Multispecific antibody that binds to both MAIT and tumor cells

Abstract

The present invention provides multispecific molecules capable of simultaneously binding to mucosal-associated invariant T (MAIT) cells and tumor cells, the multispecific molecules comprising at least one domain that specifically binds to the Vα7.2 T cell receptor (TCR) and at least one domain that specifically binds to a tumor-associated antigen (TAA).

Description

[Technical Field]

[0001] The present invention provides multispecific molecules that are useful for treating cancer. [Background technology]

[0002] T cell redirection approaches using bispecific antibodies (BsAbs) have led to significant advances in cancer immunotherapy. Two T cell-redirecting BsAbs have received regulatory approval: catumaxomab for the treatment of malignant ascites and blinatumomab for acute lymphoblastic leukemia. Many others are in clinical trials.

[0003] The recognized mechanism of action underlying T cell-redirecting BsAbs is through the formation of an immunological synapse (Offner et al., 2006; Nagorsen et al., 2011). This BsAb-mediated crosslinking of the CD3 receptor and tumor-associated antigens (TAA) on target cells leads to T cell activation, followed by the release of perforin and granzymes from cytotoxic granules into the environment of the immunological synapse, and the eventual destruction of the target cell by apoptosis. In the case of bispecific T cell induction (BiTE), the formed immunological synapse appears indistinguishable from that induced during the recognition of natural cytotoxic T cells (Offner et al., 2006). Because delivery of these apoptosis mediators is achieved by passive diffusion, the size of the synapse, defined by the distance between the anti-CD3 and anti-TAA moieties of the BsAb, is crucial for cytotoxic efficacy. The distance between the TAA epitope and the target cell membrane determines the activity of BiTEs and may explain the differences in reported cytotoxic activity between different T cell-redirecting BsAb formats, confirming that tumor cell lysis is most efficient when the two cell membranes are in closest proximity.

[0004] Furthermore, activated T cells produce interleukin (IL)-2 and interferon (IFN)-γ, which promote their proliferation and expansion at tumor sites, making them the most potent mediators of the immune response. CD8+ cells proliferate most rapidly and exert their cytotoxic activity on target cells; however, CD4+ cells contribute equally to the observed cytotoxicity, although this begins with a short delay.

[0005] The selection of optimal TAA for use in T cell redirecting mechanisms is challenging. Due to the high cytotoxicity of T cells, the therapeutic window of T cell redirecting approaches is quite narrow. Application to the treatment of solid tumors is challenging due to increased toxicity, primarily due to the widespread expression of selected tumor-associated antigens in healthy cells and tissues (on-target off-tumor effects).

[0006] Current T cell redirecting BsAbs target CD3 and therefore recruit all CD3+ T cells at the tumor site, including CD8+ (which is the major effector cell population mediating target cell killing), CD4+ (which can cause a cytokine storm, one of the major side effects of this therapy), and unwanted Tregs (which, when localized in the target tissue, reduce the immune response and suppress CD8+ effector cells by secreting immunosuppressive cytokines and activating inhibitory pathways of CTLs) (Koristka S, et al., 2012; Koristka et al., 2013). Several groups have reported that isolated Tregs can promote cytotoxic activity (Choi et al., 2013). However, it has also been demonstrated that the presence of Tregs promotes in vivo tumor growth during treatment with a T cell-redirecting BsAb (PSCA / CD3) targeting prostate hepatocyte antigen in a xenograft model (Koristka et al., 2012). Although one report showed that no Treg expansion was observed in human ex vivo studies of CD33 / CD3 T cell-redirecting BsAb (Krupka et al., 2014), redirection of CTLs alone may provide therapeutic benefit and further enhance the clinical efficacy of this class of drug. To this end, one study (Michalk et al., 2014) demonstrated that a PSCA / CD8 BiTE molecule can elicit potent antitumor responses, even though preactivated CD8+ T cells only displayed cytotoxicity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2008 / 087219 [Patent Document 2] International Publication No. 2007 / 147901 [Patent Document 3] US Patent No. 2014 / 0200331 [Patent Document 4] US Patent No. 2014 / 150973 [Patent Document 5] US Patent No. 2014 / 0154254 Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, new approaches for more efficient and safer T cell redirection are needed. [Means for solving the problem]

[0009] The present invention provides a T cell redirection approach that targets immune cells bearing invariant / semi-invariant T cell receptors (TCRs), such as mucosal-associated invariant T (MAIT) cells, and redirects these specific T cells to kill tumor cells.

[0010] More particularly, the present invention provides multispecific molecules capable of simultaneously binding to MAIT cells and tumor cells, the multispecific molecules comprising at least one anti-Vα7.2 domain, i.e., a domain that specifically binds to the Vα7.2 TCR, and at least one anti-tumor-associated antigen domain (TAA), i.e., a domain that specifically binds to a TAA.

[0011] According to the present invention, cross-linking of T cell receptors with the multispecific molecules activates MAIT cells to kill tumor cells. See Figure 1.

[0012] This approach has the advantages of a) activating only cytotoxic cells against target cells, b) not activating CD4+ T cells, thus reducing the risk of cytokine storm and autoreactivity, and c) not redirecting Tregs to the tumor site. Furthermore, MAIT cells are abundant in human peripheral tissues, particularly the liver and mucosal tissues such as the lung and intestine, favoring their migration to solid tumors.

[0013] The molecule is preferably a multispecific, preferably bispecific, antibody or antigen-binding fragment thereof. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing how bispecific antibodies according to the invention target the TAA and invariant TCR, alpha chain Vα7.2, on the surface of tumor cells. [Figure 2]FIG. 2 is a schematic diagram of an example of an antibody of the present invention. [Figure 3] Figure 3A shows the binding profile of anti-Vα7.2 / anti-CD19 Fab-Fab on CD19+ Raji cells, representative of four independent experiments, and Figure 3B shows the binding profile of anti-Vα7.2 / anti-CD19 Fab-Fab on Vα7.2+ cells, representative of three different donors. [Figure 4] Figure 4A shows the flow cytometry gating strategy for determining CD8+ T cell activation. Figure 4B shows the percentage of CD25+, CD69+, and double-positive (CD25+CD69+) CD8+TCRγδ- T cells in wells coated with different molar concentrations of either anti-Vα7.2 / anti-CD19 Fab-Fab, anti-CD3, or anti-Vα7.2, representing two donors. [Figure 5] Figure 5A shows the flow cytometry gating strategy for determining MAIT cell activation. Figure 5B shows the percentage of CD25+, CD69+, and double-positive (CD25+CD69+) MAIT (CD8+TCRγδ-CD161hiIL18RA+) cells in wells coated with different molar concentrations of either anti-Vα7.2 / anti-CD19 Fab-Fab, or anti-CD3 or anti-Vα7.2, representing two donors. [Figure 6] FIG. 6 shows the percentage of specific lysis of Raji cells when co-cultured for 48 hours with different concentrations of anti-Vα7.2 / anti-CD19 Fab-Fab and different effector:target ratios. [Figure 7]Figure 7A shows the binding profiles of anti-Vα7.2 / anti-CD19 Fab-Fab and anti-CD19 / anti-Vα7.2 Fab-Fab antibodies, as well as the binding profile of a negative control Fab-Fab antibody, on CD19+ NALM-6 tumor cells. Median values ​​from three independent experiments are shown. Figure 7B shows the binding profiles of anti-Vα7.2 / anti-CD19 BiXAb and anti-CD19 / anti-Vα7.2 BiXAb antibodies, as well as the binding profile of a negative control BiXAb antibody, on CD19+ NALM-6 tumor cells. Median values ​​from three independent experiments are shown. [Figure 8] Figure 8 shows the flow cytometry gating strategy for determining MAIT cell binding within CD8+ enriched cells. One representative experiment is shown for the Vα7.2 / CD19 BiXAb antibody. [Figure 9] Figure 9 shows the binding profiles of anti-Vα7.2 / anti-CD19 BiXAb and anti-CD19 / anti-Vα7.2 BiXAb antibodies, as well as the binding profile of a negative control BiXAb antibody, on Vα7.2+ CD8+ MAIT cells. Median values ​​from three independent experiments are shown. [Figure 10] The percentage of CD69+ MAIT cells in wells coated with anti-Vα7.2 / anti-CD19 BiXAb or anti-CD19 / anti-Vα7.2 BiXAb antibodies, or negative control BiXAb antibodies, is shown. One representative experiment out of two independent experiments is shown. [Figure 11] FIG. 11 is a schematic diagram of the cytotoxicity assay. [Figure 12] Figure 12 shows the percentage of CD69+ MAIT cells during a cytotoxicity assay using enriched CD8 T cells and A-549 tumor cells in the presence of anti-Vα7.2 / anti-CD19 BiXAb or anti-CD19 / anti-Vα7.2 BiXAb antibodies, or negative control BiXAb antibodies. One representative experiment of two independent experiments is shown. [Figure 13]Figure 13 shows the percentage of specific lysis of A-549 tumor cells in the presence of anti-Vα7.2 / anti-CD19 Fab-Fab or anti-CD19 / anti-Vα7.2 Fab-Fab antibodies, or a negative control Fab-Fab antibody, when co-cultured with CD8+ T cells for 48 hours in assay medium containing rhIL-12. Assays were performed at an effector:target ratio of 6:1. One representative experiment of two independent experiments is shown. [Figure 14] Figure 14A shows the binding profiles of the anti-Her2 / anti-Vα7.2 Fab-Fab antibody and the negative control Fab-Fab antibody on Her2+ A-549 tumor cells. Median values ​​from three independent experiments are shown. Figure 14B shows the binding profiles of the anti-Vα7.2 / anti-Her2 BiXAb and anti-Her2 / anti-Vα7.2 BiXAb antibodies, as well as the negative control BiXAb antibody, on Her2+ A-549 tumor cells. Median values ​​from three independent experiments are shown. [Figure 15] Figure 15 shows the binding profiles of anti-Vα7.2 / anti-Her2 BiXAb and anti-Her2 / anti-Vα7.2 BiXAb antibodies, as well as the binding profile of a negative control BiXAb antibody, on Vα7.2+ CD8+ MAIT cells. Median values ​​from three independent experiments are shown. [Figure 16] Figure 16A shows the percentage of double-positive CD69+CD25+ MAIT cells in a cytotoxicity assay using A-549 tumor cells and anti-Vα7.2 / anti-Her2 Fab-Fab or anti-Her2 / anti-Vα7.2 Fab-Fab antibodies, or a negative control Fab-Fab antibody. One representative experiment of three independent experiments is shown. Figure 16B shows the percentage of CD69+ MAIT cells in a cytotoxicity assay using A-549 tumor cells and anti-Vα7.2 / anti-Her2 BiXAb or anti-Her2 / anti-Vα7.2 BiXAb antibodies, or a negative control BiXAb antibody. One representative experiment of three independent experiments is shown. [Figure 17]Figure 17A shows the percentage of specific lysis of A-549 tumor cells in the presence of anti-Vα7.2 / anti-Her2 Fab-Fab or anti-Her2 / anti-Vα7.2 Fab-Fab antibodies, or a negative control Fab-Fab antibody, when co-cultured with CD8+ T cells in assay medium containing rhIL-12 for 48 hours. The assay was performed at an effector:target ratio of 6:1. One representative experiment of three independent experiments is shown. Figure 17B shows the percentage of specific lysis of A-549 tumor cells in the presence of anti-Vα7.2 / anti-Her2 BiXAb or anti-Her2 / anti-Vα7.2 BiXAb antibodies, or a negative control BiXAb antibody, when co-cultured with CD8+ T cells in assay medium containing rhIL-12 for 48 hours. The assay was performed at an effector:target ratio of 6:1. One representative experiment of three independent experiments is shown. [Figure 18] Figure 18 shows the binding profiles of anti-Vα7.2 / anti-EGFR BiXAb, anti-EGFR / anti-Vα7.2 BiXAb, and negative control BiXAb antibodies on EGFR+ A-549 tumor cells. Median values ​​from two independent experiments are shown. [Figure 19] 19 shows the binding profiles of anti-Vα7.2 / anti-EGFR BiXAb, anti-EGFR / anti-Vα7.2 BiXAb, and negative control BiXAb antibodies on Vα7.2+ CD8+ MAIT cells. Median values ​​from two independent experiments are shown. [Figure 20] FIG. 20 is a schematic diagram of the in vivo experimental design. [Figure 21]Figure 21A shows the in vivo efficacy of anti-Vα7.2 / anti-CD19 Fab-Fab or anti-Vα7.2 / anti-HER2 Fab-Fab antibodies in NSG mice; animals were inoculated with an A-549 / luciferase tumor cell line expressing HER2 and CD19 on day 0, followed by PBMCs on days 1 and 4. Data are reported as the average bioluminescence signal from each mouse. Figure 21B shows the in vivo efficacy of anti-Vα7.2 / anti-CD19 BiXAb or anti-Vα7.2 / anti-HER2 BiXAb antibodies in NSG mice; animals were inoculated with an A-549 / luciferase tumor cell line expressing HER2 and CD19 on day 0, followed by PBMCs on days 1 and 4. Data are reported as the average bioluminescence signal from each mouse. DETAILED DESCRIPTION OF THE INVENTION

