Multispecific anti-TCR delta variable 1 antibody

Abstract

The present invention relates to multispecific antibodies, particularly bispecific antibodies, that specifically bind to the T cell receptor of gamma delta T cells and one other antigen, as well as fragments and variants thereof. More particularly, the present invention provides at least two new classes of bispecific antibodies: bispecific T cell engagers that bind the variable delta 1 (V51) chain of the gamma delta T cell receptor (TCR) and a cancer antigen or a cancer-associated antigen, and bispecific dual immunomodulators (DIs) that bind the variable delta 1 (V51) chain of the gamma delta T cell receptor (TCR) and an immunomodulatory antigen.

Description

[Technical Field]

[0001] The present invention relates to a multispecific antibody, particularly a bispecific antibody, that specifically binds to the T cell receptor of gamma delta T cells and one other antigen, as well as fragments and variants thereof. [Background technology]

[0002] The growing interest in T-cell immunotherapy for cancer focuses on the apparent ability of subsets of CD8+ and CD4+ alpha-beta (αβ) T cells to recognize cancer cells and mediate host-protective functionality, particularly when suppression is released through clinically mediated antagonism of inhibitory pathways exerted by PD-1, CTLA-4, and other receptors. However, αβ T cells are MHC-restrictive, which can lead to graft-versus-host disease.

[0003] Gamma delta T cells (γδ T cells) represent a subset of T cells that express distinct, typical γδ T cell receptors (TCRs) on their surface. These TCRs consist of one gamma (γ) chain and one delta (δ) chain, each undergoing chain rearrangement, but with a limited number of V genes compared to αβ T cells. The main TRGV gene segments encoding Vγ are TRGV2, TRGV3, TRGV4, TRGV5, TRGV8, TRGV9, and TRGV11, as well as the non-functional genes TRGV10, TRGV11, TRGVA, and TRGVB. The most frequent TRDV gene segments encode Vδ1, Vδ2, and Vδ3, and also encode several V segments with both Vδ and Vα names (Adams et al., 296:30-40 (2015) Cell Immunol.). Human γδ T cells can be broadly classified based on their TCR chains. This is because certain γ and δ types are more commonly found in cells in one or more tissue types, though not exclusively. For example, most blood-resident γδ T cells express Vδ2 TCR, commonly Vγ9Vδ2, which is less common in tissue-resident γδ T cells, such as those found in the skin, which more frequently use Vδ1 TCR, which is paired with the gamma chain, often Vγ4 in the gut, for example.

[0004] γδT cells play a crucial role in immune surveillance, recognizing malignant or transformed cells (such as cancer cells) through patterns of stress markers, and subsequently exerting potent and selective cytotoxicity. Therefore, γδT cells can act as regulators of the immune response. In situ regulation of these cells may lead to increased immunogenicity even in tumors with low mutational weighting, an achievement that has proven difficult with other immunotherapies. γδT cell recognition of tumors is not dependent on any single tumor antigen or γδT cell regulator, and therefore has potential in a wide range of disease manifestations, including both hematological and solid malignancies. The γδT cell recognition mechanism is not MHC-restricted.

[0005] The authors of WO2019147735 hypothesize that some γδ cells have tumorigenic activity or inhibit the anti-cancer immune response mediated by αβ T cells. They assume that γδ T cells are immunosuppressants and therefore suggest that in cancer situations, they should be depleted, inhibited, or blocked using antibodies.

[0006] However, despite the prevailing view that anti-γδ antibodies negatively modulate the function of γδ cells by blocking or killing them, a positive correlation has been found between γδT cell infiltration and patient prognosis and / or survival.

[0007] Compared to αβ TCR receptor / ligand interactions, our understanding of vδ1 TCR receptor / ligand interactions is limited. Given this lack of understanding, antibodies that recognize vδ1 TCRs have primarily served as exploratory tools to investigate this interaction. Such tools are typically unpurified blocking antibodies, suggesting that TCR receptor / ligand interactions result in the blocking, suppression, or ablation of vδ1+ cells. For example, the tool antibodies TS8.2 and TS-1 have been used as anti-γδ blocking antibodies in multiple studies, suggesting that these antibodies reduce the cytotoxicity of vδ1 cells. Combining these findings with others, it is unlikely that such anti-vδ1 antibodies would effectively regulate vδ1 cell cytotoxicity in an in situ disease setting; therefore, antibodies that increase, rather than decrease, vδ1 cytotoxicity are needed.

[0008] To utilize γδ T cells for immunotherapy, a means of amplifying the cells either in situ or ex vivo before harvesting and reinjecting them is required. The latter approach, using the addition of exogenous cytokines, has been previously reported (see, for example, WO2017 / 072367 and WO2018 / 212808). Methods for amplifying a patient's own γδ T cells using pharmacologically modified forms of hydroxy-methylbuta-2-enylpyrophosphate (HMBPP) or clinically approved aminobisphosphonates have been described. While these approaches have seemingly safely treated over 250 cancer patients, complete remission is extremely rare. Nevertheless, there is still a need for activators that have demonstrated the ability to amplify a large number of γδ T cells.

[0009] Furthermore, conjugates or activators that can preferentially target, bind to, recognize, specifically modulate, or increase the number of Vδ1+ cells in situ may be highly desirable as drugs.

[0010] However, while there are drugs that can potentially modulate Vδ2+ cells, including aminobisphosphonates such as Zometa® (zoledronic acid), these drugs are primarily designed to slow bone resorption. Furthermore, regardless of the modulation of Vδ2+, there is a need to develop drugs specifically designed to bind to, target, modulate, activate, or increase the number of Vδ1+ cells. This is because, for example, repeated modulation of Vδ2+ can lead to a persistent and progressively depleting phenotype.

[0011] Furthermore, given the dominant tissue habituation of Vδ1+ cells, an ideal drug capable of modulating Vδ1+ would also exhibit less "off-target" undesirable effects and rapid renal clearance. Typically, the aforementioned undesirable effects can manifest when small molecule chemicals are used. For example, the aforementioned aminobisphosphonates, which have been shown to modulate a distinct class of Vδ2+ cells (as a secondary effect to their primary modulatory effect on bone), are associated with nephrotoxicity that manifests as worsening renal function and potential renal failure (e.g., Markowitz et al. (2003) Kidney Int. 64(1):281-289). Additional undesirable effects listed by the European Medicines Agency for Zometa include anemia, hypersensitivity reactions, hypertension, atrial fibrillation, myalgia, generalized pain, malaise, increased blood urea, vomiting, joint swelling, and chest pain.

[0012] Further investigation is needed into the in situ environment in which vδ1+ cells themselves are found. For example, it has been previously shown that non-hematopoietic tissue-resident γδ T cells exhibit a strong proliferative response when initially isolated from tissue, but this is limited to cases where the cells are not in direct contact with autologous fibroblasts. It has been found that non-hematopoietic tissue-resident T cells (γδ T cells) must be isolated from non-hematopoietic cells (e.g., stromal cells, especially fibroblasts) in order to function. This is because direct contact between lymphocytes and stromal or epithelial cells appears to inhibit the growth of tissue-resident γδ T cells. The observation that pre-activation cells exist in an even more suppressed state in situ is another reason why vδ1 cells have not been considered a promising therapeutic target until now. In fact, until the findings described herein, no method has been devised to effectively and selectively regulate blood and tissue vδ1+ cells in situ when these cells are typically considered to be in a “resting,” “pre-activation,” or “inactivated” state.

[0013] Various formats of bispecific and polyspecific antibodies have been developed for a wide range of therapeutic applications. Bispecific and polyspecific antibodies can be divided into overlapping but distinct classes based on the type of biological target and mode of action. For example, such polyspecific antibodies can be divided into classes such as cytotoxic effector cell redirectors (also known as bispecific T cell recruiting antibodies, bispecific T cell engagers, TCEs, or BiTEs) and dual immunomodulators (DIs).

[0014] TCEs are intended to enhance a patient's immune response to tumors by targeting T cells to tumor cells or vice versa. They work by targeting the first epitope (usually CD3) of the T cell receptor complex on T cells with a second epitope, which is a cancer antigen or tumor-associated antigen (TAA). Such antibodies co-localize with tumor cells and T cells to promote tumor cell killing. Examples of BiTEs include the CD3 x CD19 bispecific antibody blinatumomab, the CD3 x EpCAM bispecific antibody catumaxomab, and the CD3 x HER2 bispecific antibody erzmaxomab. TCEs such as BiTEs are commonly supplied in scFv format, but other formats are also available. For example, BiKE is similar to BiTE but targets CD16 on NK cells instead of CD3.

[0015] The T cell receptor is described as having the most complex receptor structure in the mammalian immune system. It involves a transmembrane multiprotein receptor complex containing several CD3 chains in close proximity to the T cell receptor. For example, in mammals, a typical such complex includes the T cell receptor, CD3γ chain, CD3δ chain, and two CD3ε chains. These chains, along with the combined ζ (zeta) chain, associate with the T cell receptor (TCR), which then generates a typical activation signal in T lymphocytes. However, alternative complexes have also been reported. For example, T cell receptor complexes containing the T cell receptor and zeta chain homodimers have been described. Additional co-receptors, such as CD4 and CD8, can also assist TCR function.

[0016] Regardless of the composition of the receptor complex, it is well established that the complex translates cell surface-bound events into intracellular phosphorylation signaling cascades. These phosphorylation events ultimately lead to the activation of transcription factors such as NFAT and NFκB, resulting in increased expression of cytokines and effector proteins such as granzymes and perforins. However, while treating cancer with such TCEs remains a compelling concept, even after 30 years of coordinated efforts to advance TCEs in early clinical development, many of these bispecific antibodies have shown lackluster safety, efficacy, and manufacturability profiles. In fact, as of January 2020, blinatumomab remains the only TCE that has not been withdrawn after approval. This TCE bispecific antibody fragment binds to the T cell receptor complex with its first binding arm and to the CD19 target with its second binding arm.

[0017] Bispecific T-cell recruiting antibodies are described in Lejeune et al., 2020, Front Immunol., 11:762. However, existing bispecific antibodies in this category, particularly those that recruit T cells via CD3 binding, have significant off-target effects that result in severe adverse effects, given the potency of the CD3 antigen as a signaling factor and its ubiquity in the patient's T-cell population. Therefore, systemic delivery (e.g., intravenously) is practically impossible for examples of CD3-targeting bispecific drugs such as catumaxomab (now retracted). Instead, more restricted delivery, such as intraoperative, intraperitoneal, or intra-abdominal, is more frequently attempted. This limits the selectivity and use of the aforementioned bispecific drugs as pharmaceuticals. In fact, even effector-attenuated anti-CD3 antibodies (i.e., CD3-targeting T-cell complex engagers but not bispecific drugs) are difficult to deliver intravenously (IV) due to associated toxicity. For example, to limit exposure and reduce toxicity, the anti-CD3 antibody foralumab is now almost always intended for oral delivery (e.g., in the treatment of intestinal diseases).

[0018] Many of the current problems observed with such TCEs in early clinical trials are often attributed to the high-affinity T cell complex binding domains used. Furthermore, it has been suggested that this is because the designers of these TCEs did not adequately consider the low affinity of the innate TCR complex binding event, and severe dose-limiting toxicity hindered their development, resulting in a very narrow therapeutic range. In this regard, it has become clear that many early TCE drug developers relied on three anti-CD3 T cell complex binding domains derived from OKT3, SP34, and UCHT1. All of these original binding domains bind with relatively high affinity in the single- to low double-digit nM range, which is nearly 1,000 times higher affinity than the innate binding event. As a result, it has been suggested that T cell activation is affected in a significantly different (and often unfavorable) way compared to the innate binding of the T cell receptor complex. For example, TCE developers using platforms based on the higher-affinity OKT3 may be confused by the fact that OKT3 induces T cell apoptosis in the presence of IL-2.

[0019] For these reasons, the binding of lower affinity T cell complexes has become an important consideration in determining the design parameters for bispecific antibody therapeutics that associate with T cells.

[0020] Another issue in designing the aforementioned TCEs is the need to attenuate Fc function. Indeed, since the binding of Fc to the Fc gamma receptor (FcγR) leads to the activation of immune effector cells, TCEs generally require complete suppression of Fc-mediated effector function to maximize therapeutic efficacy and minimize off-target toxicity. In fact, the vast majority of CD3-targeting bispecific antibodies currently used clinically either have an Fc domain with reduced binding activity to FcγR or are bispecific fragments that intentionally omit the Fc region. Generally, TCEs with unattenuated Fc function would be expected to induce antibody-dependent cell-mediated cytotoxicity (ADCC) effects, thereby depleting the γδ T cell population recognized by the antibody. However, again, attenuating such functionality to avoid toxicity / safety complexities may also attenuate important efficacy considerations, for example, by association with CD16+ or CD32+ or CD64+ immune cells, or by reducing the half-life of bispecific drugs (e.g., when using smaller bispecific antibody fragments such as BITE). Methods that reduce the interaction between FcγR and TCE (e.g., by using an IgG format designed to reduce such interaction) are expected to reduce Fc-mediated immobilization of TCE and reduce TCR clustering by crosslinking with immobilized TCE.

[0021] To address some, though not all, of these problems, many companies, including Xencor (Pasadena, CA), Macrogenics (Gaithersburg, MD), and Genentech (San Francisco, CA), have recently reported reducing the binding affinity of the T cell receptor complex binding arm in their respective TCE platforms. However, reducing this binding affinity can lead to decreased efficacy and selectivity in terms of TCE design and functionality. For example, it has now been demonstrated that the affinity of the binding domain in such TCEs drives the in vivo distribution profile. Specifically, the distribution of TCEs is usually observed to be biased towards the target with the highest affinity. Therefore, reducing the affinity of the TCE binding domain to the T cell complex generally moves the distribution away from the T cells that are precisely the cells necessary to drive the efficacy of such TCEs. This is partly why the therapeutic range of TCEs is said to be "extremely narrow."

[0022] Therefore, there is a need for improved TCE-type drugs that address these issues with existing and candidate therapies, including a platform that provides safer therapeutics with reduced adverse side effects, offering greater selectivity and independence from inactivation or attenuation of the Fc domain or specific affinity to the TCR complex.

[0023] Dual immunomodulatory antibodies (DIs) are antibodies that bind to two different immunomodulatory targets. Immunomodulatory targets include, for example, immune checkpoint inhibitors such as PD-L1, PD-1, OX40, CTLA-4, LAG-3, TIM-3, TIGIT, and VISTA (see, e.g., Qin et al., 2019, Molecular Cancer, 18:155). Typical examples of compounds in this class combine targeting of two immunomodulatory signaling pathways. Such DIs typically contain at least two binding domains, each targeting an epitope on two distinct target proteins or complex proteins present on immune cells such as T cells. To date, it has never been conceived, nor considered practically possible, to generate a DI that targets at least two immunomodulatory targets that help overcome the immunosuppressive tumor microenvironment and transform it from "cold" to "hot," promoting the accumulation of pro-inflammatory cytokines and T cell infiltration, as well as tumor cell killing, with one such target being the TCRδ1 chain. Existing DI approaches have been studied in attempts to reduce or overcome the high toxicity associated with combination therapies (and improve efficacy compared to combination treatments), but many require careful monitoring and have mixed success rates. There is still a need for alternative immunomodulatory approaches to treat conditions such as cancer, and this invention provides a completely novel alternative approach to DI antibodies that has never been attempted before.

[0024] Therefore, there is a need for improved drugs, particularly multispecific and bispecific antibodies, to treat infectious diseases, autoimmune conditions, and cancer. [Overview of the project]

[0025] The present invention relates to a multispecific antibody that targets γδT cells and another antigen. Accordingly, a first aspect of the present invention provides a multispecific antibody or fragment thereof that specifically binds to a first target epitope, which is an epitope of the variable delta (Vδ) chain of the γδT cell receptor (TCR), and a second target epitope. In a preferred embodiment, the multispecific antibody binds to a first target epitope, which is an epitope of the variable delta 1 (Vδ1) chain of the γδT cell receptor (TCR), and a second target epitope.

[0026] The polyspecific antibodies of the present invention can be divided into two classes. The first is polyspecific antibodies that are T cell engagers. The second is polyspecific antibodies that are dual immunomodulators.

[0027] Accordingly, a second aspect of the present invention provides a polyspecific antibody that specifically binds to a first target epitope and a second target epitope, wherein the first target epitope is an epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR), and the second target epitope is an epitope of a cancer antigen or cancer-associated antigen.

[0028] In a third aspect of the present invention, a polyspecific antibody is provided that specifically binds to a first target epitope and a second target epitope, wherein the first target epitope is an epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR), and the second target epitope is an epitope of an immunomodulatory antigen.

[0029] A fourth aspect of the present invention provides a polynucleotide sequence encoding a polyspecific antibody or a fragment thereof.

[0030] A fifth aspect of the present invention provides an expression vector comprising the polynucleotide sequence of the present invention. A host cell comprising the polynucleotide sequence of the present invention or the expression vector of the present invention is also provided. A method for producing any of the polyspecific antibodies or fragments thereof of the present invention is also provided, comprising culturing the host cell of the present invention in a cell culture medium.

[0031] In a further embodiment of the present invention, a composition comprising the polyspecific antibody or a fragment thereof is provided. A pharmaceutical composition comprising the polyspecific antibody or a fragment thereof and a pharmaceutically acceptable diluent or carrier is also provided.

[0032] In a further embodiment of the present invention, a kit is provided comprising the multispecific antibody or fragment of the present invention or the pharmaceutical composition of the present invention, and optionally comprising instructions for use and / or additional therapeutic agents.

[0033] A further embodiment of the present invention provides a method for treating a target disease or disorder, comprising administering to a subject a multispecific antibody or fragment of the present invention, or a pharmaceutical composition of the present invention. Also provided is a method for modulating the immune response of a subject, comprising administering to a subject a multispecific antibody or fragment of the present invention, or a pharmaceutical composition of the present invention.

[0034] In a further embodiment of the present invention, a method for preparing a pharmaceutical composition is provided, comprising preparing an antibody prepared according to the method for preparing a polyspecific antibody or fragment of the present invention, and combining the polyspecific antibody with at least one pharmaceutically acceptable diluent or carrier.

[0035] In a further embodiment of the present invention, a method for generating a polyspecific antibody of the present invention is provided, comprising the steps of: selecting a first monospecific antibody that specifically binds to a first target epitope, wherein the first target epitope is an epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR); and combining the antibody or an antigen-binding fragment of the first antibody with an antibody or a fragment thereof that includes a binding domain targeting a second epitope to generate a recombinant polyspecific antibody.

[0036] Further embodiments of the present invention provide a multispecific antibody or fragment thereof, a pharmaceutical composition, or a kit for use in pharmaceuticals. The use of the multispecific antibody or fragment thereof in the manufacture of drugs is also provided. [Brief explanation of the drawing]

[0037] [Figure 1] ELISA detection of directly coated antigens using anti-Vδ1Ab (REA173, Miltenyi Biotec). Detection was observed only with antigens containing the Vδ1 domain. The leucine zipper (LZ) format appeared to be more potent than the Fc format, which is consistent with cell-based flow-competitive assays (data not shown). [Figure 2] Polyclonal phage DELFIA data for DV1 selection. (A) Heterodimer selection: Heterodimer LZ TCR format in rounds 1 and 2, deselection of heterodimer LZ TCR in both rounds. (B) Homodimer selection: Round 1 was performed using homodimer Fc fusion TCR to deselect human IgG1 Fc, followed by Round 2 for heterodimer LZ TCR to deselect heterodimer LZ TCR. Each graph contains two bars for each target to represent selection from different libraries. [Figure 3] IgG capture: Left) Sensorgram of the interaction between anti-L1 IgG and L1, Right) Steady-state fit when available. All experiments were performed at room temperature using a MASS-2 instrument. Steady-state fitting follows Langmuir 1:1 binding. [Figure 4] Results of TCR downregulation assays for clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07 and 1251_P02_C05(A), or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09, 1251_P02_G10 and 1252_P01_C08(B). [Figure 5] Results of T cell degranulation assays for clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07, and 1251_P02_C05(A), or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09, and 1251_P02_G10(B). [Figure 6] Results of a killing assay (THP-1 flow-based assay) for clones 1245_P01_E07, 1252_P01_C08, 1245_P02_G04, 1245_P01_B07 and 1251_P02_C05(A), or clones 1139_P01_E04, 1245_P02_F07, 1245_P01_G06, 1245_P01_G09, 1138_P01_B09 and 1251_P02_G10(B). [Figure 7] Total cell count in Experiment 1 of Example 10. Samples were cultured with various concentrations of the anti-Vδ1 antibody described herein and compared with samples cultured with a comparator antibody or a control. The graphs show the total cell counts on (A) day 7, (B) day 14, and (C) day 18. [Figure 8] Analysis of Vδ1 T cells in Experiment 1 of Example 10. The graph shows (A) the percentage of Vδ1 T cells, (B) the number of Vδ1 T cells, and (C) the Vδ1 change ratio in the sample on day 18. [Figure 9] Total cell count in Experiment 2 of Example 10. Samples were cultured with various concentrations of the anti-Vδ1 antibody described herein and compared with samples cultured with a comparator antibody or a control. The graphs show the total cell count on (A) day 7, (B) day 11, (C) day 14, and (D) day 17. [Figure 10] Analysis of Vδ1 T cells in Experiment 2 of Example 10. The graph shows (A) the percentage of Vδ1 T cells, (B) the number of Vδ1 T cells, and (C) the Vδ1 change ratio in the sample on day 17. [Figure 11]Cell composition analysis. The cell types present in the sample (including non-Vδ1 cells) were measured on day 17 of Experiment 2. Cells were collected, and the surface expression of Vδ1, Vδ2, and αβTCR was analyzed by flow cytometry. Percentage values ​​are also shown in Table 7. [Figure 12] SYTOX flow chemotylation assay results. Cell functionality was tested using the SYTOX flow chemotylation assay, and results are presented for (A) Experiment 1 at day 14 using cells with a 10:1 effector-to-target (E:T) ratio, and (B) Experiment 2 at day 17 (after freeze-thaw) using cells with 1:1 and 10:1 E:T ratios. [Figure 13] Total cell count after freeze-thawing. The graph shows the total cell count after 7 days of cell culture following freeze-thawing for cultures that were contacted with B07, C08, E07, G04, or OKT-3 antibodies before freezing. [Figure 14] Monitoring of cell growth. The total cell count of cells cultured after freezing and thawing was monitored until day 42. [Figure 15] Binding equivalence study of modified anti-Vδ1 antibody. [Figure 16] Equivalence studies of anti-Vδ1 antibody binding to human germline Vδ1 antigen and its polymorphic variants. [Figure 17] Anti-Vδ1 antibody induced elevated cytokine secretion levels in Vδ1+ cells. Tissue-derived γδT cells were incubated with the indicated antibody. (A) Observed TNF-alpha levels. (B) Observed IFN-gamma levels. [Figure 18] Anti-Vδ1 antibodies induced elevated granzyme B levels / activity in Vδ1+ cells. Cancer cells were co-cultured for 1 hour with tissue-derived γδT cells in a constant 1:20 T:E ratio and with the indicated antibodies. The results reveal the amount of granzyme B detected in cancer cells at the end of co-culture. [Figure 19]Anti-Vδ1 antibodies conferred regulation and proliferation of immune cells in human tissue. Human skin punch biopsies (from 5 different donors) were incubated with the indicated antibodies in culture for 21 days. (A) Number of viable pan-γδ+ cells. (B) Number of viable Vδ1+ cells. (C) Percentage of viable double-positive Vδ1+CD25+ cells. [Figure 20] Anti-Vδ1 antibodies conferred regulation and proliferation of tumor-infiltrating lymphocytes (TILs) in human tumors. Study on renal cell carcinoma (RCC) ± antibodies. (A) Increase ratio of TIL Vδ1+ cells. (B) Total number of TIL Vδ1+ cells. (C) Example of a gating strategy. (D) Comparison of cell surface phenotypic profiles of TIL Vδ1+ cells. (E) Analysis of TIL Vδ1-negative, gated fractions. [Figure 21] Anti-Vδ1 antibodies conferred enhanced Vδ1+-mediated cytotoxicity and disease-cell-specific cytotoxicity. Cytotoxicity / efficacy assays in a model system including tripartite cultures of Vδ1+ effector cells, THP-1 monocytic cancer cells, and healthy, non-disease primary monocytes. (A) Quantification of the number of THP-1 and monocyte cells in triple co-culture using γδT cells in the presence of anti-Vδ1 mAb or control. (B) Bar graphs highlighting the window between disease-specific killing and non-disease healthy cells: Left bar graph; increase in killing of disease cells (THP-1) compared to killing of non-disease cells (primary human monocytes); Right bar graph; same data, but expressed as the rate of increased killing compared to control. (C) Tabular results summarizing the rate of improvement in Vδ1+ effector cell killing efficacy of THP-1 target cells ± mAb. (D) A tabular result of the EC50 value calculated from Figure (A), expressed as the number of γδ T cells required to induce 50% THP-1 cell toxicity. [Figure 22]The polyspecific antibody conferred enhanced Vδ1+ effector cell-mediated cytotoxicity. Targeting of tissue-centric disease-associated antigens: (A-D) Examples of co-culture of Vδ1+ effector cells and A-431 cancer cells with or without (+ / -) polyspecific antibodies containing an anti-Vδ1 × anti-TAA(EGFR) bispecific binding site, which combines the anti-Vδ1 VL+VH binding domain (for the first target) with the CH1-CH2-CH3 domain of the anti-EGFR binding site (for the second target). (E~H) Examples of co-culture of Vδ1+ effector cells and A-431 cancer cells with or without a multispecific antibody containing an anti-Vδ1 × anti-TAA(EGFR) bispecific binding site, in which the anti-Vδ1 binding domain (for the first target) contains a full-length antibody (VH-CH1-CH2-CH3 / VL-CL), which is then combined with an anti-EGFR cetuximab-derived scFv binding site (for the second target). (I~J) Alternative approaches to express the data: Percentage of improvement in Vδ1+ effector cytotoxicity against EGFR+ cells relative to the constituent parts, as conferred by the multispecific antibody. [Figure 23] The polyspecific antibody conferred enhanced Vδ1+-mediated cytotoxicity and disease cell-specific cytotoxicity. Targeting of hematopoietic disease-related antigens. (A) E:T ratio required to induce 50% Raji cell toxicity. (B) Percentage improvement by addition of Vδ1-CD19 polyspecific antibody. [Figure 24] The anti-Vδ1 / CD19 bispecific antibody exhibits high affinity binding to human Vδ1 and cynomolgus monkey Vδ1, comparable to that of the parental monoclonal Vδ1 mAb. Surface plasmon resonance (SPR) analysis was performed using the Vδ1 / CD19 bispecific agent to evaluate its binding to human Vδ1 and CD19 antigens. The bispecific antibody bound to both human and cynomolgus monkey Vδ1, with only a slight reduction in affinity for both antigens. [Figure 25]The Vδ1-CD19 bispecific T cell engager enhances CD19+ target cytotoxicity and γδ T cell activation while preserving healthy CD19+ B cells. (A) CD19 expression in cancerous NALM-6, Raji, B cells, and Vδ1γδ T cells as determined by flow cytometry. (B-D) Effect of anti-Vδ1-CD19 BiTE on NALM-6 cells (B), Raji cells (C), and B cell toxicity (D). Antibodies were titrated over 12 hours in the presence of Vδ1γδ T cells with a 1:1 E:T ratio. Viability was calculated by high-content confocal microscopy and normalized to the number of viable cells in the absence of Vδ1γδ T cells. (E) Bar graphs showing the percentage of viable cells after 12 hours of co-culture of Vδ1γδT cells with either NALM-6, Raji, or B cells in the presence of Vδ1 BiTE and a control, as shown in (B), (C), and (D). (F~G) Effect of Vδ1-CD19 BiTE on Vδ1 TCR surface expression in the presence of NALM-6 target cells (F) and healthy B cells (G), as determined by flow cytometry after 4 hours of co-culture. (H) Bar graphs showing the maximum percentage of TCR downregulation shown in (F) and (G) at the highest concentration (3 μg / ml). (I~J) Effect of the Vδ1-CD19 bispecific drug on upregulation of CD107a on the surface of Vδ1 cells in the presence of NALM-6 target cells (I) and healthy B cells (J), as determined by flow cytometry after 4 hours of co-culture. (K) Upregulation of CD107a in B cells and NALM-6 cells. All data are shown as mean ± standard deviation and are representative of n=3. Blinatumomab (CD3-CD19 BiTE) was included as a control in all assays. [Figure 26]Bispecificity format affinity-matured Vδ1 clones bind to Her2+ target cells, exhibiting enhanced binding to Vδ1γδT cells and enhanced cytotoxicity of Her2+ target cells. Cell surface expression of Her2(A) and Vδ1(B) in breast cancer cell lines and Vδ1γδT cells. (C~E) Binding of Vδ1-Her2 bispecificity antibody and Her2 mAb control (trastuzumab) to Her2+ (SK-BR-3(C), BT-474(D)), Her2- (MDA-MD-231(E)), and Vδ1+ cells (F). (G~I) Viability of cells remaining after 24 hours of co-culture of Vδ1γδT cells with SK-BR-3 cells (G), BT-474(H), and MDA-MB-231 cells (I) in a 1:1 E:T ratio in the presence of Vδ1-Her2 bispecificity antibody. (J) Bar graph showing the rate of increase in cytotoxicity in Vδ1γδT cells with Vδ1-Her2 bispecific antibody after 24 hours. [Figure 27A] Anti-Vδ1 / EGFR bispecific antibodies exhibit high affinity binding to human EGFR and human Vδ1 binding affinity comparable to their parent mAb. Surface plasmon resonance (SPR) analysis was performed using Vδ1 / EGFR bispecific drugs to evaluate their binding to human Vδ1. For comparison, the parent mAb, cetuximab, and a negative control mAb were included. [Figure 27B] Anti-Vδ1 / EGFR bispecific antibodies exhibit high affinity binding to human EGFR and human Vδ1 binding affinity comparable to their parent mAb. Surface plasmon resonance (SPR) analysis was performed using Vδ1 / EGFR bispecific drugs to evaluate their binding to the human EGFR antigen. For comparison, the parent mAb, cetuximab, and a negative control mAb were included. [Figure 28]The Vδ1 / EGFR bispecific antibody binds to EGFR+A431 target cells and Vδ1γδT cells. (A-D) Cell surface expression of EGFR in A431 cell lines and primary Vδ1γδT cells. (E, F) Level of binding of anti-Vδ1 / EGFR bispecific antibody to A431 cell lines or primary Vδ1γδT cells. Target cells were stained with various concentrations of antibody and then stained with a fluorescent anti-human IgG detection antibody. All incubation steps were performed at 4°C, and mAb binding was determined by measuring the median fluorescence level using flow cytometry. Log-scale 4-parameter dose-response curve fitting was performed using GraphPad Prism 9. [Figure 29] The Vδ1 / EGFR bispecific antibody induces activation and degranulation of EGFR-specific T cells, resulting in increased γδ T cell-mediated cytotoxicity of A431 target cells. (A) Cell surface expression of γδ TCR in primary Vδ1γδ T cells after 24 hours of culture with the bispecific antibody in or without A431 cells. (B, C) Number of viable A431 cells (B) and activation status of primary Vδ1γδ T cells after 24 hours of co-culture with various concentrations of antibody in a 1:1 ratio. Viability was measured using a viability-determinating dye, and activation status was measured using CD25 antibody. (D) Degranulation of primary Vδ1γδ T cells after 4 hours of co-culture with A431 cells in a 1:1 ratio with various concentrations of antibody. Degranulation was determined by directly adding anti-CD107α antibody conjugated to a fluorophore to the cell-antibody mix at the start of co-culture. (E) Number of viable A431 cells after 24 hours co-culture with 10 pM antibody and varying amounts of primary Vδ1γδT cells. (A-E) In all cases, fluorescence was determined using flow cytometry to measure the median fluorescence level. Log-scale 4-parameter dose-response curve fitting was performed using GraphPad Prism 9. Data are expressed as mean ± SD of two biological replicas. [Figure 30]Downregulation of CD3. (A) Shows MFI of vδ1 TCR upon antibody stimulation as an indicator of mAb target association. (B) Shows MFI of CD3 expression in positively gated vδ1 cells. Stimulation with vδ1 antibody associated vδ1 cells and resulted in downregulation of both vδ1 and CD3 on vδ1 cells. [Figure 31] The Vδ1×FAPα bispecific antibody enhances the activation of Vδ1γδT cells and the lysis of FAPα+ fibroblasts. (A) Shows the binding kinetics of anti-Vδ1, anti-FAPα monoclonal, and anti-Vδ1×FAPα bispecific antibodies to recombinant human Vδ1 and FAPα, as determined by surface plasmon resonance (SPR). (B, C) Binding of anti-Vδ1 antibody and anti-FAPα antibody to FAPα+ fibroblasts (B) and Vδ1γδT cells (C). (D, E) Effects of anti-Vδ1×FAPα bispecific antibody and monoclonal control on the downregulation of Vδ1 TCR in Vδ1γδT cells in the absence (D) or presence (E) of FAPα+ fibroblasts. (F~G) Effects of anti-Vδ1×FAPα bispecific antibody and monoclonal control on the upregulation of CD107a in Vδ1γδT cells in the absence (F) or presence (G) of FAPα+ fibroblasts. (H) Effects of anti-Vδ1×FAPα bispecific antibody and monoclonal control on the lysis of fibroblasts by Vδ1γδT cells after 24 hours of co-culture. Cell viability was calculated by high-content confocal microscopy and normalized to the number of viable cells in the absence of Vδ1γδT cells. [Figure 32]Vδ1×MSLN bispecific antibodies enhance the activation of Vδ1γδT cells and the lysis of MSLN+ target cells. (A) Binding kinetics of anti-Vδ1, anti-MSLN monoclonal, and anti-Vδ1×MSLN bispecific antibodies to recombinant human Vδ1 and MSLN, as determined by surface plasmon resonance (SPR). (B, C) Binding of anti-Vδ1 and anti-MSLN antibodies to MSLN+ HeLa cells (B) and Vδ1γδT cells (C). (D, E) Effects of anti-Vδ1×MSLN bispecific and monoclonal antibodies on the downregulation of Vδ1 TCR in Vδ1γδT cells in the absence (D) or presence (E) of MSLN+OVCAR-3 cells. (F, G) Effects of anti-Vδ1×MSLN bispecific antibody and monoclonal control on the upregulation of CD107a in Vδ1γδT cells in the absence (F) or presence (G) of MSLN+OVCAR-3 cells. (H) Effects of anti-Vδ1×MSLN bispecific antibody and monoclonal control on the lysis of HeLa cells by Vδ1γδT cells after 24 hours of co-culture. Cell viability was calculated by high-content confocal microscopy and normalized to the number of viable cells in the absence of Vδ1γδT cells. [Figure 33]Vδ1×PD-1 bispecific antibodies enhance the activation of Vδ1γδT cells and inhibit checkpoint blockade of PD-1+ T cells. A) SPR analysis of anti-Vδ1, anti-PD-1, anti-RSVIgG control × anti-PD-1, and anti-Vδ1×anti-PD-1 bispecific antibodies binding to recombinant human Vδ1 and PD-1. B) Double binding of anti-Vδ1 and anti-PD-1 bispecific antibodies to recombinant human PD-1 and Vδ1 as determined by SPR. C) Expression of PD-1 in CD4 and CD8 T cells activated with anti-CD3 / anti-CD28 Dynabeads. D) Binding of anti-Vδ1 and anti-PD-1 antibodies to PD-1+ activated CD4 and CD8 T cells and Vδ1γδT cells. E) Effects of anti-Vδ1×PD-1 bispecific antibodies and monoclonal controls on downregulation of Vδ1 TCR in Vδ1γδT cells in the absence or presence of PD-1+CD4 T cells. F) EC50 of anti-Vδ1×PD-1 bispecific antibody against downregulation of Vδ1 TCR in Vδ1γδT cells in the presence or absence of PD-1+CD4 T cells. G) Effect of Vδ1-crosslinked anti-Vδ1×PD-1 bispecific antibody on PD-1+ T cell activation. [Figure 34] The Vδ1×4-1BB bispecific antibody enhances the activation of Vδ1γδT cells and 4-1BB+ T cells. A) SPR analysis of anti-Vδ1, anti-4-1BB, anti-RSVIgG control × anti-4-1BB, and anti-Vδ1×anti-4-1BB bispecific antibodies binding to recombinant human Vδ1 and 4-1BB. B) Double binding of anti-Vδ1 and anti-4-1BB bispecific antibodies to recombinant human 4-1BB and Vδ1 as determined by SPR. C) Expression of 4-1BB in CD4 and CD8 T cells activated with anti-CD3 / anti-CD28 Dynabeads. D) Binding of anti-Vδ1 and anti-4-1BB antibodies to 4-1BB+ activated CD8 T cells and Vδ1γδT cells. E-F) Effects of anti-Vδ1×4-1BB bispecific antibody and monoclonal control on the downregulation of Vδ1 TCR in Vδ1γδT cells in the absence (E) or presence (F) of 4-1BB+CD8 T cells. G) Effects of Vδ1 crosslinked anti-Vδ1×4-1BB bispecific antibody on the activation of 4-1BB+ T cells. [Figure 35]Vδ1×OX40 bispecific antibodies enhance the activation of Vδ1γδT cells and OX40+ T cells. A) SPR analysis of anti-Vδ1, anti-OX40, anti-RSVIgG control × anti-OX40, and anti-Vδ1×anti-OX40 bispecific antibodies binding to recombinant human Vδ1 and OX40. B) Double binding of anti-Vδ1 and anti-OX40 bispecific antibodies to recombinant human OX40 and Vδ1 as determined by SPR. C) Expression of OX40 in CD4 and CD8 T cells activated with anti-CD3 / anti-CD28 Dynabeads. D) Binding of anti-Vδ1 and anti-OX40 antibodies to OX40+ activated CD4 T cells and Vδ1γδT cells. E~F) Effects of anti-Vδ1×OX40 bispecific antibodies and monoclonal controls on the downregulation of Vδ1 TCR in Vδ1γδT cells in the absence (E) or presence (F) of OX40+CD4 T cells. G) Effect of Vδ1-crosslinked anti-Vδ1×OX40 bispecific antibody on the activation of OX40+ T cells. [Figure 36] The Vδ1×TIGIT bispecific antibody enhances the activation of Vδ1γδT cells and inhibits checkpoint blockade of TIGIT+ T cells. A) SPR analysis of anti-Vδ1, anti-TIGIT, anti-RSVIgG control × anti-TIGIT, and anti-Vδ1×anti-TIGIT bispecific antibodies binding to recombinant human Vδ1 and TIGIT. B) Double binding of anti-Vδ1 and anti-TIGIT bispecific antibodies to recombinant human TIGIT and Vδ1 as determined by SPR. C) TIGIT expression in CD4 and CD8 T cells activated with anti-CD3 / anti-CD28 Dynabeads. D) Binding of anti-Vδ1 and anti-TIGIT antibodies to TIGIT+ activated CD4 and CD8 T cells and Vδ1γδT cells. E) Effects of anti-Vδ1×TIGIT bispecific antibody and monoclonal control on downregulation of Vδ1 TCR in Vδ1γδT cells in the absence or presence of TIGIT+CD8 T cells. F) EC50 of an anti-Vδ1×TIGIT bispecific antibody against the downregulation of Vδ1 TCR in Vδ1γδT cells in the presence or absence of TIGIT+CD8 T cells. G) Effect of a Vδ1 crosslinked anti-Vδ1×TIGIT bispecific antibody on the activation of TIGIT+ T cells. [Figure 37]The ADCC reporter bioassay does not show ADCC as a result of anti-vδ1 antibody. Target cells, i.e., γδ cells, were incubated with ADCC bioassay effector cells in the presence of anti-vδ1 antibody, anti-vδ1 LAGA antibody (Fc inactive), and RSV isotype control. Luminescence signals were recorded as relative light units (RLU), and induction magnification was calculated as described in the methods. N=2 γδ donors (technical duplicate) were used for "anti-vδ1 antibody," "anti-vδ1 LAGA antibody," "RSV," and "OKT3." N=1 Raji cell line was used for the "rituximab + Raji" condition (technical duplicate), and n=1 γδ donor was used for the "anti-vδ1 antibody + effector" and "anti-vδ1 LAGA antibody + effector" conditions (technical duplicate and single-dose, respectively). The effector:target ratio was 3:1. [Figure 38] The Vδ1-CD19 bispecific T cell engager enhances CD19+ target cytotoxicity and γδ T cell activation while preserving healthy CD19+ B cells. A-F) Effects of anti-Vδ1×CD19 and CD3×CD19 bispecific antibodies on γδ T cell or αβ T cell-mediated lysis of Raji cells or healthy primary B cells. The positivity of Raji cells or healthy primary B cells was determined at 24 hours by confocal microscopy in three cultures: Vδ1γδ T cells, Raji cells (A), and healthy primary B cells (B); three cultures: αβ T cells, Raji cells (C), and healthy primary B cells (D); or four cultures: Vδ1γδ T cells, αβ T cells, Raji cells (E), and healthy primary B cells (F). G-I) Quantification of IL-17A secretion from γδT cells or αβT cells 24 hours after stimulation with anti-Vδ1×CD19 and CD3×CD19 bispecific antibodies. Cell culture supernatants were collected from Raji cells, primary B cells, and co-cultures of γδT cells (G); or αβT cells (H); or γδT cells and αβT cells (I), and IL-17A secretion was determined by MSD. The dotted line represents the lowest level of quantification. [Modes for carrying out the invention]

