced by the hybridoma deposited at the CNCM under deposit number 1-4402, or an antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

In some embodiments of the method as herein disclosed, the anti-BTN3 A antibody comprises a mutant or chemically modified IgGl constant region, wherein said mutant or chemically modified IgGl constant region confers no or decreased binding to Fey receptors when compared to a corresponding antibody with wild type IgGl isotype constant region. In specific embodiments, the mutant IgGl constant region is an IgGl triple mutant L247F L248E and P350S.

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

In some embodiments of the method as herein disclosed, the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the peptide of SEQ ID NO:34 (LEDYAFNFELILEEIARLFESG) ; and

In some embodiments of the method as herein disclosed, the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein: (a) XI is a peptide 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

X4 is a peptide comprising the amino acid sequence SEQ ID NO 35

In some embodiments of the method as herein disclosed, 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 of the method as herein disclosed, the IL-2 agonist is a polypeptide that comprises an amino acid sequence that 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 to an amino acid sequence selected from any one of SEQ ID NOs: 38-49.

In some embodiments of the method as herein disclosed, the IL-2 agonist polypeptide is linked to a polyethylene glycol (“PEG”) containing moiety, optionally wherein the PEG containing moiety is linked at a cysteine residue in the polypeptide, optionally wherein the PEG containing moiety is linked to the cysteine reside via a maleimide group; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 43 and the cysteine at position 62 is present and is linked to a PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 46 and the cysteine at position 73 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 45 and the cysteine at position 69 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 44 and the cysteine at position 66 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 48 and the cysteine at position 82 is present and is linked to the PEG-containing moiety..

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

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

In some embodiments of the method as herein disclosed, the IL2 agonist is administered at a dosage comprised between 0.1 pg/kg tolOO mg/kg body weight.

In some embodiments of the method as herein disclosed, the IL2 agonist is administered prior or after administration of said anti-BTN3 A antibody.

In some embodiments of the method for treating cancer as herein disclosed, the cancer is selected among haematological cancer or solid tumor cancer, preferably selected from the group consisting of B-cell lymphoid neoplasm, T-cell lymphoid neoplasm, non-Hodgkin lymphoma (NHL), B-NHL, T-NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), NK-cell lymphoid neoplasm, myeloid cell lineage neoplasmcolon 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 the method for treating cancer as herein disclosed, the combination of the IL-2 agonist and the anti-BTN3 A antibody provides a synergistic effect in the treatment of the cancer or on the inhibition of the proliferation of tumor cells.

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

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

FIGURES

Figure 1: Resting Vy9V52 T Cells Express IL-2-RPy. (A) Example of gating strategy to identify CD4 (CD3+, Vd2-, CD4+), CD8 (CD3+, Vd2-, CD8+) and Vy9V52 (CD3+, Vd2+) T cells, Tregs (CD3+, Vd2-, CD4+, FoxP3+) and NK cells (CD3-, CD56+). (B) Surface expression of CD122 (IL-2R0), CD132 (IL-2Ry) and CD25 (IL-2Ra) were analyzed on each cell subset. Example of FACS profiles (B) and quantification of expression levels and frequency (C) for each cell subset are shown.

Figure 2: ICT01 -activated Vy9V52T Cells Express the high affinity IL-2Ra chain. Immune subsets were identified as CD8 (CD3+, Vd2-, CD8+) and Vy9V52 (CD3+, Vd2+) T cells, Tregs (CD3+, Vd2-, CD4+, FoxP3+) and NK cells (CD3-, CD56+). Surface expression of CD122 (IL-2RP), CD 132 (IL-2Ry), CD25 (IL-2Ra) and CD69 were analyzed on each cell subset at baseline (DO) and after 2 days in culture with ICT01 or its isotype control (hlgGlS). Graphs show percentage of cells positive for CD25, CD122, CD132 and CD69 expression within each subset (A) and relative mean fluorescence intensity values of each marker within the entire indicated population (B).

Figure 3: The a-independent IL-2 agonist NL-201 Induces P-STAT5 Signaling on Resting Immune Cell Populations. The Mean Fluorescence Intensity (MFI) values of P-Stat5 staining was analyzed for each cell population. Graphs represent the Mean±SEM of MFI for 3 PBMC’s donors. Data were tabulated and plotted using GraphPad Prism software. Curve fitting were obtained with sigmoidal 4PL equation.

Figure 4: The a-independent IL-2 agonist NL-201 combined with ICT01 Induces Expansion of Vy9V62T cells in vitro. Absolute count (A, C) and frequency (B, D) of Vy9V52T cells (A, B) and Tregs (C, D) after 8 days of PBMC culture with increasing concentration of IL-2 or NL-201 alone or in combination with ICT01 used at 0.01, 0.1 and 1 pg/mL. Graphs represent the Mean±SEM of values for 3 PBMC’s donors. Data were tabulated and plotted using GraphPad Prism software. Curve fitting were obtained with sigmoidal 4PL equation.

Figure 5: In vivo Pharmacology Study Design

Figure 6: ICT01+NL-201 Combination Trigger Vy9V52 T cells Expansion in the Blood of PBMC Engrafted NCG Mice. (A) Body weight of individual mice over the course of the treatment. (B) Absolute count (number of cell/ mL of blood) and frequency (% of total T cells) of Vy9V52 T cells 7 days after PBMC engraftment in NCG mice treated with ICT01 or isotype control (hlgGlS) w/o IL-2 (Proleukin) or NL-201. Graphs represent the individual values and mean for each group of animals. Data were tabulated and plotted using GraphPad Prism software.

DETAILED DESCRIPTION

Definitions

In order that the present disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

The term “agonist” is used herein for a molecule (e.g., a small 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 specific type of receptor. Binding to the receptors can be, for example, specific binding as determined by surface plasmon resonance at biologically relevant concentrations.

The term “affinity”, as used herein, means the strength of the binding of a ligand for its binding site on its receptor. Agonist binding to a receptor can be characterized both in terms of how much physiological response can be triggered (that is, the efficacy or potency) and in terms of the concentration of the agonist that is required to produce the physiological response (often measured as EC50, i.e., the concentration required to produce the half-maximal response). For example, high-affinity ligand binding implies that a relatively low concentration of a ligand is required to maximally occupy a ligand-binding site and trigger a physiological response. The binding affinity (KD) of a ligand for its receptor (see also below for a definition) can be determined by surface plasmon resonance (SPR). The term "Kon" or "Kass" (Ka), as used herein, is intended to refer to the association rate of a particular ligand-receptor interaction, whereas the term "Kdis" (Kd) or "Koff," as used herein, is intended to refer to the dissociation rate of a particular ligand-receptor interaction.

The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of k_off to k_on (i.e. koff/kon) and is expressed as a molar concentration (M). The KD value relates to the concentration of ligand (the amount of ligand needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the ligand for its receptor.

The terms "polypeptide”, “protein” or “peptide” as used herein refer to any chain of amino acid residues, regardless of its length or post-translational modification (such as glycosylation).

Within the context of this disclosure, the term “IL-2” designates any source of IL-2, including mammalian sources and may be native 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 general meaning in the art and typically refers to the 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 microbial hosts. In specific embodiments, the term “IL-2” refers to human IL-2 (interleukin 2) of SEQ ID NO:50 and disclosed for example in Genbank ref P60568.

The term IL-2 (IL-2) agonist as used herein refers to a polypeptide capable of activating IL-2 receptor-mediated signalling.

The terms “peptide mimetics” and “peptidomimetics” have herein the same meaning and refer to a polypeptide, or a modified polypeptide that biologically mimics active ligands of a biological molecule.

As used herein the term “IL-2 mimetics” refers to polypeptides which have an amino acid sequence having less than 60 %, notably less than 50; less than 40; less than 30; or less than 20 % identity with a native IL-2 sequence, in particular with the human IL-2 of SEQ ID NO: 50. In some embodiments, the IL-2 mimetic has between 10 and 60, notably between 10 and 40, or 10 and 30 % identity with human IL-2 of SEQ ID NO:50. IL-2 peptide mimetics of the present disclosure bind to the IL-2py_c receptor and are capable of activating IL-2 receptor-mediated signalling. Exemplary IL-2 mimetics to be used in the present methods induce heterodimerization of IL-2Rpy_c, leading to phosphorylation of STAT5. The term “IL- 15” has its general meaning and refers to the human interleukin-15. Like IL-2, IL-15 binds to and signals through a complex composed of IL-2/IL-15 receptor beta chain (IL- 2RP also named CD122) and the common gamma chain (IL-2RY_C, also named CD132). Within the context of this disclosure, the term “IL-15” designates any source of IL-15, including mammalian sources and may be native or obtained by recombinant or synthetic techniques, including recombinant IL-15 polypeptides produced by microbial hosts. In some exemplary aspects, the IL-15 polypeptide is derived from a human source, and includes recombinant human IL-15, particularly recombinant human IL-15 produced by microbial hosts. In specific embodiments, it refers to the human IL- 15 (interleukin 15) of SEQ ID NO:51.

As used herein, the term “BTN3 A” has its general meaning in the art. In specific embodiments, it refers to human BTN3 A polypeptides including either BTN3 Al of SEQ ID NO:24, BTN3 A2 of SEQ ID NO:25 or BTN3 A3 of SEQ ID NO:26.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The term "antibody" or "immunoglobulin" have the same meaning and will be used equally in the present disclosure. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies. The term “antibody” as used herein also includes bispecific or multispecific molecules. An antibody can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody may in fact be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites and/or target molecules; such multi-specific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule result. Additionally, for the embodiment in which the bispecific molecule is multispecific, the molecule can further include a third binding specificity, in addition to the first and second target epitope.

In natural antibodies of 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 (k) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. In typical IgG antibodies, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate in the antibody binding site, or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L- CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Accordingly, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2- FR3-CDR3-FR4.

The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NTH, USA (Kabat et al., 1992, hereafter “Kabat et al ”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H- CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L- CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested 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 produce a functional antibody.

As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions 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. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may be used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three-dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization. In some humanized forms of antibodies, some, most or all of the amino acids outside the CDR regions can be replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and 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 production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

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

The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to full length or to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a BTN3A protein as above defined). In specific embodiments, an antibody provided herein is an antibody fragment, and more particularly any protein including an antigen-binding domain of an antibody as disclosed herein. Well known- antibody fragments comprise: a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain, or any fusion proteins comprising such antigen-binding fragments; a diabody, which refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody (also shortly named herein antibody fragment). More generally antibody fragments as herein intended also encompass single-domain antibodies that are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl). These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are 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 made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells as described herein.

The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single specificity. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity which has variable and constant regions derived from or based on human germline immunoglobulin sequences or derived from completely synthetic sequences. The method of preparing the monoclonal antibody is not relevant for the binding specificity. “Recombinant antibodies” are antibodies which are produced, expressed, generated or isolated by recombinant means, such as antibodies which are expressed using a recombinant expression vector transfected into a host cell; antibodies isolated from a recombinant combinatorial antibody library; antibodies isolated from an animal (e.g. a mouse) which is transgenic due to human immunoglobulin genes; or antibodies which are produced, expressed, generated or isolated in any other way in which particular immunoglobulin gene sequences (such as human immunoglobulin gene sequences) are assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies. In some embodiments a recombinant human antibody of this disclosure has the same amino acid sequence as the corresponding naturally occurring human antibody but differs structurally from said naturally occurring human antibody. For example, in some embodiments the glycosylation pattern is different as a result of the recombinant production of the recombinant human antibody. In some embodiments the recombinant human antibody is chemically modified by addition or subtraction of at least one covalent chemical bond relative to the structure of the human antibody that occurs naturally in humans.

