Assay for T cell-dependent multispecific compounds

Abstract

The present disclosure provides reporter T cells that express a gamma-delta T cell receptor (TCR) and a reporter gene, which is expressed when the T cells are activated. Also provided are assays that utilize modified T cells to detect and / or quantify T cell-dependent binding compounds directed to gamma-delta TCR and a target antigen and to determine the relative potency of such binding compounds.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Provisional Patent Application No. 63 / 354,913, filed on June 23, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] Electronic Version of Sequence Listing The content of the electronic sequence listing (LVAT_023_01WO_SeqList_ST26.xml, size: 140,393 bytes, creation date: June 23, 2023) is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to a method for identifying and measuring the relative potency of multispecific binding compounds, such as bispecific antibodies, that can activate reporter cells expressing a T - cell receptor comprising gamma (γ) and delta (δ) chains.

Background Art

[0004] Over the past half - century, there have been great advances in the understanding of the etiology and progression of cancer. However, although cancer - related mortality has decreased significantly over the past 20 years, cancer remains an important cause of morbidity and mortality worldwide. In 2020, cancer was still the second most common cause of death in the United States.

[0005] Many treatment methods for cancer have been proposed and tried over the past few decades. Early treatments focused on toxic chemicals (i.e., chemotherapy) that can kill cancer cells, but with the increased understanding in the field of immunology, it has been realized that cells of the lymphocyte lineage, specifically T lymphocytes (T cells), can search for, identify, and kill cancer cells. Therefore, many studies have been conducted to develop cancer therapies based on T cells (e.g., CAR - T) that can more accurately target cancer cells and remove them while reducing damage to surrounding non - cancerous tissues.

[0006] Recently, there has been increasing excitement about a type of T cell known as γδ T cells. Most T cells have a T cell receptor (TCR) that includes an alpha (α) chain and a beta (β) chain (αβ T cells), whereas γδ T cells are characterized by the expression of a TCR (γδ TCR) that includes a γ chain and a δ chain. Since their discovery in 1987, it has become clear that γδ T cells function differently from the more abundant αβ T cells. For example, γδ T cells are relatively rare in lymphoid organs and are instead predominant in epithelial tissues and are abundant in the circulation. More importantly, γδ T cells can recognize target antigens in an MHC-independent manner and can thus recognize and respond to a wide range of antigens, including proteins and lipids. Finally, γδ T cells exhibit NK cell-like innate activities, including killing infected cells as well as microbes and malignant cells. Thus, γδ T cells have great promise in immunotherapy applications, such as cancer, infectious diseases, and other immune-related diseases.

[0007] There is a need in the art for an assay for selecting a potent compound that binds to and activates γδ T cells. SUMMARY OF THE INVENTION

[0008] T cells can be physically mobilized and linked to tumor surface antigens, thereby using bispecific antibodies to induce an anti-tumor response. Multispecific antibodies are engineered antibodies that have at least two different antigen-binding sites, whereby each antigen-binding site specifically binds to a unique epitope. T cell-dependent bispecific antibodies (TDbAbs) are an example of multispecific antibodies where one antigen-binding site specifically binds to a T cell signaling molecule (e.g., TCR) and the other antigen-binding site binds to a target antigen on a target cell (e.g., a tumor cell). Simultaneous binding of a TDbAb to a target antigen on a target cell and a T cell signaling molecule induces the recruitment of T cells to the target cell, resulting in T cell activation and subsequent depletion of the target cell. In clinical use, the selection of TDbAbs must balance efficacy and safety. TDbAbs that interact weakly may not have the desired therapeutic benefit, while TDbAbs that interact too strongly with T cell signaling molecules can cause undesirable side effects such as cytokine release syndrome (CRS). Furthermore, it is important that the TDbAb composition lacks non-specific T cell activation. Therefore, the development of therapeutic TDbAbs requires an assay that accurately measures the potency of potential TDbAbs to identify the ones most suitable for further development. Such an assay can also be used in the production process as a batch release assay. For example, such an assay can be used to determine whether a manufactured batch of TDbAb meets a predetermined potency criterion so that the manufactured batch can be released for clinical use (e.g., administration to patients).

[0009] The optimal assay for the potency of TDbAbs needs to be specific / selective, accurate, easy to use, and provide an output that is easy to understand. The present disclosure provides an easy-to-use, accurate, and scalable assay that enables the detection of γδ T cell activation by TDbAbs.

[0010] Provided herein is a method for detecting γδT cell receptor (γδTCR)-mediated reporter cell activation by a γδTCR-dependent multispecific binding compound (γδ-TDMBC), wherein the γδ-TDMBC comprises a target antigen-binding portion and a γδTCR-binding portion, and the method comprises: a) contacting the γδ-TDMBC with i) a reporter cell expressing γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and ii) a population of cells comprising a target antigen; and b) detecting expression of the reporter gene, wherein expression of the reporter gene indicates γδTCR-mediated activation of the reporter cell.

[0011] One aspect is a method for determining the relative potency of a γδ-TDMBC comprising a γδ-TCR-binding portion and a target antigen-binding portion, the method comprising: a) contacting a known concentration of the γδ-TDMBC with i) a reporter cell expressing γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and ii) a population of cells comprising a target antigen; and b) comparing the level of reporter gene expression resulting from a) with the level of reporter gene expression obtained by comparing the reporter cells of i) and the target antigen of ii) with a known concentration of a reference γδTCR-dependent compound that binds to the target antigen and γδTCR, thereby obtaining a measure of the relative potency of the γδ-TDMBC. In some aspects, step b) can include correlating the expression of the reporter gene as a function of the γδ-TDMBC with a standard curve generated by contacting a population of T cells and an antigen with different concentrations of the reference γδ-TDMBC.

[0012] One aspect is a method for detecting the presence of a γδ-TDMBC comprising a γδ-TCR-binding portion and a target antigen-binding portion in a composition, the method comprising contacting the composition with a) a reporter cell expressing γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and b) a population of cells comprising a target antigen, wherein expression of the reporter gene indicates the presence of the γδ-TDMBC in the composition.

[0013] One aspect is a method for quantifying a γδ-TDMBC comprising a γδ-TCR binding portion and a target antigen binding portion, comprising: a) contacting the γδ-TDMBC with i) a reporter cell expressing a γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and ii) a population of cells comprising the target antigen; and b) correlating the level of expression of the reporter gene as a function of the γδ-TDMBC concentration with a standard curve generated by contacting a population of reporter cells and the target antigen with γδ-TDMBC at different known concentrations, thereby quantifying the γδ-TDMBC.

[0014] One aspect is a method for determining the specificity of reporter cell activation by a γδ-TDMBC comprising a target antigen binding portion and a γδTCR binding portion, comprising: i) contacting the γδ-TDMBC with a) a reporter cell expressing a γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and b) a population of cells comprising the target antigen; ii) contacting the γδ-TDMBC with a) a population of cells comprising the reporter cells of i) in the absence of the antigen; and comparing the expression of the reporter gene in i) with the expression of the reporter gene in ii), wherein the ratio of the expression of the reporter gene in i) to the expression of the reporter gene in ii) indicates the specificity of the γδ-TDMBC for the target antigen.

[0015] In these methods, the reporter cell may be a reporter T cell, which may be CD3+. The γδTCR expressed by the reporter cell may contain a γ9 chain and / or a δ2 chain, and may be a γ9δ2 TCR. The reporter cell may contain one or more exogenous nucleic acid molecules encoding a TCRγ chain and / or a δ chain, and these exogenous nucleic acid molecules may be stably integrated into the genome of the reporter cell. The reporter gene may include a nucleic acid molecule containing a nucleotide sequence encoding a reporter protein operably linked to a promoter responsive to activation of the reporter cell. The promoter may be responsive to T cell activation and may be selected from the group consisting of an NFAT promoter, an AP-1 promoter, an NFKB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, and an IRF promoter. The reporter gene may include one or more response elements operably linked to the promoter.

[0016] In these methods, the reporter gene may include a nucleic acid molecule containing a nucleotide sequence encoding a reporter protein operably linked to a promoter responsive to activation of the reporter cell, and one or more response elements operably linked to the promoter. The promoter may be a minimal promoter, and the minimal promoter may be selected from the group consisting of a TK minimal promoter, a CMV minimal promoter, an SV40 minimal promoter, and an IEF la minimal promoter. The reporter gene may encode a reporter protein, and the reporter protein may be any protein that is detectable and may be selected from a fluorescent protein, a luminescent protein, a chemiluminescent protein, and an enzyme.

[0017] In these methods, the target antigen can be immobilized on a physical structure such as a plate or beads, or can be expressed by the target cells, and optionally, can be expressed on the surface of the target cells present in a population of cells. In such methods, the ratio of reporter cells to target cells can be about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:50, or about 1:100. The target antigen can be a cancer-related antigen or a tumor-related antigen and can be selected from the group consisting of EGFR, PSMA, CD1d, CD40, nectin-4, and CD123. The γδTCR-binding portion of γδ-TDMBC can specifically bind to the γ-chain of the TCR (which may be the γ9 chain). The γδTCR-binding portion of γδ-TDMBC can specifically bind to the δ-chain of the TCR (which may be the δ2 chain). γδ-TDMBC can bind to EGFR, PSMA, CD1d, CD40, nectin-4, and CD123. In these methods, γδ-TDMBC can be a bispecific antibody.

[0018] In these methods, γδ-TDMBC can be a γδ-TDbAb comprising a first single-domain antibody that specifically binds to an epitope within the γδTCR, wherein the first single-domain antibody comprises or consists of CDR1 (where X1 is G or S) comprising SEQ ID NO: 5, CDR2 comprising or consisting of SEQ ID NO: 2, and / or CDR3 comprising or consisting of SEQ ID NO: 14 (where X2 can be any amino acid and X3 is not R). The first single-domain antibody can comprise or consist of SEQ ID NO: 16, where X1 is G or S, X2 can be any amino acid, and X3 is not R. The γδ-TDbAb can be a γδ-TDbAb comprising a second single-domain antibody that specifically binds to the target antigen, wherein the second single-domain antibody can comprise CDR1 comprising or consisting of SEQ ID NO: 45, CDR2 comprising or consisting of SEQ ID NO: 46, and / or CDR3 comprising or consisting of SEQ ID NO: 47. In some embodiments, the second single-domain antibody can comprise or consist of SEQ ID NO: 48.