[0038] The present invention provides a novel class of polyspecific antibodies that target a variable delta chain, such as the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR), and a second antigen. The second antigen may be, for example, a cancer antigen or cancer-associated antigen (such as TAA), and therefore the antibody may be referred to as a T cell engager (TCE). Alternatively, the second antigen may be, for example, an immunomodulatory antigen, and therefore the antibody may be a dual immunomodulatory antibody.

[0039] The TCE of the present invention offers several advantages compared to prior art TCEs. In particular, the TCE can overcome many of the challenges associated with prior art TCEs by targeting the T cell receptor complex through an entirely novel and unique mechanism. Indeed, by specifically targeting (and activating) the T cell receptor complex solely by binding to an epitope on the TRDV1 domain, several advantages are realized, including: • They associate with only some T cells, not all of them (for example, T-reg association may be undesirable in cancerous situations); • It is primarily "tissue-resident," and its presence is often positively correlated with a favorable prognosis in cancer / tumor situations; it associates only with certain T cells (TRDV1+ T cells); • Activating the T cell receptor complex via TRDV1 association leads to greater selectivity (e.g., increasing the affinity of this binding domain). For example, by developing recombinant TCEs that associate with the T cell receptor complex solely via the TRDV1 domain and not via CD3, the increased affinity may drive more desirable functionality. For example, high-affinity TRDV1-binding TCEs may activate T cells without depleting them; and / or • By associating with the TCE complex via the novel means and via the recombinant TRDV1 binding domain, adverse effects may be reduced, thus reducing the need to attenuate the Fc functionality at the TCE site. This can then provide additional selectivity, for example, by associating with TRDV1+ cells via one binding domain to the TRDV1 TCE, associating with a second cell type (such as cancer cells) via a second binding arm, and associating with other effector cells such as CD16+ immune cells, CD32+ immune cells, or CD64+ immune cells via a functional Fc domain.

[0040] Therefore, through the discoveries described herein, the inventors have generated a novel class of recombinant TCEs. Specifically, the inventors have discovered a novel class of TCEs that associate with the T cell receptor via the TRDV1 domain rather than other domains in the T cell receptor signaling complex. More specifically, the inventors have discovered a novel class of TCEs that associate with this complex via the TRDV1 activation epitope and can bind with higher affinity without potentially conferring some of the adverse effects associated with the association of previously reported high-affinity T cell receptor complexes. Furthermore, this novel class of TCEs can associate in a manner that is also tolerant of wild-type Fc functionality, thereby offering the potential for additional efficacy.

[0041] The present invention provides a completely novel method of dual immunomodulatory target association, offering broad potential for novel therapies requiring immunomodulation, such as cancer. The present invention's DI platform provides a new class of therapeutic agents that may represent a significant alternative or improvement over existing DI approaches. Previously, it was not intended that the TRDV1-specific binding function described herein could be incorporated into a DI format.

[0042] The polyspecific antibodies of the present invention may exhibit improved properties compared to equivalent monospecific antibodies. For example, the polyspecific antibodies of the present invention may exhibit improved properties compared to monospecific antibodies having the same antigen-binding domain as the components of the polyspecific antibody. In some embodiments, for example, recombinant polyspecific antibodies confer increased gamma delta T cell-mediated cytotoxicity to disease cells expressing a second epitope compared to the cytotoxicity conferred by an equivalent amount of the first monospecific antibody. The polyspecific antibodies of the present invention may also exhibit improved cytotoxicity against disease cells while still preserving healthy cells.

[0043] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this invention pertains. As used herein, the following terms have the meanings given below.

[0044] Gamma delta (γδ) T cells represent a small subset of T cells that express distinct, typical T cell receptors (TCRs) on their surface. This TCR consists of one gamma (γ) chain and one delta (δ) chain. Each chain contains a variable (V) region, a constant (C) region, a transmembrane region, and a cytoplasmic tail. The V region contains the antigen-binding site. Human γδ T cells have two main subtypes: one dominant in peripheral blood and the other dominant in non-hematopoietic tissues. The two subtypes can be defined by the type of δ and / or γ present in the cell. For example, γδ T cells dominant in peripheral blood primarily express a delta variable 2 chain (Vδ2). γδ T cells dominant in non-hematopoietic tissues (i.e., tissue-resident) primarily express a delta variable 1 chain. References to "Vδ1 T cells" or "Vδ1+ T cells" refer to γδ T cells that possess a Vδ1 chain, i.e., Vδ1 + It refers to cells.

[0045] References to "Delta Variable 1" may also be referred to as Vδ1 or Vd1, and nucleotides encoding the TCR chain containing this region, or TCR protein complexes containing this region, may be referred to as "TRDV1". Any antibody or fragment that interacts with the Vδ1 chain of the γδTCR is, in effect, an antibody or fragment that binds to Vδ1 and may be referred to as "anti-TCR Delta Variable 1 antibody or fragment" or "anti-Vδ1 antibody or fragment" or "anti-TRDV1 antibody or fragment" or "anti-TRDV1 antibody or fragment".

[0046] This specification further refers to other delta chains, such as the “Delta Variable 2” chain. These may be referred to in a similar manner. For example, the Delta Variable 2 chain may be referred to as Vδ2, and a nucleotide encoding a TCR chain containing this region, or a TCR protein complex containing this region, may be referred to as “TRDV2”. In a preferred embodiment, an antibody or fragment thereof that interacts with the Vδ1 chain of the γδTCR does not interact with other delta chains such as Vδ2. In the present invention, the antibody is specific to TRDV1 and does not bind to other antigens such as TRDV2 (SEQ ID NO: 174) or TRDV3 (SEQ ID NO: 175).

[0047] This specification also refers to “gamma variable chains.” These may be referred to as γ chains or Vγ, and nucleotides encoding a TCR chain containing this region, or TCR protein complexes containing this region, may be referred to as TRGV. For example, TRGV4 refers to the Vγ4 chain. In preferred embodiments, an antibody or fragment that interacts with the Vδ1 chain of the γδTCR does not interact with gamma chains such as Vγ4 (TRGV4, SEQ ID NO: 173). In preferred embodiments, the antibody neither binds to nor interacts with other domains found within the γδTCR, such as TRDJ, TRDC, TRGJ, or TRGC.

[0048] The term "T cell receptor complex" refers to a complex of proteins containing the "T cell receptor" (or "TCR") found on the surface of T cells, which are involved in recognizing various antigens. While the exact composition of a T cell receptor complex can vary, it typically includes either the alpha and beta chains of the T cell receptor, or, in the case of gamma-delta T cells, the gamma and delta chains of the T cell receptor, along with up to six or more additional chains, such as CD3δ, CD3γ, CD3ε, and CD3ζ. The T cell receptor complex mediates intracellular signaling in T cells, which can lead to T cell activation.

[0049] The term "antibody" includes any antibody protein construct that includes at least one antibody variable domain, which includes at least one antigen-binding site (ABS). Antibodies include, but are not limited to, IgA, IgG, IgE, IgD, and IgM type immunoglobulins (and their subtypes). The overall structure of immunoglobulin G (IgG) antibodies assembled from two identical heavy (H) chain polypeptides and two identical light (L) chain polypeptides is well established and highly conserved in mammals (Padlan (1994) Mol.Immunol.31:169-217).

[0050] Conventional antibodies or immunoglobulins (Ig) are proteins containing four polypeptide chains, namely two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy (H) chain variable domain is abbreviated herein as VH, and the light (L) chain variable domain is abbreviated herein as VL. These domains, their associated domains, and domains derived therefrom may be referred herein as immunoglobulin chain variable domains. The VH domains and VL domains (also referred to as VH regions and VL regions) may be further divided into regions called "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions called "framework regions" ("FRs"). The framework regions and complementarity-determining regions are clearly defined (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition USD, Department of Health and Human Services, (1991), NIH Publication Number 91-3242). There are alternative numbering conventions for CDR sequences, such as those presented in Chothia et al. (1989) Nature 342:877-883, or those compiled by IMGT.org. In conventional antibodies, each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Conventional antibody tetramers, having two immunoglobulin heavy chains and two immunoglobulin light chains, are formed by immunoglobulin heavy and light chains interconnected, for example, by disulfide bonds, as well as similarly connected heavy chains. The heavy chain constant region contains three domains CH1, CH2, and CH3. The light chain constant region consists of one domain CL. The variable domains of the heavy chain and light chain are binding domains that interact with the antigen. The constant region of an antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0051] As used herein, an antibody fragment (which may also be referred to as “antibody fragment,” “immunoglobulin fragment,” “antigen-binding fragment,” or “antigen-binding polypeptide”) refers to a portion of an antibody (or a construct containing such portion) that specifically binds to the delta variable 1 (Vδ1) chain of a target γδ T cell receptor (for example, a molecule in which one or more immunoglobulin chains are not full length but specifically bind to the target). Examples of binding fragments encompassed by the term antibody fragment include: (i) Fab fragment (a monovalent fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain), (ii) F(ab')2 fragment (a divalent fragment consisting of two Fab fragments linked by disulfide bridges in the hinge region), (iii) Fd fragment (consisting of VH domain and CH1 domain), (iv) Fv fragment (consisting of the VL domain and VH domain of a single arm of the antibody), (v) Single-stranded variable fragment scFv (consisting of a VL domain and a VH domain linked by a synthetic linker that enables the VL and VH domains to pair up and form a monovalent molecule as a single protein chain using recombination), (vi) VH (variable immunoglobulin chain domain consisting of VH domains), (vii) VL (variable immunoglobulin chain domain consisting of VL domains), (viii) Domain antibody (consisting of either a dAb, VH domain, or VL domain), (ix) Minibody (consisting of a pair of scFv fragments linked via CH3 domains), (x) Diabody (consisting of a non-covalent dimer of scFv fragments, each consisting of the VH domain of one antibody connected to the VL domain of another antibody by a small peptide linker).

[0052] The fragments of polyspecific antibodies referred to herein are antigen-binding fragments. More specifically, the antibody fragments of a polyspecific antibody bind to the same antigens as the complete polyspecific antibody. For example, a fragment of a polyspecific antibody that specifically binds to TRDV1 and a second antigen also specifically binds to TRDV1 and the same second antigen.

[0053] "Human antibody" refers to an antibody having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human subjects administered with the human antibody do not produce an interspecies antibody response (e.g., referred to as the HAMA-human anti-mouse antibody response) to the primary amino acids contained in the antibody. The human antibody may contain amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis or somatic mutation) in, for example, the CDR, particularly CDR3. However, this term is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. Human antibodies prepared, expressed, produced, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, or antibodies isolated from transgenic animals (e.g., mice) for human immunoglobulin genes, or antibodies prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences, may also be referred to as "recombinant human antibodies."

[0054] Substituting at least one amino acid residue in the framework region of a non-human immunoglobulin variable domain with a corresponding residue derived from a human variable domain is called "humanization." Humanization of variable domains can reduce immunogenicity in humans.

[0055] "Specificity" refers to the number of different types of antigens or antigenic determinants to which a particular antibody or fragment can bind. Antibody specificity is the antibody's ability to recognize a particular antigen as a unique molecular entity and distinguish it from other antigens. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, more persistently, and / or with higher affinity to a particular target antigen or epitope than it reacts with other targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, higher avidity, more readily, and / or with higher persistence than it would to bind to other substances.

[0056] The antibody of the present invention is a polyspecific antibody. A "polyspecific antibody" is an antibody that can bind to multiple different epitopes simultaneously or sequentially. Generally, these epitopes do not exist on the same antigen. Therefore, a polyspecific antibody has the ability to selectively bind to epitopes present on different antigens via multiple different binding domains. This is in contrast to conventional monospecific antibodies, which do not have this ability. Rather, a "monospecific antibody" has binding specificity to only one antigen. However, it may have multiple binding sites for this one antigen (for example, a fully human IgG antibody has a titer of 2, and the titers of other antibodies may be higher, but if the antibody recognizes only one antigen, it is still classified as a monospecific antibody). Therefore, the polyspecific antibody of the present invention binds to multiple different antigens simultaneously and / or sequentially.

[0057] In preferred embodiments of the present invention, the antibody is a bispecific antibody. A "bispecific antibody" is an antibody that can bind simultaneously and / or sequentially to two different epitopes. Generally, these epitopes are not present on the same antigen. Therefore, a bispecific antibody has the ability to selectively bind to two different epitopes present on two different antigens via two different binding domains. This is in contrast to conventional monospecific antibodies that do not have this ability. Thus, the bispecific antibody of the present invention binds simultaneously and / or sequentially to two different antigens.

[0058] "Affinity," expressed by the equilibrium constant (KD) for the dissociation of an antigen and an antigen-binding polypeptide, is a measure of the binding strength between the antigenic determinant and the antigen-binding site on the antibody (or a fragment thereof). A smaller KD value indicates a stronger binding strength between the antigenic determinant and the antigen-binding polypeptide. Alternatively, affinity can be expressed as the affinity constant (KA), or 1 / KD. Affinity can be determined by known methods depending on the specific antigen of interest. For example, KD can be determined by surface plasmon resonance.

[0059] 10 -6 Any KD value less than 1 is considered to indicate binding. Specific binding of an antibody or fragment to an antigen or antigenic determinant can be determined in any preferred known manner, including, for example, scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIAs), enzyme immunoassays (EIAs), and sandwich competitive assays, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using fluorescence assays), and various variations thereof known in the Art.

[0060] Avidity is a measure of the strength of binding between an antibody or fragment of an antibody and its associated antigen. Avidity is related to both the affinity between the antigenic determinant and its antigen-binding site on the antibody, and the number of suitable binding sites present on the antibody.

[0061] "In situ" means that the cells are in their natural or original location, rather than being moved to another location. For example, in a patient, in situ Vδ1+ cells refer to in vivo Vδ1 cells, not in vitro or ex vivo cells.

[0062] "Human tissue Vδ1+ cells," as well as "hematopoietic and blood Vδ1+ cells" and "tumor-infiltrating lymphocytes (TILs) Vδ1+ cells," are defined as Vδ1+ cells contained in or derived from either human tissue, the hematopoietic blood system, or a human tumor, respectively. All of the aforementioned cell types can be identified by (i) their location or origin, and (ii) their expression of Vδ1+ TCRs.

[0063] A “modulating antibody” is an antibody that, upon contact with or binding to a cell expressing a target to which the antibody binds, confers a measurable change, including but not limited to, a measurable change in the cell cycle, and / or cell number, and / or cell viability, and / or the secretion of one or more cell surface markers, and / or one or more secretory molecules (e.g., cytokines, chemokines, leukotrienes), and / or function (e.g., cytotoxicity against target cells or diseased cells). A method for “modulating” a cell or population of cells means a method by which at least one measurable change in the cell or population of cells or their secretions is induced to produce one or more “modulated cells.”

[0064] "Immune response" is a measurable change in at least one cell or cell type of the immune system, or one endocrine or exocrine pathway, in response to the addition of a regulatory antibody (including, but not limited to, cell-mediated responses, humoral responses, cytokine responses, and chemokine responses).

[0065] "Immune cells" are defined as cells of the immune system, including but not limited to CD34+ cells, B cells, CD45+ (common lymphocyte antigen) cells, alpha-beta T cells, cytotoxic T cells, helper T cells, plasma cells, neutrophils, monocytes, macrophages, erythrocytes, platelets, dendritic cells, phagocytes, granulocytes, innate lymphoid cells, natural killer (NK) cells, and gamma-delta T cells. Typically, immune cells are classified and identified using combinatorial cell surface molecular analysis (e.g., by flow cytometry), or grouped or clustered into subpopulations. These can then be further subdivided by additional analysis. For example, CD45+ lymphocytes can be further subdivided into vδ-positive and vδ-negative populations.

[0066] A “model system” is a biological model or representation designed to help understand how a drug, such as an antibody or fragment thereof, may function as a drug in improving the signs or symptoms of a disease. Such models typically involve in vitro, ex vivo, and in vivo use of disease cells, non-disease cells, healthy cells, effector cells, and tissues to study and compare the performance of the drug.

[0067] "Disease cells" exhibit phenotypes associated with diseases such as cancer, infections such as viral infections, or inflammatory conditions or the progression of inflammatory diseases. For example, disease cells may be tumor cells, autoimmune tissues, or virus-infected cells. Therefore, disease cells may be defined as neoplastic, virus-infected, or inflammatory.

[0068] "Healthy cells" refer to normal cells that are not diseased. These may also be called "normal" cells or "non-disease" cells. Non-disease cells include non-cancerous cells, or non-infected cells, or non-inflammatory cells. These cells are often used together with relevant disease cells to determine the disease cell specificity conferred by a drug and / or to better understand the therapeutic index of a drug.

[0069] "Disease cell specificity" is a measure of how effectively effector cells or populations of effector cells (e.g., Vδ1+ cell populations) distinguish and kill disease cells, such as cancer cells, while preserving non-disease or healthy cells. This ability can be measured in a model system and may include comparing the tendency of effector cells or effector cell populations to selectively kill or lyse disease cells to the ability of said effector cells to kill or lyse non-disease or healthy cells. The disease cell specificity may provide information about the potential therapeutic index of a drug.

[0070] "Enhanced disease cell specificity" refers to the phenotype of effector cells or populations, such as Vδ1+ cells, that are regulated to further increase their ability to specifically kill disease cells. This enhancement can be measured in various ways, including by a factor of change or a percentage increase in the specificity or selectivity of disease cell killing.

[0071] "ADCC," or "antibody-dependent cell-mediated cytotoxicity," refers to an immune response to cells coated with antibodies bound to cell surface antigens. This is a cell-mediated process in which immune effector cells (e.g., NK cells) recognize the antibody bound to the cell, inducing degranulation and lysis of the target cell. Typically, this is mediated by Fc-Fcγ interactions. The Fc region of the antibody bound to the cell recruits effector cells (e.g., NK cells) expressing the Fcγ receptor, leading to degranulation of the effector cells and death of the target cells.

[0072] "Fc-effective" refers to an antibody containing a functional Fc region (fragment crystallizable region), i.e., an Fc region that has not been mutated or otherwise deactivated. Fc-effective antibodies exhibit unattenuated Fc function. Fc-effective antibodies may contain human IGHC heavy chain sequences listed in IMGT that have not been modified, engineered, or constructed to reduce binding to one or more Fc gamma receptors. For example, this may occur via IGHC hinge mutations or by constructing an antibody containing a heavy chain constant domain that is a chimeric or hybrid of an IgG1 / IgG2A or IgG1 / IgG4 IGHC sequence. Preferably, the antibody or fragment (i.e., polypeptide) of the present invention is isolated. An "isolated" polypeptide is one that has been removed from its original environment. The term "isolated" may be used to refer to an antibody that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody or fragment that specifically binds to Vδ1 substantially does not contain antibodies that specifically bind to antigens other than Vδ1). The term “isolated” may also be used to refer to preparations in which isolated antibodies, when incorporated as an active ingredient in a pharmaceutical composition, are pure enough to be administered therapeutically, or have a purity of at least 70–80% (w / w), more preferably at least 80–90% (w / w), even more preferably 90–95%, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w).

[0073] Preferably, the polynucleotides used in this invention are isolated. “Isolated” polynucleotides are those removed from their original environment. For example, a naturally occurring polynucleotide is isolated if it is separated from some or all of the materials that coexist in the natural system. A polynucleotide is considered isolated if, for example, it is cloned into a vector that is not part of its natural environment, or if it is contained within cDNA.

[0074] Antibodies or fragments thereof may be “functionally active variants,” including naturally occurring allele variants and mutants or any other non-naturally occurring variants. As is well known in the art, allele variants are alternative forms of (poly)peptides characterized by having one or more amino acid substitutions, deletions, or additions that do not essentially alter the biological function of the polypeptide. As a non-limiting example, the functionally active variant may still function even if the framework containing the CDR is modified, the CDR itself is modified, the CDR is grafted onto an alternative framework, or an N-terminal or C-terminal extension is incorporated. Furthermore, the CDR-containing binding domain may be paired with a different partner chain, such as one shared with another antibody. The binding domain may still function even when shared with a so-called “common” light chain or “common” heavy chain. Furthermore, the binding domain may function when polymerized. Furthermore, “antibodies or fragments thereof” may also include functional variants that have been modified to move away from or approach a different canonical sequence (e.g., those listed on IMGT.org) with a different VH, VL, or constant domain, and that are still functional.