An "isolated antibody", as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to BTN3 A is substantially free of antibodies that specifically bind to other antigens than BTN3A). An isolated antibody that specifically binds to BTN3A may, however, have crossreactivity to other antigens, such as related BTN3A molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The phrases "an antibody recognizing an antigen" and "an antibody having specificity for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen”. The terms “an anti-BTN3 A antibody” or “a BTN3A antibody” are also shortly used herein with the meaning of “an antibody recognizing BTN3A”.

As used herein, the term “activating antibody” refers to an antibody able to directly or indirectly induce immune functions of effector cells. In particular, as used herein, an activating anti- BTN3A antibody has at least the capacity to induce the activation of y5 T cells, typically Vy9V52 T cells, in co-culture with BTN3 expressing cells, with an EC50 below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay (see for detailed assay WO/2020/025703). As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope, notably BTN3, means typically a binding with an affinity corresponding to a KD of about 10'⁷ M or less, such as about 10'⁸ M or less, such as about 10'⁹ M or less, about IO'¹⁰ M or less, or about 10'¹¹ M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using typically a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Typically, an antibody binds to the predetermined antigen with an affinity corresponding to a Ko that is at least tenfold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen. When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen.

The term “affinity”, as used herein in the context of an antibody, means the strength of the binding of an antibody to an epitope.

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

The term "KD", as used herein, is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of k_off to k_on (i.e. koff/kon) and is expressed as a molar concentration (M). The KD value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody. KD values for antibodies can be determined using methods well established in the art. Preferred methods for determining the KD values of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. A method for determining the KD of an antibody is by using surface plasmon resonance, or by using a biosensor system such as a Biacore® (see also for detailed information regarding 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 Octet® systems. 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 - a layer of immobilized protein and an internal reference layer. The binding between a ligand immobilized on the biosensor tip surface and an analyte in solution produces an increase in optical thickness at the biosensor tip, which results in a shift in the interference pattern measured in nanometers. The wavelength shift (AZ) is a direct measure of the change in optical thickness of the biological layer, when this shift is measured over a period of time and its magnitude plotted as a function of time, a classic association/dissociation curve is obtained. This interaction is measured in real-time, allowing to monitor binding specificity, association rate and dissociation rate, and concentration, (see Abdiche et al. 2008 but also the details in the results). Affinity measurements are typically performed at 25 °C.

As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as a BTN3A. In some embodiments, it is intended to refer to an antibody that binds to human BTN3A as expressed on peripheral blood marrow cells (PBMCs), preferably with an EC50 below 50 pg/ml and more preferably below 10 pg/ml as determined in the Examples (assays and protocols are typically disclosed in WO/2020/025703, in particular with reference to Table 4). In other embodiments, it binds to an antigen recombinant polypeptide with a KD of lOOnM or lower, lOnM or lower, InM or lower, lOOpM or lower, or lOpM or lower, as measured by SPR measurements as mentioned above but see also for details Table 4 of WO/2020/025703).

An antibody that "cross-reacts with an antigen other than BTN3A" is intended to refer to an antibody that binds that antigen other than BTN3A with a KD of lOnM or lower, 1 nM or lower, or 100 pM or lower. An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen, with a KD of 1 pM or greater, or a KD of 10 pM or greater. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit essentially undetectable binding against these proteins in standard binding assays. In specific embodiment, the humanized antibody of the present disclosure, e.g., mAbl, crossreacts with cynomolgus BTN3A1, BTN3A2 and BTN3A3 of SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29 respectively for example as measured in Biacore assay (see notably the related assay exemplified in WO/2020/025703 with reference to Table 26). Specificity can further be exhibited by, e.g., an about 10:1, about 20:1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity/avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is a BTN3 A polypeptide).

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

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

As used herein, the term, "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons that are preferred in the production cell or organism, generally a eukaryotic cell, for example, a Chinese Hamster Ovary cell (CHO) or a human cell. The optimized nucleotide sequence is engineered to retain completely or as much as possible the amino acid sequence originally encoded by the starting nucleotide sequence. The amino acid sequences encoded by optimized nucleotide sequences are also referred to as optimized.

Ther term “identity” as used herein in reference to polypeptide sequences, refers to the amino acid sequence identity between two molecules. When an amino acid position in both molecules is occupied by the same amino acid, then the molecules are identical at that position. The identity between two polypeptides is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained (including gaps if necessary). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

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

When determining identity for the present disclosure, in particular for IL-2 agonists, it is also important to consider positioning of the binding interfaces with the IL-2 receptor. If amino acids are added or deleted, it should be done in such a way that doesn’t substantially interfere with presentation of the protein to its binding partner and with secondary structure.

Generally, but not necessarily, it is preferable for amino acid substitutions relative to the reference peptide domains to be conservative amino acid substitutions.

As used herein, “conservative amino acid substitution” means a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as He, Vai, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. antigen-binding activity and specificity of a native or reference polypeptide is retained. Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Gly (G), Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into H is; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into He or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Vai, into He or into Leu.

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

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

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 any measured effect of the combination which is greater than the effect predicted from a sum of the effects of the individual agents (i.e., greater than an additive effect). In some embodiments, the rate of tumor growth or tumor size (e.g., the rate of change of the size (e.g., volume, mass) of the tumor) is used to determine whether a combination of drugs is synergistic (e.g., the combination of drugs is synergistic when the rate of tumor growth is slower than would be expected if the combination of drugs produced an additive effect). In some embodiments, survival time is used to determine whether a combination of drugs is synergistic (e.g., a combination of drugs is synergistic when the survival time of a subject or population of subjects is longer than would be expected if the combination of drugs produced an additive effect). In some embodiments, T cell expansion can also be used to determine whether a combination of drugs is synergistic (e.g., a combination of drugs is synergistic when the expansion rate (determined as increased percentage of the population or increased absolute cell number compared to a baseline value) of a specific T cell subset is higher than would be expected if the combination of drugs produced an additive effect).

Activating BTN3A antibody

An activating anti-BTN3 A antibody according to the present disclosure typically exhibits one or more of the following properties:

(i) it binds to BTN3 A with a KD of lOnM or lower, preferably with a KD of InM or lower, as measured by SPR, for example as described in the Examples of patent application W02020025703;

(ii) it cross-reacts to cynomolgus BTN3A with a KD of lOOnM or lower, preferably with a KD of lOnM or lower, as measured by SPR, for example as described in the Examples of patent application W02020025703;

(iii) it binds to human PBMCs with an ECso of 50 pg/ml or below, preferably of 10 pg/ml or below, as measured in a flow cytometry assay as described in the Examples of patent application W02020025703;

(iv) it induces the activation of y5-T cells, typically Vy9V52 T cells, in co-culture with BTN3 expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as described in the Examples of patent application W02020025703.

(v) it induces in vitro activation of Vy9V52 T cells in human PBMC, with an EC50 below 0.1 pg/mL, preferably of 0.01 pg/mL or below, e.g., between lOOpg/mL and O.lpg/mL, as measured by surface expression of the activation markers CD69.

Examples anti-BTN3A activating antibodies are described in the paragraphs below. In some embodiments, the anti-BTN3 A activating antibody is selected from the group consisting of anti- BTN3A antibody such as described in the International Patent Applications W02012080769; W02012080351, and W02020025703. In some particular embodiments, BTN3 activating antibody is selected from the humanized antibodies described in W02020025703 or is a humanized version of the BTN3A agonist antibodies described in W02012080769 and W02012080351. In some embodiments, the anti-BTN3 antibody can be selected from mAb 20.1, and mAb 7.2, which are obtainable from one of the hybridomas accessible under CNCM deposit number 1-4401, and 1-4402 such as described in W02012080769 and W02012080351 or humanized version thereof, as well as from humanized mAbs 1-6 described in W02020025703.

In some embodiments, the anti-BTN3 antibody comprises the six CDRs (CDR1 (also called HCDR1), VH CDR2 (also called HCDR2), VH CDR3 (also called HCDR1), VL CDR1 (also called LCDR1), VL CDR2s (also called LCDR2), VL CDR3s (also called HCDR3)) of the antibodies 20.1, or 7.2 described in W02012080769 and W02012080351 or mAbs 1-6 as described in W02020025703. In particular embodiments, the anti-BTN3A activating antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as shown in Table 1 below:

Table 1: CDR regions of mAbl, mAb2, mAb4 and mAb5, parental murine mAb 7.2 and murine mAb 20.1 antibody according to Kabat numbering as defined in W02020025703.

In some embodiments of antibodies as herein disclosed, the 6 CDR regions are 100% identical to the 6 CDR regions of the antibodies 20.1, or 7.2 described in W02012080769 and W02012080351 or of mAbs 1-6 described in W02020025703, notably in some embodiments, the 6 CDR regions of antibodies as herein disclosed are 100% identical to the 6 CDR regions of Table 1, notably of mAbs7.2;l; 2; 4; and 5.

Other antibodies as disclosed herein include those having amino acids that have been mutated by amino acid deletion, insertion or substitution, yet have at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identity in the CDR regions as compared to the 6 CDR regions of the antibodies 20.1, or 7.2 described in W02012080769 and W02012080351 or of mAbs 1-6 described in W02020025703, notably as compared to the 6 CDR regions defined in Table 1. In some embodiments, as per the present disclosure, antibodies may have between 1, 2, 3 or 4 amino acid variations (including deletion, insertion, or substitution) in one or more CDRs, as compared to the CDR sequences of the antibodies 20.1, or 7.2 described in W02012080769 and W02012080351 or of mAbs 1-6 described in W02020025703, notably as compared to the CDR sequences of Table 1, more particularly as compared to the CDR sequences of mAbs 7.2; 1; 2; 4; and 5.

Antibodies of the present disclosure include also those having at least 90%, notably at least, 95, 96, 97, 98, 99 or 100 % identity with the VH and VL regions as defined in Table 2. More particularly, antibodies of the disclosure include the selected humanized recombinant antibodies mAbl, mAb2, mAb4 and mAb5, which are structurally characterized by their variable heavy and light chain amino acid sequences and human constant regions (isotypes) as described in the Table 2 below: Table 2: Variable heavy and light chain amino acid sequences of mAbl-mAb6

mAb3 and mAb6 are humanized antibodies of parental murine anti-BTN3 A antibody, referred as mAb 20.1 described in W02012/080351.

The corresponding amino acid and nucleotide coding sequence of the constant isotype regions of IgGl, IgG4 and their mutant versions IgGl L247F/L248E/P350S and IgG4 S241P/L248E used for generating mAbl to mAb6 are well-known in the art (Oganesyan et al., 2008; Reddy et al., 2000). The C-terminal lysine found in IgG may be naturally cleaved off and this modification does not affect the properties of the antibody; so, this residue may additionally be deleted in the constructs of mAbl to mAb6.

Full length light and heavy chains and corresponding coding sequences of mAbl, mAb2, mAb4 and mAb 5 are shown in the Table 3 below.

Table 3: Full length heavy and light chain DNA coding sequences

In certain embodiments that may be combined with the previous embodiments, an antibody provided herein is an antibody fragment of the above-defined antibodies. Antibody fragments include for example, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, Unibody, and scFv fragments, diabodies, single domain or nanobodies and other fragments. Preferably, it is a monovalent antibody, such as a Fab of scFv fragments. In some embodiments, the antibodies of the present disclosure compete for binding to BTN3 antibodies described above, in particular an antibody of the present disclosure competes for binding with an antibody selected from mAb 20.1, and mAb 7.2, which are obtainable from one of the hybridomas accessible under CNCM deposit number 1-4401, and 1-4402 such as described in W02012080769 and W02012080351, as well as from mAbs 1-6 described in W02020025703. In more particular embodiments, the antibodies of the present disclosure compete for binding with an antibody selected from mAb 7.2 as produced by the hybridomas deposited at the CNCM under deposit number 1-4402, and an antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

In some embodiments, antibodies of the present disclosure are chimeric, humanized, or human antibodies. In preferred embodiments of the present disclosure, the BTN3 antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while having at least the same affinity (or superior affinity) of the parental non- human antibody. More particularly the BTN3 antibody is a humanized form of the antibodies 20.1, or 7.2 disclosed in W02012080351. In preferred embodiments, the antibodies of the present disclosure are humanized antibodies of the parent antibody mAb 7.2 as disclosed in W02012080351. Generally, a humanized antibody comprises one or more variable domains in which, CDRs, (or portions thereof) are derived from a non-human antibody, e.g., the murine mAbs 7.2, and FRs (or portions thereof) are derived from the murine antibody sequences with mutations to reduce immunogenicity. A humanized antibody optionally will also comprise at least a portion of a human constant region. Preferably, the recombinant antibody according to the disclosure is a humanized silent antibody, typically a humanized silent IgGl or IgG4 antibody. Well-suited humanized anti-BTN3A antibodies according to the present disclosure are typically described in W02020025703 and include mAbs having VH/VL polypeptides sequences of Table 2 and mAbs having light/heavy chains of Table 3.

