Combination therapy including multispecific gamma delta TCR antibodies

Abstract

The present disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor, in combination with a common gamma chain cytokine and / or an immune checkpoint inhibitor.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [1] This application claims priority to U.S. Provisional Patent Application No. 63 / 445,171, filed February 13, 2023; U.S. Provisional Patent Application No. 63 / 471,647, filed June 7, 2023; U.S. Provisional Patent Application No. 63 / 547,960, filed November 9, 2023; U.S. Provisional Patent Application No. 63 / 624,652, filed January 24, 2024; U.S. Provisional Patent Application No. 63 / 543,364, filed October 10, 2023; and U.S. Provisional Patent Application No. 63 / 618,073, filed January 5, 2024; the contents of each of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing Reference [2] The contents of the electronic sequence listing (LVAT_013_04WO_SeqList_ST26.xml; size: 306,129 bytes; and creation date: February 13, 2024) are incorporated herein by reference in their entirety.

[0003] Field [3] The present disclosure relates to methods and medical uses for the treatment of cancer, comprising administering a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T cell receptor in combination with a common gamma chain cytokine and / or an immune checkpoint inhibitor. [Background technology]

[0004] background [4] Gamma delta (γδ) T cells are T cells that express a T cell receptor (TCR) composed of gamma and delta chains. The majority of γδ T cells express a TCR containing Vγ9 and Vδ2 chains. Vγ9Vδ2 T cells can react against a wide range of pathogens and tumor cells. This broad reactivity is understood to be conferred by phosphoantigens capable of specifically activating this T cell subset in a TCR-dependent manner. The broad antimicrobial and antitumor reactivity of Vγ9Vδ2 T cells suggests a direct involvement in the immune control of cancer and infectious diseases.

[0005] [5] Agents capable of activating Vγ9V52 T cells may be useful in the treatment of infectious diseases or cancer, as they may promote the reactivity of Vγ9V52 T cells against pathogens or infected cells or cancer cells. WO 2015 / 156673 describes antibodies that bind to the Vγ9V52 TCR and have the ability to activate Vγ9V52 T cells. WO 2020 / 060405 and WO 2022 / 008646 describe antibodies that multispecifically bind to both Vγ9V52 T cells and human cancer antigens, thereby potentially recruiting Vγ9V52 T cells to cancer cells and stimulating a therapeutic effect. Summary of the Invention [Problem to be solved by the invention]

[0006] [6] There remains a need for improved methods for treating cancer using such multispecific antibodies that minimize adverse effects while achieving effective, long-lasting responses across a broad cancer patient population. [Means for solving the problem]

[0007] overview [7] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering (i) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T cell receptor; and (ii) a dose of a common gamma chain cytokine. In some embodiments, the common gamma chain cytokine is IL-2. In some embodiments, the IL-2 is administered at a dose of less than 3 MIU daily. In some embodiments, the common gamma chain cytokine is IL-15. In some embodiments, the first antigen-binding region binds to a human cancer antigen selected from CD1d, PSMA, CD40, CD123, 5T4, nectin-4, EGFR, and CD33. In some embodiments, the first antigen-binding region binds to human PSMA.

[0008] [8] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering (i) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor; and (ii) an anti-PD1 or anti-PD-L1 antibody. In some embodiments, the method comprises administering pembrolizumab. In some embodiments, the first antigen-binding region binds to a human cancer antigen selected from CD1d, PSMA, CD40, CD123, 5T4, nectin-4, EGFR, and CD33. In some embodiments, the first antigen-binding region binds to human PSMA.

[0009] [9] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering (i) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor; (ii) an anti-PD1 or anti-PD-L1 antibody; and (iii) a common gamma chain cytokine. In some embodiments, the common gamma chain cytokine is IL-2. In some embodiments, the IL-2 is administered at a dose of less than 3 MIU daily. In some embodiments, the common gamma chain cytokine is IL-15. In some embodiments, the method comprises administering pembrolizumab. In some embodiments, the first antigen-binding region binds to a human cancer antigen selected from CD1d, PSMA, CD40, CD123, 5T4, nectin-4, EGFR, and CD33. In some embodiments, the first antigen-binding region binds to human PSMA.

[0010]

[10] In some embodiments, the present disclosure provides a kit for treating cancer, comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T cell receptor; and (b) a common gamma chain cytokine, optionally including instructions for use. In some embodiments, the common gamma chain cytokine is IL-2. In some embodiments, the dose of IL-2 is less than 3 MIU per day. In some embodiments, the common gamma chain cytokine is IL-15. In some embodiments, the first antigen-binding region binds to a human cancer antigen selected from CD1d, PSMA, CD40, CD123, 5T4, nectin-4, EGFR, and CD33. In some embodiments, the first antigen-binding region binds to human PSMA.

[0011]

[11] In some embodiments, the present disclosure provides a kit for treating cancer, comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T cell receptor; and (b) an anti-PD1 or anti-PD-L1 antibody, optionally including instructions for use. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the first antigen-binding region binds to a human cancer antigen selected from CD1d, PSMA, CD40, CD123, 5T4, nectin-4, EGFR, and CD33. In some embodiments, the first antigen-binding region binds to human PSMA.

[0012]

[12] In some embodiments, the present disclosure provides a kit for treating cancer, comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T cell receptor; (b) an anti-PD1 or anti-PD-L1 antibody; and (c) a common gamma chain cytokine, optionally including instructions for use. In some embodiments, the common gamma chain cytokine is IL-2. In some embodiments, the dose of IL-2 is less than 3 MIU per day. In some embodiments, the common gamma chain cytokine is IL-15. In some embodiments, the first antigen-binding region binds to a human cancer antigen selected from CD1d, PSMA, CD40, CD123, 5T4, nectin-4, EGFR, and CD33. In some embodiments, the first antigen-binding region binds to human PSMA. In some embodiments, the anti-PD1 antibody is pembrolizumab. [Brief explanation of the drawings]

[0013] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[13] Figure 1 shows the prostate-specific antigen (PSA) response as a percent change from baseline. For patients with the best PSA response and no PSA decline, the PSA value on day 1 of cycle 5 is shown. The dose level (DL) for each patient is indicated. [Figure 2]

[14] Figure 2 shows PSA levels over time for Pt N304. [Figure 3A]

[15] Figure 3A shows the changes in Vγ9Vδ2 T cell-related pharmacodynamic parameters over time after LAVA-1207 administration. [Figure 3B]

[15] Figure 3B shows the changes in Vγ9Vδ2 T cell-related pharmacodynamic parameters over time after LAVA-1207 administration. [Figure 3C]

[15] Figure 3C shows the changes in Vγ9Vδ2 T cell-related pharmacodynamic parameters over time after LAVA-1207 administration. [Figure 3D]

[15] Figure 3D shows the changes in Vγ9Vδ2 T cell-related pharmacodynamic parameters over time after LAVA-1207 administration. [Figure 4A]

[16] Figure 4A shows various pharmacokinetic parameters of LAVA-1207. [Figure 4B]

[16] Figure 4B shows various pharmacokinetic parameters of LAVA-1207. [Figure 5A]

[17] Figure 5A shows a summary of adverse events in the dose-escalation study of LAVA-1207. [Figure 5B]

[17] Figure 5B shows a summary of adverse events in the dose-escalation study of LAVA-1207. [Figure 6A]

[18] Figures 6A-6E show the change in PD1 expression and frequency of Vy9V52 T cells over time and the maximum observed receptor occupancy with LAVA-1207. Figure 6A shows the relative change in PD1+ Vy9V52 T cells after LAVA-1207 treatment compared to baseline for Cohort 4. [Figure 6B]

[18] Figure 6B shows the relative change in MFI of PD1 on Vγ9Vδ2 T cells compared to baseline for cohort 4. [Figure 6C]

[18] Figure 6C shows the relative change (compared to baseline, set at 100) of Vγ9Vδ2 T cells from completed cohort 6A2; the dashed line indicates administration of IL-2. [Figure 6D]

[18] Figure 6D shows Vγ9Vδ2 T cells per ml of blood over time from completed cohort 6A2; dashed lines indicate administration of IL-2. [Figure 6E]

[18] Figure 6E shows increased LAVA-1207 receptor occupancy on Vγ9Vδ2 T cells in the completed cohort. [Figure 7A]

[19] Figure 7A shows the proportion of Vγ9Vδ2 T cells in prostate cancer patient PBMCs, malignant (m), and non-malignant (nm) ranges of patient prostates. [Figure 7B]

[19] Figure 7B shows the percentage of PD1+ Vγ9Vδ2 T cells in prostate cancer patient PBMCs, malignant (m), and non-malignant (nm) ranges of patient prostates. [Figure 7C]

[19] Figure 7C shows the percentage of CD3+ T cells in prostate cancer patient PBMCs, malignant (m), and non-malignant (nm) regions of the patient's prostate. [Figure 8]

[20] Figure 8 shows upregulation of PD1 expression on Vγ9Vδ2 T cells activated with Vδ2-bsTCE. [Figure 9]

[21] Figure 9 shows an exemplary dosing schedule of the disclosed embodiments. [Figure 10]

[22] Figure 10 shows exemplary dose cohorts for LAVA-1207 and LAVA-1207 + IL-2. [Figure 11A]

[23] Figures 1 IA-B show the effect of polyspecific binding agents on the expansion of Vy9V52 T cells. Figure 1 IA shows the increase in Vy9V52 T cell frequency as a percentage of CD3+ T cells after incubation with IL-2 and either vehicle alone, pamidronate or polyspecific binding agents. [Figure 11B]

[23] Figure 1 IB shows the fold expansion of Vy9V52 T cells over baseline after incubation with IL-2 and either vehicle alone, pamidronate or a multispecific binding agent. [Figure 12A]

[24] Figures 12A-E show the frequency and phenotype of Vy9V52-T cells in patient-derived tumor and non-tumor tissues and the cytolytic activity of Vy9V52-T cells against patient-derived tumor and non-tumor tissues in the presence of LAVA-1207. Figure 12A shows the frequency of Vy9V52-T cells as a percentage of T cells in patient PBMCs, patient-derived malignant, and non-malignant tissues. [Figure 12B]

[24] Figure 12B shows the phenotype of Vy9V52 T cells and CD3+ cells from PBMCs, malignant tissues, and non-malignant tissues. [Figure 12C]

[24] Figure 12C shows LAVA-1207-mediated increased Vy9V52-T cell degranulation (as assessed by CD107a expression) when malignant tissue was incubated with LAVA-1207 and PBMCs. [Figure 12D]

[24] Figure 12D shows that there is no LAVA-1207-mediated increase in Vy9V52-T cell degranulation when non-malignant tissue is incubated with LAVA-1207 and PBMCs. [Figure 12E]

[24] Figure 12E shows increased tumor cell lysis when malignant tissues were incubated with LAVA-1207 and PBMCs, in contrast to non-malignant cells. [Figure 13A]

[25] Figures 13A-C show phenotypic analysis of ligands and corresponding co-receptors on tumor and non-tumor tissues and Vy9V52 T cells from patients. Figure 13A shows the difference in PSMA, BTN3A, and BTN2A1 expression. [Figure 13B]

[25] Figure 13B shows the differences in Nectin-2, PVR, MIC-A / MIC-B, ULPB1, ULBP3, ULBP2 / 5 / 6, and HLA-E expression. [Figure 13C]

[25] Figure 13C shows the frequency of Vy9V52 T cells expressing DNAM-1, NKG2D, or NKG2A receptors in PBMCs, malignant, and non-malignant tissues. [Figure 14A]

[26] Figures 14A-H show the effect of DNAM-1 or NKG2D receptor blockade on LAVA-1207-mediated Vy9V52 T cell cytokine production and degranulation and lysis of prostate cancer cell lines. The effect on IFNγ (Figure 14A) in the presence or absence of a DNAM-1 blocking antibody is shown. [Figure 14B]

[26] The effect on TNF (Figure 14B) in the presence or absence of DNAM-1 blocking antibodies is shown. [Figure 14C]

[26] The effect on IL-2 (FIG. 14C) in the presence or absence of DNAM-1 blocking antibodies is shown. [Figure 14D]

[26] The effect on IL-4 (Figure 14D) in the presence or absence of DNAM-1 blocking antibodies is shown. [Figure 14E]

[26] The effect on Vγ9Vδ2 T cell degranulation (Figure 14E) in the presence or absence of DNAM-1 blocking antibodies is shown. [Figure 14F]

[26] The effect on tumor cell lysis (Figure 14F) in the presence or absence of DNAM-1 blocking antibodies is shown. [Figure 14G]

[26] Figure 14G shows the effect of NKG2D receptor blockade on Vy9V52 T cell degranulation. [Figure 14H]

[26] Figure 14H shows the effect of blocking the NKG2D receptor on tumor cell lysis. [Figure 15A]

[27] Figure 15A shows the effect of blocking NKG2A (Figure 15A) on LAVA-1207-mediated T cell activation. [Figure 15B]

[27] Figure 15B shows the effect of blocking BTN3A (Figure 15B) on LAVA-1207-mediated T cell activation. [Figure 16]

[28] Figure 16 shows the effect of either anti-DNAM-1 or anti-NKG2D blocking antibodies on LAVA-1207-mediated Vy9V52 T cell degranulation using patient prostate tumor tissue. [Figure 17A]

[29] Figure 17A shows that NCG mice implanted with prostate cancer cells alone or mixed with PBMCs and treated with LAVA-1207 exhibited reduced tumor growth volume (Figure 17A) when compared to mice implanted with prostate cancer cells alone and treated with either PBMCs or PBS or PSMA-V52-Fc bsTCE. [Figure 17B]

[29] Figure 17B shows that NCG mice implanted with prostate cancer cells alone or mixed with PBMCs and treated with LAVA-1207 exhibited increased survival (Figure 17B) when compared to mice implanted with prostate cancer cells alone and treated with either PBMCs or PBS or PSMA-V52-Fc bsTCE. [Figure 18A]

[30] Figure 18A shows that labeling LAVA-1207 with FITC does not impair its binding to Vy9V52 T cells (Figure 18A). [Figure 18B]

[30] Figure 18B shows that labeling LAVA-1207 with FITC does not impair its binding to prostate cancer cells (Figure 18B). [Figure 19A]

[31] Figure 19A shows exemplary immunohistochemical staining demonstrating PSMA-positive tissue and LAVA-1207 binding (Figure 19A). Arrows indicate potential V52+ T cells (positive for LAVA-1207 binding). [Figure 19B]

[31] Figure 19B shows exemplary immunohistochemical staining demonstrating PSMA-negative tissue and the absence of LAVA-1207 binding (Figure 19B). Arrows indicate potential V52+ T cells (positive for LAVA-1207 binding). [Figure 20]

[32] Figure 20 shows the different CDR numbering systems (Kabat, Chothia, IMGT, and composite) for 6H4 VHH. DETAILED DESCRIPTION OF THE INVENTION

[0014] Detailed Description overview

[33] Vγ9Vδ2- T cells represent a conserved T cell subset (approximately 1–5% of all cluster of differentiation [CD]3+ T cells) that can induce cell death in a wide range of malignant cells in an HLA-peptide-independent manner (Lo Presti et al. 2017; de Weerdt et al. 2018; Kunzmann, Bauer, and Wilhelm 1999; Gertner-Dardenne et al. 2012).

[0015]

[34] Vγ9Vδ2 T cells possess properties of both the innate and adaptive immune systems, thus acting as a functional bridge between these two critical immune systems to influence tumor killing. Thus, upon activation, Vγ9Vδ2 T cells not only possess the capacity for immediate and potent tumor cell killing, but also potentially contribute to a cascade of events that can result in further tumor killing through the downstream activation of innate immune cells, such as natural killer (NK) cells, and adaptive immune cells, such as classical (α-β) T cells. Finally, activated Vγ9Vδ2 T cells can internalize, process, and present antigens, which can lead to priming of the adaptive immune system, potentially generating immunological memory and resulting in deep and durable antitumor responses.

[0016]

[35] Activation of Vγ9Vδ2 T cells is driven by their interaction with butyrophilin (BTN) 2A1 (BTN2A1) and BTN3A1 when phosphoantigens bind to the intracellular B30.2 domain of BTN3A1 (Harly et al. 2012; Vantourout and Hayday 2013; Rigau et al. 2020). Phosphoantigens accumulate upon dysregulation of the mevalonate pathway and are upregulated during cellular stress, such as malignant transformation, or through pharmacological treatment, such as with aminobisphosphonates. In addition, Vγ9Vδ2 T cells possess other cell surface receptors, such as NKG2D, enabling them to interact with cells expressing the NKG2D ligands MICA / B and ULBP (Vantourout and Hayday 2013; Kong et al. 2009; Rincon-Orozco et al. 2005). Following activation, Vγ9Vδ2 T cell functions include cytotoxicity, secretion of chemokines and proinflammatory cytokines, and antigen presentation (Vantourout and Hayday 2013; Brandes, Willimann, and Moser 2005; Devilder et al. 2006).

[0017]

[36] Because the presence of Vγ9Vδ2 T cells in solid tumors strongly correlates with patient survival (Gentles et al. 2015; Tosolini et al. 2017), methods to improve tumor targeting and activation of Vγ9Vδ2 T cells may lead to the development of novel, effective, and safe cancer treatments (de Bruin et al. 2016).

[0018]

[37] Interleukin (IL)-2 is a cytokine known to promote T cell proliferation and function (Nada et al. 2017; Chen et al. 2022; Li and David Pauza 2011). The rationale for exploring IL-2 as an immunomodulatory agent for LAVA-1207 is based on its known T cell stimulatory effects. IL-2 has been shown to induce rapid expansion of Vγ9Vδ2 T cells in vitro and to support the expansion of Vγ9Vδ2 T cells in both non-human primates and humans exposed to phosphoantigen stimulation (Nada et al. 2017; Sicard et al. 2005; Wilhelm et al. 2003; Dieli et al. 2007; Meraviglia et al. 2010; Bennouna et al. 2010). Thus, IL-2 and other common gamma chain cytokines, such as IL-15, may promote a more potent response against tumor cells by expanding and enhancing the availability of Vy9V52- T cells (including LAVA-1207) in patients treated with the multispecific antibodies described herein.

[0019] definition

[38] The term "human V52" as used herein refers to the rearranged 52 chain of the Vy9V52 T-cell receptor (TCR). UniProtKB-A0JD36 (A0JD36_human) is an example of a variable TRDV2 sequence.

[0020]

[39] The term "human Vy9" as used herein refers to the rearranged y9 chain of the Vy9V52-T cell receptor (TCR). UniProtKB-Q99603_human is an example of a variable TRGV9 sequence.

[0021]

[40] The term "PSMA," as used herein, refers to the human prostate-specific membrane antigen protein (UniProtKB-Q04609(FOLH1_human)).

[0022]

[41] The term "CD1d" as used herein refers to the human CD1d protein (UniProtKB-P15813(CD1D_human)).

[0023]

[42] The term "CD40," as used herein, refers to the CD40 protein, also known as tumor necrosis factor receptor superfamily member 5 (UniProtKB-P25942 (TNR5_human)), isoform I.

[0024]

[43] The term "CD123," as used herein, refers to the human CD123 protein, also known as the interleukin-3 receptor alpha chain (NCBI Reference Sequence: NP_002174.1).

[0025]

[44] The term "IL-2" or "IL2" as used herein refers to the interleukin-2 protein. UniProtKB-P60568 provides the WT human IL-2 amino acid sequence. The term IL-2 encompasses variants of the WT sequence.

[0026]

[45] The term "IL-15" or "IL15" as used herein refers to the interleukin-15 protein. UniProtKB-P40933 provides the WT human IL-15 amino acid sequence. The term IL-15 encompasses variants of the WT sequence.

[0027]

[46] The term "5T4" as used herein refers to human trophoblast glycoprotein (UniProtKB-Q13641).

[0028]

[47] The term "human nectin-4" as used herein refers to the human nectin-4 protein (UniProt Q96NY8·NECT4_human).

[0029]

[48] ​​The term "EGFR," as used herein, refers to the human EGFR protein (UniProtKB-P00533(EGFR_human)).

[0030]

[49] The term "CD33," as used herein, refers to the human CD33 protein, also known as Siglec-3 (UniProt P20138 (CD33_human)).

[0031]

[50] The term "immunoglobulin," as used herein, is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four of which may potentially be interconnected by disulfide bonds. The terms "immunoglobulin heavy chain," "immunoglobulin heavy chain," or "heavy chain," as used herein, are intended to refer to one of the chains of an immunoglobulin. A heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH), which defines the immunoglobulin isotype. The heavy chain constant region typically consists of three domains, CH1, CH2, and CH3. The heavy chain constant region further includes a hinge region. Within the structure of an immunoglobulin (e.g., IgG), the two heavy chains are interconnected by disulfide bonds at the hinge region. Like heavy chains, each light chain typically consists of several regions: a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability (or hypervariability in sequence and / or the formation of structurally defined loops), also known as complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR sequences may be determined using various methods, such as those provided by Chothia and Lesk (1987) J. Mol. Biol. 196:901 or Kabat et al. (1991) Sequence of proteins of immunological interest, fifth edition, NIH publication. A comparison of various methods for CDR determination and amino acid numbering can be found at www.abysis.org (UCL).

[0032]

[51] The term "isotype," as used herein, refers to an immunoglobulin (sub)class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f), encoded by heavy chain constant region genes. Each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain. Antibodies of the present disclosure can have any isotype.

[0033]

[52] The term "antibody" is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, that has the ability to specifically bind to an antigen under typical physiological conditions with a half-life spanning a substantial period of time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other relevant, functionally defined period of time (such as a period of time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a period of time sufficient for the antibody to recruit effector activity).

[0034]

[53] The terms "antigen-binding region" and "antigen-binding domain" are used interchangeably herein and refer to the portion of an antibody that interacts with an antigen. An antigen-binding domain can include both the heavy and light chain variable regions of an immunoglobulin molecule, or can be a single-domain antigen-binding region, e.g., only the heavy chain variable region. The constant regions of an antibody, if present, can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and components of the complement system, such as C1q, the first component of the classical pathway of complement activation.

[0035]

[54] The Fc region of an immunoglobulin is typically defined as a fragment of an antibody that can be generated after digestion of the antibody with papain, including the two CH2-CH3 regions of the immunoglobulin and a connecting region, such as the hinge region. The constant domains of the antibody heavy chain define the antibody isotype, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE. The Fc region mediates antibody effector functions through cell surface receptors called Fc receptors and proteins of the complement system.

[0036]

[55] The term "hinge region," as used herein, is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to EU numbering.

[0037]

[56] The term "CH2 region" or "CH2 domain," as used herein, is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be of any of the other subtypes as described herein.

[0038]

[57] The term "CH3 region" or "CH3 domain," as used herein, is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be of any of the other subtypes as described herein.

[0039]

[58] As noted above, the term antibody, as used herein, includes fragments of antibodies that retain the ability to specifically bind to an antigen, unless otherwise specified or clearly contradicted by the context. It has been shown that fragments of full-length antibodies can perform the antigen-binding function of an antibody. Examples of binding fragments encompassed within the term "antibody" include: (i) Fab' or Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or monovalent antibodies as described in WO 2007 / 059782; (ii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; and (iv) Fv fragments consisting essentially of the VL and VH domains on a single arm of an antibody. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods with a synthetic linker, allowing them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs); see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed within the term antibody unless the context dictates otherwise. While such fragments are generally included within the meaning of antibody, they collectively and each independently represent unique features of the present disclosure and exhibit distinct biological properties and utilities. The term antibody also includes, unless otherwise specified, polyclonal antibodies, monoclonal antibodies (mAbs), chimeric and humanized antibodies, and antibody fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

[0040]

[59] In some embodiments of the antibodies of the present disclosure, the first antigen-binding region, the second antigen-binding region, or both are single-domain antibodies. Single-domain antibodies are well known to those skilled in the art; see, for example, Hamers-Casterman et al. (1993) Nature 363:446, Roovers et al. (2007) Curr Opin Mol Ther 9:327, and Krah et al. (2016) Immunopharmacol Immunotoxicol 38:21. Single-domain antibodies contain a single CDR1, a single CDR2, and a single CDR3. Examples of single-domain antibodies include heavy-chain-only antibody variable fragments, naturally occurring antibodies without light chains, single-domain antibodies derived from conventional antibodies, and engineered antibodies. Single-domain antibodies can be derived from any species. For example, single domain antibodies can be derived from antibodies produced by Camelidae species, such as camels, dromedaries, llamas, alpacas, and guanacos. Like whole antibodies, single domain antibodies are capable of selectively binding to specific antigens. Single domain antibodies may contain only the variable domains of an immunoglobulin chain, i.e., CDR1, CDR2, and CDR3, as well as framework regions. Such antibodies are also called Nanobodies® or VHHs.