[0075] For the purpose of comparing two closely related polypeptide sequences, the "% sequence identity" between the first polypeptide sequence and the second polypeptide sequence can be calculated using NCBI BLAST v2.0 with a standard polypeptide sequence setting (BLASTP). For the purpose of comparing two closely related polynucleotide sequences, the "% sequence identity" between the first nucleotide sequence and the second nucleotide sequence can be calculated using NCBI BLAST v2.0 with a standard nucleotide sequence setting (BLASTN).

[0076] Polypeptide sequences or polynucleotide sequences are said to be the same as, or "identical to," other polypeptide or polynucleotide sequences if they share 100% sequence identity throughout their entire length. Residues in a sequence are numbered from left to right, i.e., from the N-terminus to the C-terminus in the case of polypeptides, and from the 5' end to the 3' end in the case of polynucleotides.

[0077] In some embodiments, sequence identity of any particular percentage of the sequence is calculated without the sequences of all six CDRs of the antibody. For example, an anti-Vδ1 antibody or its antigen-binding fragment may contain a variable heavy chain region sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a particular variable heavy chain region sequence, and / or a variable light chain region sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a particular variable light chain region sequence, and any amino acid mutations occur only in the framework regions of the variable heavy chain region sequence and the variable light chain region sequence. In such embodiments, an anti-Vδ1 antibody or its antigen-binding fragment having a specific sequence identity retains the complete heavy and light chain CDR1, CDR2, and CDR3 sequences of the corresponding anti-Vδ1 antibody or its antigen-binding fragment.

[0078] A “difference” between sequences refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the second sequence compared to the first sequence. Two polypeptide sequences can contain one, two, or more such amino acid differences. An insertion, deletion, or substitution in the second sequence, which is otherwise identical to the first sequence (100% sequence identity), reduces the degree of sequence identity. For example, if identical sequences are 9 amino acid residues long, one substitution in the second sequence reduces the sequence identity to 88.9%. If the first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share more than 66% identity (they share 66.7% identity).

[0079] Alternatively, for the purpose of comparing a first reference polypeptide sequence with a second comparison polypeptide sequence, the number of additions, substitutions, and / or deletions made to the first sequence to generate the second sequence can be determined. “Addition” is the addition of one amino acid residue to the sequence of the first polypeptide (including additions at either end of the first polypeptide). “Substitution” is the replacement of one amino acid residue in the sequence of the first polypeptide with one different amino acid residue. Such substitutions may be conserved or non-conservative. “Deletion” is the deletion of one amino acid residue from the sequence of the first polypeptide (including deletions at either end of the first polypeptide).

[0080] Using three-letter and one-letter codes, naturally occurring amino acids can be named as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gln), cysteine ​​(C or Cys), methionine (M or Met), serine (S or Ser), and threonine (T or Thr). If the residue may be aspartic acid or asparagine, the symbols Asx or B may be used. If the residue can be glutamic acid or glutamine, the symbols Glx or Z may be used. If the residue can be any amino acid, the symbols Xaa or X may be used. Unless otherwise specified in the context, references to aspartic acid include aspartate, and references to glutamic acid include glutamate.

[0081] A “conservative” amino acid substitution is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure and is expected to have little effect on the function, activity, or other biological properties of the polypeptide. Such conservative substitutions are preferably those in which one amino acid within the following group is replaced by another amino acid residue from the same group: [Table 1]

[0082] Preferably, the hydrophobic amino acid residue is a nonpolar amino acid. More preferably, the hydrophobic amino acid residue is selected from V, I, L, M, F, W, or C. In some embodiments, the hydrophobic amino acid residue is selected from glycine, alanine, valine, methionine, leucine, isoleucine, phenylalanine, tyrosine, or tryptophan.

[0083] As used herein, polypeptide sequence numbering and the definitions of CDR and FR are as defined in accordance with the EU and / or IMGT numbering systems, as indicated in the context. A “corresponding” amino acid residue between a first polypeptide sequence and a second polypeptide sequence is, as indicated in the context, an amino acid residue of first sequence affinity that shares the same position as an amino acid residue in the second sequence, according to the EU and / or IMGT numbering system, while the amino acid residue in the second sequence may have different identity from that of the first sequence. Preferably, corresponding residues share the same number (and letter) if the framework and CDR are of the same length according to the EU or IMGT definition. Alignment can be achieved manually or by using known computer algorithms for sequence alignment, such as NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

[0084] In this specification, the term “epitope” refers to a portion of a target to which an antibody or a fragment of it specifically binds. An epitope may also be referred to as an “antigenic determinant.” An antibody binds to “essentially the same epitope” as another antibody if both antibodies recognize the same or sterically overlapping epitope. A method commonly used to determine whether two antibodies bind to the same or overlapping epitopes is a competitive assay. This can consist of several different formats using either labeled antigens or labeled antibodies (e.g., well plates using radiolabeling or enzymatic labeling, or flow cytometry in antigen-expressing cells). An antibody binds to “the same epitope” as another antibody if both antibodies recognize the same epitope (i.e., all contact points between the antigen and the antibody are the same). For example, if all contact points in a particular region of the antigen are identified as the same using characterization methods such as antibody / antigen crosslinking MS, HDX, X-ray crystallography, cryo-electron microscopy, or mutagenesis, an antibody may bind to the same epitope as another antibody.

[0085] Furthermore, using such characterization methods, it is also possible to characterize antibodies that bind to essentially the same epitopes by recognizing some, but not all, of the same contact points. Specifically, such antibodies may share a sufficient number of identical contact points in a particular antigenic region to provide nearly equivalent technical effects and / or equivalent selectivity for antigen interaction. Moreover, in some cases where antibodies recognize essentially the same epitopes and confer nearly equivalent technical effects and / or selectivity for interaction, it may also be useful to define the epitope binding footprint by the entire antigen contact, including from the N-terminal antigen contact point to the C-terminal antigen contact point.

[0086] Epitopes found in protein targets can be defined as "linear epitopes" or "contaxial epitopes." Linear epitopes are formed by a continuous sequence of amino acids in a protein antigen. Contaxial epitopes are formed by amino acids that are discontinuous in the protein sequence but come together when the protein folds into its three-dimensional structure.

[0087] As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated. Another type of vector is a viral vector, to which an additional DNA segment can be ligated into a viral genome. Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication, as well as episomal mammalian vectors and yeast vectors). Other vectors (e.g., non-episomal mammalian vectors), upon introduction into a host cell, can be integrated into the host cell's genome and thereby replicate with the host genome. Furthermore, certain vectors can direct the expression of genes to which they are operably ligated. Such vectors are referred herein as “recombinant expression vectors” (or simply “expression vectors”). Generally, expression vectors used in recombinant DNA techniques are often in the form of plasmids. Since plasmids are the most commonly used form of vector, “plasmid” and “vector” may be used synonymously herein. However, the present invention is intended to include viral vectors that perform equivalent functions (e.g., replication-deficient retroviruses, adenoviruses and adeno-associated viruses), as well as other forms of expression vectors such as bacteriophages and phagemid systems. As used herein, the term “recombinant host cell” (or simply “host cell”) is intended to refer to a cell into which a recombinant expression vector has been introduced. Such a term is intended to refer not only to a specific target cell, but also, for example, if the offspring of such a cell are used to make a cell line or cell bank, and the cell line or cell bank is subsequently stored, provided, sold, transferred, or used to manufacture antibodies or polyspecific antibodies or fragments thereof as described herein.

[0088] References to “subject,” “patient,” or “individual” refer to the subject being treated, particularly mammalian subjects. Mammalian subjects include humans, non-human primates, livestock (such as cattle), sport animals, or companion animals, such as dogs, cats, guinea pigs, rabbits, rats, or mice. In some embodiments, the subject is human. In alternative embodiments, the subject is a non-human mammal such as a mouse.

[0089] The term "sufficient amount" means an amount sufficient to produce the desired effect. The term "therapeutic amount" is an amount that is effective in improving the symptoms of a disease or disorder. Since prevention can be considered a therapy, a therapeutic amount may also be a "preventive amount."

[0090] A disease or disorder is considered “improved” if the severity of the signs or symptoms of the disease or disorder, the frequency with which such signs or symptoms are experienced by the subject, or both, decreases.

[0091] As used herein, “treating a disease or disorder” means reducing the frequency and / or severity of at least one sign or symptom of a disease or disorder experienced by the subject.

[0092] As used herein, “cancer” refers to the abnormal proliferation or division of cells. Generally, the proliferation and / or lifespan of cancer cells is greater than and uncoordinated with the proliferation and / or lifespan of the surrounding normal cells and tissues. Cancer can be benign, premalignant, or malignant. Cancer can occur in various cells and tissues, including those of the oral cavity (e.g., mouth, tongue, pharynx), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas), respiratory system (e.g., larynx, lungs, bronchi), bones, joints, skin (e.g., basal cells, squamous cells, meningiomas), breasts, reproductive system (e.g., uterus, ovaries, prostate, testes), urinary system (e.g., bladder, kidneys, ureters), eyes, nervous system (e.g., brain), endocrine system (e.g., thyroid), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia).

[0093] As used herein, the term “about” includes values ​​that are up to 10% greater than and up to 10% less than the specified value, preferably including values ​​that are up to 5% greater than and up to 5% less than the specified value, and especially including the specified value. The term “between” includes the specified boundary values.

[0094] Polyspecific antibodies or their fragments This specification provides a polyspecific (preferably bispecific) antibody or fragment thereof that can specifically bind to the delta variable 1 chain (Vδ1) of the γδ T cell receptor (TCR) and to another antigen. The present invention relates to the use of the polyspecific antibody as a drug for administration to a subject to be treated.

[0095] In one embodiment, the polyspecific antibody or fragment thereof is an scFv, Fab, Fab', F(ab')2, Fv, a variable domain (e.g., VH or VL), a diabody, a minibody, or a monoclonal antibody. In a further embodiment, the antibody or fragment thereof is an scFv.

[0096] The polyspecific antibodies of the present invention may be of any type, e.g., IgG, IgA, IgM, IgE, IgD or isotypes thereof, and may contain kappa light chains or lambda light chains. In one embodiment, the polyspecific antibody is an IgG antibody, e.g., at least one of isotypes, IgG1, IgG2, IgG3, or IgG4. In a further embodiment, the polyspecific antibody may be in a format such as an IgG format that has been modified to confer desired properties, such as reducing effector function, extending half-life, altering ADCC, or having a mutated Fc to improve hinge stability. Such modifications are well known in the art.

[0097] In one embodiment, the polyspecific antibody or fragment is human. Therefore, the polyspecific antibody or fragment may be derived from a human immunoglobulin (Ig) sequence. The CDR, framework, and / or constant region of the antibody (or fragment) may be derived from a human Ig sequence, particularly a human IgG sequence. The CDR, framework, and / or constant region may be substantially identical with respect to a human Ig sequence, particularly a human IgG sequence. The advantage of using human polyspecific antibodies is that they are low immunogenic or non-immunogenic in humans.

[0098] The polyspecific antibody or its fragment may be a chimera, for example, a mouse-human antibody chimera.

[0099] Alternatively, the polyspecific antibody or its fragment may be derived from a non-human species such as a mouse. Such non-human antibodies can be modified to increase their similarity to naturally occurring antibody variants in humans, and thus the antibody or its fragment may be partially or completely humanized. In one embodiment, the antibody or its fragment is therefore humanized.

[0100] Sequence of polyspecific antibodies The isolated anti-Vδ1 antibodies or fragments thereof of the present invention may be described with reference to their CDR sequences that confer TRDV1 binding. However, since the present invention is based on an entirely new principle of targeting TRDV1 using a multispecific antibody to bind to TRDV1 on, for example, a TCE platform or a DI platform, the present invention is not limited to such sequences. Similarly, the present invention is not limited to specific sequences of domains that confer binding to a second antigen (such as a cancer or cancer-associated antigen or an immunomodulatory antigen).

[0101] According to one aspect of the present invention, A CDR3 containing a sequence that has at least 80% sequence identity with one of sequence numbers 2-25, CDR2 containing a sequence having at least 80% sequence identity with any one of sequence numbers 26-37 and sequence numbers 160-171, and / or CDR1 containing a sequence that has at least 80% sequence identity with one of sequence numbers 38-61 An isolated, multispecific anti-Vδ1 antibody or a fragment thereof, comprising one or more of the above, is provided.

[0102] According to one aspect of the present invention, an isolated anti-Vδ1 polyspecific antibody or fragment thereof is provided, comprising a CDR3 containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 25. In one embodiment, the polyspecific antibody or fragment thereof comprises a CDR2 containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 26 to 37 and SEQ ID NOs: 160 to 171. In one embodiment, the polyspecific antibody or fragment thereof comprises a CDR1 containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 38 to 61.

[0103] In one embodiment, the polyspecific antibody or fragment thereof includes CDR3 containing a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 2 to 25. In one embodiment, the polyspecific antibody or fragment thereof includes CDR2 containing a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 26 to 37 and SEQ ID NOs. 160 to 171. In one embodiment, the polyspecific antibody or fragment thereof includes CDR1 containing a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs. 38 to 61.

[0104] In one embodiment, the polyspecific antibody or fragment comprises CDR3, which consists of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 2 to 25. In one embodiment, the polyspecific antibody or fragment comprises CDR2, which consists of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 26 to 37 and SEQ ID NOs: 160 to 171. In one embodiment, the polyspecific antibody or fragment comprises CDR1, which consists of a sequence having at least 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 38 to 61.

[0105] A further aspect of the present invention provides a polyspecific antibody or fragment thereof comprising a VH region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 14 to 25.

[0106] According to a particular aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2-7, particularly 2-6, for example 2, 3, or 4, and / or a VL region comprising a CDR3 containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 14-19, particularly 14-18, for example 14, 15, or 16. According to another aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 2-7, particularly 2-6, for example 2, 3, or 4, and / or a VL region comprising a CDR3 containing a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 14-19, particularly 14-18, for example 14, 15, or 16.

[0107] According to a particular aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 8-13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 20-25, particularly 20, 21, 22, or 23. According to another aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 8-13, particularly 8, 9, 10, or 11, and / or a VL region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 20-25, particularly 20, 21, 22, or 23.

[0108] A further aspect of the present invention provides a polyspecific antibody or fragment thereof comprising a VH region comprising a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 14 to 25.

[0109] According to a particular aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 containing a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-7, particularly 2-6, for example 2, 3, 4, or 5, and / or a VL region comprising a CDR3 containing a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 14-19, particularly 14-18, for example 14, 15, 16, or 17. According to another aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 containing a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 2-7, particularly 2-6, for example 2, 3, 4, or 5, and / or a VL region comprising a CDR3 containing a sequence having at least 90% sequence identity with any one of SEQ ID NOs: 14-19, particularly 14-18, for example 14, 15, 16, or 17.

[0110] According to a particular aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 8, 9, 10, or 11, and / or a VL region comprising a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 20, 21, 22, or 23. According to another aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 8, 9, 10, or 11, and / or a VL region comprising a CDR3 having at least 90% sequence identity with any one of SEQ ID NOs: 20, 21, 22, or 23.

[0111] A further aspect of the present invention provides a polyspecific antibody or fragment thereof comprising a VH region comprising a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 2 to 13, and / or a VL region comprising a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 14 to 25.

[0112] According to a particular aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 containing a sequence having at least 95% sequence identity with any one of SEQ ID NOs: 2-7, particularly 2-6, for example 2, 3, 4, or 5, and / or a VL region comprising a CDR3 containing a sequence having at least 95% sequence identity with any one of SEQ ID NOs: 14-19, particularly 14-18, for example 14, 15, 16, or 17. According to another aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 containing a sequence having at least 95% sequence identity with any one of SEQ ID NOs: 2-7, particularly 2-6, for example 2, 3, 4, or 5, and / or a VL region comprising a CDR3 containing a sequence having at least 95% sequence identity with any one of SEQ ID NOs: 14-19, particularly 14-18, for example 14, 15, 16, or 17.

[0113] According to a particular aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 8, 9, 10, or 11, and / or a VL region comprising a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 20, 21, 22, or 23. According to another aspect of the present invention, a polyspecific antibody or fragment thereof is provided, comprising a VH region comprising a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 8, 9, 10, or 11, and / or a VL region comprising a CDR3 having at least 95% sequence identity with any one of SEQ ID NOs: 20, 21, 22, or 23.

[0114] A further aspect of the present invention provides a polyspecific antibody or fragment thereof comprising a VH region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 2 to 13, and a VL region comprising a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 14 to 25.

[0115] Embodiments in this specification that refer to “at least 80%” or “80% or more” will be understood to include all values ​​of sequence identity of 80% or more, such as 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In one embodiment, the antibody or fragment of the present invention contains at least 85% of sequence identity to a given sequence, for example, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.

[0116] Instead of a percentage of sequence identity, embodiments may be defined by one or more amino acid changes, e.g., one or more additions, substitutions, and / or deletions. In one embodiment, the sequence may include up to five amino acid changes, e.g., up to three amino acid changes, and in particular up to two amino acid changes. In a further embodiment, the sequence may include up to five amino acid substitutions, e.g., up to three amino acid substitutions, and in particular up to one or two amino acid substitutions. For example, the CDR3 of the polyspecific antibody or fragment of the present invention includes, or more preferably consists of, a sequence having two or fewer, more preferably one or fewer substitutions compared to any one of SEQ ID NOs: 2 to 25.

[0117] Preferably, any CDR1, CDR2, or CDR3 residue that differs from the corresponding residue in SEQ ID NOs: 2-61 and SEQ ID NOs: 160-171 is a conserved substitution with respect to that corresponding residue. For example, any CDR3 residue that differs from the corresponding residue in SEQ ID NOs: 2-25 is a conserved substitution with respect to that corresponding residue.

[0118] In one embodiment, a polyspecific antibody or a fragment thereof is (i) A VH region containing a CDR3 containing a sequence having at least 80% sequence identity with any one of sequence numbers 2 to 13, (ii) A VH region containing a CDR2 having at least 80% sequence identity with any one of sequence numbers 26-37, (iii) A VH region containing CDR1 that has at least 80% sequence identity with any one of sequence numbers 38 to 49, (iv) A VL region containing a CDR3 containing a sequence having at least 80% sequence identity with any one of sequence numbers 14-25, (v) A VL region containing a CDR2 having at least 80% sequence identity with any one of sequence numbers 160-171, and / or (vi) A VL region containing a CDR1 containing a sequence having at least 80% sequence identity with any one of sequence numbers 50 to 61.

[0119] In one embodiment, a polyspecific antibody or a fragment thereof is (i) A VH region containing a CDR3 containing a sequence having at least 80% sequence identity with any one of sequence numbers 2 to 13, (ii) A VH region including a CDR2 containing a sequence having at least 80% sequence identity with any one of sequence numbers 26 to 37, and (iii) A heavy chain comprising a VH region containing a CDR1 having at least 80% sequence identity with any one of sequence numbers 38-49.

[0120] In one embodiment, a polyspecific antibody or a fragment thereof is (i) A VL region containing a CDR3 that has at least 80% sequence identity with any one of sequence numbers 14-25, (ii) A VL region including a CDR2 containing a sequence having at least 80% sequence identity with any one of sequence numbers 160 to 171, and (iii) A light chain comprising a VL region containing a CDR1 having at least 80% sequence identity with any one of sequence numbers 50 to 61.

[0121] In one embodiment, a multispecific antibody or fragment thereof includes a VH region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 2, 3, 4, 5, or 6, e.g., 2, 3, 4, or 5, particularly 2, 3, or 4 (or the component of a multispecific antibody that confers TRDV1 binding (i.e., the binding domain) consists of this). In one embodiment, a multispecific antibody or fragment thereof includes a VH region containing a CDR2 having at least 80% sequence identity with any one of SEQ ID NOs: 26, 27, 28, 29, or 30, e.g., 26, 27, 28, or 29, particularly 26, 27, or 28 (or the component of a multispecific antibody that confers TRDV1 binding consists of this). In one embodiment, a polyspecific antibody or a fragment thereof includes a VH region containing a CDR1 having at least 80% sequence identity with any one of SEQ ID NOs. 38, 39, 40, 41, e.g., 38, 39, 40, or 41, particularly 38, 39, or 40 (or the component of the polyspecific antibody that confers TRDV1 binding consists of such a region).

[0122] In one embodiment, the multispecific antibody or fragment comprises a VH region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs: 8, 9, 10, or 11 (or the component of the multispecific antibody that confers TRDV1 binding consists of such a region). In one embodiment, the multispecific antibody or fragment comprises a VH region containing a CDR2 having at least 80% sequence identity with any one of SEQ ID NOs: 32, 33, 34, or 35 (or the component of the multispecific antibody that confers TRDV1 binding consists of such a region). In one embodiment, the multispecific antibody or fragment comprises a VH region containing a CDR1 having at least 80% sequence identity with any one of SEQ ID NOs: 44, 45, 46, or 47 (or the component of the multispecific antibody that confers TRDV1 binding consists of such a region).

[0123] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 2, CDR2 containing the sequence of sequence number 26, and CDR1 containing the sequence of sequence number 38. In one embodiment, CDR3 consists of the sequence of sequence number 2, CDR2 consists of the sequence of sequence number 26, and CDR1 consists of the sequence of sequence number 38.

[0124] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 3, CDR2 containing the sequence of sequence number 27, and CDR1 containing the sequence of sequence number 39. In one embodiment, CDR3 consists of the sequence of sequence number 3, CDR2 consists of the sequence of sequence number 27, and CDR1 consists of the sequence of sequence number 39.

[0125] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 4, CDR2 containing the sequence of sequence number 28, and CDR1 containing the sequence of sequence number 40. In one embodiment, CDR3 consists of the sequence of sequence number 4, CDR2 consists of the sequence of sequence number 28, and CDR1 consists of the sequence of sequence number 40.

[0126] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 5, CDR2 containing the sequence of sequence number 29, and CDR1 containing the sequence of sequence number 41. In one embodiment, CDR3 consists of the sequence of sequence number 5, CDR2 consists of the sequence of sequence number 29, and CDR1 consists of the sequence of sequence number 41.

[0127] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 6, CDR2 containing the sequence of sequence number 30, and CDR1 containing the sequence of sequence number 42. In one embodiment, CDR3 consists of the sequence of sequence number 6, CDR2 consists of the sequence of sequence number 30, and CDR1 consists of the sequence of sequence number 42.

[0128] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 8, CDR2 containing the sequence of sequence number 32, and CDR1 containing the sequence of sequence number 44. In one embodiment, CDR3 consists of the sequence of sequence number 8, CDR2 consists of the sequence of sequence number 32, and CDR1 consists of the sequence of sequence number 44.

[0129] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 9, CDR2 containing the sequence of sequence number 33, and CDR1 containing the sequence of sequence number 45. In one embodiment, CDR3 consists of the sequence of sequence number 9, CDR2 consists of the sequence of sequence number 33, and CDR1 consists of the sequence of sequence number 45.

[0130] In one embodiment, the VH region includes a CDR3 containing the sequence of sequence number 10, a CDR2 sequence of sequence number 34, and a CDR1 sequence of sequence number 46. In one embodiment, CDR3 consists of the sequence of sequence number 10, CDR2 consists of the sequence of sequence number 34, and CDR1 consists of the sequence of sequence number 46.

[0131] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 11, CDR2 containing the sequence of sequence number 35, and CDR1 containing the sequence of sequence number 47. In one embodiment, CDR3 consists of the sequence of sequence number 11, CDR2 consists of the sequence of sequence number 35, and CDR1 consists of the sequence of sequence number 47.

[0132] In one embodiment, a multispecific antibody or fragment thereof includes a VL region containing a CDR3 having at least 80% sequence identity with any one of SEQ ID NOs. 14-25, e.g., SEQ ID NOs. 14, 15, 16, 17, or 18, e.g., 14, 15, 16, or 17, in particular 14, 15, or 16 (or the components of a multispecific antibody that confer TRDV1 binding consist of these). In one embodiment, a multispecific antibody or fragment thereof includes a VL region containing a CDR2 having at least 80% sequence identity with any one of SEQ ID NOs. 160-171, e.g., SEQ ID NOs. 160, 161, 162, 163, or 164, e.g., 160, 161, 162, or 163, in particular 160, 161, or 162 (or the components of a multispecific antibody that confer TRDV1 binding consist of these). In one embodiment, the polyspecific antibody or a fragment thereof includes a VL region containing a CDR1 having at least 80% sequence identity with any one of SEQ ID NOs. 50-61, e.g., SEQ ID NOs. 50, 51, 52, 53, or 54, e.g., 50, 51, 52, or 53, in particular 50, 51, or 52 (or the components of the polyspecific antibody that confer TRDV1 binding consist of these).

[0133] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 14, CDR2 containing the sequence of sequence number 160, and CDR1 containing the sequence of sequence number 50. In one embodiment, CDR3 consists of the sequence of sequence number 14, CDR2 consists of the sequence of sequence number 160, and CDR1 consists of the sequence of sequence number 50.

[0134] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 15, CDR2 containing the sequence of sequence number 161, and CDR1 containing the sequence of sequence number 51. In one embodiment, CDR3 consists of the sequence of sequence number 15, CDR2 consists of the sequence of sequence number 161, and CDR1 consists of the sequence of sequence number 51.

[0135] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 16, CDR2 containing the sequence of sequence number 162, and CDR1 containing the sequence of sequence number 52. In one embodiment, CDR3 consists of the sequence of sequence number 16, CDR2 consists of the sequence of sequence number 162, and CDR1 consists of the sequence of sequence number 52.

[0136] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 17, CDR2 containing the sequence of sequence number 163, and CDR1 containing the sequence of sequence number 53. In one embodiment, CDR3 consists of the sequence of sequence number 17, CDR2 consists of the sequence of sequence number 163, and CDR1 consists of the sequence of sequence number 53.

[0137] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 18, CDR2 containing the sequence of sequence number 164, and CDR1 containing the sequence of sequence number 54. In one embodiment, CDR3 consists of the sequence of sequence number 18, CDR2 consists of the sequence of sequence number 164, and CDR1 consists of the sequence of sequence number 54.

[0138] In one embodiment, the multispecific antibody or fragment comprises a VL region containing a CDR3 having at least 80% sequence identity with any one of sequence numbers 20, 21, 22, or 23 (or the component of the multispecific antibody that confers TRDV1 binding consists of this). In one embodiment, the multispecific antibody or fragment comprises a VL region containing a CDR2 having at least 80% sequence identity with any one of sequence numbers 166, 167, 168, or 169 (or the component of the multispecific antibody that confers TRDV1 binding consists of this). In one embodiment, the multispecific antibody or fragment comprises a VL region containing a CDR1 having at least 80% sequence identity with any one of sequence numbers 56, 57, 58, or 59 (or the component of the multispecific antibody that confers TRDV1 binding consists of this).

[0139] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 20, CDR2 containing the sequence of sequence number 166, and CDR1 containing the sequence of sequence number 56. In one embodiment, CDR3 consists of the sequence of sequence number 20, CDR2 consists of the sequence of sequence number 166, and CDR1 consists of the sequence of sequence number 56.

[0140] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 21, CDR2 containing the sequence of sequence number 167, and CDR1 containing the sequence of sequence number 57. In one embodiment, CDR3 consists of the sequence of sequence number 21, CDR2 consists of the sequence of sequence number 167, and CDR1 consists of the sequence of sequence number 57.

[0141] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 22, CDR2 containing the sequence of sequence number 168, and CDR1 containing the sequence of sequence number 58. In one embodiment, CDR3 consists of the sequence of sequence number 22, CDR2 consists of the sequence of sequence number 168, and CDR1 consists of the sequence of sequence number 58.

[0142] In one embodiment, the VL region includes CDR3 containing the sequence of sequence number 23, CDR2 containing the sequence of sequence number 169, and CDR1 containing the sequence of sequence number 59. In one embodiment, CDR3 consists of the sequence of sequence number 23, CDR2 consists of the sequence of sequence number 169, and CDR1 consists of the sequence of sequence number 59.

[0143] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 2, CDR2 containing the sequence of sequence number 26, and CDR1 containing the sequence of sequence number 38; the VL region includes CDR3 containing the sequence of sequence number 14, CDR2 containing the sequence of sequence number 160, and CDR1 containing the sequence of sequence number 50. In one embodiment, HCDR3 consists of the sequence of sequence number 2, HCDR2 consists of the sequence of sequence number 26, HCDR1 consists of the sequence of sequence number 38, LCDR3 consists of the sequence of sequence number 14, LCDR2 consists of the sequence of sequence number 160, and LCDR1 consists of the sequence of sequence number 50.

[0144] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 3, CDR2 containing the sequence of sequence number 27, and CDR1 containing the sequence of sequence number 39; the VL region includes CDR3 containing the sequence of sequence number 15, CDR2 containing the sequence of sequence number 161, and CDR1 containing the sequence of sequence number 51. In one embodiment, HCDR3 consists of the sequence of sequence number 3, HCDR2 consists of the sequence of sequence number 27, HCDR1 consists of the sequence of sequence number 39, LCDR3 consists of the sequence of sequence number 15, LCDR2 consists of the sequence of sequence number 161, and LCDR1 consists of the sequence of sequence number 51.

[0145] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 4, CDR2 containing the sequence of sequence number 28, and CDR1 containing the sequence of sequence number 40; the VL region includes CDR3 containing the sequence of sequence number 16, CDR2 containing the sequence of sequence number 162, and CDR1 containing the sequence of sequence number 52. In one embodiment, HCDR3 consists of the sequence of sequence number 4, HCDR2 consists of the sequence of sequence number 28, HCDR1 consists of the sequence of sequence number 40, LCDR3 consists of the sequence of sequence number 16, LCDR2 consists of the sequence of sequence number 162, and LCDR1 consists of the sequence of sequence number 52.

[0146] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 5, CDR2 containing the sequence of sequence number 29, and CDR1 containing the sequence of sequence number 41; the VL region includes CDR3 containing the sequence of sequence number 17, CDR2 containing the sequence of sequence number 163, and CDR1 containing the sequence of sequence number 53. In one embodiment, HCDR3 consists of the sequence of sequence number 5, HCDR2 consists of the sequence of sequence number 29, HCDR1 consists of the sequence of sequence number 41, LCDR3 consists of the sequence of sequence number 17, LCDR2 consists of the sequence of sequence number 163, and LCDR1 consists of the sequence of sequence number 53.

[0147] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 6, CDR2 containing the sequence of sequence number 30, and CDR1 containing the sequence of sequence number 42; the VL region includes CDR3 containing the sequence of sequence number 18, CDR2 containing the sequence of sequence number 164, and CDR1 containing the sequence of sequence number 54. In one embodiment, HCDR3 consists of the sequence of sequence number 6, HCDR2 consists of the sequence of sequence number 30, HCDR1 consists of the sequence of sequence number 42, LCDR3 consists of the sequence of sequence number 18, LCDR2 consists of the sequence of sequence number 164, and LCDR1 consists of the sequence of sequence number 54.