As used herein, the term “silent” antibody refers to an antibody that exhibits no or low FcyR binding and/or Clq binding as measured in binding assays such as those described in W02020025703. In one embodiment, the term “no or low FcyR and/or Clq binding” means that the silent antibody exhibits an FcyR and/or Clq binding that is at least below 50%, for example below 80% of the FcyR and/or Clq binding that is observed with the corresponding antibody with wild type human IgGl or IgG4 isotype. Framework or Fc engineering

The antibodies of the disclosure can include modifications made to framework residues within VH and VL, to decrease its immunogenicity.

In some specific embodiments, the antibody of the disclosure is a humanized monoclonal antibody of the parent murine antibody mAb 7.2, including at least the following amino acid mutations in the VH framework regions: V5Q; VI IL; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and at least the following amino acid mutations in the VK framework regions: T5N; V15L; R18T; V19I; K42N; A43I; D70G; F73L; Q100G.

In other specific embodiments, the antibody of the disclosure is a humanized monoclonal antibody of the parent murine antibody mAb 7.2, including at least the following amino acid mutations in the VH framework regions as compared to mAb 7.2: V5Q; VI IL; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and at least the following amino acid mutations in the VK framework regions: T5N ; V15L ; R18T ; V19I ; K42N ; A43I ; S63T ; D70G ; F73L ; Q100G.

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

Furthermore, an antibody of the disclosure may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

As used herein, the term “isotype constant region” or “Fc region” is used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residue from position C226 or from P230 to the carboxyl-terminus of the IgG antibody wherein the numbering is according to the EU numbering system. The C- terminal lysine (residue K447) of the Fc region may be removed, for example, during production or purification of the antibody or its corresponding codon deleted in the recombinant constructs. Accordingly, a composition of antibodies of the disclosure may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

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

In other embodiments, the Fc hinge region of an antibody is mutated to decrease 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 such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by 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 functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

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

In other embodiments, the Fc region is modified to decrease the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody for an Fey receptor by modifying one or more amino acids. Such antibodies with decreased effector functions, and in particular decreased ADCC include silent antibodies. In certain embodiments, the Fc domain of the IgGl isotype is used. In some specific embodiments, a mutant variant of the IgGl Fc fragment is used, e.g. a silent IgGl Fc which reduces or eliminates the ability of the fusion polypeptide to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to bind to an Fey receptor.

In certain embodiments, the Fc domain of the IgG4 isotype is used. In some specific embodiments, a mutant variant of the IgG4 Fc fragment is used, e.g. a silent IgG4 Fc which reduces or eliminates the ability of the fusion polypeptide to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to bind to an Fey receptor.

Silenced effector functions can be obtained by mutation in the Fc constant part of the antibodies and have been described in the Art (Baudino et al., J. Immunol. 2008; Strohl, CO Biotechnology 20 2009). Examples of silent IgGl antibodies comprise the triple mutant variant IgGl L247F L248E P350S. Examples of silent IgG4 antibodies comprise the double mutant variant IgG4 S241P L248E.

In certain embodiments, the Fc domain is a silent Fc mutant preventing 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 amino acid substitution is the replacement of N314 by a glycine or an alanine.

In still other embodiments, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

Another modification of the antibodies herein that is contemplated by the present disclosure is pegylation or hesylation or related technologies. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacting with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl -CIO) alkoxy- or aryloxy -poly ethylene glycol or polyethylene glycol- maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the disclosure. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Another possibility is a fusion of at least the antigen-binding region of the antibody of the disclosure to proteins capable of binding to serum proteins, such human serum albumin to increase half-life of the resulting molecule. Such approach is for example described in Nygren et al., EP 0 486 525.

In certain embodiments, the C-terminal lysine commonly present on human IgG heavy chain constant domains, is engineered out to reduce heterogeneity due to the cleavage of this reduce commonly observed during manufacturing or storage. Such modifications do not perceptible change the desirable functions of these antibodies, while conferring the benefit of stability to these molecules.

Nucleic acid molecules encoding antibodies of the disclosure

Also disclosed herein are the nucleic acid molecules that encode the anti-BTN3 A antibodies of the present disclosure. Examples of variable light chain and heavy chain nucleotide sequences are those encoding the variable light chain and heavy chain amino acid sequences of any one of mAbl, mAb2, mAb4 and mAb5, the latter sequences being easily derived from the Table 1 and Table 2, and using the genetic code and, optionally taking into account the codon bias depending on the host cell species.

The present disclosure also pertains to nucleic acid molecules that derive from the latter sequences having been optimized for protein expression in mammalian cells, for example, CHO cell lines.

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

Nucleic acids of the disclosure 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, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to an scFv gene. In these manipulations, a VL- or VH-encoding DNA fragment (for example VL and VH as defined in Table 1) is operatively linked to another DNA molecule, or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operatively linked", as used in this context, is intended to mean that the two DNA fragments are joined in a functional manner, for example, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that the protein is expressed under control of a desired promoter.

The isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, 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 encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In some embodiments, the heavy chain constant region is selected among IgGl isotypes, for example human IgGl isotype. In other embodiments, the heavy chain constant region is selected among IgG4 isotypes, for example human IgG4 isotype. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.

The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as to a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (Kabat et al., 1992, see supra) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or a lambda constant region. To create an scFv gene, the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4 - Ser)s, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (Bird et al., 1988 see supra; Huston et al., 1988, see supra; McCafferty et al., 1990, McCafferty, J., et al, 1990. Nature 348, 552-554).

Generation of transfectomas producing monoclonal antibodies

Antibodies of the present disclosure can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, 1985; Science 229, 1202-1207).

For example, to express the antibodies, or antibody fragments thereof, DNAs encoding partial or full-length light and heavy chains can be obtained by standard molecular biology or biochemistry techniques (e.g., DNA chemical synthesis, PCR amplification or cDNA cloning using a hybridoma that expresses the antibody of interest) and the DNAs can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and 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 create full-length antibody genes of any antibody isotype by inserting them into expression vectors already encoding heavy chain constant and light chain constant regions of the desired isotype such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector. Additionally, or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors disclosed herein carry regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel’s publication ( Goeddel, D.V. (1990). [1] Systems for heterologous gene expression. In Methods in Enzymology, (Academic Press), pp. 3-7). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, nonviral regulatory sequences may be used, such as the ubiquitin promoter or P-globin promoter. Still further, regulatory elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T cell leukemia virus type 1 (Takebe et al., 1988, Mol. Cell. Biol. 8, 466-472).

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

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. It is theoretically possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, for example mammalian host cells, yeast or filamentous fungi, is discussed because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

In one specific embodiment, a cloning or expression vector according to the disclosure comprises one of the coding sequences of the heavy and light chains of any one of mAbl, mAb2, mAb4 and mAb5 operatively linked to suitable promoter sequences.

Mammalian host cells for expressing the recombinant antibodies of the disclosure include Chinese Hamster Ovary (CHO cells) including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker(as described inKaufman and Sharp, 1982), CHOK1 dhfr+ cell lines, NSO myeloma cells, COS cells and SP2 cells, for example GS CHO cell lines together with GS Xceed™ gene expression system (Lonza). When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient for expression of the antibody in the host cells and, optionally, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered and purified for example from the culture medium after their secretion using standard protein purification methods (Shukla et al., 2007, J. Chromatogr. B 848, 28-39).

In one specific embodiment, the host cell of the disclosure is a host cell transfected with an expression vector having the coding sequences suitable for the expression of mAbl, mAb2, mAb4 and mAb5 respectively, operatively linked to suitable promoter sequences.

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

The latter host cells may then be further cultured under suitable conditions for the expression and production of an antibody of the disclosure selected from the group consisting of mAbl, mAb2, mAb4 and mAb5 respectively.

Alternatively, cell free expression systems may be used for the production of any of mAbl, mAb2, mAb4 and mAb5. Typically, methods of cell-free expression of proteins or antibodies are already described (Stech et al., 2017, Sci. Rep. 7, 12030) . Anti-BTN3 A Immunoconjugates

Bispecific or multispecific anti-BTN3 A antibodies

In another aspect, it is further disclosed herein bispecific or multispecific molecules comprising an anti-BTN3 A antibody of the present disclosure. An antibody can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody may in fact be derivatized or linked to more than one other functional molecule to generate multi-specific molecules that bind to more than two different binding sites and/or target molecules; such multi-specific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, such that a bispecific molecule results.

Accordingly, the present disclosure includes bispecific molecules comprising at least one first binding specificity for BTN3A, for example, one antigen-binding portion of any one of mAbl, mAb2, mAb4 and mAb5 and a second binding specificity for a second target epitope.

Additionally, for the embodiment in which the bispecific molecule is multi-specific, the molecule can further include a third binding specificity, in addition to the first and second target epitope.

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

Other antibodies which can be employed in the bispecific molecules disclosed herein are murine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present disclosure can be prepared by conjugating the constituent binding specificities, using methods known in the art. For example, each bindingspecificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or crosslinking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2- nitrobenzoic acid) (DTNB), o-phenylenedimal eimide (oPDM), N-succinimidyl-3-(2- pyridyldithio)propi onate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane- 1-carboxylate (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 useful where the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein. A bispecific molecule of the disclosure can be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants.

Binding of the bispecific molecules to their specific targets can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassay (e.g., growth inhibition and apoptosis), or Western Blot assay. Each of these assays generally detects the presence of protein-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

IL-2 agonist

IL-2 agonists of the present disclosure include IL2 mimetic polypeptides comprising domains XI, X2, X3, and X4, wherein:

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:31);

(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO: 32); wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains. The terms “peptide mimetics” and “peptidomimetics” have herein the same meaning and refer to a polypeptide, a modified polypeptide that biologically mimics active ligands of a biological molecule. An IL-2 peptide mimetic binds to the IL-2 py_c receptor and is capable of activating IL-2py_c receptor-mediated signalling. IL-2 mimetics are described in Silva et ah, Nature 2019 Jan;565(7738): 186-191. Exemplary IL-2 mimetics to be used in the present methods induce heterodimerization of P and y chains of the IL-2 receptor, leading to phosphorylation of STAT5 (which can be assessed as previously defined notably with reference to Silva et al., 2019). Briefly cells are stimulated with an IL-2 agonist and then fixed, permeabilized and incubated with an anti-STAT5p antibody (such as Alexa Fluor® 647-conjugated anti-STAT5 pY694 (BD Biosciences)). MFI can be determined on a flow cytometer (Beckman-Coulter). Dose-response curves can be fitted to a logistic model and half-maximal effective concentration (EC50 values) and corresponding 95% confidence intervals calculated. In some embodiments, an IL-2 agonist of the present disclosure induces phosphorylation of STAT5, as measured by a STAT5 phosphorylation assay detailed above, with an EC50 of 10 nM or lower, notably of 1 nM or lower, 0.5 nM or lower, 0.1 nM or lower or 0.05 nM or lower. In some embodiments, the IL-2 agonist of the present disclosure induces phosphorylation of STAT5, as measured by a STAT5 phosphorylation assay detailed above, with an EC50 comprised between 0.001 nM and 100 nM, notably 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 particularly, the IL-2 mimetics according to the present disclosure (i) bind to the IL-2 receptor py_c heterodimer and (ii) are alpha independent. By “alpha independent” it is herein intended that the IL-2 R agonist cannot detectably bind IL-2Ra and has therefore abolished affinity for IL-2Ra (CD25). Typically, IL-2 mimetics according to the present disclosure are designed to have no IL-2Ra binding interface.

