EP4735481A2

EP4735481A2 - Trbc targeting antibody-drug conjugates

Info

Publication number: EP4735481A2
Application number: EP24833073.0A
Authority: EP
Inventors: Bert Vogelstein; Kenneth W. Kinzler; Nickolas Papadopoulos; Shibin Zhou; Suman PAUL; Tushar NICHAKAWADE; Jiaxin Ge
Original assignee: Johns Hopkins University
Current assignee: Johns Hopkins University
Priority date: 2023-06-28
Filing date: 2024-06-28
Publication date: 2026-05-06
Also published as: WO2025007013A3; WO2025007013A2; CN121443641A; AU2024305993A1

Abstract

Provided herein are antibody-drug conjugates comprising (a) an antibody or antigen-binding fragment thereof that specifically binds to a T cell receptor β chain constant region (TRBC) polypeptide; and (b) a therapeutic agent conjugated to the antibody or antigen-binding fragment thereof.

Description

TRBC TARGETING ANTIBODY-DRUG CONJUGATES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority’ to U.S. Provisional Patent Application No. 63/523,813, filed on June 28, 2023, which is incorporated herein by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers CA006973 and CA270403, awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “44807-0458WOl_ST26_SL.XML.” The XML file, created on June 26, 2024, is 57,154 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

Described herein are antibody-drug conjugates, pharmaceutical compositions, and methods for targeting TRBC-1 and treating T-cell cancer.

BACKGROUND

T-cell leukemias and lymphomas, collectively known as T-cell cancers, affect -100,000 patients each year. Patients with relapsed T-cell cancers have a 5-year survival of only 7% to 38%, which is considerably worse than patients with B-cell cancers. The increased survival in B-cell cancers is in part due to the availability of novel antibody and CAR T-cell mediated therapies targeting pan B-cell antigens. Developing analogous therapeutics targeting T-cell cancers has been challenging. Normal and neoplastic T or B cells express similar antigens on the cell surface. Pan-B cell targeting is feasible as the resulting normal B cell aplasia is well- tolerated. Targeting pan-T cell antigens, however, is infeasible, as it results in severe immunosuppression. Thus, T-cell cancers require more specific targeting of the cancerous T cells. SUMMARY

Provided herein are antibody-drug conjugates comprising: (a) an antibody or antigenbinding fragment thereof that specifically binds to a T cell receptor 0 chain constant region (TRBC) polypeptide; and (b) a therapeutic agent conjugated to the antibody or antigen-binding fragment thereof. In some embodiments, the TRBC polypeptide comprises a TRBC1 polypeptide.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 1 ; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment thereof comprises: (a) a light chain variable region sequence comprising SEQ ID NO: 1; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 2. In some embodiments, the antigenbinding fragment thereof comprises a Fab, Fab’, F(ab’)2, Fabl-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv).

In some embodiments, a heavy chain of the antigen-binding fragment thereof comprises a human immunoglobulin G1 (IgGl ) heavy chain. In some embodiments, a light chain of the antigen-binding fragment thereof comprises a human kappa light chain. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized or chimeric antibody.

In some embodiments, the therapeutic agent comprises an anti-cancer agent. In some embodiments, the therapeutic agent comprises SG3199, MMAE, DM1. SN38, or Exatecan. In some embodiments, the therapeutic agent comprises SG3199 or MMAE.

In some embodiments, the therapeutic agent is conjugated to the antibody or antigenbinding fragment thereof via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a cathepsin cleavable linker comprising the VA or VC dipeptide and the para-amino benzyloxy carbonyl (PAB) self-immolative spacer.

Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of any one of the antibody-drug conjugates described herein.

Also provided herein are methods for treating a T-cell cancer in a subject, the method comprising administering to the subject any one of the antibody-drug conjugates or any one of the pharmaceutical compositions described herein. In some embodiments, the T-cell cancer is a clonal T-cell cancer. In some embodiments, the T-cell cancer is a T-cell leukemia or lymphoma. In some embodiments, the subject is a human. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A-1G show anti-TRBCl CAR T activity is limited by normal T cell-mediated killing of the CAR T cells. FIG. 1A, NGFR-expressing CAR T cells (anti-TRBC 1 or anti-CD19) were incubated with GFP-expressing cancer cells (SUP-T1, H9 or Jurkat cells) for 48 hours. Flow cytometry was used to assess GFP (cancer cell) and NGFR (CAR T cells) expression. Numbers beside flow plots show the percentage of surviving cells. Number of biological replicates n = 3. FIG. IB, illustration depicting anti-TRBCl -CAR T cell killing TRBC1+ T-cell cancer cells. Anti-TRBCl scFv on anti-TRBCl CAR T cells bind to TRBC1 on cancerous T cells, leading to CAR T cell activation. Activated CAR T cells secrete perforins, granzymes and cytokines leading to the killing of TRBC1+ T-cell cancer cells. FIG. 1C, CAR T cells were incubated with normal T cells from human donors. After 48 hours, flow cytometry was used to assess NGFR, CD3 and TRBC1 expression. Number of biological replicates n = 3. FIG. ID, illustration depicting bi-directional killing of anti-TRBCl CAR T cells and TRBC1+ normal T cells. Anti-TRBCl -CAR T cell binding to TRBC1 on normal T cells leads to the killing of TRBC1+ normal T cells. TRBC1+ binding also leads to activation of TRBC1+ normal T cells leading to reciprocal killing of anti-TRBC 1 CAR T cells by the normal T cells. FIG. IE, CAR T cells were incubated with normal T cells and with H9 or Jurkat cells. After 48 hours, flow cytometry was used to assess GFP and NGFR expression Number of biological replicates n = 3. FIG. IF, illustration depicting the killing events that occur when anti-TRBCl CAR T cells are mixed with normal T cells and TRBC1+ T-cell cancers. Bi-directional killing leads to depletion of both anti-TRBCl CAR T cells and TRBC1+ normal T cells. The depletion of anti- TRBC1 CAR T cells lead to incomplete killing of TRBC1+ T-cell cancers and the cancer cells continue to proliferate leading to resistance. FIG. 1G. CAR T cells were incubated with SUP- T1 or H9 or Jurkat cells in the presence or absence of normal T cells. SUP-T1, Jurkat and H9 cells expressed GFP and live cell imaging was used to quantify the cancer cell lines. Black arrows indicate the time points where Jurkat cells were added to the co-culture. Data represent mean ± standard error of mean using three technical replicates. Number of biological replicates n = 2.

FIGs. 2A-2E show anti-TRBCl antibody binding to the TRBC1+ TCR leads to antibody internalization into lysosomes. FIG. 2A, illustration depicting internalization of anti-TRBCl antibody into T cells. Anti-TRBC 1 -pHrodo antibody binds to the TRBC1+ TCR leading to its endocytosis. The anti-TRBC 1 -phrodo antibody emits red fluorescence after lysosome endosome fusion. FIG. 2B, anti-TRBC 1-pHrodo antibody was added to Jurkat or Jurkat TCR knockout (TCR-KO) cells followed by live cell imaging. FIG. 2C, quantification of fluorescence overtime after addition of anti -TRCB1 -phrodo antibody to the indicated cell lines. Data represent mean ± standard error of mean using three technical replicates. Number of biological replicates, n = 3. FIGs. 2D-2E, H9 and Jurkat cells were incubated with anti-TRBC 1 antibody, followed by confocal microscopy. LAMP1 antibody and DAPI stains mark lysosomes and nucleus respectively. An anti-mouse IgG2a-Alexa568 was used to detect the location of anti-TRBCl antibody. Scale bars represent 10pm. Number of biological replicates, n = 2.

FIGs. 3A-3D show synthesis and characterization of anti-TRBCl-SG3249 ADC. FIG. 3A, schematic of the anti-TRBCl antibody conjugation to SG3249 (tesirine). FIG. 3B, hydrophobic interaction chromatography of anti-TRBCl antibody, SG3249 and anti-TRBC 1- SG3249 ADC. Number of repeated experiments, n = 4. FIG. 3C, the deconvoluted mass spectra of the anti-TRBCl antibody and the anti- TRBC1-SG3249 ADC. HC = heavy chain and LC = light chain. HC+1 and HC+2 indicate heavy chains conjugated with one or two molecules of SG3249. LC+1 indicates light chain conjugated with one molecule of SG3249. Number of repeated experiments, n = 2. FIG. 3D, size exclusions chromatography (SEC) of anti-TRBC 1-SG3249 and anti-TRBCl antibody. SEC standards include: A, thyroglobulin (MW 670 kDa); B, gamma globulin (MW 158 kDa); C, ovalbumin (MW 44 kDa); D, myoglobin (MW 17 kDa); E, vitamin B12 (MW 1.35 kDa).

FIGs. 4A-4C show anti-TRBC 1-SG3249 ADC performance in vitro. FIG. 4A, Jurkat cells or Jurkat TCR-KO cells were treated with anti-TRBCl antibody or anti-TRBCl -SG3249 ADC or the free drug SG3199 at the indicated concentration. Cell growth quantified by live cell imaging. Data represent mean ± standard error of mean using three technical replicates. Number of biological replicates, n = 3. FIG. 4B, T-cell cancer cell lines were incubated with indicated concentration of anti-TRBCl-SG3249 ADC for 5 days. The cancer cell viability was measured by luciferase assay. The m!gG2a-SG3249 ADC was used as a negative control. IC50 for anti- TRBCl -SG3249 is indicated in the graphs. Data represent mean ± standard error of mean using three technical replicates. FIG. 4C, H9 and Jurkat cells were treated with anti-TRBCl antibody or a sublethal dose of the anti-TRBCl -SG3249 ADC, followed by pH2Ax staining. The cells expressed cytosolic GFP and DAPI stain marked the nucleus. Cells were imaged using confocal microscopy. Scale bars represent 10 pm. Number of biological replicates, n = 2.

FIGs. 5A-5F show anti-TRBCl -SG3249 ADC kills cancer cells in the presence of normal T cells. FIGs. 5A-5B, T-cell cancers were cultured with normal T cells in presence of anti- TRBC1-SG3249 or mIgG2a-SG3249 (as negative control ADC). After 5 days, flow- cytometry was used to assess GFP and TRBC1 expression. Numbers adjacent to the plots indicate the numbers of cells counted by flow cytometry in a representative experiment (FIG. 5A), with data from three human T cell donors (number of biological replicates, n = 3), shown in (FIG. 5B). Bar graphs represent mean ± standard error of mean. FIG. 5C, illustration showing the effect of anti-TRBCl-SG3249 ADC added to a co-culture of T cell cancers and normal T cells. The anti-TRBCl ADC bind and kill the TRBC 1 -expressing cancer cells and normal T cells, while the TRBC2+ normal T cells survive. FIGs. 5D-5E, normal T cells incubated with anti- TRBC1-SG3249 ADC or with mIgG2a-SG3249 ADC. After 5 days, flow- cytometry was used to detect the number of TRBC1+ and TRBC2+ cells. Numbers adjacent to the plots indicate the numbers of cells counted by flow cytometry (FIG. 5D), w ith data from three human T cell donors (number of biological replicates, n = 3) shown in (FIG. 5E). Bar graphs represent mean ± standard error of mean. FIG. 5F, normal T cells from three donors were sorted to obtain TRBC1+ and TRBC2+ populations. TRBC1+ or TRBC2+ normal T cells were incubated with the indicated concentration of anti -TRBC1-SG3249 ADC for 5 days followed by viability assessment using the MTS assay (number of biological replicates, n = 2).