[0148] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 7, CDR2 containing the sequence of sequence number 31, and CDR1 containing the sequence of sequence number 43; the VL region includes CDR3 containing the sequence of sequence number 19, CDR2 containing the sequence of sequence number 165, and CDR1 containing the sequence of sequence number 55. In one embodiment, HCDR3 consists of the sequence of sequence number 7, HCDR2 consists of the sequence of sequence number 31, HCDR1 consists of the sequence of sequence number 43, LCDR3 consists of the sequence of sequence number 19, LCDR2 consists of the sequence of sequence number 165, and LCDR1 consists of the sequence of sequence number 55.

[0149] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 8, CDR2 containing the sequence of sequence number 32, and CDR1 containing the sequence of sequence number 44; the VL region includes CDR3 containing the sequence of sequence number 20, CDR2 containing the sequence of sequence number 166, and CDR1 containing the sequence of sequence number 56. In one embodiment, HCDR3 consists of the sequence of sequence number 8, HCDR2 consists of the sequence of sequence number 32, HCDR1 consists of the sequence of sequence number 44, LCDR3 consists of the sequence of sequence number 20, LCDR2 consists of the sequence of sequence number 166, and LCDR1 consists of the sequence of sequence number 56.

[0150] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 9, CDR2 containing the sequence of sequence number 33, and CDR1 containing the sequence of sequence number 45; the VL region includes CDR3 containing the sequence of sequence number 21, CDR2 containing the sequence of sequence number 167, and CDR1 containing the sequence of sequence number 57. In one embodiment, HCDR3 consists of the sequence of sequence number 9, HCDR2 consists of the sequence of sequence number 33, HCDR1 consists of the sequence of sequence number 45, LCDR3 consists of the sequence of sequence number 21, LCDR2 consists of the sequence of sequence number 167, and LCDR1 consists of the sequence of sequence number 57.

[0151] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 10, CDR2 containing the sequence of sequence number 34, and CDR1 containing the sequence of sequence number 46; the VL region includes CDR3 containing the sequence of sequence number 22, CDR2 containing the sequence of sequence number 168, and CDR1 containing the sequence of sequence number 58. In one embodiment, HCDR3 consists of the sequence of sequence number 10, HCDR2 consists of the sequence of sequence number 34, HCDR1 consists of the sequence of sequence number 46, LCDR3 consists of the sequence of sequence number 22, LCDR2 consists of the sequence of sequence number 168, and LCDR1 consists of the sequence of sequence number 58.

[0152] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 11, CDR2 containing the sequence of sequence number 35, and CDR1 containing the sequence of sequence number 47; the VL region includes CDR3 containing the sequence of sequence number 23, CDR2 containing the sequence of sequence number 169, and CDR1 containing the sequence of sequence number 59. In one embodiment, HCDR3 consists of the sequence of sequence number 11, HCDR2 consists of the sequence of sequence number 35, HCDR1 consists of the sequence of sequence number 47, LCDR3 consists of the sequence of sequence number 23, LCDR2 consists of the sequence of sequence number 169, and LCDR1 consists of the sequence of sequence number 59.

[0153] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 12, CDR2 containing the sequence of sequence number 36, and CDR1 containing the sequence of sequence number 48; the VL region includes CDR3 containing the sequence of sequence number 24, CDR2 containing the sequence of sequence number 170, and CDR1 containing the sequence of sequence number 60. In one embodiment, HCDR3 consists of the sequence of sequence number 12, HCDR2 consists of the sequence of sequence number 36, HCDR1 consists of the sequence of sequence number 48, LCDR3 consists of the sequence of sequence number 24, LCDR2 consists of the sequence of sequence number 170, and LCDR1 consists of the sequence of sequence number 60.

[0154] In one embodiment, the VH region includes CDR3 containing the sequence of sequence number 13, CDR2 containing the sequence of sequence number 37, and CDR1 containing the sequence of sequence number 49; the VL region includes CDR3 containing the sequence of sequence number 25, CDR2 containing the sequence of sequence number 171, and CDR1 containing the sequence of sequence number 61. In one embodiment, HCDR3 consists of the sequence of sequence number 13, HCDR2 consists of the sequence of sequence number 37, HCDR1 consists of the sequence of sequence number 49, LCDR3 consists of the sequence of sequence number 25, LCDR2 consists of the sequence of sequence number 171, and LCDR1 consists of the sequence of sequence number 61.

[0155] In one embodiment, the polyspecific antibody or fragment comprises one or more CDR sequences as listed in Table 3. In a further embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1252_P01_C08 as listed in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1245_P01_E07 as listed in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1245_P02_G04 as listed in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1245_P02_B07 as listed in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1251_P02_C05 as listed in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1139_P01_E04 as described in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1245_P02_F07 as described in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1245_P01_G06 as described in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1245_P01_G09 as described in Table 3. In an alternative embodiment, the polyspecific antibody or fragment comprises one or more (e.g., all) CDR sequences of clone 1138_P01_B09 as described in Table 3. In an alternative embodiment, the polyspecific antibody or fragment thereof comprises one or more (e.g., all) CDR sequences of clone 1251_P02_G10, as described in Table 3.

[0156] Preferably, the VH region and VL region described above each include four framework regions (FR1 to FR4). In one embodiment, the polyspecific antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) that includes a sequence having at least 80% sequence identity with any one of the framework regions of SEQ ID NOs. 62 to 85. In one embodiment, the polyspecific antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) that includes a sequence having at least 90%, for example, at least 95%, 97%, or 99% sequence identity with any one of the framework regions of SEQ ID NOs. 62 to 85. In one embodiment, the polyspecific antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) that includes any one of the sequences of SEQ ID NOs. 62 to 85. In one embodiment, the polyspecific antibody or fragment includes a framework region (e.g., FR1, FR2, FR3 and / or FR4) consisting of any one of the sequences of SEQ ID NOs. 62 to 85.

[0157] The antibodies described herein may be defined by a variable sequence of the complete light chain and / or heavy chain that confers TRDV1 binding. Accordingly, according to a further aspect of the present invention, an isolated multispecific anti-Vδ1 antibody or a fragment thereof is provided, comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs.62 to 85. According to a further aspect of the present invention, an isolated multispecific anti-Vδ1 antibody or a fragment thereof is provided, wherein the component of the multispecific antibody conferring TRDV1 binding consists of an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs.62 to 85.

[0158] In one embodiment, the polyspecific antibody or fragment comprises a VH region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 62-73. In another embodiment, the polyspecific antibody or fragment comprises a VH region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 62-73. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 62, 63, 64, 65, or 66, for example, 62, 63, 64, or 65, in particular 62, 63, or 64. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 62, 63, 64, 65, or 66, for example, 62, 63, 64, or 65, in particular 62, 63, or 64. In a further embodiment, the VH region comprises an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 68, 69, 70, 71, 72, or 73, for example, 68, 69, 70, or 71.

[0159] In one embodiment, the polyspecific antibody or fragment thereof includes a VL region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 74-85. In another embodiment, the polyspecific antibody or fragment thereof includes a VL region comprising an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 74-85. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 74, 75, 76, 77, or 78, for example, 74, 75, 76, or 77, in particular 74, 75, or 76. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 74, 75, 76, 77, or 78, for example, 74, 75, 76, or 77, in particular 74, 75, or 76. In a further embodiment, the VL region comprises an amino acid sequence having at least 80% sequence identity with any one of sequence numbers 80, 81, 82, 83, 84, or 85, for example, 80, 81, 82, or 83.

[0160] In a further embodiment, the polyspecific antibody or fragment comprises a VH region containing an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 62-73, and a VL region containing an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 74-85.

[0161] In a further embodiment, the polyspecific antibody or fragment comprises a VH region containing an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 62-73, and a VL region containing an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs: 74-85.

[0162] In a further embodiment, the polyspecific antibody or fragment comprises a VH region containing an amino acid sequence having at least 96% sequence identity with any one of SEQ ID NOs: 62-73, and a VL region containing an amino acid sequence having at least 96% sequence identity with any one of SEQ ID NOs: 74-85.

[0163] In one embodiment, the multispecific antibody or fragment includes a VH region (1252_P01_C08) containing the amino acid sequence of SEQ ID NO: 63. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P01_E07) containing the amino acid sequence of SEQ ID NO: 62. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P02_G04) containing the amino acid sequence of SEQ ID NO: 64. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1139_P01_E04) containing the amino acid sequence of SEQ ID NO: 68. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P02_F07) containing the amino acid sequence of SEQ ID NO: 69. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P01_G06) containing the amino acid sequence of SEQ ID NO: 70. In an alternative embodiment, the polyspecific antibody or fragment thereof includes a VH region (1245_P01_G09) containing the amino acid sequence of SEQ ID NO: 71.

[0164] In one embodiment, the multispecific antibody or fragment includes a VH region (1252_P01_C08) consisting of the amino acid sequence of SEQ ID NO: 63. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P01_E07) consisting of the amino acid sequence of SEQ ID NO: 62. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P02_G04) consisting of the amino acid sequence of SEQ ID NO: 64. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1139_P01_E04) consisting of the amino acid sequence of SEQ ID NO: 68. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P02_F07) consisting of the amino acid sequence of SEQ ID NO: 69. In an alternative embodiment, the multispecific antibody or fragment includes a VH region (1245_P01_G06) consisting of the amino acid sequence of SEQ ID NO: 70. In an alternative embodiment, the polyspecific antibody or fragment thereof includes a VH region (1245_P01_G09) consisting of the amino acid sequence of SEQ ID NO: 71.

[0165] In one embodiment, the multispecific antibody or fragment includes a VL region (1252_P01_C08) containing the amino acid sequence of SEQ ID NO: 75. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P01_E07) containing the amino acid sequence of SEQ ID NO: 74. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P02_G04) containing the amino acid sequence of SEQ ID NO: 76. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1139_P01_E04) containing the amino acid sequence of SEQ ID NO: 80. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P02_F07) containing the amino acid sequence of SEQ ID NO: 81. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P01_G06) containing the amino acid sequence of SEQ ID NO: 82. In an alternative embodiment, the polyspecific antibody or fragment thereof includes a VL region (1245_P01_G09) containing the amino acid sequence of SEQ ID NO: 83.

[0166] In one embodiment, the multispecific antibody or fragment includes a VL region (1252_P01_C08) consisting of the amino acid sequence of SEQ ID NO: 75. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P01_E07) consisting of the amino acid sequence of SEQ ID NO: 74. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P02_G04) consisting of the amino acid sequence of SEQ ID NO: 76. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1139_P01_E04) consisting of the amino acid sequence of SEQ ID NO: 80. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P02_F07) consisting of the amino acid sequence of SEQ ID NO: 81. In an alternative embodiment, the multispecific antibody or fragment includes a VL region (1245_P01_G06) consisting of the amino acid sequence of SEQ ID NO: 82. In an alternative embodiment, the polyspecific antibody or fragment thereof includes a VL region (1245_P01_G09) consisting of the amino acid sequence of SEQ ID NO: 83.

[0167] In one embodiment, the polyspecific antibody or fragment comprises a VH region (1252_P01_C08) containing the amino acid sequence of SEQ ID NO: 63 and a VL region (1252_P01_C08) containing the amino acid sequence of SEQ ID NO: 75. In an alternative embodiment, the polyspecific antibody or fragment comprises a VH region (1245_P01_E07) containing the amino acid sequence of SEQ ID NO: 62 and a VL region (1245_P01_E07) containing the amino acid sequence of SEQ ID NO: 74. In an alternative embodiment, the polyspecific antibody or fragment comprises a VH region (1245_P02_G04) containing the amino acid sequence of SEQ ID NO: 64 and a VL region (1245_P02_G04) containing the amino acid sequence of SEQ ID NO: 76. In an alternative embodiment, the polyspecific antibody or fragment comprises a VH region (1139_P01_E04) containing the amino acid sequence of SEQ ID NO: 68 and a VL region (1139_P01_E04) containing the amino acid sequence of SEQ ID NO: 80. In an alternative embodiment, the polyspecific antibody or fragment comprises a VH region (1245_P02_F07) containing the amino acid sequence of SEQ ID NO: 69 and a VL region (1245_P02_F07) containing the amino acid sequence of SEQ ID NO: 81. In an alternative embodiment, the polyspecific antibody or fragment comprises a VH region (1245_P01_G06) containing the amino acid sequence of SEQ ID NO: 70 and a VL region (1245_P01_G06) containing the amino acid sequence of SEQ ID NO: 82. In an alternative embodiment, the polyspecific antibody or fragment thereof includes a VH region (1245_P01_G06) containing the amino acid sequence of SEQ ID NO: 71 and a VL region (1245_P01_G09) containing the amino acid sequence of SEQ ID NO: 83.

[0168] In one embodiment, the polyspecific antibody or fragment includes a VH region (1252_P01_C08) consisting of the amino acid sequence of SEQ ID NO: 63 and a VL region (1252_P01_C08) consisting of the amino acid sequence of SEQ ID NO: 75. In an alternative embodiment, the polyspecific antibody or fragment includes a VH region (1245_P01_E07) consisting of the amino acid sequence of SEQ ID NO: 62 and a VL region (1245_P01_E07) consisting of the amino acid sequence of SEQ ID NO: 74. In an alternative embodiment, the polyspecific antibody or fragment includes a VH region (1245_P02_G04) consisting of the amino acid sequence of SEQ ID NO: 64 and a VL region (1245_P02_G04) consisting of the amino acid sequence of SEQ ID NO: 76. In alternative embodiments, the polyspecific antibody or fragment includes a VH region (1139_P01_E04) consisting of the amino acid sequence of SEQ ID NO: 68 and a VL region (1139_P01_E04) consisting of the amino acid sequence of SEQ ID NO: 80. In alternative embodiments, the polyspecific antibody or fragment includes a VH region (1245_P02_F07) consisting of the amino acid sequence of SEQ ID NO: 69 and a VL region (1245_P02_F07) consisting of the amino acid sequence of SEQ ID NO: 81. In alternative embodiments, the polyspecific antibody or fragment includes a VH region (1245_P01_G06) consisting of the amino acid sequence of SEQ ID NO: 70 and a VL region (1245_P01_G06) consisting of the amino acid sequence of SEQ ID NO: 82. In an alternative embodiment, the polyspecific antibody or fragment thereof includes a VH region (1245_P01_G06) consisting of the amino acid sequence of SEQ ID NO: 71 and a VL region (1245_P01_G09) consisting of the amino acid sequence of SEQ ID NO: 83.

[0169] In the case of a fragment containing both a VH region and a VL region, these may be associated either covalently (e.g., via a disulfide bond or linker) or non-covalently. The polyspecific antibody fragments described herein may include scFv, i.e., fragments containing a VH region and a VL region linked by a linker. In one embodiment, the VH region and the VL region are linked by a (e.g., synthetic) polypeptide linker. The polypeptide linker is (Gly4Ser) nIt may include a linker (where n=1-8, e.g., 2, 3, 4, 5, or 7). The polypeptide linker is [(Gly4Ser) n (Gly3AlaSer) m ] p A linker may be included (where n = 1 to 8, e.g., 2, 3, 4, 5, or 7, m = 1 to 8, e.g., 0, 1, 2, or 3, and p = 1 to 8, e.g., 1, 2, or 3). In a further embodiment, the linker includes sequence number 98. In a further embodiment, the linker consists of sequence number 98.

[0170] In one embodiment, the multispecific antibody or fragment contains an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 86 to 97. In a further embodiment, the multispecific antibody or fragment contains an amino acid sequence with any one of SEQ ID NOs: 86 to 97. In yet another embodiment, the multispecific antibody or fragment contains the amino acid sequence of SEQ ID NO: 87 (1252_P01_C08). In an alternative embodiment, the multispecific antibody or fragment contains the amino acid sequence of SEQ ID NO: 86 (1245_P01_E07). In an alternative embodiment, the multispecific antibody or fragment contains the amino acid sequence of SEQ ID NO: 88 (1245_P02_G04). In an alternative embodiment, the multispecific antibody or fragment contains the amino acid sequence of SEQ ID NO: 92 (1139_P01_E04). In an alternative embodiment, the multispecific antibody or fragment contains the amino acid sequence of SEQ ID NO: 93 (1245_P02_F07). In an alternative embodiment, the polyspecific antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). In an alternative embodiment, the polyspecific antibody or fragment thereof comprises the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09).

[0171] In one embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs: 86 to 97. In a further embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of an amino acid sequence with any one of SEQ ID NOs: 86 to 97. In yet another embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of the amino acid sequence of SEQ ID NO: 87 (1252_P01_C08). In an alternative embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of the amino acid sequence of SEQ ID NO: 86 (1245_P01_E07). In an alternative embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of the amino acid sequence of SEQ ID NO: 88 (1245_P02_G04). In an alternative embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of the amino acid sequence of SEQ ID NO: 92 (1139_P01_E04). In alternative embodiments, the component of the multispecific antibody that conjugates TRDV1 consists of the amino acid sequence of SEQ ID NO: 93 (1245_P02_F07). In alternative embodiments, the component of the multispecific antibody that conjugates TRDV1 consists of the amino acid sequence of SEQ ID NO: 94 (1245_P01_G06). In alternative embodiments, the component of the multispecific antibody that conjugates TRDV1 consists of the amino acid sequence of SEQ ID NO: 95 (1245_P01_G09).

[0172] Those skilled in the art will understand that scFv constructs can be designed and fabricated, including modifications to the N-terminus and C-terminus, to aid in translation, purification, and detection. For example, the N-terminus of the scFv sequence may include additional methionine and / or alanine amino acid residues prior to the canonical VH sequence (e.g., beginning with QVQ or EVQ). The C-terminus (i.e., the C-terminal side of the canonical mature VL domain sequence ending as defined by IMGT) may include additional sequences, such as (i) a partial sequence of the constant domain and / or (ii) additional synthetic sequences containing tags such as His tags and Flag tags, to aid in purification and detection. In one embodiment, SEQ ID NO: 124 is appended to the C-terminus of any one of SEQ ID NOs: 86, 88-90, or 92-97. In one embodiment, SEQ ID NO: 125 is appended to the C-terminus of any one of SEQ ID NOs: 86, 88-90, or 92-97. In one embodiment, SEQ ID NO: 126 is appended to the C-terminus of either SEQ ID NOs: 87 or 91. In one embodiment, sequence number 127 is appended to the C-terminus of either sequence number 87 or 91. It is well understood that the N-terminal or C-terminal sequence of the scFv is arbitrary and may be removed, modified, or substituted when alternative scFv design, translation, purification, or detection strategies are employed.

[0173] As described herein, the antibody may be in any format. In a preferred embodiment, the polyspecific antibody is in IgG1 format. Thus, in one embodiment, the polyspecific antibody or fragment comprises an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 111-122. In a further embodiment, the polyspecific antibody or fragment comprises an amino acid sequence of any one of SEQ ID NOs. 111-122. In yet another embodiment, the polyspecific antibody or fragment comprises an amino acid sequence of SEQ ID NOs. 111-116, for example, SEQ ID NOs. 111-113 and 116. In yet another embodiment, the polyspecific antibody or fragment comprises an amino acid sequence of SEQ ID NOs. 117-122, for example, SEQ ID NOs. 117-120. In yet another embodiment, the polyspecific antibody or fragment comprises an amino acid sequence of SEQ ID NOs. 111, 112, 116-120, for example, SEQ ID NOs. 111, 112 or 116, or SEQ ID NOs. 117-120.

[0174] In one embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 111 to 122. In a further embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of an amino acid sequence of any one of SEQ ID NOs. 111 to 122. In yet another embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of the amino acid sequences of SEQ ID NOs. 111 to 116, for example, SEQ ID NOs. 111 to 113 and 116. In yet another embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of the amino acid sequences of SEQ ID NOs. 117 to 122, for example, SEQ ID NOs. 117 to 120. In yet another embodiment, the component of the multispecific antibody that confers TRDV1 binding consists of the amino acid sequences of SEQ ID NOs. 111, 112, 116 to 120, for example, SEQ ID NOs. 111, 112 or 116, or SEQ ID NOs. 117 to 120.

[0175] In one embodiment, a polyspecific antibody binds to the same or essentially the same TRDV1 epitope as the antibody or fragment thereof as defined herein, or competes with the antibody or fragment thereof as defined herein. By using standard methods known in the art, it is possible to easily determine whether a polyspecific antibody binds to the same TRDV1 epitope as the reference anti-Vδ1 antibody, or whether it competes for binding with the reference anti-Vδ1 antibody. For example, to determine whether a test antibody binds to the same TRDV1 epitope as the reference anti-Vδ1 antibody of the present invention, the reference antibody is conjugated to the Vδ1 protein or peptide under saturated conditions. Next, the ability of the test polyspecific antibody to bind to the Vδ1 chain is evaluated. If the test polyspecific antibody can bind to Vδ1 after saturated binding with the reference anti-Vδ1 antibody, it can be concluded that the test polyspecific antibody binds to a different TRDV1 epitope than the reference anti-Vδ1 antibody. On the other hand, if the test multispecific antibody is unable to bind to the Vδ1 chain after saturated binding with the reference anti-Vδ1 antibody, the test multispecific antibody may bind to the same TRDV1 epitope as the reference anti-Vδ1 antibody of the present invention. Naturally, binding to the TRDV1 epitope refers to the component of the multispecific antibody that confers binding to the first target epitope, which is the epitope of TRDV1. Testing for binding to the same or different epitopes may, alternatively, be performed using the sequences of the multispecific antibodies provided herein, but in a monospecific format.

[0176] The present invention also includes a multispecific anti-Vδ1 antibody that competes for binding to Vδ1 with an antibody having a CDR sequence of any of the antibodies or fragments thereof as defined herein (in either a multispecific or monospecific format), or any of the exemplary antibodies described herein. For example, competitive assays can be performed using the antibodies of the present invention to determine which proteins, antibodies, and other antagonists compete with the antibodies of the present invention for binding to the Vδ1 chain and / or share an epitope. These assays are readily apparent to those skilled in the art. They evaluate competition between antagonists or ligands for a limited number of binding sites on a protein, such as Vδ1. The antibody (or fragment thereof) is immobilized or insolubilized before or after the competition, and the sample bound to the Vδ1 chain is separated from the unbound sample, for example, by decanting (if the antibody is insolubilized beforehand) or centrifugation (if the antibody precipitates after the competitive reaction). Competitive binding may also be determined by whether the binding or lack of binding of the antibody to the protein alters its function, for example, whether the antibody molecule inhibits or enhances the activity of enzymes such as labeling. ELISA and other functional assays known in the art and described herein can be used.

[0177] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to the target antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antibody will inhibit the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured by a competitive binding assay. Alternatively, if essentially all amino acid mutations in the target antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitope.

[0178] Subsequently, additional standard experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is actually due to binding to the same epitope as the reference antibody, or whether steric hindrance (or another phenomenon) is the cause of the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0179] In some embodiments, the antibody or fragment thereof includes an effector function modified via a mutation of a sugar linked to Asn 297 (EU numbering scheme). In further modifications, Asn 297 exhibits unfucosylated or reduced fucosylation (i.e., a defucosylated antibody or non-fucosylated antibody). Fucosylation includes the addition of sugar fucose to a molecule, e.g., the binding of fucose to N-glycans, O-glycans, and glycolipids. Therefore, in defucosylated antibodies, fucose is not bound to the carbohydrate chain in the constant region. Antibodies may be modified to prevent or inhibit antibody fucosylation. Typically, glycosylation modifications include the expression of the antibody or fragment thereof in host cells with alternative glycosylation processing functions, either by targeted engineering or by targeted or accidental selection of a host or clone (see, for example, Example 13). These and other effector modifications are further described in recent reviews, including those by Xinhua Wang et al. (2018) Protein & Cell 9:63-73 and Pereira et al. (2018) mAbs 10(5):693-711, which are incorporated herein.

[0180] Modification of antibody sequences Polyspecific antibodies and their fragments can be modified using known methods. Sequence modifications to antibody molecules described herein, particularly those to parts of polyspecific antibodies that confer Vδ1 binding, can be readily adopted by those skilled in the art. The following examples are not limiting.

[0181] During antibody discovery and sequence retrieval from phage libraries, the desired antibody variable domain can be reorganized into full-length IgG by subcloning. To accelerate the process, the variable domain is often transferred using restriction enzymes. These unique restriction sites can introduce additional / alternative amino acids and deviate from the canonical sequence (such canonical sequences can be found, for example, in the international ImMunoGeneTics [IMGT] information system; see http: / / www.imgt.org). These can be introduced as sequence modifications of the kappa or lambda light chain.

[0182] Modification of Kappa Light Chain The variable sequence of the variable kappa light chain can be cloned using a restriction site (e.g., Nhe1-Not1) when rearranged into full-length IgG. More specifically, an additional Ala-Ser sequence is introduced at the N-terminus of the kappa light chain to support cloning. Preferably, this additional AS sequence is then removed during further development, such as generating a canonical N-terminal sequence. Thus, in one embodiment, the kappa light chain-containing antibodies described herein do not contain an AS sequence at the N-terminus; that is, SEQ ID NOs. 74, 76-78 and 80-85 do not contain the first AS sequence. In a further embodiment, SEQ ID NOs. 74 and 76-78 do not contain the first AS sequence. It will be understood that this embodiment also applies to other sequences contained herein that contain this sequence (e.g., SEQ ID NOs. 86, 88-90 and 92-97).

[0183] Additional amino acid changes may be made to support cloning. For example, in the antibodies described herein, a change from valine to alanine was introduced at the boundary between the kappa light chain variable domain and the constant domain to support cloning. This modified the kappa constant domain. Specifically, this modified the constant domain [ka] It will start (from the NotI restriction site). Preferably, this sequence is [ka] Further development may be modified to generate a canonical kappa light chain constant region beginning with . Thus, in one embodiment, the kappa light chain-containing antibody described herein contains a constant domain beginning with sequence RTV. Thus, in one embodiment, sequences 111-114 and 117-122 [ka] is an array [ka] It can be replaced by [this]. See, for example, Example 13 and Sequence IDs 129 and 130.

[0184] Modification of Lambda Light Chain Similar to the kappa example described above, the lambda light chain variable domain can also be cloned by introducing a restriction site (e.g., Nhe1-Not1) during rearrangement into full-length IgG. More specifically, an additional Ala-Ser sequence may be introduced at the N-terminus of the lambda light chain to support cloning. Preferably, this additional AS sequence is then removed during further development, such as generating a canonical N-terminal sequence. Thus, in one embodiment, the lambda light chain-containing antibodies described herein do not contain an AS sequence at the N-terminus; that is, SEQ ID NOs. 75 and 79 do not contain the initial AS sequence. It will be understood that this embodiment also applies to other sequences contained herein that contain this sequence (e.g., SEQ ID NOs. 87, 91, 115 and 116). In one embodiment, SEQ ID NO. 75 does not contain the first six residues; that is, the ASSYEL sequence is removed.

[0185] As another example, in the antibodies described herein, a sequence change from lysine to alanine was introduced at the lambda light chain variable domain / constant domain boundary to support cloning. This modified the lambda constant domain. Specifically, this resulted in the constant domain being [ka] It will start (from the NotI restriction site). Preferably, this sequence is [ka] It may be modified during further development to generate a canonical lambda light chain constant region beginning with GQPK. Thus, in one embodiment, the lambda light chain-containing antibody described herein contains a constant domain beginning with the sequence GQPK. Thus, in one embodiment, the sequence of SEQ ID NO: 115 or 116 [ka] is an array [ka] It can be replaced with.

[0186] Modification of heavy chain Typically, human variable heavy chain sequences begin with either basic glutamine (Q) or acidic glutamic acid (E). However, both such sequences are known to subsequently be converted to pyroglutamic acid (pE), an acidic amino acid residue. The conversion from Q to pE alters the antibody's charge, while the conversion from E to pE does not. Therefore, one option to avoid fluctuations in charge change over time is to modify the initial Q in the starting heavy chain sequence to E. Thus, in one embodiment, the antibody heavy chain described herein contains a Q-to-E modification at its N-terminus. In particular, the first residue of SEQ ID NOs. 62, 64 and / or 67-71 can be modified from Q to E. It will be understood that this embodiment also applies to other sequences containing this sequence included herein (e.g., SEQ ID NOs. 86, 88, 91-97 and 111, 112, 115, 117-120). See, for example, Example 13 and SEQ ID NOs. 129, 130.

[0187] Furthermore, the C-terminus of the IgG1 constant domain terminates with PGK. However, the terminal basic lysine (K) is often subsequently cleaved during expression (e.g., in CHO cells). As a result, various loss of C-terminal lysine residues alters the antibody's charge. Therefore, one option is to remove the initial lysine to obtain a uniform and consistent heavy-chain C-terminal sequence ending with PG. Thus, in one embodiment, the heavy chain of the antibody described herein has terminal K removed from its C-terminus. In particular, the antibodies of the present invention may include any one of SEQ ID NOs. 111-122, in which the terminal lysine residue is removed. See, for example, SEQ ID NO. 141.

[0188] Modification of any Arotype During antibody discovery, a specific human allotype may be used. In some cases, antibodies can be switched to different human allotypes during development. As a non-limiting example, the kappa chain has three human allotypes called Km1, Km1,2, and Km3, which define three Km alleles (using allotype numbering). Km1 correlates with valine 153 (IMGT V45.1) and leucine 191 (IMGT L101), Km1,2 correlates with alanine 153 (IMGT A45.1) and leucine 191 (IMGT L101), and Km3 correlates with alanine 153 (IMGT A45.1) and valine 191 (IMGT V101). Thus, in some cases, a sequence can be modified from one allotype to another by a standard cloning approach. For example, a change in L191V (IMGT L101V) converts the Km1,2 allotype to the Km3 allotype. For details on such allotypes, see Jefferis and Lefranc (2009) MAbs 1(4):332-8. This document is incorporated herein by reference.

[0189] Therefore, in one embodiment, the polyspecific antibodies described herein contain amino acid substitutions derived from another human allotype of the same gene. In a further embodiment, the antibody contains a kappa chain L191V (IMGT L101V) substitution to convert the c-domain from km1,2 to km3 allotype. See, for example, Example 13 and SEQ ID NOs: 129, 130.

[0190] Antibodies that target the TRDV1 epitope This specification provides antibodies (or fragments thereof) that bind to an epitope on the Vδ1 chain of γδTCR (and further bind to a second epitope as described elsewhere). Such binding may, in some cases, affect γδTCR activity, such as activation. The antibodies of the present invention are preferably specific to the Vδ1 chain of γδTCR and do not bind to epitopes of other antigens, such as the Vδ2 or Vδ3 chains of γδTCR. The antibodies of the present invention can be considered agonist antibodies, at least with respect to the agonist activity conferred to Vδ1 cells upon binding.

