Methods and Materials for Treating Clonal T Cell Expansion

Abstract

The present specification relates to methods and materials for treating T cell cancer. For example, a composition containing one or more bispecific molecules targeting the T cell receptor ￡ chain constant region (TRBC) can be administered to a mammal to treat the mammal with a T cell cancer. For example, the present specification provides methods and materials for using one or more bispecific molecules to treat a mammal with a T cell cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 150,232, filed February 17, 2021. The disclosure of that prior application is considered part of (and is incorporated by reference into) the disclosure of this application.

[0002] Federal Funding Statement This invention was made with Government support under Grants AR048522, CA006973, CA009071, CA06292, CA230400, and GM007309 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0003] Sequence Listing This specification contains a Sequence Listing that has been submitted electronically as an ASCII text file named 44807-0386WO1_ST25.txt. The size of the ASCII text file, created on Feb. 10, 2022, is 24 KB. The data in the ASCII text file is incorporated herein by reference in its entirety.

[0004] 1. Technical Field The present specification relates to methods and materials for treating clonal T cell proliferation (e.g., pathogenic clonal T cell proliferation, e.g., T cell cancer). For example, a composition containing one or more bispecific molecules can be administered to a mammal to treat the mammal with a T cell cancer. For example, the present specification provides methods and materials for using one or more bispecific molecules to treat a mammal with a T cell cancer. [Background technology]

[0005] 2. Background information T-cell cancers are a heterogeneous group of malignancies that make up approximately 15% of non-Hodgkin's lymphomas (Swerdlow et al., Blood 127:2375-2390 (2016) (Non-Patent Document 1)) and 20% of acute lymphoblastic leukemias (ALL; Han et al., Cancer Causes & Control 19:841-858 (2008) (Non-Patent Document 2); and Dores et al., Blood 119:34-43 (2012) (Non-Patent Document 3)). The outcomes of T-cell lymphoma and relapsed T-cell ALL (T-ALL) are worse than the corresponding B-cell malignancies, with estimated 5-year survival rates of 32% for T-cell lymphoma (Weisenburger et al., Blood 117:3402-3408 (2011) (Non-Patent Document 4)) and only 7% for relapsed T-ALL (Fielding et al., Blood 109:944-950 (2007) (Non-Patent Document 5)).

[0006] Malignant B cells or T cells do not express different cell surface antigens from their non-cancerous counterparts. There are several targeted immunotherapeutic agents for B cell malignancies that target pan-B cell antigens, such as CD19 or CD20, and they are feasible because the associated normal B cell aplasia is clinically well tolerated. However, a similar strategy that targets pan-T cell antigens is not feasible because the resulting T cell depletion leads to clinically unacceptable levels of immunosuppression. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Swerdlow et al.,Blood 127:2375-2390(2016) [Non-Patent Document 2] Han et al., Cancer Causes & Control 19:841-858(2008) [Non-Patent Document 3] Dores et al.,Blood 119:34-43(2012) [Non-Patent Document 4] Weisenburger et al.,Blood 117:3402-3408(2011) [Non-Patent Document 5] Fielding et al.,Blood 109:944-950(2007) Summary of the Invention

[0008] overview VDJ recombination combined with allelic exclusion results in the expression of only one of the two TRBC polypeptides (i.e., TRBC1 or TRBC2 polypeptides) on the surface of each T cell. Expression in normal healthy T cells is a mixture of both TRBC1 and TRBC2. In contrast, clonal T cell cancers express only one of the two TCR β chain constant regions (e.g., express only TRBC1 or TRBC2). As described herein, bispecific molecules targeting TRBC polypeptides can selectively deplete only one of the two TRBC polypeptides while sparing one of the two TRBC polypeptides. For example, bispecific molecules targeting TRBC1 polypeptides can selectively deplete only one of the two TRBC polypeptides while sparing the remaining healthy TRBC2. + TRBC2 ensures that T cells are sufficient to maintain a functional immune system. + TRBC1 while sparing healthy human T cells + T cells (e.g., cancerous TRBC1+ T cells and healthy TRBC1 + T cells) can be selectively depleted (see, for example, FIG. 1A).

[0009] The present specification provides methods and materials for treating clonal T cell proliferation (e.g., pathogenic clonal T cell proliferation, e.g., T cell cancer). In some cases, the present specification provides bispecific molecules that can be used to treat T cell cancer. For example, a bispecific molecule comprising at least two antigen-binding domains, where a first antigen-binding domain (e.g., a first single-chain variable fragment (scFv)) can bind to a T cell receptor (TCR) β chain constant region (TRBC) polypeptide and a second antigen-binding domain (e.g., a second scFv) can bind to the same TRBC polypeptide or a T cell co-receptor polypeptide (e.g., a CD3 polypeptide), can be used to treat a mammal (e.g., a human) with a T cell cancer. In some cases, the present specification provides methods for treating T cell cancer. For example, one or more bispecific molecules provided herein (e.g., a composition containing one or more bispecific molecules provided herein) can be administered to a mammal to treat the mammal with a T cell cancer.