FIGs. 6A-6K show' ADC activity in vivo. FIG. 6A, timeline of in vivo experiment using NSG mice injected with Jurkat cells. FIGs. 6B-6C, NSG mice were intravenously injected with Jurkat cells that express luciferase and GFP. On day 8, mice were intravenously injected with one of three different ADC; m!gG2a-SG3249, anti-TRBCl -MMAE, or anti-TRBCl -SG3249. Bioluminescence imaging (BLI) performed on the indicated days with aggregate data shown in (FIG. 6C). FIGs. 6D-6E, flow cytometry to assess circulating Jurkat cells (CD3+, GFP+, at top right quadrant) on day 21, and aggregate data from 5 mice are shown in (FIG. 6E). FIG. 6F, Kaplan-Meier survival curves of Jurkat-bearing NSG mice after various treatments, with 5 mice in each group. Median survival 27 days in m!gG2a-SG3249 condition days vs. median survival 38 days with anti-TRBCl-MMAE vs. median survival not reached (undefined) with anti -TRBC1-SG3249. p = 0.0034 by Log-rank Mantle-Cox test. FIG. 6G, timeline of in vivo experiment using NSG mice injected with H9 cells. FIG. 6H, NSG mice were intravenously injected with H9 cells that express luciferase and GFP. On day 8, mice were intravenously injected with either m!gG2a-SG3249 or anti-TRBCl-SG3249 ADC. BLI performed on the indicated days. FIGs. 6I-6J, flow cytometry to assess circulating H9 cells (CD3+, GFP+, top right quadrant) on day 25, and aggregate data from 5 mice are shown in (FIG. 6J). FIG. 6K, Kaplan-Meier survival curves of H9-bearing NSG mice after various treatments, with 5 mice in each group. Median survival 24 days in mIgG2a-SG3249 condition vs. median survival not reached (undefined) in anti -TRBC1-SG3249 condition, p = 0.002 by Log-rank Mantle-Cox test. FIGs. 7A-7G show generation and testing of anti-TRBCl CAR T cells. FIGs. 7A-7B, illustration depicting the anti-TRBC 1 CAR construct. FIG. 7C, CAR T cells were stained with anti-mouse scFv-PE antibody or anti-NGFR-APC antibody followed by analysis using flow cytometry. Control T cells indicate staining in unedited T cells. FIG. 7D, the aggregate data of the experiment is shown in FIG. 1A. CAR T cells were incubated with cancer cells (SUP-T1 or H9 or Jurkat cells) for 48 hours. The percentage of surviving cancer cells is shown in the bar graphs. Bar graphs represent mean ± standard error of mean using three technical replicates. Number of biological replicates, n = 2. FIG. 7E, the aggregate data of the experiment is shown in FIG. 1C. CAR T cells were incubated with normal T cells. Flow cytometry was used to assess NGFR and TRBC1 expression after 48 hours. The percentage of surviving normal TRBC1+ or TRBC2+ T cells and CAR T cells are shown in the bar graphs. Bar graphs represent mean ± standard error of mean using three technical replicates. Number of biological replicates, n = 3. FIGs. 7F-7G, anti-TRBCl CAR T cells (2.5 x 10⁴) were incubated with 2.5 x 10⁴ (1 : 1) or 5 x 10⁴ (1 :2) or 12.5 x 10⁴ (1 :5) Jurkat cells, in the presence or absence of normal T cells. After 48 hours, flow cytometry was used to assess GFP and NGFR expression. Numbers beside flow plots show the percentage of surviving cells in each condition (FIG. 7F) and aggregate data from three technical replicates are shown in (FIG. 7G). FIGs. 8A-8B show synthesis and characterization of anti-TRBC 1 -MMAE antibody-drug conjugate (ADC). FIG. 8A, schematic representation of the anti-TRBCl antibody conjugation to

MC-VC-MMAE. FIG. 8B, hydrophobic interaction chromatography analysis of anti-TRBCl antibody, MC-VC-MMAE and anti-TRBCl -MMAE ADC.

FIGs. 9A-9C show anti-TRBC 1-SG3249 stability and binding epitope. FIG. 9A, anti-TRBC 1- SG3249 ADC was incubated in human serum at 7 pg/mL concentration for 0, 3, and 7 days at 37°C. After incubation with human serum, anti-TRBCl -ADC (at 100 ng/mL) was added to TRBC1+ cells (Jurkat and H9) and TRBC1- cells (HPB-ALL and SUP-T1). After 5 days, cancer cell viability was assessed using luminescence. Bar graphs represent mean ± standard error of mean using three technical replicates. Number of biological replicates n = 3. FIG. 9B, TRBC1 and TRBC2 amino acid sequence alignment. The distinct amino acid residues are highlighted in red. The anti-TRBCl antibody binding epitope at position 3, 4 are shown inside a box. FIG. 9C, Jurkat, Jurkat TCR-KO, Jurkat TRBC2+, HPBALL or HPB-ALL TRBC1+ cancer cell lines were stained with anti-TRBCl -PE antibody. The histograms of the anti- TRBCl -PE stain of the indicated cell lines are shown on the left. The nucleotide and amino acid sequences of the anti-TRBCl - antibody binding epitope are shown on the right.

FIGs. 10A-10C show anti-TRBC 1-SG3249 kills TRBC1+ cancer cell lines in vivo. FIG. 10A, weight measurements of the Jurkat-injected NSG mice plotted as means ± S.E.M of values from 5 mice. FIG. 10B, bioluminescence imaging of H9-injected NSG mice used in the experiment FIGs. 6G-6H. FIG. 10C, weight measurement of all mice in H9-injected NSG mice, plotted as means ± standard error of mean of values from 5 mice.

FIGs. 11A-11C show chimeric anti-TRBCl-SG3249 ADC has comparable cytotoxicity. FIG. 11A, schematic representation of the mouse IgG2a anti-TRBCl antibody. The mouse IgG2a heavy chain (HC) and kappa light chain (LC) constant regions w ere replaced with human IgGl HC and human kappa LC constant regions. FIG. 11B, hydrophobic interaction chromatography analysis of chimeric anti-TRBCl antibody and chimeric anti-TRBC 1-SG3249 ADC. Number of repeated experiments n = 2. FIG. 11C, H9 or Jurkat cells were incubated with indicated concentrations of anti-TRBC 1-SG3249 ADC or chimeric anti-TRBC 1-SG3249 ADC for 5 days. The H9 and Jurkat cells expressed luciferase and luminescence was used to assess cell viability. Data plotted as means ± standard error of mean from three technical replicates. The calculated IC50 for anti-TRBC 1-SG3249 and chimeric anti-TRBC 1-SG3249 ADC is shown at the right of the graphs. Number of repeated experiments n = 3. FIG. 12 shows an exemplary schematic of an anti-TRBC2 antibody-drug conjugate (ADC) targeting strategy’.

FIG. 13 shows flow cytometry results showing improved binding of 6R to TRBC2+ healthy T cells compared to Marengo clone.

FIG. 14 shows flow cytometry’ results showing binding of the anti-TRBC2 antibody to TRBC2+ healthy T cells.

FIG. 15 shows flow cytometry results that show 50V6R specifically binding to TRBC2 with no TRBC1 binding, and SAM.2 demonstrating suboptimal TRBC2 binding as shown by the smear in the plots.

FIG. 16 shows high-performance liquid chromatography (HPLC) runs confirming ADC conjugation.

FIGs. 17A-17D show results that demonstrate anti-TRBC2 SG3249 ADC in vitro potency.

DETAILED DESCRIPTION

The majority of T cells, normal or neoplastic, express the aP T cell receptor (TCR) on their cell surface. Each T cell expresses a unique a TCR resulting from the somatic recombination of the variable, diversity, joining, and constant gene segments. Because T-cell leukemias and lymphomas arise from the clonal outgrowth of one specific T cell, every neoplastic T cell expresses the same unique TCR sequence. Thus, targeting the unique TCR sequence in neoplastic T cells should spare enough normal T cells to maintain cellular immunity. As described herein, an approach to achieve this is through targeting the TRBC allele. T cells select one of two possible TRBC alleles: TRBC1 or TRBC2. Normal T cells are composed of about an equal number of TRBC1 and TRBC2-expressing cells, while T-cell cancers express either TRBC1 or TRBC2. Clonal T cell cancers express only one of the two TCR beta chain constant regions (e.g., TRBC1 or TRBC2), providing a potential opportunity to selectively deplete clonal T cell cancers while retaining most of the normal T cells. Specifically, preclinical studies showed that targeting the T-cell receptor beta chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity, making TRBC1 an attractive target to treat T-cell cancers.

Provided herein are antibody-drug conjugates that include (a) an antibody or antigenbinding fragment thereof that specifically binds to a T cell receptor p chain constant region (TRBC) polypeptide; and (b) a therapeutic agent conjugated to the antibody or antigen-binding fragment thereof. In some embodiments, the TRBC polypeptide includes a TRBC 1 polypeptide. In some embodiments, the TRBC polypeptide includes a TRBC2 polypeptide.

Also provided herein are pharmaceutical compositions that include a therapeutically effective amount of any one of the antibody-drug conjugates described herein.

Also provided herein are methods for treating a T-cell cancer in a subject that include administering to the subject any one of the antibody-drug conjugates or the pharmaceutical compositions described herein.

Various non-limiting aspects of these methods are described herein, and can be used in any combination without limitation.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” "an” and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term “about”, when used in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%. 19%. 18%. 17%. 16%, 15%, 14%, 13%, 12%, 11%, 10%. 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

As used herein, the term “administration” ty pically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be intravenous, intra-arterial, intratumor, intraperitoneal, intrathecal, or intraventricular. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

As used herein, the term “antibody” refers to an immunoglobulin molecule that includes one or more antigen-binding domains that specifically bind to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibodies include, but are not limited to monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, an antibody may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term “antibody” is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody utilized in accordance with the present invention can be in a format selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc ); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan. a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.]. In many embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity’ determining region (CDR); in some embodiments an antibody is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%. 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

As used herein, the term ‘'antigen” refers to a molecule or molecular structure that binds to a specific antibody, B-cell receptor, or T-cell receptor. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid sequence (e.g., DNA or RNA). a peptide, a polypeptide, a protein, a carbohydrate, a glycoprotein, a lipid or phospholipid, a lipoprotein, a polymer (including biologic polymers [e.g., nucleic acid and/or amino acid polymers] and polymers other than biologic polymers [e.g., other than a nucleic acid or amino acid polymer]), etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises aglycan. In some embodiments, an antigen is or comprises a phospholipid. In some embodiments, an antigen is or comprises a phospholipid-protein complex. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigen-containing source). In some certain embodiments, an antigen is present in a cellular context (e.g., an antigen is expressed on the surface of a cell or expressed in a cell). In some embodiments, an antigen is present on the surface of a cell in a complex with HLA. In some embodiments, an antigen is a recombinant antigen.

As used herein, the term “binding” ty pically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by w ay of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts - including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell).