[0191] In one embodiment, the epitope may be an activating epitope for γδT cells. An "activating" epitope may include, for example, stimulation of TCR function such as cell degranulation, TCR downregulation, cytotoxicity, proliferation, recruitment, increased resistance to survival or depletion, intracellular signaling, secretion of cytokines or growth factors, phenotypic changes, or altered gene expression. For example, binding of an activating epitope may stimulate an increase (i.e., proliferation) of the γδT cell population, preferably the Vδ1+ T cell population. Therefore, these multispecific antibodies can be used to modulate γδT cell activation and thereby modulate the immune response. Thus, in one embodiment, binding of an activating epitope downregulates the γδTCR. In additional or alternative embodiments, binding of an activating epitope activates γδT cell degranulation. In further additional or alternative embodiments, binding of an activating epitope promotes γδT cell-mediated killing.

[0192] In some embodiments, the activating epitope of TRDV1, upon antibody binding, results in downregulation of the receptor and, optionally, activation of Vδ1 cells. In some embodiments, this downregulation of the receptor also results in downregulation of the associated CD3 molecule. In some embodiments, the activating epitope upregulates the expression of activation markers on Vδ1 cells, such as CD107a, CD25, CD69, and / or Ki67. In some embodiments, the activating epitope upregulates the expression of activation markers on Vδ1 cells, such as CD107 and CD25, and optionally CD69 and / or Ki67. In some embodiments, upregulation of one or more activation markers (such as CD107a) may occur in the presence of cancer cells. In preferred embodiments of the present invention, a multispecific antibody binds to the activating epitope of TRDV1, particularly via the TRDV1-binding domain.

[0193] Because T cell receptors often form complexes with other proteins, downregulation of the T cell receptor via Vδ1 antibody binding can lead to downregulation of other proteins associated with the T cell receptor (i.e., binding of the Vδ1 antibody leads to downregulation of the T cell receptor complex). For example, in some embodiments, the activating epitope of TRDV1 downregulates the TCR / CD3 receptor complex upon binding. Thus, the antibodies of the present invention can indirectly downregulate cell surface proteins that are not bound to the antibody but are complexed with the T cell receptor. Considering that T cells expressing gamma delta 1 chains (i.e., Vδ1 cells) represent only a small fraction of the total T cell population, the antibodies of the present invention can be used to selectively (and indirectly) downregulate proteins in the TCR complex, such as CD3, by downregulating them only in Vδ1 cells.

[0194] In some embodiments, the activation epitope of the T cell receptor complex, upon activation, downregulates the T cell receptor complex, but does not downregulate CD3 molecules unrelated to the TRDV1 TCR complex.

[0195] This epitope preferably consists of at least one extracellular portion, soluble portion, hydrophilic portion, or outer portion of the Vδ1 chain of γδ TCR.

[0196] In particular, this epitope does not include epitopes found in the hypervariable regions of the Vδ1 chain of γδ TCR, especially CDR3 of the Vδ1 chain. In a preferred embodiment, this epitope is within the non-variable region of the Vδ1 chain of γδ TCR. It will be understood that such binding enables unique recognition of the Vδ1 chain without being restricted to highly variable TCR sequences (especially CDR3). Various γδ TCR complexes that recognize antigens can be recognized in this manner solely by the presence of the Vδ1 chain. Thus, it will be understood that any γδ TCR containing the Vδ1 chain can be recognized using the antibodies or fragments thereof defined herein, regardless of the specificity of the γδ TCR. In one embodiment, the epitope includes one or more amino acid residues within amino acid regions 1-24 and / or 35-90 of SEQ ID NO: 1, for example, a portion of the Vδ1 chain that is not part of the CDR1 and / or CDR3 sequences. In one embodiment, the epitope does not include amino acid residues within amino acid region 91-105 (CDR3) of SEQ ID NO: 1.

[0197] In some embodiments, the epitope includes amino acids in the TRDV-1 CDR2 sequence.

[0198] Similar to well-characterized αβ T cells, γδ T cells utilize a unique set of somatically rearranged variable (V) genes, diversity (D) genes, joining (J) genes, and constant (C) genes, but γδ T cells contain fewer V segments, D segments, and J segments than αβ T cells. In one embodiment, the epitope to which the antibody (or fragment thereof) binds is the J region of the Vδ1 chain (e.g., the four J regions encoded by the human delta 1 chain germline: SEQ ID NO: 152 (J1 * 0) or 153 (J2 * 0) or 154 (J3 * 0) or 155 (J4 *(one of (0)) or does not contain an epitope found in the C region of the Vδ1 chain (e.g., SEQ ID NO: 156 (C1) containing the C-terminal membrane-proximal / membrane-spanning region) * does not contain an epitope found in the N-terminal leader sequence of the Vδ1 chain (e.g., SEQ ID NO: 150). Thus, in one embodiment, the epitope to which the antibody (or fragment thereof) binds does not contain an epitope found in the N-terminal leader sequence of the Vδ1 chain. Thus, the antibody or fragment can bind only in the V region of the Vδ1 chain (e.g., SEQ ID NO: 151). Thus, in one embodiment, the epitope consists of an epitope within the V region of the γδ TCR (e.g., amino acid residues 1-90 of SEQ ID NO: 1).

[0199] References to epitopes are made in the context of the sequence shown as SEQ ID NO: 1 in Luoma et al. (2013) Immunity 39:1032-1042, and Vδ1 sequences derived from RCSB Protein Data Bank entries: 4MNH and 3OMZ. AQKVTQAQSSVSMPVRKAVTLNCLYETSWWSYYIFWYKQLPSKEMIFLIRQGSDEQNAKSGRYSVNFKKAAKSVALTISALQLEDSAKYFCALGESLTRADKLIFGKGTRVTVEPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 1)

[0200] SEQ ID NO: 1 represents a soluble TCR containing a V region (also referred to as the variable domain), a D region, a J region, and a TCR constant region. The V region contains amino acid residues 1-90, the D region contains amino acid residues 91-104, the J region contains amino acid residues 105-115, and the constant region (derived from T cell receptor alpha) contains amino acid residues 116-209. Within the V region, CDR1 is defined as amino acid residues 25-34 of SEQ ID NO: 1, CDR2 is defined as amino acid residues 50-54 of SEQ ID NO: 1, and CDR3 is defined as amino acid residues 93-104 of SEQ ID NO: 1 (Xu et al., PNAS USA 108(6):2414-2419 (2011)).

[0201] Thus, according to one aspect of the present invention, (i) amino acids 3 to 20 of SEQ ID NO: 1, and / or (ii) amino acids 37 to 77 of SEQ ID NO: 1 There is provided an isolated antibody or fragment thereof that binds to an epitope of the variable delta 1 (Vδ1) chain of a γδ T cell receptor (TCR) that includes one or more amino acid residues within the amino acid region of.

[0202] In a further embodiment, the antibody or fragment thereof further recognizes a polymorphic V region that includes an epitope of amino acid residues 1 to 90 of SEQ ID NO: 128. Thus, amino acids 1 to 90 of SEQ ID NO: 1 and the polymorphic germline variant sequence (amino acids 1 to 90 of SEQ ID NO: 128) can be considered synonymous when defining the epitopes described herein. The studies presented herein demonstrate that the antibodies of the present invention can recognize variants of both of these germline sequences. As an example, when it is described that an antibody or fragment thereof as defined herein recognizes an epitope that includes one or more amino acid residues within amino acid regions 1 to 24 and / or 35 to 90 of SEQ ID NO: 1, this also refers to the same regions of SEQ ID NO: 128, specifically amino acid regions 1 to 24 and / or 35 to 90 of SEQ ID NO: 128.

[0203] In one embodiment, the antibody or fragment thereof recognizes one or more amino acid residues within amino acid region 1 to 90 of SEQ ID NO: 1 and amino acids at equivalent positions in region 1 to 90 of SEQ ID NO: 128. More specifically, in one embodiment, the antibody or fragment thereof as defined herein recognizes a human germline epitope, wherein the germline encodes either alanine (A) or valine (V) at position 71 of SEQ ID NO: 1.

[0204] In one embodiment, the epitope includes one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues within the described region.

[0205] In a further embodiment, the epitope comprises one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 3-20 of SEQ ID NO: 1. In an alternative embodiment, the epitope comprises one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 37-77 (e.g., amino acid region 50-54) of SEQ ID NO: 1. In yet another embodiment, the epitope comprises one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 3-20 (e.g., 5-20 or 3-17) of SEQ ID NO: 1 and one or more (e.g., five or more, e.g., ten or more) amino acid residues within amino acid region 37-77 (e.g., 62-77 or 62-69).

[0206] It will be further understood that the antibody (or fragment thereof) does not need to bind to all amino acids within a defined range. Such an epitope may be called a linear epitope. For example, an antibody that binds to an epitope containing amino acid residues within the amino acid region 5-20 of SEQ ID NO: 1 may bind to one or more amino acid residues within the range, for example, only the amino acid residues at both ends of the range (i.e., amino acids 5 and 20), and possibly to amino acids within the range (i.e., amino acids 5, 9, 16, and 20).

[0207] In one embodiment, the epitope comprises at least one of the amino acid residues 3, 5, 9, 10, 12, 16, 17, 20, 37, 42, 50, 53, 59, 62, 64, 68, 69, 72, or 77 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acids selected from the amino acid residues 3, 5, 9, 10, 12, 16, 17, 20, 37, 42, 50, 53, 59, 62, 64, 68, 69, 72, or 77 of SEQ ID NO: 1.

[0208] In one embodiment, the epitope comprises one or more amino acid residues within the following amino acid region of SEQ ID NO: 1 (or SEQ ID NO: 128 as described above): (i) 3-17, (ii) 5-20, (iii) 37-53, (iv) 50-64, (v)59~72, (vi) 59-77, (vii) 62-69, and / or (viii) 62~77.

[0209] In a further embodiment, the epitope comprises one or more amino acid residues within the amino acid region of SEQ ID NO: 5-20 and 62-77, 50-64, 37-53 and 59-72, 59-77, or 3-17 and 62-69. In a further embodiment, the epitope comprises one or more amino acid residues within the amino acid region of SEQ ID NO: 5-20 and 62-77, 50-64, 37-53 and 59-72, 59-77, or 3-17 and 62-69.

[0210] In a further embodiment, the epitope includes the amino acid residues of SEQ ID NO: 3, 5, 9, 10, 12, 16, 17, 62, 64, 68, and 69 of SEQ ID NO: 1, or preferably consists of the amino acid residues of SEQ ID NO: 3, 5, 9, 10, 12, 16, 17, 62, 64, 68, and 69 of SEQ ID NO: 1. In a further embodiment, the epitope includes the amino acid residues of SEQ ID NO: 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO: 1, or preferably consists of the amino acid residues of SEQ ID NO: 1, 5, 9, 16, 20, 62, 64, 72, and 77 of SEQ ID NO: 1. In further embodiments, the epitope includes the amino acid residues of SEQ ID NO: 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77, or preferably consists of the amino acid residues of SEQ ID NO: 37, 42, 50, 53, 59, 64, 68, 69, 72, 73, and 77. In further embodiments, the epitope includes the amino acid residues of SEQ ID NO: 50, 53, 59, 62, and 64, or preferably consists of the amino acid residues of SEQ ID NO: 50, 53, 59, 62, and 64. In further embodiments, the epitope includes the amino acid residues of SEQ ID NO: 59, 60, 68, and 72, or preferably consists of the amino acid residues of SEQ ID NO: 59, 60, 68, and 72.

[0211] In one embodiment, the epitope comprises one or more amino acid residues within amino acid regions 5-20 and / or 62-77 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one or more amino acid residues within amino acid regions 5-20 and 62-77 of SEQ ID NO: 1. In an alternative further embodiment, the epitope comprises one or more amino acid residues within amino acid regions 5-20 or 62-77 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may have part or all of the sequence of 1245_P01_E07, or such an antibody or fragment thereof may be derived from 1245_P01_E07. For example, an antibody or fragment thereof having one or more CDR sequences of 1245_P01_E07 or one or both of the VH and VL sequences of 1245_P01_E07 may bind to such an epitope.

[0212] In one embodiment, the epitope comprises one or more amino acid residues within the amino acid region 50-64 of SEQ ID NO: 1. In a further embodiment, the epitope consists of one or more amino acid residues within the amino acid region 50-64 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may have part or all of the sequence of 1252_P01_C08, or such an antibody or fragment thereof may be derived from 1252_P01_C08. For example, an antibody or fragment thereof having one or more CDR sequences of 1252_P01_C08 or one or both of the VH and VL sequences of 1252_P01_C08 may bind to such an epitope.

[0213] In one embodiment, the epitope comprises one or more amino acid residues within the amino acid region 37-53 and / or 59-77 of SEQ ID NO: 1. In a further embodiment, the epitope comprises one or more amino acid residues within the amino acid region 37-53 and 59-77 of SEQ ID NO: 1. In an alternative further embodiment, the epitope comprises one or more amino acid residues within the amino acid region 37-53 or 59-77 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may have part or all of the sequence of 1245_P02_G04, or such an antibody or fragment thereof may be derived from 1245_P02_G04. For example, an antibody or fragment thereof having one or more CDR sequences of 1245_P02_G04 or one or both of the VH and VL sequences of 1245_P02_G04 may bind to such an epitope.

[0214] In one embodiment, the epitope comprises one or more amino acid residues within the amino acid region 59-72 of SEQ ID NO: 1. In a further embodiment, the epitope consists of one or more amino acid residues within the amino acid region 59-72 of SEQ ID NO: 1. An antibody or fragment thereof having such an epitope may have part or all of the sequence of 1251_P02_C05, or such an antibody or fragment thereof may be derived from 1251_P02_C05. For example, an antibody or fragment thereof having one or more CDR sequences of 1251_P02_C05 or one or both of the VH and VL sequences of 1251_P02_C05 may bind to such an epitope.

[0215] In one embodiment, the epitope does not contain amino acid residues within amino acid regions 11-21 of SEQ ID NO: 1. In one embodiment, the epitope does not contain amino acid residues within amino acid regions 21-28 of SEQ ID NO: 1. In one embodiment, the epitope does not contain amino acid residues within amino acid regions 59 and 60 of SEQ ID NO: 1. In one embodiment, the epitope does not contain amino acid residues within amino acid regions 67-82 of SEQ ID NO: 1.

[0216] In one embodiment, the epitope is not the same as the epitope to which commercially available anti-Vδ1 antibodies such as TS-1 or TS8.2 bind. As described in WO2017197347, binding of TS-1 and TS8.2 to soluble TCRs was detected when the δ1 chain contained Vδ1 J1 and Vδ1 J2 sequences, but not for the Vδ1 J3 chain, indicating that key residues within the delta J1 and delta J2 regions are involved in the binding of TS-1 and TS8.2.

[0217] In this specification, "within" includes both ends of the defined range. For example, "within amino acid region 5-20" refers to all amino acid residues from residue 5 to residue 20.

[0218] Various techniques for determining which epitope an antibody binds to are known in the art. Exemplary techniques include, for example, standard cross - blocking assays, alanine - scanning mutagenesis, peptide blot analysis, peptide cleavage analysis, crystallographic experiments, and NMR analysis. Additionally, methods such as epitope excision, epitope extraction, and chemical modification of the antigen can be used. Another method that can be used to identify amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry (as described in Example 9). Generally speaking, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium and then binding an antibody to the deuterium - labeled protein. Next, the protein / antibody complex is transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex undergo back - exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within amino acids that are not part of the interface. As a result, the amino acids forming part of the protein / antibody interface may retain deuterium and thus exhibit a relatively higher mass compared to amino acids not contained within the interface. After dissociation of the antibody, the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing the deuterium - labeled residues corresponding to the specific amino acids with which the antibody interacts.

[0219] Bispecific antibodies and their fragments preferably specifically bind via the TRDV1 - binding domain to both human TRDV1 (polymorphic variants of SEQ ID NO: 1 and SEQ ID NO: 128) and cynomolgus monkey TRDV1 (SEQ ID NO: 172).

[0220] An antibody targeting an epitope of a second antigen Since the antibodies of the present invention are bispecific antibodies (preferably bispecific antibodies), in addition to specifically binding to TRDV1, they specifically bind to a second antigen. The identity of the second antigen determines whether the antibody is one of two categories described herein: a T - cell engager (TCE) antibody or a dual immunomodulator (DI) antibody.

[0221] In embodiments relating to TCE, the second antigen is a cancer antigen or cancer-associated antigen. In such embodiments, the antibody specifically binds to a first target epitope and a second target epitope, the first target epitope being an epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR), and the second target epitope being an epitope of a cancer antigen or cancer-associated antigen. The identity of specific second antigens that may be considered in this category will be described elsewhere. However, the second antigen may be any antigen expressed by cancer cells that promotes Vδ1-T cell-mediated killing of said cancer cells (e.g., direct killing or killing via the immune licensing effect of signaling to other immune cells when bound to tumor cells). Such killing of cancer cells by Vδ1 cells is promoted by co-localization of Vδ1-T cells and cancer cells, as well as activation of Vδ1-T cells via the binding of a multispecific antibody, particularly binding to the activating epitope of the Vδ1-T cells. This invention exemplifies a completely novel platform for TCE-type antibodies.

[0222] In some embodiments, the second antigen is not an ovarian cancer antigen. In some embodiments, the second antigen is not a Mov19+ ovarian cancer antigen. In some embodiments, the polyspecific (preferably bispecific) antibody does not specifically bind to Mov19+ ovarian cancer cells. In some embodiments, the polyspecific (preferably bispecific) antibody does not specifically bind to the alpha-folate receptor (alpha-FR). Alpha-FR is also known as folate receptor 1, FOLR1, folate receptor alpha, or FRα. It is encoded by the FOLR1 gene (UniProt accession number P15328) and has the sequence of SEQ ID NO: 176. In some embodiments, the polyspecific (preferably bispecific) antibody does not specifically bind to the epitope to which scFv MOV19 binds.

[0223] In some embodiments, the antibody or its antigen-binding fragment is a bispecific antibody, and the second antigen is not an alpha-folate receptor.

[0224] In some embodiments, the polyspecific antibody is a human recombinant antibody encoded by one or more open reading frames of recombinant nucleic acids expressed from recombinant host cells. In some embodiments, the polyspecific antibody is not a rodent antibody or other non-human antibody derived from B-cell fusion hybridoma technology. In some embodiments, the polyspecific antibody does not contain non-human IgG constant domain sequences found only in non-human animal species, such as sequences found in rodent-derived hybridomas.

[0225] In embodiments relating to DI, the second antigen is an immunomodulatory antigen. In such embodiments, the antibody specifically binds to a first target epitope and a second target epitope, the first target epitope being the variable delta 1 (Vδ1) chain epitope of the γδ T cell receptor (TCR), and the second target epitope being the immunomodulatory antigen. An "immunomodulatory" antigen is an antigen that modulates (e.g., promotes) antibody-mediated and / or cell-mediated immunity. Immunomodulatory antigens are those present on the cell surface of T cells. In embodiments of the present invention where the second antigen is an immunomodulatory antigen, the second antigen is neither a T cell receptor nor a component of a T cell receptor complex. For example, in embodiments of the present invention where the second epitope is an epitope of an immunomodulatory antigen, the second epitope is not the TRDV1 epitope. In preferred embodiments of the present invention, the second epitope is an epitope of an immunomodulatory antigen, and the second epitope is not an epitope of a T cell receptor complex. For example, in some embodiments, the second epitope is not an epitope of CD3. Thus, the antibodies of these embodiments are "dual immunomodulators" because they can bind specifically to T cells via TRDV1 and also bind to T cells via a second, different epitope that is not an epitope of a T cell receptor complex. Examples of the second antigen include, for example, the immune checkpoint inhibitors PD-L1, PD-1, OX40, CTLA-4, LAG-3, TIM-3, TIGIT, and VISTA. For example, solid tumors recruit immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), and regulatory T cells (Tregs), all of which inhibit the activity of cytotoxic T cells. Therefore, to most effectively utilize DI in solid tumors, it is likely necessary to combine and target multiple T cell regulatory pathways using multispecific sites to overcome immunosuppressive TMEs and help turn "cold" tumors—immune excluded or immunodesert—into inflammatory "hot" tumors. However, the present invention presents an entirely novel platform for DI-type antibodies and is not limited to a specific second immunomodulatory antigen.

[0226] In preferred embodiments, the polyspecific antibody (preferably a bispecific antibody) of the present invention does not specifically bind to (or directly interact with) CD3. In preferred embodiments, the second antigen is not CD3.

[0227] antibody binding The multispecific antibody or fragment of the present invention, when measured by surface plasmon resonance, yields 1.5 × 10⁻⁶. -7 It can bind to the Vδ1 chain of the γδTCR with a binding affinity (KD) of less than M (i.e., 150 nM). In a preferred embodiment, the KD is 1.5 × 10⁻⁶ -7 It is less than M (i.e., 150 nM). In a further embodiment, KD is 1.3 × 10⁻⁶ -7 Less than M (i.e., 130 nM), for example, 1.0 × 10⁻⁶ -7 M (i.e., 100 nM) or less. In a further embodiment, KD is 5.0 × 10 -8 Less than M (i.e., 50 nM), for example, 4.0 × 10⁻⁶ -8 Less than M (i.e., 40 nM), 3.0 × 10 -8 Less than M (i.e., 30 nM), or 2.0 × 10⁻⁶ -8 It is less than M (i.e., 20 nM). For example, according to some embodiments, when measured by surface plasmon resonance, it is 1.5 × 10⁻⁶ -7 A human polyspecific antibody is provided that binds to the Vδ1 chain of the γδTCR with a binding affinity (KD) of less than M (i.e., 150 nM).

[0228] In one embodiment of the present invention, when measured by surface plasmon resonance, the result is 4.0 × 10⁻⁶. -8 Less than M (i.e., 40 nM), 3.0 × 10 -8 Less than M (i.e., 30 nM), or 2.0 × 10⁻⁶ -8 A polyspecific antibody or fragment thereof that binds to the Vδ1 chain of γδTCR with a binding affinity (KD) of less than M (i.e., 20 nM) is provided.

[0229] The multispecific antibody or fragment of the present invention, when measured by surface plasmon resonance, yields 1.5 × 10⁻⁶. -7It can bind to the second antigen (or the epitope of the second antigen) with a binding affinity (KD) of less than M (i.e., 150 nM). In a preferred embodiment, the KD is 1.5 × 10⁻⁶ -7 It is less than M (i.e., 150 nM). In a further embodiment, KD is 1.3 × 10⁻⁶ -7 Less than M (i.e., 130 nM), for example, 1.0 × 10⁻⁶ -7 M (i.e., 100 nM) or less. In a further embodiment, KD is 5.0 × 10 -8 Less than M (i.e., 50 nM), for example, 4.0 × 10⁻⁶ -8 Less than M (i.e., 40 nM), 3.0 × 10 -8 Less than M (i.e., 30 nM), or 2.0 × 10⁻⁶ -8 It is less than M (i.e., 20 nM). For example, according to some embodiments, when measured by surface plasmon resonance, it is 1.5 × 10⁻⁶ -7 A human polyspecific antibody is provided that binds to a second antigen (or an epitope of a second antigen) with a binding affinity (KD) of less than M (i.e., 150 nM).

[0230] In one embodiment of the present invention, when measured by surface plasmon resonance, the result is 4.0 × 10⁻⁶. -8 Less than M (i.e., 40 nM), 3.0 × 10 -8 Less than M (i.e., 30 nM), or 2.0 × 10⁻⁶ -8 A multispecific antibody or fragment thereof is provided that binds to a second antigen (or an epitope of a second antigen) with a binding affinity (KD) of less than M (i.e., 20 nM).

[0231] In one embodiment, the binding affinity of a polyspecific antibody or fragment thereof is determined by directly or indirectly coating the surface of a sensor (e.g., a high-dose amine chip or equivalent) with the antibody or fragment thereof (e.g., by capture with anti-human IgG Fc), and detecting the binding by flowing the target to which the antibody or fragment thereof is bound (e.g., the Vδ1 chain of γδTCR) onto the chip. Preferably, a MASS-2 instrument (also known as Sierra SPR-32) is used at 30 μl / min in PBS + 0.02% Tween 20 running buffer at 25°C.

[0232] This specification also describes other assays that may be used to define antibody function. For example, antibodies or fragments described herein may be evaluated by measuring the association of γδTCRs, for example, by measuring the downregulation of γδTCRs upon antibody binding. Surface expression of γδTCRs after application of the antibody or fragment (which may be presented on the cell surface) can be measured, for example, by flow cytometry. Antibodies or fragments described herein may also be evaluated by measuring γδT cell degranulation. For example, after applying the antibody or fragment (which may be presented on the cell surface) to γδT cells, the expression of CD107a, a marker of cell degranulation, can be measured, for example, by flow cytometry. Antibodies or fragments described herein may also be evaluated by measuring γδT cell killing activity (to test whether the antibody affects the killing activity of γδT cells). For example, target cells may be incubated with γδT cells in the presence of the antibody or fragment (which may be presented on the cell surface). After incubation, the culture can be stained with a cell viability-determinating dye to distinguish between living and dead target cells. Next, the percentage of dead cells can be measured, for example, by flow cytometry.

[0233] multispecific antibody The antibodies of the present invention are bispecific or polyspecific. Polyspecific antibodies may generally be specific to different epitopes of one target polypeptide, or to multiple target polypeptides. However, in the present invention, the antibodies generally bind specifically to two (or more) different antigens.

[0234] In various embodiments, the second target epitope is an epitope of a cancer antigen or cancer-associated antigen (e.g., tumor-associated antigen). In various embodiments, the cancer antigen or cancer-associated antigen is AFP, AKAP-4, ALK, alpha-fetoprotein, androgen receptor, B7H3, BAGE, BCA225, BCAA, Bcr-abl, beta-catenin, beta-HCG, beta-human chorionic gonadotropin, BORIS, BTAA, CA125, CA15-3, CA195, CA19-9, CA242, CA27.29, CA72-4, CA-50, CAM 17.1, CAM43, Carbonic anhydrase IX, Carcinoembryonic antigen, CD22, CD33 / IL3Ra, CD68\P1, CDK4, CEA, Chondroitin sulfate proteoglycan 4 (CSPG4), c-Met, CO-029, CSPG4, Cyclin B1, Cyclophilin C-related protein, CYP1B1, E2A-PRL, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, Epstein-Barr virus antigen EBVA, ERG (TMPRSS2ETS fusion gene), ETV6-AML, FAP, FGF-5, Fos-related antigen 1, Fucosyl GM1, G250, Ga733\EpCAM, GAGE-1, GAGE-2, GD2, GD3, Glioma-related antigen, GloboH, Glycolipid F77, GM3, GP 100, GP 100(Pmel 17), H4-RET, HER-2 / neu, HER-2 / Neu / ErbB-2, High Molecular Weight Melanoma-Associated Antigen (HMW-MAA), HPV E6, HPV E7, hTERT, HTgp-175, Human Telomerase Reverse Transcriptase, Idiotype, IGF-I Receptor, IGF-II, IGH-IGK, Insulin Growth Factor (IGF)-I, Intestinal Carboxysterase, K-ras, LAGE-1a, LCK, Lectin-Reactive AFP, Regmine, LMP2, M344, MA-50, Mac-2 Binding Protein, MAD-CT-1, MAD-CT-2, MAGE, MAGE A1, MAGE A3, MAGE-1, MAGE-3, MAGE-4, MAGE-5, MAGE-6, MART-1, MART-1 / MelanA, M-CSF, Melanoma-associated chondroitin sulfate proteoglycan (MCSP), Mesothelin, MG7-Ag, ML-IAP, MN-CAIX, MOV18, MUC1, Mum-1, hsp70-2, MYCN, MYL-RAR, NA17, NB / 70K, Neuron glial antigen 2 (NG2), Neutrophil elastase, nm-23H1, NuMa, NY-BR-1, NY-CO-1, NY-ESO, NY-ESO-1, NY-ESO-1, OY-TES1, p15, p16, p180erbB3, p185erbB2, p53, p53 mutant, Page4, PAX3, PAX5, PDGFR beta, PLAC1, Poly The following are selected from sialic acid, prostate cancer tumor antigen-1 (PCTA-1), prostate-specific antigen, prostatic acid phosphatase (PAP), proteinase 3 (PR1), PSA, PSCA, PSMA, RAGE-1, Ras, Ras-mutant, RCAS1, RGS5, RhoC, ROR1, RU1, RU2(AS), SART3, SDCCAG16, sLe(a), sperm protein 17, SSX2, STn, Survivin, TA-90, TAAL6, TAG-72, telomerase, thyroglobulin, Tie 2, TIGIT, TLP, Tn, TPS, TRP-1, TRP-2, TRP-2, TSP-180, tyrosinase, VEGF, VEGFR2, VISTA, WT1, XAGE 1, 43-9F, 5T4, and 791Tgp72.

[0235] In some embodiments, the second target epitope is from the ErbB subfamily. Erb-B1 (EGFR) and Erb-B2 (HER2) are members of subclass I of the receptor tyrosine kinase (RTK) superfamily. In some embodiments, the second target epitope is an RTK. RTKs share a similar protein structure consisting of an extracellular ligand-binding domain and a single transmembrane helix. They are further subdivided into separate subclasses, primarily based on the properties of their intracellular tyrosine kinase domain (TKD) and carboxyl (C)-terminal tail. The extracellular domain region of RTKs exhibits a variety of conserved elements, including immunoglobulin (Ig)-like domains or epidermal growth factor (EGF)-like domains. In some embodiments, the second epitope is an epitope of receptor tyrosine kinase. Examples of the RTK family include VEGFR2, EGFR, c-MET, IGF-I receptor, PDGFR beta, CD115, CD117, CD140A, CD140B, CD167a, CD167b, CD172g, CD220, CD246, CD303, CD331, CD332, CD333, and CD340.

[0236] For example, in some embodiments, the second target epitope is HER2 (human epidermal growth factor receptor 2). HER2 (also known as ErbB-2 or CD340) is a cancer-associated antigen and an example of a receptor tyrosine kinase of the ErbB subfamily. HER2 expression is low in healthy tissue and up to 40-100-fold increased in Her2+ cancers compared to normal tissue. Her2 overexpression is associated with many breast, gastric, esophageal, ovarian, endometrial, NSCLC, and colorectal cancers. In many cancers, HER2 dimerizes with other ErbB receptors, leading to activation of various downstream signaling pathways, resulting in uncontrolled proliferation and resistance to apoptosis. Since HER2 overexpression correlates with reduced survival, it is a target for improving prognosis and is also a tumor marker. Monoclonal antibodies that specifically bind to the HER2 epitope are well known in the art. For example, trastuzumab is a monoclonal antibody that specifically binds to the HER2 epitope.