[0019] The second single-domain antibody may comprise or consist of CDR1 comprising or consisting of SEQ ID NO: 33, CDR2 comprising or consisting of SEQ ID NO: 34, and / or CDR3 comprising or consisting of SEQ ID NO: 35. The second single-domain antibody may comprise or consist of CDR1 comprising or consisting of SEQ ID NO: 49, CDR2 comprising or consisting of SEQ ID NO: 50, and / or CDR3 comprising or consisting of SEQ ID NO: 51. The second single-domain antibody may comprise or consist of CDR1 comprising or consisting of SEQ ID NO: 41, CDR2 comprising or consisting of SEQ ID NO: 42, and / or CDR3 comprising or consisting of SEQ ID NO: 43. The second single-domain antibody may comprise or consist of CDR1 comprising or consisting of SEQ ID NO: 37 (where X4 is G or S), CDR2 comprising or consisting of SEQ ID NO: 38 (where X5 is A or T), and / or CDR3 comprising or consisting of SEQ ID NO: 39 (where X6 is Y or F). The second single-domain antibody may comprise or consist of SEQ ID NO: 48, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 44, SEQ ID NO: 40, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, or SEQ ID NO: 80.

[0020] The reporter cells may not express a functional TCRα chain and / or β chain and may contain one or more knockout mutations within the TCRα chain locus and / or TCRβ chain locus, or within the gene elements that control the expression of either or both loci. In these methods, the reporter cells may not express EGFR, PSMA, CD1d, CD40, CD123, and nectin-4.

[0021] One aspect is a reporter cell that expresses a γ9δ2 T cell receptor (γδTCR) and contains a reporter gene that responds to activation of the reporter cell. In some aspects, the reporter cell may be a reporter T cell, which may be CD3+, and / or may be Jurkat cells or CTLL-2 cells. In some aspects, the reporter cell may contain one or more exogenous nucleic acid molecules encoding the TCR γ chain and / or δ chain, and these exogenous nucleic acid molecules may be stably integrated into the genome of the reporter cell. In some aspects, the reporter gene may include a nucleic acid molecule containing a nucleotide sequence encoding a reporter protein operably linked to a promoter that responds to activation of the reporter cell. The promoter may be responsive to T cell activation and may be selected from the group consisting of NFAT promoter, AP-1 promoter, NFκB promoter, FOXO promoter, STAT3 promoter, STAT5 promoter, and IRF promoter. The reporter gene may include one or more response elements operably linked to the promoter. In some aspects, the reporter gene may include a nucleic acid molecule containing a nucleotide sequence encoding a reporter protein operably linked to a promoter that responds to activation of the reporter cell, and one or more reporter cell activation response elements operably linked to the promoter. The promoter may be a minimal promoter, and the minimal promoter may be selected from the group consisting of TK minimal promoter, CMV minimal promoter, SV40 minimal promoter, and IEFlα minimal promoter. The reporter cell activation response element may be a T cell activation response element that may be selected from the group consisting of NFAT gene response element, AP-1 gene response element, NFκB gene response element, FOXO gene response element, STAT3 gene response element, STAT5 gene response element, and IRF gene response element. In some aspects, the reporter gene may include at least two reporter cell activation response elements that may be arranged as tandem repeats.The reporter cell activation response element(s), or tandem repeats thereof, may be positioned 5' to the nucleic acid sequence encoding the reporter protein, or may be positioned 5' to the promoter. In some embodiments, the reporter gene may encode a reporter protein, and the reporter protein may be any protein that is detectable and may be selected from fluorescent proteins, luminescent proteins, chemiluminescent proteins, and enzymes. In some embodiments, the reporter cells may not express a functional TCRα chain and / or β chain and may contain one or more knockout mutations within the TCRα chain locus and / or TCRβ chain locus, or within the genetic elements that control the expression of either or both loci. In some embodiments, the reporter cells do not express EGFR, PSMA, CD1d, CD40, nectin-4, and CD123.

[0022] One embodiment is a kit comprising the reporter cells of the present disclosure, which may contain a pharmaceutically acceptable excipient.

Brief Description of the Drawings

[0023]

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Modes for Carrying Out the Invention

[0024] The present disclosure provides a method for detecting γδTCR-mediated activation of reporter cells. More specifically, the present disclosure provides an assay for measuring the ability of a γδT cell receptor (γδTCR)-dependent multispecific binding compound (γδ-TDMBC), such as a TDbAb, to activate γδ-expressing reporter cells in the presence of a target antigen recognized by the γδ-TDMBC. Such an assay can be used to measure the relative potency of a γδ-TDMBC. The reporter cells used in the disclosed assay express a γδTCR and are modified to contain a reporter gene such that activation of the γδ-expressing reporter cells results in the production of a reporter protein that enables rapid and easy detection of the activated reporter cells. Thus, the methods of the present disclosure generally can be carried out by contacting a γδ-TDMBC with a population of reporter cells that contain a reporter gene responsive to target antigen and reporter cell activation and assaying the reporter cells for expression of the reporter gene, wherein expression of the reporter gene indicates γδ-TDMBC-induced activation of the reporter cells. The level of expression of the reporter gene may be compared to the level of expression obtained by contacting a reference γδ-TDMBC with the target antigen and the reporter cells, thereby determining the relative potency of the first γδ-TDMBC.

[0025] Before further describing the present disclosure, it is to be understood that the present disclosure is not limited to the specific embodiments described, and accordingly, it goes without saying that it can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present disclosure is limited only by the claims.

[0026] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, a compound refers to one or more compound molecules. Thus, the terms "a", "an", "one or more", and "at least one" can be used interchangeably. Similarly, the terms "comprising", "including", and "having" can be used interchangeably. It should further be noted that the claims may be drafted to exclude any optional elements. Thus, this description is intended to serve as a precedent for the use of exclusive terms such as "solely", "only", etc., or the use of "negative" limitations with respect to the recitation of claim elements.

[0027] The publications disclosed in this specification are provided only for their disclosures prior to the filing date of this application. Nothing in this specification should be construed as an admission that the present disclosure is not entitled to antedate such publications by virtue of prior disclosure. Further, the dates of the publications provided may be different from the actual publication dates and may need to be independently verified. All publications mentioned in this specification are hereby incorporated by reference into this specification to disclose and describe the methods and / or materials for which those publications are cited.

[0028] γδ-TDMBC (γδ-TCR-dependent multi-specific binding compound) is a compound having two or more binding sites, each of which binds to a unique epitope, and one of the binding sites specifically binds to an epitope in the γδ TCR. In some embodiments, the second binding site of γδ-TDMBC can bind to a target antigen. An example of TDMBC is a multi-specific antibody such as a bispecific antibody. A multi-specific antibody is an engineered antibody having at least two different antigen-binding sites, whereby each antigen-binding site specifically binds to a unique epitope. The γδ T cell-dependent bispecific antibody (γδ-TDbAb) disclosed herein is a bispecific antibody in which one antigen-binding site specifically binds to the γδ TCR and the second antigen-binding site specifically binds to a target antigen. In some embodiments, γδ-TDMBC may be a multi-specific antibody comprising at least two single-domain antibodies, wherein at least one single-domain antibody specifically binds to the γδ TCR, preferably the Vδ chain (e.g., Vδ2 chain), and at least one other single-domain antibody specifically binds to a target antigen (e.g., CD1d, CD40, CD123, PSMA, nectin-4, and EGFR). In some embodiments, γδ-TDMBC may be a bispecific antibody comprising two single-domain antibodies, wherein one single-domain antibody specifically binds to the γδ TCR, preferably the Vδ chain (e.g., Vδ2 chain), and the other single-domain antibody specifically binds to a target antigen (e.g., CD1d, CD40, CD123, PSMA, nectin-4, and EGFR). As used herein, "specifically binds" refers to the differential binding of an antigen-binding site to at least two different epitopes. "Specifically binds" means that the antigen-binding site binds to the target molecule with a significantly higher affinity or avidity than its affinity or avidity for molecules not related to the target molecule. For example, an antigen-binding site that specifically binds to a TCRγ chain protein means that the affinity or avidity of the antigen-binding site for the TCRγ chain protein is significantly higher than its affinity or avidity for a protein unrelated to the TCRγ chain protein, such as the TCRα chain.In some aspects of the present disclosure, the antigen-binding site of TDMBC binds to an epitope with a KD of about 1 μM or less, 0.1 μM or less, 0.01 μM or less, or about 1 nM or less. In some aspects, γδ-TDMBC specifically binds to the γ chain of the TCR. In some aspects, γδ-TDMBC specifically binds to the δ chain of the TCR. In some aspects, γδ-TDMBC specifically binds to the γ9 chain of the TCR. In some aspects, γδ-TDMBC specifically binds to the δ2 chain of the TCR.

[0029] As used herein, "target antigen", "antigen target", "antigen", etc. refer to any biomolecule such as a protein, glycoprotein, lipoprotein, or sugar that can be expressed by a cell in which it is desirable to induce a T cell response. In the methods of the present disclosure, the target antigen can be expressed by a target cell, such as on the surface of the target cell, but this is not necessary. In some aspects, the antigen may be immobilized on a surface such as a plate (e.g., a microtiter plate) or beads (e.g., latex beads). Useful target antigens can be found, for example, on the surface of tumor cells, virus-infected cells, bacteria-infected cells, and other diseased cells. The target antigen can be a biomolecule (e.g., a protein) derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), or a virus or bacteria source. When referring to a specific target antigen herein, the term encompasses the "full-length" untreated target antigen, as well as any form of the target antigen resulting from processing in the target cell. The term also encompasses naturally occurring variants of the target antigen, such as splice variants or allelic variants. Exemplary human target proteins useful as antigens include, but are not limited to, CD1d, epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), cluster of differentiation (CD) 40 (CD40), nectin-4, and CD123.

[0030] "Reporter cells" refer to cells that are genetically modified by human hands to express γδ TCR and have a reporter gene that responds to γδ TCR-mediated activation of the reporter cells. The reporter cells of the present disclosure may naturally express γδ TCR or may express γδ TCR as a result of genetic manipulation of the cells by human hands (e.g., introduction of exogenous nucleic acid molecules encoding the γ chain and δ TCR chain into the cells). "Activation of reporter cells", "reporter cell activation", etc. refer to changes in the physiological state and / or composition of reporter cells upon binding of at least γδ TCR by γδ-TDMBC. Reporter cell activation includes a γδ TCR-mediated signaling cascade and can bind to regulatory elements (e.g., promoters, enhancers, operators, etc.) in the genome, thereby regulating (e.g., activating) gene expression, including increased, decreased, and / or new production and / or activation of cellular proteins and other molecules. Thus, reporter cell activation results in one or more cellular responses, examples of which include changes in gene expression, cell proliferation, cell differentiation, cytokine production and / or secretion, qualitative or quantitative changes in the composition of cell surface proteins, production and / or release of cytotoxic effector molecules, and cytotoxic activity, but are not limited thereto. In some embodiments of the present disclosure, the reporter cells may be produced by genetically modifying T cells. In such embodiments, the reporter cells may be referred to as reporter T cells.