As used herein, the terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and “carcinoma”, refer to cells that exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g.. benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. Exemplary cancers that may be treated with a compound or method provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, Medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, cancer of the head, Hodgkin's Disease, and Non-Hodgkin's Lymphomas. Exemplary cancers that may be treated with a compound or method provided herein include cancer of the thyroid, endocrine system, brain, breast, cervix, colon, head & neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus. Additional examples include, thyroid carcinoma, cholangiocarcinoma, pancreatic adenocarcinoma, skin cutaneous melanoma, colon adenocarcinoma, rectum adenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung carcinoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobuhnemia. primary brain tumors, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and nonHodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

As used herein, a “cell” can refer to either a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source. As used herein, an “effective amount” or a “therapeutically effective amount” is an amount sufficient for a compound to accomplish a stated purpose relative to the absence of the compound (e.g. achieve the effect for which it is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signaling pathway, or reduce one or more symptoms of a disease or condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease. A “therapeutically effective amount.” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate the disorder. For example, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2- fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

For any compound (e.g., antibody-drug conjugate) described herein, the therapeutically effective amount can be initially determined from cell culture assays. Target concentrations will be those concentrations of active compound(s) that are capable of achieving the methods described herein, as measured using the methods described herein or known in the art.

As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a dose for humans can be formulated to achieve a concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring compounds effectiveness and adjusting the dosage upwards or downwards, as described above. Adjusting the dose to achieve maximal efficacy in humans based on the methods described above and other methods is w ell within the capabilities of the ordinarily skilled artisan.

As used herein, a “patient” or “subject” refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In some embodiments, a patient is human.

As used herein, the terms “treating”, or “treatment” can refer to any indicia of success in the therapy or amelioration of an injury, disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric exams, and/or a psychiatric evaluation. The term "treating" and conjugations thereof, may include prevention of an injury, pathology, condition, or disease. In embodiments, treating is preventing. In embodiments, treating does not include preventing.

‘Treating” or “treatment” as used herein (and as well-understood in the art) can also broadly include any approach for obtaining beneficial or desired results in a subject’s condition, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (z. e. , not worsening) the state of disease, prevention of a disease’ s transmission or spread, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable. In some embodiments, "treatment" as used herein includes any cure, amelioration, or prevention of a disease. Treatment may prevent the disease from occurring; inhibit the disease’s spread; relieve the disease’s symptoms, fully or partially remove the disease’s underlying cause, shorten a disease’s duration, or do a combination of these things.

A. Antibody-drug conjugate (ADC)

As used herein, an “antibody-drug conjugate” refers to an agent comprising an antibody chemically linked to a therapeutic agent (e.g., a drug). In some embodiments, the antibody binds to a specific protein or receptor found on certain t pes of cells (e.g.. cancer cells, T-cells, B-cells).

In some embodiments, provided herein are antibody-drug conjugates that include (a) an antibody or antigen-binding fragment thereof that specifically binds to a T cell receptor chain constant region (TRBC) polypeptide; and (b) a therapeutic agent conjugated to the antibody or antigen-binding fragment thereof. In some embodiments, the TRBC polypeptide comprises a TRBC1 polypeptide. In some embodiments, the TRBC polypeptide comprises a TRBC2 poly peptide.

A T-cell receptor (TCR) is a protein complex found on the surface of T cells or T lymphocytes, wherein the TCR is responsible for recognizing a presented antigen, immune synapse formation, inducing intracellular signaling, and initiating target cell killing. The TCR can include an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the TCR includes a CD3 gamma (C D3v) chain, a CD3 epsilon (CD3E) chain, a CD3 delta (CD35) chain, a T cell receptor (TCR) alpha chain (with variable [V] and/or constant [C] regions), a T cell receptor (TCR) beta chain (with variable [V] and/or constant [C] regions), a T cell receptor (TCR) gamma chain (with variable [V] and/or constant [C] regions), and a T cell receptor (TCR) delta chain (with variable [V] and/or constant [C] regions). In some embodiments, the TCR can further include a CD3 zeta (CD3Q chain.

T cells select one of two possible TRBC alleles: TRBC1 or TRBC2. While normal T cells are composed of about an equal number of TRBC1 and TRBC2-expressing cells, T-cell cancers express either TRBC1 or TRBC2. In some embodiments, targeting the T-cell receptor beta chain constant region 1 (TRBC1) can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity, making TRBC1 an attractive target to treat T-cell cancers. In some embodiments, targeting the TRBC2 can kill cancerous T cells while preserving sufficient healthy T cells to maintain immunity, also making TRBC2 an attractive target to treat T-cell cancers.

Antibody targeting TRBC1 polypeptide

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that specifically binds to a TRBC1 polypeptide. In some embodiments, an antibody-drug conjugate includes an antibody or antigen-binding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 1; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 1; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 2. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 1 ; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 1 ; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 1; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 2.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 5; and (b) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 6. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a heavy chain variable region sequence having at least 90% sequence identity' to SEQ ID NO: 5; and (b) a light chain variable region sequence having at least 90% sequence identity' to SEQ ID NO: 6. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 5; and (b) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 6. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a heavy' chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 5; and (b) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 6. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a heavy chain variable region sequence comprising SEQ ID NO: 5; and (b) a light chain variable region sequence comprising SEQ ID NO: 6.

In some embodiments, an antigen-binding fragment comprises a Fab, Fab’, F(ab’)2, Fabl-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv). In some embodiments, a heavy chain of the antigen-binding fragment thereof comprises a human immunoglobulin G1 (IgGl ) heavy chain. In some embodiments, a heavy chain of the antigenbinding fragment thereof comprises SEQ ID NO: 3. In some embodiments, a heavy chain of the antigen-binding fragment thereof comprises a mouse immunoglobulin G2a (IgG2a) heavy chain. In some embodiments, a light chain of the antigen-binding fragment thereof comprises a human kappa light chain. In some embodiments, a light chain of the antigen-binding fragment thereof comprises SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized or chimeric antibody. SEQ ID NO: 1 - VL TRBC1 scFv

DVVMTQSPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR

SEQ ID NO: 2 - VH TRBC1 scFv

EVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYND DIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFFDF WGQGTTLTVSS

SEQ ID NO: 3 - Human IgGl heavy chain

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL

QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP

ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO: 4 - Human kappa light chain

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT

EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 5 - VH Humanized TRBC1 scFv

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYVMHWVRQAPGQGLEWMG

FINPYNDDIQSNERFRGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGAGYNFDG AYRFFDFWGQGTMVTVSS

SEQ ID NO: 6 - VL Humanized TRBC1 scFv

DIVMTQSPLSLPVTPGEPASISCRSSQRLVHSNGNTYLHWYLQKPGQSPRLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGQGTKLEIK Antibody targeting TRBC2 polypeptide

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that specifically binds to a TRBC2 polypeptide. In some embodiments, an antibody-drug conjugate includes an antibody or antigen-binding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 7; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%, 95%, 96%, 96%, 98%, 99%. or 100%) sequence identity to SEQ ID NO: 8. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) alight chain variable region sequence having at least 90% sequence identity' to SEQ ID NO: 7; and (b) a heavy' chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 8. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 7; and (b) aheavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 8. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 7; and (b) aheavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 7; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 8.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%. 98%, 99%, or 100%) sequence identity to SEQ ID NO: 9; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 10. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 9; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 10. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity' to SEQ ID NO: 9; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 9; and (b) aheavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 9; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 10.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 96%. 98%. 99%, or 100%) sequence identity to SEQ ID NO: 11; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 12. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 11; and (b) aheavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 11; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity⁷ to SEQ ID NO: 11; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 1 1; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 12.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%. 83%. 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 13: and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 14. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 13; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 14. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 13; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 13; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 13; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 14.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 96%. 98%. 99%, or 100%) sequence identity to SEQ ID NO: 15; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 16. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 15; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 15; and (b) a heavy' chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 16. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity' to SEQ ID NO: 15; and (b) a heavy' chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 15; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 16.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 17; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 18. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 17; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 18. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 17; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 18. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 17; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 18. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 17; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 18.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%. 99%. or 100%) sequence identity to SEQ ID NO: 19; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 20. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 19; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 20. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 19; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 20. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 19; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 19; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 20. In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 21; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 22. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity⁷ to SEQ ID NO: 21; and (b) a heavy chain variable region sequence having at least 90% sequence identity' to SEQ ID NO: 22. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 21; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 22. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 21; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 22. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 21; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 22.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%. 99%. or 100%) sequence identity to SEQ ID NO: 23; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 24. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 23; and (b) a heavy’ chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 24. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 23; and (b) a heavy chain variable region sequence having at least 95% sequence identity' to SEQ ID NO: 24. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 23; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 24. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 23; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 24.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%. 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%. 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 25; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 26. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 25; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 26. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity- to SEQ ID NO: 25; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 26. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 25; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 26. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 25; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 26.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 27; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 28. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 27; and (b) a heavy- chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 28. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 27; and (b) a heavy' chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity' to SEQ ID NO: 27; and (b) a heavy' chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 28. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 27; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 28.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%. 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 29; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 30. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity⁷ to SEQ ID NO: 29; and (b) a heavy chain variable region sequence having at least 90% sequence identity' to SEQ ID NO: 30. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 29; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 30. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 29; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 30. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 29; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 30.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 31; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%. 89%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity’ to SEQ ID NO: 32. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 31; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 32. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 31; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 32. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 31 ; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 32. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 31; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 32.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%. 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 96%. 98%. 99%. or 100%) sequence identity to SEQ ID NO: 33; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 34. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 33; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 34. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 33; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 34. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 33; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 34. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 33; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 34.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%. 99%. or 100%) sequence identity to SEQ ID NO: 35; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 36. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 35; and (b) a heavy’ chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 36. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 35; and (b) a heavy chain variable region sequence having at least 95% sequence identity' to SEQ ID NO: 36. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity⁷ to SEQ ID NO: 35; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 36. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 35; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 36.

In some embodiments, an antibody-drug conjugate includes an antibody or antigenbinding fragment thereof that includes (a) a light chain variable region sequence having at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 96%. 98%. 99%, or 100%) sequence identity to SEQ ID NO: 37; and (b) a heavy chain variable region sequence having at least 80% (e.g., 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 96%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 38. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 37; and (b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 38. In some embodiments, an antibody or antigen-binding fragment thereof can include (a) a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 37; and (b) a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 38. In some embodiments, an antibody or antigenbinding fragment thereof can include (a) a light chain variable region sequence having at least 98% sequence identity⁷ to SEQ ID NO: 37; and (b) a heavy chain variable region sequence having at least 98% sequence identity to SEQ ID NO: 38. In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a light chain variable region sequence comprising SEQ ID NO: 37; and (b) a heavy chain variable region sequence comprising SEQ ID NO: 38.

In some embodiments, an antigen-binding fragment comprises a Fab, Fab’, F(ab’)2, Fabl-SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv). In some embodiments, a heavy chain of the antigen-binding fragment thereof comprises a human immunoglobulin GI (IgGl) heavy chain. In some embodiments, a heavy chain of the antigenbinding fragment thereof comprises SEQ ID NO: 3. In some embodiments, a heavy chain of the antigen-binding fragment thereof comprises a mouse immunoglobulin G2a (IgG2a) heavy chain. In some embodiments, a light chain of the antigen-binding fragment thereof comprises a human kappa light chain. In some embodiments, a light chain of the antigen-binding fragment thereof comprises SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized or chimeric antibody.