In some preferred embodiments, IL-2 mimetics according to the present disclosure have an affinity for IL-2Rpy_cthat is increased by at least 5-fold, 10-fold, 20-fold, 30-fold, or 50-fold as compared to native IL-2. In some embodiments, the IL-2 agonist has a binding affinity for IL- 2Rpy_c (i.e., KD as typically determined by surface plasmon resonance, SPR) of 200 nm or lower, 100 nm or lower, 50 nM or lower, or 25 nM or lower. Typically, the 11-2 agonist of the presently disclosed combination has a binding affinity comprised between 0.1 and 100 nM, notably between 1 and 100 nM, 1 and 50 nM, 5 and 100 nM, 5 and 50 nM, or 10 and 100 nM.

In some embodiments of the present disclosure, the IL-2 mimetic is a long-acting IL-2 receptor agonist. By “long-acting”, it is meant that the IL-2 mimetic has a plasma or serum half-life of 3 hours or greater, preferably 4 hours or greater. In some aspects, the IL-2 mimetic will have a serum or plasma half-life of 9 or 10 hours or greater or 12 hours or greater. The half-life of a polypeptide refers to the time necessary for the concentration of the polypeptide to decrease by 50% as measured by an appropriate assay. The reduction can be caused by in vivo degradation, clearance, or sequestration of the polypeptide. The half-life of an IL-2 mimetic can be determined by any manner known in the art in view of the present disclosure, such as by measuring the concentration of the IL-2 mimetic in the blood. For example, to measure the halflife of a polypeptide in vivo, a suitable dose of the polypeptide is administered to a warmblooded animal (i.e. to a human or to another suitable mammal, such as a mouse, rabbit, rat, pig, dog, or a primate); blood samples or other samples from the animal are collected; the level or concentration of the polypeptide in the sample is determined; and the time until the level or concentration of the polypeptide has been reduced by 50% is calculated based on measured data. See, e.g., Kenneth, A et ah, Chemical Half-life of Pharmaceuticals: A Handbook for Pharmacists and Peters et ah, Pharmacokinetic analysis: A Practical Approach (1996). As used herein, “an increase in half-life” or “longer half-life” refers to an increase in any one or more of the parameters used to describe the protein half-life, such as the tl/2-alpha, 11/2-beta and the area under the curve (AUC), as compared to a control. The long-acting nature of the IL-2 mimetic can be due to a moiety that it is conjugated or fused to the IL-2 mimetic.

IL-2 mimetics may thus be further linked to other compounds to promote an increased half-life in vivo (also referred to as half-life extension technologies). Any such compounds can be used in the present disclosure provided that they are sufficiently safe to administer to a human subject.

Representative technologies to improve half-life of proteins 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 linkage include albumin coupling, including chemical conjugation or genetic fusion to albumin (e.g., human serum albumin, HAS) or derivatives thereof (e.g., albumin binding domain, ABD), chemical coupling with one or more synthetic polymer chains such as PEG (to produce a pegylated IL-2 mimetic) or polypropylene glycol (see e.g., for more details WO 87/00056, and Harris, J., Chess, R. “Nat Rev Drug Discov 2, 214-221 (2003)), chemical coupling to the biodegradable hydroxyethyl starch (HES - to produce typically an HESylated IL-2 mimetic) (see for details Liebner R, Mathaes R, Meyer M, Hey T, Winter G, Besheer A. Eur J Pharm Biopharm. 2014;87(2):378-385), , glycosylation (typically achieved by the introduction of additional N-glycosylation or modules containing glycosylation sites such as a carboxy-terminal peptide) and IgG Fc fusions (including dimeric or monomeric Fc regions and/or Fc variants).

A “PEG” is a poly(ethylene glycol) molecule which is a water-soluble polymer of ethylene glycol. PEGs can be obtained in different sizes, and can also be obtained commercially in chemically activated forms that are derivatized with chemically reactive groups to enable covalent conjugation to proteins. Linear PEGs are produced in various molecular weights, such as PEG polymers of weight- average molecular weights of 5,000 daltons, 10,000 daltons, 20,000 daltons, 30,000 daltons, and 40,000 daltons. Branched PEG polymers have also been developed. Commonly used activated PEG polymers are those derivatized with N- hydroxysuccinimide groups (for conjugation to primary amines such as lysine residues and protein N-termini), with aldehyde groups (for conjugation to N-termini), and with maleimide or iodoacetamide groups (for coupling to thiols such as cysteine residues). Methods of designing IL-2 mimetic moieties for conjugation to PEG are known in the art. For example, addition of polyethylene glycol (“PEG”) containing moieties may comprise attachment of a PEG group linked to maleimide group (e.g., PEG-MAL") to a cysteine residue of the polypeptide. Suitable examples of PEG- MAL include, but are not limited to, methoxy PEG- MAL 5 kD; methoxy PEG- MAL 20 kD; methoxy (PEG) 2 -MAL 40 kD; methoxy PEG(MAL)2 5 kD; methoxy PEG( MAL)220 kD; methoxy PEG( MAL)240 kD; or any combination thereof. See also US Patent No. 8,148,109.

Such linkages can be covalent or non-covalent.

Typically, IL-2 agonist of the present disclosure has less than 60 % identity notably less than 50, 40, 35, 30, 25, 20, 15 or 10 % identity with a native IL-15 polypeptide such as the human IL-15 of SEQ ID 51.

In some embodiments, the IL-2 agonist according to the present disclosure does not bind IL- 15Ra (CD215), in particular the human IL-15Ra (CD215) of SEQ ID 52.

In some embodiments, the IL-2 agonist according to the present disclosure does not comprise an amino acid sequence having more than 60 %, notably more than 65; 70; 75; 80; 85; 90; 91; 92; 93; 94; 95; 96, 97; 98; or 99 % identity with IL-15Ra (CD215), in particular the human 15Ra (CD215) of SEQ ID 52. Exemplary IL2 mimetics

The amino acid linkers may be of any length as deemed appropriate for an intended use. Exemplary lengths of amino acids include linkers between 1-200, 1-100, 1-50, 1-20, 1-15, 1- 10, 2-20, 2-15, or 2-10 amino acids in length.

In some embodiments, XI is a peptide comprising the amino acid sequence EHALYDAL (SEQ ID NO:30); X2 is a peptide of at least 8 amino acids in length; X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:31); and X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO:32).

XI, X3, and X4 may be of any suitable length, meaning each domain may contain any suitable number of additional amino acids in addition to the amino acids of SEQ ID NOS:30, 31, and 32, respectively. Typically, each of XI, X3 and X4 comprise at least 8 amino acids. In some aspects, each of XI, X3 and X4 comprise at least 19 amino acids. In some such aspects, each of XI, X3 and X4 is no more than 200 or 100 or 50 amino acids in length.

In some embodiments, XI 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); X2 is a peptide of at least 8 amino acids in length; 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); and 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).

For all of these embodiments, XI, X2, X3, and X4 may be in any order in the polypeptide. An exemplary order of domains is X1-X3-X2-X4.

In some embodiments, XI includes 1, 2, 3, 4, or all 5 of the following: L at residue 4, H at residue 5, H at residue 8, Y at residue 11; 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, Y at residue 4, F at residue 6, N at residue 7, L at residue 10, 1 at residue 11, E at residue 13, or E at residue 14. In a further embodiment, (iii) X4 includes I at residue 19. The noted positions for XI are numbered in reference to SEQ ID NO:33; the noted positions for X3 are numbered in reference to SEQ ID NO:34; and the noted positions for X4 are numbered in reference to SEQ ID NO:35.

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 positions IL, 4Y, 7N, 10L, 111 and 151; amino acid substitutions relative to SEQ ID NO:35 do not occur at positions 121, 16Q, and 18W.

In some embodiments, XI 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); X2 is a peptide of at least 8 amino acids in length; X3 is a peptide comprising an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence LEDYAFNFELILEEIARLFESG (SEQ ID NO:34); and X4 is a peptide comprising an amino acid sequence at least 90%, at least 95%, or 100% identical to the amino acid sequence EDEQEEMANAIITILQSWIFS(SEQ ID NO:35); wherein an amino acid sequence identical to EHALYDAL (SEQ ID NO: 30) is comprised within SEQ ID NO:33; an amino acid sequence identical to YAFNFELI (SEQ ID NO:31) is comprising within SEQ ID NO:34; and an amino acid sequence identical to ITILQSWIF (SEQ ID NO:32) is comprised within SEQ ID NO:35.

As noted herein, domain X2 is a structural domain, and thus any amino acid sequence that connects the relevant other domains (depending on domain order) and allows them to fold can be used. The length required will depend on the structure of the protein being made and can be 8 amino acids or longer and, in some aspects, is 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 of the embodiments provided herein for the IL-2 mimetics, 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, an 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.

An exemplary IL-2 receptor agonist of the present disclosure comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NO:37.

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 a further 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 py_c heterodimer (IL-2Rpy_c) with a binding affinity (KD as measured by SPR as detailed in Silva et al, Nature 2019) of 200 nm or lower, 100 nm or lower, 50 nM or lower, or 25 nM or lower. In some aspects, the binding affinity is comprised between 1 and 200 nM, notably between 5 and 100 nM, more particularly between 10 and 100 nM, between 10 and 50 nM or between 5 and 50 nM.

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

Exemplary IL-2 receptor agonists of the present disclosure comprise an amino acid sequence that 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 to an amino acid sequence set forth in any one of SEQ ID NOs:38-49 as shown in Table 4 below.

Table 4

IL-2 receptor agonists of the present disclosure include polypeptides comprising an amino acid sequence that 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 to an amino acid sequence set forth in any one of SEQ ID NOs.38-49. In illustrative such embodiments, the mutated cysteine is present (i.e. D56C; K58C; D59C; R66C; T77C; E85C; R50C; E53C; E62C; E69C; R73C; and/or E82C) and is optionally attached to a stability moiety such as, for example, a water stabilizing moiety such as a PEG-containing moiety, as set forth 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 is in reference to any one of SEQ ID NO:38- 49). In a further embodiment, one or both of the following are not true: position 68 is I and position 98 is F (numbering is in reference to any one of SEQ ID NO:38-49).

Exemplary PEGylated IL-2 receptor agonists of the present disclosure 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 PEGlyated. The polyethylene group can be attached via any suitable attachment chemistry, including, for example, with maleimide (e.g., maleimide- modified PEG (PEG-MAL) 5 kD; PEG-MAL 20 kD; or PEG-MAL 40 kD). In some embodiments, the PEGylation is with PEG-MAL 30 kD. In some embodiments, the PEGylation is with modified PEG-MAL 40 kD. In some embodiments, the range for repeating PEG units in the PEGylated peptide of SEQ ID NO:43 is 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. One of skill in the art will understand that PEG portions can be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the present disclosure 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 PEGlyated. The polyethylene group can 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, the PEGylation is with PEG-MAL 30 kD. In some embodiments, the PEGylation is with PEG-MAL 40 kD. In some embodiments, the range for repeating PEG units in the PEGylated peptide of SEQ ID NO:48 is 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. One of skill in the art will understand that PEG portions can be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the present disclosure 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 PEGlyated. The polyethylene group can be attached via any suitable attachment chemistry, including, for example, with maleimide-modified PEG (e.g., PEG-MAL 5 kD; PEG-MAL 20 kD; or PEG-MAL 40 kD). In some embodiments, the PEGylation is with PEG-MAL 30 kD. In some embodiments, the PEGylation is with modified PEG-MAL 40 kD. In some embodiments, the range for repeating PEG units in the PEGylated peptide of SEQ ID NO:45 is 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. One of skill in the art will understand that PEG portions can be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the present disclosure include polypeptides comprising the amino acid sequence set forth in SEQ ID NO:46 (NEO 2-15 R73C), wherein the cysteine at position 73 is PEGlyated. The polyethylene group can 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, the PEGylation is with PEG-MAL 30 kD. In some embodiments, the PEGylation is with modified PEG-MAL 40 kD. In some embodiments, the range for repeating PEG units in the PEGylated peptide of SEQ ID NO:46 is 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. One of skill in the art will understand that PEG portions can be linear or branched.