[0015] definition The basic structure of a naturally occurring antibody molecule is a Y-shaped tetramer of four identical heavy chains and two identical light chains held together by noncovalent interactions and interchain disulfide bonds.

[0016] In mammalian species, there are five types of heavy chains: α, δ, ε, γ, and μ, which determine the immunoglobulin class (isotype): IgA, IgD, IgE, IgG, and IgM, respectively. The N-terminal variable domain (VH) of the heavy chain is followed by a constant region, which contains three domains (numbered CH1, CH2, and CH3 from N-terminus to C-terminus) in the γ, α, and δ heavy chains, while the constant region of the μ and ε heavy chains is composed of four domains (numbered CH1, CH2, CH3, and CH4 from N-terminus to C-terminus). The CH1 and CH2 domains of IgA, IgG, and IgD are separated by a flexible hinge, which varies in length among different classes; in the case of IgA and IgG, among different subtypes, IgG1, IgG2, IgG3, and IgG4 have hinges of 15, 12, 62 (or 77), and 12 amino acids, respectively, and IgA1 and IgA2 have hinges of 20 and 7 amino acids, respectively.

[0017] There are two types of light chains: lambda and kappa, which can combine with any heavy chain isotype, but which are both of the same type in a given antibody molecule. Both light chains appear to be functionally identical. Their N-terminal variable domain (VL) is followed by a constant region consisting of one domain, designated CL.

[0018] Heavy and light chains pair through protein / protein interactions between the CH1 and CL domains and between the VH and VL domains, and the two heavy chains are bound by protein / protein interactions between their CH3 domains.

[0019] The antigen-binding regions corresponding to the arms of the Y-shaped structure, each consisting of an intact light chain paired with the VH and CH1 domains of a heavy chain, are called Fab fragments (for fragment antigen binding). Fab fragments are produced from native immunoglobulin molecules by papain digestion, which first cleaves the antibody molecule at the hinge region, amino-terminal to the interchain disulfide bond, releasing two identical antigen-binding arms. Other proteases, such as pepsin, also cleave antibody molecules at the hinge region, but carboxy-terminal to the interchain disulfide bond, releasing a fragment consisting of two identical Fab fragments, still linked via disulfide bonds; reduction of the disulfide bond in the F(ab')2 fragment produces Fab' fragments.

[0020] The part of the antigen-binding region corresponding to the VH and VL domains is called the Fv fragment (for Fragment variable); it contains the CDRs (complementarity-determining regions) and forms the antigen-binding site (also called the paratope).

[0021] The effector region of an antibody, responsible for binding to effector molecules on immune cells, corresponds to the stem of the Y-shaped structure and comprises the paired CH2 and CH3 domains of the heavy chain (or CH2, CH3 and CH4 domains, depending on the type of antibody) and is called the Fc (for Fragment crystallisable) region.

[0022] Due to the identity of the two heavy chains and the two light chains, a naturally occurring antibody molecule has two identical antigen-binding sites and therefore binds simultaneously to two identical epitopes.

[0023] In the context of the present invention, a "multispecific antigen-binding fragment" is defined herein as a molecule having two or more antigen-binding regions, each recognizing a different epitope. The different epitopes can be carried by the same or different antigen molecules. The terms "recognizing" or "recognizes" mean that the fragment specifically binds to the target antigen.

[0024] An antibody "specifically binds" to a target antigen if it binds with greater affinity, avidity, more readily, and / or with greater persistence than it binds to other substances. "Specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding implies preferential binding. Preferably, the molecule exhibits no significant binding (e.g., about 100-fold lower affinity) to ligands other than its specific target, i.e., minimal cross-reactivity.

[0025] "Affinity" is defined as the strength of the binding interaction between two molecules, e.g., an antigen and an antibody, and it is defined as the strength of binding of a ligand at one defined binding site to an antibody and other molecules that have more than one binding site. While non-covalent binding of a ligand to an antibody is typically not as strong as covalent binding, "high affinity" is defined as a strength of about 10 6 to 10 11 M -1 of interest is a ligand that binds to an antibody with an affinity constant (Ka) of

[0026] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or treated. The subject can be a human, but can also be other mammals, particularly mammals useful as research models for human disease, such as mice, rats, rabbits, dogs, etc.

[0027] The term "treatment" or "treating" refers to a procedure, application, or therapy in which a subject, including a human, receives medical assistance for the purpose of directly or indirectly improving the subject's condition. In particular, the term, in some embodiments, refers to reducing the occurrence or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing the occurrence, improving symptoms, improving prognosis, or a combination thereof. One of skill in the art will understand that treatment does not necessarily result in the complete absence or elimination of symptoms. For example, with respect to cancer, "treatment" or "treating" can refer to slowing the growth, proliferation, or metastasis of neoplastic or malignant cells, preventing or delaying the development of neoplastic or malignant cell growth, proliferation, or metastasis, or some combination thereof.

[0028] Mucosal-associated invariant T (MAIT) cells are non-conventional T cells not restricted by standard MHC and found in blood vessels and tissues, where they contribute to the immune barrier. They have the ability to redirect cytotoxicity based on expression studies (Salou et al., 2019) and in vitro assays (Le Bourhis et al., 2013). MAIT cells express a semi-invariant TCR (called Vα7.2) that recognizes the vitamin B2 precursor expressed by MR1, a highly evolutionarily conserved MHC class Ib molecule (Franciszkiewicz et al., 2016; Salou et al., 2017). This receptor is also designated TRAV1 / TRAJ in the WHO-IUIS nomenclature for the T cell receptor (TCR) gene division of the immune system, as reported in Bull World Health Organ. 1993; 71(1): 113-115.

[0029] MAIT cells represent approximately 1 to 10% of T cells in the blood, but are also present in tissues and organs such as the lung, liver, skin, and colon. Furthermore, MAIT cells possess cytotoxic activity and can produce IFNγ and TNFα upon activation (Dusseaux et al., 2011).

[0030] [Vα7.2] is the α chain of the T cell receptor expressed by MAIT cells. This term includes the α chains Vα7.2-Jα33, Vα7.2-Jα20, or α7.2-Jα12. In humans, they consist of TRAV1-2 linked to TRAJ33, TRAJ20, or TRAJ12 with no or few n nucleotide additions at the TCR-α complementarity-determining region 3 (CDR3α) junction. As used herein, "Vα7.2-Jα33 / 20 / 12" includes any variant, derivative, or rearranged isoform of the Vα7.2-Jα33 / 20 / 12 gene or encoded protein. The amino acid sequences of human and mouse Vα7.2-Jα33 are described in Tilloy et al., 1999, while Vα7.2-Jα20 and Va7.2-Jα12 are described in Reantragoon et al., 2013. The sequence of human Vα7.2-Jα33 is set forth in SEQ ID NO: 1. The sequence of Vα7.2-Jα12 is set forth in SEQ ID NO: 2, and Jα20 is set forth in SEQ ID NO: 3.

[0031] The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0032] The term "tumor" is used interchangeably with the term "cancer" herein, e.g., both terms encompass solid and liquid, e.g., spreading or circulating, tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0033] As used herein, the term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, sugar, lipid, or combination thereof) that is expressed on the surface of a cancer cell (or is overexpressed relative to normal tissue), either entirely or as a fragment (e.g., MHC / peptide). As used herein, the term "cancerous cell" refers to a cell that is undergoing or has undergone uncontrolled proliferation. In some embodiments, a TAA is a marker expressed by both normal cells and cancer cells, such as CD19, described in more detail below. In some embodiments, a TAA is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., two-fold overexpression, three-fold overexpression, or more compared to normal cells / tissues. In some embodiments, a TAA is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule with a deletion, addition, or mutation compared to the molecule expressed in normal cells (e.g., EGFRvIII). In some embodiments, a TAA, either entirely or as a fragment (e.g., MHC / peptide), is expressed exclusively on the cell surface of cancer cells and is not normally synthesized or expressed on the surface of cells. Thus, the term "TAA" refers to a cellular antigen specific to cancer cells, sometimes known in the art as a tumor-specific antigen ("TSA").

[0034] Anti-Vα7.2 domain The multispecific molecules of the invention comprise at least one domain that binds to Vα7.2, such as Vα7.2-Jα33, Vα7.2-Jα20 and / or Vα7.2-Jα12.

[0035] The binding domain can be derived from any anti-Vα7.2 antibody. Methods for producing such antibodies are known in the art. Examples of such antibodies are published in WO 2008 / 087219.

[0036] It will be understood that the multispecific molecules of the present invention can recognize any site of a Vα7.2-Jα33, Vα7.2-Jα20, and / or Vα7.2-Jα12 polypeptide, e.g., a Vα7.2-Jα33 / Vβ2 or Vα7.2-Jα33 / Vβ2 polypeptide. For example, Voc7, Voc7.2, Joc33, fragments thereof, or any combination of any of these polypeptides or fragments can be used as an immunogen to generate antibodies, and the antibodies of the present invention can recognize an epitope anywhere within the Vα7.2-Joc33 (or, for example, Vα7.2-Jα33 / Vβ2 or Vα7.2-Jα33 / Vβ2) polypeptide. Preferably, the recognized epitopes are present on the cell surface, i.e., they are accessible to antibodies present outside the cell.