SEQ ID NO: 7 - VL anti-TRBC2 antibody 6R

DVVMTQSPLSLPVTPGEPASISCRSSQNLRHSNGRTYLQWYLQKPGQSPQLLIYRVSN RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 8 - VH anti-TRBC2 antibody 6R

QVQLVQSGAEVKKPGASVKVSCKASPRGFHGYHMHWVRQAPGQGLEWMGFINPY NDDIQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S

SEQ ID NO: 9 - VL anti-TRBC2 antibody 6R8K

DVVMTQSPLSLPVTPGEPASISCRSSQNLRHKNGRTYLQWYLQKPGQSPQLLIYRVS NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 10 - VH anti-TRBC2 antibody 6R8K

QVQLVQSGAEVKKPGASVKVSCKASPRGFHGYHMHWVRQAPGQGLEWMGFINPY NDDIQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S SEQ ID NO: 11 - VL anti-TRBC2 antibody 6R8R

DVVMTQSPLSLPVTPGEPASISCRSSQNLRHRNGRTYLQWYLQKPGQSPQLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 12 - VH anti-TRBC2 antibody 6R8R

SEQ ID NO: 13 - VL anti-TRBC2 antibody 6R33Y

DVVMTQSPLSLPVTPGEPASISCRSSQNLRHSNGRTYLQWYLQKPGQSPQLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 14 - VH anti-TRBC2 antibody 6R33Y

QVQLVQSGAEVKKPGASVKVSCKASPYGFHGYHMHWVRQAPGQGLEWMGFINPY NDDIQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S

SEQ ID NO: 15 - VL anti-TRBC2 antibody 6R48G

DVVMTQSPLSLPVTPGEPASISCRSSQNLRHSNGRTYLQWYLQKPGQSPQLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 16 - VH anti-TRBC2 antibody 6R48G

QVQLVQSGAEVKKPGASVKVSCKASPRGFHGYHMHWVRQAPGQGLEWMGFINPY NGDIQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S

SEQ ID NO: 17 - VL anti-TRBC2 antibody 6R50V

DVVMTQSPLSLPVTPGEPASISCRSSQNLRHSNGRTYLQWYLQKPGQSPQLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK SEQ ID NO: 18 - VH anti-TRBC2 antibody 6R50V

QVQLVQSGAEVKKPGASVKVSCKASPRGFHGYHMHWVRQAPGQGLEWMGFINPY NDDVQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S

SEQ ID NO: 19 - VL anti-TRBC2 antibody 6K

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHSNGRTYLQWYLQKPGQSPQLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 20 - VH anti-TRBC2 antibody 6K

SEQ ID NO: 21 - VL anti-TRBC2 antibody 6K8K

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHKNGRTYLQWYLQKPGQSPQLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 22 - VH anti-TRBC2 antibody 6K8K

SEQ ID NO: 23 - VL anti-TRBC2 antibody 6K8R

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHRNGRTYLQWYLQKPGQSPQLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 24 - VH anti-TRBC2 antibody 6K8R

QVQLVQSGAEVKKPGASVKVSCKASPRGFHGYHMHWVRQAPGQGLEWMGFINPY NDDIQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S SEQ ID NO: 25 - VL anti-TRBC2 antibody 6K48G

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHSNGRTYLQWYLQKPGQSPQLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 26 - VH anti-TRBC2 antibody 6K48G

SEQ ID NO: 27 - VL anti-TRBC2 antibody 6K50V

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHSNGRTYLQWYLQKPGQSPQLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 28 - VH anti-TRBC2 antibody 6K50V

SEQ ID NO: 29 - VL anti-TRBC2 antibody 6K33Y

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHKNGRTYLQWYLQKPGQSPQLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 30 - VH anti-TRBC2 antibody 6K33Y

SEQ ID NO: 31 - VL anti-TRBC2 antibody 6K8R33Y (JX1.1)

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHRNGRTYLQWYLQKPGQSPQLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK SEQ ID NO: 32 - VH anti-TRBC2 antibody 6K8R33Y (JX1.1)

QVQLVQSGAEVKKPGASVKVSCKASPYGFHGYHMHWVRQAPGQGLEWMGFINPY

NDDIQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S

SEQ ID NO: 33 - VL anti-TRBC2 antibody 6K8R48G (JX1.2)

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHRNGRTYLQWYLQKPGQSPQLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 34 - VH anti-TRBC2 antibody 6K8R48G (JX1.2)

QVQLVQSGAEVKKPGASVKVSCKASPRGFHGYHMHWVRQAPGQGLEWMGFINPY

NGDIQSNERFRGRVTMTSDKSTTTAYMELSSLRSEDTAVYYCARGNGKWGDGAYR FFDFWGQGTLVTVS S

SEQ ID NO: 35 - VL anti-TRBC2 antibody 6K8R50V (JX1.3)

DVVMTQSPLSLPVTPGEPASISCRSSQNLKHRNGRTYLQWYLQKPGQSPQLLIYRVS

NRFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 36 - VH anti-TRBC2 antibody 6K8R50V (JX1.3)

SEQ ID NO: 37 - VL anti-TRBC2 antibody 50V

DVVMTQSPLSLPVTPGEPASISCRSSQNLVHSNGRTYLQWYLQKPGQSPQLLIYRVSN

RFPGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSTHVPYTFGGGTKVEIK

SEQ ID NO: 38 - VH anti-TRBC2 antibody 50V

In some embodiments, an antibody-drug conjugate comprises a therapeutic agent, wherein the therapeutic agent can include an anti-cancer agent. Examples of therapeutic agents that can be attached to an antibody-drug conjugate described herein can include, without limitation, anti-cancer agents such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, mertansine/emtansine (DM1), ravtansine/soravtansine (DM4), SN-38, calicheamicin, D6.5, dimeric pyrrolobenzodiazepines (PBDs), a-amantin (AAMT), PNU- 159682, ricin, pseudomonas exotoxin A, diphtheria toxin, and gelonin. In some embodiments, the therapeutic agent comprises SG3199. MMAE, DM1. SN38, or Exatecan. In some embodiments, the therapeutic agent comprises SG3199 or MMAE.

In some embodiments, the therapeutic agent can be conjugated to the antibody or antigen-binding fragment thereof via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a cathepsin cleavable linker with a VA or VC dipeptide, and the para-amino benzyloxy carbonyl (PAB) space. In some embodiments, the linker further comprises a poly(ethylene glycol)(PEG) spacer.

In some embodiments, the antibody-drug conjugate comprises SG3249. In some embodiments, the antibody-drug conjugate comprises a compound of:

B. Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions provided herein are formulated with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier’⁷ can refer to a substance that aids the administration of an active agent to and absorption by a subject and can be included in the compositions of the present disclosure without causing a significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds of the disclosure. In some embodiments, a pharmaceutically acceptable carrier can comprise Kollifor EL (e.g.. cremaphor) and glycerol. One of skill in the art will recognize that other pharmaceutically acceptable carriers are useful in the present disclosure.

Pharmaceutical compositions provided herein can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

Pharmaceutical compositions provided herein can be administered alone or as a component of a pharmaceutical formulation. Pharmaceutical compositions provided herein may be formulated for administration, in any convenient way for use in human or veterinary medicine. Pharmaceutical composition provided herein may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form can vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

Pharmaceutical compositions described herein can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such compositions can contain, for example, preserving agents. A composition can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Compositions may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, controlled release formulations, on patches, in implants, etc. Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Aqueous suspensions can contain an active agent (e.g., one or more the compounds of Formula I, Formula II, Formula III, or pharmaceutically acceptable salts thereof) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include, without limitation, a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from Patty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate. one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity⁷.

Pharmaceutical compositions can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as egg or soybean lecithin, esters or partial esters derived from fatty⁷ acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In some embodiments, these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.

In some embodiments, pharmaceutical compositions provided herein can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In some embodiments, pharmaceutical compositions provided herein can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed include, without limitation, water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. Formulations provided herein may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in formulations provided herein can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally - acceptable diluent or solvent, such as a solution of 1,3-butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).

In some embodiments, pharmaceutical compounds and formulations provided herein can be lyophilized. Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g.. mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.

Compositions and formulations provided herein can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587. As used herein, the term “liposome’' means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH- sensitive or negatively -charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

Liposomes can also include “sterically stabilized” liposomes, i.e., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety⁷. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference in its entirety .

In some embodiments, pharmaceutical compositions provided herein can be formulated into any appropriate dosage form. Examples of dosage forms include solid or liquid forms including, without limitation, pills, capsules, tablets, gels, liquids, suspensions, solutions (e.g, sterile solutions), sustained-release formulations, and delay ed-release formulations.

In some embodiments, pharmaceutical compositions provided herein can be designed for parenteral (e.g., topical, subcutaneous, intravenous, intraperitoneal, intrathecal, and intraventricular) administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient: and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. In some embodiments, pharmaceutical compositions provided herein can be administered locally or systemically. For example, a composition containing one or more bispecific molecules provided herein can be administered systemically by an intravenous injection to a mammal (e.g., a human). For example, a composition containing one or more bispecific molecules provided herein can be administered systemically by a subcutaneous injection to a mammal (e.g. , a human).

C. Methods of Treatment

Also provided herein are methods for treating a T-cell cancer in a subject that include administering to the subject any one of the antibody-drug conjugates or pharmaceutical compositions described herein. In some embodiments, the T-cell cancer is a clonal T-cell cancer. In some embodiments, the T-cell cancer is a T-cell leukemia or lymphoma.

In some embodiments, the materials and methods described herein can be used to treat a subject having any type of T cell cancer. In some embodiments, a T cell cancer treated as described herein can include one or more solid tumors. In some embodiments, a T cell cancer treated as described herein can be a blood cancer. In some embodiments, a T cell cancer treated as described herein can be a primary cancer. In some embodiments, a T cell cancer treated as described herein can be a metastatic cancer. In some embodiments, a T cell cancer treated as described herein can be a refractory cancer. In some embodiments, a T cell cancer treated as described herein can be a non-Hodgkin’s lymphoma. In some embodiments, a T cell cancer treated as described herein can be a Hodgkin’s lymphoma. Examples of T cell cancers that can be treated as described herein include, but are not limited to, ALL, PTCL, AITL, T-PLL, ATLL, EATL, MEITL, FTCL, nodal PTCL, CTCL, ALCL, T-LGL, NKTL, and hepatosplenic T- cell lymphoma.

In some embodiments, the materials and methods provided herein can be used to reduce or eliminate the number of cancer cells present within a subject having a T cell cancer. For example, a subject in need thereof (e.g., a subject having a T cell cancer) can be administered any one of the antibody-drug conjugates or pharmaceutical compositions described herein to reduce or eliminate the number of cancer cells present within the subject. For example, any one of the antibody-drug conjugates or pharmaceutical compositions described herein can be used to reduce the number of cancer cells present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the antibody-drug conjugates or pharmaceutical compositions described herein can be used to reduce the size (e.g. , volume) of one or more tumors present within a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80. 90. 95, or more percent. In some embodiments, the number of cancer cells present within a subject being treated can be monitored. Any appropriate method can be used to determine whether or not the number of cancer cells present within a subject is reduced. For example, imaging techniques can be used to assess the number of cancer cells present within a subject.