[0031] "T cells" refer to a type of blood cell known as T lymphocytes that mature in the thymus and are distinguished from other lymphocytes, such as B cells, by the presence of at least a T cell receptor on the cell surface. T cells useful for producing the reporter cells of the present disclosure include any cell that expresses a functional TCR and has a TCR activation signaling cascade that regulates transcription. Examples of such cells include T helper cells (CD4 + cells), cytotoxic T cells (CD8 +Cells), natural killer T cells, T regulatory cells (Tregs), and γδ T cells, but are not limited thereto. The T cells used in the embodiments of the present disclosure can be isolated from humans or animals, obtained from cultures, or obtained from commercial sources. Non-limiting examples of commercially available T cell lines include BCL2(AAA)Jurkat (ATCC® CRL-290™), BCL2(S70A)Jurkat (ATCC® CRL-290™), BCL2(S87A)Jurkat (ATCC® CRL-290™), BCL2 Jurkat (ATCC® CRL-2899™), Neo Jurkat (ATCC® CRL-2898™), TALL-104 cytotoxic human T cell line (ATCC® #CRL-11386), and CTLL-2 (ATCC® TIB-214™).As further examples, mature T cell lines such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1 and T34; and immature T cell lines such as ALL-SIL, Be13, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-T1, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1, TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, TK-6, TLBR-1, -2, -3, and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC® TIB-153), J45.01 (ATCC® CRL-1990), J.CaM1.6 (ATCC® CRL-2063), RS4;11 (ATCC® CRL-1873), CCRF-CEM (ATCC® CRM-CCL-119); and cutaneous T cell lymphoma lines such as HuT78 (ATCC® CRM-TIB-161), MJ[G11] (ATCC® CRL-8294), HuT102 (ATCC® TIB-162), etc. are included, but not limited thereto. In some embodiments, the reporter T cells of the present disclosure are CD4. + (i.e., expressing the CD4 protein), CD3+ and / or CD8 + can be. In some embodiments, the reporter T cells of the present disclosure are CD4 - (i.e., not expressing the CD4 protein), and / or CD8 -It can be a T cell. In some embodiments, the reporter T cells of the present disclosure may be produced from immobilized T cells (e.g., T cell lines). In some embodiments, the reporter T cells of the present disclosure can be produced from Jurkat cells. In some embodiments, the reporter T cells of the present disclosure can be produced from CTLL-2 T cells.

[0032] As used herein, "T cell receptor" refers to a T cell receptor as generally understood in the field of immunology. The TCR is a heterodimer composed of two different peptide chains, an α chain and a β chain, or a γ chain and a δ chain. The reporter cells of the present disclosure express a TCR receptor that includes a γ chain and a δ chain. The TCR-γ locus is known to contain at least 12 functional variable (V) gene segments, each encoding a γ chain variable region, while the TCR-δ locus is known to contain at least 8 V gene segments, each encoding a δ chain variable region. In some embodiments, the reporter cells of the present disclosure can express a γδ TCR that includes a V region from any Vγ gene segment. In some embodiments, the reporter cells of the present disclosure can express a γδ TCR that includes a V region from any Vδ gene segment. In some embodiments, the reporter cells of the present disclosure can express a γδ TCR that includes a V region from the Vγ9 gene segment. In some embodiments, the reporter cells of the present disclosure can express a γδ TCR that includes a V region from the Vδ2 gene segment. In some embodiments, the reporter cells of the present disclosure include a TCR (Vγ9Vδ2 TCR) that includes a Vγ9 region and a Vδ2 region.

[0033] As used herein, "reporter gene" refers to a nucleic acid molecule comprising a nucleotide sequence encoding a reporter protein, the presence or activity of which can be detected or measured when operably linked to a promoter and optionally an activated reporter cell response element. The phrase "reporter gene" as used herein does not necessarily indicate the presence of gene elements such as exons, introns, splicing signals, etc. In some embodiments, such elements may be present, but the reporter genes of the present disclosure may include open reading frames lacking introns and exons. For example, the reporter genes of the present disclosure may include a single open reading frame encoding a fluorescent protein operably linked to a promoter and optionally an activated reporter cell response element. In some embodiments, the reporter protein may generate a detectable signal that enables detection to indicate the presence and / or amount of the reporter protein under appropriate conditions. Examples of suitable reporter proteins include, but are not limited to, fluorescent molecules such as fluorescent proteins, luminescent molecules such as luminescent proteins, chemiluminescent molecules such as chemiluminescent proteins, and enzymes such as alkaline phosphatase or β-galactosidase. An example of a luminescent protein is luciferase. Luciferase is a class of luminescent proteins derived from many sources, including firefly luciferase (from the species Photinus pyralis), Renilla luciferase from sea pansy (e.g., Renilla reniformis), click beetle luciferase (e.g., from Pyrearinus termitilluminans), marine copepod Gaussia luciferase (e.g., from Gaussia princeps), and deep sea shrimp nanoluciferase (e.g., from Oplophorus gracilirostris). Firefly luciferase catalyzes the oxygenation of luciferin to oxyluciferin, resulting in the emission of photons of light, while other luciferases such as Renilla emit light by catalyzing the oxygenation of coelenterazine.The wavelengths of light emitted from different luciferase forms and variants can be read using different filter systems, thereby facilitating multiplexing. The amount of luminescence is proportional to the amount of luciferase expressed within the cell. In some embodiments, the reporter gene can be polycistronic, meaning that it encodes two or more reporter proteins that can be fusion proteins. The use of a polycistronic reporter gene enables the use of dual reporter proteins (e.g., fluorescent proteins having two different colors) and / or enables the use of destabilizing sequences (e.g., PEST sequences) that can reduce the half-life of the fusion protein, thereby reducing "leaky" expression. In some embodiments, the reporter protein can be an intracellular protein (i.e., it remains within the cell). In some embodiments, the reporter protein can be a secreted protein.

[0034] Phrases such as "responsive to reporter cell activation", "responsive to activation of reporter cells", etc. are used with reference to genetic elements (e.g., regulatory elements such as genes, promoters, etc., response elements, etc.), and refer to the fact that the state or activity of the element being referred to changes upon activation of the reporter cell. For example, a gene responsive to reporter cell activation means that the gene is expressed (i.e., transcribed) when the reporter cell is in an activated state. Similarly, a promoter responsive to reporter cell activation is a promoter that becomes active (e.g., promotes transcription of a gene operably linked thereto) when the reporter cell is in an activated state. Similarly, an activation reporter cell response element is a response element that affects (e.g., enhances transcription) promoter / transcription activity when the reporter cell is activated.

[0035] "Operably linked" refers to the relative positioning of two or more nucleotide sequences, with or without an intervening sequence such as a spacer or linker sequence, such that an event (i.e., a molecular binding) in one or more of the nucleotide sequences causes an effect in one or more different nucleotide sequences. For example, a promoter operably linked to a coding sequence such as an open reading frame can drive the expression of the coding sequence. Such a coding sequence can also be said to be "under the control of" or "regulated by" the promoter. As a further example, a response element operably linked to a coding sequence, or a promoter that drives the expression of a coding sequence, enables or enhances the expression of the linked coding sequence.

[0036] "Response element" refers to a cis-acting DNA sequence that confers responsiveness to a gene, such responsiveness being mediated through interaction with the DNA-binding domain of a cellular molecule such as a transcription factor. "Activated reporter cell response element", "activated cell response element", "activation response element", etc. refer to a response element that affects (e.g., enhances) promoter / transcriptional activity when the reporter cell is activated. An example of a response element is an enhancer. In the present disclosure, operably linking a response element to a reporter gene can enhance the activity of the operably linked promoter when the reporter cell is in an activated state. Accordingly, suitable response elements useful for practicing aspects of the present disclosure include any response element that causes the expression of the reporter gene to respond to activation of the reporter cell when operably linked to the reporter gene. In some aspects, the response element can include a T cell activation response element. Examples of such response elements include the NFAT gene response element, the AP-1 gene response element, NF KExamples of response elements include, but are not limited to, B gene response elements, FOXO gene response elements, STAT3 gene response elements, STAT5 gene response elements, and IRF gene response elements. The response elements can be arranged as tandem repeats (such as any of about 2, 3, 4, 5, 6, 7, 8, or more tandem repeats), which can increase the responsiveness of an operably linked promoter or gene. The response element(s) can be positioned 5' or 3' relative to the reporter gene. The response element(s) can be located 5' from the site of the promoter.

[0037] One aspect of the present disclosure is a method for detecting γδTCR-mediated reporter cell activation by a γδ-TDMBC having a γδTCR binding site and a target antigen binding site, the method comprising: a) contacting the γδ-TDMBC with i) a reporter cell of the present disclosure that expresses γδTCR and comprises a reporter gene that responds to activation of the reporter cell, and ii) a population of cells comprising a target antigen; and b) detecting expression of the reporter gene, wherein expression of the reporter gene indicates γδTCR-mediated activation of the reporter cell. In some aspects, detecting expression of the reporter gene comprises detecting a reporter protein encoded by the reporter gene.

[0038] One aspect of the present disclosure is a method for determining the relative potency of a γδ-TDMBC comprising a γδ-TCR binding moiety and a target antigen binding moiety, the method comprising: a) contacting a known concentration of the γδ-TDMBC with i) a reporter cell expressing a γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and ii) a population of cells comprising the target antigen; and b) comparing the level of reporter gene expression resulting therefrom to the level of reporter gene expression obtained by comparing the level of reporter gene expression as a function of γδ-TDMBC concentration to a standard curve generated by contacting a population of T cells and antigen with different concentrations of a reference γδ-TDMBC that binds to the target antigen and γδ TCR, thereby obtaining a measure of the relative potency of the first γδ-TDMBC. In some embodiments, the method comprises detecting the expression of the reporter gene by detecting a reporter protein encoded by the reporter gene. In some embodiments, step b) comprises correlating the expression of the reporter gene as a function of γδ-TDMBC concentration to a standard curve generated by contacting a population of T cells and antigen with different concentrations of a reference γδ-TDMBC.

[0039] One aspect of the present disclosure is a method for detecting the presence of a γδ-TDMBC comprising a γδ-TCR binding site and a target antigen binding site in a composition, the method comprising contacting the composition with a) a reporter cell of the present disclosure expressing a γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and b) a population of cells comprising the target antigen, wherein expression of the reporter gene indicates the presence of the γδ-TDMBC in the composition. In some embodiments, the method comprises detecting the expression of the reporter gene by detecting a reporter protein encoded by the reporter gene.