[0037] In certain embodiments, the Vα7.2-binding domain is an antigen-binding fragment derived from an anti-Vα7.2 antibody that can compete with the monoclonal antibody 3C10 described in WO 2008 / 087219 or that can bind to the same or substantially the same epitope of the Vα7.2-Jα33 polypeptide as said antibody. When an antibody or agent is said to "compete" with or "bind to substantially the same epitope" as a particular monoclonal antibody (e.g., 3C10), it means that the antibody or agent competes with the monoclonal antibody in a binding assay using either a recombinant Vα7.2-Joc33 molecule or a surface-expressed Vα7.2-Joc33 molecule. For example, if a test antibody or agent reduces the binding of 3C10 to the Vα7.2-Joc33 polypeptide in a binding assay, the antibody or agent is said to "compete" with 3C10 or 1A6, respectively.

[0038] In certain embodiments, the multispecific molecules of the invention comprise: The following CDRs of the 3C10 antibody: GFNIKDTH (SEQ ID NO: 4); TDPASGDT (SEQ ID NO: 5) and CAHYYRDDVNYAMDY (SEQ ID NO: 6) and / or a heavy chain variable chain comprising The following CDRs of the 3C10 antibody: QNVGSN (SEQ ID NO: 7); SSS, and QQYNTYPYT (SEQ ID NO: 8) The light chain variable chain comprises:

[0039] Anti-TAA domain The multispecific molecules of the invention comprise at least one domain that binds to a TAA, particular examples of which include CD19, CD20, CD38, EGFR, HER2, VEGF, CD52, CD33, RANK-L, GD2, CD33, the CEA family (including CEACAM antigens, e.g., CEACAM1, CEACAM5; or PSG antigens), MUC1, PSCA, PSMA, GPA33, CA9, PRAME, CLDN1, HER3, and glypican-3, as well as CD22, CD25, CD40, CD30, CD79b, CD138 (syndecan-1), BCMA, SLAMF7 (CS1, CD319), CD56, CCR4, EpCAM, PDGFR-α, Apo2L / TRAIL, and PD-L1.

[0040] CD19, EGFR, and HER2 are particularly preferred.

[0041] The multispecific antibodies that bind to CD19, EGFR or HER2 are described in more detail below.

[0042] Generally speaking, any person skilled in the art knows how to produce antibodies that specifically bind to any of the above-mentioned TAAs. Many are commercially available.

[0043] In a preferred embodiment, the multispecific molecules of the invention comprise humanized or chimeric antigen-binding fragments.

[0044] Multispecific antibody design Provided herein are multispecific antigen-binding fragments and multispecific antibody constructs comprising said fragments, each multispecific antigen-binding fragment consisting essentially of tandemly arranged Fab fragments.

[0045] The fragments and constructs preferably comprise chains derived from a human immunoglobulin, preferably IgG, more preferably IgG1.

[0046] In the case of multispecific antigen-binding fragments comprising more than two different Fab fragments, the polypeptide linkers separating the Fab fragments can be the same or different.

[0047] According to preferred embodiments, there is provided a multispecific antibody comprising two identical antigen-binding arms, each consisting of a multispecific antigen-binding fragment as defined above. The antigen-binding arms can be connected to each other in a variety of ways.

[0048] If one wishes to obtain an antibody without Fc-mediated effects or a monovalent antibody for each of two target antigens, the antibody will not contain an Fc region at all. In this case, the two antigen-binding arms would be, for example: - by homodimerization of the antigen-binding arms via interchain disulfide bonds provided by the polypeptide linker separating the Fab fragments; and / or - via the addition at the C-terminal end of each antigen-binding arm of a polypeptide extension comprising cysteine ​​residues resulting in the formation of interchain disulfide bonds and homodimerization of said polypeptide extension resulting in a hinge-like structure (by way of non-limiting example, said polypeptide extension can be, for example, the hinge sequence of IgG1, IgG2 or IgG3); - connecting the C-terminal ends of the heavy chains of the two antigen-binding arms to form one polypeptide chain, via a linker, preferably a semi-rigid linker, that maintains the antigen-binding arms at a sufficient distance from each other; They can be connected to each other.

[0049] Alternatively, if effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent phagocytosis (ADP), or bivalent binding to each of two antigens, are desired, the multispecific antibodies of the invention can further comprise an Fc domain that provides these effector functions. The choice of Fc domain will depend on the type of effector function desired.

[0050] In this case, the multispecific antibody of the invention has an immunoglobulin-like structure and is - two identical multispecific antigen-binding arms as defined above; - dimerized CH2 and CH3 domains of immunoglobulins; - either an IgA, IgG, or IgD hinge region that connects the C-terminal end of the CH1 domain of the antigen-binding arm to the N-terminal end of the CH2 domain (or alternatively, when the CH4 domain following the CH3 domain is from IgM or IgE, the C-terminal end of the CH1 domain of the antigen-binding arm can be connected directly to the N-terminal end of the CH2 domain); Includes:

[0051] Preferably, the CH2 and CH3 domains, the hinge region and / or the CH4 domain are derived from the same immunoglobulin or an immunoglobulin of the same isotype and subclass as the CH1 domain of the antigen-binding arm.

[0052] The CH2, CH3, and optionally CH4 domains and hinge region from a native immunoglobulin can be used. If desired, they can also be mutated, for example, to adjust the effector functions of the antibody. In some instances, all or part of the CH2 or CH3 domain can be deleted.

[0053] The present invention more particularly provides bispecific tetravalent antibodies comprising two binding sites for each of the targets and a functional Fc domain that provides effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and phagocytosis activation. Preferred antibodies are full-length antibodies. However, preferred antibodies carry mutations in the Fc domain to avoid or reduce binding to Fc gamma receptors.

[0054] The antibody preferably comprises heavy and light chains derived from a human immunoglobulin, preferably IgG, more preferably IgG1.

[0055] The light chains can be lambda or kappa light chains; they are preferably kappa light chains.

[0056] In a preferred embodiment, the linker joins IgG Fab domains in a tetra-Fab bispecific antibody format, the amino acid sequence of which comprises at least two Fab domains connected by said polypeptide linker, followed by a natural hinge sequence, followed by an IgG Fc sequence, a heavy chain sequence which is co-expressed with an appropriate IgG light chain sequence.

[0057] An example of an antibody of the invention, termed a BiXAb antibody and having an IgG-like structure, is illustrated in FIG.

[0058] In certain embodiments, the bispecific antibody of the invention comprises: - Fc (hinge-CH2-CH3) followed by - the Fab heavy chain (CH1-VH) of antibody 1 and the Fab heavy chain (CH1-VH) of antibody 2 (the latter connected by a polypeptide linker sequence, such as a linker described in more detail below); a continuous heavy chain consisting of - during protein expression, the resulting heavy chains dimerize with the co-expressed Antibody 1 and Antibody 2 light chains (VL-CL) associating with their cognate heavy chains to form the final tandem F(ab)'2-Fc molecule; Antibody 1 (Ab1) and antibody 2 (Ab2) are different.

[0059] In a preferred embodiment, a) a Fab fragment containing the CH1 and C-Kappa domains derived from human IgG1 / Kappa and the VH and VL domains of Ab1; b) CH1 and C-Kappa domains derived from human IgG1 / Kappa and VH and VL domains of Ab2 a Fab fragment having c) a mutated light chain CL constant domain derived from the human Kappa constant region d) Mutated heavy chain CH1 constant domain The present invention describes a bispecific antibody comprising two Fab fragments having CH1 and CL domains, consisting of: The Fab fragments are in the following order: - the C-terminal end of the CH1 domain of the Ab1 Fab fragment connected to the N-terminal end of the VH domain of the Ab2 Fab fragment through a polypeptide linker - the hinge region of human IgG1, which connects the C-terminal end of the CH1 domain of the Ab2 fragment to the N-terminus of the CH2 domain - the dimerized CH2 and CH3 domains of human IgG1, preferably with one or more mutations that reduce or eliminate interaction with Fc gamma receptors. and Fab fragments arranged in tandem.

[0060] According to the present invention, Ab1 and Ab2 are independently antibodies that specifically bind to Vα7.2 (e.g., antibodies described in more detail above) and antibodies that specifically bind to a tumor-associated antigen, or vice versa.

[0061] A preferred construct of the invention is a multispecific antigen-binding fragment Fab-Fab, which does not contain an Fc domain. A particular Fab-Fab construct according to the invention is described in Example 1.

[0062] The Fab-Fab constructs typically contain two different Fab domains, which have the same light chains as in the corresponding BiXAb antibody; however, the heavy chains of the Fab-Fab are shortened in this manner so that their most C-terminal residue is cysteine-220 (in EU numbering).

[0063] The association of the Fab domains is achieved through the natural pairing of the light and heavy chains without the use of a peptide linker.

[0064] To maximize the propensity for cognate pairing between the light and heavy chains, one can consider introducing mutations at the light and heavy chain interface (CL / CH1 interface) in the Fab fragment.

[0065] In a preferred embodiment, each CH1 domain has at least one mutation and each CL1 domain also has at least one mutation, the mutations being selected to improve correct homologous pairing of the CH1 and CL1 domains.

[0066] These mutations are listed below: - de novo introduced ion pairs or mutations that result in oppositely charged natural ion pairs already present at the interface of the heavy and light chains of the Fab fragment; - "knobs-into-holes" mutation; - Mutations that reshape the surfaces of the constant regions facing the heavy and light chain interface in the Fab fragment, changing them from highly polar to highly hydrophobic, or vice versa can be selected from:

[0067] Several sets of mutations are therefore suitable, as described in more detail below.

[0068] In particular, throughout this description, the amino acid sequences and sequence position numbers used herein for the CH1 and CL domains are defined by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0069] Residues that may be mutated in the VL domain may, for example, be selected from the group consisting of D1, W36, Q38, A43, P44, T85, F98 and Q100 (e.g. Q100C).

[0070] Residues that may be mutated in the CL kappa domain may, for example, be selected from the group consisting of S 114, F 116, F 118, E 123 (e.g., E123K), Q 124, T 129, S 131, V 133, L 135, N 137, Q 160, S 162, S 174, S 176, T 178, and T 180.

[0071] Residues that may be mutated in the CL lambda domain may, for example, be selected from the group consisting of S 114, T 116, F 118, E 123, E 124, K 129, T 131, V 133, L 135, S 137, V 160, T 162, A 174, S 176, Y 178 and S 180.

[0072] Residues that may be mutated in the VH domain may for example be selected from the group consisting of V 37, Q 39, G 44 (eg G44C), R 62, F 100, W 103 and Q 105.

[0073] Residues that may be mutated in the CH1 domain may be selected from the group consisting of L 124, A 139, L 143, D 144, K 145, D 146, H 172, F 174, P 175, Q 179, S 188, V 190, T 192, and K 221 (e.g., K221E).