[0237] In some embodiments, the second target epitope is EGFR. EGFR (epidermal growth factor receptor) is a cancer-associated antigen and an example of a receptor tyrosine kinase in the ErbB subfamily. EGFR is expressed in multiple organs and plays a crucial role in initiating signaling that governs the behavior of epithelial cells and epithelial-derived tumors. EGFR-mediated signaling is also involved in controlling cell proliferation, migration, survival, and metastasis by regulating diverse cellular pathways. As with other receptor tyrosine kinases, mutations that affect EGFR activity or lead to EGFR upregulation are associated with many cancers. In fact, EGFR gene mutations are observed in up to 30% of solid tumors and are typically associated with poor prognosis. Disrupting EGFR signaling by inhibiting the binding of EGF to the extracellular domain or by inhibiting intracellular tyrosine kinase activity can limit the growth of EGFR-expressing tumors. Therefore, EGFR inhibitors can be anticancer agents. Indeed, since certain tumor cells are dependent on EGFR signaling and thus have "oncogene toxicity," this receptor is an attractive target for therapy. Monoclonal antibodies that specifically bind to the EGFR epitope are well known in this art. For example, cetuximab is a monoclonal antibody that specifically binds to the EGFR epitope.

[0238] In some embodiments, the second target epitope is an epitope of a B lymphocyte antigen. Examples of antigens particularly expressed on B cells include CD1d, CD5, CD10, CD11b, CD19, CD20, CD21, CD22, CD23, CD24, CD32A, and CD32B. Examples include CD37, CD39, CD40, CD45, CD52, CD72, CD79a, CD79b, CD138, CD166, CD179A, CD179B, CD180, CD185, CD150, CD213a1, CD213a2, CD217, CD244, CD255, CD229, CD232, CD267, CD268, CD269, CD274, CD277, CD279, CD290, CD300A, CD300C, CD305, CD307a, CD307b, CD307c, CD307d, CD307e, CD316, CD319, CD327, CD352, and CD361. For example, in some embodiments, the second target epitope is CD19. CD19 (cell membrane differentiation antigen 19) is a cancer-associated antigen. CD19 is also an example of a type I transmembrane glycoprotein in the immunoglobulin superfamily. In some embodiments, the second target is an epitope present in members of the immunoglobulin superfamily (IgSF). Examples of this family include CD2, CD3, CD4, CD7, CD8, CD19, CD79A, CD79B, CD28, CD48, CD58, CD80, CD86, CD90, CD96, CD147, CD150, CD155, CD229, CD244, CD273, CD274, and CD276. CD19 is widely expressed in B cells during their development, and its surface density increases as B cells mature. CD19 is involved in the recruitment of signaling proteins from the cytoplasm. CD19 is also involved in the B cell receptor signaling pathway and is essential for the function of the B cell receptor. Because it is expressed on B cells, CD19 is not only a useful target for leukemia and neoplastic lymphocytes, but also a diagnostic biomarker for cancers originating from B cells. CD19 mutations can lead to reduced antibody production and immunodeficiency, and therefore CD19 can also be a target for the treatment of autoimmune diseases. Monoclonal antibodies that specifically bind to the CD19 epitope are well known in the art.For example, blinatumomab is a monoclonal antibody that specifically binds to the CD19 epitope.

[0239] In some embodiments, the second target epitope is present in tumor stromal antigens. Examples of tumor stromal antigens include FAP alpha, CD29, CD44, CD73, CD105, and CD166. For example, in some embodiments, the second target epitope is FAPα (fibroblast-activating protein α). FAPα is a cancer-associated antigen also known as seplace or prolyl endopeptidase FAP, and is selectively expressed in the stroma of various epithelial cancers. FAPα is an example of a cell surface serine protease of the dipeptidyl peptidase family. FAPα is expressed by cancer-associated fibroblasts (CAFs), which play an important role in the tumor microenvironment. Other molecules selectively expressed in CAFs include CD10, CD90, CD140A, and CD140B. In some embodiments, the second epitope is an epitope present in molecules selectively expressed in CAFs. It is known that more than 90% of epithelial cancers (breast cancer, CRC skin cancer, and pancreatic cancer) express FAPα on the surface of CAFs in the surrounding stroma. CAFs secrete the immunosuppressive chemokine CXCL12, which binds to CXCR4 on T cells. FAPα is known to be expressed in invasive melanoma cell lines and is significantly increased in patients with poor outcomes and survival rates in breast cancer. FAPα possesses both collagenase and dipeptidase activity and promotes tumor growth, migration, invasion, metastasis, and ECM degradation. In normal, healthy adult tissues, there is no detectable FAPα expression outside the areas of tissue remodeling or wound healing, and therefore, FAPα expression is almost exclusively limited to the tumor stroma. Because FAPα plays a direct role in various aspects of cancer progression, FAPα is a promising anti-cancer target. Monoclonal antibodies that specifically bind to the FAPα epitope are well known in the art. For example, cibrotuzumab is a monoclonal antibody that specifically binds to the FAPα epitope.

[0240] In some embodiments, the second target epitope is located on cell surface glycoproteins. Protein glycosylation is an important and common post-translational modification. It is thought that more than 50% of human proteins are glycosylated to regulate protein functionality. Abnormal glycosylation is associated with several diseases, including inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's disease and prion diseases, and cancer. Examples of cell surface glycoproteins include CD1a, CD1b, CD1c, CD1d, CD1e, CD3d, CD3e, CD3g, CD8a, CD8b, CD11a, CD21, CD36, CD42a, CD42b, CD42c, CD42d, CD43, CD66a, CD66f, CD177, CD235a, CD235b, CD236, CD238, CD243, CD227, and CD301. For example, in some embodiments, the second target epitope is mesothelin (MSLN). MSLN is a cancer-associated antigen and an example of a cell surface glycoprotein. MSLN is expressed in several cancers (malignant mesothelioma and pancreatic, cholangiocarcinoma, ovarian and lung adenocarcinoma, malignant mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, biliary tract cancer, gastric cancer, and pediatric acute myeloid leukemia), although its expression is limited in healthy cells. MSLN is an example of a tumor differentiation antigen. Although the physiological function of MSLN is unknown, MSLN is a useful target for limiting therapy to MSLN+ tumors or can be used as a tumor marker. Monoclonal antibodies that specifically bind to the MSLN epitope are well known in the art. For example, anetumab is a monoclonal antibody that specifically binds to the MSLN epitope.

[0241] In various embodiments, the second target epitope is an immunomodulatory antigen epitope. An immunomodulatory antigen is an antigen that modulates (activates or suppresses) the immune system. In some embodiments, the immunomodulatory antigen is a cell surface protein (i.e., an antigen expressed on the surface of a cell, particularly an antigen expressed on the surface of immune cells such as lymphocytes, neutrophils, monocytes, or macrophages). The immunomodulatory antigen may be expressed on Vδ1+ T cells, or on different cells, such as CD4+ cells, CD8+ cells, or different immune cells. The immunomodulatory antigens are B7-1 (CD80), B7-2 (CD86), B7-DC (CD273), B7-H1 (CD274), B7-H2 (CD275), B7-H3 (CD276), and B7-H4 (VTCN1). , B7-H5(VISTA), BTLA(CD272), 4-1BB(CD137), CD137L, CD24, CD27, CD28, CD38, CD40, CD40L(CD154), CD54, CD59, CD The group may be selected from 70, CTLA4 (CD152), CXCL9, GITR (CD357), HVEM (CD270), ICAM-1 (CD54), ICOS (CD278), LAG-3 (CD223), OX40 (CD134), OX40L (CD252), PD-1 (CD279), PD-L1 (CD274), TIGIT, CD314, CD334, CD335, CD337, and TIM-3 (CD366).

[0242] In some embodiments, the second target epitope is an epitope of a stimulative immune checkpoint molecule. For example, in some embodiments, the second target epitope is OX40 (CD134). OX40 is an immunomodulatory antigen and an example of a member of the TNFR superfamily (TNFRSF). In some embodiments, the second target epitope is an epitope present in the TNFRSF molecule. These proteins are a superfamily of cytokine receptors characterized by their ability to bind to tumor necrosis factor (TNF) via an extracellular cysteine-rich domain. Examples include CD18, CD27, CD30, CD40, CD95, CD120a, CD120b, CD134, CD137, CD265, CD268, CD269, CD270, CD271, CD357, and CD358. One such example is OX40 (CD134), a late-stage costimulatory immune checkpoint receptor expressed on CD4+ and CD8+ T cells. OX40 is more highly expressed on CD4+ T cells, and its expression depends on the full activation of T cells; therefore, it is not constitutively expressed on naive T cells. When activated (e.g., by OX40L), OX40 promotes the activation of CD4+ / CD8+ T cells, the survival and proliferation of effector and memory T cells, and suppresses Treg activity. This suppresses immune evasion by tumors. Because OX40 is a stimulative target, therapies targeting OX40 activate OX40-expressing immune cells to stimulate an immune response against tumors (e.g., CD48+ T cell response). Monoclonal antibodies that specifically bind to the OX40 epitope are well known in the art. For example, pogalizumab is a monoclonal antibody that specifically binds to the OX40 epitope.

[0243] In some embodiments, the second target epitope is an epitope present in members of the TNF superfamily. For example, in some embodiments, the second target epitope is the 4-1BB(CD137) epitope. 4-1BB is an immunomodulatory antigen and is also an example of a member of the TNF superfamily. This is a protein superfamily of type II transmembrane proteins containing TNF homology domains, including CD70, CD137, CD153, CD154, CD252, CD253, CD254, CD256, CD257, and CD258. 4-1BB(CD137) is an inducible costimulatory immune checkpoint receptor expressed on T cells, as well as NK cells, dendritic cells (DCs), monocytes, neutrophils, and B cells. 4-1BB is a stimulatory antigen and is particularly expressed on activated CD8+ T cells. In vitro, 4-1BB stimulates the proliferation of CD4+ T cells, CD8+ T cells, macrophages, and dendritic cells, as well as cytokine production. In vivo, 4-1BB exhibits potent antitumor activity, particularly in activating CD8+ T cells, and cross-linking of 4-1BB has been shown to enhance T cell proliferation, IL-2 secretion, survival, and cytolytic activity. Because 4-1BB is a stimulant target, therapies targeting 4-1BB activate 4-1BB-expressing immune cells, stimulating an immune response against tumors (e.g., a cytolytic CD8+ T cell response). Monoclonal antibodies that specifically bind to the 4-1BB epitope are well known in the art. For example, utomirumab is a monoclonal antibody that specifically binds to the 4-1BB epitope.

[0244] In some embodiments, the second target epitope is an immune checkpoint inhibitor molecule. For example, in some embodiments, the second target epitope is TIGIT (a T cell immune receptor having Ig and ITIM domains). TIGIT is an immunomodulatory antigen and an example of an immune checkpoint inhibitor expressed on T cells, including γδ T cells, and NK cells. TIGIT is involved in immune homeostasis and suppresses T cells by preventing autoimmunity through binding to its ligand (PVR / CD155). TIGIT is overexpressed in tumor-infiltrating lymphocytes. Therapeutic blockade of TIGIT is desirable because it increases T cell proliferation, cytokine production, and degranulation. Monoclonal antibodies that specifically bind to the TIGIT epitope are well known in the art. For example, tiragolumab is a monoclonal antibody that specifically binds to the TIGIT epitope.

[0245] In some embodiments, the second target epitope is an immune checkpoint inhibitor molecule. For example, in some embodiments, the second target epitope is PD-1 (programmed cell death protein 1). PD-1 is an immunomodulatory antigen and an example of a cell surface receptor member of the immunoglobulin superfamily. PD-1 is an example of an immune checkpoint inhibitor expressed on activated CD4+ / CD8+ T cells, as well as other types of immune cells such as γδ T cells, B cells, and macrophages. When PD-1 binds to its ligand, T cell activation is inhibited. Under normal circumstances, this is involved in immune homeostasis, protecting against autoimmunity by reducing apoptosis of Tregs and increasing apoptosis of antigen-specific T cells. In cancerous situations, this results in immune evasion of tumor cells by inactivating cytolytic CD8+ T cells. Therefore, blocking PD-1 is a promising therapeutic target. Monoclonal antibodies that specifically bind to the PD-1 epitope are well known in the art. For example, pembrolizumab is a monoclonal antibody that specifically binds to the PD-1 epitope.

[0246] In some embodiments, the second target epitope is a stimulating immune checkpoint molecule. Stimulating immune checkpoint molecules include, for example, OX40, OX40L, 4-1BB(CD137), CD137L, CD27, CD70, CD28, GITR, ICOS, CD40, and CD40L. In some embodiments, the second target epitope is one or more selected from OX40, OX40L, 4-1BB(CD137), CD137L, CD27, CD70, CD28, GITR, ICOS, CD40, and CD40L. In some embodiments, the second target epitope is one or more selected from OX40 and 4-1BB.

[0247] In some embodiments, the second target epitope is an immune checkpoint inhibitor molecule. Immune checkpoint inhibitor molecules include, for example, TIGIT, CD155, PD-1, PD-L1, CTLA-4, B7-H3, B7-H4, BTLA, LAG-3, VISTA, and TIM-3. In some embodiments, the second target epitope is one or more selected from TIGIT, CD155, PD-1, PD-L1, CTLA-4, B7-H3, B7-H4, BTLA, LAG-3, VISTA, and TIM-3. In some embodiments, the second target epitope is one or more selected from TIGIT and PD-1.

[0248] In this specification, references to antigens "on" cells refer to antigens that are expressed on the cell surface membrane or associated with the cell surface membrane (extracellular space) of such cells.

[0249] In various embodiments, the second target epitope is an epitope of a cell membrane differentiation (CD) antigen. The nomenclature of cell membrane differentiation antigens (CDs) is a unified system for identifying and naming cell surface molecules. Typically, a cell surface protein is not assigned a CD number until at least two monoclonal antibodies against that cell surface protein are produced. Therefore, this system ensures that all cell surface proteins assigned a CD number are readily recognized and bound by specific monoclonal antibodies or fragments thereof. In various embodiments, the CD antigens include CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD13, CD14, CD15, CD16, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, and CD22. , CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32A, CD32B, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD 40, CD41, CD42, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49 e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD 62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD7 4, CD75, CD75s, CD77, CD79A, CD79B, CD80, CD81, CD82, CD83, CD84, CD85A, CD85B, CD85C, CD85D, CD85F, CD85G, CD85 H, CD85I, CD85J, CD85K, CD85M, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99,CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107、CD107a、CD107b、CD108、CD109、CD110、CD111、CD112、CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120、CD120a、CD120b、CD121a、CD121b、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140A、CD140B、CD141、CD142、CD143、CD144、CDw145、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156、CD156a、CD156b、CD156c、CD157、CD158、CD158A、CD158B1、CD158B2、CD158C、CD158D、CD158E1、CD158E2、CD158F1、CD158F2、CD158G、CD158H、CD158I、CD158J、CD158K、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD187、CD188、CD189、CD190、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CDw199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210a、CDw210b、CD211、CD212、CD213a1、CD213a2、CD214、CD215、CD216、CD217、CD218a、CD218b、CD219、CD220、CD221、CD222、CD223、CD224、CD225、CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD237, CD238, CD239, CD240CE, CD240D, CD24 1, CD242, CD243, CD244, CD245

[17] , CD246, CD247, CD248, CD249, CD250, CD251, CD252, CD253, CD254, CD255, CD256, CD257, CD258, CD25 9, CD260, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD 278, CD279, CD280, CD281, CD282, CD283, CD284, CD285, CD286, CD287, CD288, CD289, CD290, CD291, CD292, CDw293, CD294, CD295, CD296 , CD297, CD298, CD299, CD300A, CD300C, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD307a, CD307b, CD307c, CD307d, CD307e, CD 308, CD309, CD310, CD311, CD312, CD313, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD323, CD324, CD325, CD326, It is selected from CD327, CD328, CD329, CD330, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, and CD371.

[0250] Preservation of healthy cells Although the mechanisms by which γδ T cells recognize antigens and distinguish between healthy and diseased cells are not fully understood (Ming Heng and Madalene Heng, Antigen Recognition by γδ T-Cells. Madame Curie Bioscience Database [Internet], Austin (TX): Landes Bioscience; 2000-2013), the fact that γδ T cells can distinguish between healthy and diseased cells and exhibit significant multicellular toxicity of diseased cells (see non-limiting examples of cell types in Table 1) means that these capabilities can be leveraged to provide improved drugs with enhanced therapeutic ranges. Furthermore, by utilizing these capabilities of γδ T cells, even when specific cancer antigens, inflammatory antigens, or pathogenic antigens are unknown or present on the healthy cells of a particular patient, it is possible to treat disease while preserving healthy cells by co-localizing γδ T cells with diseased cells. [Table 2-1] [Table 2-2]

[0251] As one non-limiting example, recent research on CD3×HER2 multispecific drugs highlights the challenges of current or conventional approaches. Specifically, the use of such conventional approaches can result in less favorable toxicity profiles. This is because, like many other tumor-associated antigens (TAAs), the HER2 antigen is expressed not only in cancers such as breast cancer, but also in healthy tissues such as cardiac cells. Therefore, the use of CD3×HER2 drugs that associate with all T cells and co-localize with HER2-positive cells may result in less favorable therapeutic ranges or therapeutic indices. This is because such drugs associate with all T cells, the majority of which are αβ T cells (CD4+ positive, CD8+ positive, etc.) in circulation. And when αβ T cells co-localize with HER2-positive cells, such conventional αβ T cells exhibit limited ability to preserve HER2+ healthy cells and limited ability to kill only affected HER2+ disease cells. Therefore, and according to this example, in studies of cynomolgus monkeys administered such CD3×HER2 bispecific drugs, early euthanasia was necessary in some situations (even on the day of administration). Furthermore, this study (see Staffin et al. (2020) JCI Insight 5(7):e133757) concluded that T cell retargeting to kill HER2-expressing cells can induce adverse effects on HER2-expressing tissues. With the exception of the liver, all affected or damaged tissues were found to express HER2.

[0252] In a further, less restrictive example, a second binding specificity may be directed towards tumor-associated sites that are also involved in the control or regulation of immune cell function. For example, a second specificity could be designed to target so-called "checkpoint inhibitors" such as PD-L1 (CD274) or CD155. Again, neither PD-L1 nor CD155 is 100% disease-specific. Both proteins can be expressed in healthy cells. However, a polyspecific antibody designed to specifically co-localize Vδ1+ cells to either PD-L1-positive or CD155-positive cells could result in the selective killing of PD-L1 or CD155-positive disease or cancer cells. Further targeting of disease-related checkpoint inhibitors present on disease cells such as cancer cells can not only co-localize Vδ1+ cells to such tumors, but can also confer additional desirable effects, for example, by modulating or suppressing PD-1 / PD-L1 or TIGIT / CD155 signaling, which could otherwise negatively regulate the T cell-mediated immune response to the disease.

[0253] Therefore, instead of using such conventional approaches, what is provided herein is a polyspecific antibody that has at least one first binding specificity capable of binding to Vδ1+ cells and at least one second binding specificity capable of binding to targets present on diseased tissues and cells. Using such a polyspecific antibody in this manner can lead to the co-localization of Vδ1+ cells to diseased cells expressing the second target. Furthermore, given that such disease-related targets are often not 100% disease-specific, this approach of specifically targeting and co-localizing Vδ1+ effector cells may be preferable to conventional approaches. This is because Vδ1+ effector cells may be able to recognize stress patterns in diseased or infected cells and thus selectively kill diseased cells while preserving healthy cells that similarly express the same targets.

[0254] Therefore, while the multispecific antibodies presented herein can associate with the TCR of vδ1 cells, complete activation does not occur unless tumor cells are also present. Complete association of the antibodies presented herein with the TCR results in partial downregulation, and it is believed that vδ1 cells bound to these antibodies are fully activated and cytotoxic only in the presence of stressed cells such as tumor cells. This is illustrated, for example, in Figures 25, I, J, K, and 38A-F. This represents another important safety advantage of the approach presented herein, because off-target cytotoxicity is reduced, and the full potency of the multispecific antibodies activating vδ1 cells is released only in the presence of tumor cells, thus preserving healthy cells (and even healthy cells expressing the second target antigen of the multispecific antibodies).

[0255] One mechanism by which γδT cells can detect stress signals from tumor cells is thought to be due to the NCRs (innate cytotoxic receptors) they express. NCRs can associate with NCR ligands on tumor cells. Therefore, a dual activation mechanism may be employed, in which γδT cells are activated via TCR stimulation, including an NCR-mediated mechanism that can sense tumor cells and enable full activation and cytotoxicity.

[0256] This is in contrast to, for example, CD3-mediated stimulation of αβ T cells, where all stimulation is mediated through the TCR. Therefore, such cells do not possess mechanisms such as antigen-independent sensing of tumor cells via the NCR, making it nearly impossible to distinguish them from healthy or transformed cells. Consequently, if CD3 antibodies are Fc-effective, they attract other immune cells, which can trigger a cascade of unexpected but desirable events such as cytokine storms, immune cell depletion, and even hyperactivation, leading to, for example, T cell killing by NK cells. In the present approach, γδ T cells can distinguish between healthy and tumor cells, including those mediated through their NCR sensing mechanisms, and therefore, due to this disease cell specificity, they can selectively kill stressed cells such as cancer cells or virus-infected cells. Thus, the stimulation of γδ T cells with multispecific antibodies presented in this book does not raise such concerns.

[0257] In a further non-limiting example, the patient may have liver cancer, and the liver cancer-specific antigen is unknown in the patient. In this case, the second specificity of the multispecific antibody may be against an epitope present on many or all liver cells, such as asialoglycoprotein receptor 1. This then co-localizes γδT cells in the liver, where γδT cells can kill liver cancer cells while preserving healthy liver cells. This is shown, for example, in Figures 25, I, J, K and 38A-f. In a third non-limiting example, if the patient has lung cancer, and the lung cancer antigen is unknown in the patient, the second specificity of the multispecific antibody may be against an epitope on normal lung cells, such as SP-1. This co-localizes γδT cells in the lung, where γδT cells can kill lung cancer cells while preserving healthy lung cells. As a fourth, non-limiting example, if a patient has B-cell lymphoma and the B-cell lymphoma antigen is unknown in the patient, the second specificity of the multispecific antibody may be against an epitope on normal B cells, such as CD19. This co-localizes γδT cells to B cells, where the γδT cells can kill lymphoma cells while preserving healthy B cells. Cell-specific antigens, cell-associated antigens, tissue-specific antigens, and tissue-associated antigens are well known in the art, and any of these antigens can be targeted by the second specificity of the multispecific antibody of the present invention.

[0258] The second binding specificity allows for targeting antigens on the same cells as Vδ1, or on different cells of the same or different tissue types. In certain embodiments, the target epitopes may be on different cells, including different T cells, B cells, tumor cells, autoimmune tissue cells, or virus-infected cells. Alternatively, the target epitopes may be on the same cells.

[0259] A polyspecific antibody or fragment thereof can be prepared in any format, as long as the antibody or fragment has multiple specificities. Generally, the polyspecific (preferably bispecific) antibody of the present invention comprises at least two binding domains: a first binding domain and a second binding domain. The first binding domain confers binding specificity to a first target antigen (or an epitope of the first target antigen), where the first target epitope is an epitope of the variable delta 1 (Vδ1 or TRDV1) chain of the γδ T cell receptor (TCR). The second binding domain confers binding specificity to a second target antigen (or an epitope of the second target antigen). The second target antigen may be a cancer antigen or a cancer-associated antigen, or the second antigen may be an immunomodulatory antigen.

[0260] Examples of multispecific antibody formats include CrossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, Knob-in-Hole (KIH), Knob-in-Hole (common light chain), Charge pair, Fab-arm exchange, SEED body, Triomab, LUZ-Y, Fcab, κλ body, Orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, and KIH. IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG (Four-in-One), Nanobody, Nanoby-HAS, BiTE, Diabody, DART, TandAb, scDiabody, scDiabody-CH3, Diabody-CH3, Triplebody, Morrison format, Mini antibody, Minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab)2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, tandem scFv-Fc, Intrabody, Dock and Lock, ImmTAC, HSAbody, scDiabody-HAS, tandem scFv-toxin, IgG-IgG, ov-X-Body, Duobody, mab 2 This includes, but is not limited to, scFv1-PEG-scFv2 (see Spiess et al. (2015) Molecular Immunology 67:95-106).

[0261] The antibodies or fragments described herein can also be evaluated by measuring their ability to enhance functionality in multispecific formats, such as bispecific or triplicate formats. Surprisingly, through such studies, it is possible to identify further functional improvements in the performance of the antibodies or fragments described herein (see Examples 20 and 21).

[0262] Various antibody-derived multispecific formats have been reported and are typically constructed empirically from component-binding moieties. Typically, once a multispecific or multi-target binding format, such as those described herein, is constructed, its performance can be measured in one or more of the aforementioned model systems (e.g., cell toxicology, cell proliferation, healthy cell preservation / disease cell-specific models). These are also, as may be, compared to the aforementioned components and other comparator molecules.

[0263] While not limited to this approach, when constructing antibodies as polyspecific antibodies, the binding domain modules for each target (first, second, third, etc.) are generally constructed from scFv, Fab, Fab', F(ab')2, Fv, variable domains (e.g., VH or VL), diabodies, minibodies, or full-length antibodies, depending on the case. For example, each of the binding domains or modules is produced in one or more of the following non-limiting formats, in which binding domains containing variable domains, and / or full-length antibodies, and / or antibody fragments are sequentially and operably linked to produce a polyspecific antibody.

[0264] Notably, a polyspecific antibody comprising at least one (first) binding domain targeting the Vδ1 chain of the γδTCR, as described herein, is further enhanced when the first binding domain is formatted in a polyspecific antibody format comprising at least one second binding domain to either a tissue ("solid") or hematopoietic system ("liquid") disease or cell type-related target.

[0265] Polyspecific antibodies - non-specific examples: To outline the applicability of the approach, we constructed a series of non-limiting examples of polyspecific antibodies. These polyspecific antibodies contained at least one (first) binding domain that targeted the Vδ1 chain of the γδTCR and at least one (second) binding domain that targeted disease-related targets.

[0266] First example; Vδ1-EGFR polyspecific antibody: In this example, one binding domain (for the first target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and its congeneral VL-CL partner, while the second binding domain (for the second target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. The resulting bispecific format is sometimes called the "Morrison format." In this example, the first binding domain targets the Vδ1 chain of the γδTCR, and the second binding domain targets EGFR (see Example 20).

[0267] Second example; Vδ1-EGFR polyspecific antibody: In this example, one binding domain (for the first target) includes an antibody variable domain (specifically, the VH and the related VL domain), and the second binding domain (for the second target) includes a binding domain within the heavy chain constant domain (CH1-CH2-CH3) (see also Table 1 of EP2546268 A1 / EP3487885 A1). The resulting bispecificity includes a first binding domain targeting the Vδ1 chain of the γδTCR and a second binding domain targeting the EGF receptor (see Example 20).

[0268] Third example; Vδ1-CD19 polyspecific antibody: In this example, one binding domain (for the first target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and its congeneral VL-CL partner, and the second binding domain (for the second target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. In this example, the resulting bispecificity included a first binding domain targeting the Vδ1 chain of the γδTCR and a second binding domain targeting CD19 (see Example 21).

[0269] Fourth example; further Vδ1-CD19 polyspecific antibodies In this example, additional polyspecific antibodies that specifically bind to both TRDV1 and CD19 were prepared and tested for antigen binding (including both human and cynomolgus monkey TRDV1), Vδ1 cell activation, and Vδ1 cytotoxicity (see Examples 22 and 23).

[0270] Fifth example: Vδ1-Her2 polyspecific antibody In this example, a polyspecific antibody that specifically binds to both TRDV1 and Her2 was prepared and tested for antigen binding, Vδ1 cell activation, and Vδ1 cytotoxicity (see Example 24).

[0271] Sixth example; further Vδ1-EGFR polyspecific antibodies In this example, additional polyspecific antibodies that specifically bind to both TRDV1 and EGFR were prepared and tested for antigen binding, Vδ1 cell activation, and Vδ1 cytotoxicity (see Examples 26 and 27).

[0272] Example 7: Vδ1-FAPα polyspecific antibody In this example, one binding domain (for the first target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and a congeneral VL-CL partner, and the second binding domain (for the second target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. In this example, the resulting bispecificity included a first binding domain targeting the Vδ1 chain of the γδTCR and a second binding domain targeting FAPα (see Example 29).

[0273] Example 8: Vδ1-mesothelin polyspecific antibody In this example, one binding domain (for the first target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and a congeneral VL-CL partner, and the second binding domain (for the second target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. In this example, the resulting bispecificity included a first binding domain targeting the Vδ1 chain of the γδTCR and a second binding domain targeting mesothelin (see Example 30).

[0274] Example 9: Vδ1-PD-1 polyspecific antibody In this example, one binding domain (for the second target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and a congeneral VL-CL partner, and the second binding domain (for the first target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. In this example, the resulting bispecificity included a first binding domain targeting PD-1 and a second binding domain targeting the Vδ1 chain of the γδTCR (see Example 32).

[0275] Example 10: Vδ1-4-1BB polyspecific antibody In this example, one binding domain (for the first target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and a congeneral VL-CL partner, and the second binding domain (for the second target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. In this example, the resulting bispecificity included a first binding domain targeting the Vδ1 chain of the γδTCR and a second binding domain targeting 4-1BB (see Example 33).

[0276] Example 11: Vδ1-OX40 polyspecific antibody In this example, one binding domain (for the first target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and a congeneral VL-CL partner, and the second binding domain (for the second target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. In this example, the resulting bispecificity included a first binding domain targeting the Vδ1 chain of the γδTCR and a second binding domain targeting OX40 (see Example 34).

[0277] Example 12: Vδ1-TIGIT polyspecific antibody In this example, one binding domain (for the second target) contained an intact antibody site, specifically VH-CH1-CH2-CH3 and its congeneral VL-CL partner, and the second binding domain (for the first target) contained an antibody fragment, specifically the scFv format. The two binding modules were then fused using a linker. In this example, the resulting bispecificity included a first binding domain targeting TIGIT and a second binding domain targeting the Vδ1 chain of the γδTCR (see Example 35).

[0278] Notably, in all of the aforementioned examples, which included at least one (first) binding domain targeting the Vδ1 chain of γδTCR and at least one second domain targeting a second epitope, enhanced functionality was observed compared to the control and components (see Examples 20-35 of this specification).

[0279] In summary, these non-limiting examples demonstrate the flexibility of the polyspecific antibodies or fragments described herein. These non-limiting examples outline a polyspecific antibody approach that can be further enhanced by combining an antibody or fragment targeting the germline Vδ1 chain (amino acids 1-90 of SEQ ID NO: 1) with a second binding domain to form a polyspecific antibody. As a non-limiting example, this specification provides polyspecific antibodies containing a binding domain comprising an enhanced functional and intact antibody (VH-CH1-CH2-CH3 and VL-CL), and / or a variable domain (VH and its congener VL or VH-CH1 and its congener VL-CL), and / or an antibody fragment (scFv).