[0040] One aspect of the present disclosure is a method for quantifying a γδ-TDMBC comprising a γδ-TCR binding site and a target antigen binding site, the method comprising: a) contacting the γδ-TDMBC with i) a reporter cell of the present disclosure expressing a γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and ii) a population of cells comprising a target antigen; and b) correlating the expression of the reporter gene as a function of the γδ-TDMBC concentration with a standard curve generated by contacting a population of reporter cells and the target antigen with different concentrations of γδ-TDMBC, thereby quantifying the γδ-TDMBC. In some aspects, the method comprises detecting the expression of the reporter gene by detecting a reporter protein encoded by the reporter gene. In some aspects, step b) comprises correlating the expression of the reporter gene as a function of the γδ-TDMBC concentration with a standard curve generated by contacting a population of T cells and the antigen with different concentrations of a reference γδ-TDMBC.

[0041] One aspect of the present disclosure is a method for determining the specificity of reporter cell activation by a γδ-TDMBC comprising a target antigen binding site and a γδTCR binding site, the method comprising: i) contacting the γδ-TDMBC with a) a reporter cell of the present disclosure expressing a γδTCR and comprising a reporter gene responsive to activation of the reporter cell, and b) a population of cells comprising a target antigen; and ii) comparing the expression of the reporter gene in i) with the expression of the reporter gene in the reporter cells when the reporter cells are contacted with the γδ-TDMBC in the absence of the antigen, wherein the ratio of the expression of the reporter gene in i) to the expression of the reporter gene in ii) indicates the specificity of the γδ-TDMBC for the target antigen. In some aspects, the method comprises detecting the expression of the reporter gene by detecting a reporter protein encoded by the reporter gene.

[0042] In these methods, the reporter cell may be a reporter T cell. In these methods, the reporter T cell can be produced by modification of Jurkat cells or CTLL-2 T cells. In these methods, the reporter T cell may contain one or more knockout mutations within the TCRα chain locus, or the TCRβ chain locus, or within the gene elements controlling the expression of either or both loci. In these methods, the reporter cell may lack a functional TCRα chain gene and / or TCRβ chain gene. In these methods, the reporter cell may lack a functional αβ TCR.

[0043] In these methods, the γδ TCR expressed by the reporter cell may contain the γ9 chain. In these methods, the γδ TCR expressed by the reporter cell may contain the δ2 chain. In these methods, the γδ TCR expressed by the reporter cell may contain the γ9δ2 TCR. In these methods, the γδ TCR may contain the γ9δ2 TCR, and the γδ-TCR binding site may specifically bind to the γ9 chain of the TCR. In these methods, the γδ TCR may contain the γ9δ2 TCR, and the γδ-TCR binding site may specifically bind to the δ2 chain of the γδ TCR. In these methods, the reporter cell may contain one or more exogenous nucleic acid molecules encoding the γ and δ chains of the TCR. In these methods, one or more exogenous nucleic acid molecules can be stably integrated into the genome of the reporter cell.

[0044] In these methods, the target antigen can be a cancer-related antigen or a tumor-related antigen. In these methods, the target antigen can be selected from the group consisting of CD1d, CD40, CD123, PSMA, nectin-4, and EGFR. In these methods, the target antigen can be immobilized on a physical structure such as beads, tubes, or microtiter plates, or can be aggregated by multimerization and cross-linking techniques (e.g., chemical or multivalent proteins). In these methods, the cell population may contain target cells expressing the target antigen. In these methods, the target antigen can be displayed on the surface of the target cells.

[0045] In these methods, the target antigen-binding site can specifically bind to a cancer-related antigen or a tumor-related antigen. In these methods, the target antigen-binding site can specifically bind to a target antigen selected from the group consisting of CD1d, CD40, CD123, PSMA, nectin-4, and EGFR. In these methods, γδ-TDMBC can include TDbAb.

[0046] In these methods, the reporter gene can include a nucleic acid molecule encoding a reporter protein operably linked to a promoter that responds to reporter cell activation, and optionally one or more additional response elements. In these methods, the promoter can be selected from any promoter known to respond to the activation of reporter cells. For example, in embodiments where the reporter cells are reporter T cells, the promoter can be any promoter that responds to T cell activation. In these methods, the promoter may be selected from the group consisting of the nuclear factor of activated T cells (NFAT) gene promoter, the interleukin-2 (IL-2) gene promoter, the activator protein 1 (AP-1) gene promoter, the nuclear factor kappa B subunit (NFκB) gene promoter, the forkhead box subfamily O (FOXO) gene promoter, the signal transducer and activator of transcription 3 (STAT3) gene promoter, the signal transducer and activator of transcription 5 (STAT5) gene promoter, and the interferon regulatory factor (IRF) gene promoter.

[0047] In these methods, the reporter gene can include a nucleotide sequence encoding a reporter protein under the control of a promoter and a nucleic acid molecule operably linked to one or more response elements that respond to reporter cell activation. In such methods, the promoter controlling the nucleotide sequence encoding the reporter protein can be a minimal promoter. A "minimal promoter" refers to the minimal nucleotide sequence derived from a promoter that is necessary for the expression of the coding sequence under the control of the promoter. A commercially available minimal promoter is a synthetic promoter designed to provide minimal (i.e., no or very low level) transcription of a nucleic acid sequence operably linked to it in the absence of stimulatory signals such as enhancers. The use of minimal promoters is known in the art and is also disclosed in US2020 / 0182882 and US10690678, both of which are hereby incorporated by reference in their entirety. In these methods, the minimal promoter can be a thymidine kinase (TK) minimal promoter, a cytomegalovirus (CMV) minimal promoter, a simian virus (SV) 40 minimal promoter, or an elongation factor (1EF1α) minimal promoter.

[0048] In these methods, the response element can be any response element that causes the reporter gene to respond to the activation of the reporter cell when operably linked to the reporter gene. In these methods, one or more response elements can include a T cell activation response element. In these methods, one or more response elements can include an NFAT gene response element, an AP-1 gene response element, NF KIt may be selected from the group consisting of a B gene response element, a FOXO gene response element, a STAT3 gene response element, a STAT5 gene response element, or an IRF gene response element. In some embodiments, the response element may be arranged as a tandem repeat (e.g., any of about 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). In these methods, the response element(s) can be positioned 5' or 3' relative to the reporter gene. In these methods, the response element(s) can be located 5' from the site of the promoter. In these methods, the nucleic acid molecule containing the reporter gene may be stably integrated into the genome of the reporter cell. In these methods, the reporter cell does not express the target antigen.

[0049] In these methods, the reporter gene can encode a fluorescent protein, a luminescent protein, a chemiluminescent protein, or an enzyme. In some embodiments, the luminescent protein may be luciferase, and the luciferase may be firefly luciferase, Renilla luciferase, Gaussia luciferase, or NanoLuc luciferase.

[0050] The reporter cells of the present disclosure may include any additional changes that improve their suitability for use in the assays of the present disclosure. For example, in some methods of the present disclosure, it may be desirable for the reporter cells not to express a target antigen, or a protein that immunologically cross-reacts with the target protein. In these methods, the reporter cells of the present disclosure can be modified so that they do not express the target antigen, or a protein that cross-reacts with the target antigen. Thus, in these methods, the reporter cells of the present disclosure may not express the target antigen or a molecule that immunologically cross-reacts with the target antigen. In these methods, the reporter cells of the present disclosure may not express an antigen selected from the group consisting of CD1d, CD40, CD123, PSMA, nectin-4, and EGFR.

[0051] In these methods, when the cell population includes target cells expressing a target antigen, the ratio of reporter cells to target cells can be about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:50, or about 1:100.

[0052] In these methods, γδ-TDMBC can be at least about 1 picomolar (pM), about 5 pM, about 25 pM, about 50 pM, about 100 pM, about 250 pM, about 500 pM, 750 pM, about 1,000 pM, about 1,500 pM, about 2,000 pM, about 2,500 pM, about 5,000 pM, about 10,000 pM, about 15,000 pM, or about 20,000 pM, or can be within that concentration range.

[0053] In these methods, reporter gene expression can be detected after the cells are contacted with a T cell-dependent bispecific binding molecule for any time exceeding about 5 minutes, 15 minutes, 10 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 20 hours, or 24 hours. In these methods, the reporter gene or molecule can be detected between any of about 5 - 15 minutes, 15 - 20 minutes, 30 minutes - 1 hour, 1 hour - about 24 hours, about 1 hour - about 12 hours, about 1 hour - about 8 hours, about 1 hour - about 6 hours, about 1 hour - about 4 hours, about 1 hour - about 2 hours, about 4 hours - about 24 hours, about 4 hours - about 12 hours, about 4 hours - about 8 hours, about 8 hours - about 24 hours, about 8 hours - about 12 hours, about 16 hours - about 24 hours, about 16 hours - about 20 hours, or about 20 hours - about 24 hours after the cells are contacted with a T cell-dependent bispecific binding molecule.

[0054] In these methods, the γδ-TDbAb can include a first single-domain antibody and a second single-domain antibody, where the first single-domain antibody specifically binds to an epitope in the γδ TCR, and the second single-domain antibody specifically binds to a target antigen. In some embodiments, the first single-domain antibody specifically binds to the TCRγ chain or the TCRδ chain. In some embodiments, the first single-domain antibody specifically binds to the TCRδ chain. In some embodiments, the first single-domain antibody specifically binds to the TCRδ2 chain. In some embodiments, the first single-domain antibody may include or consist of a CDR1 that includes SEQ ID NO: 5 (where X1 is G or S), a CDR2 that includes or consists of SEQ ID NO: 2, and / or a CDR3 that includes or consists of SEQ ID NO: 3 (where X2 can be any amino acid and X3 is not R), and the first single-domain antibody specifically binds to a TCRδ chain such as the δ2 chain. In some embodiments, the first single-domain antibody may include or consist of an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to SEQ ID NO: 16, where the amino acid sequence includes a CDR1 that includes or consists of SEQ ID NO: 5 (where X1 is G or S), a CDR2 that includes or consists of SEQ ID NO: 2, and a CDR3 that includes or consists of SEQ ID NO: 3 (where X2 can be any amino acid and X3 is not R), and the first single-domain antibody specifically binds to a TCRδ chain, such as the δ2 chain. In some embodiments, the first single-domain antibody may include or consist of SEQ ID NO: 16, where X1 is S or G, X2 can be any amino acid, and X3 is not R.