[0074] Particular mutations are described in U.S. Patent No. 2014 / 0200331, U.S. Patent No. 2014 / 150973, U.S. Patent No. 2014 / 0154254, and WO 2007 / 147901, all of which are incorporated herein by reference.

[0075] In a preferred embodiment, a pair of interacting polar interface residues is exchanged for a pair of neutral and salt-bridge forming residues: Substitution of Thr192 in the CH1 chain with glutamic or aspartic acid and substitution of Asn137 in the CL chain with Lys can be selected, optionally with substitution of serine residue 114 of the CL domain with an alanine residue.

[0076] In one set of mutations, Leu143 in the CHI domain can be replaced by a Gin residue, while the opposing residue, Val33, in the CL chain is replaced by a Thr residue. This first double mutation constitutes a switch of interaction from hydrophobic to polar. Simultaneously, mutation of two interacting serines (Ser188 in the CH1 chain and Ser176 in the CL chain) to valine residues can achieve a switch of interaction from polar to hydrophobic.

[0077] In further embodiments, the mutations can include a substitution of a leucine residue at position 124 of the CH1 domain with a glutamine and a substitution of a serine residue at position 188 of the CH1 domain with a valine residue; and a substitution of a valine residue at position 133 of the CL domain with a threonine residue and a substitution of a serine residue at position 176 of the CL domain with a valine residue.

[0078] The "knob into holes" mutations include a set of mutations (KH1) in which Leu 124 and Leu 143 in the CH1 domain are substituted by Ala and Glu residues, respectively, while Val 33 in the CL chain is substituted by a Trp residue, while in a set of mutations called H2, Val 90 in the CH1 domain is substituted by an Ala residue, and Leu 135 and Asn 137 in the CL chain are substituted by Trp and Ala residues, respectively.

[0079] The preferred mutations are published below:

[0080] [Table 1]

[0081] In certain embodiments, the multispecific antibody can have a double mutation, for example, one arm with a CR3 mutation and the other arm with a Mut4 mutation.

[0082] In certain embodiments, the multispecific antibody further comprises an immunoglobulin Fc region comprising hinge-CH2-CH3 domains, wherein the Fc region connects both antigen-binding arms by said hinge domain connecting the C-terminal end of the CH1 domain to the N-terminal end of the CH2 domain of the antigen-binding arms.

[0083] Specific mutations at the interface in the CH3 or CH2 domain of the Fc can be considered to result in heterodimerization of the two heavy chains instead of their natural homodimerization.

[0084] The mutations are selected from the following list: - de novo introduced ion pairs or oppositely charged mutations in natural ion pairs already present at the interface of the two heavy chains of the Fc domain; - knobs-into-holes mutations, which are well known and described in the art; - mutations that reshape the interface between the two facing heavy chains into a new interface (e.g., changing them from highly polar to highly hydrophobic, or vice versa) can be selected from:

[0085] Also, specific mutations in the IgG1 Fc domain that reduce or eliminate binding to Fc gamma receptors can be utilized, including but not limited to the following: - L234A / L235A - N297A (removes an N-linked glycosylation site) - L234A / L235A / G237A / P238S / H268A / A330S / P331S or specific combinations of position substitutions of any of the following residues: L234A, L235A, G236R, G237A, P238S, H268A, L328R, A330S, P331S (EU numbering) Includes:

[0086] Any of the molecules described herein can be modified to contain additional nonproteinaceous moieties known and readily available in the art, for example, by PEGylation, hyperglycosylation, etc. Modifications that can increase serum half-life or stability against proteolysis are of interest.

[0087] The antibodies of the invention can be glycosylated or non-glycosylated, or can exhibit diverse glycosylation profiles. In a preferred embodiment, the antibodies are non-glycosylated in the variable region of the heavy chain, but glycosylated in the Fc region.

[0088] Humanized forms of the reference non-human antibody can be used. In the humanization approach, the complementarity-determining regions (CDRs) and certain other amino acids from the donor variable region are grafted onto a human variable receptor region, which is then joined to a human constant region. See, e.g., Riechmann et al., Nature 332:323-327 (1988); U.S. Pat. No. 5,225,539.

[0089] Linker Design In certain embodiments, a polypeptide linker is used to connect the Fab fragment that binds to Vα7.2 and the Fab fragment that binds to a tumor-associated antigen.

[0090] It is also called a "hinge-derived polypeptide linker sequence" or "pseudo hinge linker" and comprises all or part of the sequence of the hinge region of one or more immunoglobulins, preferably selected from IgA, IgG, and IgD of human origin. The polypeptide linker may comprise all or part of the sequence of the hinge region of only one immunoglobulin. In this case, the immunoglobulin may belong to the same isotype and subclass as the immunoglobulin from which the adjacent CH1 domain is derived, or to a different isotype or subclass. Alternatively, the polypeptide linker may comprise all or part of the sequence of the hinge regions of at least two immunoglobulins of different isotypes or subclasses. In this case, the N-terminal portion of the polypeptide linker directly follows the CH1 domain and preferably consists of all or part of the hinge region of an immunoglobulin from the same isotype and subclass as the immunoglobulin from which the CH1 domain is derived.

[0091] Optionally, the polypeptide linker may further comprise a sequence of 2 to 15, preferably 5 to 10, N-terminal amino acids of an immunoglobulin CH2 domain.

[0092] The polypeptide linker sequence typically consists of fewer than 80 amino acids, preferably fewer than 60 amino acids, and more preferably fewer than 40 amino acids.

[0093] In some cases, sequences derived from the native hinge region can be used; in other cases, point mutations can be introduced into these sequences, particularly substitutions of one or more cysteine ​​residues in the native IgG1, IgG2 or IgG3 hinge sequences with alanine or serine to avoid unnecessary intra- or inter-chain disulfide bonds.

[0094] In certain embodiments, the polypeptide linker sequence comprises or consists of the amino acid sequence EPKX1CDKX2HX3X4PPX5PAPELLGGPX6X7PPX8PX9PX10GG (SEQ ID NO: 9), where X1, X2, X3, X4, X5, X6, X7, X8, X9, X10 are the same or different and are any amino acid. EPKSCDKTHTSPPAPAPELLGGPGGPPGPGPGGG (SEQ ID NO: 10); EPKSCDKTHTSPPAPAPELLGGPAAPPAPAPAGG (SEQ ID NO: 11); EPKSCDKTHTSPPAPAPELLGGPAAPPGPAPGGG (SEQ ID NO: 12); EPKSCDKTHTCPPCPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 13) and EPKSCDKTHTSPPSPAPELLGGPSTPPTPSPSGG (SEQ ID NO: 14) It may comprise or consist of a sequence selected from the group consisting of:

[0095] In certain embodiments, X1, X2, and X3 are the same or different and are threonine (T) or serine (S).

[0096] In certain embodiments, X1, X2, and X3 are the same or different and are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D), and Ile (I), and more preferably, X1, X2, and X3 are the same or different and can be Ala (A) or Gly (G).

[0097] Alternatively, X1, X2 and X3 may be the same or different and represent Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), Trp (W), preferably Leu (L), Glu (E) or Gln (Q).

[0098] In certain embodiments, X4 and X5 are the same or different and are any amino acid selected from the group consisting of serine (S), cysteine ​​(C), alanine (A) and glycine (G).

[0099] In a preferred embodiment, X4 is serine (S) or cysteine ​​(C).

[0100] In preferred embodiments, X5 is alanine (A) or cysteine ​​(C).

[0101] In certain embodiments, X6, X7, X8, X9, and X10 are the same or different and are any amino acid other than threonine (T) or serine (S). Preferably, X6, X7, X8, X9, and X10 are the same or different and are selected from the group consisting of Ala (A), Gly (G), Val (V), Asn (N), Asp (D), and Ile (I).

[0102] Alternatively, X6, X7, X8, X9, and X10 may be the same or different and represent Leu (L), Glu (E), Gln (Q), Met (M), Lys (K), Arg (R), Phe (F), Tyr (T), His (H), or Trp (W), preferably Leu (L), Glu (E), or Gln (Q).

[0103] In a preferred embodiment, X6, X7, X8, X9, and X10 are the same or different and are selected from the group consisting of Ala (A) and Gly (G).

[0104] In a further preferred embodiment, X6 and X7 are the same and are preferably selected from the group consisting of Ala (A) and Gly (G).

[0105] In a preferred embodiment, the polypeptide linker sequence has the sequence of SEQ ID NO:9. (In the sequence, X1, X2 and X3 are the same or different and are threonine (T) or serine (S); X4 is serine (S) or cysteine ​​(C); X5 is alanine (A) or cysteine ​​(C); X6, X7, X8, X9, and X10 may be the same or different and are selected from the group consisting of Ala (A) and Gly (G). It comprises or consists of:

[0106] In a preferred embodiment, the polypeptide linker sequence is SEQ ID NO:9 (In the sequence, X1, X2, and X3 are the same or different and are Ala (A) or Gly (G); X4 is serine (S) or cysteine ​​(C); X5 is alanine (A) or cysteine ​​(C); X6, X7, X8, X9, and X10 may be the same or different and are selected from the group consisting of Ala (A) and Gly (G). It comprises or consists of:

[0107] In embodiments in which the antibody comprises different Fab fragments, the polypeptide linkers separating the Fab fragments can be the same or different.

[0108] Production of multispecific antibodies Nucleic acids encoding the heavy and light chains of the antibody of the present invention are inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. The DNA fragments encoding the immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters, enhancers, and transcription termination sequences. Expression vectors are typically capable of replicating in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors will contain selectable markers, such as tetracycline or neomycin, to allow detection of those cells transformed with the desired DNA sequences.

[0109] In one example, sequences encoding both the heavy and light chains (e.g., VH and VL, VH-CH1, or VL-CL) are contained in a single expression vector. In some examples, each of the heavy and light chains of an antibody is cloned into a separate vector. In the latter case, expression vectors encoding the heavy and light chains can be transfected into a single host cell for expression of both chains, and the heavy and light chains can associate to form an intact antibody either in vivo or in vitro.

[0110] In certain embodiments, the host cell is co-transfected with three independent expression vectors, e.g., plasmids, resulting in the co-production of all three chains (i.e., the heavy chain HC and each of the two light chains LC1 and LC2) and secretion of the multispecific antibody.

[0111] More particularly, the three vectors can be advantageously used in the following molecular ratio: 3:2:2 (HC:LC1:LC2).

[0112] Recombinant vectors for expressing the antibodies described herein typically contain a nucleic acid encoding the antibody amino acid sequence operably linked to either a constitutive or inducible promoter. Vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the antibody-encoding nucleic acid. Vectors optionally contain at least one independent terminator sequence, a sequence that allows replication of the cassette in both eukaryotes and prokaryotes, i.e., a shuttle vector, and a generic expression cassette containing a selectable marker for both prokaryotic and eukaryotic systems.