[0010] As described herein, clonal T cell proliferation (e.g., pathogenic clonal T cell proliferation, e.g., T cell cancer) can be treated by targeting specific subsets of TCR antigens. For example, TRBC1 + T-cell cancers, which have malignant proliferation of T cells, can be treated using bispecific antibodies (BsAbs) that target TRBC1 polypeptides (e.g., BsAbs that include a first antigen-binding domain capable of binding to a TRBC1 polypeptide and a second antigen-binding domain capable of binding to the same TRBC1 polypeptide). BsAbs that target TRBC1 can bind to TRBC2 in a mammalian body. + While preserving T cells (e.g., about half of normal T cells), + TRBC1 (including T cells) +Healthy T cells can be stimulated to specifically lyse T cells (see, for example, FIG. 1A). As demonstrated herein, residual malignant TRBC1 that may still remain after treatment with BsAb targeting TRBC1 polypeptide can be suppressed. + Treatments can be performed to target T cells. For example, a BsAb capable of binding to a TRBC1 polypeptide and capable of binding to a CD3 polypeptide can be used to target residual malignant TRBC1 + Healthy TRBC2 as effector T cells to target (e.g., target and destroy) T cells + To recruit T cells, one or more antibody-drug conjugates targeting TRBC1 (TRBC1-ADCs) can be used to target residual malignant TRBC1. + It may be used to target (eg, target and destroy) T cells (see, eg, FIG. 1C).

[0011] Similarly, TRBC2 + T cell cancers having malignant proliferation of T cells may be treated using a BsAb targeting a TRBC2 polypeptide (e.g., a BsAb comprising a first antigen binding domain capable of binding to a TRBC2 polypeptide and a second antigen binding domain capable of binding to the same TRBC2 polypeptide), and such treatment with a BsAb targeting a TRBC2 polypeptide may optionally be followed by treatment with a BsAb capable of binding to a TRBC2 polypeptide and capable of binding to a CD3 polypeptide, and / or treatment with one or more TRBC2-ADCs.

[0012] Having the ability to treat clonal T cell proliferations (e.g., T cell cancers) as described herein (e.g., by administering one or more bispecific molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to the same TRBC polypeptide, and optionally one or more bispecific molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) provides a unique and as yet unrealized opportunity to selectively deplete clonal T cell cancers while retaining approximately half of normal T cells (e.g., sufficient healthy T cells to maintain adequate T cell immunity and a functional immune system). Furthermore, the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to the same TRBC polypeptide, and optionally, bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be used as cost-effective, off-the-shelf targeted therapeutic agents for T cell cancers.

[0013] In general, one aspect of the present disclosure features a bispecific molecule comprising a polypeptide comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising a second antigen-binding domain capable of binding to a TRBC polypeptide. The polypeptide comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and the polypeptide comprising a second antigen-binding domain capable of binding to a TRBC polypeptide can each independently be a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a F(ab')2 fragment, or any biologically active fragment thereof. The binding affinity of the first antigen-binding domain capable of binding to a TRBC polypeptide can be lower than the binding affinity of the second antigen-binding domain capable of binding to a TRBC polypeptide. The TRBC polypeptide can be a TRBC1 polypeptide or a TRBC2 polypeptide. The TRBC polypeptide can be a TRBC1 polypeptide. The first antigen-binding domain capable of binding to a TRBC1 polypeptide or the second antigen-binding domain capable of binding to a TRBC1 polypeptide can be a VFV ... L CDR1 and V having the amino acid sequence set forth in SEQ ID NO:2 L CDR2 and V having the amino acid sequence set forth in SEQ ID NO:3 L and a light chain comprising a V CDR3 and a V CDR4 having the amino acid sequence set forth in SEQ ID NO:4. H CDR1 and V having the amino acid sequence set forth in SEQ ID NO:5 H CDR2 and V having the amino acid sequence set forth in SEQ ID NO:6 HThe bispecific molecule may comprise a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:3 and a CDR3. The light chain may comprise or consist essentially of the amino acid sequence set forth in SEQ ID NO:7 and the heavy chain may comprise or consist essentially of the amino acid sequence set forth in SEQ ID NO:8. The light chain may comprise or consist essentially of the amino acid sequence set forth in SEQ ID NO:48 and the heavy chain may comprise or consist essentially of the amino acid sequence set forth in SEQ ID NO:49. The bispecific molecule may also comprise a molecule capable of improving the stability of the bispecific molecule.