[0055] In some embodiments, the second single-domain antibody may comprise or consist of a CDR1 comprising SEQ ID NO: 45, a CDR2 comprising SEQ ID NO: 46, and / or a CDR3 comprising SEQ ID NO: 47, and the second single-domain antibody specifically binds to CD1d. In some embodiments, the second single-domain antibody may comprise or consist of an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to SEQ ID NO: 48, and the amino acid sequence comprises a CDR1 comprising or consisting of SEQ ID NO: 45, a CDR2 comprising or consisting of SEQ ID NO: 46, and a CDR3 comprising or consisting of SEQ ID NO: 47, and the second single-domain antibody specifically binds to CD1d. In some embodiments, the second single-domain antibody may comprise or consist of SEQ ID NO: 48. In some embodiments, the second single-domain antibody may comprise or consist of a CDR1 comprising SEQ ID NO: 33, a CDR2 comprising SEQ ID NO: 34, and / or a CDR3 comprising SEQ ID NO: 35, and the second single-domain antibody specifically binds to EGFR. In some embodiments, the second single-domain antibody may comprise or consist of an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 97% identical, or at least 99% identical to SEQ ID NO: 36, and the amino acid sequence comprises a CDR1 comprising or consisting of SEQ ID NO: 33, a CDR2 comprising or consisting of SEQ ID NO: 34, and a CDR3 comprising or consisting of SEQ ID NO: 35, and the second single-domain antibody specifically binds to EGFR. In some embodiments, the second single-domain antibody may comprise or consist of SEQ ID NO: 36. In some embodiments, the second single-domain antibody may comprise or consist of a CDR1 comprising SEQ ID NO: 49, a CDR2 comprising SEQ ID NO: 50, and / or a CDR3 comprising SEQ ID NO: 51, and the second single-domain antibody specifically binds to CD40.In some embodiments, the second single-domain antibody may comprise or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 52, the amino acid sequence comprising or consisting of CDR1 comprising or consisting of SEQ ID NO: 49, CDR2 comprising or consisting of SEQ ID NO: 50, and CDR3 comprising or consisting of SEQ ID NO: 51, and the second single-domain antibody specifically binds to CD40. In some embodiments, the second single-domain antibody may comprise or consist of SEQ ID NO: 52. In some embodiments, the second single-domain antibody may comprise or consist of CDR1 comprising or consisting of SEQ ID NO: 41, CDR2 comprising or consisting of SEQ ID NO: 42, and / or CDR3 comprising or consisting of SEQ ID NO: 43, and the second single-domain antibody specifically binds to PSMA. In some embodiments, the second single-domain antibody may comprise or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 44, the amino acid sequence comprising or consisting of CDR1 comprising or consisting of SEQ ID NO: 41, CDR2 comprising or consisting of SEQ ID NO: 42, and CDR3 comprising or consisting of SEQ ID NO: 43, and the second single-domain antibody specifically binds to PSMA. In some embodiments, the second single-domain antibody may comprise or consist of SEQ ID NO: 44. In some embodiments, the second single-domain antibody may comprise or consist of CDR1 (where X4 is G or S) comprising or consisting of SEQ ID NO: 37, CDR2 (where X5 is A or T) comprising or consisting of SEQ ID NO: 38, and / or CDR3 (where X6 is Y or F) comprising or consisting of SEQ ID NO: 39, and the second single-domain antibody specifically binds to CD123.In some embodiments, the second single-domain antibody can comprise or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 40, the amino acid sequence comprising or consisting of CDR1 (where X4 is G or S) comprising or consisting of SEQ ID NO: 37, CDR2 (where X5 is A or T) comprising or consisting of SEQ ID NO: 38, and / or CDR3 (where X6 is Y or F) comprising or consisting of SEQ ID NO: 39, and the second single-domain antibody specifically binds to CD123. In some embodiments, the second single-domain antibody can comprise or consist of SEQ ID NO: 40. In some embodiments, the second single-domain antibody can comprise or consist of a CDR1 comprising any one of SEQ ID NOs: 53, 57, 61, 65, 69, 73, and 77, a CDR2 comprising any one of SEQ ID NOs: 54, 58, 62, 66, 70, 74, and 78, and / or a CDR3 comprising any one of SEQ ID NOs: 55, 59, 63, 67, 71, 75, and 79, and the second single-domain antibody specifically binds to nectin-4. In some embodiments, the second single-domain antibody can comprise or consist of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80, the amino acid sequence comprising a CDR1 comprising any one of SEQ ID NOs: 53, 57, 61, 65, 69, 73, and 77, a CDR2 comprising any one of SEQ ID NOs: 54, 58, 62, 66, 70, 74, and 78, and / or a CDR3 comprising or consisting of SEQ ID NOs: 55, 59, 63, 67, 71, 75, and 79, and the second single-domain antibody specifically binds to nectin-4. In some embodiments, the second single-domain antibody can comprise or consist of any one of SEQ ID NOs: 56, 60, 64, 68, 72, 76, and 80.

[0056] One aspect of the present disclosure is a reporter cell that expresses a γδ TCR and contains a reporter gene that responds to the activation of the reporter cell. In some aspects, the reporter cell is a reporter T cell. In some aspects, the reporter T cell is produced by modifying Jurkat cells or CTLL-2 T cells. In some aspects, the γδ TCR may include a Vγ9 chain and / or a Vδ2 chain. In some aspects, the γδ TCR may include a Vγ9Vδ2 TCR. In some aspects, the reporter gene may encode a reporter protein that can be a fluorescent protein, a luminescent protein, a chemiluminescent protein, and an enzyme. In some aspects, the luminescent protein may be luciferase, and the luciferase may be firefly luciferase, Renilla luciferase, Gaussia luciferase, or NanoLuc luciferase. In some aspects, the reporter gene may include a nucleic acid molecule that includes a promoter that responds to the activation of the reporter cell and a nucleotide sequence encoding a reporter protein operably linked to one or more response elements optionally. In some aspects, the promoter may respond to T cell activation. In some aspects, the promoter may be an NFAT promoter, an AP-1 promoter, an NF K κB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. In one aspect, the reporter gene may include a nucleotide sequence encoding a reporter protein under the control of a promoter and a nucleic acid molecule operably linked to one or more elements that respond to the activation of the reporter cell. In some aspects, the promoter controlling the nucleotide sequence encoding the reporter protein may be a minimal promoter. In some aspects, the minimal promoter may be a TK minimal promoter, a CMV minimal promoter, an SV40 minimal promoter, or an EF1α minimal promoter. In some aspects, the response element is an NFAT gene response element, an AP-1 gene response element, an NF KIt may be selected from the group consisting of a B gene response element, a FOXO gene response element, a STAT3 gene response element, a STAT5 gene response element, or an IRF gene response element. In some embodiments, the response element may be arranged as a tandem repeat (e.g., any of about 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). In some embodiments, the response element(s) may be positioned 5' or 3' relative to the reporter gene. In some embodiments, the response element(s) may be located 5' from the promoter site. In some embodiments, the nucleic acid molecule may be stably integrated into the genome of the reporter cell. In one embodiment, the reporter cell does not express the target antigen. In one embodiment, the modified T cell does not express CD1d, CD40, CD123, PSMA, nectin-4, or EGFR.

[0057] As described herein, T cells can be used to produce the reporter cells of the present disclosure, thereby producing reporter T cells. Since the cells used to produce the reporter T cells are T cells, it will be apparent to those skilled in the art that T cell receptors such as the T cell receptor having an alpha (α) chain and a (β) beta chain (i.e., αβ TCR) may already be expressed. In some embodiments, it may be undesirable to have T cells that express both types of receptors (i.e., αβ TCR and γδ TCR). Thus, in some embodiments, the reporter T cells, or the T cells used to produce the reporter T cells, can be engineered to exclude the expression of αβ TCR. Excluding the expression of αβ TCR can be achieved using any known method for silencing gene expression, including "knocking out" the genes encoding the TCRα chain protein and / or the TCRβ chain protein. Knocking out the alpha or beta chain gene can involve making a change, or a deletion or insertion mutation, at any position within the alpha chain locus or beta chain locus, or within the gene elements that control the expression of either or both loci. Methods for knocking out a gene can include, for example, inserting a nucleic acid into the gene, deleting all or part of the gene, or interrupting, deleting, or editing a control element such as a promoter. Methods for silencing genes within a cell are known in the art. Thus, in one embodiment, the reporter T cells of the present disclosure may not express a functional TCRα chain or a functional TCRβ chain. In one embodiment, the reporter T cells of the present disclosure may not express a functional TCRα chain and / or a functional TCRβ chain. In one embodiment, the reporter T cells of the present disclosure may contain a mutation within the endogenous alpha or beta chain locus, or within the gene elements that control the expression of either or both loci such that the reporter cells do not express a functional αβ TCR.

[0058] One aspect of the present disclosure is a reporter T cell that expresses a γδ TCR and includes a T cell activation-responsive reporter gene, and the reporter T cell does not express a functional αβ TCR. In some aspects, the reporter T cell is a modified Jurkat cell or a modified CTLL-2 T cell. In some aspects, the γδ TCR may include a Vγ9 chain and / or a Vδ2 chain. In some aspects, the γδ TCR may include a Vγ9Vδ2 TCR. In some aspects, the reporter gene can be any gene that encodes a reporter protein, the presence or activity of which is detectable. In some aspects, the reporter gene can encode a reporter protein that can be selected from the group consisting of a fluorescent protein, a luminescent protein, a chemiluminescent protein, and an enzyme. In some aspects, the luminescent protein may be luciferase, and the luciferase may be firefly luciferase, Renilla luciferase, Gaussia luciferase, or NanoLuc luciferase. In one aspect, the reporter gene can include a nucleic acid molecule that encodes a reporter protein operably linked to a T cell activation-responsive promoter and optionally one or more T cell activation-responsive elements. In some aspects, the T cell activation-responsive promoter may be selected from promoters known in the art to respond to T cell activation. In some aspects, the promoter is an NFAT promoter, an AP-1 promoter, NF KIt may be a B promoter, FOXO promoter, STAT3 promoter, STAT5 promoter, or IRF promoter. In one aspect, the reporter gene may comprise a nucleic acid molecule that includes a nucleotide sequence encoding a reporter protein under the control of a promoter and is operably linked to one or more T cell activation response elements. In some aspects, the promoter controlling the nucleotide sequence encoding the reporter protein may be a minimal promoter. In some aspects, the minimal promoter may be a TK minimal promoter, CMV minimal promoter, SV40 minimal promoter, or 1EF1α minimal promoter. In some aspects, one or more T cell activation response elements may include a T cell activation response element selected from the group consisting of an NFAT gene response element, an AP-1 gene response element, an NF K B gene response element, FOXO gene response element, STAT3 gene response element, STAT5 gene response element, or IRF gene response element. In some aspects, one or more T cell activation response elements may be arranged as tandem repeats (e.g., any of about 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). In some aspects, one or more T cell activation response elements may be positioned 5' or 3' relative to the reporter gene. In some aspects, one or more T cell activation response elements may be located 5' from the promoter site. In some aspects, the nucleic acid molecule may be stably integrated into the T cell genome. In one aspect, the modified T cell may include one or more knockout mutations within its α-chain locus, or β-chain locus, or within the gene elements controlling the expression of either or both loci. In one aspect, the modified T cell lacks a functional α-chain gene and / or β-chain gene. In one aspect, the modified T cell does not express a target antigen expressed by a target cell in an assay in which the T cell is intended to be used. In one aspect, the modified T cell does not express CD1d, CD40, CD123, PSMA, nectin-4, or EGFR.