[0113] The multispecific antibodies described herein can be produced in prokaryotic or eukaryotic expression systems, such as bacteria, yeast, filamentous fungi, insect, and mammalian cells. The recombinant antibodies of the present invention do not need to be glycosylated or expressed in eukaryotic cells; however, expression in mammalian cells is generally preferred. Examples of useful mammalian host cell lines are the human embryonic kidney line (293 cells), baby hamster kidney cells (BHK cells), Chinese hamster ovary cells / - or + DHFR (CHO, CHO-S, CHO-DG44, Flp-in CHO cells), African green monkey kidney cells (VERO cells), and human hepatocytes (Hep G2 cells).

[0114] Mammalian tissue cell culture is preferred for expressing and producing polypeptides because a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed in the art, including CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines, or transformed B cells or hybridomas.

[0115] In a most preferred embodiment, the multispecific, preferably bispecific, antibodies of the invention are produced using a CHO cell line, most advantageously a CHO-S cell line.

[0116] Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Preferred expression control sequences include immunoglobulin genes, SV40, adenovirus, bovine papillomavirus, cytomegalovirus, etc.

[0117] Vectors containing the polynucleotide sequences of interest (e.g., heavy and light chain encoding sequences and expression control sequences) can be transferred into host cells by known methods, which vary depending on the type of cellular host. For example, calcium phosphate treatment or electroporation can be used for other cellular hosts. (See generally, Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)). When the heavy and light chains are cloned into separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins.

[0118] Host cells are transformed or transfected with the vector (e.g., by chemical transfection or electroporation) and cultured in conventional nutrient media (or appropriately modified) to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0119] Once expressed, whole antibodies of the invention, their dimers, their respective light and heavy chains, or other immunoglobulin forms can be further isolated or purified to obtain preparations that are substantially homogeneous for further analysis and applications. Standard protein purification methods known in the art can be used. For example, suitable purification procedures can include fractionation using immunoaffinity or ion exchange columns, ethanol precipitation, high-performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation, and gel filtration (see generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)). For pharmaceutical uses, substantially pure immunoglobulins having at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred.

[0120] In vitro production allows for scale-up to provide large quantities of the desired multispecific, preferably bispecific, antibodies of the invention. The methods can use homogenous suspension cultures, e.g., in airlift reactors or continuous stirrer reactors, or immobilized or entrapped cell cultures, e.g., in hollow fibers, in microcapsules, on agarose microbeads, or on ceramic cartridges.

[0121] Therapeutic applications A further aspect of the invention is a pharmaceutical composition comprising a multispecific molecule, more particularly an antibody, according to the invention. An aspect of the invention is the use of a multispecific molecule, more particularly an antibody, according to the invention for the preparation of a pharmaceutical composition. A further aspect of the invention is a method for the preparation of a pharmaceutical composition comprising a multispecific molecule, more particularly an antibody, according to the invention.

[0122] In one aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising a multispecific molecule, more particularly an antibody, as defined herein, formulated together with a pharmaceutical carrier.

[0123] The compositions of the present invention can be administered by a variety of methods known in the art. Any suitable route of administration is encompassed, including intravenous, oral, subcutaneous, intradermal, or mucosal administration. In certain embodiments, direct injection at or near the tumor site is contemplated.

[0124] The compositions of the present invention are useful for treating tumors, particularly solid tumors, such as lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), skin cancer, melanoma, breast cancer, colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, renal cancer, liver cancer, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer, urothelial cancer, gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal gland cancer, and sarcoma. Hematological malignancies (e.g., lymphoma, leukemia, multiple myeloma) are also included.

[0125] The invention, thus generally described above, will be more readily understood by reference to the following examples, which are provided for purposes of illustration and are not intended to be limiting of the invention. [Example]

[0126] [Example] Examples of multispecific constructs according to the invention have been produced. The sequences of the constructs produced and tested as described in the examples below are shown in Table 2 below.

[0127] [Table 2]

[0128] Example 1: Production of anti-Vα7.2 / anti-CD19 IgG1 and Fab-Fab [Gene synthesis] The amino acid sequences of the variable regions of anti-Vα7.2 and anti-CD19 monoclonal antibodies were used to design DNA sequences after codon optimization for mammalian expression using the GeneScript program. For the heavy chain, DNA encoding the signal peptide, variable region and constant CH1 domain of Fab1, followed by a hinge linker and variable region and constant CH1 domain of Fab2, along with flanking sequences for restriction enzyme cleavage, was synthesized by GeneScript. For the light chain, DNA encoding the signal peptide and variable and constant kappa regions was synthesized by GeneScript.

[0129] PCR reactions using PfuTurbo Hot Start were performed to amplify the insert fragments, which were subsequently digested with NotI + ApaI and NotI + HindIII for the heavy and light chains, respectively. For Fc-containing molecules, the double-digested heavy chain fragment was ligated into the Icosagen-proprietary pQMCF expression vector, into which the human IgG1 CH1 + hinge + CH2 + CH3 domains had already been inserted, digested with NotI + ApaI. For expression of Fab-Fab molecules, a stop codon was inserted immediately downstream of C201 (Kabat numbering). The double-digested light chain fragment was ligated into the Icosagen-proprietary vector, digested with NotI + HindIII. Plasmid DNA was verified by double-stranded DNA sequencing.

[0130] Expression, purification and characterization For 50 mL-scale expression, a total of 50 μg of plasmid DNA in Icosagen's proprietary pQMCF vector (25 μg heavy chain + 12.5 μg each of LC1 and LC2 light chains) was mixed in 1 mL of CHO TF (Xell AG) growth medium containing Icosagen's proprietary transfection Reagent 007 in a 1.5 mL Eppendorf tube and incubated for 20 minutes at RT. The mixture was then grown at a concentration of 1-2 x 10 in CHO TF (Xell AG) growth medium in a 125 mL shake flask. 6 The solution was loaded onto 49 mL of CHOEBNALT85 1E9 cells at 1000 cells / mL. The cells were shaken at 37°C for 4 days and at 30°C for another 6 days. The supernatant was collected by centrifuging the cells at 3,000 rpm for 15 minutes. The supernatant from the Fc-containing BiXAb antibody was purified with Protein A resin (MabSelect SuRe 5 mL column), and the supernatant from the Fab-Fab antibody was purified with CaptureSelect IgGCH1 resin. We further purified the BiXAb Fc-containing antibody using gel filtration chromatography on a Superdex 200 HiLoad 26 / 60 pg preparative column, while the Fab-Fab antibody was purified using Superdex 200 Increase 10 / 300 GL; all antibodies were buffer-exchanged into PBS pH 7.4. All samples were sterile filtered using a 0.2 μm ULTRA Capsule GF. Electrophoresis was performed under reducing and non-reducing conditions using 10% SDS-PAGE. Samples were prepared by combining purified antibody with 2X SDS sample buffer and heating to 95°C for 5 minutes. Preparation of reduced samples included the addition of DTT to a final concentration of 100 mM prior to heating. Apparent MW was determined using Ladder Precision Plus Protein Unstained Standards (Biorad).

[0131] The Vα7.2 3C10-ML1 light chain is as shown in SEQ ID NO:15.

[0132] The Vα7.2 3C10-ML1-AP-CD19-CC1 heavy chain is as shown in SEQ ID NO:16.

[0133] The CD19 light chain is as shown in SEQ ID NO:17.

[0134] and Vα7.2 3C10-ML1-AP-CD19-CC1 heavy chain as shown in SEQ ID NO: 18.

[0135] Example 2: CD19 + cells and Vα7.2 + Anti-Vα7.2 / anti-CD19 Fab-Fab binding in T cells] The anti-Vα7.2 / anti-CD19 Fab-Fab produced in Example 1 was first synthesized by immunoprecipitation of CD19 + Binding was tested in Raji cells. The assay was performed by flow cytometry. Briefly, Raji cells were washed with PBS and stained with Fixable Viability Dye (eFluorescent TM The cells were stained with a 780 (ThermoFisher) and human Fc block reagent (BD) in PBS for 25 minutes at 4°C. The cells were then washed with FACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and stained with different concentrations of anti-Vα7.2 / anti-CD19 Fab-Fab (Table 1, shown in both nM and μg / ml) for 1 hour at 4°C. An irrelevant Fab-Fab was used as a negative control. Following washing (5x), Raji cells were stained with a secondary goat anti-human antibody conjugated with phycoerythrin (Jackson Immunoresearch) for 45 minutes at 4°C. The cells were then analyzed on a MACSquant flow cytometer (Miltenyi Biotec).

[0136] The results showed that the anti-Vα7.2 / anti-CD19 Fab-Fab bound to CD19, whereas the irrelevant Fab-Fab showed no binding. +Figure 3A shows dose-dependent binding of CD19 IgG to Raji cells, expressed as normalized geometric mean fluorescence intensity, representative of four independent experiments. + 1 shows the binding profile of anti-Vα7.2 / anti-CD19 Fab-Fab on Raji cells.

[0137] Anti-Vα7.2 / anti-CD19 Fab-Fab was then purified to Vα7.2 + CD8 + Binding to human CD8 T cells was tested. The assay was performed by flow cytometry. + T cells were isolated from purified peripheral blood mononuclear cells (PBMCs). Briefly, leukapheresis packs from healthy donors were centrifuged on a Ficoll gradient and PBMCs were collected. CD8 + T cells were then isolated using a commercial negative selection kit (Miltenyi Biotec). These cells were then transfected with Vα7.2 + Anti-Vα7.2 / anti-CD19 antibodies were used to determine binding of peripheral blood mononuclear cells. Cells were washed with PBS and stained with Fixable Viability Dye (eFluorescent TM The Raji cells were stained with a 780 (ThermoFisher) and human Fc block reagent (BD) in PBS for 25 minutes at 4°C. The cells were then washed with FACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and stained with different concentrations of anti-Vα7.2 / anti-CD19 Fab-Fab (Table 3, shown in both nM and μg / ml) for 1 hour at 4°C. An irrelevant Fab-Fab was used as a negative control. Following washing (5x), Raji cells were stained with secondary goat anti-human and anti-CD8 antibodies (Biolegend) conjugated with phycoerythrin (Jackson Immunoresearch) for 45 minutes at 4°C. The cells were then analyzed on a MACSquant flow cytometer.

[0138] Vα7.2 depending on the donor + The cells are CD8 + The results show that the anti-Vα7.2 / anti-CD19 Fab-Fab expresses Vα7.2, whereas the irrelevant Fab-Fab is undetectable. + Figure 3B shows the Vα7.2 population, expressed as normalized geometric mean fluorescence intensity, representative of three independent donors. + Figure 1 shows the binding profile of anti-Vα7.2 / anti-CD19 Fab-Fab on cells. The EC50 calculated from two experiments was 3.49±0.2 nM.

[0139] In conclusion, the anti-Vα7.2 / anti-CD19 Fab-Fab is able to specifically bind to both molecular targets (CD19 and Vα7.2) expressed on the surface of living cells.