[0041]

[60] The term "parent antibody" should be understood to be an antibody identical to the antibody of the present disclosure, but where the parent antibody does not have one or more of the specified mutations. An "antibody variant" or "variant of a parent antibody" of the present disclosure is an antibody molecule that contains one or more mutations compared to the "parent antibody." Amino acid substitutions may replace a native amino acid with another naturally occurring amino acid or with a non-naturally occurring amino acid derivative. Amino acid substitutions may be conservative or non-conservative. In the context of the present disclosure, conservative substitutions may be defined by substitutions within amino acid classes reflected in one or more of Tables 1, 2, and 3.

[0042] [Table 1]

[0043] [Table 2]

[0044] [Table 3]

[0045]

[61] The term "parent protein" should be understood to be a protein identical to the protein of the present disclosure, but where the parent protein does not have one or more of the specified mutations. A "variant" or "protein variant" of the present disclosure is a protein that contains one or more amino acid mutations compared to the "parent protein."

[0046]

[62] In the context of this disclosure, substitutions in variants are designated original amino acid-position-substituted amino acid; the three-letter code or one-letter code is used to designate amino acid residues, including the codes Xaa and X. Thus, the designation "T366W" means a variant containing a substitution of threonine with tryptophan at the variant amino acid position corresponding to amino acid 366 of the parent antibody.

[0047]

[63] Furthermore, the term "substitution" encompasses substitution with any one of the other 19 naturally occurring amino acids, or substitution with other amino acids, such as unnatural amino acids. For example, substitution of the amino acid T at position 366 includes each of the following substitutions: 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L, 366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y.

[0048]

[64] The term "full-length antibody," as used herein, refers to an antibody that contains all of the heavy and light chain constant and variable domains corresponding to those normally found in a wild-type antibody of that isotype.

[0049]

[65] The term "chimeric antibody" refers to an antibody whose variable regions are derived from a non-human species (e.g., from a rodent) and whose constant regions are derived from another species, such as human. Chimeric antibodies can be produced by genetic engineering. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce the immunogenicity of antibodies.

[0050]

[66] The term "humanized antibody" refers to a genetically engineered non-human antibody with a human antibody constant domain and a non-human variable domain that has been modified to have a high degree of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen-binding site, onto homologous human acceptor framework regions (FRs). To fully reproduce the binding affinity and specificity of the parent antibody, it may be necessary to substitute framework residues from the parent antibody (i.e., non-human antibody) into human framework regions (back mutations). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may contain non-human CDR sequences, primarily human framework regions optionally containing one or more amino acid back mutations to non-human amino acid sequences, and, optionally, fully human constant regions. Optionally, additional amino acid modifications, not necessarily back mutations, can be introduced to obtain a humanized antibody with desirable characteristics, such as affinity and biochemical properties. Humanization of non-human therapeutic antibodies is performed so that such humanized antibodies retain the specificity and binding affinity of the antibody of non-human origin, while at the same time minimizing its immunogenicity in humans.

[0051]

[67] The term "multispecific antibody" or "MSAb" refers to an antibody having specificity for at least two different, e.g., at least three, typically non-overlapping epitopes. Such epitopes may be on the same or different target antigens. When the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. A multispecific antibody may comprise one or more single domain antibodies.

[0052]

[68] The term "bispecific antibody" refers to an antibody having specificity for two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. A bispecific antibody may comprise one or two single domain antibodies.

[0053]

[69] The term "bispecific T cell engager" or "bsTCE" refers to a bispecific antibody that has specificity for two different epitopes, one of which is expressed by T cells.

[0054]

[70] (ii) recombinant IgG-like dual-targeting molecules, each flanked by Fab fragments or portions of Fab fragments of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or portion of an Fab fragment; (iv) Fc fusion molecules, in which a single-chain Fv molecule or a stabilized diabody is fused to a heavy-chain constant domain, Fc region, or portion thereof; (v) Fab fusion molecules, in which different Fab fragments are fused together, fused to a heavy-chain constant domain, Fc region, or portion thereof; and (vi) ScFv- and diabody-based heavy-chain antibodies (e.g., domain antibodies, Nanobodies®), in which different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, Nanobodies®) are fused to each other or to another protein or carrier molecule that is fused to a heavy-chain constant domain, Fc region, or portion thereof.

[0055]

[71] Examples of IgG-like molecules with complementary CH3 domain molecules include, but are not limited to, Triomab® (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and Electrostatically Matched (Amgen, Chugai, Oncomed), LUZ-Y (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG body and PIG body (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEED body) (EMD Serono), Biclonics (Merus, WO 2013157953), FcΔAdp (Regeneron), bispecific IgG1 and IgG2 (Pfizer / Rinat), Azymetric scaffolds (Zymeworks / Merck), mAb-Fv (Xencor), bivalent bispecific antibodies (Roche, WO 2009 / 080254) and DuoBody® molecules (Genmab).

[0056]

[72] Examples of recombinant IgG-like dual-targeting molecules include, but are not limited to, Dual Targeting (DT)-Ig (GSK / Domantis, WO 2009 / 058383), Two-in-one antibodies (Genentech, Bostrom, et al 2009. Science 323, 1610-1614), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star), Zybodies™ (Zyngenia, LaFleur et al. MAbs. 2013 Mar-Apr; 5(2):208-18), common light chain approach, κλBodies (NovImmune, WO 2012 / 023053), and CovX-body® (CovX / Pfizer, Doppalapudi, VR, et al 2007. Bioorg. Med. Chem. Lett. 17,501-506).

[0057]

[73] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig (Abbott), dual domain dual-head antibody (Unilever; Sanofi Aventis), IgG-like bispecific (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb; 32(2):191-8), Ts2Ab (MedImmune / AZ, Dimasi et al. J Mol Biol. 2009 Oct 30; 393(3):672-92), and BsAb (Zymogenetics, WO 2010111625), HERCULES (Biogen Idec), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc), and TvAb (Roche).

[0058]

[74] Examples of Fc-fusion molecules include, but are not limited to, ScFv / Fc fusions (Academic Institution, Pearce et al. Biochem Mol Biol Int. 1997 Sep;42(6):1179), SCORPION (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics / BMS, WO 2010111625), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics), and Dual (ScFv)2-Fab (National Research Center for Antibody Medicine - China).

[0059]

[75] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 (Medarex / AMGEN), dual action or bis-Fab (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech).

[0060]

[76] Examples of ScFv-based, diabody-based and domain antibodies include, but are not limited to, bispecific T cell engagers (BiTE®) (Micromet, Tandem diabodies (Affimed), Dual Affinity Retargeting Technology (DART™) (MacroGenics), single chain diabodies (Academic, Lawrence FEBS Lett. 1998 Apr 3; 425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO 2010059315) and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug; 88(6):667-75), dual-targeting nanobodies® (Ablynx, Hmila et al., FASEB J. 2010), and dual-targeting heavy chain-only domain antibodies.

[0061]

[77] In some embodiments, the multispecific antibodies used in the present disclosure are in the VHH-Fc format, i.e., the antibodies comprise two or more single-domain antigen-binding regions linked together by a human Fc region dimer. In this format, each single-domain antigen-binding region is fused to an Fc region polypeptide, and these two fusion polypeptides form a dimeric bispecific antibody via disulfide bridges in the hinge region. Such constructs typically do not contain the complete CH1 or light chain sequence, or any of them at all. Figure 12B of WO 06064136 provides an illustration of one example of this format.

[0062]

[78] In the context of antibody binding to an antigen, the terms "bind," "capable of binding," or "specifically bind" refer to a binding activity of approximately 10, as determined, for example, using flow cytometry. -6 M or less, e.g. 10 -7 M or less, about 10-8 M or less, about 10 -9 M or less, about 10 -10 M or less, or about 10- 11 Specific binding refers to the binding of an antibody to a predetermined antigen or target (e.g., human V52 or human PSMA), typically with an apparent affinity corresponding to a KD such as M or even lower. Alternatively, KD values ​​can be determined using, for example, surface plasmon resonance (SPR) technology on a BIAcore T200 or biolayer interferometry (BLI) on an Octet RED96 instrument using the antigen as the ligand and the binding moiety or binding molecule as the analyte. Specific binding means that an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least 10-fold lower, such as at least 100-fold lower, such as at least 1,000-fold lower, such as at least 10,000-fold lower, such as at least 100,000-fold lower, such as at least 100,000-fold lower, than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The extent to which affinity is reduced depends on the KD of the binding moiety or binding molecule, so that when the KD of a binding moiety or binding molecule is very low (i.e., the binding moiety or binding molecule is highly specific), the affinity for the antigen may be at least 10,000-fold reduced compared to the affinity for a nonspecific antigen. The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction between an antigen and a binding moiety or binding molecule.

[0063]

[79] "Ability to bind to the V52 chain of Vy9V52-TCR" or "binds to the V52 chain of Vy9V52-TCR" etc. means that the antibody can bind to the V52 chain as a separate molecule and / or the V52 chain as part of the Vy9V52-TCR. However, the antibody does not bind to the Vy9 chain as a separate molecule.

[0064]

[80] "Ability to bind to the Vγ9 chain of Vγ9Vδ2-TCR" or "binds to the Vγ9 chain of Vγ9Vδ2-TCR" etc. means that the antibody can bind to the Vγ9 chain as a separate molecule and / or the Vγ9 chain as part of the Vγ9Vδ2-TCR. However, the antibody does not bind to the Vδ2 chain as a separate molecule.

[0065]

[81] In the context of this disclosure, "compete" or "capable of competing" or "competing" refers to any detectably greater decrease in the tendency of a particular binding molecule (e.g., a PSMA-binding antibody) to bind to a particular binding partner (e.g., PSMA) in the presence of another molecule (e.g., a different PSMA antibody) that binds to the binding partner. Typically, competition means that the presence of another molecule, such as an antibody, causes at least about a 25 percent decrease, such as at least about a 50 percent decrease, e.g., at least about a 75 percent decrease, at least about a 90 percent decrease, etc., in binding, as determined, for example, by ELISA analysis or flow cytometry using sufficient amounts of two or more competing molecules, e.g., antibodies. For further methods of determining binding specificity by competitive inhibition, see, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley InterScience NY, (1992, 1993), and Muller, Meth. Enzymol. 92, 589-601 (1983)).

[0066]

[82] The disclosed methods or uses may involve the use of antibodies that bind to the same epitope on a target (e.g., Vδ2 or PSMA) as the antibodies described herein. Several methods known in the art for mapping antibody epitopes on target antigens are available, including, but not limited to, cross-linking coupling mass spectrometry, which allows identification of peptides that are part of the epitope, and X-ray crystallography, which identifies individual residues on the antigen that form the epitope. Epitope residues can be determined as all amino acid residues with at least one atom within 5 Å of the antibody. 5 Å was selected as the epitope cutoff distance, allowing for atoms within the van der Waals radius plus potential hydrogen bonds via water. Epitope residues can then be determined as all amino acid residues with at least one atom within 8 Å. 8 Å was selected as the epitope cutoff distance, allowing for the extended length of arginine amino acids. Cross-linking coupling mass spectrometry begins with conjugating an antibody and an antigen with a mass-tagged chemical cross-linker. The presence of the complex is then confirmed using high-mass MALDI detection. After cross-linking chemistry, the Ab / Ag complex is extremely stable, allowing the complex to be subjected to many different enzymes and digestion conditions, providing many different overlapping peptides. Identification of these peptides is performed using high-resolution mass spectrometry and MS / MS techniques. The identity of the cross-linked peptides is determined using mass tags attached to the cross-linking reagents. After MS / MS fragmentation and data analysis, the cross-linked and antigen-derived peptides are part of the epitope, while the antibody-derived peptides are part of the paratope. All residues between the most N- and C-terminal cross-linked residues from each identified cross-linked peptide are considered part of the epitope or paratope.

[0067]

[83] The terms "first" and "second" antigen-binding regions, as used herein, do not refer to their orientation / position on the antibody, i.e., they have no meaning in relation to the N-terminus or C-terminus. The terms "first" and "second" merely serve to correctly and consistently refer to two different antigen-binding regions in the claims and the description herein.

[0068]

[84] "Percent sequence identity," as used herein, refers to the number of identical nucleotide or amino acid positions shared by different sequences, taking into account the number of gaps that need to be introduced for optimal alignment and the length of each gap (i.e., % identity = number of identical positions / total number of positions x 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0069]

[85] "Treatment" or "treating" refers to the administration of an effective amount of an antibody, common gamma chain cytokine, and / or immune checkpoint inhibitor according to the present disclosure to alleviate, ameliorate, halt, eradicate (cure), or prevent a symptom or disease state.

[0070]

[86] An "effective amount" or "therapeutically effective amount" refers to an amount of an agent described herein (e.g., a multispecific antibody, a common gamma chain cytokine, and / or an immune checkpoint inhibitor, either alone or in combination) effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., prevent or treat cancer in an individual). An effective amount of a polypeptide, such as an antibody, can vary depending on factors such as the stage, age, sex, and weight of the individual, the timing and route of administration; the duration of treatment; drugs used in combination; the judgment of the prescribing physician; and similar factors known in the medical arts. An effective amount is also one in which any toxic or detrimental effects of the antibody are outweighed by the therapeutically beneficial effects. Administration can be carried out by any suitable route, but is typically parenteral, such as intravenous, intramuscular, or subcutaneous. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular active ingredient, as well as the desired physiological effect. It will be appreciated by those skilled in the art that for various diseases or disorders, long-term treatment may be required, possibly involving multiple administrations using various rounds of administration.

[0071]

[87] As used herein, "dosing interval" refers to the frequency of administration of a particular agent (e.g., the frequency of administration of a multispecific antibody, a common gamma chain cytokine, or an immune checkpoint inhibitor) during the course of treatment.

[0072] Combination therapy

[88] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject (i) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor, and (ii) a common gamma chain cytokine.

[0073]

[89] The term "common gamma chain cytokine," as used herein, refers to a cytokine that binds to the common gamma chain receptor (common γc), also known as CD132. Common gamma chain cytokines include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. In some embodiments, the methods and kits described herein utilize IL-2 or IL-15. IL-2 and IL-15 bind to a common heterodimeric receptor composed of IL-2 / 15Rβ (CD122) and the common γ chain. The specificity of action of both cytokines is conferred by their corresponding α receptor chains, IL-2Rα (CD25) and IL-15Rα (CD215).

[0074] IL-2 combination therapy

[90] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject (i) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor, and (ii) IL-2.

[0075]

[91] The IL-2 used in the methods of the present disclosure can be recombinant IL-2. The IL-2 can also be aldesleukin (Proleukin®), e.g., 1.3 mg aldesleukin / Proleukin in a 5 mL vial (22 x 10 per vial). 6IU (International Units), and this formulation can be reconstituted using sterile water for injection. Other suitable forms of IL-2 include NKTR-214 (bempegaldesleukin / Nektar), ALK 4230 (nemvaleukin / Aalkermes-Reliant), SAR444245-THOR 707 / Synthorx / Sanofi), and XTX-202 (Xilio). In some embodiments, the IL-2 is a mutant IL-2. In some embodiments, the mutant IL-2 binds to the IL-2R beta and gamma chains but does not bind or demonstrates reduced / absent binding to the IL-2R alpha chain (e.g., NL201 from Neoleukin, Silva et al. De novo design of potent and selective mimics of IL-2 and IL-15. Nature 565, 186-191 (2019), and MDNA11 from Medicenna). In some embodiments, the IL-2 and / or variants thereof are pegylated. The IL-2 may be administered by any suitable route of administration, for example, subcutaneously.

[0076]

[92] In some embodiments, the multispecific antibody is administered prior to administration of IL-2. In some embodiments, IL-2 is administered at least once, at least twice, at least three times, at least four times, at least five times, at least six times, or at least seven times after administration of the multispecific antibody. For example, in some embodiments, the method comprises administration of a multispecific antibody followed by 1, 2, 3, 4, 5, 6, 7, or more doses of IL-2 daily. In some embodiments, the multispecific antibody is administered simultaneously with administration of IL-2. In some embodiments, IL-2 is administered prior to administration of the multispecific antibody.

[0077] IL-2 Dosage Administration

[93] IL-2 or its variants can be administered at any suitable dose. IL-2 can be administered at a dose of, for example, 0.1 to 5 MIU (0.1 to 5 × 10 6IU), 0.1-2.5 MIU / day, 0.1-3.5 MIU / day, 0.1-4.5 MIU, 0.5-3.5 MIU / day, 0.5-2.5 MIU / day, or 0.5-1.5 MIU / day. In some embodiments, IL-2 is given at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 MIU. In some embodiments, IL-2 is given at a daily dose of 1.0 MIU. In some embodiments, IL-2 is given at a dose of 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 MIU. In some embodiments, IL-2 is given at a dose of 2.0 MIU. In some embodiments, IL-2 is given at a dose of 3.0 MIU.

[0078]

[94] Currently, low-dose subcutaneous interleukin-2 (LDSC IL-2) is being used and studied in the management of immune-mediated diseases and is well tolerated when administered at low doses (1 MIU / day or less (Mahmoudpour et al. 2019; de Bono et al. 2022). In an ongoing study of the BTN3A-specific monoclonal antibody ICT01, which stimulates Vγ9Vδ2-T cell activation in patients with advanced solid tumors (EVICTION-2 trial), it was reported that adding LDSC IL-2 at a dose of 1 MIU / m2 (administered on days 1, 2, 3, 4, and 5 after ICT01) was safe and induced the expansion of Vγ9Vδ2-T cells in 6 out of 6 evaluable patients (de Bono et al. al. 2022). Thus, in some embodiments, IL-2 is given as a "low dose." A low dose of IL-2 refers to a dose of less than 3 MIU. In some embodiments, IL-2 is given at a dose of less than 3.0 MIU. In some embodiments, IL-2 is given at a dose of less than 2.0 MIU. In some embodiments, IL-2 is given at a dose of less than 1.0 MIU. IL-2 can also be given at doses of 0.1 to 5 MIU (0.1 to 5 x 10), such as 0.2 to 2 MIU, e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 MIU. 6 IU) multiple times daily, for example, twice daily. In some embodiments, IL-2 is administered in different amounts depending on the treatment cycle. In some embodiments, IL-2 is administered at a low dose during the first treatment cycle and then increased during subsequent treatment cycles.

[0079] IL-2 can be administered at any suitable dosing interval, e.g., between 1 day and 1 month, e.g., between 1-21 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or between 7-21 days, e.g., 7 or 14 days. In some embodiments, the IL-2 is administered multiple times daily, e.g., IL-2 doses can be administered 1, 2, 3, 4, 5, or 6 times daily. In some embodiments, IL-2 is administered once daily. In some embodiments, IL-2 is administered twice daily.

[0080] IL-15 combination therapy

[0009] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject (i) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor, and (ii) IL-15.

[0081]

[97] Suitable forms of IL-15 are known in the art. See, e.g., Int J Mol Sci. 2022 Jul;23(13):7311, which is incorporated herein by reference in its entirety. In some embodiments, the IL-15 is a heterodimer of IL-15 and IL-15 receptor alpha (hetIL-15), also known as NIZ985. In some embodiments, the IL-15 is N-803, formerly known as ALT-803, which is an IL-15 variant complexed with a human IL-15Rα sushi domain-Fc fusion protein. In some embodiments, the IL-15 is SOT101, also known as Nanrilkefusp alfa, which is a human fusion protein comprising the cytokine IL-15 and the high-affinity binding sushi domain of IL-15 receptor alpha (IL-15Rα). In some embodiments, the IL-15 is NKTR-255, which is a polyethylene glycol conjugate of rhIL-15. In some embodiments, the NKTR-255 is administered at 1.5 μg / kg, 3.0 μg / kg, or 3.0 / 6.0 μg / kg. In some embodiments, the IL-15 and / or variants thereof are pegylated.

[0082]

[98] In some embodiments, the multispecific antibody is administered prior to administration of IL-15. In some embodiments, IL-15 is administered at least once, at least twice, at least three times, at least four times, at least five times, at least six times, or at least seven times after administration of the multispecific antibody. For example, in some embodiments, the method comprises administration of a multispecific antibody followed by 1, 2, 3, 4, 5, 6, 7, or more daily doses of IL-15. In some embodiments, the multispecific antibody is administered simultaneously with administration of IL-15. In some embodiments, IL-15 is administered prior to administration of the multispecific antibody.

[0083] IL-15 Dosage Administration

[99] IL-15 can be administered at any suitable dose. In some embodiments, IL-15 is administered at a dose of 0.001 to 1000 MIU (0.001 to 1000 × 10 6 IU). In some embodiments, the dose of IL-15 is 0.01 to 100 MIU. In some embodiments, the dose of IL-15 is 0.1 to 10 MIU. In some embodiments, the dose of IL-15 is 0.5 to 5 MIU. In some embodiments, the dose of IL-15 is 1 to 5 MIU. In some embodiments, the dose of IL-15 is 2 to 5 MIU. In some embodiments, IL-15 is given at a dose of 0.5 to 1.5 MIU / day, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5 MIU / day. IL-15 may also be administered multiple times per day, such as 0.2-2 MIU / day, such as 0.5-1.5 MIU, for example, at doses of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or 1.5 MIU, or at doses of 0.1-5 MIU (0.1-5 10 6 IU) twice daily.

[0084]

[0100] In some embodiments, IL-15 is administered at a dose of 0.01 to 40 μg / kg. In some embodiments, the dose of IL-15 is 0.05 to 20 μg / kg. In some embodiments, the dose of IL-15 is 0.1 to 10 μg / kg. In some embodiments, the dose of IL-15 is 1 to 15 μg / kg. In some embodiments, the dose is any dose between 1 and 15 μg / kg, such as 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 2.5 μg / kg, 3 μg / kg, 3.5 μg / kg, 4 μg / kg, 4.5 μg / kg, 5 μg / kg, 5.5 μg / kg, 6 μg / kg, 6.5 μg / kg, 7 μg / kg, 7.5 μg / kg, 8 μg / kg, 8.5 μg / kg, 9 μg / kg, 9.5 μg / kg, 10 μg / kg, 10.5 μg / kg, 11 μg / kg, 11.5 μg / kg, 12 μg / kg, 12.5 μg / kg, 13 μg / kg, 13.5 μg / kg, 14 μg / kg, 14.5 μg / kg, or 15 μg / kg. In some embodiments, IL-15 is administered in different amounts depending on the treatment cycle: hi some embodiments, IL-15 is administered at a low dose during treatment cycle 1 and the dose is increased during subsequent treatment cycles.

[0085]

[0101] IL-15 can be administered at any suitable dosing interval, e.g., between 1 day and 1 month, e.g., between 1-21 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or between 7-21 days, e.g., 7 or 14 days. In some embodiments, the IL-15 dosing regimen is multiple times daily, e.g., IL-15 doses can be administered 1, 2, 3, 4, 5, or 6 times daily. In some embodiments, IL-15 is administered once daily. In some embodiments, IL-15 is administered twice daily.

[0086] Immune checkpoint inhibitor combination therapy

[0102] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject (i) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor, and (ii) an immune checkpoint inhibitor.

[0087]

[0103] Immune checkpoint inhibitors are known in the art, including agents that target PD-1, PD-L1, CTLA4, LAG-3, TIM-3, TIGIT, and / or NKG2A. In some embodiments, the immune checkpoint inhibitor targets PD-1 or PD-L1.