Exemplary PEGylated IL-2 receptor agonists of the present disclosure 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 PEGlyated. The polyethylene group can 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, the PEGylation is with PEG-MAL 30 kD. In some embodiments, the PEGylation is with modified PEG-MAL 40 kD. In some embodiments, the range for repeating PEG units in the PEGylated peptide of SEQ ID NO:46 is 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. One of skill in the art will understand that PEG portions can be linear or branched.

The polypeptides and peptide domains described herein may include additional residues at the N-terminus, C-terminus, or both; these additional residues are not included in determining the percent identity of the polypeptides or peptide domains of the disclosure relative to the reference polypeptide. Such residues may be any residues suitable for an intended use, including but not limited to detection tags (i.e.: fluorescent proteins, antibody epitope tags, etc.), adaptors, ligands suitable for purposes of purification (His tags, etc.), other peptide domains that add functionality to the polypeptides, etc. Residues suitable for attachment of such groups may include cysteine, lysine or p-acetylphenylalanine residues or can be tags, such as amino acid tags suitable for reaction with transglutaminases as disclosed in U.S. Patent Nos. 9,676,871 and 9,777,070.

Combination kits and compositions

Combination kits

The combination of the present disclosure comprising an anti-BTN3A antibody and an IL-2 agonist as previously defined may be presented as a combination kit. By the term "combination kit" "or kit of parts" as used herein is meant the pharmaceutical composition or compositions that are used to administer, the anti-BTN3A antibody and the IL-2 agonist, according to the disclosure. When both compounds are administered simultaneously, the combination kit can contain, for example, the components, suitably the anti-BTN3 A antibody and the IL-2 agonist in separate pharmaceutical compositions. When the components, suitably the anti-BTN3A antibody and the IL-2 agonist are not administered simultaneously, the combination kit will contain the actives in separate pharmaceutical compositions either in a single package or in separate pharmaceutical compositions in separate packages.

In one aspect there is provided a kit of parts comprising: the anti-BTN3 A antibody in association with a pharmaceutically acceptable excipients, diluents or carrier, typically a composition comprising the anti-BTN3 A antibody as previously defined ; and the IL-2 agonist in association with a pharmaceutically acceptable excipients, diluents and/or carriers, typically a composition comprising the IL-2 agonist as previously defined.

In one embodiment of the present disclosure the kit of parts comprises: the anti-BTN3 A antibody in association with a pharmaceutically acceptable excipients, diluents and/or carriers, typically a composition comprising the anti-BTN3A antibody as previously defined; and the IL-2 agonist in association with a pharmaceutically acceptable excipients, diluents and/or carriers, typically a composition comprising the IL-2 agonist as previously defined wherein the components are provided in a form which is suitable for sequential, separate and/or simultaneous administration.

In one embodiment the kit of parts comprises: a first container comprising the anti-BTN3A antibody in association with pharmaceutically acceptable excipients, diluents and/or carrier, typically a composition comprising the anti- BTN3A antibody as previously defined; and a second container comprising the IL-2 agonist in association with pharmaceutically acceptable excipients, diluents and/or carriers, typically a composition comprising the IL-2R agonist as previously defined, and a container means for containing said first and second containers.

The combination kit can also be provided by instruction, such as dosage and administration instructions. Such dosage and administration instructions can be of the kind that is provided to a doctor, or they can be of the kind that are provided by a doctor, such as instructions to a patient.

Compositions

Therefore, according to the present disclosure, the anti-BTN3 A antibody as herein defined and the IL-2 agonist as herein defined can be, for example, formulated individually into separate compositions, e.g., pharmaceutical compositions, wherein said anti-BTN3A antibody and/or IL-2 agonist are formulated together with a pharmaceutically acceptable carrier.

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

The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., 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 the action of acids and other natural conditions that may inactivate the compound.

Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well-known to those in the art. (Remington and Gennaro, 1995) Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the disclosure can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

Preferably, the pharmaceutical compositions contain vehicles, which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

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

An injectable composition is preferably sterile. The 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 the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions of the active compounds as 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 glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of 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 desired ingredient from a previously sterile-filtered solution thereof.

The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. A parenteral composition can be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently used. When the composition is in the form of a capsule, e.g., a gelatin capsule, it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil. The composition can be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. Also contemplated are delayed release capsule, including those with an enteric coating.

In certain embodiments, the use of liposomes and/or nanoparticles is contemplated for the introduction of antibodies into host cells. The formation and use of liposomes and/or nanoparticles are known to those of skill in the art.

Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present disclosure, and such particles may be are easily made. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

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

Compositions comprising an anti-BTN3A antibody and dosage regimen

In some embodiments the anti-BTN3 A antibody as herein defined may thus be formulated in a composition, e.g., a pharmaceutical composition as defined above, containing one or a combination of antibodies disclosed herein, for example, one antibody selected from the group consisting of mAbl, mAb2, mAb4 and mAb5 or their antigen-binding portions, formulated together with a pharmaceutically acceptable carrier.

An antibody of the disclosure can be formulated into a composition as above defined in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Suitable formulation for solution for infusion or subcutaneous injection of antibodies have been described in the art and for example are reviewed in Cui et al (Drug Dev Ind Pharm 2017, 43(4): 519-530). In preferred embodiments, the anti-BTN3A antibody is formulated for intravenous infusion as above defined.

The pharmaceutical composition comprising the activating anti-BTN3A antibody can be formulated at various concentrations. For example, the formulation may comprise the activating anti-BTN3A antibody at a concentration of between 0.1 pM and 1 mM, more preferably between 1 pM and 500 pM, between 500 pM and 1 mM, between 300 pM and 700 pM, between 1 pM and 200 pM, between 100 pM and 200 pM, between 200 pM and 300 pM, between 300 pM and 400 pM, between 400 pM and 500 pM, between 500 pM and 600 pM, between 600 pM and 700 pM, between 800 pM and 900 pM or between 900 pM and 1 mM. Typically, the formulation comprises the activating anti-BTN3A antibody at a concentration of between 300 pM and 700 pM.

Typically, the therapeutic dose of the activating anti-BTN3 A antibody in a human patient will be in the range of 100 pg to 700 mg per administration (based on a body weight of 70kg). For example, the maximum therapeutic dose may be in the range of 0.1 to 10 mg/kg per administration, e.g. between 0.1 and 5 mg/kg or between 1 and 5 mg/kg or between 0.1 and 2 mg/kg. It will be appreciated that such a dose may be administered at different intervals, as determined by the oncologist/physician; for example, a dose may be administered daily, twice- weekly, weekly, bi-weekly, every three weeks or monthly.

Typically, in specific embodiments, said activating anti-BTN3A antibody is administered intravenously at a dose comprised between 20 pg and 200 mg, notably between 1 mg and 200 mg or between 7 and 200 mg each dose, typically every 21 days.

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

In specific embodiments, the activating anti-BTN3 A antibody for use according to the methods of the disclosure (preferably mAbl as described hereafter) is administered intravenously at a dose comprised between 20 pg and 200 mg each dose, preferably a second dose being administered at least 15 days after the first dose, typically after about 21 days.

Compositions comprising an IL-2 agonist and dosage regimen

Pharmaceutical compositions comprising the 11-2 agonist of the present disclosure can be formulated so as to allow the IL-2 agonists to be bioavailable upon administration of the composition to a patient. The IL-2 agonists can take the form of solutions, suspensions, emulsion, microparticles, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the IL-2 receptor agonists, the manner of administration, and the composition employed. The IL-2 agonists as herein defined can be administered by any convenient route, for example by infusion or bolus injection. Administration can be systemic or local. Typical routes of administration include, without limitation, oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intra-tumor, and intranasal. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. In one aspect, the IL- 2 agonists are administered parenterally. In yet another aspect, the IL-2 agonists are administered intravenously or subcutaneously. In specific embodiments, it can be desirable to administer an IL-2 agonist locally to the area in need of treatment. In one embodiment, administration can be by direct injection at the site (or former site) of a cancer, tumor or neoplastic or pre neoplastic tissue. In another embodiment, administration can be by direct injection at the site (or former site) of a manifestation of an autoimmune disease. An example of local administration is infusion via a catheter, e.g., intravesical infusion.

A suitable dosage range for the IL-2R agonist and notably for IL-2 mimetics as herein defined may, for instance, be 0.1 ug/kg-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, exemplary doses are from about 2 ug/mg to about 15 ug/kg. In some embodiments, administration regimens include providing the IL-2 receptor agonist on a 21 day cycle wherein dosing is once or twice during the 21 day cycle.

Uses and methods of the combination of the disclosure

The present disclosure provides a therapeutic combination comprising an anti BTN3 A antibody and an IL-2 agonist as previously defined for use in the treatment of cancer.

The present disclosure also provides a method of treatment of cancer in a patient in need thereof comprising administering to said patient a therapeutically effective amount of an anti BTN3 A activating antibody, in combination, simultaneously, sequentially, or separately with a therapeutically effective amount of an IL2 agonist which binds to the IL-2 receptor Pye heterodimer and either (i) has reduced affinity for IL-2Ra (CD25) or (ii) has no binding site for IL-2Ra.

As used herein, the term “treat” "treating" or "treatment" refers to one or more of (1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease. In particular, with reference to the treatment of a tumor, the term “treatment” may refer to the inhibition of the growth of the tumor, or the reduction of the size of the tumor.

The antibodies of the disclosure are anti-BTN3A activating antibodies and can activate the cytolytic function, cytokine production and/or proliferation of Vy9V52 T cells, and thereby may be used to overcome the immunosuppressive mechanisms observed in cancer patients (see notably W02012080769, W02012080351, and W02020025703) and during chronic infections. The results of the present disclosure now show that the present combination promotes further synergistic and specific Vy9V52 T cell expansion in human PBMCs highlighting its therapeutic interest, notably for the treatment of cancer.

As used herein, the terms "cancer", "hyperproliferative" and "neoplastic" refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.

The terms "cancer" or "neoplasms" include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

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

Examples of non-hematological 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 (i.e., the anti BTN3A antibody and the IL-2 agonist as previously defined) in the combination therapy of the present disclosure may be administered simultaneously (i.e., in the same medicament), concurrently (i.e., in separate medicaments administered one right after the other in any order) or sequentially in any order. The anti- BTN3A antibody and the IL-2 agonist are typically formulated into separate compositions as previously described. The one or more composition(s) can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration as detailed previously.

In the combination therapy of the present disclosure, the BTN3 A antibody and the IL-2 agonist can be independently administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal, topical, and transdermal routes of administration.

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

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

Selecting a dosage regimen (also referred to herein as an administration regimen) for a combination therapy of the disclosure depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissue or organ in the individual being treated. Preferably, a dosage regimen maximizes the amount of each therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Accordingly, the dose amount and dosing frequency of each biotherapeutic and chemotherapeutic agent in the combination depends in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., 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; 57th edition (November 2002). Determination of the appropriate dosage regimen may be made 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, the patient's clinical history (e.g., previous therapy), the type and stage of the cancer to be treated and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

Any suitable dosage range may be used as determined by attending medical personnel. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). A suitable dosage range for the IL-2 agonist and notably for IL-2 mimetics as herein defined may, for instance, be 0.1 ug/kg-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, exemplary doses are from about 2 ug/mg to about 15 ug/kg. In some embodiments, administration regimens include providing the IL-2 receptor agonist on a 21 day cycle wherein dosing is once or twice during the 21 day cycle.