[0140] [Table 3]

[0141] Example 3: Anti-Vα7.2 / anti-CD19 Fab-Fab activates specific MAIT cells, but minimally total CD8 + T cell activation] CD8 + T cells and specific MAIT cells (which are CD8 + TCRγδ - CD161 hi IL18RA +The effect of anti-Vα7.2 / anti-CD19 Fab-Fab on mediating activation of T cells (Vα7.2 / anti-CD19 Fab-Fab) was evaluated in vitro. Briefly, anti-Vα7.2 / anti-CD19 Fab-Fab and two antibodies, anti-Vα7.2 and anti-CD3, were coated onto flat-bottom 96-well plates (in PBS overnight at 4°C) at the molar concentrations shown in Table 3. The anti-Vα7.2 antibody was the 3C10 clone (described in WO 2008 / 087219), from which the anti-Vα7.2 sequence of the anti-Vα7.2 / anti-CD19 Fab-Fab was derived. The anti-CD3 antibody was the OKT3 clone, an antibody commonly used in T cell activation assays (Saitakis et al., 2017). Before adding cells, wells were washed at least twice with PBS.

[0142] CD8 + T cells were isolated as in Example 2 and added to flat-bottom 96-well plates (100,000 cells per well in 100 μl of RPMI 1640, 10% FBS). Wells were coated with different molar concentrations of either anti-Vα7.2 / anti-CD19 Fab-Fab, or anti-CD3 or anti-Vα7.2 antibodies (Table 3). Plates were placed in an incubator at 37°C and 5% CO2 for 16 hours. Following incubation, cells were harvested, washed with PBS, and first stained with Fixable Viability Dye (eFluorescent TM Cells were stained with BD human Fc Block reagent (BD 780, ThermoFisher) and human Fc Block reagent (BD 780, ThermoFisher) (in PBS, 25 min, 4°C), followed by the following antibodies (1 / 100 dilution in FACS buffer, 45 min, 4°C): anti-CD8-PerCP-Cy5.5, anti-TCRγδ-FITC, anti-CD161-PE, anti-IL18RA-APC, anti-CD25-PE-Cy5, and anti-CD69-APC-Cy7 (Biolegend). Cells were then analyzed on a MACSquant flow cytometer (Miltenyi Biotec).

[0143] Upregulation of CD25 and CD69 is a metric for assessing T cell activation. Therefore, following activation, we examined the percentage of cells that expressed CD25, CD69, or both. Figure 4A shows the flow cytometry gating strategy for determining overall CD8+ T cell activation. Figure 4B shows the upregulation of CD25 in wells coated with different molar concentrations of anti-Vα7.2 / anti-CD19 Fab-Fab, or either anti-CD3 or anti-Vα7.2 antibodies, representative of two donors. + , CD69 + and double positive (CD25 + CD69 + ) CD8 + TCRγδ - The percentage of T cells is shown. Anti-CD3 antibody inhibits CD25 + , CD69 + and double-positive T cells, whereas anti-Vα7.2 / anti-CD19 Fab-Fab increased overall CD8 + TCRγδ - They showed at best 4-5 times less activation of T cells.

[0144] Figure 5A shows the flow cytometry gating strategy for determining MAIT cell activation. Figure 5B shows the CD25 expression level in wells coated with different molar concentrations of anti-Vα7.2 / anti-CD19 Fab-Fab, or anti-CD3 or anti-Vα7.2 antibodies, representative of two donors. + , CD69 + and double positive (CD25 + CD69 + ) MAIT (CD8 + TCRγδ - CD161 hi IL18RA + The percentage of CD25 cells is shown. Anti-Vα7.2 / anti-CD19 Fab-Fab inhibited CD25 more than both monospecific antibodies. + , CD69 + and was more efficient in increasing the fraction of double-positive MAIT cells.

[0145] In conclusion, anti-Vα7.2 / anti-CD19 Fab-Fab specifically activated MAIT cells and overall CD8 + Minimally activates T cells.

[0146] Example 4: Anti-Vα7.2 / anti-CD19 Fab-Fab-mediated cytotoxicity A cytotoxicity assay was set up to evaluate the cytotoxic potential of MAIT cell redirection using anti-Vα7.2 / anti-CD19 Fab-Fab. Briefly, human CD8 + T cells were isolated from purified PBMCs as described in Example 2. These cells were engineered to express luciferase, CD19 + These cells were used in coculture with Raji cells. 50,000 Raji cells were first added to a U-bottom 96-well plate in 50 μl of RPMI 1640 10% FBS. Different numbers of T cells (in 100 μl of RPMI 1640 10% FBS) corresponding to different effector:target cell ratios were then added (Table 4). Finally, 50 μl of RPMI 1640 10% FBS containing different concentrations of anti-Vα7.2 / anti-CD19 Fab-Fab (final molar concentrations as shown in Table 3) was added, and the coculture was incubated at 37°C with 5% CO2 for 48 hours. The wells were mixed with a multi-pipette, and 100 μl was transferred to a white polystyrene 96-well plate. 50 μl of PBS with a final concentration of 0.1 mg / ml luciferine (Pierce) was added to each well, and bioluminescence was measured in a SpectraMax ID3 plate reader (BioTek).

[0147] CD8 +In a donor with 9% MAIT cells among T cells, anti-Vα7.2 / anti-CD19 Fab-Fab promoted specific cytotoxicity with increasing dose and effector:target ratio. Figure 6 shows the percentage of specific lysis after culturing Raji cells for 48 hours with different concentrations of anti-Vα7.2 / anti-CD19 Fab-Fab and different effector:target ratios.

[0148] In conclusion, anti-Vα7.2 / anti-CD19 Fab-Fab inhibits CD19 + It can promote the in vitro cytotoxicity of MAIT cells against Raji cells.

[0149] [Table 4]

[0150] Example 5: Binding of anti-CD19 / anti-Vα7.2 based bispecific antibodies to CD19 on tumor cells or Vα7.2 TCR chain on T cells The binding ability of anti-CD19 / anti-Vα7.2-based bispecific antibodies, i.e., anti-CD19 / anti-Vα7.2 Fab-Fab, anti-Vα7.2 / anti-CD19 Fab-Fab, anti-CD19 / anti-Vα7.2 BiXAb, and anti-Vα7.2 / anti-CD19 BiXAb, to CD19 protein expressed on the cell surface of NALM-6 tumor cells was measured using flow cytometry. Briefly, tumor cells were harvested and washed with RPMI 1640 (Gibco), 10% FBS (Eurobio), and 0.1% penicillin / streptomycin (P / S) (Gibco). The cells were then washed with FACS buffer (PBS, 2 mM EDTA, 0.5% BSA), plated, and incubated for 45 minutes at 4°C with serial dilutions (concentrations ranging from 0 to 66 nM) of anti-CD19 / anti-Vα7.2-based or negative control bispecific antibodies. The cells were washed and incubated for 1 hour at 4°C with a phycoerythrin-conjugated secondary antibody (Jackson ImmunoResearch) to detect bound bispecific antibodies. A phycoerythrin-conjugated anti-human Fc (Jackson ImmunoResearch, 109-116-098) secondary antibody was used to detect bound BiXAb molecules, and a phycoerythrin-conjugated anti-human Fab (Jackson ImmunoResearch, 109-116-097) antibody was used to detect bound Fab-Fab molecules. Cells were washed and resuspended in FACS buffer containing DAPI (Sigma) and analyzed using a MACSquant flow cytometer (Miltenyi Biotec).

[0151] The results of the binding assays are shown in Figures 7A and 7B for the Fab-Fab and BiXAb molecules, respectively. Data are expressed as the percentage of positive cells. The results demonstrated that the anti-CD19 / anti-Vα7.2-based Fab-Fab and BiXAb bispecific antibodies bound to CD19 expressed on NALM-6 cells in a dose-dependent manner. No binding was observed with the negative control Fab-Fab or BiXAb antibodies.

[0152] Anti-CD19 / anti-Vα7.2 based BiXAb antibodies—namely, anti-CD19 / anti-Vα7.2 BiXAb and anti-Vα7.2 / anti-CD19 BiXAb—were subsequently shown to inhibit Vα7.2 + CD8 + Binding to the Vα7.2 TCR chain expressed on MAIT cells was determined using flow cytometry. + T cells were isolated from purified peripheral blood mononuclear cells (PBMCs). Briefly, leukapheresis packs obtained from healthy donors were centrifuged on a Ficoll gradient and PBMCs were collected. CD8 + T cells were isolated from PBMCs using a positive selection kit (REAlease CD8 microbead kit, Human, Miltenyi Biotec, 130-117-036) according to the manufacturer's instructions. These cells were then transfected with Vα7.2 + CD8 +This was used to evaluate the binding of different BiXAb antibodies to MAIT cells. To this end, cells were washed with FACS buffer (PBS, 2 mM EDTA, 0.5% BSA) and incubated with serial dilutions (concentrations ranging from 0 to 66 nM) of BiXAb or negative control bispecific antibodies for 45 min at 4°C. Cells were washed and incubated with a phycoerythrin-conjugated secondary antibody (Jackson ImmunoResearch) for 1 h at 4°C to detect bound bispecific antibodies. Cells were washed, incubated in FACS buffer containing mouse serum for 30 min at room temperature, washed again, and stained with the following antibody panel for 30 min at 4°C: anti-human CD161-PE / Cy7 (Biolegend, HP-3G10), anti-human Vα7.2-APC / Cy7 (Biolegend, 3C10), and anti-human IL18Ra-APC (Biolegend, H44). The cells were then washed, stained with DAPI (Sigma), and analyzed using a MACSquant flow cytometer (Miltenyi Biotec). The binding results were shown in Figure 8. + CD161 + Binding was obtained by gating on IL-18RA cells. + None were observed outside the cell population.

[0153] The results, expressed as the percentage of positive cells, are presented in Figure 9. The anti-CD19 / anti-Vα7.2 and anti-Vα7.2 / anti-CD19 BiXAbs were found to bind to Vα7.2+ CD8+ MAIT cells in a dose-dependent manner. The negative control BiXAb antibody showed no binding.

[0154] The results of the binding assays showed that the anti-CD19 / anti-Vα7.2-based bispecific antibody was able to specifically bind to both CD19 and TCR Vα7.2 chains expressed on the surface of live cells.