[0088]

[0104] In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody or an anti-PD-L1 antibody. In some embodiments, the anti-PD1 antibody is selected from pembrolizumab (Keytruda®), nivolumab (Opdivo®), Zynz™ (retifanlimab), Jemperli™ (dostarlimuab), cemiplimab (Libtayo®), tislelizumab (BGB-A317), and sintilimab (IBI308). In some embodiments, the anti-PD-L1 antibody is selected from atezolizumab (Tecentriq®), durvalumab (Imfinzi®), and avelumab (Bavencio®). In some embodiments, the anti-PD1 antibody is pembrolizumab.

[0089]

[0105] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody is ipilimumab (Yervoy®). In some embodiments, the immune checkpoint inhibitor is an inhibitor of LAG3. In some embodiments, the inhibitor is an anti-LAG3 antibody selected from leratolimab, tebotelimab, or favezelimab. Additional LAG-3 antibodies and inhibitors are summarized in Huo et al., The promising immune checkpoint LAG-3 in cancer immunotherapy: from basic research to clinical application. Front Immunol. 2022 Jul 26;13:956090 (incorporated herein by reference). In some embodiments, the immune checkpoint inhibitor is an inhibitor of TIM3 or TIGIT. Antibodies and inhibitors of TIM3 or TIGIT are described in Cai et al. Targeting LAG-3, TIM-3, and TIGIT for cancer immunotherapy. J Hematol Oncol 16, 101 (2023) (incorporated herein by reference). In some embodiments, the immune checkpoint inhibitor is an NKG2A inhibitor. In some embodiments, the NKG2A inhibitor is monalizumab.

[0090]

[0106] In some embodiments, the multispecific antibody is administered prior to administration of the immune checkpoint inhibitor (e.g., an anti-PD1 antibody). In some embodiments, the multispecific antibody is administered simultaneously with administration of the immune checkpoint inhibitor (e.g., an anti-PD1 antibody). In some embodiments, the immune checkpoint inhibitor (e.g., an anti-PD1 antibody) is administered prior to administration of the multispecific antibody.

[0091] Dosage regimens for immune checkpoint inhibitors

[0107] In some embodiments, the immune checkpoint inhibitor is administered at any suitable dose and interval consistent with the agent being used. In some embodiments, the immune checkpoint inhibitor is an antibody and is administered in doses of 100-1000 mg, such as 200-800 mg, such as 300-500 mg, such as 400 mg. In some embodiments, the doses are administered every six weeks. For example, in some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody. In some embodiments, the immune checkpoint inhibitor antibody is administered as directed in its approved labeling.

[0092]

[0108] In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, pembrolizumab is administered at a dose specified in the drug labeling for the appropriate indication. Pembrolizumab labeling is available at accessdata.fda.gov / drugsatfda_docs / label / 2021 / 125514s096lbl.pdf. In some embodiments, pembrolizumab is administered at 400 mg IV over 30 minutes every 6 weeks. Nivolumab labeling is available at accessdata.fda.gov / drugsatfda_docs / label / 2018 / 125554s058lbl.pdf. Additional drug labeling for approved formulations is available on the FDA website.

[0093]

[0109] The anti-PD1 or anti-PD-L1 antibody may be administered at any suitable dosing interval, for example, between 1 day and 2 months, between 1 and 50 days, such as between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 35, 42, or 49 days, or between 7 and 49 days, such as between 7, 14, 21, 28, 35, or 42 days. In some embodiments, the anti-PD1 antibody is administered every 42 days (e.g., every 6 weeks).

[0094]

[0110] In some embodiments, the multispecific antibody is administered prior to administration of the immune checkpoint inhibitor (e.g., an anti-PD1 antibody). In some embodiments, the multispecific antibody is administered simultaneously with administration of the immune checkpoint inhibitor (e.g., an anti-PD1 antibody). In some embodiments, the immune checkpoint inhibitor (e.g., an anti-PD1 antibody) is administered prior to administration of the multispecific antibody.

[0095]

[0111] In some embodiments, an immune checkpoint inhibitor (e.g., an anti-PD1 antibody) is used in combination with a common gamma chain cytokine (e.g., IL-2 or IL-15) as described above. In some embodiments, the multispecific antibody is administered prior to administration of the immune checkpoint inhibitor (e.g., an anti-PD1 antibody) and IL-2 or IL-15. In some embodiments, the multispecific antibody is administered simultaneously with administration of the immune checkpoint inhibitor (e.g., an anti-PD1 antibody) and IL-2 or IL-15. In some embodiments, the immune checkpoint inhibitor (e.g., an anti-PD1 antibody) and IL-2 or IL-15 are administered prior to administration of the multispecific antibody. For example, IL-2 or IL-15 and / or anti-PD1 or anti-PD-L1 antibody may be administered daily for at least one day, such as 2, 3, 4, 5, 6, 7 or more days, following administration of the multispecific antibody, e.g., starting the day after administration of the multispecific antibody. For example, the multispecific antibody may be administered on day 1, and IL-2 or IL-15 and / or anti-PD1 or anti-PD-L1 antibody may be administered on day 2, or on days 2, 3 and 4. Such a cycle may be repeated at least once, such as 2, 3, 4, 5, 6, 7, 8 or more times, e.g., with an interval of 14 days between multispecific antibody administrations.

[0096] Multispecific antibody dosing regimen

[0112] The multispecific antibody may be administered at any suitable interval between doses, for example, between 1 day and 1 month, for example, between 1 and 21 days, between 7 and 21 days, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or between 7 or 14 days. In some embodiments, the multispecific antibody is administered every 14 days.

[0097]

[0113] The multispecific antibody may be administered in any suitable dose. In some embodiments, the multispecific antibody is administered in a dose of at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 6 μg, at least 7 μg, at least 8 μg, at least 9 μg, at least 10 μg, at least 11 μg, at least 12 μg, at least 13 μg, at least 14 μg, at least 15 μg, at least 16 μg, at least 17 μg, at least 18 μg, at least 19 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, or at least 45 μg. In some embodiments, the multispecific antibody is administered in a dose of at least 100 μg, at least 110 μg, at least 120 μg, or at least 130 μg. In some embodiments, the multispecific antibody is administered at a dose of at least 300 μg, at least 310 μg, at least 320 μg, at least 330 μg, at least 340 μg, at least 350 μg, at least 360 μg, at least 370 μg, at least 380 μg, at least 390 μg, or at least 400 μg. In some embodiments, the multispecific antibody is administered at a dose of at least 500 μg, at least 510 μg, at least 520 μg, at least 530 μg, at least 540 μg, at least 550 μg, at least 560 μg, at least 570 μg, at least 580 μg, at least 590 μg, or at least 600 μg. In some embodiments, the multispecific antibody is administered at a dose of at least 750 μg, at least 760 μg, at least 770 μg, at least 780 μg, at least 790 μg, at least 800 μg, at least 810 μg, at least 820 μg, at least 830 μg, at least 840 μg, or at least 850 μg. In some embodiments, the multispecific antibody is administered at a dose of at least 1000 μg, at least 1.1 mg, at least 1.2 mg, at least 1.3 mg, at least 1.4 mg, at least 1.5 mg, at least 1.6 mg, at least 1.7 mg, at least 1.8 mg, at least 1.9 mg, or at least 2 mg.In some embodiments, the multispecific antibody is administered at a dose of at least 2.5 mg, at least 3 mg, at least 3.5 mg, at least 4 mg, at least 4.5 mg, at least 5 mg, at least 5.5 mg, at least 6 mg, at least 6.5 mg, at least 7 mg, at least 7.5 mg, at least 8 mg, at least 8.5 mg, at least 9 mg, at least 9.5 mg, or at least 10 mg. In some embodiments, the multispecific antibody is administered at a dose of at least 20 mg, at least 30 mg, at least 40 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, or at least 100 mg. In some embodiments, the multispecific antibody is administered at a dose of at least 110 mg, at least 120 mg, at least 130 mg, at least 140 mg, at least 150 mg, at least 160 mg, at least 170 mg, at least 180 mg, at least 190 mg, or at least 200 mg. In some embodiments, the multispecific antibody is administered at a dose of 360 micrograms to 10 mg per administration. In some embodiments, the multispecific antibody is administered at a dose of 10 mg to 100 mg per administration.

[0098]

[0114] In some embodiments, the multispecific antibody is administered at a dose of at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 6 μg, at least 7 μg, at least 8 μg, at least 9 μg, at least 10 μg, at least 11 μg, at least 12 μg, at least 13 μg, at least 14 μg, at least 15 μg, at least 16 μg, at least 17 μg, at least 18 μg, at least 19 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, or at least 45 μg once every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 100 μg, at least 110 μg, at least 120 μg, or at least 130 μg once every 14 days. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 300 μg, at least 310 μg, at least 320 μg, at least 330 μg, at least 340 μg, at least 350 μg, at least 360 μg, at least 370 μg, at least 380 μg, at least 390 μg, or at least 400 μg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 500 μg, at least 510 μg, at least 520 μg, at least 530 μg, at least 540 μg, at least 550 μg, at least 560 μg, at least 570 μg, at least 580 μg, at least 590 μg, or at least 600 μg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 750 μg, at least 760 μg, at least 770 μg, at least 780 μg, at least 790 μg, at least 800 μg, at least 810 μg, at least 820 μg, at least 830 μg, at least 840 μg, or at least 850 μg.In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 1000 μg, at least 1.1 mg, at least 1.2 mg, at least 1.3 mg, at least 1.4 mg, at least 1.5 mg, at least 1.6 mg, at least 1.7 mg, at least 1.8 mg, at least 1.9 mg, or at least 2 mg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 2.5 mg, at least 3 mg, at least 3.5 mg, at least 4 mg, at least 4.5 mg, at least 5 mg, at least 5.5 mg, at least 6 mg, at least 6.5 mg, at least 7 mg, at least 7.5 mg, at least 8 mg, at least 8.5 mg, at least 9 mg, at least 9.5 mg, or at least 10 mg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of 360 micrograms to 10 mg per administration.

[0099]

[0115] In some embodiments, the multispecific antibody is administered at a dose of at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 6 μg, at least 7 μg, at least 8 μg, at least 9 μg, at least 10 μg, at least 11 μg, at least 12 μg, at least 13 μg, at least 14 μg, at least 15 μg, at least 16 μg, at least 17 μg, at least 18 μg, at least 19 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, or at least 45 μg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 100 μg, at least 110 μg, at least 120 μg, or at least 130 μg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 300 μg, at least 310 μg, at least 320 μg, at least 330 μg, at least 340 μg, at least 350 μg, at least 360 μg, at least 370 μg, at least 380 μg, at least 390 μg, or at least 400 μg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 500 μg, at least 510 μg, at least 520 μg, at least 530 μg, at least 540 μg, at least 550 μg, at least 560 μg, at least 570 μg, at least 580 μg, at least 590 μg, or at least 600 μg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 750 μg, at least 760 μg, at least 770 μg, at least 780 μg, at least 790 μg, at least 800 μg, at least 810 μg, at least 820 μg, at least 830 μg, at least 840 μg, or at least 850 μg twice every 14 days.In some embodiments, the multispecific antibody is administered at a dose of at least 1000 μg, at least 1.1 mg, at least 1.2 mg, at least 1.3 mg, at least 1.4 mg, at least 1.5 mg, at least 1.6 mg, at least 1.7 mg, at least 1.8 mg, at least 1.9 mg, or at least 2 mg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 2.5 mg, at least 3 mg, at least 3.5 mg, at least 4 mg, at least 4.5 mg, at least 5 mg, at least 5.5 mg, at least 6 mg, at least 6.5 mg, at least 7 mg, at least 7.5 mg, at least 8 mg, at least 8.5 mg, at least 9 mg, at least 9.5 mg, or at least 10 mg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of 360 micrograms to 10 mg per administration twice every 14 days.

[0100]

[0116] In some embodiments, the multispecific antibody is administered at a dose of at least 1 μg / kg, at least 2 μg / kg, at least 3 μg / kg, at least 4 μg / kg, at least 5 μg / kg, at least 6 μg / kg, at least 7 μg / kg, at least 8 μg / kg, at least 9 μg / kg, at least 10 μg / kg, at least 11 μg / kg, at least 12 μg / kg, at least 13 μg / kg, at least 14 μg / kg, at least 15 μg / kg, at least 16 μg / kg, at least 17 μg / kg, at least 18 μg / kg, at least 19 μg / kg, at least 20 μg / kg, at least 25 μg / kg, at least 30 μg / kg, at least 35 μg / kg, at least 40 μg / kg, or at least 45 μg / kg. In some embodiments, the multispecific antibody is administered at a dose of at least 100 μg / kg, at least 110 μg / kg, at least 120 μg / kg, or at least 130 μg / kg, hi some embodiments, the multispecific antibody is administered at a dose of at least 300 μg / kg, at least 310 μg / kg, at least 320 μg / kg, at least 330 μg / kg, at least 340 μg / kg, at least 350 μg / kg, at least 360 μg / kg, at least 370 μg / kg, at least 380 μg / kg, at least 390 μg / kg, or at least 400 μg / kg. In some embodiments, the multispecific antibody is administered at a dose of at least 500 μg / kg, at least 510 μg / kg, at least 520 μg / kg, at least 530 μg / kg, at least 540 μg / kg, at least 550 μg / kg, at least 560 μg / kg, at least 570 μg / kg, at least 580 μg / kg, at least 590 μg / kg, or at least 600 μg / kg. In some embodiments, the multispecific antibody is administered at a dose of at least 750 μg / kg, at least 760 μg / kg, at least 770 μg / kg, at least 780 μg / kg, at least 790 μg / kg, at least 800 μg / kg, at least 810 μg / kg, at least 820 μg / kg, at least 830 μg / kg, at least 840 μg / kg, or at least 850 μg / kg.In some embodiments, the multispecific antibody is administered at a dose of at least 1000 μg / kg, at least 1.1 mg / kg, at least 1.2 mg / kg, at least 1.3 mg / kg, at least 1.4 mg / kg, at least 1.5 mg / kg, at least 1.6 mg / kg, at least 1.7 mg / kg, at least 1.8 mg / kg, at least 1.9 mg / kg, or at least 2 mg / kg. In some embodiments, the multispecific antibody is administered at a dose of at least 2.5 mg / kg, at least 3 mg / kg, at least 3.5 mg / kg, at least 4 mg / kg, at least 4.5 mg / kg, at least 5 mg / kg, at least 5.5 mg / kg, at least 6 mg / kg, at least 6.5 mg / kg, at least 7 mg / kg, at least 7.5 mg / kg, at least 8 mg / kg, at least 8.5 mg / kg, at least 9 mg / kg, at least 9.5 mg / kg, or at least 10 mg / kg.

[0101]

[0117] In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 1 μg / kg, at least 2 μg / kg, at least 3 μg / kg, at least 4 μg / kg, at least 5 μg / kg, at least 6 μg / kg, at least 7 μg / kg, at least 8 μg / kg, at least 9 μg / kg, at least 10 μg / kg, at least 11 μg / kg, at least 12 μg / kg, at least 13 μg / kg, at least 14 μg / kg, at least 15 μg / kg, at least 16 μg / kg, at least 17 μg / kg, at least 18 μg / kg, at least 19 μg / kg, at least 20 μg / kg, at least 25 μg / kg, at least 30 μg / kg, at least 35 μg / kg, at least 40 μg / kg, or at least 45 μg / kg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 100 μg / kg, at least 110 μg / kg, at least 120 μg / kg, or at least 130 μg / kg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 300 μg / kg, at least 310 μg / kg, at least 320 μg / kg, at least 330 μg / kg, at least 340 μg / kg, at least 350 μg / kg, at least 360 μg / kg, at least 370 μg / kg, at least 380 μg / kg, at least 390 μg / kg, or at least 400 μg / kg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 500 μg / kg, at least 510 μg / kg, at least 520 μg / kg, at least 530 μg / kg, at least 540 μg / kg, at least 550 μg / kg, at least 560 μg / kg, at least 570 μg / kg, at least 580 μg / kg, at least 590 μg / kg, or at least 600 μg / kg.In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 750 μg / kg, at least 760 μg / kg, at least 770 μg / kg, at least 780 μg / kg, at least 790 μg / kg, at least 800 μg / kg, at least 810 μg / kg, at least 820 μg / kg, at least 830 μg / kg, at least 840 μg / kg, or at least 850 μg / kg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 1000 μg / kg, at least 1.1 mg / kg, at least 1.2 mg / kg, at least 1.3 mg / kg, at least 1.4 mg / kg, at least 1.5 mg / kg, at least 1.6 mg / kg, at least 1.7 mg / kg, at least 1.8 mg / kg, at least 1.9 mg / kg, or at least 2 mg / kg. In some embodiments, the multispecific antibody is administered once every 14 days at a dose of at least 2.5 mg / kg, at least 3 mg / kg, at least 3.5 mg / kg, at least 4 mg / kg, at least 4.5 mg / kg, at least 5 mg / kg, at least 5.5 mg / kg, at least 6 mg / kg, at least 6.5 mg / kg, at least 7 mg / kg, at least 7.5 mg / kg, at least 8 mg / kg, at least 8.5 mg / kg, at least 9 mg / kg, at least 9.5 mg / kg, or at least 10 mg / kg.

[0102]

[0118] In some embodiments, the multispecific antibody is administered at a dose of at least 1 μg / kg, at least 2 μg / kg, at least 3 μg / kg, at least 4 μg / kg, at least 5 μg / kg, at least 6 μg / kg, at least 7 μg / kg, at least 8 μg / kg, at least 9 μg / kg, at least 10 μg / kg, at least 11 μg / kg, at least 12 μg / kg, at least 13 μg / kg, at least 14 μg / kg, at least 15 μg / kg, at least 16 μg / kg, at least 17 μg / kg, at least 18 μg / kg, at least 19 μg / kg, at least 20 μg / kg, at least 25 μg / kg, at least 30 μg / kg, at least 35 μg / kg, at least 40 μg / kg, or at least 45 μg / kg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 100 μg / kg, at least 110 μg / kg, at least 120 μg / kg, or at least 130 μg / kg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 300 μg / kg, at least 310 μg / kg, at least 320 μg / kg, at least 330 μg / kg, at least 340 μg / kg, at least 350 μg / kg, at least 360 μg / kg, at least 370 μg / kg, at least 380 μg / kg, at least 390 μg / kg, or at least 400 μg / kg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 500 μg / kg, at least 510 μg / kg, at least 520 μg / kg, at least 530 μg / kg, at least 540 μg / kg, at least 550 μg / kg, at least 560 μg / kg, at least 570 μg / kg, at least 580 μg / kg, at least 590 μg / kg, or at least 600 μg / kg twice every 14 days.In some embodiments, the multispecific antibody is administered at a dose of at least 750 μg / kg, at least 760 μg / kg, at least 770 μg / kg, at least 780 μg / kg, at least 790 μg / kg, at least 800 μg / kg, at least 810 μg / kg, at least 820 μg / kg, at least 830 μg / kg, at least 840 μg / kg, or at least 850 μg / kg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 1000 μg / kg, at least 1.1 mg / kg, at least 1.2 mg / kg, at least 1.3 mg / kg, at least 1.4 mg / kg, at least 1.5 mg / kg, at least 1.6 mg / kg, at least 1.7 mg / kg, at least 1.8 mg / kg, at least 1.9 mg / kg, or at least 2 mg / kg twice every 14 days. In some embodiments, the multispecific antibody is administered at a dose of at least 2.5 mg / kg, at least 3 mg / kg, at least 3.5 mg / kg, at least 4 mg / kg, at least 4.5 mg / kg, at least 5 mg / kg, at least 5.5 mg / kg, at least 6 mg / kg, at least 6.5 mg / kg, at least 7 mg / kg, at least 7.5 mg / kg, at least 8 mg / kg, at least 8.5 mg / kg, at least 9 mg / kg, at least 9.5 mg / kg, or at least 10 mg / kg twice every 14 days.

[0103] Gradual dose administration

[0119] Multispecific antibodies (including bispecific T cell engagers) hold great promise for cancer treatment, but their use can be limited by treatment-related toxicities, including dose-dependent cytokine release syndrome (CRS). CRS is a systemic inflammatory response that can be triggered by immunotherapeutic agents, such as bispecific T cell engager antibodies. CRS events typically occur during the first treatment cycle, with the highest cytokine production levels often observed after the first dose of antibody. For example, compounds in clinical development, such as AMG 160 and HPN424, aim to direct cytotoxic T cells to prostate tumors by simultaneously targeting CD3 and PSMA. Interim results from phase 1 clinical trials of these molecules have shown initial signs of efficacy (Tran 2020; Bendell et al. 2020). Consistent with other CD3-based T cell engagers (Khadka et al. 2019), CRS was observed as the most frequent adverse event (AE) for AMG 160 and HPN424, and prophylactic risk mitigation for CRS was implemented.

[0104]

[0120] To mitigate the risk of CRS, a step-dose approach can be used. Step-dose administration involves administering one or more priming doses of a multispecific antibody prior to the target dose. The priming dose is a lower dose of the multispecific antibody than the target dose. This allows for priming of the immune system in a stepwise manner to prevent an uncontrolled inflammatory response at the target dose (Ball et al., 2023).

[0105]

[0121] Thus, the term "priming dose" or as used herein refers to a dose of a multispecific antibody described herein that primes a subject for administration of a target dose of the multispecific antibody such that the target dose does not result in one or more treatment-related toxicities, such as CRS. A "target dose," as used herein, refers to a therapeutically effective dose of a multispecific antibody described herein or a dose of a multispecific antibody under evaluation for use as a therapeutically effective dose (e.g., in a dose escalation study described herein).

[0106]

[0122] In some embodiments, the priming dose is 25% or less of the target dose. For example, if the target dose of the multispecific antibody is 100 mg, the priming dose would be 25 mg. In some embodiments, the priming dose is 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less of the target dose. In some embodiments, the priming dose is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the target dose. In some embodiments, the priming dose is 15% of the target dose. In some embodiments, the priming dose is 50% of the target dose.

[0107]

[0123] In some embodiments, the priming dose is 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg, 130 μg, 140 μg, 150 μg, 160 μg, 170 μg, 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270μg, 280μg, 290μg, 300μg, 310μg, 320μg, 330μg, 340μg, 350μg, 360μg, 370μg, 380μg, 390μg, 400μg, 410μg, 420μg, 430μg, 440μg, 450μg, 460μg, 470μg, 480μg, 490μg, 500μg, 510μg, 520μg, 530μg, 540μg, 550μg, 560μg, 570μg, 580μg, 590μg, or 600μg.

[0108]

[0124] In some embodiments, the first priming dose is 120 μg and the second priming dose is 360 μg.

[0109]

[0125] In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 120 μg and the priming dose is 18 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 540 μg and the priming dose is 81 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 800 μg and the priming dose is 120 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 800 μg and the priming doses are 120 μg and 360 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 1200 μg and the priming dose is 180 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 1200 μg and the priming doses are 120 μg and 360 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 1800 μg and the priming dose is 180 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 1800 μg and the priming doses are 120 μg and 360 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 1800 μg and the priming dose is 180 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 1800 μg and the priming doses are 120 μg and 360 μg. In some embodiments, the target dose of the multispecific antibody (e.g., LAVA-1207) is 3600 μg and the priming dose is 180 μg. In some embodiments, the target dose of a multispecific antibody (e.g., LAVA-1207) is 3600 μg and the priming doses are 120 μg and 360 μg.

[0110]

[0126] In some embodiments, there are two or more priming doses. In some embodiments, the two or more priming doses are administered one day apart. In some embodiments, the two or more priming doses are administered two days apart. In some embodiments, the two or more priming doses are administered three days apart. In some embodiments, the two or more priming doses are administered four days apart. In some embodiments, the two or more priming doses are administered five days apart. In some embodiments, the two or more priming doses are administered six days apart. In some embodiments, the two or more priming doses are administered seven days apart. In some embodiments, the two or more priming doses are administered eight days apart. In some embodiments, the two or more priming doses are administered nine days apart. In some embodiments, the two or more priming doses are administered ten days apart. In some embodiments, the two or more priming doses are administered eleven days apart. In some embodiments, the two or more priming doses are administered twelve days apart. In some embodiments, two or more priming doses are administered 13 days apart. In some embodiments, two or more priming doses are administered 14 days apart. In some embodiments, the first priming dose is administered on day 1 and the second priming dose is administered on day 4. In some embodiments, the first priming dose is administered on day 1 and the second priming dose is administered on day 8.