In some embodiments, any one of the antibody-drug conjugates or pharmaceutical compositions provided herein can be used to improve survival of a subject having a T cell cancer. For example, a subject in need thereof can be administered any one of the antibodydrug conjugates or pharmaceutical compositions described herein to improve survival of the subject. For example, any one of the antibody -drug conjugates or pharmaceutical compositions described herein can be used to improve the survival of a subject having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, any one of the antibodydrug conjugates or pharmaceutical compositions described herein can be used to improve the survival of a subject having cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).

An effective amount (e.g, effective dose) of any one of the antibody-drug conjugates or pharmaceutical compositions described herein can vary depending on the severity of the T cell cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and/or the judgment of the treating physician.

An effective amount of a composition (e.g, a pharmaceutical composition) containing any one of the antibody-drug conjugates or pharmaceutical compositions described herein can be any amount that can treat a subject having a T cell cancer without producing significant toxicity to the subject. An effective amount of any one of the antibody-drug conjugates or pharmaceutical compositions described herein can be any appropriate amount. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g, a T cell cancer) may require an increase or decrease in the actual effective amount administered. The frequency of administration of a composition (e.g., a pharmaceutical composition) containing any one of the antibody-drug conjugates or pharmaceutical compositions described herein can be any frequency that can treat a subject having a T cell cancer without producing significant toxicity to the subject. For example, the frequency of administration can be once a day, once a week, once every 2 weeks, or once every 4 weeks. In some embodiments, an administration can include a continuous infusion of a composition containing any one of the antibody-drug conjugates or pharmaceutical compositions described herein. The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing any one of the antibody-drug conjugates or pharmaceutical compositions described herein can include rest periods. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., a T cell cancer) may require an increase or decrease in administration frequency.

An effective duration for administering a composition (e.g, a pharmaceutical composition) containing any one of the antibody-drug conjugates or pharmaceutical compositions described herein can be any duration that treat a subject having a T cell cancer without producing significant toxicity to the subject. For example, the effective duration can vary from several days to several w eeks, months, or years. In some embodiments, the effective duration for the treatment of a subject can range in duration from about one month to about 10 years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition (e.g.. a T cell cancer) being treated.

In some embodiments, any one of the antibody-drug conjugates or pharmaceutical compositions described herein can be used as the sole active agent to treat a subject having a T cell cancer.

In some embodiments, one or more (e.g. , one, two, three, four, five or more) additional therapeutic agents can be used to treat a subject having a T cell cancer. For example, a subject in need thereof (e.g, a subject having a T cell cancer) can be administered any one of the antibody-drug conjugates or pharmaceutical compositions described herein in combination with one or more additional therapeutic agents. In some embodiments, an additional therapeutic agent can be an alkylating agent. In some embodiments, an additional therapeutic agent can be a platinum compound. In some embodiments, an additional therapeutic agent can be a taxane. In some embodiments, an additional therapeutic agent can be a luteinizing- hormone-releasing hormone (LHRH) agonist. In some embodiments, an additional therapeutic agent can be an anti-estrogen. In some embodiments, an additional therapeutic agent can be an aromatase inhibitor. In some embodiments, an additional therapeutic agent can be an angiogenesis inhibitor. In some embodiments, an additional therapeutic agent can be a poly(ADP)-ribose polymerase (PARP) inhibitor. In some embodiments, an additional therapeutic agent can be a topoisomerase inhibitor. In some embodiments, an additional therapeutic agent can be a corticosteroid. In some embodiments, an additional therapeutic agent can be an antibody. In some embodiments, an additional therapeutic agent can be an antibody drug conjugate. Examples of an additional therapeutic agent include, without limitation, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt- 11), melphalan, pemetrexed, topotecan, vinorelbine, goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, cyclophosphamide, doxorubicin (e.g, liposomal doxorubicin), prednisone, prednisolone, dexamethasone, mogamuhzumab, brentuximab, and any combinations thereof. In some cases, the one or more additional therapeutic agents can be administered together with any one of the antibody-drug conjugates or pharmaceutical compositions described herein. In some embodiments, the one or more additional therapeutic agents can be administered independent of any one of the antibody-drug conjugates or pharmaceutical compositions described herein. When the one or more additional therapeutic agents are administered independent of any one of the antibodydrug conjugates or pharmaceutical compositions described herein, any one of the antibodydrug conjugates or pharmaceutical compositions described herein can be administered first, and the one or more additional therapeutic agents administered second, or vice versa.

In some embodiments, one or more (e.g. , one, two, three, four, five or more) additional treatments (e g., therapeutic interventions) that are effective to treat T cell cancers can be used. For example, a subject in need thereof (e.g, a subject having a T cell cancer) can be administered any one of the antibody -drug conjugates or pharmaceutical compositions described herein in combination with one or more therapeutic interventions. Examples of therapeutic interventions that can be used as described herein to treat a T cell cancer include, without limitation, cancer surgeries, radiation therapies, chemotherapies, and any combinations thereof. In some embodiments, the one or more additional treatments that are effective to treat T cell cancers can be performed at the same time as the administration of any one of the antibody-drug conjugates or pharmaceutical compositions described herein. In some embodiments, the one or more additional treatments that are effective to treat T cell cancers can be performed before and/or after the administration of any one of the antibody-drug conjugates or pharmaceutical compositions described herein.

EXAMPLES

Methods

Primary' human T cells and cell lines

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque Plus (GE Healthcare. GE17-1440-02) density gradient centrifugation from leukapheresis samples (Stemcell Technologies, Cellero). Human T cells were expanded from peripheral blood mononuclear cells either with Human T-Activator CD3/CD28 Dynabeads (Thermo Fisher Scientific, 1113 ID) for 3 days at a bead: cell ratio of 1 : 1 or with the addition of the anti -human CD3 antibody (clone 0KT3, BioLegend) at 15 ng/ml or with. T cells were cultured in RPMI 1640 with 10% FBS, 1% penicillin-streptomycin, recombinant human IL-2 (100 lU/ml) (aldesleukin, Prometheus Therapeutics and Diagnostics), and recombinant human IL-7 (5 ng/ml) (BioLegend, 581906). Jurkat (Clone E6-1, [American Type Culture Collection (ATCC)], H9 (ATCC). SUP-T1 (ATCC), HPB-ALL [Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ)], and CML-T1 (DSMZ) were cultured in RPMI 1640 (ATCC, 30-2001) supplemented with 10% HyClone fetal bovine serum (FBS; GE Healthcare SH30071.03) and 1% penicillin-streptomycin (Thermo Fisher Scientific). Retroviral transduction was used to enable the expression of GFP and luciferase in the desired cell lines. Transduced cells were sorted using a BD FACSAria II based on GFP expression. Cells were grown in a humidified incubator at 37°C in 5% CO2.

CRISPR gene editing of cell lines

The IDT Alt-R CRISPR system (IDT) was used to convert the TRBC1 allele of Jurkat to TRBC2 to generate the Jurkat-TRBC2+ cell line, and to convert the TRBC2 allele of HPB- ALL to TRBC1 to generate the HPB-ALL-TRBC1+ cell line. Homology-directed repair templates (HDRT) encoded the TRBC1 or TRBC2 sequences. A TRBC-targeted Cas9 crRNA was duplexed with tracrRNA at a 1: 1 molar ratio at 95°C for 5 mins. After cooling to room temperature, 100 pmols of crtracrRNA duplex was complexed with 50 pmol of Cas9 nuclease for 15 minutes at room temperature. The resulting ribonucleoprotein was mixed with 60 pmol of a single-stranded HDRT (IDT, Alt-R HDR Donor Oligo) encoding the TRBC2 sequence (to edit Jurkat cells) or the TRBC 1 sequence (to edit the HPB-ALL cells) along with 5 x 10⁵ Jurkat or HPB-ALL cells in 20 .1 of OptiMEM (Thermo Fisher Scientific, 31985062) in a 0.1 cm electroporation cuvette (Bio-Rad, 1652089). The mixture was then electroporated at 100 V for 10 ms using an ECM 2001 (BTX).

Cells were immediately recovered with a warm culture medium. After 1 week of culture, the electroporated cells were plated by limiting dilution. Individual clones were screened by Sanger sequencing a PCR amplicon encompassing the edit site to assess the TRBC sequence. Sequences of the gRNA, HDRT, and primers are included in Table 1.

Cell staining, flow cvtometrv. and cell sorting

Cells were suspended at a concentration of 1 x 10⁶ cells/ml in flow sorting buffer [PBS (phosphate buffered saline containing 4% FBS] or flow stain buffer (PBS and 0.5% bovine serum albumin, 2 mM EDTA, 0.1% sodium azide), and incubated with desired antibodies at a concentration of 1 pg/ml for 20 min at 4°C, protected from light. The antibodies used were as follows: Brilliant Violet (BV)-711 anti-human CD3 (clone OKT3 BioLegend. #317328). phycoerythrin (PE)-anti-human CD4 (clone RPA-T4, BioLegend, #300508), BV-421 antihuman CD3, PE-anti-human Cpi TCR (clone JOVI. 1 BD Biosciences, #565776) APC-anti- human CD7, APC-anti-human CD26, R-PE AffiniPure F(ab’)2 Fragment Goat Anti-Mouse IgG, F(ab’)2 fragment specific (Jackson ImmunoResearch. #115-116-072), APC-anti-human NGFR. Stained cells were analyzed using a BD LSRII (Becton Dickinson) flow cytometer or with an IntelliCyt iQue Screener PLUS (Sartorius). Cell sorting was performed using BD FACSAria II. Gating on single live cells was performed with the use of viability dyes (LIVE/DEAD Fixable Near-IR Kit L10119 Invitrogen) and forward and side scatter characteristics. Flow cytometry data were analyzed with either the iQue Forecyt Software (Sartorius) or FlowJo v. 10. 1 software (BD) or BD FACSDiva™ Software.