[0155] Example 6: MAIT cells are activated following incubation with plate-bound anti-CD19 / anti-Vα7.2-based BiXAb. The ability of anti-CD19 / anti-Vα7.2-based BiXAb antibodies—namely, anti-CD19 / anti-Vα7.2 BiXAb and anti-Vα7.2 / anti-CD19 BiXAb—to induce MAIT cell activation was assessed by examining the surface expression of the activation marker CD69 after in vitro stimulation with plate-bound BiXAb antibodies. Briefly, anti-CD19 / anti-Vα7.2-based BiXAbs were coated onto flat-bottom 96-well plates at concentrations ranging from 0 to 66 nM (in PBS at 37°C for 2 h). Before adding cells, the plates were washed with PBS (x4) to remove unbound antibody. CD8+ T cells were isolated from PBMCs of healthy donors as in Example 5 and added to pre-coated flat-bottom 96-well plates (100,000 cells per well in 100 μl of RPMI 1640 (Gibco), 10% FBS (EUROBIO), 0.1% P / S (Gibco)). After 16 hours of incubation at 37°C and 5% CO2, cells were harvested, washed with FACS buffer, and stained for 30 minutes at 4°C with Fixable Viability Dye (Aqua, eBioscience, 65-0866-14) and the following antibody panel: anti-CD3-BUV395 (BDBiosciences, UCHT1), anti-CD4-BUV737 (BDBiosciences, SK3), anti-CD8-PerCP-Cy5.5 (Biolegend, SK1), anti-TCRγδ-FITC (Biolegend, B1), anti-CD161-PE (Biolegend, HP-3G10), anti-IL18RA-APC (Biolegend, H44), anti-CD25-BV421 (Biolegend, BC96), and anti-CD69-PE / Cy7 (BDBiosciences, L78). Cells were then washed and analyzed for expression of the activation marker CD69 using a Cytoflex flow cytometer (Beckman Coulter). As expected, T cell activation in this assay setting resulted in downregulation of TCR from the cell surface. As a result, MAIT cells expressed CD3 + CD8 + CD161hi Vα7.2 + MAIT cells were identified as CD69+ MAIT cells. The activation profile of this subset is shown in Figure 10. Results are presented as the percentage of CD69+ MAIT cells. The negative control antibody did not induce upregulation of CD69 on MAIT cells. In contrast, the anti-CD19 / anti-Vα7.2-based BiXAb bispecific antibody induced a dose-dependent increase in CD69 expression on MAIT cells, as shown in Figure 10. In conclusion, plate-bound anti-CD19 / anti-Vα7.2 BiXAb and anti-Vα7.2 / anti-CD19 BiXAb antibodies activated MAIT cells through engagement of the anti-Vα7.2 arm of the bispecific antibody with the Vα7.2 TCR chain on MAIT cells.

[0156] Example 7: Redirection of MAIT cell cytotoxicity to kill CD19+ tumor cells upon cross-linking of the Vα7.2 TCR chain on MAIT cells and CD19 on tumor cells with an anti-CD19 / anti-Vα7.2-based bispecific antibody. Anti-CD19 / anti-Vα7.2-based bispecific antibodies, i.e., anti-CD19 / anti-Vα7.2 Fab-Fab, anti-Vα7.2 / anti-CD19 Fab-Fab, anti-CD19 / anti-Vα7.2 BiXAb, and anti-Vα7.2 / anti-CD19 BiXAb, were analyzed for their ability to induce MAIT cell-mediated apoptosis in CD19-expressing tumor cells when the constructs were cross-linked via binding of the anti-CD19 moiety to CD19 on A-549 tumor cells. In addition, the ability of the bispecific antibodies to induce MAIT cell activation was assessed by considering the surface expression of the activation markers CD69 and CD25. Briefly, human CD8 + T cells were isolated from purified PBMCs as described in Example 5. These cells were co-cultured with A-549 tumor cells engineered to express CD19 and luciferase. 5A-549 tumor cells were first plated in 50 μl of RPMI 1640 (Gibco), 10% FBS (EUROBIO), 0.1% P / S (Gibco) in a white polystyrene 96-well plate, and then 6x10 cells were plated in 100 μl of RPMI 1640 (Gibco), 10% FBS (EUROBIO), 0.1% P / S (Gibco), recombinant human interleukin-12 (rhIL-12) 30 ng / mL (Peprotech). 5 CD8 +T cells were added at a 6:1 effector:target cell ratio. Finally, 50 μl of RPMI 1640 (Gibco), 10% FBS (Eurobio), 0.1% P / S (Gibco), and IL-12 30 ng / mL (Peprotech) containing different concentrations of bispecific antibodies (final molar concentrations ranging from 0 to 66 nM) was added. The plates were incubated at 37°C and 5% CO for 48 hours. The supernatant was discarded, and the cells were washed with PBS. The cells were then resuspended in 50 μl of RPMI 1640 (Gibco), 10% FBS (Eurobio), and 0.1% P / S (Gibco) in a white polystyrene 96-well plate. 50 μl of PBS containing luciferin (Perkin Elmer) at a final concentration of 0.1 mg / ml was added to each well, and bioluminescence was measured on a SpectraMax ID3 plate reader (BioTek). An overview of the experimental setup is shown in Figure 11. Cocultures of CD8+ T cells and tumor cells were also analyzed using flow cytometry. For this purpose, cells were harvested, washed with FACS buffer, and stained for 30 min at 4°C with Fixable Viability Dye (Aqua, eBioscience, 65-0866-14) and the following antibody panel: anti-CD3-BUV395 (BDBiosciences UCHT1), anti-CD4-BUV737 (BDBiosciences, SK3), anti-CD8-PerCP-Cy5.5 (Biolegend, SK1), anti-TCRγδ-FITC (Biolegend, B1), anti-CD161-PE (Biolegend, HP-3G10), anti-IL18RA-APC (Biolegend, H44), anti-CD25-BV421 (Biolegend, BC96), and anti-CD69-PE / Cy7 (BDBiosciences, L78). The cells were then washed and analyzed by flow cytometry (Cytoflex, Beckman Coulter) to measure the expression of activation markers CD69 and CD25 in MAIT cells.

[0157] MAIT cell activation following coculture was analyzed as described in Example 6. The results for each BiXAb antibody are reported in Figure 12. Results are shown as the percentage of single-positive CD69 MAIT cells. Addition of a negative control BiXAb antibody to the coculture did not activate MAIT cells, as indicated by the lack of upregulation of CD69 on MAIT cells. As shown in Figure 12, addition of an anti-CD19 / anti-Vα7.2-based BiXAb promoted MAIT cell activation at the tested concentrations. Similarly, an anti-CD19 / anti-Vα7.2-based Fab-Fab induced upregulation of the activation markers CD69 and CD25 on MAIT cells.

[0158] In addition, the percentage of lysis of CD19+ A-549 tumor cells was assessed by adding luciferin to the culture medium and measuring the level of luciferase activity in viable tumor cells in coculture wells. The percentage of lysis is reported in Figure 13 for the Fab-Fab bispecific antibodies. A maximum percentage of lysis of 30% was reached at concentrations as low as 0.06 nM for the anti-CD19 / anti-Vα7.2 Fab-Fab and anti-Vα7.2 / anti-CD19 Fab-Fab. Similarly, the addition of anti-CD19 / anti-Vα7.2 BiXAb and anti-Vα7.2 / anti-CD19 BiXAb to the coculture induced a maximum of 30% tumor lysis at concentrations as low as 0.06 nM.

[0159] Taken together, these results demonstrate that anti-CD19-based bispecific antibodies target the cytotoxicity of MAIT cells against tumor cells expressing CD19.

[0160] Example 8: Production of IgG1 (BiXAb) and Fab-Fab targeting Vα7.2, EGFR, or HER2 The amino acid sequences of the variable regions of the anti-Vα7.2, anti-EGFR and anti-HER2 monoclonal antibodies are shown in Table 1 for the following IgG1 BiXAb and Fab-Fab bispecific antibodies: ·Anti-Vα7.2 / Anti-EGFR Fab-Fab ·Anti-EGFR / anti-Vα7.2 Fab-Fab ·Anti-Vα7.2 / anti-EGFR BiXAb ·Anti-EGFR / anti-Vα7.2 BiXAb ·Anti-Vα7.2 / Anti-HER2 Fab-Fab ·Anti-HER2 / anti-Vα7.2 Fab-Fab ·Anti-Vα7.2 / anti-HER2 BiXAb ·Anti-HER2 / anti-Vα7.2 BiXAb was used to design the

[0161] The names reflect the location of the respective binding sites: for example, anti-Vα7.2 / anti-TAA Fab-Fab or BixAb means that the anti-Vα7.2 binding fragment is located at the N-terminus (see Figure 1), whereas anti-TAA / anti-Vα7.2 Fab-Fab or BixAb means that the anti-TAA binding fragment is located at the N-terminus.

[0162] See Table 2 for sequence reference.

[0163] Additionally, negative control BiXAb and Fab-Fab antibodies were generated using the variable region sequences of the humanized monoclonal anti-RSV antibody MEDI-493. All BiXAbs contained a LALA mutation in the CH2 domain. Introduction of a LALA mutation in the CH2 domain of human IgG1 is known to reduce Fcγ receptor binding (Bruhns et al., 2009 and Hezareh et al., 2001).

[0164] The methods used to perform the gene synthesis, expression, purification and characterization of these bispecific antibodies are described in Example 1.

[0165] Example 9: Binding of anti-HER2 / anti-Vα7.2 based bispecific antibodies to HER2 on tumor cells or Vα7.2 TCR chain on T cells. Anti-HER2 / anti-Vα7.2 based bispecific antibodies, i.e., anti-HER2 / anti-Vα7.2 Fab-Fab, anti-HER2 / anti-Vα7.2 BiXAb and anti-Vα7.2 / anti-HER2 BiXAb, bind to HER2 protein and Vα7.2 protein expressed on the cell surface of A-549 tumor cells. + CD8 + The ability to bind to the Vα7.2 TCR chain expressed in MAIT cells was measured using flow cytometry. The experiment was performed as described in Example 5.

[0166] The binding results of anti-HER2 / anti-Vα7.2-based bispecific molecules to HER2-expressing tumor cells are shown in Figures 14A and 14B for Fab-Fab and BiXAb molecules, respectively. Results are presented as the percentage of positive cells. All anti-HER2 / anti-Vα7.2-based bispecific antibodies show dose-dependent binding to HER2+ A-549 cells, while the negative control Fab-Fab or BiXAb antibodies showed no binding.

[0167] Additionally, as shown in Figure 15, both anti-HER2 / anti-Vα7.2-based BiXAb bispecific antibodies demonstrated dose-dependent binding to Vα7.2+CD8+ MAIT cells via the anti-Vα7.2 arm of the antibody. The negative control BiXAb antibody showed no cell binding. Results are presented as the percentage of positive cells.

[0168] Taken together, the results of the binding assays demonstrated that the anti-HER2 / anti-Vα7.2-based bispecific antibody can specifically bind to both HER2 and TCR Vα7.2 chains expressed on the surface of HER2-expressing tumor cells and MAIT cells, respectively.

[0169] Example 10: MAIT cells are activated following incubation with plate-bound anti-HER2 / anti-Vα7.2-based BiXAb. The ability of anti-HER2 / anti-Vα7.2-based BiXAbs, i.e., anti-HER2 / anti-Vα7.2 BiXAb and anti-Vα7.2 / anti-HER2 BiXAb, to activate MAIT cells was assessed in vitro using plate-bound BiXAb antibodies as described in Example 6.

[0170] Stimulation of MAIT cells with anti-HER2 / anti-Vα7.2-based BiXAbs induced dose-dependent upregulation of activation markers CD69 and CD25, demonstrating that plate-bound anti-HER2 / anti-Vα7.2 BiXAb and anti-Vα7.2 / anti-HER2 BiXAb antibodies activated MAIT cells ex vivo through engagement of the anti-Vα7.2 arm of the bispecific antibody with the Vα7.2 TCR chain on MAIT cells.