[0059] In some embodiments, the cells used to produce the reporter cells of the present disclosure can be engineered to express a γδ TCR. In some embodiments, the cells can be engineered to express a γδ TCR by introducing into the cells one or more exogenous nucleic acid molecules encoding the γ-chain protein and / or δ-chain protein of the TCR. The one or more exogenous nucleic acids may or may not be stably inserted into the genome of the cell. Expression of the γ-chain and / or δ-chain protein of the TCR can be placed under the control of any endogenous, exogenous, or heterologous promoter capable of driving gene expression in the cell. Examples of suitable promoters for driving expression of the encoded γ-chain gene and / or δ-chain gene include, but are not limited to, the thymidine kinase (TK) promoter, the cytomegalovirus (CMV) promoter, and the simian virus (SV) 40 promoter.

[0060] One aspect of the present disclosure is a reporter cell that expresses a γδ TCR and contains a reporter gene that responds to activation of the reporter cell, and the reporter cell contains one or more exogenous nucleic acid molecules encoding TCRγ and δ chain proteins. One aspect of the present disclosure is a reporter T cell that expresses a γδ TCR and contains a T cell activation-responsive reporter gene, the reporter T cell contains one or more exogenous nucleic acid molecules encoding TCRγ and δ chain proteins, and the reporter T cell does not express a functional αβ TCR. In some aspects, the reporter T cell is a modified Jurkat cell or a modified CTLL-2 T cell. In some aspects, the γδ TCR may include a Vγ9 chain and / or a Vδ2 chain. In some aspects, the γδ TCR may include a Vγ9Vδ2 TCR. In some aspects, the reporter gene can be any gene encoding a reporter protein, the presence or activity of which is detectable. In some aspects, the reporter protein can be a fluorescent protein, a luminescent protein, a chemiluminescent protein, or an enzyme such as alkaline phosphatase or beta-galactosidase. In some aspects, the luminescent protein may be luciferase, and the luciferase may be firefly luciferase, Renilla luciferase, Gaussia luciferase, or NanoLuc luciferase. In some aspects, the reporter gene includes a nucleic acid molecule containing a promoter that responds to activation of the reporter cell and, optionally, a nucleotide sequence encoding a reporter protein operably linked to one or more response elements. In some aspects, the promoter may be selected from promoters known in the art to respond to activation of the reporter cell. In some aspects, the promoter can be an NFAT promoter, an AP-1 promoter, an NFκ3 promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. In one aspect, the reporter gene includes a nucleotide sequence encoding a reporter protein under the control of a promoter and a nucleic acid molecule operably linked to one or more response elements.In some embodiments, the promoter controlling the nucleotide sequence encoding the reporter protein can be a minimal promoter. In some embodiments, the minimal promoter can be a TK minimal promoter, a CMV minimal promoter, an SV40 minimal promoter, or an EF1α minimal promoter. In some embodiments, one or more T cell activation response elements can include a T cell activation response element selected from the group consisting of an NFAT gene response element, an AP-1 gene response element, an NF. K κ B gene response element, a FOXO gene response element, a STAT3 gene response element, a STAT5 gene response element, or an IRF gene response element. In some embodiments, one or more response elements may be arranged as tandem repeats (e.g., any of about 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). In some embodiments, one or more response elements may be positioned 5' or 3' relative to the reporter gene. In some embodiments, one or more response elements may be located 5' from the promoter site. In some embodiments, the nucleic acid molecule can be stably integrated into the genome of the reporter cell. In some embodiments, one or more exogenous nucleic acid molecules can be stably integrated into the T cell genome. In one embodiment, the reporter cell can contain one or more knockout mutations within its α-chain locus, or β-chain locus, or within the gene elements controlling the expression of either or both loci.

[0061] In one aspect, the reporter cell lacks a functional alpha chain and / or beta chain gene. In one aspect, the reporter cell does not express a target antigen expressed by a target cell in an assay in which the reporter is intended to be used. In one aspect, the modified T cell does not express CD1d, CD40, CD123, PSMA, nectin-4, or EGFR. In some embodiments, the reporter cells of the present disclosure may include one or more modifications that reduce the expression of a target antigen (or a protein that immunologically cross-reacts with the target protein). Thus, in these methods, the reporter cells of the present disclosure may not express a target antigen or a molecule that immunologically cross-reacts with the target antigen. For example, the reporter cells of the present disclosure may not express an antigen selected from the group consisting of CD1d, CD40, CD123, PSMA, nectin-4, and EGFR. Such embodiments are useful for reducing background signal resulting from engagement of the target antigen binding site of the γδ-TDbAb by the target antigen expressed on the reporter cell.

[0062] In some embodiments, the reporter cells of the present disclosure comprise one or more genomic DNA modifications (e.g., gene knockouts) that eliminate or substantially reduce the expression of a target antigen. Systems for generating gene knockouts are known in the art and include, for example, Cre-Lox or FLP-FRT recombination systems, TALENs, zinc finger nucleases, and endonucleases. In some embodiments, an endonuclease is used to eliminate the expression of a target antigen. Exemplary endonucleases are known in the art and include, for example, Cas endonucleases for use with guide RNAs in CRISPR-Cas systems. In some embodiments, the reporter cells comprise a Cas endonuclease and a guide RNA that binds to a target sequence within the target antigen gene (e.g., a target sequence within the CD1d gene, PSMA gene, CD40 gene, EGFR gene, CD123 gene, and / or nectin 4 gene). In some embodiments, the reporter cells of the present disclosure may comprise oligonucleotides such as siRNA or shRNA that inhibit the translation of mRNA encoding the target antigen.

[0063] In some embodiments, the reporter cells of the present disclosure stably express a Cas endonuclease (e.g., Cas9 endonuclease). In such embodiments, the expression of the target antigen can be eliminated by selection of an appropriate guide RNA (gRNA) and introduction of the gRNA into the reporter cells. The reporter cells can then be assayed by means known in the art (e.g., flow cytometry, Western blot, etc.) and selected for successful knockout of the target antigen. The selected knockout reporter cells can be expanded for use according to the methods described herein.

[0064] One aspect of the present disclosure is a reporter T cell that expresses a γδ TCR and contains a reporter gene that responds to T cell activation. The reporter T cell contains one or more exogenous nucleic acid molecules encoding TCR γ and δ chain proteins, the reporter T cell does not express a functional αβ TCR, and the reporter T cell does not express a target antigen. In some aspects, the reporter T cell is a modified Jurkat cell or a modified CTLL-2 T cell. In some aspects, the γδ TCR may include a Vγ9 chain and / or a Vδ2 chain. In some aspects, the γδ TCR may include a Vγ9Vδ2 TCR. In some aspects, the reporter gene can be any gene encoding a reporter protein, the presence or activity of which is detectable. In some aspects, the reporter protein can be a fluorescent protein, a luminescent protein, a chemiluminescent protein, or an enzyme such as alkaline phosphatase or beta-galactosidase. In some aspects, the luminescent protein may be luciferase, and the luciferase may be firefly luciferase, Renilla luciferase, Gaussia luciferase, or NanoLuc luciferase. In one aspect, the reporter gene includes a nucleic acid molecule containing a nucleotide sequence encoding a reporter protein operably linked to a T cell activation-responsive promoter and optionally one or more T cell activation-responsive elements. In some aspects, the T cell activation-responsive promoter can be selected from promoters known in the art to respond to T cell activation. In some aspects, the promoter can be an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, or an IRF promoter. In one aspect, the reporter gene includes a nucleic acid molecule containing a nucleotide sequence encoding a reporter protein under the control of a promoter and operably linked to one or more T cell activation-responsive elements. In some aspects, the promoter can be a minimal promoter. In some aspects, the minimal promoter can be a TK minimal promoter, a CMV minimal promoter, an SV40 minimal promoter, or an EF1α minimal promoter.In some embodiments, the T cell activation response element comprises a T cell activation response element selected from the group consisting of an NFAT gene response element, an AP-1 gene response element, an NFκB gene response element, a FOXO gene response element, a STAT3 gene response element, a STAT5 gene response element, or an IRF gene response element. In some embodiments, one or more T cell activation response elements may be arranged as tandem repeats (e.g., any of about 2, 3, 4, 5, 6, 7, 8, or more tandem repeats). In some embodiments, one or more T cell activation response elements may be positioned 5' or 3' relative to the reporter gene. In some embodiments, one or more T cell activation response elements may be located 5' from the promoter site. In some embodiments, the nucleic acid molecule may be stably integrated into the T cell genome. In some embodiments, one or more exogenous nucleic acid molecules may be stably integrated into the T cell genome. In one embodiment, the modified T cell may comprise one or more knockout mutations within its alpha chain locus, or beta chain locus, or within the gene elements that control the expression of either or both loci. In one embodiment, the modified T cell lacks a functional alpha chain gene and / or beta chain gene. In one embodiment, the modified T cell does not express a target antigen expressed by the target cell, or a molecule that immunologically cross-reacts with the target antigen, in an assay in which the T cell is intended to be used. In one embodiment, the modified T cell does not express CD1d, CD40, CD123, PSMA, nectin-4, or EGFR. K

[0065] In some embodiments, knockout or knockdown of the target gene removes the background signal from the signal. "Background signal" or "background noise" is the signal detected when the recipient cell is incubated with γδ-TDMBC in the absence of the target cell.

[0066]

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

[0067] The specification of the document uses examples to disclose the present invention, including the best mode, and to enable the practice of the present invention, including the making and using of any device or system by any person skilled in the art and the carrying out of any incorporated method. The patentable scope of the present invention is defined by the claims and may include other examples contemplated by persons skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal words of the claims or if they include structural elements that are equivalent without substantial difference from the literal words of the claims.