[0111]

[0127] In some embodiments, the final priming dose is administered one day before the target dose. In some embodiments, the final priming dose is administered two days before the target dose. In some embodiments, the final priming dose is administered three days before the target dose. In some embodiments, the final priming dose is administered four days before the target dose. In some embodiments, the final priming dose is administered five days before the target dose. In some embodiments, the final priming dose is administered six days before the target dose. In some embodiments, the final priming dose is administered seven days before the target dose. In some embodiments, the final priming dose is administered eight days before the target dose. In some embodiments, the final priming dose is administered nine days before the target dose. In some embodiments, the final priming dose is administered ten days before the target dose. In some embodiments, the final priming dose is administered eleven days before the target dose. In some embodiments, the final priming dose is administered twelfth days before the target dose. In some embodiments, the final priming dose is administered 13 days before the target dose, hi some embodiments, the final priming dose is administered 14 days before the target dose.

[0112]

[0128] In some embodiments, the first priming dose is administered on day 1, the second priming dose is administered on day 4, and the target dose is administered on day 8. In some embodiments, the first priming dose is administered on day 1, the second priming dose is administered on day 8, and the target dose is administered on day 15.

[0113]

[0129] In some embodiments, the multispecific antibody is LAVA-1207, and the first treatment cycle comprises administration of one or more priming doses and one or more target doses. In some embodiments, the first treatment cycle comprises administration of one priming dose and one or more target doses. In some embodiments, the first treatment cycle comprises administration of two priming doses and one or more target doses. In some embodiments, the first treatment cycle comprises administration of three priming doses and one or more target doses. In some embodiments, the first treatment cycle comprises administration of one priming dose and one target dose. In some embodiments, the first treatment cycle comprises administration of two priming doses and one target dose. In some embodiments, the first treatment cycle comprises administration of three priming doses and one target dose.

[0114]

[0130] In some embodiments, the multispecific antibody is LAVA-1207, and the second treatment cycle comprises administration of one or more priming doses and one or more target doses. In some embodiments, the second treatment cycle comprises administration of one priming dose and one or more target doses. In some embodiments, the second treatment cycle comprises administration of two priming doses and one or more target doses. In some embodiments, the second treatment cycle comprises administration of three priming doses and one or more target doses. In some embodiments, the second treatment cycle comprises administration of one priming dose and one target dose. In some embodiments, the second treatment cycle comprises administration of two priming doses and one target dose. In some embodiments, the second treatment cycle comprises administration of three priming doses and one target dose.

[0115] In some embodiments, the multispecific antibody is LAVA-1207, and the first and second treatment cycles comprise administration of one or more priming doses and one or more target doses. In some embodiments, the first and second treatment cycles comprise administration of one priming dose and one or more target doses. In some embodiments, the first and second treatment cycles comprise administration of two priming doses and one or more target doses. In some embodiments, the first and second treatment cycles comprise administration of three priming doses and one or more target doses. In some embodiments, the first and second treatment cycles comprise administration of one priming dose and one target dose. In some embodiments, the first and second treatment cycles comprise administration of two priming doses and one target dose. In some embodiments, the first and second treatment cycles comprise administration of three priming doses and one target dose.

[0116] Treatment regimen

[0131] Table 4 below provides exemplary dosing regimens for the multispecific antibody, IL-2 or IL-15, and immune checkpoint inhibitor within a particular treatment cycle. One of skill in the art will appreciate that the following regimens may be combined. For example, regimen A may be utilized for the first treatment cycle, followed by regimen B for the second treatment cycle, followed by regimen C for the third treatment cycle. In some embodiments, any of the regimens described below may be repeated. For example, regimen A may be utilized for the first, second, third, or subsequent treatment cycle, followed by regimen B for one or more treatment cycles, followed by regimen C for one or more treatment cycles.

[0117]

[0132] Furthermore, although Table 4 below refers to a "first agent," a "second agent," and a "third agent," one of skill in the art will understand that each agent can be administered more than once. For example, in Regimen A, the administration of a multispecific antibody (MSAb) as the first agent can be a single dose of the MSAb or multiple doses (e.g., 2, 3, 4, 5, 6, or more doses) of the MSAb over the course of one or more days, and the administration of a cytokine as the second agent can be a single dose of the cytokine or multiple doses (e.g., 2, 3, 4, 5, 6, or more doses) of the cytokine over the course of one or more days. References to "cytokine" in Table 4 are intended to refer to common gamma chain cytokines (e.g., IL-2 or IL-15). References to MSAb include bispecific T cell engagers (bsTCEs). The inclusion of two agents as either the first or second agents is intended to refer to simultaneous administration of the listed agents.

[0118] [Table 4]

[0119]

[0133] The agents described herein may be administered over the course of one or more treatment cycles. As used herein, a "treatment cycle" refers to a period of treatment (e.g., administration of a multispecific antibody, a common gamma chain cytokine, or an immune checkpoint inhibitor), optionally followed by a rest period (e.g., during which no therapeutic agent is administered). A full course of treatment may include one or more treatment cycles, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more treatment cycles. A treatment cycle may be 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days or longer.

[0120]

[0134] Thus, an exemplary course of treatment may include: (a) administration of the multispecific antibody as an intravenous infusion on day 1 of a 14-day treatment cycle; and (b) once-daily administration of a common gamma chain cytokine (e.g., IL-2 or IL-15) on days 2, 3, and 4 of a 14-day treatment cycle; (c) wherein the total course of treatment comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) treatment cycles.

[0121]

[0135] In this exemplary embodiment, the interval between administrations of the multispecific antibody over the entire course of treatment is every 14 days.

[0122]

[0136] Exemplary treatment cycles and total treatment course for LAVA-1207 are provided in Table 5.

[0123]

[0137] Exemplary treatment regimens including a multispecific antibody that binds to human PSMA (e.g., LAVA-1207) are provided below in Table 5. Although not shown in Table 5, an immune checkpoint inhibitor (e.g., an anti-PD1 antibody) can be administered at various times throughout the course of treatment. For example, the anti-PD1 antibody can be administered before, simultaneously with, or after a dose of LAVA-1207. In some embodiments, the anti-PD1 antibody can be administered before, simultaneously with, or after a dose of IL-2 and / or IL-15.

[0124]

[0138] In some embodiments, the LAVA-1207 treatment regimen includes administration of one or more premedications. The premedications are typically administered before the administration of the target dose of LAVA-1207. In some embodiments, the premedications are administered together with a priming dose of LAVA-1207. The premedications can include one or more of an antipyretic (e.g., paracetamol or acetaminophen), an antihistamine (e.g., diphenhydramine), and a corticosteroid (e.g., dexamethasone or methylprednisolone). In some embodiments, the premedications include each of paracetamol or acetaminophen, diphenhydramine, and dexamethasone or methylprednisolone. In some embodiments, the premedications are given in combination with hydration (e.g., 1000 mL or more of infusion fluids).

[0125]

[0139] In some embodiments, methylprednisolone is administered at a dose of 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg. In some embodiments, methylprednisolone is administered at a dose of 20 mg. In some embodiments, dexamethasone is administered at a dose of 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In some embodiments, dexamethasone is administered at a dose of 4 mg. In some embodiments, acetaminophen is administered in a dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, or 2000 mg. In some embodiments, diphenhydramine is administered at a dose of 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, or 150 mg.

[0126]

[0140] In some embodiments, the premedication is administered 4, 3, 2, or 1 hour before the first dose of LAVA-1207. One or more of the premedications may be administered based on a physician's opinion after administration of LAVA-1207 has begun. In some embodiments, the premedication is administered 4, 3, 2, or 1 hour before any of the LAVA-1207 doses (priming dose or target dose) shown in Table 5.

[0127] [Table 5]

[0128]

[0141] A subject's responsiveness to LAVA-1207 treatment can be monitored by sampling the subject before and during LAVA-1207 treatment. For example, a blood sample is collected from the subject before initiation of LAVA-1207 treatment, and additional blood samples are collected at various time points after treatment begins. These blood samples can be analyzed for pharmacodynamic characteristics, including LAVA-1207 binding to Vy9V52 T cells, Vy9V52 T cell activation status, general immune cell analysis (e.g., activation status and frequency of B cells, T cells, NK cells, and monocytes), cytokine levels, and anti-drug antibodies. In some embodiments, additional blood samples are collected for use in tumor mutation burden, tumor burden, and / or cell-free DNA analysis of circulating tumor cells.

[0129]

[0142] In some embodiments, the methods of the disclosure comprise administering LAVA-1207 at doses of 1.5 μg, 4.5 μg, 13.5 μg, 40 μg, 120 μg, 360 μg, 540 μg, 800 μg, 1.2 mg, 1.8 mg, 3.6 mg, 5.4 mg, 7.2 mg, 8.1 mg, 12 mg, 21 mg, 65 mg, or 200 mg once every two weeks for a total of 12 treatment cycles.

[0130]

[0143] In some embodiments, the methods of the disclosure comprise twelve two-week treatment cycles, wherein the first and second treatment cycles comprise administration of LAVA-1207 at a dose of 1.5 μg, 4.5 μg, 13.5 μg, 40 μg, 120 μg, 360 μg, 540 μg, 800 μg, 1.2 mg, 1.8 mg, 3.6 mg, 5.4 mg, 7.2 mg, 8.1 mg, 12 mg, 21 mg, 65 mg, or 200 mg on day 1 of the treatment cycle, followed by a once-daily dose of IL-2 at 1 MIU for one or three days, for a total of 12 cycles. Cycles 5-12 involve administration of LAVA-1207 at doses of 1.5 μg, 4.5 μg, 13.5 μg, 40 μg, 120 μg, 360 μg, 540 μg, 800 μg, 1.2 mg, 1.8 mg, 3.6 mg, 5.4 mg, 7.2 mg, 8.1 mg, 12 mg, 21 mg, 65 mg, or 200 mg on the first day of the treatment cycle.

[0131]

[0144] In some embodiments, the disclosed methods comprise twelve two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose of 120 μg on days 1 and 2 of the treatment cycle, followed by administration of a target dose of 800 μg on day 3 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 800 μg on day 1 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 800 μg on day 1 of the treatment cycle.

[0132]

[0145] In some embodiments, the disclosed methods include 12 two-week treatment cycles, wherein the first treatment cycle includes administration of LAVA-1207 at a priming dose of 120 μg on day 1 of the treatment cycle and a priming dose of 360 μg on day 2 of the treatment cycle, followed by administration of a target dose of 800 μg on day 3 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for one or three days. The second, third, and fourth treatment cycles include administration of LAVA-1207 at a target dose of 800 μg on day 1 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for three days. Cycles 5-12 include administration of LAVA-1207 at a target dose of 800 μg on day 1 of the treatment cycle.

[0133]

[0146] In some embodiments, the disclosed methods comprise 12 two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 1200 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 1200 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 1200 μg on day 1 of the treatment cycle.

[0134]

[0147] In some embodiments, the disclosed methods include 12 two-week treatment cycles, wherein the first treatment cycle includes administration of LAVA-1207 at a priming dose of 120 μg on day 1 of the treatment cycle and a priming dose of 360 μg on day 2 of the treatment cycle, followed by a target dose of 1200 μg on day 3 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for one or three days. The second, third, and fourth treatment cycles include administration of LAVA-1207 at a target dose of 1200 μg on day 1 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for three days. Cycles 5-12 include administration of LAVA-1207 at a target dose of 1200 μg on day 1 of the treatment cycle.

[0135]

[0148] In some embodiments, the disclosed methods include 12 two-week treatment cycles, wherein the first treatment cycle includes administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by administration of a target dose of 1800 μg on day 3 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for one or three days. The second, third, and fourth treatment cycles include administration of LAVA-1207 at a target dose of 1800 μg on day 1 of the treatment cycle, followed by once-daily doses of IL-2 at 1 MIU for three days. Cycles 5 through 12 include administration of LAVA-1207 at a target dose of 1800 μg on day 1 of the treatment cycle.

[0136]

[0149] In some embodiments, the disclosed methods comprise twelve two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 3600 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 3600 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 3600 μg on day 1 of the treatment cycle.

[0137]

[0150] In some embodiments, the disclosed methods comprise twelve two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 5400 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 5400 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 5400 μg on day 1 of the treatment cycle.

[0138]

[0151] In some embodiments, the disclosed methods include 12 two-week treatment cycles, wherein the first treatment cycle includes administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 7200 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles include administration of LAVA-1207 at a target dose of 7200 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 include administration of LAVA-1207 at a target dose of 7200 μg on day 1 of the treatment cycle.

[0139]

[0152] In some embodiments, the disclosed methods comprise twelve two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 8100 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 8100 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 8100 μg on day 1 of the treatment cycle.

[0140]

[0153] In some embodiments, the disclosed methods comprise twelve two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 12,000 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 12,000 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 12,000 μg on day 1 of the treatment cycle.

[0141]

[0154] In some embodiments, the disclosed methods comprise twelve two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 65,000 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 65,000 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 65,000 μg on day 1 of the treatment cycle.

[0142]

[0155] In some embodiments, the disclosed methods comprise twelve two-week treatment cycles, wherein the first treatment cycle comprises administration of LAVA-1207 at a priming dose on days 1 and 2 of the treatment cycle, followed by a target dose of 200,000 μg on day 3 of the treatment cycle, followed by IL-2 at 1 MIU once daily for one or three days. The second, third, and fourth treatment cycles comprise administration of LAVA-1207 at a target dose of 200,000 μg on day 1 of the treatment cycle, followed by IL-2 at 1 MIU once daily for three days. Cycles 5 through 12 comprise administration of LAVA-1207 at a target dose of 200,000 μg on day 1 of the treatment cycle.

[0143]

[0156] In some embodiments, the methods of the disclosure comprise at least four treatment cycles, wherein the first cycle is four weeks long and the second through fourth cycles are each two weeks long. In some such embodiments, the first cycle comprises administration of an initial priming dose of 120 μg LAVA-1207 on day 1 and a target dose of 800 μg LAVA-1207 on day 15, wherein the target dose is administered on day 1 of each of cycles 2 through 4. In some such embodiments, the first cycle comprises administration of an initial priming dose of 120 μg LAVA-1207 on day 1, a second priming dose of 360 μg LAVA-1207 on day 8, and a target dose of 800 μg LAVA-1207 on day 15, wherein the target dose is administered on day 1 of each of cycles 2 through 4. In some such embodiments, the first cycle comprises administration of an initial priming dose of 120 μg LAVA-1207 on day 1, a second priming dose of 360 μg LAVA-1207 on day 8, and a target dose of 1200 μg LAVA-1207 on day 15, where the target dose is administered on day 1 of each of cycles 2-4.

[0144]

[0157] In some such embodiments, the first cycle comprises administration of an initial priming dose of 120 μg LAVA-1207 on day 1, a second priming dose of 360 μg LAVA-1207 on day 8, and a target dose of 800 μg LAVA-1207 on day 15, followed by 1 MIU of IL-2 on day 16, where the target dose is administered on day 1 of each of cycles 2-4, followed by 1 MIU of IL-2 on day 2 of each of cycles 2-4. In some such embodiments, the first cycle comprises administration of an initial priming dose of 120 μg LAVA-1207 on day 1, a second priming dose of 360 μg LAVA-1207 on day 8, and a target dose of 800 μg LAVA-1207 on day 15, followed by 1 MIU of IL-2 on days 16, 17, and 18, where the target dose is administered on day 1 of each of cycles 2-4, followed by 1 MIU of IL-2 on days 2, 3, and 4 of each of cycles 2-4.

[0145]

[0158] In some such embodiments, the first cycle comprises administration of an initial priming dose of 120 μg LAVA-1207 on day 1, a second priming dose of 360 μg LAVA-1207 on day 8, and a target dose of 1200 μg LAVA-1207 on day 15, followed by 1 MIU of IL-2 on day 16, where the target dose is administered on day 1 of each of cycles 2-4, followed by 1 MIU of IL-2 on day 2 of each of cycles 2-4. In some such embodiments, the first cycle comprises administration of an initial priming dose of 120 μg LAVA-1207 on day 1, a second priming dose of 360 μg LAVA-1207 on day 8, and a target dose of 1200 μg LAVA-1207 on day 15, followed by 1 MIU of IL-2 on days 16, 17, and 18, where the target dose is administered on day 1 of each of cycles 2-4, followed by 1 MIU of IL-2 on days 2, 3, and 4 of each of cycles 2-4.

[0146] Indications and methods of treatment

[0159] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering a polyspecific binding agent as described herein or a composition comprising same and (i) a common gamma chain cytokine (e.g., IL-2 or IL-15) and / or (ii) an immune checkpoint inhibitor (e.g., an anti-PD1 or anti-PDL1 antibody).

[0147]

[0160] In some embodiments, treating refers to the treatment of cancer in a mammal, e.g., a human, including (a) inhibiting cancer, i.e., halting the onset of cancer or preventing the progression of cancer; (b) alleviating cancer, i.e., causing regression of the cancerous condition or alleviating one or more symptoms of cancer; and (c) curing cancer, i.e., ameliorating one or more symptoms of cancer. In some embodiments, treating can refer to short-term (e.g., temporary and / or acute) and / or long-term (e.g., sustained) reduction of one or more symptoms of cancer. In some embodiments, treatment results in improvement or remediation of cancer symptoms. Improvement can be an observable or measurable improvement, or an improvement in the subject's overall sense of well-being.

[0148]

[0161] In some embodiments, the subject may be a neonate, a juvenile, or an adult. Of particular interest are mammalian subjects. Mammalian species that may be treated with the present method include dogs and cats; horses; cows; sheep, etc., and primates, particularly humans. Animal models, particularly small mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, etc.), may be used in experimental studies. In some embodiments, the subject is a human.

[0149]

[0162] In some embodiments, the cancer is one in which the target antigen is expressed on tumor neovasculature cells or tumor-associated endothelial cells of primary or metastatic tumors, including colorectal cancer, lung cancer, non-small cell lung cancer, endometrial and ovarian cancer, uterine cancer, uterine endometrial cancer, gastric cancer, urothelial carcinoma, hepatocellular carcinoma, oral squamous cell carcinoma, thyroid tumor and glioblastoma, or head and neck adenoid cystic carcinoma, head and neck squamous cell carcinoma, prostate cancer, non-metastatic prostate cancer, metastatic prostate cancer, treatment-refractory metastatic castration-resistant prostate cancer, glioblastoma multiforme, or blastic plasmacytic dendritic cell neoplasm.

[0150]

[0163] In some embodiments, the cancer is a hematological malignancy such as T-cell lymphoma, multiple myeloma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, myelodysplastic syndrome pre-B cell acute lymphoblastic leukemia, B cell lymphoma, smoldering myeloma, myelomonocytic leukemia, lymphoplasmacytic lymphoma, hairy cell leukemia, splenic marginal zone lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, pancreatic cancer, colon cancer, B cell acute lymphoblastic leukemia, B cell lymphoma / leukemia, B cell chronic lymphoproliferative disorder, Burkitt's lymphoma, or B cell acute lymphoblastic leukemia.

[0151]

[0164] In some embodiments, the cancer is a solid tumor cancer such as renal cell carcinoma, melanoma, breast cancer (including triple-negative breast cancer), gastroesophageal cancer, small intestine cancer, central nervous system tumors, medulloblastoma, hepatocellular carcinoma, glioma, neuroblastoma, bladder cancer, sarcoma, penile cancer, basal cell carcinoma, Merkel cell carcinoma, neuroendocrine carcinoma, neuroendocrine tumor, carcinoma of unknown primary (CUP), thymoma, vulvar cancer, cervical cancer, testicular cancer, bile duct cancer, appendix cancer, mesothelioma, ampullary carcinoma, anal cancer, or choriocarcinoma.

[0152]

[0165] Administration of the agents described herein (e.g., multispecific antibodies, common gamma chain cytokines, and / or immune checkpoint inhibitors, either alone or in combination) may be by injection, irrigation, inhalation, ingestion, electroosmosis, hemodialysis, iontophoresis, and other methods known in the art. In some embodiments, the route of administration is local or systemic. In some embodiments, the route of administration is intra-arterial, intracranial, intradermal, intraduodenal, intramammary, intrathecal, intraperitoneal, intrathecal, intramuscular, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, ​​vaginal, or intrauterine.

[0153]

[0166] In some embodiments, the administration route is via injection (e.g., continuous or bolus). Examples of local administration methods, i.e., delivery to the site of injury or disease, include delivery from an Ommaya reservoir, for example, for intrathecal delivery (see, e.g., U.S. Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); delivery by bolus injection, for example, via a syringe, for example, intra-articular delivery; delivery by continuous infusion, for example, via cannulation, such as with convection (see, e.g., U.S. Patent Application Publication No. 2007-0254842, incorporated herein by reference); or delivery via implantation of a device to which the drug is reversibly loaded (see, e.g., U.S. Patent Application Publication Nos. 2008-0081064 and 2009-0196903, incorporated herein by reference). In some embodiments, the administration route is via local administration or direct injection.

[0154]

[0167] In some embodiments, subjects are selected for treatment according to the methods described herein by one or more exclusion and / or inclusion criteria, which may include age, sex, infectious status (e.g., hepatitis B or C), previous treatment regimens (e.g., immunosuppressants or aminobisphosphonates), presence of a malignancy other than the cancer intended to be treated, and general health.

[0155]

[0168] In some embodiments, subjects are selected for treatment with LAVA-1207 by one or more of the following inclusion criteria: (a) Men with metastatic castration-resistant prostate cancer (mCRPC) as defined by PCWG3 criteria (histologically confirmed adenocarcinoma; adenocarcinoma with 10% or less small cell or neuroendocrine features is acceptable, but brain metastases are acceptable as long as the subject's symptoms are adequately controlled); (b) failure of at least one line of taxane-based chemotherapy or deemed medically unsuitable for treatment with a taxane regimen; (c) taking second-generation or later androgen receptor-targeted therapy / androgen biosynthesis inhibitors (e.g., abiraterone, enzalutamide, and / or apalutamide) or being deemed inappropriate for such treatment; (d) evidence of progressive disease as defined by an increase in one or more PSA levels to ≥1 ng / mL on at least two consecutive occasions separated by at least 1 week; (e) Computed tomography (CT) or magnetic resonance imaging (MRI) scan showing nodal or visceral progression as defined by RECIST 1.1; and / or (f) Bone scintigraphy showing two or more new metastatic lesions.

[0156]

[0169] In some embodiments, a subject is excluded from treatment if any one or more of the following criteria apply: (a) Diagnosis of other malignancies within the past 2 years, excluding adequately treated carcinoma in situ, basal cell carcinoma of the skin, or squamous cell carcinoma; (b) Concurrent uncontrolled or severe medical conditions (c) positive serological test for human immunodeficiency virus (HIV) antibodies; (d) Positive serology for hepatitis B surface antigen (HBsAg). Subjects with anti-HBc or hepatitis C antibody positive may be included if they have a negative polymerase chain reaction (PCR) within 6 weeks prior to their first multispecific antibody dose. PCR-positive subjects will be excluded. (e) Active, uncontrolled, or suspected infection; (f) known clinically relevant immunodeficiency disorder; (g) Significant history of renal, neurological, psychiatric, pulmonary, endocrine, metabolic, immunological, cardiovascular, or hepatic disease that, in the opinion of the investigator, may adversely affect participation in this study. Unstable cardiovascular function is defined as (a) symptomatic ischemia, or (b) uncontrolled clinically significant conduction abnormalities (i.e., ventricular tachycardia while taking antiarrhythmic medications is excluded; first-degree atrioventricular block or asymptomatic left anterior fascicular block / right bundle branch block is not excluded), or (c) congestive heart failure New York Heart Association class 3 or greater, or (d) myocardial infarction within 3 months; (h) History of antitumor therapy within 2 weeks prior to the first multispecific antibody for radiation therapy and androgen receptor-targeted therapy / androgen biosynthesis inhibitors, and within 4 weeks prior to systemic chemotherapy or targeted / immunotherapy; (i) History of treatment with live or live-attenuated vaccines within 2 weeks prior to the first administration of the multispecific antibody (new vaccines need to be evaluated for their mode of action); (j) treatment with other investigational drugs in the 4 weeks prior to the first multispecific antibody; (k) major surgery within 4 weeks before the first multispecific antibody administration; (l) hypersensitivity to LAVA-1207, IL-2, IL-15, or any of the excipients present in pembrolizumab (if applicable); (m) History of treatment with any systemic immunosuppressant within 4 weeks prior to the first multispecific antibody administration, except for systemic corticosteroid use of up to 10 mg oral doses of prednisone daily (or equivalent doses for other steroids); (n) History of treatment with an IV aminobisphosphonate (e.g., ibandronate, pamidronate, zoledronate, etc.) within 4 weeks or 1 year prior to the first IMP; and / or (o) Known drug and alcohol abuse in the opinion of the investigator.