[Table 1]

CAR T cell generation

CAR T cells were engineered using non-viral knock-in of CAR transgenes into the TRAC locus. The anti-TRBCl CAR T was based on CAR designs incorporating CD28 and 0X40 co- stimulation (FIGs. 7A-7B and Table 2). The anti-CD19 CAR was based on a CD28 hinge, transmembrane, and co-stim format using the FMC63 scFv45. CAR molecules were expressed under the control of a human elongation factor-la (EFla) promoter in a bicistronic gene that also co-expressed a truncated nerve growth factor receptor (tNGFR) tag for magnetically activated cell sorting (MACS) of the edited T cells. CAR genes were synthesized as human codon-optimized DNA fragments (GeneArt Gene Strings) and cloned into a modified pUC19 vector backbone, containing 300 bp TRAC homology arms, via NEBuilder HiFi DNA Assembly (NEB, E2621L). Cloned plasmids were sequence verified by sanger sequencing (Genewiz, Azenta Life Sciences). To generate homology directed repair templates (HDRTs) for CRISPR mediated knock-in, plasmid templates were linearized with primers specific to the Ml 3 forw ard and reverse sites using the High-Fidelity 2X Master Mix (New' England BioLabs,

M0494L). HDRT PCR amplicons were purified with lx AMPure XP Reagent (Beckman Coulter Life Sciences. A63880). eluted in sterile water (to 0.5-2 ug/uL), and quantified with a NanoDrop Spectrophotometer (Thermo Fisher Scientific). [Table 2]

To engineer CAR T cells, CD3+ T cells were isolated from PBMCs (described above) by negative selection (StemCell Technologies, 17951) and activated with Dynabeads Human TActivator CD3/CD28 (Thermo Fisher Scientific, 11132D) at a 1: 1 bead-to-cell ratio in T cell media, which consisted of RPMI-1640 (ATCC, 30-2001) supplemented with 10% FBS (Cytiva. SH30070.03), 1% penicillin-streptomycin (Thermo Fisher Scientific, 15140163), 100 lU/mL recombinant human IL-2 (Proleukin, Prometheus Laboratories), and 5 ng/mL recombinant human IL-7 (BioLegend, 581908). After 48-56 hours of activation, T cells were separated from CD3/CD28 beads by two passes over a magnet. For each electroporation condition of simultaneous TRAC knock-in and TRBC knock-out, 50 pmols of each crRNA were independently mixed with 25 pmols of Alt-R A.s. Casl2a (Cpfl) Ultra (IDT, 10001273) and 37.5 pmols of Alt-R Cpfl Electroporation Enhancer (IDT, 1076301) in a total volume of 1.27 pl ofNuclease Free Duplex Buffer (IDT, l l-01-03-01) forat least 15 minutes before combining the two RNPs at a 1 : 1 volume ratio. The TRAC and TRBC RNPs were then mixed with 0.5 pg HDRT diluted in 2.4 uL OptiMEM for a final volume of 5 pL per electroporation condition. Immediately before electroporation, T cells were centrifuged at 90g for 10 minutes, then resuspended at 0.75-1.25 x 10⁶ T cells in 20 pl P3 buffer (Lonza, V4XP-3032) and combined with the 5uL Cpfl RNP and HDRT mixture. Cells were nucleofected in 16-well cuvettes (Lonza, V4XP-3032) with a 4D Nucleofector X-Unit (Lonza, AAF-1003X) using pulse code EH115. After nucleofection, 80 pl pre-warmed cytokine-free T cell media was added to cells and the cuvette strip was placed in a 37°C incubator for 20 min. The cells were then diluted in 1 rnL of T cell media and transferred to a 24-well plate. CAR T cells were purified using Easy Sep™ Release Human PSC-Derived Neural Crest Cell Positive Selection Kit (Stemcell, 100-0047). which selects for NGFR (CD271)-expressing CAR T cells. T cell media was changed every 2 or 3 days until functional assays on days 11 or later after the initial activation. The expression and purity (>95%) of CAR T cells were verified via flow cytometry. CRISPR reagents and primers are listed on Table 1. CAR T cell co-cultures and multiple antigen challenge assays

For CAR T cell co-cultures with cancer cells; CAR T cells (2.5 x 10⁴) were incubated with cancer cells (2.5 x 10⁴ SUP-T1, H9, Jurkat cells) in a 96 well plate. After 48 hours, flow cytometry was used to assess GFP (to detect SUP-T1, H9 or Jurkat cells) and NGFR (to detect CAR T cells) expression. For CAR T cell co-cultures with normal T cells; CAR T cells (2.5 x 10⁴) were incubated with normal T cells from human donors (5 x 10⁴ cells) to ensure an approximate anti-TRBCl CAR T:TRBC1+ normal T cell ratio of 1:1. After 48 hours, flow cytometry was used to assess NGFR, CD3 and TRBC1 expression. For CAR T cell co-cultures with cancer cells and normal T cells; CAR T cells (2.5 x 10⁴) were incubated with normal T cells (5 x 10⁴ cells) and with TRBC1+ T-cell cancers (2.5 x 10⁴ H9 or Jurkat cells). After 48 hours, flow cytometry was used to assess GFP and NGFR expression. For live cell imaging studies, CAR T cells (2.5 x 10⁴) were incubated with cancer cells (2.5 x 10⁴ SUP-T1, H9 or Jurkat cells) in the presence or absence of normal T cells (5 x 10⁴). Cells were suspended in 200 pl of cytokine-free RPMI-1640 media supplemented with 10% FBS and 1% PS in 96-well flat-bottom tissue culture-treated plates. SUP-T1, Jurkat and H9 cells expressed GFP and live cell imaging with an Incucyte SX5 (Sartorius) was used to quantify the number of GFP+ cancer cells every 6-8 hrs. For multiple antigen challenge assay, after approximately 48 hours 100 pl of supernatant was removed and 2.5 x 10⁴ Jurkat cells in 100 pl of fresh media were added to the co-culture. This process was repeated for the duration of the experiments. The normalized number of cancer cells (SUP-T1. H9 and Jurkat cells) indicates the total sum of the GFP fluorescent intensity from the cancer cell.

Drug cytotoxicity assays

1 x 10⁴ T-cell cancer cell lines or the normal human T cells were suspended in 200 pl of RPMI- 1640 media supplemented with 10% FBS and 1% penicillin-streptomycin in 96-well flatbottom tissue culture-treated plates. The following drugs were resuspended in DMSO solution to make 10 mM stock solution; Exatecan mesylate (MedChemExpress ITY-13631A), DM1 (MedChemExpress HY-19792), SN38 (MedChemExpress HY-13704), MMAE (MedChemExpress HY-15162). SG3199 (MedChemExpress HY-101 161). The drugs were added to target cells at the concentrations specified in the text for 5 days at 37°C. For luciferaseexpressing cells, cell viability was assayed by the ONE-Glo™ luciferase assay (E6110, Promega), per the manufacturer’s instructions. Viability was calculated as the ratio of the luminescence signal to the no antibody or control antibody condition: (antibody well luminescence)/(no antibody or control antibody well luminescence). Mouse anti-TRBCl antibody and chimeric anti-TRBCl antibody generation

Mouse IgG2a kappa anti-TRBC 1 antibody was expressed and purified by BD and was stored in a buffer containing PBS with <0.09% sodium azide. The chimeric anti-TRBC 1 antibody was generated by grafting the human IgGl Fc segment to the murine anti-TRBCl variable region. The chimeric anti-TRBC l antibody was expressed by GeneArt (ThermoFisher) using ExpiCHO cells and purified using HisTrapTM column (Millipore Sigma) followed by size exclusion chromatography with HiLoad Superdex200 16/600 column (Millipore Sigma). Generation of purified chimeric antibody was confirmed by analytic size exclusion chromatography using TOSOH TSKgel G3000SWxl column and 50 mM sodium phosphate, 300 mM sodium chloride, pH 7.0 running buffer. The chimeric antibody was stored in PBS.

Antibody internalization and confocal microscopy

Anti-TRBCl antibody was labeled using pHrodo™ iFL Red Microscale Protein Labeling Kit (ThermoFisher P36014). The indicated cancer cell lines (3 x 10⁴) were plated on 96-well plates, followed by the addition of anti-TRBCl -pHrodo antibody at a final concentration of 10 pg/mL. Live cell imaging with an Incucyte SX5 (Sartorius) was used to quantify the integrated red fluorescence intensity which is the sum of all red fluorescence intensity within the cell multiplied by pixel area. For anti-TRBCl and lysosome co-localization studies, H9 and Jurkat cells were treated with anti-TRBC l antibody for 30 mins or 240 minutes, followed by a secondary stain with anti-mouse IgG2a-Alexa568 antibody (ThermoFisher A-21134) to detect the location of the anti-TRBCl antibody. Anti-LAMP1-Alexa647 antibody (Cell Signaling, 73589) and DAPI stains were used to detect the location of lysosomes and nucleus respectively. For pH2Ax detection studies, H9 and Jurkat cells were treated with the anti-TRBCl antibody (5 ng/mL) or with a sublethal dose of the anti-TRBC 1-SG3249 ADC (5 ng/mL) for 12 hours. Cells were stained with anti-pH2Ax antibody (Cell Signaling, 2577) followed by secondarystaining with anti-rabbit-Alexa647 antibody (ThermoFisher A27040). The H9 and Jurkat cells expressed GFP in the cytosol, and DAPI was used to mark the nucleus. Coverslips were mounted onto slides using ProLong^1M Gold Antifade with DAPI (ThermoFisher P36941). Confocal images were obtained with a 40X/1.30 PlanNeofluar oil or a 63X/1.4 PlanApo oil objective, a Zeiss LSM700 confocal microscope, and Zen software. Images were cropped and adjusted for contrast in the Zen software (Zeiss). Antibody-drug conjugate production

The antibodies were partially reduced using 2.2 molar excess Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, ThermoFisher 77720) for 2 hours at 37°C with rotation, followed by TCEP removal by buffer exchange into PBS using ZebaTM Spin 7K MWCO columns. The following drug-linker solutions for conjugation were prepared by diluting the drug-linkers from DMSO stock solution at 10 mM concentration in sufficient DMSO to make the final conjugation reaction mixture 10% organic and 90% aqueous; SG3249 (MedChemExpress HY- 128952) MC-VC-PAB-MMAE (MedChemExpress HY- 15575). The drug-linker solutions were added to the partially reduced antibodies at 5-fold molar excess and incubated for 2 hours at room temperature with rotation. The excess unreacted drug linkers were removed by buffer exchange into PBS using ZebaTM Spin 7K MWCO columns. The antibody-drug conjugates were analyzed for concentration using Pierce™ BCA Protein Assay (ThermoFisher 23227), aggregation using size-exclusion chromatography, drug-antibody conjugation and presence of the residual free drug by HPLC.

High-Performance Liquid Chromatography

High-performance liquid chromatography was performed using an Agilent 1260 Infinity I LC system. For analytical size exclusion chromatography the Yarra SEC-2000 column (Phenomenex, 3 um, 300 x 7.8 mm) was used with IX PBS pH 7.2 as the mobile phase at room temperature (0.2 mL/min). SEC column calibration was performed using a reconstituted gel filtration standard (Biorad, #151 1901). The TSKgel Butyl-NPR column (Tosoh BioSciences, 2.5 um, 4.6x35 mm) was used for analytical hydrophobic interaction chromatography to assess antibody-drug conjugation. Antibody conjugate species of increasing DAR were gradient eluted over 12 minutes (0.8 mL/min) from 0% 1.5 M ammonium sulfate. 25 mM sodium phosphate pH 7.0 to 100% 25 mM sodium phosphate, 25% isopropanol pH 7.0 at room temperature. ADC DAR was estimated by taking the weighted average of the integrated peak densities for each A280 peak as follows:

Where DARMK is the drug loading of the ADC product mixture, DAR\ is the average drug loading of HIC fraction i, A is the integrated area of fraction i, and A is the total integrated peak area. When necessary for plotting, A280 chromatograms were baseline corrected using the BaselineRemoval python package with adaptive iteratively reweighted penalized least squares via the ZhangFit function with default parameters.

Mass spectrometry

The non-conjugated and the drug-conjugated antibodies were diluted to 0.3 pg/pl in PBS, pH 7.4, and treated with 100 mM of DTT at 60°C for 30 mins. The treated samples were loaded into the 96-well plate. Heavy and light chains in the samples were separated using HPLC (Vanquish, Thermo Fisher Scientific) with a 10-minute gradient from 15% to 55% CAN, and an analytical column (MAbPac™ Reversed Phase HPLC Columns, Thermo Fisher Scientific). Fractions were analyzed by mass spectrometry to quantify DAR. An Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific) was used to collect the experimental data. The experimental data collected were deconvoluted using the Unidec deconvolution software and was performed by Rapid Novor. After deconvoluting the m/z spectra, the mass spectra were further analyzed to calculate the peak area using Origin Pro software. Finally, the DAR values were calculated from the mass spectra peak areas using the standard formula.