Examples

[0068] Example 1. Construction of Reporter Cells Generation of J.RT3-T3.5 Vγ9Vδ2-TCR Cells Parental J.RT3-T3.5 cells were co-transfected by electroporation using plasmids encoding CD3 (pExoIN3-CD3 containing zeocin resistance marker (SEQ ID NO: 137)) and Vγ9Vδ2-TCR receptor (pExoIN2 Vγ9Vδ2-TCR containing puromycin resistance marker (SEQ ID NO: 140)). At 24 hours post-transfection, cells were stained for Vγ9Vδ2-TCR expression and Vγ9Vδ2-TCR positive cells were isolated by FACS sorting. Twenty-four hours after recovery, the sorted cell population was seeded onto soft agar and subjected to antibiotic selection. Growing cells were transferred to standard culture conditions and expanded, and then the stable cells were stained with specific antibodies against TCR-Vδ2 chain (TCR Vδ2-FITC, clone IMMU 389, #IM1464, Beckman Coulter), TCR-Vγ9 chain (PE anti-human TCR Vγ9, mouse IgG1, kappa, clone: B3, #BLD-331308, Biozol), and CD3 (APC anti-human CD3, mouse IgG2a, kappa, clone: OKT3, #BLD-317318, Biozol), and target expression was analyzed by flow cytometry, resulting in the isolation of CD3+Vγ9Vδ2TCR-expressing cells. Parental cells were used as a control. Stable CD3+Vγ9Vδ2-TCR-expressing cells were enriched by flow cytometry-based cell sorting to remove any non-expressing cells and enrich the fluorescent cell population for high-expressing cells.

[0069] Generation of a recombinant J.RT3-T3.5 Vg9Vd2-TCR cell pool for luciferase The concentrated CD3+Vγ9Vδ2 TCR-expressing cells were transfected by electroporation using a plasmid containing a luciferase coding sequence under the control of an NFAT response element, and a hygromycin resistance gene (pGL4.30[luc2P / NFAT-RE / Hygro] vector (Promega, catalog number E8481)). At 24 hours after transfection, the transiently transfected cells were stained for Vγ9Vδ2-TCR expression, and Vγ9Vδ2-TCR positive cells were separated by FACS sorting to enhance the possibility of co-expression of Vγ9Vδ2-TCR and NFAT-RE-Luc. For antibiotic selection, the sorted cells were seeded on soft agar and subjected to three different hygromycin concentrations (150 μg / mL, 300 μg / mL, and 450 μg / mL hygromycin). In addition, conventional pool generation was initiated by applying 300 μg / mL hygromycin for selection. The growing cells were tested for luciferase activity upon stimulation (4 hours) with an ionomycin / PMA mixture (50 ng / mL PMA and 500 ng / mL ionomycin). Parental cells were used as a negative control, and a stable Jurkat-NFAT control cell line was used as a positive control. A significant luminescence signal could be detected 4 hours after stimulation.

[0070] Generation of the clone J.RT3-T3.5-Vγ9Vδ2 NFAT-RE Luc cell line For the generation of a clone J.RT3-T3.5 cell line stably expressing Vγ9Vδ2-TCR and NFAT-RE_Luc, single cell cloning was performed by limiting dilution with cells from a stable J.RT3-T3.5-Vγ9Vδ2_NFAT-RE_Luc cell pool. The cells were deposited singly into the wells of a 96-well plate. The growing clones were transferred to a higher culture format, expanded, and cryopreserved. After thawing and expansion, 24 clones were analyzed for luciferase activity and Vγ9Vδ2-TCR expression, and the highest expressing clones were preserved.

[0071] Example 2. Protocol for reporter cell assay This example outlines a protocol used to test bispecific binding compounds using an assay that includes Jurkat cell (J.RT3-T3.5)-based reporter cells (also known as reporter cells) expressing Vγ9Vδ2-TCR generated according to Example 1, and PSMA-expressing target cells (LNCaP) (referred to herein as target cells).

[0072] 1×10 5 Reporter and 5×10 4 50 μL of RPMI complete medium (RPMI 1640, 10% fetal bovine serum (FBS), 1% pen / strep [penicillin / streptomycin (10,000 U / mL)]) containing reporter and target cells (at a ratio of 2:1) was added to the wells of a microtiter plate. 50 μL of bispecific binding compounds (having SEQ ID NOs: 5, 2, 14, and 16 (X2 = Y), and SEQ ID NOs: 41-44) were added to each well at the desired concentration, and the plate was incubated at 37 °C and 5% CO2 for 18 hours. After incubation, 100 μL of ONE-Glo™ Luciferase Assay Reagent (Promega) was added, and the plate was incubated in the dark at room temperature (RT) for 15 minutes. Luminescence was then measured using a SpektraMax® M3 plate reader.

[0073] Example 3. Assay Validity Criteria To determine the potency of the bispecific compounds of Example 2, the luminescence values obtained from the plate reader were analyzed in SoftMax Pro software version 7.1 using 4PL fit and subtraction of the "group blank" value. To confirm the validity of the assay, the parameters listed in the following table were evaluated.

[0074]

Table 2

[0075] Example 4. Relative Potency Evaluation When the assay was considered valid, additional relative potency analysis was performed. To evaluate whether a constrained fit could be applied, dose-response curves were analyzed for their "equivalence" or parallelism using an F-test. In this test, a value of ≥ 0.8 was considered acceptable. If the results were valid, a constrained curve fit / global fit (PLA) was performed using SoftMax Pro version 7.1 software. This allowed for the acceptance of values for relative potency (0.8 (80%) to 1.2 (120%)) compared to the reference control set as the standard, i.e., the bispecific compound of Example 2, batch 54-PS-PBG, EC 50 compared to which. The parallelism of the dose-response curves compared to the control was the sample acceptance criterion. If the curves were not parallel, the run was not accepted.

[0076] Example 5. Stability Study This example demonstrates the use of the reporter cell assay of the present disclosure to measure the stability of TDbAb stored under various conditions.

[0077] TDbAb specific for prostate-specific membrane antigen (PSMA) and TCR delta (δ) 2 chain was stored at 5 °C or 20 °C for 1 month, 3 months or 6 months, or at 40 °C for 2 weeks or 1 month. After storage, the TDbAb was tested for its ability to activate T cells in the presence of PSMA-expressing target cells using the protocol described in Example 2. The results are shown in Figures 2 to 4. Figure 2 shows the estimated relative potency of TDbAb after storage at 5 °C for various periods. Figure 3 shows the estimated relative potency of TDbAb after storage at 20 °C for various periods. Figure 4 shows the estimated relative potency of TDbAb when stored at 40 °C over various periods.

[0078] Example 6. Detection of Immobilized Antigen Wells of polystyrene plates were coated with either CD1d or PSMA at various concentrations and the plates were incubated overnight. The next day, the immobilized antigen was detected using a TDbAb specific for each antigen. Binding of the TDbAb to the immobilized antigen was detected by addition of a reporter cell line and detection of the luciferase signal. The results are shown in Figure 5A (CD1d) and Figure 5B (PSMA).

[0079] Example 7. Generation of knockout reporter cell lines As described herein, expression of the target antigen on the reporter cell line can be determined by incubating the reporter cells with γδ-TDMBC in the absence of target cells to obtain a background luciferase signal. See, for example, Figure 6. As shown, co-culture of reporter cells with target cells in the presence of γδ-TDMBC results in a luciferase signal (circles), indicating activation of the reporter cells by γδ-TDMBC. However, incubation of reporter cells with γδ-TDMBC in the absence of target cells (triangles) results in a background luciferase signal. Subtracting the background signal from the signal generated in the presence of target cells allows determination of the on-target activation of the reporter cells (squares).

[0080] Knockout reporter cells were generated to eliminate expression of the target antigen on the reporter cells and reduce the background signal due to γδ-TDMBC binding to the target antigen on the reporter cells. Figure 7 shows successful knockout of the CD1d target antigen in the reporter cells. Furthermore, as shown in Figure 8, elimination of CD1d in the reporter cells also eliminated the background signal seen in the absence of target cells.

Claims

1. A method for detecting γδ T cell receptor (γδ TCR)-mediated reporter cell activation by a γδ T cell receptor (TCR)-dependent multispecific binding compound (γδ-TDMBC), wherein the γδ-TDMBC comprises a target antigen binding moiety and a γδ TCR binding moiety, and the method comprises: a) contacting the γδ-TDMBC with i) a population of cells comprising the reporter cells expressing γδ TCR and comprising a reporter gene responsive to activation of the reporter cells, and ii) a target antigen; b) detecting the expression of the reporter gene, wherein the expression of the reporter gene indicates γδ TCR-mediated activation of the reporter cells.

2. A method for determining the relative potency of a γδ-TDMBC comprising a γδ-TCR binding moiety and a target antigen binding moiety, the method comprising: a) contacting a known concentration of the γδ-TDMBC with i) a population of cells comprising the reporter cells expressing γδ TCR and comprising a reporter gene responsive to activation of the reporter cells, and ii) a target antigen; b) comparing the level of reporter gene expression resulting from a) with the level of reporter gene expression resulting from contacting the reporter cells of i) and the target antigen of ii) with a known concentration of a reference γδ TCR-dependent compound that binds to the target antigen and the γδ TCR, thereby obtaining a measure of the relative potency of the γδ-TDMBC.

3. The method of claim 2, wherein step b) comprises correlating the expression of the reporter gene as a function of the γδ-TDMBC with a standard curve generated by contacting the population of reporter cells and the antigen with different concentrations of a reference γδ-TDMBC.

4. A method for detecting the presence of a γδ-TDMBC comprising a γδ-TCR binding moiety and a target antigen binding moiety in a composition, the method comprising contacting the composition with a) the reporter cells expressing γδ TCR and comprising a reporter gene responsive to activation of the reporter cells, and b) a population of cells comprising a target antigen, wherein the expression of the reporter gene indicates the presence of the γδ-TDMBC in the composition.

5. A method for quantifying the amount of a γδ-TDMBC comprising a γδ-TCR binding moiety and a target antigen binding moiety in a sample, the method comprising: a) contacting the sample with i) A population of cells comprising said reporter cells expressing a γδ TCR and containing a reporter gene responsive to activation of the reporter cells, and ii) Contacting with a target antigen, b) Correlating the level of expression of said reporter gene as a function of the γδ-TDMBC concentration with a standard curve generated by contacting the population of reporter cells and said target antigen with said γδ-TDMBC at different known concentrations, thereby quantifying said γδ-TDMBC, said method comprising.

6. A method for determining the specificity of reporter cell activation by a γδ-TDMBC comprising a target antigen-binding portion and a γδ TCR-binding portion, i) Contacting said γδ-TDMBC with a) a population of cells comprising said reporter cells expressing a γδ TCR and containing a reporter gene responsive to activation of the reporter cells, and b) a target antigen, ii) Contacting said γδ-TDMBC with a) a population of cells comprising the reporter cells of i) in the absence of said antigen, iii) Comparing the expression of said reporter gene in i) with the expression of said reporter gene in ii), wherein the ratio of the expression of said reporter gene in i) to the expression of said reporter gene in ii) indicates the specificity of said γδ-TDMBC for said target antigen, said comparing.