[0157] multispecific antibodies

[0170] Multispecific antibodies suitable for use in the methods described herein are described below. In some embodiments, the multispecific antibody comprises a first antigen-binding domain capable of binding to a human cancer antigen and a second antigen-binding domain capable of binding to the human Vy9V52 T cell receptor by binding to either the Vy9 chain or the V52 chain (e.g., a bispecific antibody). In some embodiments, the multispecific antibody comprises a first antigen-binding domain capable of binding to a human cancer antigen, a second antigen-binding domain capable of binding to the human Vy9V52 T cell receptor, and a third antigen-binding domain capable of binding to a third target antigen or epitope. In some embodiments, the multispecific antibody further comprises a fourth, fifth, or sixth antigen-binding domain.

[0158]

[0171] In some embodiments, any of the first, second, third, fourth, fifth, or sixth antigen-binding domains of the multispecific antibody may be a single domain antibody, such as a VHH. In some embodiments, the first and second antigen-binding domains of the multispecific antibody are VHHs.

[0159]

[0172] Antibodies may be formulated with pharmaceutically acceptable excipients according to conventional techniques, such as those disclosed in Rowe et al. 2012 Handbook of Pharmaceutical Excipients, ISBN 9780857110275. Pharmaceutically acceptable excipients, as well as any other carriers, diluents, or adjuvants, must be suitable for the antibody and the selected mode of administration. Suitability of excipients and other components of the pharmaceutical composition is determined based on their lack of significant negative impact on the desired biological properties of the selected antibody or pharmaceutical composition of the present disclosure (e.g., no substantial effect on antigen binding (10% or less relative inhibition, 5% or less relative inhibition, etc.)). Pharmaceutical compositions may include diluents, bulking agents, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in pharmaceutical compositions. Additional pharmaceutically acceptable excipients include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents that are physiologically compatible with the antibodies of the present disclosure.

[0160]

[0173] Suitable formulations of multispecific antibodies for use in the present disclosure are described in WO 2022 / 008646. In some embodiments, the multispecific antibodies may be formulated at a strength of about 1 mg / mL. In some embodiments, the formulation includes 1-20 mM histidine. In some embodiments, the formulation includes 0.1-10 mM methionine. In some embodiments, the formulation includes 50-500 mM sucrose. In some embodiments, the formulation includes 0.01-0.05% polysorbate 80. In some embodiments, the formulation has a pH of 5.0-7.5. In some embodiments, the multispecific antibodies are formulated at a strength of 1 mg / mL. In some embodiments, the formulation includes 10 mM histidine. In some embodiments, the formulation includes 1 mM methionine. In some embodiments, the formulation includes 280 mM sucrose. In some embodiments, the formulation includes 0.02% polysorbate 80. In some embodiments, the formulation has a pH of 6.0. For example, the multispecific antibody may be formulated at a strength of 1 mg / mL in 10 mM histidine, 1 mM methionine, 280 mM sucrose, 0.02% polysorbate 80, pH 6.0. The multispecific antibody may be administered by any suitable route of administration, for example, intravenously or subcutaneously.

[0161]

[0174] Multispecific antibodies for use in the present disclosure are typically produced recombinantly, i.e., by expression of a nucleic acid construct encoding the antibody in a suitable host cell, followed by purification of the produced recombinant antibody from the cell culture. The nucleic acid construct can be produced by standard molecular biology techniques well known in the art. The construct is typically introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art. Suitable host cells for recombinant expression of antibodies are well known in the art and include CHO, HEK-293, Expi293F, PER-C6, NS / 0, and Sp2 / 0 cells.

[0162]

[0175] In some embodiments, any of the multispecific antibodies of this disclosure is modified to reduce or eliminate the formation of pyroglutamic acid at the N-terminus of the modified multispecific antibody when compared to an unmodified multispecific antibody of this disclosure. In some embodiments, a glutamic acid (E) or glutamine (Q) residue is modified to become an aspartic acid (D) residue. In some embodiments, any of the multispecific antibodies of this disclosure may include an aspartic acid (D) residue at the first position.

[0163]

[0176] In some embodiments, the first and second antigen-binding regions of a multispecific antibody used in the present disclosure may be covalently linked via a peptide linker, e.g., a linker having a length of 1 to 20 amino acids, e.g., 1 to 10 amino acids, such as 2, 3, 4, 5, 6, 7, 8, or 10 amino acids. The peptide linker may comprise or consist of four glycine residues followed by a serine residue. The first antigen-binding region may be located N-terminally or C-terminally of the second antigen-binding region. In some embodiments, the first and second antigen-binding regions may be linked or directly fused to an Fc domain monomer.

[0164]

[0177] The multispecific or bispecific antibodies used in the present disclosure are capable of binding to the human Vy9V52 T cell receptor. In some embodiments, the multispecific antibodies are capable of binding to human V52. The term "human V52" as used herein refers to the rearranged 52 chain of the Vy9V52 T cell receptor (TCR). UniProtKB-A0JD36 (A0JD36_human) is an example of a variable TRDV2 sequence. Antigen-binding domains that specifically bind to V52+ T cell receptors are known in the art. See, for example, WO 2015 / 156673, WO 2022 / 008646, WO 2022 / 122973, and WO 2023 / 242319 (each of which discloses an antigen-binding domain that specifically binds to V52, each of which is incorporated herein by reference). Exemplary V52 antigen-binding domain sequences are shown in Table 6A, Table 6B, Table 6C, and Table 6D.

[0165] [Table 6]

[0166] [Table 7]

[0167] [Table 8]

[0168] [Table 9]

[0169]

[0178] Those skilled in the art will recognize that there are many numbering systems known in the art for defining antibody CDR sequences, including the AbM, Kabat, Chothia, and IMGT systems. A summary of these systems is provided in Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition. Front Immunol. 2018 Oct 16;9:2278. The CDR sequences of the V52 antigen-binding domain as defined by Kabat, IMGT, and Chothia are provided in Tables 6A, 6B, and 6C above. Descriptions of binders herein refer to CDRs as defined by the Kabat numbering system. However, those skilled in the art will be able to correlate CDR sequences defined by one numbering system to the same CDR sequences as defined by another system.

[0170]

[0179] In some embodiments, a combination of CDR numbering systems may be used. In such embodiments, the CDR sequences of a given binder as determined by multiple numbering systems (e.g., the CDR1 sequences as determined by the Kabat, IMGT, and Chothia numbering systems) are compiled into a single sequence that encompasses each of the entire CDR amino acid ranges in the variable region. As an illustrative example, the CDRs of the 6H4 VHH listed in Tables 6A, 6B, and 6C are shown in Figure 20. The N- and C-terminal ranges of each CDR region are indicated by dashed lines. The combined CDR sequences covering these ranges are shown in the last row. Exemplary combined CDRs for the V52 antigen-binding domain are shown in Table 6D.

[0171]

[0180] As an illustrative example, a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO:26, a CDR2 amino acid sequence comprising SEQ ID NO:27, and a CDR3 amino acid sequence comprising SEQ ID NO:28, as defined by the Kabat numbering system, will be understood by those skilled in the art to be the functional equivalent of: (1) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO:45, a CDR2 amino acid sequence comprising SEQ ID NO:46, and a CDR3 amino acid sequence comprising SEQ ID NO:47, as defined by the IMGT numbering system; (2) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO:63, a CDR2 amino acid sequence comprising SEQ ID NO:64, and a CDR3 amino acid sequence comprising SEQ ID NO:65, as defined by the Chothia numbering system; and (3) a 6H4 VHH comprising a CDR1 amino acid sequence comprising SEQ ID NO:84, a CDR2 amino acid sequence comprising SEQ ID NO:85, and a CDR3 amino acid sequence comprising SEQ ID NO:86.

[0172]

[0181] In some embodiments, the multispecific antibody is capable of binding to human Vγ9. The term "human Vγ9" as used herein refers to the rearranged Vγ9 chain of the Vγ9Vδ2-T cell receptor (TCR). UniProtKB-Q99603 (TRGV9_human) is an example of a variable Vγ9 sequence. Exemplary Vγ9Vδ2 TCR antigen-binding domain sequences are known in the art, see, for example, WO 2015 / 156673.

[0173]

[0182] In some embodiments, the (second) antigen-binding domain capable of binding to the human Vy9V52 T-cell receptor may comprise the VH CDR1 sequence of SEQ ID NO: 5, the VH CDR2 sequence of SEQ ID NO: 6, and the VH CDR3 sequence of SEQ ID NO: 7. In some embodiments, the second antigen-binding region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In some embodiments, the second antigen-binding region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the second antigen-binding domain comprises or consists of SEQ ID NO: 8. In some embodiments, the second antigen-binding domain comprises or consists of SEQ ID NO: 9.

[0174]

[0183] In some embodiments, the (second) antigen-binding domain capable of binding to the human Vy9V52 T-cell receptor may comprise the VH CDR1 sequence of SEQ ID NO: 10, the VH CDR2 sequence of SEQ ID NO: 11 and the VH CDR3 sequence of SEQ ID NO: 12. In some embodiments, the second antigen-binding region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some embodiments, the second antigen-binding domain comprises or consists of SEQ ID NO: 13. In some embodiments, the (second) antigen-binding domain capable of binding to the human Vy9V52 T-cell receptor may comprise the VH CDR1 sequence of SEQ ID NO: 14, the VH CDR2 sequence of SEQ ID NO: 15 and the VH CDR3 sequence of SEQ ID NO: 16. In some embodiments, the second antigen-binding region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17. In some embodiments, the second antigen-binding domain comprises or consists of SEQ ID NO: 17.

[0175]

[0184] In some embodiments, the (second) antigen-binding domain capable of binding to the human Vy9V52 T-cell receptor may comprise the VH CDR1 sequence of SEQ ID NO: 18, the VH CDR2 sequence of SEQ ID NO: 19 and the VH CDR3 sequence of SEQ ID NO: 20. In some embodiments, the second antigen-binding region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 21. In some embodiments, the second antigen-binding domain comprises or consists of SEQ ID NO: 21.

[0176]

[0185] In some embodiments, the (second) antigen-binding domain capable of binding to the human Vy9V52 T-cell receptor may comprise the VH CDR1 sequence of SEQ ID NO: 22, the VH CDR2 sequence of SEQ ID NO: 23 and the VH CDR3 sequence of SEQ ID NO: 24. In some embodiments, the second antigen-binding region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25. In some embodiments, the second antigen-binding domain comprises or consists of SEQ ID NO: 25.

[0177]

[0186] In some embodiments, the multispecific antibody used in the methods of the disclosure competes (i.e. is capable of competing) for binding to V52 with an antibody having a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 25, and 29. The multispecific antibody used in the methods of the disclosure may bind to the same epitope on human V52 as an antibody having a sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 25, and 29.

[0178]

[0187] The multispecific or bispecific antibodies used in the present disclosure are capable of binding to a human cancer antigen. The human cancer antigen may be, for example, an antigen associated with a solid tumor or an antigen associated with a hematological cancer disease. The human cancer antigen may be exclusively expressed on malignant cells or may be overexpressed on malignant cells compared to healthy cells.

[0179]

[0188] In some embodiments, the human cancer antigen is HER2 / neu, EGFR, CA-125, PSA, CD44, EpCAM, CEACAM5, GD2, CD1d, CD40, CD123, 5T4, nectin-4, PSMA, B7-H3, RAGE, CA72-4, HE4, glypican-3, PSMA, MUC1, CD133, EGFRvIII, CAIX, CD24, CD166, CD47, CD70, CD133, C In some embodiments, the human cancer antigen is selected from the group consisting of D276 (B7-H3), CD271, CD146, CD164, CD123, CD38, CD166, CD24, CD29, CD49f, CD56, CD20, CD71, CD98, CD99, CD147, CD166, CD200, CD184 (CXCR4), CD44v6, CD271, CD276, CD304 (BDCA-4), CD326 (EpCAM), and CD338 (ABCB5). In some embodiments, the human cancer antigen is selected from the group consisting of PSMA, CD1d, CD40, CD123, 5T4, EGFR, and Nectin-4. In some embodiments, the human cancer antigen is selected from PSMA, CD1d, CD40, CD123, 5T4, EGFR, CD33, and Nectin-4. Exemplary cancer antigen-specific CDR and VHH sequences are provided below in Tables 7A, 7B, 7C, and 7D.

[0180] [Table 10]

[0181] [Table 11]

[0182] [Table 12]

[0183] [Table 13]

[0184] [Table 14]

[0185] [Table 15]

[0186] [Table 16]

[0187] [Table 17]

[0188] [Table 18]

[0189] [Table 19]

[0190] [Table 20]

[0191] [Table 21]

[0192] PSMA multispecific antibody In some embodiments, the human cancer antigen may be PSMA. Multispecific antibodies having an antigen-binding region capable of binding to human PSMA suitable for use in the present disclosure are described, for example, in WO 2022 / 008646 (incorporated herein by reference).

[0193] In some embodiments, the (first) antigen-binding region of a multispecific antibody used in the present disclosure may comprise the VH CDR1 sequence of SEQ ID NO: 87, the VH CDR2 sequence of SEQ ID NO: 88, and the VH CDR3 sequence of SEQ ID NO: 89. In some embodiments, the first antigen-binding region may comprise an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 90. In some embodiments, the first antigen-binding region comprises or consists of SEQ ID NO: 90.

[0194] The multispecific antibody used in the methods of the present disclosure may compete (i.e., may be capable of competing) for binding to PSMA with the antibody having the sequence set forth in SEQ ID NO: 90. The multispecific antibody may bind to the same epitope on human PSMA as the antibody having the sequence set forth in SEQ ID NO: 90.

[0195] In some embodiments, a multispecific antibody used in the present disclosure may comprise a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 87, the CDR2 sequence set forth in SEQ ID NO: 88, and the CDR3 sequence set forth in SEQ ID NO: 89, and the second antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 5, the CDR2 sequence set forth in SEQ ID NO: 6, and the CDR3 sequence set forth in SEQ ID NO: 7. In some embodiments, a multispecific antibody used in the present disclosure may comprise a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the sequence set forth in SEQ ID NO: 90, and the second antigen-binding region comprises the sequence set forth in SEQ ID NO: 8. In some embodiments, the second antigen-binding domain comprises any of the CDR combinations listed in Tables 6A to 6D.

[0196] The multispecific antibody may comprise or consist of the sequences set forth in SEQ ID NO: 334 and SEQ ID NO: 335. Such a multispecific antibody is referred to herein as LAVA-1207 or PSMA-Vδ2-Fc. The sequence of LAVA-1207 is provided in Table 8 below. The antigen-binding domain is shown in underlined text; the hinge region is in bold and italic text; the DEL sequence is shown in boxed text; the Fc domain is in regular text, and mutations relative to WT IgG1 are in bold and underlined text.

[0197] [Table 22]

[0198] Multispecific antibodies of the present disclosure may be capable of mediating the killing of PSMA-expressing cells, such as LNCaP cells, 22Rv1 cells, or VCaP cells, through activation of Vy9V52 T cells. For example, the multispecific antibodies may be capable of inducing the killing of LNCaP cells through activation of Vy9V52 T cells with an EC50 value of 50 pM or less, such as 25 pM or less, such as 20 pM or less, such as 15 pM or less, such as 10 pM or less, or even 5 pM or less, 2 pM or less, or 1 pM or less, when tested as described in Example 6 of WO 2022 / 008646.

[0199]

[0195] The multispecific antibody may have the ability to induce killing of LNCaP, 22Rv1 or VCaP cells through activation of Vγ9Vδ2 T cells with an EC50 value of 50 pM or less, such as 25 pM or less, for example 20 pM or less, 15 pM or less, when tested after 24 hours as described in Example 13 of WO 2022 / 008646, preferably at both effector to target cell ratios of 1:1 and 1:10.

[0200] Multispecific antibodies of the present disclosure may be capable of binding to the PSMA-positive prostate cancer cell line LNCaP with an EC50 of 50 nM or less, such as 20 nM or less, for example 10 nM or less, when tested as described in Example 7 of WO 2022 / 008646. Multispecific antibodies of the present disclosure may also, or alternatively, be capable of binding to Vy9V52 T cells with an EC50 of 10 nM or less, such as 5 nM or less, for example 2 nM or less, when tested as described in Example 7 of WO 2022 / 008646.

[0201] The multispecific antibody may be capable of binding to recombinant human PSMA protein with a KD value of 100 nM or less, such as 50 nM or less, when tested as described in Example 11 of WO 2022 / 008646. The multispecific antibody of the present disclosure may also, or alternatively, be capable of binding to human Vy9V52-Fc with a KD value of 10 nM or less, such as 5 nM or less, for example 2 nM or less, such as 1 nM or less, when tested as described in Example 11 of WO 2022 / 008646.

[0202] Multispecific antibodies may be capable of mediating the killing of human PSMA-expressing cells from prostate cancer patients. Killing of human PSMA-expressing cells from prostate cancer patients may be determined, for example, as described in Example 10 of WO 2022 / 008646. Multispecific antibodies of the present disclosure may also, or alternatively, be capable of mediating greater than 25%, greater than 50%, etc. specific cell death at a concentration of 50 nM as determined in the assay described in Example 10 or Example 14 of WO 2022 / 008646.

[0203] The multispecific antibody may be capable of mediating the killing of PSMA-negative cells, such as PSMA-negative human cells. The multispecific antibody may not induce IL-2, IL-4, IL-6, IL-10, or TNFα in whole blood from healthy donors at concentrations up to 280 nM when tested as described in Example 16 of WO 2022 / 008646. Alternatively, or in addition, the multispecific antibody may induce at least 10-fold less IL-8 and / or at least 50-fold less IFNγ than Campath® in whole blood from healthy donors when tested as described in Example 16 of WO 2022 / 008646.

[0204] The multispecific antibody used in the present disclosure comprises a first antigen-binding region capable of binding to PSMA and may be for use in the treatment of prostate cancer, non-metastatic or metastatic prostate cancer, such as metastatic castration-resistant prostate cancer or treatment-refractory metastatic castration-resistant prostate cancer. For example, the multispecific antibody comprises a first antigen-binding region capable of binding to PSMA and may be used to treat prostate cancer in patients who have previously received at least one or more treatments for prostate cancer. In some embodiments, the one or more treatments are selected from taxane chemotherapy, androgen receptor-targeted therapy treatment with an androgen biosynthesis inhibitor (e.g., abiraterone, enzalutamide, and / or apalutamide), treatment with a PARP inhibitor, and treatment with lutetium / radiolabelled PSMA.

[0205] Furthermore, multispecific antibodies capable of binding to PSMA may be for use in the treatment of cancers in which PSMA is expressed in tumor neovasculature or tumor-associated endothelial cells of primary or metastatic tumors, including those from colorectal cancer, lung cancer, breast cancer, endometrial and ovarian cancer, gastric cancer, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, oral squamous cell carcinoma, thyroid tumors and glioblastoma. Moreover, multispecific antibodies capable of binding to PSMA may be for use in the treatment of head and neck adenoid cystic carcinoma.

[0206] CD1d multispecific antibody In some embodiments, the human cancer antigen is CD1d. Antibodies having an antigen-binding region capable of binding to human CD1d suitable for incorporation into multispecific antibodies used in the present disclosure are described, for example, in WO 2016 / 122320 and WO 2020 / 060405 (both of which are incorporated herein by reference).

[0207] The (first) antigen-binding region of a multispecific antibody used in the present disclosure may comprise the VH CDR1 sequence of SEQ ID NO: 95, the VH CDR2 sequence of SEQ ID NO: 96, and the VH CDR3 sequence of SEQ ID NO: 97. In some embodiments, the first antigen-binding region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 98. In some embodiments, the first antigen-binding region comprises an amino acid sequence comprising or consisting of SEQ ID NO: 98.

[0208] A multispecific antibody used in the methods of the present disclosure may compete (i.e., may be capable of competing) for binding to CD1d with an antibody having the sequence set forth in SEQ ID NO: 98. The multispecific antibody may bind to the same epitope on human CD1d as the antibody having the sequence set forth in SEQ ID NO: 98.

[0209] A multispecific antibody used in the present disclosure may comprise a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 95, the CDR2 sequence set forth in SEQ ID NO: 96, and the CDR3 sequence set forth in SEQ ID NO: 97, and the second antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 5, the CDR2 sequence set forth in SEQ ID NO: 6, and the CDR3 sequence set forth in SEQ ID NO: 7. In some embodiments, the second antigen-binding domain comprises any of the CDR combinations set forth in Tables 6A to 6D.

[0210] The multispecific antibodies used in the present disclosure comprise a first antigen-binding region capable of binding to CD1d, and are intended to treat hematological malignancies such as T-cell lymphoma, multiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, B-cell lymphoma, smoldering myeloma, Hodgkin's lymphoma, myelomonocytic leukemia, lymphoplasmacytic lymphoma, hairy cell leukemia, or splenic marginal zone lymphoma, or renal cell carcinoma, melanoma, It may be for use in the treatment of a solid tumor, such as colorectal cancer, head and neck cancer, breast cancer, prostate cancer, lung cancer, pancreatic cancer, gastroesophageal cancer, small intestine cancer, central nervous system tumors, medulloblastoma, hepatocellular carcinoma, ovarian cancer, glioma, neuroblastoma, urothelial carcinoma, bladder cancer, sarcoma, penile cancer, basal cell carcinoma, Merkel cell carcinoma, neuroendocrine carcinoma, neuroendocrine tumor, carcinoma of unknown primary (CUP), thymoma, vulvar cancer, cervical cancer, testicular cancer, bile duct cancer, appendix cancer, mesothelioma, ampullary cancer, anal cancer or choriocarcinoma.

[0211] CD40 multispecific antibody In another embodiment, the human cancer antigen is CD40. Antibodies having an antigen-binding region capable of binding to human CD40 suitable for incorporation into multispecific antibodies used in the present disclosure are described, for example, in WO 2020 / 159368 (incorporated herein by reference).

[0212] The (first) antigen-binding region of a multispecific antibody used in the present disclosure may comprise the VH CDR1 sequence of SEQ ID NO: 99, the VH CDR2 sequence of SEQ ID NO: 100, and the VH CDR3 sequence of SEQ ID NO: 101. In some embodiments, the first antigen-binding region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 102. In some embodiments, the first antigen-binding region comprises an amino acid sequence comprising or consisting of SEQ ID NO: 102.

[0213] In some embodiments, a multispecific antibody used in the methods of the present disclosure may compete (i.e., may be capable of competing) for binding to CD40 with an antibody having the sequence set forth in SEQ ID NO: 102. The multispecific antibody may bind to the same epitope on human CD40 as the antibody having the sequence set forth in SEQ ID NO: 102.

[0214] A multispecific antibody used in the present disclosure may comprise a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 99, the CDR2 sequence set forth in SEQ ID NO: 100, and the CDR3 sequence set forth in SEQ ID NO: 101, and the second antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 5, the CDR2 sequence set forth in SEQ ID NO: 6, and the CDR3 sequence set forth in SEQ ID NO: 7. In some embodiments, the second antigen-binding domain comprises any of the CDR combinations set forth in Tables 6A to 6D.