[0280] In one embodiment, the multispecific antibody-binding domain that targets the Vδ1 chain (first target) of γδTCR may include (i) one or more antibody-binding domains each containing a heavy chain (VH-CH1-CH2-CH3) and a congenerate light chain partner (VL-CL), and / or (ii) one or more antibody-binding domains each containing a heavy chain variable domain (VH, or VH-CH1) and a congenerate light chain variable domain partner (VL, or VL-VC), and / or (iii) one or more antibody-binding domains each containing a CDR-containing antibody fragment.

[0281] In one embodiment, a polyspecific antibody is provided comprising at least one first antibody-derived binding domain that targets the Vδ1 chain of the γδTCR, the first being operably linked to at least one second antibody-binding domain that targets a second epitope. Optionally, the binding domain comprises at least one or more VH and congeneral VL binding domains, or one or more VH-CH1-CH2-CH3 and congeneral VL-CL binding domains, or one or more antibody fragment binding domains. Optionally, the second binding domain targets a second epitope associated with or expressed on the cell surface of a cell. Optionally, the second epitope is located on a cell surface polypeptide associated with disease cells or tumor cells or virus-infected cells or autoimmune tissue cells. In some cases, the second or more epitopes are located on disease and cell type-associated CD19, EGFR, Her2, FAPα, mesothelin, PD-1, 4-1BB, OX40, or TIGIT antigens. In some cases, the multispecific antibody, comprising at least one first antibody-derived binding domain targeting the Vδ1 chain of the γδTCR, binds to the EGF receptor and is operably linked to a second binding domain comprising one or more of the following heavy chain modifications according to EU nomenclature: L358T and / or T359D and / or K360D and / or N361G and / or Q362P and / or N384T and / or G385Y and / or Q386G and / or D413S and / or K414Y and / or S415W and / or Q418Y and / or Q419K.

[0282] In some cases, a polyspecific antibody comprising at least one first antibody-derived binding domain targeting the Vδ1 chain of γδTCR includes SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 157, or functionally equivalent binding variants thereof, operably linked to a second binding domain targeting EGFR, CD19, Her2, FAPα, mesothelin, PD-1, 4-1BB, OX40, or TIGIT. In some cases, the resulting polyspecific antibody includes SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 158, or SEQ ID NO: 159. The entities are included herein as non-limiting novel compositions to aid understanding.

[0283] In one embodiment of the present invention, the polyspecific antibody of the present invention may be used in a therapeutically effective amount to treat a disease or disorder such as to improve at least one sign or symptom of the disease or disorder.

[0284] In one embodiment, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR in a multispecific antibody format, wherein the multispecific antibody is applied to Vδ1+ cells to measure the effect conferred to Vδ1+ cells by the multispecific entity (e.g., to the Vδ1+ phenotype and / or cytotoxicity and / or disease cell specificity and / or enhancement thereof).

[0285] Immunoconjugate The multispecific antibodies or fragments thereof of the present invention can be conjugated to therapeutic sites such as cytotoxins or chemotherapeutic agents. Such conjugates may be referred to as immunoconjugates. As used herein, the term “immunoconjugate” refers to an antibody chemically or biologically linked to another site such as a cytotoxin, radiopharmaceutical, cytokine, interferon, target site or reporter site, enzyme, toxin, peptide or protein, or therapeutic agent. An antibody may be conjugated to a cytotoxin, radiopharmaceutical, cytokine, interferon, target site or reporter site, enzyme, toxin, peptide, or therapeutic agent at any position along the molecule, as long as it can bind to its target. Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment, the drug may be a second, different antibody against Vδ1. In certain embodiments, the antibody may be conjugated to a drug specific to tumor cells or virus-infected cells. The type of therapeutic site that can be conjugated to the anti-Vδ1 antibody will take into consideration the condition to be treated and the desired therapeutic effect to be achieved. In one embodiment, the drug may be a second antibody or a fragment thereof that binds to a molecule other than Vδ1.

[0286] Drugs to regulate γδT cells The multispecific antibodies or fragments described herein may be used, or may be useful, to modulate patients' delta-variable single-chain (Vδ1) T cells in situ (i.e., in vivo) via the TRDV1 binding domain. The multispecific antibodies or fragments may be included in drugs for such purposes.

[0287] Regulation of Vδ1 T cells may include the following: - For example, increasing the number of Vδ1 T cells by selectively increasing their number, or promoting the survival of Vδ1 T cells; - For example, stimulating Vδ1 T cells by increasing Vδ1 T cell efficacy, i.e., increasing the killing of target cells; - For example, preventing Vδ1 T cell depletion by increasing the persistence of Vδ1 T cells; - Degranulation of Vδ1 T cells; - Increased NCR expression; - For example, immunomodulation of Vδ1 T cells by downregulating Vδ1 TCR cell surface expression, i.e., by causing internal translocation of Vδ1 TCR or a decrease in Vδ1 TCR protein expression, or by blocking Vδ1 TCR binding; and / or - Downregulation of the TCR / CD3 complex.

[0288] Unlike prior art anti-Vδ1 antibodies that focus on Vδ1 T cell depletion, the multispecific antibodies of the present invention are useful for activating Vδ1 T cells via the TRDV1 binding domain. These antibodies may induce downregulation of the TCR on the binding T cells, but do not cause Vδ1 T cell depletion; rather, they stimulate T cells, making them potentially useful in therapeutic situations where activation of this T cell compartment is beneficial. Vδ1 T cell activation is evident from TCR downregulation, CD3 downregulation, changes in activation markers such as CD25 and Ki67, and the degranulation marker CD107a. Vδ1 T cell activation consequently promotes immune licensing by inducing the release of inflammatory cytokines such as INFγ and TNFα.

[0289] In one embodiment of the present invention, a. Causes downregulation of the TCR on Vδ1 T cells, b. Not to indicate CDC or ADCC, and c. Do not deplete Vδ1 T cells. A multispecific anti-Vδ1 antibody or its antigen-binding fragment is provided, characterized by the above.

[0290] In some embodiments, the multispecific anti-Vδ1 antibody or antigen-binding fragment also stimulates the proliferation of Vδ1 T cells.

[0291] T cell depletion is a process of eliminating or reducing T cell death. References to multispecific antibodies or antigen-binding fragments that do not deplete Vδ1 T cells refer to depletion of less than 30% or less than 20% (preferably less than 10%) of the viable Vδ1 T+ cell population during incubation by one or more of the antibodies of the present invention described herein, as measured by any suitable means in a controlled study (e.g., by a controlled flow cytometry method or by another established controlled assay).

[0292] ADCC and CDC are mechanisms that can cause T cell depletion. In this specification, references to antibodies or antigen-binding fragments that do not cause ADCC or CDC refer to the depletion of a viable Vδ1 T+ cell population by ADCC and / or CDC during incubation, mediated by one or more of the antibodies of the present invention described herein, as measured by any suitable means (e.g., by controlled flow cytometry or by other established controlled assays), by ADCC and / or CDC during incubation, of less than 30% or less than 20% (preferably less than 10%).

[0293] In one embodiment, a multispecific anti-Vδ1 antibody or its antigen-binding fragment is provided, characterized by not inducing IL-17A secretion. IL-17A (interleukin-17A) is a pro-tumoric cytokine produced by activated T cells. IL-17A can enhance tumor growth and suppress anti-cancer immune responses. As shown in Figure 38, G-I, the anti-vδ1 antibody does not induce IL-17A secretion, while the anti-CD3 antibody does. In this specification, references to an antibody or antigen-binding fragment that does not induce IL-17A secretion refer to induction of less than approximately 30%, less than approximately 20%, or less than approximately 10% of IL-17A secretion induced by an equivalent CD3 multispecific antibody.

[0294] Part of the following sections relate to anti-Vδ1 antibodies, which are provided in monospecific format, and which are particularly intended for use in a multispecific (preferably bispecific) format in this invention. Preferably, the functional properties of the antibody when provided in monospecific format are shared by the multispecific antibody of this invention which further specifically binds to a second antigen.

[0295] Drugs that modulate immune cell markers on Vδ1+ cells When administered to a patient, polyspecific antibodies or fragments thereof may modulate immune cell markers in Vδ1+ cells.

[0296] The multispecific antibodies or fragments described herein may also be evaluated for their suitability for therapeutic use by measuring γδT regulation, and such evaluation can be performed when the antibody is provided in a monospecific format. For example, by measuring changes in the levels of CD25, CD69, or CD107a present in Vδ1+ T cells or cells of a model system. Such markers are often used as markers of lymphocyte regulation (e.g., proliferation or degranulation) and can be measured, for example, by flow cytometry after application of the antibodies or fragments described herein. Surprisingly, in such evaluations (see, for example, Examples 7, 17, 18, etc.), it has been observed that the monospecific versions of the multispecific antibodies described herein conferred measurably higher levels of CD25, CD69, or CD107a in target Vδ1+ T cells. In some cases, the phenotypic changes of Vδ1+ cells or populations tested in a model system may be compared to the phenotypic changes when alternative comparator antibodies (e.g., OKT-3, TS8.2, etc.) are applied to the equivalent γδT cells.

[0297] Accordingly, in one aspect of the present invention, a method is provided for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of a γδTCR for therapeutic use, comprising administering the antibody or fragment thereof to a cell population containing Vδ1+ cells and determining the effect on the levels of CD25 and / or CD69 and / or CD107a on the surface of the Vδ1+ cells. The effect on the levels of CD25, CD69 and / or CD107a may be determined / measured over a period of time. It will be understood that this effect may be measured by comparing it to the levels of CD25 and / or CD69 and / or CD107a on the surface of Vδ1+ cells without applying the antibody to the cells over the same period of time. In a further aspect of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR, which is carried out by adding the antibody to a cell population containing Vδ1+ cells and then measuring the level (or expression) of CD25 or CD69 or CD107a on the surface of the Vδ1+ cells.

[0298] Drugs that modulate the proliferation characteristics or number of Vδ1+ cells When administered to a patient, multispecific antibodies or fragments thereof may modulate the proliferative characteristics of Vδ1+ cells. For example, multispecific antibodies or fragments thereof may increase the number of Vδ1+ cells.

[0299] An alternative approach to measuring γδT proliferation may involve measuring the change over time in the relative number of Vδ1+ cells when a model system containing Vδ1+ cells is applied to the antibody or fragment thereof described herein. Surprisingly, in such evaluations, it has been observed that the antibodies described herein, when provided in monospecific format, were able to measurably increase the number of Vδ1+ T cells (see, e.g., Examples 10, 17, and 18). In some cases, this change in number may be compared to the change in number observed when an alternative comparator antibody (e.g., anti-OKT3) is applied to the model system.

[0300] Therefore, in another aspect of the present invention, a method is provided for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of a γδTCR, comprising administering the antibody or fragment thereof to a cell population containing Vδ1+ cells and determining the effect on the number of Vδ1+ cells in the population. The effect on the cell number may be determined / measured over a period of time. It will be understood that this effect may be measured by comparing it to the effect on the cell number observed if the antibody is not applied to the cell population over the same period of time. In a further aspect of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR, which is carried out by applying the antibody to a cell population containing Vδ1+ cells and then measuring the number of the cells over time.

[0301] Drugs that regulate the proliferative capacity and number of Vδ1+ cells An ideal therapeutic multispecific antibody or fragment thereof, as described herein, that binds to the Vδ1 chain of the γδTCR may be capable of enhancing the proliferation of Vδ1+ cells in vivo. Such an antibody can be used as a drug designed to specifically increase the number of Vδ1+ cells in a subject or patient. Examples are given below.

[0302] cancer: A relative increase in the number of Vδ1+ cells has been reported as a positive prognostic indicator associated with improved outcomes for many cancers (see, for example, Gentles et al (2015) Nature Immunology 21:938-945, Wu et al. (2019) Sci.Trans.Med.11(513):eaax9364, and Catellani et al. (2007) Blood 109(5):2078-2085). In one embodiment, the present invention relates to a drug that can increase the relative or absolute number of Vδ1+ cells in situ in cancer patients.

[0303] Pathogenic / parasitic / viral infections: The enrichment of Vδ1+ cells is observed during host defense against numerous acquired pathogenic / parasitic / viral infections. For a recent review, see Zhao et al. (2018) Immunol. Res. 2018:5081634. Furthermore, an increase in the number of Vδ1+ cells is thought to protect against infection by various DNA and RNA viruses. For example, the increase in number is thought to be protective even in CMV infection associated with allogeneic grafts (see van Dorp et al. (2011) Biology of Blood and Marrow Transplantation 17(2):S217). In addition, the number of Vδ+ cells increases in patients with coronavirus infection (Poccia et al. (2006) J. Infect. Dis. 193(9):1244-1249).

[0304] In another embodiment, the present invention relates to a drug capable of increasing the relative or absolute number of Vδ1+ cells in a subject or patient having a pathogenic infection.

[0305] Stem cell transplant: An increase in Vδ1+ cell count is associated with reduced disease recurrence, decreased viral infection, increased overall survival and disease-free survival, and generally favorable clinical outcomes during hematopoietic stem cell transplantation (see, for example, Aruda et al. (2019) Blood 3(21):3436-3448 and Godder et al. (2007) Bone Marrow Transplantation 39:751-757). Thus, in another embodiment, what is presented herein is a drug that can increase the relative or absolute number of Vδ1+ cells in a subject as part of a treatment regimen supporting stem cell transplantation.

[0306] Therefore, drugs that can preferentially or specifically increase the number of Vδ1+ cells in situ are highly desirable.

[0307] Drugs that maintain, induce, or increase cytokine secretion from Vδ1+ cells Cytokines are a large group of proteins, peptides, or glycoproteins secreted by specific cells of the immune system. They are a category of signaling molecules that mediate and regulate immunity, inflammation, and hematopoiesis. Some cytokines have been associated with improving the signs and symptoms of disease through direct or indirect regulation of the tumor and cellular microenvironment, autoimmune tissue and associated microenvironment, or the environment of virus-infected tissue or cells. Exemplary pro-inflammatory cytokines include tumor necrosis factor alpha (TNFα) and interferon-gamma (IFNγ).

[0308] However, many such cytokines exhibit undesirable toxicity when administered systemically. For example, TNFα can induce hemorrhagic necrosis of transplanted tumors and has been reported to exert synergistic antitumor effects when combined with other chemotherapy drugs, but various clinical trials using systemic recombinant human TNFα (rhTNFα) have revealed serious dose-limiting side effects, including hypotension, chills, phlebitis, thrombocytopenia, leukopenia and hepatotoxicity, fever, fatigue, nausea / vomiting, malaise and weakness, headache, chest tightness, back pain, diarrhea and shortness of breath.

[0309] The use of recombinant IFNγ also presents similar systemic toxicity challenges. For example, in cancer settings, IFNγ can exert favorable multifaceted effects, including upregulation of MHC classes I and II, stimulating antitumor immunity, increasing T cell infiltration, conferring anti-angiogenic effects, inducing chemokine / cytokine secretion, and directly inhibiting cancer cell proliferation; however, adverse side effects have also been observed. These include fever, headache, chills, fatigue, diarrhea, nausea, vomiting, loss of appetite, a temporary increase in hepatic transaminases, and a temporary decrease in granulocyte and white blood cell counts.

[0310] For a recent overview of both the potential and limitations of systemic recombinant TNFα and IFNγ, see Shen et al. (2018) Cell Prolif. 51(4):e12441.

[0311] Therefore, there is a need for more in situ-controlled, more localized, and more tissue-specific or cell-specific production of such cytokines. For example, more controlled expression or induction of pro-inflammatory cytokines has been proposed as one approach that can transform "cold" tumors into "hot" ones. Hot tumors are sometimes called "T-cell inflammatory" because an increase in the number or density of CD45+ T cells is also observed. For a recent overview, see Bonaventura et al. (2019) Front.Immunol.10:168.

[0312] For these reasons, an ideal multispecific antibody or fragment thereof, as described herein, that binds to the Vδ1 chain of the γδTCR, may be capable of maintaining, enhancing, or inducing cytokine secretion in Vδ1+ cells in vivo. Such an antibody may be used as a drug designed to specifically increase or induce cytokines in a subject or patient in a more localized, non-systemic manner, and which better correlates with the distribution of Vδ1+ cells in said subject or patient.

[0313] Notably, when a monospecific version of the multispecific antibody described herein, which binds to the Vδ1 chain of the γδTCR, is applied to Vδ1+ cells, remarkably high levels of secreted cytokines are observed. More specifically, and as a non-limiting example, remarkably high levels of TNFα and IFNγ are observed. See, for example, Example 15.

[0314] Therefore, in another aspect of the present invention, a method is provided for evaluating an antibody or a polyspecific antibody or a fragment thereof that binds to the Vδ1 chain of γδTCR, comprising administering the antibody or a fragment thereof to a cell population containing Vδ1+ cells, and determining the amount of at least one cytokine produced by the cell population. The amount of cytokine produced may be determined / measured over a period of time and may optionally be compared to the amount observed when the antibody is not applied to the cell population over the same period. In one embodiment, the observed level of cytokine produced when the antibody is administered to the cell population is about 10%, about 20%, about 30%, about 50%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, about 500%, and about 1000% compared to the level of cytokine produced when the antibody is not applied. In a further aspect of the present invention, the cytokine is a pro-inflammatory cytokine. In a further aspect of the present invention, the cytokine is a TNF-α cytokine. In a further aspect of the present invention, the cytokine is an IFN-γ cytokine.

[0315] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing antibodies or polyspecific antibodies or fragments thereof that bind to the Vδ1 chain of γδTCR as described herein, the method being carried out by applying the antibodies to a cell population comprising Vδ1+ cells and then measuring the level of at least one cytokine produced. In a further embodiment of the present invention, the cytokine to be measured is a TNF-α cytokine and / or an IFN-γ cytokine.

[0316] In a further embodiment of the present invention, a method is provided for evaluating antibodies or polyspecific antibodies or fragments thereof that bind to the Vδ1 chain of γδTCR, the method being carried out by applying the antibody or fragment thereof to a cell population containing Vδ1+ cells and determining the amount of pro-inflammatory cytokines produced and / or the number or density of CD45+ T cells present in the tumor or tumor microenvironment, thereby measuring the effect of the antibody in modulating a colder or colder tumor to a hotter or hotter tumor.

[0317] Drugs that maintain, induce, or increase granzyme B activity in Vδ1+ cells Granzyme B is a serine protease commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. Granzyme B is secreted by these cells along with the pore-forming protein perforin and mediates apoptosis in target cells such as diseased cells.

[0318] In a model system, incubating Vδ1+ cells in a co-culture with target disease cells (such as cancer cells) allows for the measurement of granzyme B levels and activity levels in the target disease cells before lysis. Notably, in such a model system, applying the antibodies or fragments thereof described herein, which bind to the Vδ1 chain of the γδTCR, to such a co-culture of Vδ1+ cells and cancer cells results in higher granzyme B levels and activity in disease cancer cells before cell death (see, for example, Example 16).

[0319] Therefore, in another aspect of the present invention, a method is provided for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of γδTCR, comprising administering the antibody or fragment thereof to a co-culture containing Vδ1+ cells and disease cells (such as cancer cells), and measuring the effect on the amount of granzyme B produced by the disease cells in the co-culture. The amount of cytokine produced may be determined / measured over a period of time and may optionally be compared to the amount observed when the antibody is not applied to the co-culture over the same period. In one embodiment, the level of granzyme B measured when the antibody is applied to the co-culture is about 10%, about 20%, about 30%, about 40%, about 50%, about 70%, about 80%, about 90%, about 100%, and about 200% compared to the level of granzyme B observed when the antibody is not applied.

[0320] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of γδTCR, the method being carried out by applying the antibody to a co-culture comprising Vδ1+ cells and disease cells, and then measuring the amount or activity of granzyme B in the disease cells.

[0321] Drugs that increase the polyclonal Vδ1+ cell population An ideal multispecific antibody drug could also be designed to ensure that the proliferation of Vδ1+ cells does not become too clonally concentrated at the hypervariable CDR3 sequence level. Therefore, an ideal antibody drug could be designed to avoid inducing Vδ1+ cell proliferation by binding to specific or "private" δ1+CDR3 sequence paratopes. Rather, the antibody could bind via conserved germline sequences present in all Vδ1+ T cell receptors, and in a gamma-chain-independent manner, rather than binding to sequences presented on only some Vδ1+ cells.

[0322] Therefore, an ideal antibody drug can stimulate the growth of Vδ1+ cells, generating multiple Vδ1+ cells containing a mixture of CDR3 sequences. This results in an in vivo augmented heterologous polyclonal population of Vδ1+ cells presenting different CDR3 sequences on the delta variable single strand. Notably, a high degree of polyclonality has been observed during analysis of the augmented Vδ1+ cell population generated by the method of adding the antibody or fragment thereof described herein to a starting population of immune cells containing Vδ1+ cells, using an RNAseq-based method designed to sequence through the CDR3 hypervariable region of the extracted RNA (see, for example, Example 10).

[0323] Accordingly, one embodiment provides a method for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of a γδTCR, comprising administering the antibody or fragment thereof to a cell population containing Vδ1+ cells, and determining the polyclonality of the enlarged Vδ1+ cells. It is desirable that the antibody drug generates an enlarged polyclonal population containing multiple Vδ1+CDR3 sequences. Polyclonality can be determined using methods known in the art, for example, by a nucleic acid sequencing approach that can analyze the Vδ1 chain hypervariable CDR3 content of the Vδ1+ cells.

[0324] Drugs that prolong the growth of polyclonal Vδ1+ cells An ideal multispecific antibody drug could potentially enhance, promote, or stimulate the proliferation of primary Vδ1+ cells without depleting such cells in vivo. For example, in comparison, anti-CD3 drugs such as OKT3 (e.g., muronomab) can increase CD3-positive T cells, but may also deplete or induce anergy. To evaluate the ability of monospecific versions of the multispecific antibodies that bind to the Vδ1 chain of the γδTCR described herein to drive sustained cell division of viable Vδ1+ cells, longer-term proliferation studies were conducted using monospecific antibodies. Notably, these studies revealed that monospecific versions of the multispecific antibodies that bind to the Vδ1 chain of the γδTCR described herein can drive cell division / proliferation of viable and still functionally cytotoxic Vδ1+ cells for more than 40 days (see, for example, Example 10).

[0325] In one embodiment, a method is provided for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of a γδTCR, comprising applying the antibody or fragment thereof to a cell population and monitoring the length of time over which division of Vδ1+ cells occurs. Ideally, the antibody should be able to stimulate division of Vδ1+ cells for 5 to 60 days, for example, at least 7 to 45 days, 7 to 21 days, or 7 to 18 days.

[0326] In a further embodiment, there are provided multispecific antibodies or fragments thereof described herein that bind to the Vδ1 chain of γδTCR and, when administered to a patient, stimulate the division of Vδ1+ cells, thereby increasing their number by at least 2, at least 5, at least 10, at least 25, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, and 600 times, and by at least 1,000 times.

[0327] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR, the method being carried out by applying the antibody to Vδ1+ cells or a mixed cell population containing Vδ1+ cells, and then measuring the number of Vδ1+ cells over time.

[0328] Drugs that modulate non-Vδ1+ immune cells by targeting Vδ1+ immune cells The multispecific antibodies or fragments described herein can also be evaluated by measuring the Vδ1+ cell-mediated regulation of other immune cells. For example, changes observed in the non-γδT cell "fraction" can be measured after applying the antibodies or fragments described herein to a model system containing a mixed population of immune cells, such as one containing human tissue αβ cells and γδT cells. Furthermore, the effect on the non-γδ cell type in the model can be measured by flow cytometry, for example, by measuring the relative change in the number of CD8+ αβT cells when the antibodies or fragments described herein are added to a mixed culture containing γδT cells and non-γδT cells. Optionally, changes observed in the number or phenotype of the non-γδT cell CD8+ lymphocyte population may be compared to changes in the number when an alternative comparator antibody (e.g., OKT-3) is applied to the mixed population.

[0329] Therefore, in another aspect of the present invention, a method is provided for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of γδTCR, comprising administering the antibody or fragment thereof to a mixed population of immune cells or tissues including Vδ1+ cells and Vδ1-negative immune cells, and measuring the effect on Vδ1-negative immune cells. This effect may be determined / measured over a period of time and may optionally be compared to the effect observed in Vδ1-negative cells when the antibody is not applied over the same period. This effect can be measured as a change in the number of Vδ1-negative immune cells. For example, the antibody may increase the number of Vδ1-negative immune cells by more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, or more than 500% compared to the level observed when the antibody is not applied.

[0330] In a further embodiment of the present invention, the regulated Vδ1-negative cells are CD45+ cells. In a further embodiment of the present invention, the regulated cells are αβT cells. In a further embodiment of the present invention, the regulated αβ+ cells are CD8+ lymphocytes. In a further embodiment of the present invention, the regulated αβT cells or population exhibit evidence of enhanced cell division. In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR, the method being performed by administering the antibody to a mixed population of immune cells including Vδ1+ cells and Vδ1-negative immune cells, and then measuring the effect conferred to the Vδ1-negative cell population by the Vδ1+ cells regulated by the antibody or fragment thereof.

[0331] In some cases, a concomitant increase in the number of Vδ1+ cells is observed during the "Vδ1+ cell-mediated immune system regulation" conferred by the antibodies or fragments described herein. And, although not bound by this theory, the aforementioned increase in the number of Vδ1+ cells may be responsible for the simultaneous increase of coexisting Vδ1-negative immune cells, such as αβ T cells. An alternative hypothesis may be that antibody-induced cytokine secretion from Vδ1+ T cells stimulates the increase of Vδ1-negative immune cells.

[0332] In a further embodiment of the present invention, the increase observed in the αβ+CD8+ lymphocyte population is compared to a comparator antibody such as an OKT3 antibody or an alternative anti-Vδ1 antibody. In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR, which is carried out by applying the antibody to a mixed population of immune cells including Vδ1+ T cells and αβT cells, and then measuring the number of CD8+αβ+ T cell lymphocytes over time.

[0333] Drugs that regulate tumor-infiltrating lymphocytes (TILs) The multispecific antibodies or fragments described herein can also be evaluated by measuring the effect they confer to tumor infiltration populations (TILs) in a model system. Surprisingly (see, for example, Example 18), in such evaluations, the monospecific versions of the multispecific antibodies described herein measurably modified the TIL population in human tumors. For example, changes in either the number or phenotype of the γδ+ lymphocyte TIL population or the non-γδ lymphocyte TIL population are measured after applying the antibodies or fragments described herein to human tumors such as human renal cell carcinoma. Optionally, the changes observed in either the number or phenotype of the γδ+ lymphocyte TIL population or the non-γδ lymphocyte TIL population may be compared to the changes observed when an alternative comparator antibody (e.g., OKT-3) is applied to the model system.

[0334] Therefore, in another aspect of the present invention, a method is provided for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of a γδTCR, comprising administering the antibody or fragment thereof to TILs located in or originating from a human tumor, and determining the effect on the number of TILs. This effect may be determined / measured over a period of time and may optionally be compared to the number of TILs observed when the antibody is not applied over the same period. This effect may be an increase in the number of TILs. For example, the antibody may increase the number of TILs by more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 70%, more than about 80%, more than about 90%, or more than 100% compared to the number of TILs observed when the antibody is not applied. In a further aspect, the TILs whose number is observed are γδ+ lymphocyte TIL cells and / or non-γδ lymphocyte TIL cells.

[0335] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR cell antibody, the method being performed by applying the antibody to a TIL or a group of TILs located within or originating from a human tumor, and then measuring the change in the number of the TIL or group of TILs over time.

[0336] Drugs that modulate human Vδ1+ cytotoxicity The polyspecific antibodies or fragments thereof described herein can also be evaluated by measuring the effect they have on Vδ1+-mediated cytotoxicity. Surprisingly, in such evaluations of monospecific versions of the polyspecific antibodies described herein and some of the polyspecific antibodies themselves (see, for example, Examples 19, 23, 24, and 27, etc.), measurably enhanced Vδ1+-mediated cytotoxicity was observed. For example, a reduction in the number of cancer cells or an increase in the number of cancer cells killed is observed after applying the antibody or fragment thereof to a model system comprising Vδ1+ cells and a mixed culture containing the cancer cells. In some cases, the reduction in the number of cancer cells or the increase in the number of cancer cells killed may be compared to the outcome when an alternative comparator antibody (e.g., OKT-3) is applied to the model system.

[0337] Therefore, in another aspect of the present invention, a method is provided for evaluating an antibody or fragment thereof that binds to the Vδ1 chain of γδTCR, comprising applying the antibody or fragment thereof to a mixed population of cells including Vδ1+ cells and cancer cells, and measuring the cytotoxicity of Vδ1+ cells against cancer cells. Cytotoxicity can be measured by an increase in the number of dead cancer cells over a period of time, which may optionally be compared to the number of dead cancer cells observed when the mixed population of cells is not applied with the antibody over the same period. For example, the increase observed in dead cells when the antibody is applied may be about 10%, about 20%, about 30%, about 40%, about 50%, about 70%, about 80%, about 90%, about 100%, about 200%, or about 500% compared to the number of dead cells observed when the antibody is not applied.

[0338] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of γδTCR cells, the method being carried out by adding the antibody to a mixed population of immune cells, including human Vδ1+ cells and cancer cells, and then measuring the increase in dead cancer cells over time.

[0339] Drugs that modulate the Vδ1+ cell target to effector cell ratio (T:E ratio) The multispecific antibodies or fragments described herein can also be evaluated as potential drugs by measuring how they enhance Vδ1+-mediated cancer cytotoxicity by determining the target cell-to-effector cell ratio at which 50% (EC50) of target cells are killed in a model system, for example, a mixed culture containing target cancer cells and human Vδ1+ effector cells. Surprisingly, in such evaluations (see, e.g., Examples 19s and 23), the multispecific antibodies described herein (and monospecific versions of the multispecific antibodies described herein) favorably modify the EC50 T:E ratio in the model system. Such modification can be measured as the number of Vδ1+ cells required to observe 50% killing of cancer cells over a period of time. This can also be reported as a change or enhancement ratio or rate of cytotoxicity against the cancer cells. Optionally, the T:E ratio conferred by the antibodies of the present invention may be compared to the T:E ratio when an alternative comparator antibody (e.g., OKT-3) is applied to the model system. In some scenarios, the multispecific antibodies of the present invention offer the opportunity to enhance cancer cell toxicity even at lower E:T ratios compared to monospecific antibodies.