7. The method according to any one of claims 1 to 6, wherein said reporter cells are reporter T cells.

8. The method according to claim 7, wherein said reporter T cells are CD3+.

9. The method according to any one of claims 1 to 8, wherein said γδ TCR expressed by said reporter cells is a γ9δ2 TCR.

10. The method according to any one of claims 1 to 9, wherein said reporter cells contain one or more exogenous nucleic acid molecules encoding a TCR γ chain and / or a TCR δ chain.

11. The method according to claim 10, wherein said one or more exogenous nucleic acid molecules are stably integrated into the genome of said reporter cells.

12. The method according to any one of claims 1 to 11, wherein said reporter gene comprises a nucleotide sequence encoding a reporter protein operably linked to a promoter responsive to activation of said reporter cells.

13. The method according to claim 12, wherein the promoter is responsive to T cell activation.

14. The promoter is selected from the group consisting of an NFAT promoter, an AP-1 promoter, an NF Ｋ B promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, and an IRF promoter, the method according to claim 12 or 13.

15. The method according to any one of claims 12 to 14, wherein the reporter gene comprises one or more response elements operably linked to the promoter, and optionally, one or more reporter cell activation response elements are T cell activation response element(s).

16. The method according to any one of claims 1 to 11, wherein the reporter gene comprises a nucleic acid molecule encoding a reporter protein operably linked to a promoter responsive to activation of the reporter cell, and one or more response elements operably linked to the promoter.

17. The method according to claim 16, wherein the promoter is a minimal promoter.

18. The method according to claim 16 or 17, wherein the promoter is selected from the group consisting of a TK minimal promoter, a CMV minimal promoter, an SV40 minimal promoter, and an EF1α minimal promoter.

19. The method according to any one of claims 15 to 18, wherein the one or more reporter cell activation response elements are T cell activation response element(s).

20. The one or more reporter cell activation response elements are NFAT gene response elements, AP-1 gene response elements, NF Ｋ B gene response elements, FOXO gene response elements, STAT3 gene response elements, STAT5 gene response elements, and IRF gene response elements, and the method according to any one of claims 15 to 19, which is selected from the group consisting of.

21. The method according to any one of claims 1 to 20, wherein the reporter gene encodes a reporter protein.

22. The method according to claim 21, wherein the reporter protein is a fluorescent protein, a luminescent protein, a chemiluminescent protein, or an enzyme.

23. The method according to any one of claims 1 to 22, wherein the target antigen is immobilized on a physical structure.

24. The method according to any one of claims 1 to 22, wherein the target antigen is expressed on the surface of a target cell.

25. The method according to claim 24, wherein the reporter cells and the target cells are incubated together at a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:50, or about 1:

100.

26. The method according to any one of claims 1 to 25, wherein the target antigen is a cancer-related antigen or a tumor-related antigen.

27. The method according to any one of claims 1 to 26, wherein the target antigen is selected from the group consisting of nectin-4, PSMA, CD1d, CD40, and CD123.

28. The method according to any one of claims 1 to 27, wherein the γδTCR-binding portion of the γδ-TDMBC specifically binds to the γ chain of the TCR.

29. The method according to any one of claims 1 to 29, wherein the γδTCR-binding portion of the γδ-TDMBC specifically binds to the δ chain of the TCR.

30. The method according to any one of claims 1 to 29, wherein the target antigen-binding portion of the γδ-TDMBC specifically binds to nectin-4, PSMA, CD1d, CD40, or CD123.

31. The method according to any one of claims 1 to 30, wherein the γδ-TDMBC is a bispecific antibody.

32. The γδ-TDMBC comprises a CDR1 (X １ is G or S), a CDR2 comprising or consisting of SEQ ID NO: 2, and / or a CDR3 (X ２ can be any amino acid, X ３ is not R) comprising a first single-chain domain, the method according to claim 31.

33. The first single domain antibody can comprise or consist of SEQ ID NO: 16, wherein X １ is S or G, X ２ can be any amino acid, X ３ is not R, the method according to claim 32.

34. The γδ-TDMBC is a. CDR1 comprising or consisting of SEQ ID NO: 45, CDR2 comprising or consisting of SEQ ID NO: 46, and / or CDR3 comprising or consisting of SEQ ID NO: 47, b. CDR1 comprising or consisting of SEQ ID NO: 49, CDR2 comprising or consisting of SEQ ID NO: 50, and / or CDR3 comprising or consisting of SEQ ID NO: 51, c. CDR1 comprising or consisting of SEQ ID NO: 41, CDR2 comprising or consisting of SEQ ID NO: 42, and / or CDR3 comprising or consisting of SEQ ID NO: 43, d. CDR1 that includes or consists of SEQ ID NO: 37 (where ４ X is G or S), CDR2 that includes or consists of SEQ ID NO: 38 (where ５ X is A or T), and / or CDR3 that includes or consists of SEQ ID NO: 39 (where ６ X is Y or F). e. CDR1 comprising or consisting of SEQ ID NO: 53, CDR2 comprising or consisting of SEQ ID NO: 54, and / or CDR3 comprising or consisting of SEQ ID NO: 55, f. CDR1 comprising or consisting of SEQ ID NO: 57, CDR2 comprising or consisting of SEQ ID NO: 58, and / or CDR3 comprising or consisting of SEQ ID NO: 59, g. CDR1 comprising or consisting of SEQ ID NO: 61, CDR2 comprising or consisting of SEQ ID NO: 62, and / or CDR3 comprising or consisting of SEQ ID NO: 63, h. CDR1 comprising or consisting of SEQ ID NO: 65, CDR2 comprising or consisting of SEQ ID NO: 66, and / or CDR3 comprising or consisting of SEQ ID NO:

67. i. A CDR1 comprising or consisting of SEQ ID NO: 69, a CDR2 comprising or consisting of SEQ ID NO: 70, and / or a CDR3 comprising or consisting of SEQ ID NO: 71 j. A CDR1 comprising or consisting of SEQ ID NO: 73, a CDR2 comprising or consisting of SEQ ID NO: 74, and / or a CDR3 comprising or consisting of SEQ ID NO: 75, or k. A method according to any one of claims 31 to 33, comprising a second single-chain domain comprising a CDR1 comprising or consisting of SEQ ID NO: 77, a CDR2 comprising or consisting of SEQ ID NO: 78, and / or a CDR3 comprising or consisting of SEQ ID NO: 79

35. The method according to claim 34, wherein the second single-domain antibody comprises or consists of SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 44, SEQ ID NO: 40, SEQ ID NO: 56, SEQ ID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, or SEQ ID NO: 80

36. The method according to any one of claims 1 to 35, wherein the reporter cell does not express a functional TCRα chain and / or a functional TCRβ chain

37. The method according to any one of claims 1 to 36, wherein the reporter cell comprises one or more knockout mutations within the TCRα chain locus and / or the TCRβ chain locus, or within a gene element controlling the expression of either or both loci

38. The method according to any one of claims 1 to 37, wherein the reporter cell does not express the target antigen

39. A reporter cell expressing a γ9δ2 T cell receptor (γδ TCR) and comprising a reporter gene responsive to activation of the reporter cell

40. The reporter cell according to claim 39, wherein the reporter cell is a reporter T cell

41. The reporter cell according to claim 39 or 40, wherein the reporter cell is CD3+

42. The reporter cell according to any one of claims 39 to 41, wherein the T cell is a Jurkat cell or a CTLL-2 cell

43. The reporter cell according to any one of claims 39 to 42, wherein the reporter cell comprises one or more exogenous nucleic acid molecules encoding the TCRγ chain and / or the TCRδ chain

44. The reporter gene comprises a nucleic acid molecule comprising a nucleotide sequence encoding a reporter protein operably linked to a promoter responsive to activation of the reporter cell, and optionally, the reporter gene comprises one or more reporter cell activation response elements operably linked to the promoter. The reporter cell according to any one of claims 39 to 43.

45. The reporter cell according to claim 44, wherein the promoter is responsive to T cell activation.

46. The promoter is selected from the group consisting of an NFAT promoter, an AP-1 promoter, an NF Ｋ B promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter, and an IRF promoter, the reporter cell according to claim 44 or 45.

47. The reporter cell according to any one of claims 39 to 43, wherein the reporter gene comprises a nucleic acid molecule comprising a nucleotide sequence encoding a reporter protein operably linked to a promoter.

48. The reporter cell according to claim 47, wherein the promoter is a minimal promoter.

49. The reporter cell according to claim 47 or 48, wherein the promoter is selected from the group consisting of a TK minimal promoter, a CMV minimal promoter, an SV40 minimal promoter, and an EF1α minimal promoter.

50. The reporter cell according to any one of claims 44 to 49, wherein the reporter gene comprises one or more reporter cell activation response elements operably linked to the promoter.

51. The reporter cell according to claim 50, wherein the one or more reporter cell activation response elements are T cell activation response element(s).

52. The reporter cell according to claim 50 or 51, wherein the one or more reporter cell activation response elements are selected from the group consisting of an NFAT gene response element, an AP-1 gene response element, an NFκB gene response element, a FOXO gene response element, a STAT3 gene response element, a STAT5 gene response element, and an IRF gene response element.

53. The reporter cell according to any one of claims 50 to 52, wherein the reporter gene comprises at least two reporter cell activation response elements, and the at least two reporter cell activation response elements are arranged as tandem repeats.

54. The reporter cell activation response element, or tandem repeats thereof, is located 5' of the nucleic acid sequence encoding the reporter protein, the reporter cell according to claim 53.

55. The reporter cell activation response element or tandem repeat thereof is located 5' of the promoter, the reporter cell according to any one of claims 53 or 54.

56. The reporter gene encodes a reporter protein, the reporter cell according to any one of claims 39 to 55.

57. The reporter protein is a fluorescent protein, a luminescent protein, a chemiluminescent protein, or an enzyme, the reporter cell according to claim 56.

58. The reporter cell does not express a functional TCR α chain and / or a functional TCR β chain, the method according to any one of claims 39 to 57.

59. The reporter cell contains one or more knockout mutations within the TCR α chain locus and / or the TCR β chain locus, or within a gene element controlling the expression of either or both loci, the reporter cell according to claim 58.

60. The reporter cell does not express the target antigen, the reporter cell according to any one of claims 39 to 59.

61. A kit comprising the reporter cell according to any one of claims 39 to 60.

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