[0215] The multispecific antibodies used in the present disclosure comprise a first antigen-binding region capable of binding to CD40 and may be for use in the treatment of chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, head and neck cancer, pancreatic cancer, ovarian cancer, lung cancer, breast cancer, colon cancer, prostate cancer, B-cell lymphoma / leukemia, Burkitt's lymphoma or B-cell acute lymphoblastic leukemia.

[0216] CD123 multispecific antibody In another embodiment, the human cancer antigen is CD 123. Antibodies having an antigen-binding region capable of binding to human CD 123 suitable for incorporation into multispecific antibodies used in the present disclosure are described, for example, in WO 2022 / 180271 (incorporated herein by reference).

[0217] The (first) antigen-binding region of a multispecific antibody used in the present disclosure may comprise the VH CDR1 sequence of SEQ ID NO: 103, the VH CDR2 sequence of SEQ ID NO: 104, and the VH CDR3 sequence of SEQ ID NO: 105. In some embodiments, the first antigen-binding region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 106. In some embodiments, the first antigen-binding region comprises an amino acid sequence comprising or consisting of SEQ ID NO: 106.

[0218] In some embodiments, a multispecific antibody used in the methods of the present disclosure may compete (i.e., may be capable of competing) for binding to CD123 with an antibody having the sequence set forth in SEQ ID NO: 106. The multispecific antibody may bind to the same epitope on human CD123 as the antibody having the sequence set forth in SEQ ID NO: 106.

[0219] A multispecific antibody used in the present disclosure may comprise a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 103, the CDR2 sequence set forth in SEQ ID NO: 104, and the CDR3 sequence set forth in SEQ ID NO: 105, and the second antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 5, the CDR2 sequence set forth in SEQ ID NO: 6, and the CDR3 sequence set forth in SEQ ID NO: 7. In some embodiments, the second antigen-binding domain comprises any of the CDR combinations set forth in Tables 6A to 6D.

[0220] The multispecific antibodies used in the present disclosure comprise a first antigen-binding region capable of binding to CD123 and may be for use in the treatment of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic cell neoplasm, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disorder or myelodysplastic syndrome.

[0221] Nectin 4 multispecific antibody In another embodiment, the human cancer antigen is Nectin-4. The (first) antigen-binding region of a multispecific antibody used in the present disclosure may comprise a VH CDR1 sequence selected from SEQ ID NOs: 107, 11, 115, 119, 123, 127, and 131, a VH CDR2 sequence selected from SEQ ID NOs: 108, 112, 116, 120, 124, 128, and 132, and a VH CDR3 sequence selected from SEQ ID NOs: 109, 113, 117, 121, 125, 129, 133. In some embodiments, the first antigen-binding region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 110, 114, 118, 122, 126, 130, and 134. In some embodiments, the first antigen-binding region comprises an amino acid sequence comprising or consisting of a sequence selected from SEQ ID NOs: 110, 114, 118, 122, 126, 130, and 134.

[0222] In some embodiments, the multispecific antibody used in the methods of the present disclosure may compete (i.e., may be capable of competing) for binding to Nectin-4 with an antibody having a sequence selected from SEQ ID NOs: 110, 114, 118, 122, 126, 130, and 134. The multispecific antibody may bind to the same epitope on human Nectin-4 as an antibody having a sequence selected from SEQ ID NOs: 110, 114, 118, 122, 126, 130, and 134.

[0223]

[0219] The multispecific antibodies used in the present disclosure comprise a first antigen-binding region capable of binding to Nectin-4 and may be for use in the treatment of bladder cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, esophageal cancer, head and neck cancer, lung cancer, pancreatic cancer, gastric cancer, thyroid cancer, colorectal cancer, bile duct cancer, or uterine endometrial cancer.

[0224] 5T4 multispecific antibody In another embodiment, the human cancer antigen is 5T4. The (first) antigen-binding region of a multispecific antibody used in the present disclosure may comprise a VH CDR1 sequence selected from SEQ ID NOs: 135, 139, 143, 147, 151, and 159, a VH CDR2 sequence selected from SEQ ID NOs: 136, 140, 144, 148, 152, 156, and 160, and a VH CDR3 sequence selected from SEQ ID NOs: 137, 141, 145, 149, 153, 157, and 161. In some embodiments, the first antigen-binding region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NOs: 138, 142, 146, 150, 154, 158, and 162. In some embodiments, the first antigen-binding region comprises an amino acid sequence comprising or consisting of a sequence selected from SEQ ID NOs: 138, 142, 146, 150, 154, 158, and 162.

[0225] In some embodiments, a multispecific antibody used in the methods of the disclosure may compete (i.e., may be capable of competing) for binding to 5T4 with an antibody having a sequence selected from SEQ ID NOs: 138, 142, 146, 150, 154, 158, and 162. The multispecific antibody may bind to the same epitope on human 5T4 as an antibody having a sequence selected from SEQ ID NOs: 138, 142, 146, 150, 154, 158, and 162.

[0226] The multispecific antibodies used in the methods of the present disclosure comprise a first antigen-binding region capable of binding to 5T4 and may be for use in the treatment of bladder cancer, cervical cancer, non-small cell lung cancer, mesothelioma, squamous cell carcinoma of the head and neck, glioblastoma multiforme, esophageal cancer, pancreatic cancer, breast cancer including triple-negative breast cancer, colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, renal cancer, esophageal cancer or pre-B-cell acute lymphoblastic leukemia.

[0227] EGFR multispecific antibody In some embodiments, the human cancer antigen is EGFR. The (first) antigen-binding region of a multispecific antibody used in the present disclosure may comprise the VH CDR1 sequence of SEQ ID NO: 91, the VH CDR2 sequence of SEQ ID NO: 92, and the VH CDR3 sequence of SEQ ID NO: 93. In some embodiments, the first antigen-binding region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected from SEQ ID NO: 94. In some embodiments, the first antigen-binding region comprises an amino acid sequence comprising or consisting of SEQ ID NO: 94.

[0228] In some embodiments, a multispecific antibody used in the methods of the present disclosure may be capable of competing, i.e., competing, for binding to EGFR with an antibody having the sequence set forth in SEQ ID NO: 94. The multispecific antibody may bind to the same epitope on human EGFR as the antibody having the sequence set forth in SEQ ID NO: 94.

[0229] A multispecific antibody used in the present disclosure may comprise a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 91, the CDR2 sequence set forth in SEQ ID NO: 92, and the CDR3 sequence set forth in SEQ ID NO: 93, and the second antigen-binding region comprises the CDR1 sequence set forth in SEQ ID NO: 5, the CDR2 sequence set forth in SEQ ID NO: 6, and the CDR3 sequence set forth in SEQ ID NO: 7. In some embodiments, the second antigen-binding region comprises a combination of CDRs selected from Tables 6A to 6D.

[0230] The multispecific antibodies used in the methods of the present disclosure comprise a first antigen-binding region capable of binding to EGFR and may be for use in the treatment of primary or metastatic colon or colorectal cancer, peritoneal cancer, liver cancer, head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), cutaneous squamous cell carcinoma.

[0231] EGFR multispecific antibodies may be capable of activating human Vy9V52 T cells. Activation of Vy9V52 T cells may be measured by measuring changes in gene expression and / or (surface) marker expression (e.g., activation markers such as CD25, CD69, or CD107a) and / or secreted protein (e.g., cytokine or chemokine) profile. EGFR multispecific antibodies may be capable of increasing the number of CD107a-positive cells by at least two-fold, at least five-fold, etc., when tested at a concentration of, for example, 1 nM, preferably 100 pM, preferably 10 pM, preferably 1 pM, even more preferably 100 fM, as described, for example, in Example 9 of WO 2022 / 122973 (incorporated herein by reference). The EGFR multispecific antibodies of the disclosure may have an EC50 value of 100 pM or less, such as 50 pM or less, such as 25 pM or less, such as 20 pM or less, for example 15 pM or less for increasing the percentage of CD107a positive cells, when tested, for example, using Vy9V52 T cells and A431 target cells as described in Example 9 of WO 2022 / 122973.

[0232] Half-life extension domain In some embodiments, a multispecific antibody used in the present disclosure may further comprise a half-life-prolonging domain that extends the serum half-life of the multispecific antibody. Examples of means for extending the serum half-life of a binding agent of the present disclosure include peptides, proteins, or protein domains that are fused or otherwise attached to the multispecific antibody. Examples of peptide, protein, or protein domain groups include peptides that bind to other proteins with favorable pharmacokinetic profiles in the human body, such as serum albumin (see WO 2009 / 127691). As used herein, the term "human serum albumin" refers to the albumin protein present in human plasma. Human serum albumin is the most abundant protein in blood, accounting for approximately half of serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID number: P02768. In some embodiments, a multispecific antibody may have a terminal half-life of greater than about 168 hours upon administration to a human subject. The terminal half-life can be 336 hours or longer. The "terminal half-life" of an antibody, as used herein, refers to the time it takes for the serum concentration of a polypeptide to decline by 50% during the terminal elimination phase in vivo.

[0233] Half-life extending domains include larger protein domains or complete proteins, such as fusions of human serum albumin, variants or mutants of human serum albumin (see WO 2011 / 051489, WO 2012 / 059486, WO 2012 / 150319, WO 2013 / 135896, WO 2014 / 072481, WO 2013 / 075066) or domains thereof with the constant region of an immunoglobulin (Fc domain) and variants thereof as described herein. Such variants of the Fc domain may be optimized / modified to abolish Fc receptor binding (e.g., Fcg receptor), enhance binding to FcRn, or for other reasons, by allowing the desired pairing of dimers or multimers. A further concept known in the art for increasing the half-life of small protein compounds in the human body is the pegylation of such compounds, such as the polypeptides or binding agents of the present disclosure.

[0234] In some embodiments, the half-life extending domain can be an Fc domain (also referred to as an "Fc region"). As used herein, "Fc domain" refers to the minimal region (in the context of a larger polypeptide) or smallest protein fold (in the context of an isolated protein) capable of binding to or being bound by an Fc receptor (FcR). As used herein, "Fc domain monomer" refers to a single-chain protein that forms a functional Fc domain when associated with another Fc domain monomer. The association of two Fc domain monomers creates an Fc domain. When two Fc domain monomers associate, the resulting Fc domain has Fc receptor binding activity. Thus, the Fc domain is a dimeric structure capable of binding to an Fc receptor. Unless otherwise noted, all references herein to "variant Fc domains" should be understood to refer to a dimeric Fc domain in which each Fc domain monomer contains the referenced mutation.

[0235] As used herein, the term "Fc domain" will be understood to include polypeptides comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, the term "Fc domain" refers to the last two constant region immunoglobulin domains (CH2, CH3) of an IgG and, optionally, the flexible hinge at the N-terminus of those domains. Unless otherwise noted, all references to amino acid positions in Fc domains and Fc domain monomers are according to the EU index as set forth in Kabat (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). It is noted that polymorphisms have been observed at several Fc domain positions, including, but not limited to, Kabat positions 270, 272, 312, 315, 356, and 358, and therefore slight differences may exist between the sequences provided herein and those in the art.

[0236] The Fc domain can be derived from any of a variety of different antibody isotypes, including, but not limited to, wild-type or modified IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. In some embodiments, the Fc domain is derived from human IgG1.

[0237] There are many known polymorphisms in the IgG1 Fc domain, including the "DEL" polymorphism and the "EEM" polymorphism. The DEL polymorphism contains the amino acids DEL at positions 356, 357, and 358, respectively (e.g., SEQ ID NO: 337). The EEM polymorphism contains the amino acids EEM at positions 356, 357, and 358, respectively (e.g., SEQ ID NO: 336). Two binding agents that are otherwise identical except for the presence of a DEL or EEM Fc domain are expected to exhibit similar properties in terms of ligand binding and therapeutic efficacy. In some embodiments, the Fc domain is a DEL Fc domain. In some embodiments, the Fc domain is an EEM Fc domain.

[0238] In some embodiments, the Fc domain is a variant Fc domain, forming a variant Fc domain with desirable properties, such as increased half-life, compared to a naturally occurring (wild-type) Fc sequence. As used herein, "variant Fc domain" refers to a non-naturally occurring Fc domain, e.g., an Fc domain that comprises one or more non-naturally occurring amino acid residues, one or more amino acid substitutions relative to a wild-type human constant domain, or one or more amino acid deletions, additions, and / or modifications.

[0239] The serum half-life of a binding agent comprising an Fc domain can be increased by increasing the binding affinity of the Fc domain for FcRn. In some embodiments, the Fc domain variant exhibits enhanced serum half-life compared to a comparable molecule. In particular embodiments, the Fc domain variant comprises at least one amino acid substitution at one or more positions selected from the group consisting of M252Y, S254T, and T256E (referred to herein as "YTE"; e.g., SEQ ID NOs: 338 and 339). In another embodiment, the Fc domain variant comprises a Y at position 252 (referred to herein as "Y"; e.g., SEQ ID NOs: 340 and 341). Optionally, Fc domain variants may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Patent Nos. 5,624,821; 6,277,375; 6,737,056; WO 01 / 58957; WO 02 / 06919; WO 04 / 016750; WO 04 / 029207; WO 04 / 035752; WO 04 / 074455; WO 04 / 099249; WO 04 / 063351; WO 05 / 070963; WO 05 / 040217, WO 05 / 092925, and WO 06 / 020114).

[0240] In exemplary embodiments, Fc domain monomers allow two or more polypeptide chains to assemble in a covalent manner, for example, via disulfide bonds between cysteine ​​residues. In this manner, the Fc domain monomers act as dimerization domains, allowing two polypeptide chains to assemble to form a dimer. In some embodiments, such dimers comprise two polypeptides, each comprising an antigen-binding domain described herein linked to an Fc domain monomer, thereby forming a bivalent binder. In some embodiments, such dimers comprise two polypeptides, one comprising an antigen-binding domain described herein linked to an Fc domain monomer, and one comprising an Fc domain monomer, thereby forming a monovalent binder.

[0241]

[0237] The Fc domain may be a heterodimer comprising two Fc monomers, wherein a first antigen-binding domain is fused to the first Fc monomer and a second antigen-binding domain is fused to the second Fc monomer, wherein the first and second Fc monomers contain asymmetric amino acid mutations that favor heterodimer formation over homodimer formation (see, e.g., Ridgway et al. (1996) 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng 9:617). The CH3 regions of the Fc monomers may comprise the asymmetric amino acid mutations, for example, a first Fc polypeptide may comprise a T366W substitution (e.g., a "knob" mutation) and a second Fc polypeptide may comprise T366S, L368A, and Y407V substitutions (e.g., a "hole" mutation), or vice versa (wherein the amino acid positions correspond to those of human IgG1 according to the EU numbering system). Furthermore, the cysteine ​​residue at position 220 in the first and second Fc polypeptides may be deleted or substituted (wherein the amino acid positions correspond to those of human IgG1 according to the EU numbering system). See, for example, SEQ ID NOs: 343 and 344.

[0242] Furthermore, the first and / or second Fc monomer may include mutations that result in an inactive antibody, i.e., an antibody that is unable to mediate Fc effector functions or has a reduced ability to do so. An inactive Fc domain may additionally be one that is unable to bind C1q. The first and second Fc monomers may include mutations at positions 234 and / or 235, for example, the first and second Fc monomers may include L234F and L235E substitutions (wherein the amino acid positions correspond to those of human IgG1 according to the EU numbering system). See, for example, SEQ ID NO: 342.

[0243] Exemplary Fc domain monomer sequences are shown in Table 9. Hinge sequences are shown in bold and italic text; DEL and EEM polymorphisms are shown in boxed text; mutations relative to WT IgG1 are shown in bold and underlined text.

[0244] [Table 23]

[0245] [Table 24]

[0246] In some embodiments, the Fc domains listed in Table 9 comprise a wild-type IgG1 hinge (e.g., SEQ ID NO: 347-EPKSCDKTHTCPPCP). In some embodiments, the Fc domains listed in Table 9 comprise a modified IgG1 hinge (e.g., SEQ ID NO: 348-AAASDKTHTCPPCP).

[0247] kit In some embodiments, the present disclosure provides a kit for treating cancer comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vγ9Vδ2 T-cell receptor, as described herein; and (b) a dose of a common gamma cytokine (e.g., IL-2, IL-15, or a variant thereof), and optionally instructions for use.

[0248] In some embodiments, the present disclosure provides a kit for treating cancer, comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vy9V52 T-cell receptor, as described herein; and (b) an immune checkpoint inhibitor, optionally comprising instructions for use. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody (e.g., pembrolizumab).

[0249] In a further aspect, the present disclosure relates to a kit for treating cancer comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vγ9Vδ2 T-cell receptor, as described herein; (b) an immune checkpoint inhibitor (e.g., an anti-PD1 antibody such as pembrolizumab); and (c) a common gamma cytokine (e.g., IL-2 or IL-15), and optionally instructions for use.

[0250]

[0244] The kit may further comprise a solvent for diluting the multispecific antibody, common gamma chain cytokine, and / or immune checkpoint inhibitor.

[0251] Incorporation by Reference

[0245] All references, articles, publications, patents, patent application publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent application publication, or patent application herein is not, and should not be construed as, an admission or any form of suggestion that they constitute prior art or form part of the common general knowledge in any country in the world. [Example]

[0252] Example Example 1 - Dose Escalation of LAVA-1207 in Patients with Metastatic Castration-Resistant Prostate Cancer (mCRPC) background LAVA-1207 is a 78 kDa bispecific humanized antibody that associates with Vy9V52- T cells and PSMA+ tumor cells. LAVA-1207 is a disulfide-linked heterodimer of two fusion proteins set forth in SEQ ID NO:334 and SEQ ID NO:335, and is further described and characterized in WO 2022 / 008646 (incorporated herein by reference).

[0253] Study design and objectives

[0247] This study was an open-label, multicenter, phase 1 / 2a dose-escalation study in patients with treatment-refractory metastatic castration-resistant prostate cancer (mCRPC).

[0254] LAVA-1207 is administered by intravenous infusion every two weeks at the following dose levels: 1.5 μg (starting dose) (dose level (DL) 1), 4.5 μg (DL2), 13.5 μg (DL3), 40 μg (DL4), 120 μg (DL5), 360 μg (DL6), 540 μg (DL7), and 800 μg (DL8).

[0255] The objectives of this study were to investigate the safety and tolerability of LAVA-1207 and to evaluate PK, PD, immunogenicity, and preliminary antitumor activity. Exemplary baseline patient characteristics are provided in Table 10 below. Patients were treated with LAVA-1207 for treatment durations ranging from 4 to 38 weeks.

[0256] [Table 25]

[0257] result

[0250] Safety: At doses of 120 μg or less, the frequency and severity of adverse effects did not appear to be dose-related. The most common adverse effects were not suspected to be treatment-related.

[0258] Antitumor Activity: Antitumor activity was determined by assessing changes in prostate-specific antigen (PSA) in patient blood samples upon treatment with LAVA-1207. Some patients showed stable or declining PSA levels after LAVA-1207 treatment (Figure 1). Additionally, baseline Vy9V52 T cell frequency as a percentage of total T cells was determined for each patient (not shown). At least in some cases, a decline in PSA was observed in patients with a high Vy9V52 T cell count as a percentage of total T cells. See Figure 2 for N304 PSA levels and Figure 3A for relative changes in N304 Vy9V52 T cells. Figure 3A shows a significant decline in Vy9V52 T cell frequency in peripheral blood 2 hours post-dose, likely reflecting Vy9V52 T cell migration / redistribution and subsequent recovery. Figures 3C and 3D also show that Vγ9Vδ2 T cell activation markers (CD25 - Figure 3C and CD69 - Figure 3D) were upregulated after administration and receptor occupancy (RO - Figure 3B) was detectable up to 14 days post-EoI, with peak levels ranging from 6.1% to 12.6% at dose level 4 (40 μg).

[0259] Example 2 - Evaluation of the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and antitumor activity of a PSMA-targeting bispecific γδ-T cell engager in patients with treatment-refractory metastatic castration-resistant prostate cancer Study design:

[0252] This study is an open-label, multicenter, Phase 1 and Phase 2a dose-escalation study with expansion cohorts to investigate the safety, tolerability, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary antitumor activity of LAVA-1207 in patients with treatment-refractory metastatic castration-resistant prostate cancer (mCRPC).

[0260] The study will begin with an open-label dose-escalation part (Part 1) to determine the recommended Phase 2 dose (RP2D). The second part of the study (Part 2) will be an open-label expansion cohort at the RP2D and schedule, which will expand the number of patients to confirm safety in a patient population with treatment-refractory mCRPC and measurable disease. LAVA-1207 will be administered with or without low-dose subcutaneous IL-2 (referred to as LDSC IL-2), with the option of pembrolizumab.

[0261] Part 1 - Dose Escalation Dose Cohorts and Dose Administration Regimen The study anticipates dose escalation, including a cohort with a priming dose. Eligible patients will receive successively higher doses of LAVA-1207 in subsequent dose cohorts and will continue to receive LAVA-1207 for up to 24 weeks or until disease progression, unacceptable toxicity, or withdrawal of consent, or as otherwise specified in the investigational medicinal product (IMP) discontinuation criteria.

[0262] Patients in a given dose cohort will receive LAVA-1207 as an intravenous (IV) infusion at the same dose continuously and uninterruptedly during a two-week dosing interval (equivalent to one cycle), except as needed to manage adverse events (AEs). The biologic will be infused over two hours for the first cycle, one hour for the second cycle, and 30 minutes for subsequent cycles.

[0263]

[0256] In all cohorts, at least three patients will be enrolled at the same dose level unless one patient develops a dose-limiting toxicity (DLT) within the first 28 days of treatment (defined as the DLT period).

[0264] Dose escalation and RP2D determination

[0257] The planned dose cohorts for Part 1 of this study are presented in Table 11 below:

[0265] [Table 26]

[0266] Dose escalation will continue until the RP2D can be defined by the optimal biologically active dose of LAVA-1207 or until the maximum tolerated dose (MTD) is determined, whichever occurs first. The optional biologically active dose will be defined as the safe dose that demonstrates the highest pharmacological activity of LAVA-1207. Pharmacological activity considerations include assessment of effects on Vy9V52 T cells, such as binding by LAVA-1207 to Vy9V52 T cells and changes in the frequency or activation status of Vy9V52 T cells as assessed in peripheral blood. The final RP2D determination will take into account safety (including any delayed effects in the post-treatment phase, including potential AEs occurring throughout the entire treatment period), preliminary antitumor activity, pharmacokinetics, and pharmacodynamics data.

[0267] Preliminary data on adverse events are provided in Figure 5. Suspected related treatment-emergent AEs (TEAEs) were grade 1 or 2. There was no increase in severity or frequency of TEAEs with increasing dose, and no patients discontinued treatment due to an AE.

[0268] Exploratory dose cohort with LAVA-1207 and low-dose SC IL-2 (LDSC IL-2)

[0260] For LAVA-1207 dose level 6, two exploratory cohorts (n=3 per cohort) will be initiated in which IL-2 will be administered subcutaneously (SC) as a single dose (Cohort A1) or as three doses (Cohort A2) starting 24 hours after the start of LAVA-1207 infusion (see Table 12).

[0269] [Table 27]

[0270] [Table 28]

[0271] LDSC IL-2 will be administered only during the first four cycles of LAVA-1207 treatment (e.g., up to 8 weeks). IL-2 is likely to increase the number of Vy9V52 T cells, thereby increasing the total number of Vy9V52 T cells available for LAVA-1207-mediated lysis of PSMA-expressing prostate cancer cells.

[0272] Cohort initiation will begin at a dose level determined safe and well-tolerated by DEC, with the multispecific antibody target dose (second target dose) starting in cycle 2, along with 400 mg of anti-PD-L1 antibody administered intravenously over 30 minutes every six weeks (Q6W). Treatment will be administered for a period not to exceed two years (18 cycles of anti-PD-L1 antibody or until one or more discontinuation criteria are met).

[0273]

[0263] The dose of pembrolizumab will remain constant at 400 mg Q6W for each dose level of the multispecific antibody.

[0274] Part 2 - Expansion Cohort

[0264] This study will include additional cohorts to further explore the safety, tolerability, immunogenicity, pharmacokinetics, and pharmacodynamics of LAVA-1207 in combination with IL-2, and to investigate preliminary antitumor activity in patients with treatment-refractory mCRPC with measurable disease.