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

Regarding the dosage regimen of the anti-BTN3A antibody and of the IL-2 agonist of the present therapeutic combination any suitable dosage range may be used as determined by attending medical personnel. Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). The antibodies of the disclosure may be formulated within a therapeutic mixture to comprise about 0.0001 to 100.0 milligrams, or about 0.001 to 10 milligrams, or about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 milligrams or even 1.0 to about 10 milligrams per dose. Multiple doses can also be administered. Typically, the therapeutic dose of the activating anti-BTN3 A antibody in a human patient will be in the range of 100 pg to 700 mg per administration (based on a body weight of 70kg). For example, the maximum therapeutic dose may be in the range of 0.1 to 10 mg/kg per administration, e.g., between 0.1 and 5 mg/kg or between 1 and 5 mg/kg or between 0.1 and 2 mg/kg. It will be appreciated that such a dose may be administered at different intervals, as determined by the oncologist/physician; for example, a dose may be administered daily, twice- weekly, weekly, bi-weekly, every three weeks or monthly.

Typically, in specific embodiments, said activating anti-BTN3A antibody is administered intravenously at a dose comprised between 20 pg and 200 mg each dose, typically every 21 days.

For the IL-2 agonist, a suitable dosage range for the polypeptides may, for instance, be 0.1 ug/kg-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, exemplary doses are from about 2 ug/mg to about 15 ug/kg. In some embodiments, administration regimens include providing the IL-2 receptor agonist on a 21 day cycle wherein dosing is once or twice during the 21 day cycle.

The combination therapy comprising the anti-BTN3A antibody and the IL-2 agonist as previously defined, whether in a mixed single composition or in separate compositions, can further be administered in conjunction with other drugs e.g., for the treatment or prevention of diseases mentioned above. In specific embodiments, the combination therapy as herein disclosed (typically mAbl as previously described and an IL-2 agonist, notably an IL-2 mimetic) may be administered in combination with anti -neoplastic agents.

In other specific embodiments, the combination therapy (typically mAbl as previously described and an IL-2 agonist, notably an IL-2 mimetic) as herein disclosed may be administered in combination with cell therapy (in particular y6T cell therapy).

In other specific embodiments, the combination therapy as herein disclosed (typically mAbl as previously described and an IL-2 agonist, notably an IL-2 mimetic) may be administered with immunotherapeutic drugs, such as immune checkpoint inhibitors (in particular, anti-PD-1, anti- PD-L1, and anti-CTLA-4antibody).

As used herein, the term “cell therapy” refers to a therapy comprising the in vivo administration of at least a therapeutically efficient amount of a cell composition to a subject in need thereof. The cells administered to the patient may be allogenic or autologous. The term “y6 T cell therapy” refers to a cell therapy wherein the cell composition includes, as the active principle, y5 T cells, in particular Vy9V52 T cells. In specific embodiments, said Vy9V52 T cells have been expanded and/or activated ex vivo.

A cell therapy product refers to the cell composition which is administered to said patient for therapeutic purposes. Said cell therapy product include a therapeutically efficient dose of cells and optionally, additional excipients, adjuvants or other pharmaceutically acceptable carriers.

The term “PD-1” has its general meaning in the art and refers to the programmed death- 1 receptor. The term “PD-1” also refers to a type I transmembrane protein, belonging to the CD28-B7 signalling family of receptors that 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 pp 515- 548).

The term “anti-PD-1 antibody” or “anti-PD-Ll” has its general meaning in the art and refers to an antibody with binding affinity to PD-1 or PD-L1 respectively, and antagonist activity to PD- 1, i.e., it inhibits the signal transduction cascade related to the PD-1 and inhibits PD-1 ligand binding (PD-L1; PD-L2). Such anti-PD-1 antibody or anti-PD-Ll antibody preferentially inactivates PD-1 with a greater affinity and potency, respectively, than its interaction with the other sub-types or isoforms of the CD28-B7 signalling family of receptors (CD28; CTLA-4; ICOS; BTLA). Tests and assays for determining whether a compound is a PD-1 antagonist are well known by the skilled person in the art such as described in Shaabani S, et al (2015-2018). Expert Opin Ther Pat. 2018 Sep;28(9):665-678; Seliger, B. J. Clin. Med. 2019, 8, 2168.

Examples of such anti-PDl or anti-PDLl antibody includes without limitation, nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

Examples of such anti-CTLA4 antibody includes without limitation, ipilimumab.

Another therapeutic strategy is based on the use of the property of the herein disclosed combination as agents which selectively expand and/or activate Vy9V52 T cells isolated from a sample of a human subject.

The disclosure thus relates to a method for treating a subject in need thereof, comprising:

(a) isolating blood cells comprising Vy9V52 T cells, for example PBMCs from a blood sample of a subject,

(b) culturing said isolated blood cells in an appropriate cell culture medium with an efficient amount of the anti-BTN3A antibody and of the IL-2 agonist can be added simultaneously, concurrently or sequentially to the cell culture, with optionally, other tumor or accessory cells, and thereby obtaining expanded Vy9V52 T cells,

(c) collecting the expanded Vy9V52 T cells,

(d) optionally, formulating the expanded Vy9V52 T cells and administering a therapeutically efficient amount of said Vy9V52 T cells to the subject.

The disclosure further relates to the use of the combination disclosed herein to selectively expand Chimeric Antigen Receptor (CAR) Vy9V52 T cells. CAR y6 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 disclosure also relates the combination as herein defined for use in vivo to potentiate tumor cells in a y5 T cell therapy in a subject in need thereof, typically suffering from cancer, wherein the anti-BTN3A antibody and the IL-2 agonist can be administered simultaneously, concurrently or sequentially to the subject.

As used herein, the term y6 T cell therapy refers to a therapy which comprises the administration to a subject in need thereof of at least an efficient amount of y5 T cells. Such y6 T cells may be allogeneic or autologous. In specific embodiments, the y5 T cells can be genetically engineered by deletion or knock-out or insertion or knock-in of specific genes. In specific embodiments, said y5 T cells include y5 T cells expressing chimeric antigen receptor. The y5 T cells may have been expanded and/or purified ex vivo. Alternatively, the y5 T cells may also be comprised in a cell composition comprising other blood cells, and for example other cells of the immune system. For references regarding y6 T cell therapy, please 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 disclosure thus relates to a method of treatment of a subject suffering from cancer including solid tumors or hematological malignancies, in particular, leukemias such as acute myeloid leukemia, and having tumor cells, for example blood tumor cells, said method comprising: i. administering in said subject an efficient amount of anti-BTN3A antibodies as disclosed herein, typically mAb 1 , mAb2, mAb4 or mAb5 in combination with an efficient amount of an IL-2 agonist, wherein said anti-BN3A antibody and IL-2 agonist can be administered simultaneously, concurrently or sequentially, and, ii. administering an efficient amount of y5 T cell composition in said subject, wherein the combination of said efficient amount of anti-BTN3 A antibodies and said efficient amount of IL-2 agonist has the capacity to potentiate antitumor cytolysis mediated by said y5 T cell composition against said tumor cells.

The disclosure further relates to a method for treating a subject suffering from cancer with solid tumor cells, e.g. ovarian cancer cells, said method comprising: i. administering in said subject an efficient amount of anti-BTN3A antibodies as disclosed herein, typically mAbl, mAb2, mAb4 or mAb5, in combination with an efficient amount of an IL-2 agonist as herein defined, wherein said anti-BN3 A antibody and IL- 2 agonist can be administered simultaneously, concurrently or sequentially, and, ii. administering an efficient amount of y5 T cell composition in said subject, wherein the combination of said efficient amount of anti-BTN3 A antibodies with said efficient amount of 11-2 agonist has the capacity to potentiate antitumor cytolysis mediated by said y5 T cell composition against said tumor cells.

The disclosure having been fully described is now further illustrated by the following examples, which are illustrative only and are not meant to be further limiting. Table 5: sequences used in the present disclosure

EXAMPLES

Methods and assays of the disclosure

Methods to characterize anti-BTN3A activating antibodies for use according to the present disclosure

1.1 Binding Affinity Assay : Multi-cycle kinetic assay (SPR)

Multi-cycle kinetic analysis can be performed on anti-BTN3A antibodies using a Biacore T200 (serial no. 1909913) instrument running Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden).

Purified antibodies are diluted to a concentration of 2 pg/ml in 2 % BSA/PBS. At the start of each cycle, each antibody is captured on the Protein A at a density (RL) of ~ 146.5 RU (theoretical value to obtain an RMax of - 50 RU). Following capture, the surface is allowed to stabilize before injection of the BTN3A1 antigen (Sino Biological cat. no. 15973-H08H). BTN3A1 is titrated in 0.1% BSA/HBS-P+ (running buffer) in a two-fold dilution range from 25 to 0.78 nM. The association phase is monitored for 400 seconds and the dissociation phase for 35 minutes (2100 seconds). Kinetic data is obtained using a flow rate of 50 pl/min to minimize any potential mass transfer effects. Regeneration of the Protein A surface is conducted using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle. Two blanks (no BTN3A1) and a repeat of a single concentration of the analyte are performed for each tested antibody to check the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fcl is subtracted from that of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to a reference surface. Additionally, blank runs are subtracted for each Fc to correct any antigen-independent signal variation, such as drift. Sensorgrams are fitted using a one-to-one binding mathematical model with a global RMax parameter and no bulk signal (Constant RI = 0 RU).

1.2 Binding assay by flow cytometry on human PBMCs

Anti-BTN3A antibodies for use according to the present disclosure may also be characterized for their binding to human PBMCs, isolated from blood of healthy donors. PBMCs are isolated from buffy coats using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. PBMCs are then frozen and stored at -80°C or in liquid nitrogen until required.

100 pl cells at 1 xlO⁶ cells/ml are transferred to each well of a fresh U-shaped bottom 96-well plate, then the plate was centrifuged and supernatant discarded.

A serial dilution of the antibodies, 0.001 pg/ml to 150 pg/ml is prepared in PBS 2 mM EDTA. Human PBMCs were resuspended in 50 pl of the diluted test antibody titration series prepared.

After incubation for 30 minutes at 4°C in the dark, the plate was centrifuged and washed twice with 150 pl/well of PBS 2 mM EDTA following which the wells are resuspended in 50 pl of a mix composed of goat anti-human antibody (PE labelled) diluted 1/100 and Live/dead neat IR diluted 1/500 in PBS 2 mM EDTA.

After incubation for 15 minutes at 4°C in the dark, the plate is centrifuged and washed once with 150 pl/well PBS 2 mM EDTA following which the wells are resuspended in 200 pl PBS 2 mM EDTA. Cells are analyzed on a BD LSR Fortessa Cytometer. Data is analyzed using a FlowJo software (Version 10, FlowJo, LLC, Ashland, USA).

Same protocol may be performed on cynomolgus PBMCs and on Daudi Burkitt's lymphoma cell line.