[0171] Example 11: Redirected MAIT cell cytotoxicity to kill HER2+ cells when anti-HER2 / anti-Vα7.2-based bispecific antibodies cross-link both Vα7.2 on MAIT cells and HER2 on tumor cells. Following the same protocol as described in Example 7, cytotoxicity assays were performed to evaluate the ability of different anti-HER2 / anti-Vα7.2-based bispecific antibodies, i.e., anti-HER2 / anti-Vα7.2 Fab-Fab, anti-Vα7.2 / anti-HER2 Fab-Fab, anti-HER2 / anti-Vα7.2 BiXAb, and anti-Vα7.2 / anti-HER2 BiXAb, to activate and redirect the cytotoxic activity of MAIT cells against tumor target cells. The A-549 tumor cell line, engineered to express luciferase, was used as the target cell line.

[0172] Activation of MAIT cells following co-culture was confirmed by CD8 +T cells were analyzed as described above. Figures 16A and 16B show the results for Fab-Fab and BiXAb antibodies, respectively. Results are presented as the percentage of double-positive CD25+CD69+ or single-positive CD69+ MAIT cells. Addition of negative control Fab-Fab or BiXAb antibodies to coculture did not activate MAIT cells, as indicated by the lack of upregulation of CD69 and CD25 in MAIT cells. In contrast, addition of an anti-HER2 / anti-Vα7.2-based bispecific antibody to coculture promoted MAIT cell activation at the doses tested. BiXAb molecules induced maximal responses at concentrations as low as 0.06 nM.

[0173] In addition, the rate of HER2+ A-549 tumor cell lysis was assessed by adding luciferin to live tumor cells in co-culture wells and measuring luciferase activity. The rate of lysis is reported in Figures 17A and 17B for Fab-Fab and BiXAb, respectively. A maximum rate of lysis of 31% was reached with concentrations as low as 0.06 nM of anti-HER2 / anti-Vα7.2 Fab-Fab or BiXAb, anti-Vα7.2 / anti-HER2 Fab-Fab or BiXAb.

[0174] Taken together, these results demonstrate that anti-HER2 / anti-Vα7.2-based bispecific antibodies redirect MAIT cell-mediated cytotoxicity toward HER2-expressing tumor cells.

[0175] Example 12: Binding of anti-EGFR / anti-Vα7.2-based bispecific antibodies to EGFR on tumor cells or Vα7.2 TCR chain on T cells. Anti-EGFR / anti-Vα7.2 based bispecific antibodies, i.e., anti-Vα7.2 / anti-EGFR BiXAb and anti-EGFR / anti-Vα7.2 BiXAb, bind to EGFR protein and Vα7.2 protein expressed on the cell surface of A-549 tumor cells. + CD8 +The ability to bind to the Vα7.2 TCR chain expressed on T cells was measured using flow cytometry. The experiment was performed as described in Example 5.

[0176] The results of binding of anti-EGFR / anti-Vα7.2-based bispecific antibodies to EGFR-expressing tumor cells are shown in Figure 18. Results are presented as the percentage of positive cells. The anti-EGFR / anti-Vα7.2-based bispecific antibodies were found to bind to cell surface-expressed EGFR in a dose-dependent manner. The negative control BiXAb antibody showed no binding.

[0177] Additionally, as shown in Figure 19, the anti-EGFR / anti-Vα7.2-based BiXAb bispecific antibody was found to bind to the Vα7.2 TCR chain expressed by CD8+ MAIT cells. The negative control BiXAb antibody showed no binding. Results are presented as the percentage of positive cells.

[0178] The results of the binding assays showed that the anti-EGFR / anti-Vα7.2 based bispecific antibody was able to specifically bind to both the EGFR and TCR Vα7.2 chains expressed on the surface of living cells.

[0179] Example 13: Redirected MAIT cell cytotoxicity to kill EGFR+ tumor cells when anti-EGFR / anti-Vα7.2-based bispecific antibodies cross-link both Vα7.2 on MAIT cells and EGFR on tumor cells. Cytotoxicity assays were performed to assess the ability of anti-EGFR / anti-Vα7.2-based bispecific antibodies to activate and redirect the cytotoxic activity of MAIT cells against tumor target cells, following the same protocol as described in Example 7. The EGFR-expressing A-549 tumor cell line engineered to express luciferase was used as the target cell line.

[0180] Addition of anti-Vα7.2 / anti-EGFR BiXAb to cocultures induced the cytolytic function of MAIT cells by redirecting them to kill tumor cells at concentrations as low as 0.6 nM. Maximum specific lysis of up to 49% was achieved at a concentration of 6 nM.

[0181] Example 14: MAIT cells exhibited cytotoxic effects against tumor cells in vivo. Six 8- to 12-week-old female NSG mice (non-obese diabetic severe combined immunodeficiency gamma [NOD.Cg-Prkdcscid IL2rgtm1Wjl / SzJ]) were used for each group. All mice from the same treatment group were housed together in the same cage. For this experiment, PBMCs were obtained from a single healthy donor. After tumor implantation (1 x 10 cells expressing CD19 and luciferase), 6 HER2+ A-549 tumor cells, 100 μl in PBS, injected into the tail vein), and mice received 5×10 6 Mice were treated by intravenous injection of human PBMCs and intraperitoneal injection of antibody (10 μg antibody per injection in 100 μL of PBS, for a total of five injections on days 2, 5, 7, 9, 10, and 18). Mice were monitored for weight loss every 2–3 days and for general health daily. Tumor progression was monitored twice weekly by monitoring luciferase activity in implanted tumor cells. Briefly, mice were intraperitoneally injected with 100 μL of D-luciferin firefly potassium salt in PBS (30 mg / kg, Perkin Elmer Ref. 122 799). Bioluminescence images were acquired using an IVIS® Lumina II In Vivo Imaging System, and luciferase expression was analyzed using Living Image® software (Perkin Elmer). Mice were under general anesthesia throughout the procedure. Mice were sacrificed when body weight loss exceeded 20%. No treatment-related toxicity was observed in the mice throughout the study.

[0182] As shown in Figures 21A and 21B, in tumor-bearing mice injected with PBMCs without any antibody treatment, the bioluminescence signal increased over time in most mice. In contrast, in animals treated with anti-Vα7.2 / anti-CD19 Fab-Fab, anti-Vα7.2 / anti-HER2 Fab-Fab, anti-Vα7.2 / anti-CD19 BiXAb, or anti-Vα7.2 / anti-HER2 BiXAb, tumors grew more slowly before regressing on day 17 after tumor implantation. On day 17, the majority of animals treated with bispecific antibodies showed weak or no bioluminescence signals.

[0183] [References] TIFF0007809639000005.tif204170

Claims

1. A multispecific molecule capable of simultaneously binding to mucosal-associated invariant T (MAIT) cells and tumor cells, said multispecific molecule comprising at least one domain that specifically binds to the Vα7.2 T cell receptor (TCR) and at least one domain that specifically binds to a tumor-associated antigen (TAA); or a domain that binds to the Vα7.2 TCR comprising a heavy variable chain comprising the following CDRs of the 3C10 antibody: GFNIKDTH (SEQ ID NO: 4) as CDR1, TDPASGDT (SEQ ID NO: 5) as CDR2, and CAHYYRDDVNYAMDY (SEQ ID NO: 6) as CDR3; and a light variable chain comprising the following CDRs of the 3C10 antibody: QNVGSN (SEQ ID NO: 7) as CDR1, SSS as CDR2, and QQYNTYPYT (SEQ ID NO: 8) as CDR3; or A multispecific molecule, wherein the domain that binds to the Vα7.2 TCR is a humanized variant of the 3C10 antibody, which is capable of competing with the 3C10 antibody for the same or substantially the same epitope on the Vα7.2-Jα33 polypeptide.

2. 2. The multispecific molecule of claim 1, which is a multispecific antibody, preferably a bispecific antibody, or an antigen-binding fragment thereof.

3. 3. The multispecific molecule of claim 1, comprising at least one multispecific antigen-binding fragment comprising at least two Fab fragments with different CH1 and CL domains, the Fab fragments being arranged in tandem in any order, the C-terminal end of the CH1 domain of a first Fab fragment being linked via a polypeptide linker to the N-terminal end of the VH domain of the next Fab fragment, and wherein at least one Fab fragment binds to Vα7.2 and at least one other Fab fragment binds to TAA.

4. 4. The multispecific molecule of claim 3, consisting of a multispecific antigen-binding fragment as defined in claim 3.

5. 4. The multispecific molecule of claim 3, comprising two identical antigen-binding arms, each consisting of a multispecific antigen-binding fragment as defined in claim 3, preferably comprising: - two identical antigen-binding arms, each consisting of a multispecific antigen-binding fragment as defined in claim 3; - dimerized CH2 and CH3 domains of immunoglobulins; - an IgA, IgG, or IgD hinge region linking the C-terminal end of the CH1 domain to the N-terminal end of the CH2 domain of the antigen-binding arm; and more preferably, the multispecific molecule is a bispecific antibody comprising at least two heavy chains and four light chains, each heavy chain further comprising an immunoglobulin Fc region comprising a hinge-CH2-CH3 domain.

6. 6. The multispecific molecule of any one of claims 1 to 5, wherein the TAA is a tumor cell surface antigen expressed on hematological malignant cells or solid tumor cells.

7. 7. The multispecific molecule of claim 6, wherein the TAAs are selected from the group consisting of CD19, CD20, CD38, EGFR, HER2, VEGF, CD52, CD33, RANK-L, GD2, CD33, CEA family (including CEACAM antigens, e.g., CEACAM1, CEACAM5; or PSG antigens), MUC1, PSCA, PSMA, GPA33, CA9, PRAME, CLDN1, HER3, glypican-3, CD22, CD25, CD40, CD30, CD79b, CD138 (syndecan-1), BCMA, SLAMF7 (CS1, CD319), CD56, CCR4, EpCAM, PDGFR-α, Apo2L / TRAIL, and PD-L1.

8. 8. The multispecific molecule of claim 7, wherein the TAA is CD19.

9. 8. The multispecific molecule of claim 7, wherein the TAA is EGFR.

10. 8. The multispecific molecule of claim 7, wherein the TAA is HER2.

11. 11. A host cell transfected with an expression vector comprising a polynucleotide encoding a heavy chain of a multispecific molecule as defined in any one of claims 5 to 10, wherein the host cell has further been transformed with at least two polynucleotides encoding two different light chains of a multispecific molecule as defined in any one of claims 5 to 10: a first light chain that specifically pairs with a first VH / CH1 region of said heavy chain; a second light chain that specifically pairs with a second VH / CH1 region of said heavy chain.

12. 11. A method for producing a multispecific molecule as defined in any one of claims 5 to 10, comprising the steps of: a) culturing in a suitable culture medium and conditions a host cell expressing the heavy chain of an antibody defined in any one of claims 5 to 10 and the light chain of an antibody defined in any one of claims 5 to 10; and b) recovering the produced multispecific molecules from the culture medium or from the cultured cells. A method comprising:

13. 11. A composition for use in treating a tumor in a patient, comprising a multispecific molecule as defined in any one of claims 1 to 10.

14. The composition of claim 13, wherein the tumor is a solid tumor.

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