[0275]

[0265] The primary objectives of this dose escalation cohort will include: (a) To investigate the safety and tolerability of LAVA-1207 in patients with treatment-refractory mCRPC. (b) Determine the RP2D of LAVA-1207 in patients with treatment-refractory mCRPC for LAVA-1207 monotherapy, LAVA-1207 + LDSC IL-2, and LAVA-1207 + pembrolizumab.

[0276]

[0266] Primary objectives of the expansion cohort will include: (a) Examine the safety and tolerability of LAVA-1207 at RP2D in treatment-refractory mCRPC patients with measurable and non-measurable disease.

[0277]

[0267] Secondary objectives for both cohorts will include: (a) Explore the preliminary antitumor activity of LAVA-1207. (b) Evaluate the pharmacokinetics of LAVA-1207. (c) Evaluate the pharmacodynamics of LAVA-1207. (d) Evaluate the immunogenicity of LAVA-1207.

[0278] Exploratory objectives for both cohorts will include: (a) To investigate the safety and tolerability of LAVA-1207 and LDSC IL-2 in patients with treatment-refractory mCRPC. (b) Explore the preliminary pharmacokinetics of LAVA-1207 and LDSC IL-2. (c) Explore the preliminary pharmacodynamics of LAVA-1207 and LDSC IL-2. (d) Explore the preliminary immunogenicity of LAVA-1207 and LDSC IL-2. (e) Explore the preliminary antitumor activity of LAVA-1207 and LDSC IL-2. (f) To assess the effect of study treatments on circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA).

[0279] Primary endpoints for dose escalation for LAVA-1207 alone, LAVA-1207 + LDSC IL-2, and LAVA-1207 + pembrolizumab will include: (a) Frequency and severity of AEs using the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 and the ASTCT grading system for CRS. (b) Frequency and type of DLTs.

[0280] For LAVA-1207 alone, LAVA-1207 + LDSC IL-2, and LAVA-1207 + pembrolizumab, primary endpoints of the expansion cohorts will include: (a) Frequency and severity of AEs using the CTCAE 5.0 version and CRS ASTCT grading classification at RP2D.

[0281] Secondary endpoints for both cohorts will include: (a) Number of participants with anti-tumor response according to immune response evaluation criteria in solid tumors (iRECIST) in patients with measurable disease. (b) Duration of response. (c) Disease control rate (DCR) for patients with measurable disease at 8, 16 and 24 weeks. (d) The number of participants experiencing any decline in prostate-specific antigen (PSA) and the number of participants experiencing a 50% or greater decline in PSA from baseline. (e) Progression-free survival (using Prostate Cancer Working Group (PCWG3) criteria for bone lesions and / or iRECIST criteria for soft tissue lesions). (f) Pharmacokinetic parameters. (g) Pharmacodynamic markers. (h) Incidence and prevalence of anti-LAVA-1207 antibodies.

[0282] Exploratory endpoints for both cohorts include: (a) Frequency and severity of AEs using CTCAE 5.0 and ASTCT grading for CRS. (b) Pharmacokinetic parameters. (c) Pharmacodynamic markers. (d) Incidence and prevalence of anti-LAVA-1207 antibodies. (e) Number of participants with anti-tumor response according to immune response evaluation criteria in solid tumors (iRECIST) in patients with measurable disease. (f) The number of participants experiencing any decline in PSA from baseline and the number of participants experiencing a decline of 50% or more from baseline. (g) DCR for patients with measurable disease at 8, 16, and 24 weeks. (h) Determine any relationship between change in CTC / ctDNA from baseline and treatment dose and clinical response.

[0283] Inclusion Criteria: Patients are eligible for inclusion in this study only if all of the following criteria are met: (a) Patients must be 18 years of age or older at the time of signing the informed consent. (b) Male patients with mCRPC as defined by PCWG3 criteria (histologically confirmed adenocarcinoma; adenocarcinoma with ≤10% small cell or neuroendocrine features is acceptable). Brain metastases are acceptable as long as the patient's symptoms are well controlled. (c) Patients must have failed at least one line of taxane-based chemotherapy or are deemed medically unsuitable for treatment with a taxane regimen. (d) Patients must have received second-generation or later androgen receptor-targeted therapy / androgen biosynthesis inhibitors (e.g., abiraterone, enzalutamide, and / or apalutamide). In the non-metastatic CRPC setting, progression on novel antiandrogen therapy may have occurred. (e) The patient is unlikely to tolerate or derive clinically meaningful benefit from other available therapies. (f) The patient must have resolved all drug-related toxic adverse effects of any prior cancer therapy to Grade 1 or less according to CTCAE v5.0 or to baseline severity levels. (g) Patients with evidence of progressive disease, defined as one or more of the following criteria: (i) Elevated PSA levels ≥ 1 ng / mL on at least two consecutive occasions separated by at least one week. (ii) Computed tomography (CT) or magnetic resonance imaging (MRI) scan: nodal or visceral progression as defined by RECIST 1.1. (iii) Bone scintigraphy: Appearance of two or more new metastatic lesions. (h) Patients must have undergone bilateral orchiectomy or be undergoing ongoing androgen deprivation therapy (ADT) with gonadotropin-releasing hormone agonists or antagonists (surgical or medical castration). (i) Total serum testosterone ≤ 50 ng / dL or 1.73 nmol / L. (j) Evaluable (measurable or non-measurable) disease for prostate cancer. (k) predicted life expectancy of 6 months or more; (l) Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. (m) Adequate renal function (estimated glomerular filtration rate [eGFR] > 40 mL / min / 1.73 m2 by in-house laboratory), liver function (bilirubin ≤ 2 times upper limit of normal (ULN), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 3.0 times ULN). In the presence of liver metastases, AST and ALT ≤ 5.0 times ULN are acceptable. Adequate hematologic function (neutrophils > 1 × 10 9 / L, platelet count >75×10 9 / L, Hb>9g / dL) and no history of blood transfusion within the past 2 weeks. (n) A male; (i) Sterile surgery completed (bilateral orchiectomy, vasectomy). (ii) Adherence to an effective contraceptive regimen from the time of signing the Informed Consent Form (ICF) until 90 days after the final IMP dose (i.e., use of male condoms with female partners, plus ensuring the use of a highly effective method of contraception with a failure rate of less than 1% per year when intercourse with a woman of childbearing potential who is not currently pregnant). Abstinence is not considered an adequate contraceptive regimen. (iii) refrain from donating sperm from the time of signing the ICF until 90 days after the final IMP administration; (o) Be capable of providing signed and dated informed consent, including adherence to the requirements and restrictions set forth in the ICF and protocol, prior to the initiation of any study-related procedures that are not considered standard of care.

[0284] Exclusion criteria:

[0274] Patients will be excluded from this study if any of the following criteria apply: (a) Other malignancies within the past two years, excluding adequately treated carcinoma in situ, basal cell carcinoma of the skin, or squamous cell carcinoma. (b) Concurrent uncontrolled or serious medical conditions. (c) positive serological test for human immunodeficiency virus (HIV) antibodies; (d) Hepatitis B surface antigen [HBsAg] serology positive and hepatitis B core antibody (anti-HBc) negative, and hepatitis C virus antibody. Patients who are positive for anti-HBc or hepatitis C antibody may be included if they have a negative polymerase chain reaction (PCR) within 6 weeks prior to their first IMP dose. Patients who are PCR positive will be excluded. (e) The patient has any active, uncontrolled, or suspected infection. (f) known clinically relevant immunodeficiency disorder; (g) A significant history of renal, neurological, psychiatric, pulmonary, endocrine, metabolic, immunological, cardiovascular, or hepatic disease that, in the opinion of the investigator, may adversely affect participation in this study. (h) Unstable cardiovascular function, defined as (a) symptomatic ischemia, or (b) uncontrolled clinically significant conduction abnormalities (i.e., ventricular tachycardia while taking antiarrhythmic medications is excluded; first-degree atrioventricular block or asymptomatic left anterior fascicular block / right bundle branch block is not excluded), or (c) congestive heart failure New York Heart Association grade 3 or greater, or (d) myocardial infarction within 3 months. (i) Prior treatment with anti-tumor therapy within 2 weeks prior to the first IMP for radiation therapy and androgen receptor targeted therapy / androgen biosynthesis inhibitors, and within 4 weeks prior to systemic chemotherapy or targeted / immunotherapy. (j) History of treatment with live or live-attenuated vaccines within 2 weeks prior to the first IMP dose. New vaccines need to be evaluated for their mode of action. (k) Treatment with other investigational drugs in the 4 weeks prior to the first IMP. (l) Major surgery within 4 weeks prior to the first IMP administration. (m) Hypersensitivity to LAVA-1207, IL-2, or any of the excipients present in pembrolizumab (if applicable). (n) History of treatment with any systemic immunosuppressant within 4 weeks prior to the first IMP dose, except for systemic corticosteroid use of up to 10 mg oral dose of prednisone daily (or equivalent for other steroids). (o) History of treatment with an IV aminobisphosphonate (e.g., ibandronate, pamidronate, zoledronate, etc.) within 4 weeks or 1 year prior to the first IMP. (p) Known drug and alcohol abuse in the opinion of the investigator.

[0285] Additional exclusion criteria for pembrolizumab trials: Patients will be excluded from the LAVA-1207 + pembrolizumab study if any of the following criteria apply: (a) Active infection requiring systemic therapy. (b) A history of (non-infectious) pneumonitis / interstitial lung disease that required steroid medication or current pneumonitis / interstitial lung disease. (c) Have a diagnosis of immunodeficiency within 7 days prior to the first dose of study drug or are receiving chronic systemic steroid therapy (dosed at greater than 10 mg prednisone equivalent daily) or any other form of immunosuppressive therapy. (d) Have an active autoimmune disease requiring systemic treatment (i.e., involving the use of disease-modifying agents, corticosteroids, or immunosuppressants) for the past two years. Replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment and is permitted. (e) had previously received therapy with an anti-programmed cell death 1 (PD1), anti-programmed cell death ligand 1 (PD-L1), or anti-programmed cell death ligand 2 (PD-L2) agent, or with an agent directed against another stimulatory or co-inhibitory T cell receptor (e.g., CTLA-4, OX40, CD137), and discontinued that treatment due to a grade 3 or higher immune-related AE; (f) Any significant history of renal, neurological, psychiatric, pulmonary, endocrine, metabolic, immunological, cardiovascular, or hepatic disease that, in the opinion of the investigator, may adversely affect participation in the study. (g) Known active central nervous system (CNS) metastases and / or carcinomatous meningitis. Participants with previously treated brain metastases may participate provided they have been radiologically stable for at least 4 weeks with repeat imaging at least 14 days prior to the first dose of study treatment, i.e., no evidence of progression (note that repeat imaging must be performed during study screening), are clinically stable, and do not require steroid treatment. (h) Have received prior systemic anti-cancer therapy, including the investigational drug, within 4 weeks prior to treatment (shorter intervals may be considered for kinase inhibitors or other short half-life drugs). Note: Participants must have recovered from all AEs attributable to prior therapy to Grade 1 or below or to baseline. Participants with Grade 2 or below neuropathy may be eligible. Participants with Grade 2 or below endocrine-related AEs requiring treatment or hormone replacement may be eligible. Note: If a participant has undergone major surgery, they must have adequately recovered from the procedure and / or any complications from the surgery before the start of the study intervention. (i) Previous radiation therapy within 2 weeks of starting study treatment or a history of radiation pneumonitis. Note: Participants must have recovered from all radiation-related toxicities and must not require corticosteroids. A 1-week washout is allowed for palliative radiation therapy for non-CNS disease (radiation therapy for ≤2 weeks). (j) Receipt of a live or live-attenuated vaccine within 30 days prior to the first dose of the study intervention. Note: Administration of killed vaccines is permitted. (k) Currently participating in or have participated in an investigational drug study within 4 weeks prior to the first dose of investigational treatment or have used an investigational device. Note: Participants in the follow-up phase of an investigational study may participate as long as 4 weeks have passed since their last dose of the previous investigational drug. (l) Has history or current evidence of any medical condition, therapy, laboratory abnormality, or other condition that, in the opinion of the treating investigator, may confound the results of the study or prevent the participant from participating for the full duration of the study, such that participation is not in the participant's best interest. (m) The participant has a known mental or substance abuse disorder that would interfere with the participant's ability to cooperate with the requirements of the study.

[0286] Patient participation period:

[0276] Screening phase: Within 28 days prior to first IMP.

[0287] Treatment Phase: A 24-week treatment period is planned. At the request of the treating physician, and in consultation with the sponsor, continued use of IMP alone or in combination with pembrolizumab beyond the planned treatment phase may be offered for individual patients with ongoing disease control.

[0288]

[0278] For patients in the LAVA-1207 + pembrolizumab arm, treatment duration will be for up to 18 cycles of pembrolizumab (approximately 24 months) or until any discontinuation criteria are met.

[0289] Post-Treatment Phase: A 120-day follow-up phase following the last dose of IMP for each patient with an End of Treatment (EoT) visit 14-42 days after the last dose of IMP for each patient.

[0290] IMP, dose, mode of administration and dosage regimen: LAVA-1207 is an infusion solution concentrate and will be administered as an intravenous infusion with a 14-day dosing interval. The infusion period will be 2 hours (+15 minutes) for any one or more priming doses and for the first target dose, followed by 1 hour for the second target dose and 30 minutes for subsequent cycles. The escalating dosing regimen in cycle 1 will begin in expansion cohort 8 or later.

[0291] Patients in the dose-escalation arm with pembrolizumab will receive pembrolizumab 400 mg IV Q6W, starting with the second target LAVA-1207 dose. Patients receiving LAVA-1207 and pembrolizumab on the same day will wait at least 30 minutes after the end of the pembrolizumab infusion before administering LAVA-1207.

[0292]

[0282] LDSC IL-2 will be administered as a single dose or as three daily doses starting 24 hours (-1 hour to +2 hours) after the start of the LAVA-1207 infusion for a total of four cycles.

[0293]

[0283] In Part 2, patients will receive an intravenous infusion of LAVA 1207 at the RP2D (dose and schedule) as established in the dose escalation part of the study (with or without LDSC IL-2).

[0294] In the Part 2 expansion arm with pembrolizumab, patients will receive an intravenous infusion of LAVA-1207 plus pembrolizumab at the RP2D (dose(s) and schedule) as established in the dose escalation part of the study. Pembrolizumab will be administered at least 30 minutes before LAVA-1207 administration.

[0295]

[0285] In Part 1 of this study, patients who experience DLT according to protocol or grade 3 non-hematologic toxicity may require a dose reduction or dose delay.

[0296]

[0286] For Part 2, specific toxicities requiring dose interruption or reduction will be entered into the protocol prior to the initiation of dose expansion cohorts.

[0297]

[0287] In general, patients may continue treatment or be withdrawn from the study if the (serious) (S)AE resolves to a Grade 1 AE.

[0298] Evaluation and analysis criteria: Safety of LAVA-1207 alone, LAVA-1207 + LDSC IL-2, and LAVA-1207 + pembrolizumab Vital signs, physical examination, clinical chemistry, hematology, urinalysis, electrocardiogram (ECG), and documentation of any reported AEs will be recorded. AE severity will be graded according to the National Cancer Institute's CTCAE Version 5.0 (NCI CTCAE) grading scale (for details, see the NCI CTCAE webpage, http: / / ctep.cancer.gov), except that CRS will evaluate and grade according to the ASTCT consensus criteria.

[0299]

[0289] LAVA-1207 was safely achieved at a dose of 1200 μg in patients with treatment-refractory mCRPC.

[0300] Pharmacodynamics of LAVA-1207 alone, LAVA-1207 + LDSC IL-2, and LAVA-1207 + pembrolizumab

[0290] Blood samples will be collected to determine LAVA-1207 concentrations and the following pharmacodynamic markers will be assessed: binding of LAVA-1207 to Vγ9Vδ2-T cells, Vγ9Vδ2-T cells (activation status and frequency) and general immune cell analysis (activation status and frequency of B cells, T cell subsets (CD4+, CD8+ and Treg), natural killer (NK) cells and monocytes), and induction of cytokines (e.g., interleukin [IL]-1β, IL-2, IL 6, IL-8, tumor necrosis factor [TNF]-α, interferon [IFN]-γ and granulocyte-macrophage colony-stimulating factor [GM-CSF]).

[0301] Biopsies (optional, only for patients with biopsy-accessible metastatic sites) will be taken to assess the following pharmacodynamic markers: Vy9V52-T cell infiltration and activation status / PSMA expression / butyrophilin expression / tumor cell viability. Preliminary pharmacokinetic results of LAVA-1207 are shown in Figure 4. Figure 4 shows that the pharmacokinetics of LAVA-1207 appear to be linear.

[0302] immunogenicity

[0292] Blood samples will be taken to determine the possible development of anti-drug antibodies to LAVA-1207.

[0303] Antitumor activity of LAVA-1207 alone, LAVA-1207 + LDSC IL-2, and LAVA-1207 + pembrolizumab

[0293] Blood samples will be taken to determine the following: (a) PSA assessment. (b) CTC and / or ctDNA evaluation.

[0304] Imaging will be performed for the following: (a) PSMA-positron emission tomography (PET). (b) Tumor evaluation by (contrast-enhanced) CT scan of the chest, abdomen, and pelvis. (c) Bone scintigraphy.

[0305] Statistical considerations: In general, data will be summarized by dose level (and separately, with or without LDSC IL-2 and with or without pembrolizumab) and for the overall population. Baseline and safety data will be presented based on the safety analysis set, which will include all patients who received at least one dose of IMP. Anti-tumor data summaries will be based on the evaluable analysis set, which will include all patients who received at least one dose of IMP and had a disease evaluation performed to assess response, and the intention-to-treat analysis set (all enrolled patients). Pharmacokinetic and pharmacodynamic analyses will include all patients who received at least one dose of IMP and had at least one available post-dose measurement.

[0306] Safety: All safety outcomes will be summarized using descriptive statistics. AEs that began or worsened at or after the first dose of IMP (treatment-emergent AEs [TEAEs]) will be presented separately from those that began before the first dose of IMP or those observed during the post-treatment phase beyond the end-of-treatment (EoT) visit. The number and percentage of patients with TEAEs will be summarized by major Medical Dictionary for Regulatory Activities (MedDRA) system organ class (SOC) and preferred term (PT). Separate tables will be generated showing all TEAEs suspected to be related to IMP, all serious TEAEs, and all TEAEs. The number and percentage of patients with DLTs will be summarized; DLTs will be presented in patient-specific tables.

[0307]

[0297] Vital signs, ECG, physical examination findings, and safety laboratory parameters will be analyzed using descriptive statistics.

[0308]

[0298] Abnormal laboratory findings will be summarized by parameter in a frequency distribution table and a table of change from baseline values.

[0309] Pharmacokinetics: Individual patient serum LAVA-1207 concentrations will be used to determine pharmacokinetic parameters using standard non-compartmental methods.

[0310] Pharmacodynamics: Pharmacodynamic markers and potential cytokine induction will be analyzed descriptively.

[0311] Immunogenicity: The incidence and individual serum titers and isotypes of anti-drug antibodies to LAVA-1207 will be summarized.

[0312] Antitumor activity and disease evaluation

[0302] For patients with measurable disease (Parts 1 and 2), tumor response will be summarized according to iRECIST.

[0313] DCR (complete response (CR) + partial response (PR) + stable disease (SD)) at 8, 16 and 24 weeks for patients with measurable disease (Parts 1 and 2).

[0314]

[0304] For patients evaluable for PSA response, any PSA decline (defined as any decrease in PSA level compared to baseline) and PSA response (defined as a 50% or greater decrease in PSA level compared to baseline) will be analyzed descriptively.

[0315] Progression-free survival will be analyzed descriptively using the Kaplan-Meier method. Progression-free survival is defined as the time from the start of treatment to progression (soft tissue disease progression [per iRECIST], bone disease progression [per PCWG3 criteria]), or death.

[0316]

[0306] Exploratory endpoints (e.g., change from baseline in CTC / ctDNA) will be analyzed descriptively.

[0317] Sample size justification:

[0307] The sample size for Part 1 of this study will be determined by clinical evaluation and predefined dose escalation rather than statistical considerations.

[0318] The sample size for Part 2 (n=30 for LAVA-1207 or LAVA-1207 + LDSC IL-2 and n=40 for the LAVA-1207 + pembrolizumab arm) will allow for an overall safety evaluation of LAVA-1207 at the recommended dose and schedule. At least 20 patients with measurable disease will be enrolled to receive LAVA-1207 or LAVA-1207 + LDSC IL-2, thereby allowing for a preliminary efficacy evaluation of LAVA-1207 in two relatively homogenous populations of patients with measurable and non-measurable disease.

[0319] Dose Escalation Committee (DEC):

[0309] The DEC will consist, at a minimum, of a medical monitor, at a minimum, one of the primary investigators, and a representative from the sponsor.

[0320]

[0310] The study design outlined in Example 2 can be repeated, but with IL-15 replacing IL-2.

[0321] Example 3: Anti-PD1 Combination Therapy Figures 6A-6B show increased PD-1 expression on Vy9V52 T cells after treatment with LAVA-1207. Figure 7A shows the percentage of Vy9V52 T cells in untreated non-malignant (nm) and malignant (m) areas of the prostate from a prostate cancer patient, and in a prostate cancer patient PBMC sample. Figures 7B and 7C show PD-1 expression on Vy9V52 T cells and CD3+ T cells from the same prostate cancer patient sample. Figure 8 shows that Vy9V52 T cells activated in vitro by V52-bsTCE (bispecific T cell engager) upregulate PD-1. Each of these data suggests that combination therapy with anti-PD1 or anti-PD-L1 antibodies may be beneficial.

[0322] Example 4: Exemplary LAVA-1207 and IL-2 Dosing Regimen 9 and 10 show exemplary dosing regimens for LAVA-1207 and IL-2. Each of these exemplary regimens may also include pembrolizumab.

[0323] In the exemplary dosing regimen of Figure 9, in cycle 1, the patient receives a priming dose of LAVA-1207 on days -14 and -7. Then, on day 0, the patient receives a target dose of LAVA-1207. Then, on days 1, 2, and 3, the patient receives a low dose of IL-2. This cycle is repeated every two weeks three more times without the priming dose, and then, for each subsequent cycle, the patient receives only the target dose of LAVA-1207.

[0324] The rationale for including IL-2 in this clinical protocol can be seen in Figures 11A and 11B, which show that expansion of Vy9V52 T cells relative to baseline with an exemplary bsTCE or pamidronate is highly IL-2 dependent. In the absence of IL-2, little to no expansion was observed.

[0325]

[0315] Similar exemplary dosing regimens can be performed using IL-15 in place of IL-2.

[0326] Example 5: LAVA-1207 activates Vγ9Vδ2-T cells in patient-derived tumor tissue and PBMCs and induces selective prostate tumor cell lysis Peripheral blood mononuclear cells (PBMCs) were derived from healthy donor and prostate cancer patient blood and separated using Lymphoprep™ (AXI-1114547, Fresenius) density gradient centrifugation. Blood samples were obtained from Sanquin (Amsterdam, The Netherlands) for healthy donors or from prostate cancer patients with written informed consent from Amsterdam UMC (location VUmc, Amsterdam, The Netherlands). PBMCs were processed for phenotypic analysis using flow cytometry or resuspended in RPMI complete medium for functional experiments.

[0327] Vy9V52 T cells from healthy donors were isolated from PBMCs and expanded. If the purity of Vy9V52 T cells was greater than 95% of the total cells (referred to as expanded Vy9V52 T cells), they were then used for experiments. Alternatively, Vy9V52 T cells were isolated from PBMCs using the EasySep™ Human Gamma / Delta T Cell Isolation Kit (19255, STEMCELL Technologies) and used directly for functional experiments (referred to as non-expanded Vy9V52 T cells).