[0340] Therefore, in another aspect of the present invention, a method is provided for evaluating a polyspecific antibody or fragment thereof that binds to the Vδ1 chain of γδTCR, comprising applying the antibody or fragment thereof to a mixed population of cells including human Vδ1+ cells and cancer cells, and measuring the number of Vδ1+ cells required to kill 50% of the cancer cells. This may be measured in comparison to the number of Vδ1+ cells required to kill 50% of the cancer cells without application of the antibody over the same period. For example, the reduction in the number of Vδ1+ cells required to kill 50% of the cancer cells when the antibody is applied may be about 10%, about 20%, about 30%, about 40%, about 50%, about 70%, about 80%, about 90%, about 100%, about 200%, or about 500% compared to the number of Vδ1+ cells required to kill 50% of the cancer cells without application of the antibody.

[0341] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to a Vδ1 chain, the method being carried out by adding the antibody to a cell population comprising Vδ1+ cells and cancer cells, and then measuring the number of Vδ1+ cells required to kill 50% of the cancer cells.

[0342] Drugs that enhance EC50 cytotoxicity of Vδ1+ cells An alternative method for measuring the observed enhancement of cytotoxicity in human Vδ1+ cells or populations is to measure the number of cells required to kill 50% of cancer cells over a period of time under condition A (e.g., starting control) and compare this to the number of cells required to kill 50% of cancer cells over a period of time under condition B (e.g., when the antibody of the present invention described herein is applied).

[0343] While it is recognized that there are various methods for measuring such parameters, the following non-restrictive hypothetical examples will be outlined to aid understanding.

[0344] Hypothetically, the enhancement of effector cytotoxicity can be measured as follows: Under condition A (control treatment), it is observed that 1000 Vδ1+ cells are required to kill 50% of cancer cells over a certain period (e.g., 5 hours). Under condition B (e.g., application of the multispecific antibody of the present invention as described herein), it is observed that 500 Vδ1+ cells are required to kill 50% of cancer cells over the same period. Therefore, in this example, the application of the antibody enhanced the cytotoxicity of the Vδ1+ cell population by 200%. (1000 / 500) × 100 = 200%

[0345] For example (see, for example, Example 19), surprisingly, such a percentage enhancement was observed with respect to the antibodies of the present invention described herein.

[0346] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing an antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR, the method being carried out by adding the antibody to a mixed population of immune cells, including Vδ1+ cells and cancer cells, and determining the relative change or rate of change in cytotoxicity compared to an equivalent or control experiment in which the mixture of cells is not treated with the antibody.

[0347] Drugs that enhance disease cell specificity of Vδ1+ cells while preserving healthy cells. Another approach to evaluating the multispecific antibodies described herein is to measure how the antibodies modulate disease-cell-specific cytotoxicity. Surprisingly, in such studies, it has been found that monospecific and multispecific antibodies can specifically enhance Vδ1+ cell-specific killing of disease cells, such as cancer cells, while preserving healthy or non-disease cells (see, for example, Examples 19 and 23). An ideal antibody drug administered to a patient to improve cancer symptoms would confer enhanced cytotoxicity, particularly against disease cells, while preserving healthy cells. Drugs that enhance effector cytotoxicity, particularly against disease cells, such as cancer cells, can be said to exhibit a higher therapeutic index (TI) than drugs that do not selectively enhance effector cytotoxicity, particularly against said disease cells. The therapeutic index, also called the therapeutic ratio, is a quantitative measure of the relative safety of a drug. It is a comparison between the amount of a therapeutic agent that produces a therapeutic effect and the amount that causes toxicity, for example, by causing undesirable death in the relevant or related healthy cell population. The multispecific antibodies or fragments described herein can be evaluated by measuring their ability to alter, enhance, or double the ability of Vδ1+ cells to selectively kill disease cells more effectively than healthy cells in a model system. For example, the model system may include Vδ1+ effector cells, cancer cells, and control cells (such as healthy cells). Optionally, the improvement in selective disease cell killing conferred by the multispecific antibodies of the present invention may be compared to the improvement observed when an alternative comparator antibody (e.g., OKT-3) is applied to the model system.

[0348] The disease cell specificity and enhancement of disease cell specificity of Vδ1+ cells can be measured in cultures containing Vδ1+ cells, disease cells, and healthy cells. For example, Vδ1+ specificity for disease cells can be measured by observing the number of cancer cells killed by Vδ1+ cells and then comparing this to the number of healthy cells killed by Vδ1+ cells. Such comparisons can be controlled by including equal numbers of disease and healthy cells in a model system that also contains Vδ1+ cells, for example, a "three-cell culture." If, for example, analytical or instrumental limitations reduce the ability to distinguish and track all three or more cell types in parallel in a single assay (including Vδ1+ cells, disease cells, and non-disease cells), alternative comparison methods may be considered. In the above example, an alternative approach to such research is provided by comparing Vδ1+ cytotoxicity against disease cells in one experiment and then comparing Vδ1+ cytotoxicity against healthy cells in a separate, equivalent experiment.

[0349] Another aspect of the present invention provides a method for evaluating a polyspecific antibody or fragment thereof that binds to the Vδ1 chain of γδTCR, comprising administering the polyspecific antibody or fragment thereof to a cell population including Vδ1+ cells and target cells, and measuring the cytotoxic specificity against the target cells. In one embodiment, the cytotoxic specificity against a first target cell type may be compared to the cytotoxicity observed against a second target cell type, and thus the method may be repeated using different target cell types. In a further aspect of the present invention, the first target cell type is diseased cells, and the second target cell type is a control cell such as healthy cells or cells having a disease different from that of the first target cell type.

[0350] In a further embodiment of the present invention, a method is provided for selecting, characterizing, or comparing a polyspecific antibody or fragment thereof described herein that binds to the Vδ1 chain of a γδTCR, wherein the effect of the antibody on conferring Vδ1+ cytotoxicity to (i) a first cell type and (ii) a second cell type is measured and compared. In a further embodiment of the present invention, an antibody that enhances specific cytotoxicity to the first cell type more than that to the second cell type is selected thereby. In a further embodiment of the present invention, the first cell type is diseased cells and the second cell type is healthy cells.

[0351] As described herein, the multispecific antibody or fragment used in the assay may be presented on the surface of cells, such as cells containing an Fc receptor. For example, the antibody or fragment may be presented on the surface of THP-1 cells, such as TIB-202® cells (available from the American Type Culture Collection (ATCC)). Alternatively, the multispecific antibody or fragment may be used directly in the assay.

[0352] In such functional assays, the output can be measured by calculating the maximum half-volume concentration, also referred to as "EC50" or "50 percent effective concentration." The term "IC50" refers to the inhibitory concentration. Both EC50 and IC50 can be measured using methods known in the art, such as flow cytometry. In some cases, EC50 and IC50 can be considered the same value or equivalent. For example, the effective concentration (EC) of effector cells required to inhibit (e.g., kill) 50% of a particular cell type can also be considered the 50% inhibitory concentration (IC). To avoid any doubt, in the case of antibodies, the EC50 values ​​in this application are provided using antibodies in IgG1 format. Such values ​​can be readily converted to equivalent values ​​based on the molecular weight of the antibody format, as follows: (μg / ml) / (MW in kDa units) = μM

[0353] The EC50 for downregulation of γδTCR upon antibody (or fragment) binding may be less than 0.50 μg / ml, for example, less than 0.40 μg / ml, less than 0.30 μg / ml, less than 0.20 μg / ml, less than 0.15 μg / ml, less than 0.10 μg / ml, less than 0.06 μg / ml, or less than 0.05 μg / ml. In a preferred embodiment, the EC50 for downregulation of γδTCR upon antibody (or fragment) binding is less than 0.10 μg / ml. In particular, the EC50 for downregulation of γδTCR upon antibody (or fragment) binding may be less than 0.06 μg / ml, for example, less than 0.05 μg / ml, less than 0.04 μg / ml, or less than 0.03 μg / ml. In particular, the EC50 values ​​are those when the antibody is measured in IgG1 format. For example, the EC50 value for γδTCR downregulation can be measured using flow cytometry (as described in assays such as Examples 6 and 23).

[0354] The EC50 for γδ T cell degranulation upon antibody (or fragment) binding may be less than 0.050 μg / ml, for example, less than 0.040 μg / ml, less than 0.030 μg / ml, less than 0.020 μg / ml, less than 0.015 μg / ml, less than 0.010 μg / ml, or less than 0.008 μg / ml. In particular, the EC50 for γδ T cell degranulation upon antibody (or fragment) binding may be less than 0.005 μg / ml, for example, less than 0.002 μg / ml. In a preferred embodiment, the EC50 for γδ T cell degranulation upon antibody (or fragment) binding is less than 0.007 μg / ml. In particular, the EC50 values ​​are those when the antibody is measured in IgG1 format. For example, the EC50 value of γδ T cell degranulation can be measured by detecting CD107a expression (i.e., a marker of cell degranulation) using flow cytometry (as described, for example, in the assay of Example 7). In one embodiment, CD107a expression is measured using an anti-CD107a antibody such as anti-human CD107a BV421 (clone H4A3) (BD Biosciences).

[0355] The EC50 for γδ T cell killing upon antibody (or fragment) binding may be less than 0.50 μg / ml, for example less than 0.40 μg / ml, less than 0.30 μg / ml, less than 0.20 μg / ml, less than 0.15 μg / ml, less than 0.10 μg / ml, or less than 0.07 μg / ml. In a preferred embodiment, the EC50 for γδ T cell killing upon antibody (or fragment) binding is less than 0.10 μg / ml. In particular, the EC50 for γδ T cell killing upon antibody (or fragment) binding may be less than 0.060 μg / ml, for example less than 0.055 μg / ml, and especially less than 0.020 μg / ml. In particular, the EC50 values ​​are those when the antibody is measured in IgG1 format. For example, the EC50 value of γδT cell killing can be measured by detecting the percentage of dead cells (i.e., using a cell viability-determinating dye) using flow cytometry after incubation of the antibody, γδT cells, and target cells (as described, for example, in the assay of Example 8). In one embodiment, target cell death is measured using the cell viability-determinating dye Viability Dye eFluor® 520 (ThermoFisher).

[0356] In the assays described in these embodiments, the antibody or a fragment thereof may be presented on the surface of cells such as THP-1 cells, for example, TIB-202® (ATCC). The THP-1 cells may be optionally labeled with a dye such as CellTracker® Orange CMTMR (ThermoFisher).

[0357] A drug that downregulates the CD3 molecule associated with the Vδ1 TCR. Current multispecific TCE (transcatheter cell activator) drugs typically associate with and activate T cells via CD3 binding events. This can lead to downregulation of the CD3 molecular complex from the T cell surface. However, it is also well understood that TCEs can overstimulate T cells through such association and downregulation. Because the CD3 molecular complex is not specific to a single class of T cells, it is not a precisely targeted drug. Stimulating all T cells (primarily αβ subtypes) via CD3 can result in cytokine overproduction, potentially leading to an acute cytokine flare (a so-called cytokine storm). Furthermore, non-targeted approaches that associate with and activate all T cells via CD3 can paradoxically lead to T cell overactivation, which can result in chronic T cell depletion and / or T cell death. Indeed, the approach presented herein specifically interacts with the effector population, whereas this nonspecific pan-T cell activation leads to the activation of both effector and regulatory T cells. Therefore, this "sledgehammer" approach is by no means ideal when it is desirable to upregulate only selective T cells.

[0358] In contrast, what is presented herein are multispecific antibodies, particularly T cell engagers, that associate with the T cell / CD3 complex in a different manner. Specifically, these TCEs can associate via the TRDV1 domain of the Vδ1 TCR, which is expressed only on Vδ1+ cells. In this way, such TCE-based drugs function differently. First, these TCEs can downregulate the TCR through association with the TRDV-1 epitope. As a result, this association event can downregulate the TCR-associated CD3 molecule complex. Such downregulation of CD3 is synonymous with T cell activation. However, by associating with the T cell / CD3 complex via the TRDV1 domain in this way, only the CDR3 molecule associated with the Vδ1 TCR is downregulated. This approach, which specifically targets and activates Vδ1 cells, can avoid many of the aforementioned problems (e.g., cytokine storm, T cell depletion / exhaustion, and ADCC). This mechanism is illustrated in Figure 30.

[0359] T cell stimulation via the CD3 "sledgehammer" approach can also contribute to T cell depletion through mechanisms driven by Fc gamma receptors such as ADCC. Therefore, the majority of CD3-targeting bispecific antibodies currently used clinically either have an Fc domain with reduced binding activity to FcγR, or are bispecific fragments that intentionally do not contain an Fc region. In CD3-targeting therapy, the binding affinity of the T cell receptor complex binding arm may also be reduced.

[0360] This reduction in affinity can lead to decreased efficacy and selectivity in terms of TCE design and functionality. For example, the affinity of the binding domain in such TCEs is known to drive the in vivo distribution profile. Specifically, the distribution of TCEs is usually observed to be biased towards the target with the highest affinity. Therefore, reducing the affinity of the TCE binding domain to the T cell complex generally moves the distribution away from the T cells that are precisely the cells necessary to drive the efficacy of such TCEs. This is partly why the therapeutic range of TCEs is said to be "extremely narrow."

[0361] The approach presented here specifically targets and activates vδ1 cells, avoiding the need to remove or reduce Fc function in some cases.

[0362] Furthermore, in the approach presented herein, the antibody associates with the TCR of vδ1 cells, but complete activation does not occur unless tumor cells are also present. When the antibody presented herein fully associates with the TCR, partial downregulation occurs, and vδ1 cells bound to the antibody presented herein are fully activated and cytotoxic only in the tumor microenvironment. This represents another important safety advantage of the present approach, because off-target cytotoxicity is reduced and the full potency of the antibody in activating vδ1 cells is released only in the presence of tumor cells.

[0363] One mechanism by which γδT cells can detect stress signals from tumor cells is thought to be due to the NCR (Natural Cytotoxic Receptor) they express. NCR can associate with NCR ligands on tumor cells. Therefore, a dual activation mechanism is employed: γδT cells are activated via TCR stimulation, and NCR senses tumor cells, enabling full activation and cytotoxicity.

[0364] This is in contrast to CD3-mediated stimulation of αβ T cells, where all stimulation is mediated through the TCR. Therefore, such cells do not sense tumor cells independently of antigen presentation via the NCR, making it nearly impossible to distinguish between healthy and transformed cells. Consequently, if the CD3 antibody is Fc, it attracts other immune cells, which can trigger a cascade of unintended but desirable events such as cytokine storms, immune cell depletion, and even hyperactivation, including, for example, T cell killing by NK cells. In the present approach, since both γδ T cells (and other immune cells such as NK cells) can distinguish between healthy and tumor cells, the stimulation of γδ T cells with the antibodies presented herein does not raise such concerns. For example, they do not kill each other via their NCR sensing mechanisms.

[0365] For example, in early cynomolgus monkey studies using the anti-vδ1 Fc effective multispecific antibody presented herein, which has specificity for both human and cynomolgus monkey vδ1 antigens (SEQ ID NO: 1 and SEQ ID NO: 172), it was found to be safe and well-tolerated in dose-escalation repeated-dose in vivo studies, according to all parameters measured. Adverse effects typically associated with T cell activation, such as cytokine release or weight loss, were not observed.

[0366] These findings also highlight another advantage of the approach described herein. Specifically, unlike TCEs represented by CD3 engagers, the TCE bispecific agents of the present invention may be designed as full-length antibodies containing, in some cases, a heavy chain in the VH-CH1-CH2-CH3 format accompanied by a homologous light chain in the VL-CL format. Unlike smaller bispecific formats (e.g., less than 70 kDa), such full-length bispecific formats can exhibit a longer half-life in vivo, thus reducing the frequency of required drug regimens. The longer half-life observed by such formats is due to a variety of reasons, including increased size (>70 kDa), which means that such formats are not filtered by the kidney (the glomerular pore size cutoff is 60-70 kDa). In one embodiment, the polyspecific antibody may be larger than about 70 kDa and may contain human IGHC sequences listed in IMGT (e.g., IGHA, IGHD, IGHD, IGHM, IHG sequences). Such IgG1 formats can also be recycled via the FcRn mechanism. A clear drawback of such full-length antibody formats, particularly with respect to TCE bispecific drugs, is that they exhibit an unfavorable safety profile due to reduced clearance rates, increased exposure, and more chronic exposure.

[0367] Therefore, this approach enables the possibility of Fc functionality without concerns about off-target effects, such as NK cells killing γδT cells or vice versa. Thus, this approach is superior to CD3-directed approaches, which have the constraint that measures such as reducing CD3 affinity and eliminating Fc function are necessary to limit incidental damage outside the tumor environment. The multispecific antibodies presented here, particularly T cell engagers, can bind to vδ1 cells without the possibility of damage, and full activation and enhanced cytotoxicity occur only when vδ1 cells are in close contact with tumor cells.

[0368] Therefore, in one embodiment, a method for downregulating the TRDV1-containing Vδ1 TCR and associated CD3 molecular complex on the cell surface with a TCE-polyspecific antibody is provided, as well as the use of such a polyspecific antibody for this purpose.

[0369] Polynucleotides and expression vectors In one embodiment of the present invention, a polynucleotide encoding a polyspecific antibody or fragment of the present invention is provided. In one embodiment, the polynucleotide comprises or comprises a sequence having at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 99% sequence identity with any one of SEQ ID NOs. 99 to 110. In one embodiment, the expression vector comprises the VH region of any one of SEQ ID NOs. 99 to 110. In another embodiment, the expression vector comprises the VL region of any one of SEQ ID NOs. 99 to 110. In a further embodiment, the polynucleotide comprises or comprises SEQ ID NOs. 99 to 110. In a further embodiment, a cDNA comprising the polynucleotide is provided. The polynucleotide may further comprise a sequence encoding a second binding domain, the second binding domain may confer specificity to a second antigen.

[0370] The polynucleotides and expression vectors of the present invention may also be described with reference to the encoded amino acid sequence. Thus, in one embodiment, the polynucleotide comprises or consists of a sequence encoding any one of the amino acid sequences of SEQ ID NOs. 62 to 85. In one embodiment, the expression vector comprises a sequence encoding any one of the amino acid sequences of SEQ ID NOs. 62 to 73. In another embodiment, the expression vector comprises a sequence encoding any one of the amino acid sequences of SEQ ID NOs. 74 to 85. The polynucleotides and expression vectors may further comprise sequences encoding additional binding domains, which may confer specificity to a second antigen.

[0371] To express an antibody or a fragment thereof, polynucleotides encoding part or the full length of the light and heavy chains, as described herein, are inserted into an expression vector so that the gene is operably linked to transcriptional and translational regulatory sequences. Thus, in one embodiment of the present invention, an expression vector comprising a polynucleotide sequence as defined herein is provided.

[0372] The nucleotide sequences described herein may include additional sequences encoding amino acid residues to aid in translation, purification, and detection, but it should be understood that alternative sequences may be used depending on the expression system used. For example, the first (5' end) nine nucleotides of SEQ ID NOs. 99–110, and the last (3' end) 36 nucleotides of SEQ ID NOs. 99–100, 102–103, 105–110, or the last (3' end) 39 nucleotides of SEQ ID NOs. 101 and 104 are optional sequences. These optional sequences may be removed, modified, or substituted if alternative design, translation, purification, or detection strategies are employed.

[0373] Mutations may be induced in the DNA or cDNA encoding a polypeptide that are silent with respect to the polypeptide's amino acid sequence but provide codons preferred for translation in a particular host. For example, preferred codons for nucleic acid translation in E. coli and S. cerevisiae, as well as in mammals, particularly humans, are known.

[0374] Polypeptide mutations can be achieved, for example, by substitution, addition, or deletion of the nucleic acid encoding the polypeptide. Substitutions, additions, or deletions of polypeptide-encoding nucleic acids can be introduced by many methods, including, for example, error-prone PCR, shuffling, oligonucleotide-designated mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recurrent ensemble mutagenesis, exponential ensemble mutagenesis, site-directed mutagenesis, gene rearrangement, artificial gene synthesis, site-saturated mutagenesis (GSSM), synthetic ligation rearrangement (SLR), or combinations thereof. Modifications, additions, or deletions of nucleic acids can also be introduced by methods including recombination, recurrent sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gap double-strand mutagenesis, point mismatch repair mutagenesis, repair-deficient host-strain mutagenesis, chemical mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimerization, or combinations thereof.

[0375] In particular, artificial gene synthesis can be used. The gene encoding the polypeptide of the present invention can be synthesized synthetically, for example, by solid-phase DNA synthesis. The entire gene can be synthesized de novo without the need for a precursor template DNA. To obtain the desired oligonucleotide, the components are sequentially attached to a growing oligonucleotide chain in the order required by the product sequence. Once chain construction is complete, the product is released from the solid phase into solution, deprotected, and collected. The product can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotide in high purity.

[0376] Expression vectors include, for example, plasmids, retroviruses, cosmids, yeast artificial chromosomes (YACs), and Epstein-Barr virus (EBV)-derived episomes. Polynucleotides are ligated into the vector so that transcriptional and translational regulatory sequences within the vector perform their intended functions of controlling the transcription and translation of the polynucleotides. Expression and / or regulatory sequences may include promoters, enhancers, transcriptional terminators, start codons (i.e., ATG) at the 5' end of the coding sequence, intron splicing signals, and stop codons. Expression vectors and expression regulatory sequences are selected to be compatible with the expression host cells used. Sequence IDs 99-110 contain nucleotide sequences encoding single-stranded variable fragments that may be included in the polyspecific antibodies of the present invention, including VH and VL regions linked by a synthetic linker (encoding Sequence ID 98). It will be understood that the polynucleotides or expression vectors of the present invention may include the VH region, the VL region, or both (including the linker, as may be included). Therefore, the polynucleotides encoding the VH and VL regions may be inserted into separate vectors, or the sequences encoding both regions may be inserted into the same expression vector. The polynucleotide(s) are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites of the polynucleotide and vector, or blunt-end ligation if no restriction sites exist).

[0377] A convenient vector encodes a functionally complete human CH or CL immunoglobulin sequence and features appropriate restriction sites engineered to facilitate the insertion and expression of any VH or VL sequence, as described herein. Expression vectors can also encode signal peptides that promote the secretion of antibodies (or fragments thereof) from host cells. Polynucleotides can be cloned into vectors such that the signal peptide is in-frame linked to the amino terminus of the antibody. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0378] In one embodiment of the present invention, a cell (e.g., a host cell such as a recombinant host cell) containing a polynucleotide or expression vector as defined herein is provided. It will be understood that the cell may contain a first vector encoding the light chain of an antibody or fragment thereof, and a second vector encoding the heavy chain of an antibody or fragment thereof. Alternatively, both the heavy and light chains may be encoded on the same expression vector introduced into the cell.

[0379] In one embodiment, a polynucleotide or expression vector encodes a membrane anchor or transmembrane domain fused to an antibody or fragment thereof, and the antibody or fragment thereof is presented on the extracellular surface of a cell.

[0380] Transformation can be carried out by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybren-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides into liposomes, microparticle gun injection, and direct microinjection of DNA into the nucleus. Furthermore, nucleic acid molecules may be introduced into mammalian cells by viral vectors.

[0381] Mammalian cell lines available as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels. Other usable cell lines include insect cell lines such as Sf9 cells, amphibian cells, bacterial cells, plant cells, and fungal cells. Antigen-binding fragments of antibodies, such as scFv and Fv fragments, can be isolated using methods known in the art and expressed in E. coli.

[0382] Antibodies are produced by culturing host cells for a period of time sufficient to allow antibody expression in the host cells, or more preferably, the secretion of antibodies into the culture medium in which the host cells grow. Antibodies can be recovered from the culture medium using standard protein purification methods.

[0383] The antibody (or fragment) of the present invention can be obtained and manipulated using, for example, the techniques disclosed in Green and Sambrook, Molecular Cloning: A Laboratory Manual (2012), 4th Edition, Cold Spring Harbour Laboratory Press.

[0384] Monoclonal antibodies can be produced by using hybridoma technology to fuse specific antibody-producing B cells with myeloma (B-cell cancer) cells selected for their ability to proliferate in tissue culture and their lack of antibody chain synthesis.

[0385] Monoclonal antibodies against the determined antigen are, for example, a) Immortalizing lymphocytes obtained from the peripheral blood of animals previously immunized with immortal cells and preferably myeloma cells with a determined antigen in order to form hybridomas. b) Culturing the formed immortalized cells (hybridoms) and recovering cells that produce antibodies with the desired specificity. It can be obtained by [method].

[0386] Alternatively, the use of hybridoma cells is not required. Antibodies capable of binding to the target antigens described herein can be isolated from a suitable antibody library via standard methods, for example, using phage display, yeast display, ribosome display, or mammalian display techniques known in the art. Thus, monoclonal antibodies can be, for example, a) A step of cloning a DNA or cDNA sequence obtained from lymphocytes, particularly peripheral blood lymphocytes of animals (preferably previously immunized with a determined antigen), into a vector, particularly phages and more specifically filamentous bacteriophages. b) A step of transforming prokaryotic cells with the above vector under conditions that enable antibody production, c) A step of selecting antibodies by subjecting them to antigen affinity selection, d) Step of recovering an antibody having the desired specificity. It can be obtained through a process that includes this.

[0387] In some cases, isolated polynucleotides encoding the polyspecific antibodies or fragments thereof described herein can be readily manufactured to produce quantities sufficient for use as drugs to improve the signs or symptoms of a disease. When used as a drug in this manner, the polynucleotide of interest is typically first operably ligated to an expression vector or expression cassette designed to express the antibody or fragment thereof in a subject or patient. Methods for delivering such expression cassettes and polynucleotides, or often referred to as "nucleoside" drugs, are well known in the art. For a recent overview, see Hollevoet and Declerck (2017) J.Transl.Med. 15(1):131.

[0388] A method for producing a polyspecific antibody or its antigen-binding fragment or variant is also provided, comprising culturing recombinant host cells of the present invention in a cell culture medium under conditions that express the encoding nucleic acid sequence of a plasmid or vector within the cells. If both binding domains are produced in the same recombinant cell, the method may further include obtaining the polyspecific antibody or its antigen-binding fragment or variant from the cell culture supernatant. The obtained polyspecific antibody or fragment can then be further processed and / or formulated into a pharmaceutical composition. Alternatively, if the binding domain of the polyspecific antibody is produced in separate recombinant host cells, the polyspecific antibody may be obtained by first collecting the separate cell supernatants from each recombinant host cell, then combining the binding domains to form a polyspecific antibody, which can then be further processed and / or formulated into a pharmaceutical composition. Furthermore, a method for producing recombinant host cells expressing a polyspecific antibody or its fragment or variant is provided, comprising transfecting the cells with the plasmid or vector of the present invention. The cells can then be cultured to produce the polyspecific antibody or its fragment or variant.

[0389] Pharmaceutical composition Further embodiments of the present invention provide compositions comprising a polyspecific antibody or a fragment thereof as defined herein. In such embodiments, the composition may comprise the polyspecific antibody in combination with other excipients as optional. Compositions comprising one or more additional activators (e.g., activators suitable for treating diseases referred to herein) are also provided.

[0390] A further aspect of the present invention provides a pharmaceutical composition comprising a multispecific antibody or fragment thereof as defined herein, together with a pharmaceutically acceptable diluent or carrier. The multispecific antibody of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a target. Typically, the pharmaceutical composition comprises the antibody of the present invention and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent, and absorption retarder. Examples of pharmaceutically acceptable carriers include one or more of water, physiological saline, salt, phosphate-buffered saline, dextrose, glycerol, ethanol, and combinations thereof. ...

Claims

1. a. The first target epitope, which is an epitope of the variable delta 1 (Vδ1) chain of the γδ T cell receptor (TCR), and b. Second target epitope A polyspecific antibody or fragment thereof that specifically binds to and the first target epitope is the activation epitope of γδ T cells, A polyspecific antibody or fragment thereof, wherein the second epitope is selected from the group consisting of the epitopes CD19, Her2 (CD340), EGFR, FAPα, mesothelin, PD-1, PD-L1, 4-1BB, OX40, and TIGIT.

2. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is the CD19 epitope.

3. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is the Her2 (CD340) epitope.

4. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is an EGFR epitope.

5. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is the epitope of FAPα.

6. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is a mesothelin epitope.

7. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is the epitope of PD-1.

8. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is the 4-1BB epitope.

9. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is OX40.

10. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is the TIGIT epitope.

11. The polyspecific antibody or fragment thereof according to claim 1, wherein the second epitope is the PD-L1 epitope.

12. The first target epitope is, (i) Sequence IDs 1-3 to 1-20 and / or (ii) Sequence ID 1, numbers 37-77 A polyspecific antibody or fragment thereof according to any one of claims 1 to 11, which is an epitope comprising one or more amino acid residues within the amino acid region of the antibody.

13. The multispecific antibody or antigen-binding fragment thereof according to claim 12, wherein the binding of the activated epitope (i) downregulates the γδTCR, (ii) activates degranulation of the γδT cells, and / or (iii) promotes γδT cell-mediated killing.

14. The polyspecific antibody or fragment thereof according to any one of claims 1 to 13, wherein the first target epitope is an epitope that upregulates the expression of CD107a, CD25, CD69 and / or Ki67.

15. The aforementioned polyspecific antibody or fragment yielded 1.5 × 10⁻¹⁴ when measured by surface plasmon resonance. －７ Less than M, 1.3 x 10 －７ Less than M, 1.0 x 10 －７ Less than M, or 5.0 x 10 －８ A polyspecific antibody or fragment thereof according to any one of claims 1 to 14, which binds to the first epitope with a binding affinity (KD) of less than M.

16. The polyspecific antibody or fragment thereof according to any one of claims 1 to 15, wherein the antibody is a bispecific antibody.

17. The multispecific antibody or fragment thereof according to any one of claims 1 to 16, wherein the multispecific antibody or fragment thereof is a human antibody or an antigen-binding fragment thereof.

18. The polyspecific antibody or its antigen-binding fragment according to any one of claims 1 to 17, wherein the polyspecific antibody or its antigen-binding fragment is an IgG polyspecific antibody, and optionally the polyspecific antibody or its antigen-binding fragment is an IgG1 polyspecific antibody.

19. The polyspecific antibody or a fragment thereof according to any one of claims 1 to 18, wherein the polyspecific antibody is of the Fc-effective type.

20. The aforementioned polyspecific antibody a. Inducing downregulation of TCR on Vδ1 T cells, b. Not showing CDC or ADCC, and c. Avoid depleting Vδ1 T cells. A polyspecific antibody or fragment thereof according to any one of claims 1 to 19, characterized by the above.

21. The multispecific antibody or fragment thereof according to any one of claims 1 to 20, wherein the multispecific antibody causes depletion of less than 30%, less than 20%, or less than 10% of the viable Vδ1+ T cell population via ADCC and / or CDC.

22. A pharmaceutical composition comprising a polyspecific antibody or a fragment thereof as defined in any one of claims 1 to 21, and a pharmaceutically acceptable diluent or carrier.

23. The pharmaceutical composition according to claim 22 for use in a method of treating a target disease or disorder.

24. The pharmaceutical composition according to claim 23, wherein the disease or disorder is cancer.

Images