ADC cytotoxicity assays and ADC co-cultures

For ADC cytotoxicity assay, 1 x 10⁴ T-cell cancer cell lines or the normal human T cells were suspended in 200 pl of RPMI-1640 media supplemented with 10% FBS and 1% penicillinstreptomycin in 96-well flat-bottom tissue culture-treated plates. ADCs were added at the concentration indicated in the figure legends. After 5 days, cell viability was assayed by the ONE-Glo™ luciferase assay (E6110, Promega) for luciferase-expressing cells or by flow cytometry for GFP-expressing cells. For normal human T cells, cell viability was assessed by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (G3580. Promega). For co-cultures, 1 x 10⁴ cancer cells were incubated with 2 x 10⁴ normal T cells in 96-well flatbottom tissue culture-treated plates in a total 100-pl volume RPMI media. The ADCs were added at concentrations indicated in the figure legend. The co-cultures were incubated at 37°C for the duration indicated in the text. Target T-cell cancer cell lines and normal human T cells were quantified by flow cytometry -based GFP expression (for GFP-expressing tumor cell lines) or distinct TRBC1 expression.

Animal experiments

Six- to eight-week-old female NOD.Cg-Prkdc^scld I12rg^tmlwjl/SzJ (NSG) mice acquired from the Jackson Laboratory or the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center Animal Resources facility were maintained according to the JHU Animal Care and Use Committee-approved research protocol MO21M43. Cancer cell lines or human T cells were injected via the tail vein. Appropriate antibody-drug conjugates were injected via the tail vein on the indicated days. For survival studies, animals were followed until death or until day 80. Mouse bioluminescence was measured using the IVIS system (PerkinElmer). For flow-based detection of circulating tumor cells from mouse blood, 100 to 200 pl of blood was collected in EDTA-treated microvettes (Sarstedt Inc., NC9299309) by mouse cheek bleed. Ten microliters of counting beads (Precision Count Beads, BioLegend, 424902) were added to an equal volume of cell suspension in each tube. The number of tumor cells (GFP+, CD3+) or T cells (GFP- CD3+) was counted based on the acquisition of 500 beads for each sample.

Statistical analyses

Data were summarized using mean ± standard error of the mean. One-way analysis of variance (ANOVA) with Sidak s multiple comparison test (when comparing two select groups) were used to compare means between three or more samples, with a = 0.05. The Kaplan-Meier method was used to generate median survival, and p values were estimated by log-rank test. The exact p values were reported except when p was less than < 0.0001. Prism version 9.2.0 software (GraphPad) was used for graph production and statistical analysis.

Example 1 - Anti-TRBCl CAR T cytotoxicity is limited by normal T cell-mediated killing of the CAR T-cells

A classic method of normal T cell activation is to stimulate the TCR-CD3 complex with antibodies bound to cells or beads. Accordingly, bispecific antibodies that crosslink TCRs on two adjacent T cells induce activation and death of both T cells. Similarly, normal yo T cells can kill anti-yd CAR T cells. To test whether normal T cells could kill anti-TRBCl CAR T cells, anti-TRBCl CAR T cells were generated (FIGs. 7A-7C). Anti-CD19 CAR T cells were used as negative controls. To test functionality⁷, purified anti-TRBCl CAR T cells were cocultured with two TRBC1+ cancer cell lines: H9 and Jurkat cells, and one TRBC1- T cell cancer cell line: SUP-T1 as a negative control, in an E:T ratio of 1 : 1 (FIG. 1A). All cancer cell lines were virally transduced to express GFP to monitor target cell death via flow cytometry. After a 48-hour co-culture, it was observed that the anti-TRBCl CAR T cells mediated complete lysis of TRBC1+ cells H9 and Jurkat cells, but not TRBC1- SUP-T1 cells (FIG. 1A and FIG. 7D). The persistence of anti-TRBCl CAR T cells indicated that CAR T cell killing was unidirectional, as T-cell cancers lack cytotoxic capabilities (FIG. IB).

Next, the anti-TRBCl -CAR T cells were co-cultured with normal T cells from healthy human donors. This mimics the situation after the infusion of anti-TRBCl CAR T cells into patients. After 48 hours in co-culture, the anti-TRBC 1 CAR T cells were eliminated (FIG. 1C). Staining of the co-cultures with anti-TRBC 1 antibody revealed that the remaining normal T cells express TRBC2+ TCRs as they lack TRBC1+ expression (FIG. 1C and FIG. 7E). This result suggested that the ligation of anti-TRBCl CARs to TRBC1+ TCRs on the unmodified normal T-cells was sufficient to induce potent bidirectional killing by stimulating T cell activation in both the CAR T cells and the unmodified normal T cells (FIG. ID).

It was then tested whether the presence of normal T cells could reduce the ability of anti-TRBCl CAR T cells to kill cancer cells. For this purpose, anti-TRBCl CAR T cells, normal T cells and TRBC1+ cancer cells (Jurkat or H9 cells) were co-cultured. As expected from the data in Fig. 1c, complete ablation of anti-TRBCl CART cells was observed (Fig. le). Furthermore, the anti-TRBCl CAR T cells failed to eradicate the TRBC1+ cancer cells. In patients with a high cancer burden, cancer cells can outnumber the CAR T cells. To replicate such a scenario, the co-culture with anti-TRBCl CAR T cells was repeated, with and without normal T cells, and with vary ing numbers of target Jurkat cancer cells. The anti-TRBCl CAR T cells efficiently killed the cancer cells at all tested CAR T: cancer cell ratios in the absence of normal T cells (FIGs. 7F-7G). However, in the presence of added normal T cells, a dramatic loss of the CAR T cell population occurred, leading to progressively less killing of the cancer cells (FIGs. 7F-7G). To confirm the results of flow cytometry, live cell imaging was used to track the growth of cancer cells exposed to the anti-TRBCl CAR T or anti-CD19 CAR T cells. Similar results were obtained, showing that normal T cells could substantially impede the killing of H9 cancer cells by the anti-TRBCl CAR T cells (FIG. 1G top right panel) with the single antigen challenge and the Jurkat cells with the repeated antigen challenge (FIG. 1G bottom right panel). Collectively, these results suggest that the therapeutic benefit of anti- TRBCl CAR T cells is limited by fratricide resulting from engagement with normal TRBC1+ T cells (FIG. IF).

Example 2 - Anti-TRBCl antibody binding to the TRBC1+ TCR leads to antibody internalization into lysosomes

Based on the challenges with anti-TRBC 1 CAR T cells noted above, it was sought to develop a different strategy, still using the anti-TRBCl antibody but not employing CAR T cells. Antibody-drug conjugates (ADC) can kill target cells through the internalization and release of cytotoxic drugs. It has been previously shown that TCR activation by peptide-MHC complexes results in rapid TCR endocytosis. Thus, it was tested whether the anti-TRBCl antibody is also internalized upon binding TRBC1+ TCRs on T cells. To visualize antibody internalization, the anti-TRBCl antibody was conjugated with a pH-sensitive dye (pHrodoTM) that emits fluorescence in the acidic environment of lysosomes (FIG. 2A). After the addition of the anti-TRBCl -pHrodo antibody, there was a steady increase in fluorescence in the TRBC1+ cancer cells (Jurkat and H9), but not in the TRBC2+ cancer cell line (HPB-ALL) or in the cancer cell line in which the TRBC gene was inactivated using CRISPR-Cas912 (Jurkat TCR-KO; FIGs. 2B-2C). To confirm trafficking into lysosomes, cells were stained with an antibody targeting lysosomal-associated membrane protein 1 (LAMP1). At 30 minutes, it was observed that the anti-TRBCl antibody bound to the surface of TRBC 1+ cells (H9 and Jurkat cell), and at 240 minutes, the anti-TRBCl antibody co-localized with the LAMP1 (FIGs. 2D- 2E). This indicated anti-TRBCl antibody was efficiently internalized and trafficked into lysosomes and could be used for the delivery of antineoplastic drugs.

Example 3 - Synthesis, characterization, and performance of anti-TRBCl antibody-drug conjugates

To identify potent antineoplastic drugs for conjugation to the anti-TRBCl antibody, five drugs were evaluated for their ability to kill various T-cell cancer cell lines (Table 3). All five drugs were previously used to generate ADCs. The two best-performing drugs (SG3199 and monomethyl auristatin e; MMAE) were conjugated to the anti-TRBCl antibody (FIG. 3A, FIG. 8A). For both conjugates, drug formats with cathepsin cleavable linkers, either VC-PAB or VA-P AB, linkers were chosen to enhance drug release upon internalization into lysosomes. Following ADC synthesis, a variety of chromatographic methods and mass spectrometry were used to characterize the conjugates (FIGs. 3B-3D, FIG. 8B). Size exclusion chromatography showed that the ADC existed in monomeric form (FIG. 3D). The drug-antibody-ratio (DAR) was estimated to be between 2.4 to 2.6 for anti-TRBCl -SG3249 and 3.2 for anti-TRBCl- MMAE based on hydrophobic interaction chromatography and mass spectrometry (FIGs. 3B- 3C, FIG. SB and Table 4). Initial experiments showed that the anti-TRBCl -SG3249 was more potent. [Table 3] Cytotoxicity of indicated drugs in different T-cell cancer cell lines

Dose-dependent cytotoxicity of the anti-TRBCl-SG3249 ADC was observed in TRBC1+ cancer cells (FIGs. 4A-4B). An IC50 of 7.6 and 4.3 ng/mL was achieved for the anti- TRBC1-SG2349 (FIG. 4B and Table 5) in Jurkat and H9 cells, respectively which is comparable to clinically used ADCs that target CD30 or CD 19 in other hematologic malignancies. Minimal killing was observed with the control ADC (mIgG2a-SG3249) made with an antibody of the same isotype as the anti-TRBCl antibody. Cells devoid of TRBC1 [Jurkat TCR-KO, HPB-ALL (TRBC2+), SUP-T1 (TRBC1-), CML-T1 (TRBC1-)] provided a specificity control and were refractory to the anti-TRBCl -SG3249 mediated killing while retaining sensitivity to the free drug SG3199 (FIGs. 4A-4B and Tables 3 and 5). The resistance of the cells devoid of TRBC1 -expression indicated that the ADC was stable under the culture conditions. To confirm this stability, the ADC was incubated in human serum for 7 days at 37°C. Cytotoxicity assays over the subsequent five 5 days showed that anti-TRBCl -SG3249 retained its potency and selectivity for TRBC1+ cells (FIG. 9A). The MMAE-based ADC required a higher concentration to kill TRBC1+ cells, with an IC50 of 115.3 and 232.5 ng/mL in Jurkat and H9 cells, respectively (Table 5).