[0328] Non-malignant and malignant tissue samples were collected from radical prostatectomy patients with non-metastatic prostate cancer. A pathologist assessed tissue sections for malignancy through macroscopic analysis, combined with clinical and radiological findings provided by the treating physician. The tissues were finely minced and resuspended in dissociation medium consisting of Iscove's Modified Dulbecco's Medium (IMDM, 12440053, Gibco) supplemented with 0.1% DNase I (10104159001, Roche), 0.14% Collagenase A (10103586001, Roche), 100 IU / mL PSG, and 5% FCS. The tissues were transferred to sterile flasks and incubated at 37°C for 45 minutes on a magnetic stirrer. The cell suspension was then strained through a 100 μM cell strainer (352360, BD Falcon).

[0329] The dissociation step was repeated for 1 to 3 rounds. Single cell suspensions derived from these tissues were either processed for phenotypic analysis using flow cytometry or resuspended in RPMI complete medium for functional experiments.

[0330]

[0320] LNCaP (PSMA+ clone FGC, CRL-1740) and PC3 (PSMA-, CRL-1435) were obtained from the American Type Culture Collection (ATCC), and VCaP (PSMA+, 06020201-1VL) and 22Rv1 (PSMA+, 5092802) were obtained from the European Collection of Authenticated Cell Cultures (ECACC). Cell lines were maintained in Roswell Park Memorial Institute (RPMI) medium (22400089, Gibco) supplemented with 10% (v / v) fetal calf serum (FCS, 04-007-1A, Biological Industries), 0.05 mM β-mercaptoethanol (200-646-6, Merck), 100 IU / mL sodium penicillin, 100 μg / mL streptomycin sulfate, and 2.0 mM L-glutamine (PSG, 10378-016, Life Technologies), referred to as RPMI complete medium. Cell lines were kept at 37°C in a humidified atmosphere containing 5% CO.

[0331]

[0321] Figure 12A shows the frequency of Vγ9Vδ2 T cells relative to total CD3+ cells in PBMCs (2.6±0.7% (mean±SEM; n=40)), non-malignant tissues (1.4±0.4% (mean±SEM; n=31)), and malignant tissues (1.6±0.5% (mean±SEM; n=31)).

[0332] Phenotypic analysis of Vγ9Vδ2 T cells and CD3+ non-Vγ9Vδ2 T cells showed that CD69, an early activation and tissue-resident marker, tended to be more highly expressed in non-malignant and malignant prostate tissues compared with PBMCs (68.1% ± 4.5% of Vγ9Vδ2 T cells in PBMCs; CD69 mean ± SEM: 81.4% ± 8.9% of Vγ9Vδ2 T cells in non-malignant prostate tissues; and 87.7% ± 6.0% of Vγ9Vδ2 T cells in malignant prostate tissues). Malignant prostate tissue Vγ9Vδ2 T cells also highly expressed the costimulatory marker 4-1BB and the terminal differentiation marker CD57. However, PD-1 expression did not differ between PBMCs, non-malignant, and malignant prostate tissue Vγ9Vδ2 T cells. In comparison, the percentage of all CD3+ cells that expressed CD69, 4-1BB, and PD-1 was significantly higher in non-malignant and malignant prostate tissues compared to PBMCs (see Figure 12B).

[0333] To test whether Vy9V52-T cells from prostate cancer patients were reactive, LAVA-1207 (PSMA-V52-Fc) was incubated with malignant (PSMA expression MFI mean ± SEM; 5.7 ± 2.4) and non-malignant prostate tissue (PSMA expression MFI mean ± SEM; 0.7 ± 0.3) alone or co-cultured with patient-specific PBMCs for 24 hours. When incubated with co-cultures of malignant prostate tissue and PBMCs, 50 nM LAVA-1207 increased degranulation in both malignant prostate tissue (TI tissue infiltrate) and PBMCs (Figure 12C). In contrast, LAVA-1207 did not enhance degranulation in non-malignant prostate tissue when incubated alone or co-cultured with PBMCs (see Figure 12D). Furthermore, 24-hour co-culture of patient-derived PBMCs, prostate tissue (PBMC:target cell ratio = 10:1; Vγ9Vδ2-T cells:target cell ratio = 1:25 (donor 1), 1:9 (donor 2), and 1:5 (donor 3)), and LAVA-1207 resulted in non-tumor cytolytic (CD45 - EpCAM - tumor cell lysis (CD45-EpCAM cells) compared with dim / +There was an increase in tumor cell counts (as defined by intratumoral Vy9V52 T cells) in the LAVA-1207-treated donors. See Figure 12E. Tumor lysis was observed in two of three donors with LAVA-1207 alone, indicating that tumor cell lysis was mediated by intratumoral Vy9V52 T cells.

[0334] Example 6: NKG2D and DNAM-1 receptor-ligand interaction contributes to LAVA-1207-induced Vy9V52-T cell degranulation and tumor cell lysis Because LAVA-1207 induced Vγ9Vδ2 T cell degranulation and lysis in the presence of malignant tissue but not in the presence of non-malignant tissue, we analyzed factors that could potentially contribute to this tumor-preferential activity. PSMA (unpaired and paired), BTN3A, BTN2A1, nectin-2 (a DNAM-1 ligand), PVR (a DNAM-1 ligand), MIC-A / B (an NKG2D ligand), ULBP-3 (an NKG2D ligand), ULBP-2 / 5 / 6 (an NKG2D ligand), and HLA-E (an NKG2A ligand) were all more highly expressed in malignant prostate tissue compared to non-malignant prostate tissue. Only ULBP-1 (an NKG2D ligand) was expressed at similar levels between malignant and non-malignant prostate tissue. However, the frequency of NKG2D and NKG2A receptor expression on Vy9V52 T cells did not vary significantly between malignant and non-malignant prostate tissues, and the proportion of DNAM-1 receptor-positive Vy9V52 T cells was lower in malignant than in non-malignant prostate tissues (see Figures 13A-C).

[0335] After preincubation with 10 μg / ml Fc receptor blocker (130-059-901, Miltenyi Biotec) and α-DNAM-1 (clone DX11, 559787, BD Bioscience) for 30 min, expanded healthy donor-derived Vγ9Vδ2 T cells were cultured with prostate cell lines (LNCaP, VCaP, or 22Rv1) for 4 h, and α-NKG2D (clone 149810, MAB139-100, R&D Systems) or α-NKG2A (monalizumab, PX-TA1392-100UG, Proteogenics) blocking antibodies were added for 30 min, followed by incubation with 0.001–5 nM LAVA-1207.

[0336] Vy9V52-T cell degranulation (as measured by CD107a expression), cytokine production (IFN-γ, TNF, IL-2, and IL-4), and tumor cell lysis all increased with increasing LAVA-1207 concentrations. However, in the presence of neutralizing antibodies against DNAM-1 or NKG2D receptors, this increase could be partially attenuated, except at the highest (saturating) concentrations of LAVA-1207. See Figures 14A-H. Neutralizing antibodies against NKG2A and BTN3A did not significantly affect LAVA-1207-mediated Vy9V52-T cell degranulation. See Figures 15A-B.

[0337] Next, to assess whether these receptor interactions also influence Vy9V52 T cell reactivity using patient tumor samples, dissociated prostate tumor samples from three patients were cultured for 4 hours in the presence of 0.05 nM LAVA-1207 and either DNAM-1 or NKG2D blocking antibodies. LAVA-1207 induced an increase in Vy9V52 T cell degranulation, which was significantly reduced in the presence of DNAM-1 blocking antibodies. Degranulation was reduced in only one of three donors in the presence of NKG2D blocking antibodies. See Figure 16.

[0338]

[0328] In conclusion, DNAM-1 and NKG2D receptor interaction may contribute to Vγ9Vδ2-T cell reactivity and prostate cancer lysis at non-saturating LAVA-1207 concentrations.

[0339] Example 7: LAVA-1207 elicits in vivo antitumor activity in a xenograft prostate cancer model using human PBMCs To evaluate the in vivo therapeutic efficacy of LAVA-1207, male immunodeficient NCG mice were inoculated with 5x10 22Rv1 6 Tumor cells were inoculated subcutaneously with PBMCs from healthy human donors at a 2:1 ratio (22Rv1:PBMC, n=2 PBMC donors: Vy9V52 T cell frequencies of 21.9% and 8.8% of total CD3+ cells, respectively, resulting in 22Rv1:Vy9V52 T ratios of 22:1 and 60:1). Mice were treated weekly with LAV A-1207 (0.2 and 2.0 mg / kg) or PBS IV on days 7, 14, and 21, starting on the day of tumor cell+PBMC inoculation. Tumor volumes were measured twice weekly using calipers and calculated as a volume in mm using the formula: 3 The tumor growth inhibition (TGI) was expressed in units of: V = (L × W × W) / 2, where V was the tumor volume, L was the tumor length (longest tumor dimension), and W was the tumor width (longest tumor dimension perpendicular to L). At the end of the follow-up period, tumor growth inhibition (TGI) was calculated as ((mm 3 Tumor volume in LAVA-1207-treated mice in mm 3 Tumor volume of PBMC-treated mice in units) / mm 3 The tumor volume was calculated as the tumor volume of the PBMC-treated mice in units of 1000 mm × 100%. 3 When the serotonin concentration exceeded 100kJ / kg, the animals were sacrificed.

[0340] As seen in Figures 17A-B, inoculation of PBMCs did not affect tumor growth, but when combined with either 0.2 or 2.0 mg / kg LAVA-1207, there was a significant inhibition of tumor growth (TGI) (0.2 mg / kg: P=<0.0001, TGI 61% and 2.0 mg / kg: P=<0.0001 and TGI 60%) and an increase in overall survival (0.2 mg / kg: P=<0.0001 and 2.0 mg / kg: P=<0.0001). Median overall survival was 51 days in both LAVA-1207 + PBMC treatment groups and 37 days in the PBMC treatment group.

[0341] Example 8: LAVA-1207 tissue cross-reactivity To confirm the tissue cross-reactivity of LAVA-1207 in humans, LAVA-1207 was FITC-labeled by Squarix GmbH (Marl, Germany) using a controlled reaction with NHS-coupled FITC. The average number of fluorophores conjugated per protein (F / P) was 4.3. Tissue reactivity was assessed by immunohistochemistry (IHC) using FITC-labeled LAVA-1207 in a panel of 41 different frozen normal human tissues (adrenal gland, bone marrow, breast / mammary gland, cecum, cerebellum, cerebral cortex, colon, duodenum, endothelium [vascular], eye, esophagus, fallopian tube [fallopian tube], gallbladder, heart [ventricle], ileum, jejunum, kidney [cortex], liver, lung, lymph node, muscle [striated and skeletal], peripheral nerve, ovary, pancreas, parotid gland, parathyroid gland, pituitary gland, placenta, prostate, rectum, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, tonsil, ureter, bladder, uterus [cervix], and uterus [endometrium]) and peripheral blood smears (three donors per tissue or blood smear) by Charles River Laboratories (Evreux, France). Frozen sections were air-dried, fixed in zinc formalin (4087236, Microm Microtech), and rinsed with Millipore water. Test samples were incubated with 0, 3, or 20 μg / ml FITC-labeled LAVA-1207, followed by 2 μg / ml goat α-FITC antibody (01-40-01, KPL), followed by application of antibody blocking agent (760-4204, Discovery). Detection was performed using a ChromoMab DAB kit (760-159, Discovery) according to the manufacturer's recommendations. Test samples were then stained with hematoxylin II (790-2208, Ventana) and bluing reagent (760-2037, Ventana), washed, dehydrated, and mounted. Slides were evaluated by a pathologist using a light microscope (Olympus BX51).

[0342] Labeling of LAVA-1207 with FITC did not affect the affinity of LAVA-1207 for either Vy9V52-T cells (Vy9V52-T cell binding EC50 = 1.41 nM (FITC-labeled LAVA-1207) vs. 0.97 nM (LAVA-1207)) (Figure 18A) or PSMA-expressing LNCaP cells (LNCaP binding EC50 6.00 nM (FITC-labeled LAVA-1207) vs. 6.28 nM (LAVA-1207)) (Figure 18B).

[0343]

[0333] Next, the reactivity of LAVA-1207 against a panel of 42 different frozen normal human tissues and blood smears (3 donors for each tissue / blood smear) was tested.

[0344] Moderate to prominent membrane staining (with some variable cytoplasmic staining) was observed in prostate acinar cells. Low-intensity, primarily cytoplasmic staining was observed in parotid acinar cells in 3 of 3 donors. Minimal staining of vascular and placental villi spindle cells, likely corresponding to endothelial cells, was observed in the placenta of 1 of 3 donors (representative images shown in Figure 19A). No reactivity was detected in a wide range of other tissue samples, including adrenal gland, blood smear, breast, small and large intestine, gallbladder, kidney, liver, lung, lymph node, nerve, ovary, fallopian tube, spleen, testis, thymus, tonsil, ureter, bladder, cervix, and endometrium, with the exception of occasional membrane reactivity of lymphoid cells, most likely Vγ9Vδ2 T cells (representative images shown in Figure 19B). This tissue reactivity analysis of LAVA-1207 was consistent with the known tissue distribution of PSMA expression (i.e., prostate, parotid gland, and placenta) and Vγ9Vδ2-T cells and did not reveal any unexpected reactivity.

Claims

1. 1. A method of treating cancer in a subject in need thereof, comprising administering to said subject a. a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vγ9Vδ2 T cell receptor; and b. Immune checkpoint inhibitors Administering

2. 1. A method of treating cancer in a subject in need thereof, comprising administering to said subject a. a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vγ9Vδ2 T cell receptor; and b. Common gamma chain cytokines Administering

3. 3. The method of claim 2, further comprising administering an immune checkpoint inhibitor.

4. The method of claim 1 or 3, wherein the immune checkpoint inhibitor is an anti-PD1 antibody or an anti-PDL1 antibody.

5. The method of any one of claims 1 to 3, wherein the common gamma chain cytokine is IL-2 or a variant thereof.

6. 6. The method of claim 5, wherein the IL-2 is administered at a dose of less than 3 MIU / day or less than 2 MIU / day.

7. The method of any one of claims 1 to 3, wherein the common gamma chain cytokine is IL-15.

8. 8. The method of claim 7, wherein the IL-15 is administered at a dose of 0.5 to 5 MIU / day.

9. The method of claim 8, wherein the IL-2 or IL-15 is pegylated.

10. The method of any one of claims 1 to 9, wherein the multispecific antibody is administered intravenously or subcutaneously.

11. The method of any one of claims 1 to 10, wherein IL-2 or IL-15 is administered subcutaneously.

12. The method of any one of claims 1 to 11, wherein the multispecific antibody is administered with an administration interval of 7 to 21 days.

13. The method of any one of claims 1 to 12, wherein the multispecific antibody is administered at an interval of 14 days.

14. administering the multispecific antibody a. administering one or more priming doses of said multispecific antibody; and b. Administering one or more target doses of said multispecific antibody. The method according to any one of claims 1 to 13, comprising:

15. The method of any one of claims 1 to 14, wherein the IL-2 or IL-15 is administered daily for at least one day after administration of the multispecific antibody.

16. The method of any one of claims 1 to 15, wherein the IL-2 or IL-15 is administered daily for at least 3 days following administration of the multispecific antibody.

17. The method of any one of claims 1 to 16, wherein the IL-2 or IL-15 is administered at a dose of 0.5 to 1.5 MIU / day.

18. The method of any one of claims 1 to 17, wherein the IL-2 or IL-15 is administered at a dose of 1 MIU / day.

19. 19. The method of any one of claims 1 to 18, wherein the multispecific antibody is administered at a dose of at least 1.5, 4.5, 13.5, 120, 360, 540, 800, or 1200 micrograms.

20. The method of any one of claims 1 to 19, wherein the human cancer antigen is selected from the group consisting of PSMA, CD1d, CD40, CD123, 5T4, and nectin-4.

21. The method according to any one of claims 1 to 20, wherein the multispecific antibody has the ability to bind to human Vδ2.

22. the second antigen-binding region comprises: a. a VH CDR1 sequence of SEQ ID NO: 1, a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3; b. A VH CDR1 sequence of SEQ ID NO:5, a VH CDR2 sequence of SEQ ID NO:6, and a VH CDR3 sequence of SEQ ID NO:7; c. A VH CDR1 sequence of SEQ ID NO: 10, a VH CDR2 sequence of SEQ ID NO: 11, and a VH CDR3 sequence of SEQ ID NO: 12; d. A VH CDR1 sequence of SEQ ID NO: 14, a VH CDR2 sequence of SEQ ID NO: 15, and a VH CDR3 sequence of SEQ ID NO: 16; e. the VH CDR1 sequence of SEQ ID NO: 18, the VH CDR2 sequence of SEQ ID NO: 19, and the VH CDR3 sequence of SEQ ID NO: 20; f. the VH CDR1 sequence of SEQ ID NO: 22, the VH CDR2 sequence of SEQ ID NO: 23, and the VH CDR3 sequence of SEQ ID NO: 24; or g. The VH CDR1 sequence of SEQ ID NO: 26, the VH CDR2 sequence of SEQ ID NO: 27, and the VH CDR3 sequence of SEQ ID NO: 28 The method of any one of claims 1 to 21, comprising:

23. 23. The method of any one of claims 1 to 22, wherein the second antigen-binding region comprises an amino acid sequence comprising at least 90% sequence identity to an amino acid sequence selected from SEQ ID NOs: 4, 8, 9, 13, 17, 21, 25, and 29.

24. a. the human cancer antigen is PSMA, and the first antigen-binding region comprises a VH CDR1 sequence of SEQ ID NO: 87, a VH CDR2 sequence of SEQ ID NO: 88, and a VH CDR3 sequence of SEQ ID NO: 89; b. the human cancer antigen is CD1d, and the first antigen-binding region comprises a VH CDR1 sequence of SEQ ID NO: 95, a VH CDR2 sequence of SEQ ID NO: 96, and a VH CDR3 sequence of SEQ ID NO: 97; c. the human cancer antigen is CD40, and the first antigen-binding region comprises a VH CDR1 sequence of SEQ ID NO: 99, a VH CDR2 sequence of SEQ ID NO: 100, and a VH CDR3 sequence of SEQ ID NO: 101; d. the human cancer antigen is CD123, and the first antigen-binding region comprises the VH CDR1 sequence of SEQ ID NO: 103, the VH CDR2 sequence of SEQ ID NO: 104, and the VH CDR3 sequence of SEQ ID NO: 105; e. the human cancer antigen is Nectin-4, and the first antigen-binding region comprises a VH CDR1 sequence selected from SEQ ID NOs: 107, 11, 115, 119, 123, 127, and 131, a VH CDR2 sequence selected from SEQ ID NOs: 108, 112, 116, 120, 124, 128, and 132, and a VH CDR3 sequence selected from SEQ ID NOs: 109, 113, 117, 121, 125, 129, and 133; or f. The method of any one of claims 1 to 23, wherein the human cancer antigen is 5T4 and the first antigen-binding region comprises the VH CDR1 sequences of SEQ ID NOs: 135, 139, 143, 147, 151, 155, and 159, the VH CDR2 sequences of SEQ ID NOs: 136, 140, 144, 148, 152, 156, and 160, and the VH CDR3 sequences of SEQ ID NOs: 137, 141, 145, 149, 153, 157, and 161.

25. a. the human cancer antigen is PSMA and the first antigen-binding region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 90; b. the human cancer antigen is CD1d, and the first antigen-binding region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 98; c. the human cancer antigen is CD40 and the first antigen-binding region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 102; d. the human cancer antigen is CD123, and the first antigen-binding region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 106; e. the human cancer antigen is Nectin-4, and the first antigen-binding region comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NOs: 110, 114, 118, 122, 126, 130, and 134; or f. The method of any one of claims 1 to 24, wherein the human cancer antigen is 5T4 and the first antigen-binding region comprises an amino acid sequence having at least 90% sequence identity to a sequence selected from SEQ ID NOs: 138, 142, 146, 150, 154, 158, and 162.

26. The method of any one of claims 1 to 25, wherein the multispecific antibody comprises an Fc region.

27. 27. The method of claim 26, wherein the Fc region is a heterodimeric Fc region comprising a first and a second Fc monomer polypeptide.

28. 28. The method of claim 27, wherein (a) the first Fc polypeptide comprises the sequence set forth in SEQ ID NO: 345 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO: 346, or (b) the first Fc polypeptide comprises the sequence set forth in SEQ ID NO: 346 and the second Fc polypeptide comprises the sequence set forth in SEQ ID NO:

345.

29. 29. The method of any one of claims 1 to 28, wherein the multispecific antibody is formulated at a strength of 1 mg / mL in 10 mM histidine, 1 mM methionine, 280 mM sucrose, 0.02% polysorbate 80, pH 6.

0.

30. 30. The method of any one of claims 1 to 29, wherein the cancer is (i) prostate cancer, such as non-metastatic or metastatic prostate cancer, e.g., metastatic castration-resistant prostate cancer, such as treatment-refractory metastatic castration-resistant prostate cancer; (ii) a cancer in which PSMA is expressed in tumor neovasculature or tumor-associated endothelial cells of primary or metastatic tumors, including those from colorectal cancer, lung cancer, breast cancer, endometrial and ovarian cancer, gastric cancer, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, oral squamous cell carcinoma, thyroid tumors, and glioblastoma; or (iii) adenoid cystic carcinoma of the head and neck.

31. The cancer is (i) a hematological malignancy such as T-cell lymphoma, multiple myeloma, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, B-cell lymphoma, smoldering myeloma, Hodgkin's lymphoma, myelomonocytic leukemia, lymphoplasmacytic lymphoma, hairy cell leukemia, or splenic marginal zone lymphoma, or (ii) renal cell carcinoma, melanoma, colorectal cancer, head and neck cancer, lung cancer, The method of any one of claims 1 to 30, wherein the cancer is a solid tumor cancer such as pancreatic cancer, gastroesophageal cancer, small intestine cancer, central nervous system tumor, medulloblastoma, hepatocellular carcinoma, glioma, neuroblastoma, urothelial carcinoma, bladder cancer, sarcoma, penile cancer, basal cell carcinoma, Merkel cell carcinoma, neuroendocrine carcinoma, neuroendocrine tumor, carcinoma of unknown primary (CUP), thymoma, vulvar cancer, cervical cancer, testicular cancer, bile duct cancer, appendix cancer, mesothelioma, ampullary cancer, anal cancer, or choriocarcinoma.

32. 32. The method of any one of claims 1 to 31, wherein the cancer is non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, pancreatic cancer, lung cancer, colon cancer, B-cell lymphoma / leukemia, Burkitt's lymphoma, or B-cell acute lymphoblastic leukemia.

33. 33. The method of any one of claims 1 to 32, wherein the cancer is B-cell acute lymphoblastic leukemia, blastic plasmacytic dendritic cell neoplasm, chronic myeloid leukemia, B-cell chronic lymphoproliferative disorder, or myelodysplastic syndrome.

34. 34. The method of any one of claims 1 to 33, wherein the cancer is bladder cancer, kidney cancer, esophageal cancer, lung cancer, pancreatic cancer, thyroid cancer, colorectal cancer, bile duct cancer, or uterine endometrial cancer.

35. 35. The method of any one of claims 1 to 34, wherein the cancer is bladder cancer, cervical cancer, non-small cell lung cancer, mesothelioma, squamous cell carcinoma of the head and neck, glioblastoma multiforme, esophageal cancer, pancreatic cancer, colorectal cancer, uterine cancer, renal cancer, esophageal cancer, or pre-B cell acute lymphoblastic leukemia.

36. 1. A kit for treating cancer, comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vγ9Vδ2 T-cell receptor; and (b) IL-2 and / or an anti-PD1 or anti-PD-L1 antibody at a dose of less than 3 MIU, and optionally instructions for use.

37. 1. A kit for treating cancer, comprising: (a) a multispecific antibody comprising a first antigen-binding region capable of binding to a human cancer antigen and a second antigen-binding region capable of binding to a human Vγ9Vδ2 T-cell receptor; and (b) IL-2 and / or an anti-PD1 or anti-PD-L1 antibody at a dose of less than 3 MIU, and optionally instructions for use.

38. 38. The kit of claim 37, further comprising a solvent for diluting the multispecific antibody, the dose of IL-2 and / or IL-15, and / or the anti-PD1 or anti-PD-L1 antibody.

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