1.3 In vitro functional efficacy: y6-T cell degranulation assay

The assay consists of measuring activating or inhibitory effect of anti-BTN3 A antibodies on y5 -T cell degranulation against Daudi Burkitt's lymphoma cell line (Harly et al., 2012). y5-T cells are expanded from PBMCs of healthy donors by culturing with zoledronic acid (1 pM) and IL2 (200 Ui/ml) for 11-13 days. IL2 is added at day 5, day 8 and every 2 days thereafter. The percentage of y5-T cells is determined at the initiation of culture and assessed for the time of culture by flow cytometry until it reached at least 80%. Frozen or fresh y5-T cells are then used in degranulation assays against Daudi cell line (E:T ratio of 1 : 1), whereby the cells are co- cultured for 4 hours at 37°C in presence of 10 pg/ml of the 7.2 and/or 20.1 humanized variants and/or their chimeric versions. Activation by PMA (20 ng/ml) plus lonomycin (1 pg/ml) served as positive control for y5-T cell degranulation, and medium alone as negative control. At the end of 4 hours co-incubation, cells are analyzed by flow cytometry to evaluate the percentage of y5-T cells positive for CD107a (LAMP-1, lysosomal-associated membrane protein-1) + CD 107b (LAMP -2). CD 107 is mobilized to the cell surface following activation-induced granule exocytosis, thus measurement of surface CD 107 is a sensitive marker for identifying recently degranulated cytolytic T cells.

The same protocol may be performed using AML blasts isolated from patients as target cells, in place of Daudi cells.

1.4 In vitro functional efficacy: Vy9V62 T cell activation in PBMC

The assay consists of measuring the activating effect of anti-BTN3 A antibodies on Vy9V52 T cells in PBMC. Human PBMCs were isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA-buffy coats or heparinized whole blood). When using whole blood, RBC were depleted using IX RBC Lysis Buffer (eBioscience) for 10 minutes at room temperature and then washed with PBS 1% FBS.

PBMCs or RBC-depleted cells were cultured in RPMI 1640 10% FBS, 1% P/S at 1.5 to 3x106 cells/mL, with increasing concentration of anti-BTN3A antibodies (dose range 0.00001 to 100 pg/mL) at 37°C, 5% CO2 in a volume of 200 pL in 96 round-bottom well plates. Activation status was monitored after two days of culture by flow cytometry analysis of activation marker surface expression. Cells were washed in in PBS 2% FBS 2 mM EDTA (FACS buffer). Cells were centrifuged at 1800rpm for 5 minutes and then incubated 10 minutes at RT with 10 pl of FcR blocking Reagent (Miltenyi Biotec) before adding 30 to 50 pL of the appropriate antibody mix prepared in FACS buffer and containing at least fluorescently-conjugated anti-CD3, anti- Vy9 or V52 TCR and anti-CD69 antibodies. A viability marker (LIVE/DEAD Fixable Dead Cell Stain) was added in all experiments in order to exclude dead cells from the analysis. Cells were incubated 30 minutes at 4°C and washed 2 times in FACS buffer before fixation in Cytofix Fixation Buffer (BD Bioscience) and flow cytometry analysis. Data were analyzed with flowjo V-10.6 software. Activated Vy9V52 T cells were defined as CD3+V52+ (or Vy9+ or V52+Vy9+) CD69+.

Methods to characterize IL-2 agonists for use according to the present disclosure 2.1 Preparation of exemplary PEGylated IL-2 mimetic:

NL-201 was developed from Neo-2/15 by introducing one cysteine residue that is subsequently conjugated to a single 40kDa maleimide-modified polyethylene glycol. Neo-2/15 stocks with a single mutation at position 62 are dialyzed into phosphate buffer, pH7.0 and adjusted to 1.0- 2.0mg/ml. TCEP is added at a molar ratio of 10:1 to protein and incubated for 10 minutes at RT to reduce disulfides. Maleimide-modified PEG40k (PEG40k-MA) or PEG30k (PEG30k- MA) powder is added directly to the reduced protein solution at a molar ratio of 10:1 PEG cysteine and incubated for 2 hours with stirring. Aliquots for SDS-PAGE are taken directly from the reaction mixture. Rapid, spontaneous, and near-quantitative covalent linkages between PEG40k-MA or PEG30k-MA and Neo-2/15 cysteine mutants are formed.

2.2 STAT5 phosphorylation studies:

In vitro studies: Approximately 2* 10⁵ YT-1, IL-2Ra⁺ YT-l, or starved CTLL-2 cells were plated in each well of a 96-well plate and re-suspended in RPMI complete medium containing serial dilutions of hIL-2, or engineered IL-2 mimetics. Cells were stimulated for 15 min at 37°C and immediately fixed by addition of formaldehyde to 1.5% and 10 min incubation at room temperature. Permeabilization of cells was achieved by resuspension 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 incubated with Alexa Fluor® 647-conjugated anti-STAT5 pY694 (BD Biosciences) diluted 1 :50 in FACS buffer for 2 hr at room temperature. Cells were then washed twice in FACS buffer and MFI was determined on a CytoFLEX flow cytometer (Beckman-Coulter). Dose-response curves were fitted to a logistic model and half-maximal effective concentration (ECso values) and corresponding 95% confidence intervals were calculated using GraphPad Prism data analysis software after subtraction of the mean fluorescence intensity (MFI) of unstimulated cells and normalization to the maximum signal intensity. Experiments were conducted in triplicate and performed three times with similar results.

2.3 Binding assays

Surface plasmon resonance (SPR): For IL-2 receptor affinity titration studies, biotinylated human or mouse IL-2Ra, IL-2RP, and IL-2Ry_c receptors were immobilized to streptavidin- coated chips for analysis on a Biacore T100 instrument (GE Healthcare). An irrelevant biotinylated protein was immobilized in the reference channel to subtract non-specific binding. Less than 100 response units (RU) of each ligand was immobilized to minimize mass transfer effects. Three-fold serial dilutions of hIL-2, or engineered IL-2 mimetics, were flowed over the immobilized ligands for 60 s and dissociation was measured for 240 s. For IL-2Rpy_c binding studies, saturating concentration of hIL-2Rp (3 uM) was added to the indicated concentrations of hIL-2. Surface regeneration for all interactions was conducted using 15 s exposure to 1 M MgC12 in 10 mM sodium acetate pH 5.5. SPR experiments were carried out in HBS-P+ buffer (GE Healthcare) supplemented with 0.2% bovine serum albumin (BSA) at 25°C and all binding studies were performed at a flow rate of 50 L/min to prevent analyte rebinding. Data was visualized and processed using the Biacore T100 evaluation software version 2.0 (GE Healthcare). Equilibrium titration curve fitting and equilibrium binding dissociation (KD) value determination was implemented using GraphPad Prism assuming all binding interactions to be first order. SPR experiments were reproduced three times with similar results. Biolayer interferometry: binding data were collected in a 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-2Ra, IL-2RP or y_c were functionalized to streptavidin-coated biosensors (SA ForteBio) at Ipg/ml in binding buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat dry milk) for 300 seconds. Analyte proteins were diluted from concentrated stocks into binding buffer. After baseline measurement in binding buffer alone, the binding kinetics were monitored by dipping the biosensors in wells containing the target protein at the indicated concentration (association step) and then dipping the sensors back into baseline/buffer (dissociation). For heterodimeric receptor binding experiments for IL-2Rpy_c, y_c was bound to the sensor while IL-2RP was in solution at saturating concentrations (i.e., at least ~2.5 fold molar excess over the Ka).

Results

1. Resting Vy9V52 T Cells Express IL-2R B and y chains

The 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 composed of different combinations of the a (CD25), P (CD122), and y (CD132) chains. The three receptor chains are expressed separately and differently on various cell types and can assemble in different combinations and orders to generate intermediate and high affinity IL-2 receptors. The combination of P and y chains form a complex that binds IL-2 with intermediate affinity and is primarily expressed on memory y6 T cells and NK cells, whereas the combination of all three receptor chains (a, P, and y) form a complex that binds IL-2 with high affinity (Kd ~ 10-11 M) generally expressed on activated T cells and regulatory T cells (Tregs). The expression of IL-2R chains on resting Vy9V52 T cells in comparison to aP CD4 and CD8 T cells, Tregs and NK cells was monitored using antibodies specific to CD25, CD122 and CD 132 and flow cytometry analysis.

Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA -buffy coats) from healthy donors (HD; n=3). Cells were centrifuged at 1800rpm for 5 minutes and then incubated 10 minutes at room temperature with 10 pl of FcR blocking Reagent (Miltenyi Biotec) before adding 50 pL of the following antibody mix prepared in PBS 2% FBS 2 mM EDTA (FACS buffer): CD3-APC-A700, CD8- BV395, CD4-V660, CD56-V605, Vd2-FITC, FoxP3-PE for identification of immune subsets and CD25-APC, CD122-PerCPCy5 and CD132-mCherry for assessment of surface expression of IL-2 receptor-a, -P and -y respectively. CD69-PC7 was added to monitor activation status and a viability marker (LIVE/DEAD™ Fixable Dead Cell Stain) was added to exclude dead cells. Cells were incubated 30 minutes at 4°C and washed 2 times in FACS buffer before fixation in Cytofix Fixation Buffer (BD Bioscience) and flow cytometry analysis.

Figure 1 shows that IL-2R-y (CD 132) is expressed at similar level on most of the T cell subsets and NK cells. IL-2R-P (CD122) is expressed at low level on T cell subsets and highly expressed on NK cells while the high affinity IL-2R-a (CD25) is not detected on resting CD8, CD4, Vy9V52 T cells and NK cells but highly expressed on resting Tregs.

2. ICTOl-Mediated Activation of Vy9V52 T Cells Induces Increase Expression of IL- 2-R a chain (CD25)

ICTOl(mAbl) is an anti-BTN3A therapeutic antibody that specifically activates Vy9V52 T cells. The effect of ICT01 on the expression of IL-2R chains -a, -P and -y on Vy9V52T cells, aP CD8 T cells, Tregs and NK cells was monitored using antibodies specific to CD25, CD122 and CD 132 and flow cytometry analysis.

Human PBMC from healthy donors (HD, n=3) were cultured in complete medium (RPMI 1640 Medium supplemented with 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin) and treated with ICT01 or its isotype control (hlgGlS) used at 1 pg/mL. Resting cells (DO) or cells activated for 2 days were harvested, centrifuged at 1800rpm for 5 minutes and then incubated 10 minutes at RT with 10 pl of FcR blocking Reagent (Miltenyi Biotec) before adding 50 pL of the following antibody mix prepared in in PBS 2% FBS 2 mM EDTA (FACS buffer): CD3- APC-A700, CD8-BV395, CD4-V660, CD56-V605, Vd2-FITC, FoxP3-PE for identification of immune subsets and CD25-APC, CD122-PerCPCy5 and CD132-mCherry for assessment of 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 Stain) was added to exclude dead cells. Cells were incubated 30 minutes at 4°C and washed 2 times in FACS buffer before fixation in Cytofix Fixation Buffer (BD Bioscience) and flow cytometry analysis.

As expected, ICT01 induces specific activation of Vy9V52 T cells as shown by increase CD69 expression after 2 days of treatment. In addition, ICT01 triggers increase expression of CD25 on Vy9V52 T cells whereas it does not affect expression of CD122 and CD132 on this immune cell subset. No consistent activation or modulation of IL-2R subunits were noted on CD8 T cells, Tregs and NK cells. This result suggests that ICT01 could change the Vy9V52 T cell response to IL-2 by increasing expression of the high affinity IL-2R-a subunit.

3. The IL-2-receptor a-independent agonist NL-201 Induces pSTAT5 Signaling on Immune Effector Cell Populations including Vy9V52 T Cells

IL-2R signaling in response to IL-2 (Proleukin) or NL-201, an a-independent IL-2 agonist, was assessed in Vy9V52 T cells, CD4 and CD8 T cells, Tregs and NK cells by following the phosphorylation of tyrosine 694 (Y694) on Stat5 (Signal transducer and activators of transcription-5), a transcription factor well known to mediate the biological activity of IL-2 upon binding to its receptor.