TRBC1 and TRBC2 differ in only 4 amino acid residues and two of these (N and K at positions 3 and 4) are required for binding to the anti-TRBCl antibody (FIG. 9B). To rigorously test the specificity of the ADC in isogenic cells, CRISPR-Cas9 homology-directed repair was used to convert residues NKto KN at positions 3 and 4 in wild-type TRBC1+ Jurkat cells, creating “Jurkat (TRBC2+)” cells. Similarly, wild-type HPB-ALL cells that express TRBC2, were gene-edited to convert KN to NK at positions 3 and 4 to generate “HPB-ALL (TRBC I-) cells. Successful gene-editing was confirmed by sequencing and flow' cytometry (FIG. 9C). Cytotoxicity assay showed that the Jurkat (TRBC2+) cells became resistant to anti- TRBCl ADCs while the HPB-ALL (TRBC1+) cells became sensitive to the anti- TRBC1 ADCs (FIG. 4B, and Table 5) It was sought to confirm that the potent killing observed with anti-TRBCl-SG3249 ADC was due to the SG3199 pay load rather than some other property of the antibody. Cathepsin cleavage of SG3249 in the lysosome has been shown to release SG3199, a pyrrolobenzodiazepine (PDB) dimer that binds to the DNA minor grove and promotes inter-strand cross-links that trigger phosphorylation of H2AX (pH2Ax). pH2Ax is a histone variant that marks the site of the DNA damage for subsequent repair. To determine whether such damage occurred in cancer cells exposed to the anti-TRBCl SG3249 ADC. treated cells were evaluated with an antibody to pH2Ax via confocal microscopy. Imaging revealed distinct nuclear pH2Ax staining, which was not observed with the unconjugated antibody (FIG. 4C).

[Table 4] The calculated drug antibody ratio (DAR) for anti-TRBCl-SG3249, anti-TRBCl-

MMAE and chimeric anti-TRBCl -SG3249 antibodies [Table 5]

Example 4 - Anti-TRBC1-SG3249 ADC kills cancer cells in the presence of normal T cells

Given the ability of normal T cells to compromise the therapeutic effectiveness of anti- TRBC1 CAR T cells, it was important to determine whether the ADC generated with the same antibody was similarly compromised. For this purpose, T cells from human donors were cultured with cancer cell lines in the presence of the anti-TRBCl -SG3249 ADC. Normal T cells did not impact the toxicity of the ADC against any of the three TRBC1+ cancer cell lines tested (FIGs. 5A-5C). Additionally, no cytotoxicity was observed against four TRBC1- cancer cell lines in the presence of normal T cells (FIGs. 5A-5C). Dose-dependent cytotoxicity of the ADC against the TRBC1+ subset of the normal T cells was also observed, but minimal toxicity was exerted on the TRBC2+ subset (FIGs. 5A, 5D, 5E). To confirm that anti-TRBC 1 antibody- mediated TCR internalization or TCR epitope blocking were not interfering with subsequent antibody-based detection of T cell subtypes, TRBC1+ and TRBC2+ T cells were flow-sorted. Subsequent viability assay again showed the specific killing of the TRBC1+ normal T cells with anti-TRBC 1-SG3249 ADC (FIG. 5F). It was notable that the ADC more potently killed TRBC1+ cancer cells (IC50 of 4 to 8 ng/mL; FIG. 4B) than TRBC1+ normal T cells (IC50 of 28 ng/mL; FIG. 5F).

Example 5 - ADC activity in vivo

To assess in vivo efficacy, the ADC was tested in advanced, widely disseminated TRBC1+ T-cell cancer xenografts generated from patient-derived cell lines. Immunodeficient mice were injected intravenously with Jurkat (TRBC1+) cancer cells, imaged to confirm engraftment, then treated with a single dose of an ADC 8 days following injection (FIG. 6A). Imaging of the mice treated with a control ADC (mIgG2a-SG3249) showed the expected expansion of the cancer cells (FIG. 6B, left column). When the mice were treated with the anti- TRBC 1 -MMAE ADC, the cancers regressed, within a week after treatment initiation (FIG. 6B, middle column, Day 14). But the cancers recurred thereafter (FIG. 6B. middle column, and FIG. 6C). When the mice were treated with the anti-TRBCl-SG3249 ADC, the cancers became undetectable within a week after treatment initiation, and no recurrences were observed throughout the 80 days of the experiment (FIG. 6B, right columns, and FIG. 6C). The cancer regression observed in FIGs. 6B-6C was confirmed by flow cytometric evaluation of blood cells from the mice 14 days after treatment was initiated. Abundant circulating cancer cells were observed in the mice treated with the control ADC, but the cancer cell burden was reduced by >90% in the mice treated with anti-TRBC 1 -MMAE ADC (FIGs. 6D-6E). There were no detectable cancer cells in the blood of mice treated with the anti-TRBC 1-SG3249 ADC. These results were consistent with Kaplan-Meyer analysis of survival; the anti-TRBC 1 -MMAE ADC prolonged survival of the mice and the anti-TRBC 1- SG3249 ADC appeared to cure them (FIG. 6F). Weight loss or other signs of toxicity were not observed in the mice treated with anti- TRBC 1-SG3249 ADC (FIG. 10A).

The anti-TRBC 1-SG3249 ADC was additionally tested in a disseminated T-cell cancer xenograft model derived from TRBC1+ H9 cells, another patient-derived cell line. As in the first model, the ADC vastly reduced tumor cells in all mice as assessed by imaging (FIG. 6H and FIG. 10B), no circulating cancer cells were detectable in the blood (FIGs. 6I-6J). and the mice were seemingly cured and maintained a stable body weight (FIG. 6K and FIG. 10C).

Example 6 - Chimeric anti-TRBC 1-SG3249 ADC has comparable cytotoxicity

Food and Drug Administration (FDA)-approved ADCs are engineered as chimeric or humanized antibodies to reduce the risk of generating an immune response in human patients against the ADCs. The anti-TRBC 1 antibody used in the examples described above was of mouse origin. To prepare for a human clinical trial a human IgGl heavy chain was grafted along with a human kappa light chain to create the chimeric anti-TRBC 1-SG3249 ADC such as those already used in clinical trials (FIGs. 11A-11B). This chimeric anti-TRBCl-SG3249 and original anti-TRBC 1-SG3249 ADC had similar potencies when tested against two TRBC1+ cancer cell lines (FIG. 11C).

Example 7 - Method of TRBC2 antibody generation

Single site variation library (Libi) was generated from Marengo clone, wherein the framework and CDR regions were mapped. All CDR positions included single site amino acid mutagenesis, and also 74 single site mutations to other 19 amino acids to generate a total diversity of 1406 unique sequences. These linear fragments were contained in a phage display vector (pADL). The plasmids pool was then electroporated into a bacteria system, generating a phage pool. Sequencing-linked immunosorbent assay (SLISY) and phage display were performed as a readout for enriched clones. For SLISY, a cell-based approach was used and the following cell lines were used: HPB-ALL isogenic TRBC1+/TRBC2+/TCR-KO cell lines; Jurkat isogenic TRBC1+/TRBC2+/TCR-KO cell lines (Table 6). Then, phage bound to cells were eluted off and the DNA w as extracted for MiSeq runs.

[Table 6]

Sequencing reads pertaining to each cell lines were acquired, wherein 95% confidence Interval was calculated with all reads to get the lower bound for TRBC2 cells and upper bound for TRBC1/K0 cells. Ratios of upper bound from cells that are TRBC2+ over lower bound from cell that are TRBC1+ (namely C2/C1) were obtained. Ratios of upper bound from cells that are TRBC2+ over lower bound from cell that are TCR-K.0 (namely C2/KO) were also obtained (Table 7). The ratios were then ranked from highest to lowest, wherein the top hits were 6R and 6K. The scfv sequences were turned into full-length antibodies and flow⁷ cytometry assay was used to confirm specific binding to C2 cells (FIGs. 13-14).

[Table 7]

One of the clones, 50V6R, was compared to the Thermo clone (SAM.2), wherein 50V6R showed specific TRBC2 binding with no TRBC1 binding. The results show SAM.2 demonstrating suboptimal TRBC2 binding as shown by the smear in the plots (FIG. 15).

Example 8 - TRBC2 antibody drug conjugate (ADC) Generation

The top anti-TRBC2 antibody clones were selected for ADC generation wherein TRBC2 antibodies were expressed as human IgGl and mouse IgG2a antibodies. Antibodies were linked to payload SG3249 using maleimide conjugation, and ADC conjugation was confirmed by hydrophobic interaction chromatography (FIG. 16). ADC activity was then tested in vitro using TRBC1+ and TRBC2+ cell lines (FIGs. 17A-17D).

Claims

WHAT IS CLAIMED IS:

1. An antibody-drug conjugate comprising:

(a) an antibody or antigen-binding fragment thereof that specifically binds to a T cell receptor p chain constant region (TRBC) polypeptide; and

(b) a therapeutic agent conjugated to the antibody or antigen-binding fragment thereof.

2. The antibody-drug conjugate of claim 1, wherein the TRBC polypeptide comprises a TRBC1 polypeptide.

3. The antibody-drug conjugate of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 1; and

(b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 2.

4. The antibody-drug conjugate of claim 3, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a light chain variable region sequence comprising SEQ ID NO: 1; and

(b) a heavy chain variable region sequence comprising SEQ ID NO: 2.

5. The antibody-drug conjugate of claim 1, wherein the TRBC polypeptide comprises a TRBC2 polypeptide.

6. The antibody-drug conjugate of claim 1 or 5, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a light chain variable region sequence having at least 90% sequence identity⁷ to SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; and

(b) a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 8. 10. 12. 14. 16. 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38, respectively.

7. The antibody-drug conjugate of claim 6, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a light chain variable region sequence comprising SEQ ID NO: 7, 9, 1 1, 13, 15,

17, 19, 21, 23, 25, 27, 29, 31, 33, 35, or 37; and

(b) a heavy chain variable region sequence comprising SEQ ID NO: 8, 10, 12, 14, 16,

18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38, respectively.

8. The antibody-drug conjugate of any one of claims 1-7, wherein the antigen -bin ding fragment thereof comprises a Fab, Fab’, F(ab’)2, Fab^SH, Fv, diabody, linear antibody or single-chain variable fragment (scFv).

9. The antibody-drug conjugate of any one of claims 1 -8, wherein a heavy chain of the antigen-binding fragment thereof comprises a human immunoglobulin G1 (IgGl) heavy chain.

10. The antibody-drug conjugate of any one of claims 1-9, wherein a light chain of the antigen-binding fragment thereof comprises a human kappa light chain.

11. The antibody-drug conjugate of any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof is a humanized or chimeric antibody.

12. The antibody-drug conjugate of any one of claims 1-11, w herein the therapeutic agent comprises an anti-cancer agent.

13. The antibody-drug conjugate of claim 12, wherein the therapeutic agent comprises SG3199, MMAE, DM1, SN38, or Exatecan.

14. The antibody-drug conjugate of claim 13, wherein the therapeutic agent comprises SG3199 or MMAE.

15. The antibody-drug conjugate of any one of claims 1-14, wherein the therapeutic agent is conjugated to the antibody or antigen-binding fragment thereof via a linker.

16. The antibody-drug conjugate of claim 15, wherein the linker is a cleavable linker.

17. The antibody-drug conjugate of claim 16, wherein the linker comprises a cathepsin cleavable linker comprising a VA or VC protease sensitive site and self-immolative para-amino benzyloxy carbonyl (PAB) group.

18. A pharmaceutical composition comprising a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-17.

19. A method for treating a T-cell cancer in a subject, the method comprising administering to the subject the antibody-drug conjugate of any one of claims 1-17 or the pharmaceutical composition of claim 18.

20. The method of claim 19, wherein the T-cell cancer is a clonal T-cell cancer.

21. The method of claim 20, wherein the T-cell cancer is a T-cell leukemia or lymphoma.

22. The method of any one of claims 19-21, wherein the subject is a human.