Human PBMC from healthy donors (HD, n=3) were incubated 1 hour in PBS before culture in 100 pL of complete medium (20 M cells/mL) in presence of increasing concentration of IL-2 (Proleukin) or NL-201 with ICT01 or its isotype control (hlgGlS) used at 1 pg/mL. After 20 minutes, 100 pL of pre warmed CytoFix solution (BD Bioscience) was added, and cells were incubated for 20 minutes at room temperature. Cells were washed, resuspended in cold permeabilization solution (Permlll; BD Bioscience) and incubated 30 minutes on ice. After washing in FACS buffer, cells were stained with the following antibody mix: Vd2-FITC, CD56-BV605, CD4-BV785, CD3-PeCy7, FoxP3-PE, Pstat5-APC, and FcR blocking Reagent (Miltenyi Biotec) for 30 minutes and analyzed by flow cytometry. The response of Vy9V52 T cells, CD4 and CD8 T cells, Tregs and NK cells to IL-2 or NL-201 was assessed by monitoring the staining intensity (MFI) of P-Stat5 in each population.

Results showed a concentration dependent increase of P-Stat5 signal in response to both IL-2 and the a-independent IL-2 agonist NL-201 in all immune subsets. However, NL-201 appeared to be -100X more potent than IL-2 to trigger IL-2R signaling in Vy9V52 T cells, ~50X more potent than IL-2 to trigger IL-2R signaling in CD8 T cells and NK cells and -100X less potent than IL-2 to trigger IL-2R signaling in Tregs. NL-201 and IL-2 have similar activity on conventional CD4 T cells. ICT01 has no significant effect on IL-2R signaling at resting state (Figure 3 and Table 6).

Similar experiment was performed on PBMC activated with ICT01 for 2 days prior to incubation with increasing concentration of IL-2 or NL-201. In these conditions, as shown in Figure 2, Vy9V52 T cells demonstrated an increase surface expression of the high affinity IL- 2R a subunit (CD25). Results showed that (i) all cell subsets are equally sensitive to NL-201 at resting state and after 2 days in culture w/o ICT01, (ii) PBMCs culture for 2 days lead to an increase sensitivity of all subsets to IL-2 independently of ICT01, (iii) Tregs remain more than -150X less sensitive to NL-201 compared to IL-2, (iv) ICT01 tends to decrease sensitivity to IL-2 on CD4 T cells, CD8 T cells and NK cells. This last effect is less marked with NL-201.

Table 6 : EC50 of resting g9d2 T cells, CD4 T cells, CD8 T cells, Tregs and NK cells response to IL-2 and NL-201

Table 7 : EC50 of ICTOl-activated g9d2 T cells, CD4 T cells, CD8 T cells, Tregs and NK cells response to IL-2 and NL-201

4. The IL-2-receptor a-independent agonist NL-201 induces VY9V52 T Cell Expansion in vitro

To further demonstrate the activity of a-independent IL-2 agonist NL-201 on Vy9V52 T cells, the expansion of Vy9V52 T cells was monitored using human PBMC cultured for 8 days with increasing concentration of IL-2 or NL-201 (7 doses starting from 12nM, serial dilution 3X) in presence of hlgGlS (1 pg/mL) or ICT01 (0.01, 0.1 and 1 pg/mL). Cells were cultured in complete medium. ICT01 and IL-2 or NL-201 were added at day 0 and cytokines were renewed at day 4. After 8 days, cells were stained with a cocktail of antibodies to identify Vy9V52 T cells and Tregs before flow cytometry analysis. Counting beads were added to each well in order to extrapolate absolute cell numbers.

As shown in Figure 4A and B, the combination of ICT01 with IL-2 or NL-201 induces a concentration dependent synergistic expansion of Vy9V52 T cells, with this specific T cell subset reaching up to 50% of the total T cell compartment (versus less than 5% with IL-2 or NL-201 used as single agent and -20% with ICT01 alone) (Figure 4B). In addition, at doses >0.2 nM, NL-201 combined with ICT01 demonstrated superior capacity to trigger Vy9V52 T cells expansion as compared to IL-2 with 1.2 to 3.8-fold more Vy9V52 T cells (absolute number) recovered at the end of the culture with ICT01 combined with NL-201 as compared to IL-2 used at the same concentration (Figure 4A). In regard to the Treg compartment, IL-2 is -100X more potent than NL-201 to induce Treg expansion. ICT01 consistently blunted IL-2 and NL-201 -mediated expansion and activation of Tregs. In particular, the combination of NL- 201 with ICT01 appeared to completely inhibits Treg expansion even at high concentration whereas Treg expansion is persisting when ICT01 was combined with IL-2 at concentration >1 nM.

Therefore, by inducing a robust expansion of cytotoxic Vy9V52 T cells together with avoiding the immunosuppressive Tregs, the combination of ICT01 with NL201 could be a novel approach to potentiate Vy9V52 T cell-mediated cancer immunotherapy.

5. The IL-2-receptor a-independent agonist NL-201 induces VY9V52 T Cell Expansion in vivo in mice models

To confirm the pharmacology of ICT01 + NL-201 combination on Vy9V52 T cells, human PBMCs from two different healthy donors were engrafted in NOD CRISPR Prkdc I12r Gamma (NCG) mice. One day later, mice were treated with ICT01 alone (IV 1 mg/kg at Day 1) or in combination with IL-2 (IP 0.3 M lU/kg at Day 1, 2, 3 and 4) or NL-201 (IV 1, 3 and 10 pg/kg at Day 1) (Figure 5). The treatment was repeated one week later. Mice were monitored daily for unexpected signs of distress. Body weight and complete clinical scoring were monitored weekly. Flow cytometry analysis of blood cells was performed at Day 7 to monitor Vy9V52 T cell number and frequency. Combination of ICT01 with IL-2 or NL-201 in PBMC engrafted NCG mice does not trigger apparent toxicity (Figure 6A). Immunophenotyping of mice blood samples withdraw at Day 7 post-PBMC engraftment demonstrated a modest expansion of Vy9V52 T cell compartment in ICT01+IL-2 combination group (~1.5 more cells in ICT01+ IL-2 group compared to ICT01 alone). In contrast, ICT01+NL-201 combination induces a robust dose dependent expansion of Vy9V52 T cell compartment with -4.3, 20.8 and 23 more cells in ICT01+NL-201 at 1, 3 and 10 pg/kg respectively compare to ICT01 alone, and Vy9V52 T cell frequency reaching a mean of 22, 34 and 42% of the total T cells in ICT01+NL-201 at 1, 3 and 10 pg/kg groups respectively (Figure 6B).

Claims

1. An anti BTN3A activating antibody, for use in the treatment of cancer in a subject in need thereof, wherein said anti-BTN3A antibody is administered in combination simultaneously, sequentially or separately with an IL2 agonist which binds to IL-2 receptor py_c heterodimer (IL-2RPYc); wherein said IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:31);

(d) X4 is a peptide comprising the amino acid sequence ITILQSWIF (SEQ ID NO: 32); wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; or wherein said IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(b) X2 is a peptide of at least 8 amino acids in length;

(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); wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; or wherein said IL-2 agonist comprises an amino acid sequence that 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

82 least 98%, at least 99%, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs:37-49.

2. The anti BTN3A antibody for use according to Claim 1, wherein said anti-BTN3A antibody binds to human BTN3A with a KD of 10 nM or lower, preferably with a KD of 5 nM or lower, as measured by surface plasmon resonance.

3. The anti BTN3 A antibody for use according to any one of Claim 1 or 2, wherein said anti-BTN3A antibody induces the activation of y5 T cells, typically Vy9V52 T cells, in coculture with BTN3A expressing cells, with an ECso below 5 pg/ml, preferably of 1 pg/ml or below, as measured in a degranulation assay.

4. The anti BTN3 A antibody for use according to any one of Claims 1-3, wherein said anti-BTN3A antibody: comprises (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to of SEQ ID NO: 2 or SEQ ID NO: 3;

- comprises HCDRs 1-3 of SEQ ID NO: 12- 14 and LCDRs 1-3 of SEQ ID NO: 15-17

- comprises HCDRs 1-3 of SEQ ID NO: 18-20 and LCDRsl-3 of SEQ ID NO:21-23, or competes for binding with an antibody selected from mAb 20.1 as produced by the hybridoma deposited at the CNCM under deposit number 1-4401, mAb 7.2 as produced by the hybridoma deposited at the CNCM under deposit number 1-4402, and an antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

5. The anti BTN3 A antibody for use according to any one of Claims 1-4, wherein said anti-BTN3A antibody comprises a mutant or chemically modified IgGl constant region, wherein said mutant or chemically modified IgGl constant region confers no or decreased binding to Fey receptors when compared to a corresponding antibody with wild type IgGl isotype constant region.

6. The anti BTN3 A antibody for use according to any one of Claims 1-5, wherein said mutant IgGl constant region is the IgGl triple mutant L247F L248E and P350S.

7. The anti BTN3A antibody for use according to any one of Claims 1-6, wherein the anti-BTN3A antibody is a mAb comprising a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6.

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8. The anti BTN3 A for use according to any one of Claims 1-7, wherein the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising the amino acid sequence YAFNFELI (SEQ ID NO:31);

9. The anti BTN3A antibody for use according to any one of Claims 1-8, wherein the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

(b) X2 is a peptide of at least 8 amino acids in length;

(c) X3 is a peptide comprising an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the peptide of SEQ ID NO:34 (LEDYAFNFELILEEIARLFESG); and

(d) X4 is a peptide comprising an amino acid sequence that is at least 90%, at least 95%, or 100% identical to the peptide EDEQEEMANAIITILQSWIFS (SEQ ID NO: 35); and wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide binds to IL-2 receptor Py_c heterodimer (IL-2Rpy_c).

10. The anti BTN3 A antibody for use according to any one of Claims 1-8, wherein the IL-2 agonist is a polypeptide comprising domains XI, X2, X3, and X4, wherein:

84 (a) XI is a peptide 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 the amino acid sequence SEQ ID NO:35 (EDEQEEMANAIITILQSWIFS) wherein XI, X2, X3, and X4 may be in any order in the polypeptide; wherein amino acid linkers may be present between any of the domains; and wherein the polypeptide binds to IL-2 receptor PYc heterodimer (IL-2RPYc).

11. The anti BTN3A antibody for use according to any one of Claims 1-10, wherein the X2 domain of the IL-2 agonist is a peptide comprising the amino acid sequence that is at least 90%, at least 94%or 100% identical to SEQ ID NO:36 (KDEAEKAKRMKEWMKRIKT).

12. The anti BTN3A antibody for use according to any one of Claims 1-11, wherein the IL-2 agonist comprises a polypeptide that 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 to an amino acid sequence selected from any one of SEQ ID NOs: 38-49.

13. The anti BTN3 A antibody for use according to any one of Claims 1-12, wherein the IL-2 agonist polypeptide is linked to a polyethylene glycol (“PEG”) containing moiety, optionally wherein the PEG containing moiety is linked at a cysteine residue in the polypeptide, optionally wherein the PEG containing moiety is linked to the cysteine reside via a maleimide group; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 43 and the cysteine at position 62 is present and is linked to a PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at leas 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NOs: 46 and the cysteine at position 73 is present and is linked to the PEG-containing moiety;

85 optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 45 and the cysteine at position 69 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 44 and the cysteine at position 66 is present and is linked to the PEG-containing moiety; optionally wherein the IL-2 agonist comprises an amino acid sequence that 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 to the amino acid sequence set forth in SEQ ID NOs: 48 and the cysteine at position 82 is present and is linked to the PEG-containing moiety.

14. The anti BTN3 A antibody for use according to any one of Claims 1-13, wherein the anti-BTN3A antibody is administered at a dosage comprised between 0.1 and 10 mg/kg body weight.

15. The anti BTN3 A antibody for use according to any one of Claims 1-14, wherein the IL-2 agonist is administered at a dosage comprised between 0.1 pg/kg tolOO mg/kg body weight.

16. The anti BTN3 A antibody for use according to any one of Claims 1-15, wherein the IL-2 agonist is NL-201.

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