[0014] In another aspect, the present specification features a method for treating a mammal with T-cell cancer. The method can comprise or consist essentially of administering a bispecific molecule comprising a polypeptide comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising a second antigen-binding domain capable of binding to a TRBC polypeptide to a mammal with T-cell cancer. The mammal can be a human. The T-cell cancer can be a clonal T-cell cancer. The T-cell cancer may be acute lymphoblastic leukemia (ALL), peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), enteropathy-associated T-cell lymphoma (EATL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), follicular T-cell lymphoma (FTCL), nodal peripheral T-cell lymphoma (nodal PTCL), cutaneous T-cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL), T-cell large granular lymphocytic leukemia (T-LGL), extranodal NK / T-cell lymphoma (NKTL), or hepatosplenic T-cell lymphoma. The cancer cells in the mammal's body may be reduced by at least 50%. The method may be effective in improving the survival of the mammal. The method may also include, after administration of the bispecific molecule, administering to the mammal a second bispecific molecule comprising a polypeptide comprising a third antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising an antigen-binding domain capable of binding to a CD3 polypeptide. The CD3 polypeptide may be a CD3γ polypeptide, a CD3δ polypeptide, or a CD3ε polypeptide. The method may also include, after administration of the bispecific molecule, administering to the mammal a molecule comprising a polypeptide comprising a third antigen-binding domain capable of binding to a TRBC polypeptide and an anti-cancer agent.

[0015] In another aspect, the present disclosure features a method for treating a mammal with T-cell cancer. The method can comprise or consist essentially of administering a first bispecific molecule to a mammal with T-cell cancer, the first bispecific molecule comprising a polypeptide comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising a second antigen-binding domain capable of binding to a TRBC polypeptide; and administering a second bispecific molecule to the mammal, the second bispecific molecule comprising a polypeptide comprising a third antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising an antigen-binding domain capable of binding to a CD3 polypeptide. The CD3 polypeptide can be a CD3γ polypeptide, a CD3δ polypeptide, or a CD3ε polypeptide. The mammal can be a human. The T-cell cancer can be a clonal T-cell cancer. The T-cell cancer can be ALL, PTCL, AITL, T-PLL, ATLL, EATL, MEITL, FTCL, nodal PTCL, CTCL, ALCL, T-LGL, NKTL, or hepatosplenic T-cell lymphoma. The cancer cells in the mammal can be reduced by at least 50%. The method may be effective in improving the survival of a mammal.

[0016] In another aspect, the present specification features a method for treating a mammal with T-cell cancer. The method can include or consist of administering to a mammal with T-cell cancer a first bispecific molecule comprising a polypeptide comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising a second antigen-binding domain capable of binding to a TRBC polypeptide; and administering to the mammal a molecule comprising a polypeptide comprising a third antigen-binding domain capable of binding to a TRBC polypeptide and an anti-cancer agent. The mammal can be a human. The T-cell cancer can be a clonal T-cell cancer. The T-cell cancer can be ALL, PTCL, AITL, T-PLL, ATLL, EATL, MEITL, FTCL, nodal PTCL, CTCL, ALCL, T-LGL, NKTL, or hepatosplenic T-cell lymphoma. The cancer cells in the mammal can be reduced by at least 50%. The method can be effective in improving the survival of the mammal.

[0017] In another aspect, the present disclosure features a method for treating a mammal having a disease, disorder, or condition associated with clonal T cell proliferation. The method can comprise or consist essentially of administering a bispecific molecule comprising a polypeptide comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising a second antigen-binding domain capable of binding to a TRBC polypeptide to a mammal having a disease, disorder, or condition associated with clonal T cell proliferation. The mammal can be a human. The disease, disorder, or condition associated with clonal T cell proliferation can be graft-versus-host disease (GVHD), celiac disease, Felty's syndrome, Sjogren's syndrome, scleroderma, eosinophilic fasciitis, sclerosing myxedema, myositis, multiple sclerosis, Rasmussen's encephalitis, autoimmune thyroid disease, neuromyelitis optica, aplastic anemia, paroxysmal nocturnal hemoglobinuria, Alzheimer's disease, narcolepsy, or aging. The method may also include, after administration of the bispecific molecule, administering to the mammal a second bispecific molecule comprising a polypeptide comprising a third antigen-binding domain capable of binding to a TRBC polypeptide and a polypeptide comprising an antigen-binding domain capable of binding to a CD3 polypeptide. The CD3 polypeptide may be a CD3γ polypeptide, a CD3δ polypeptide, or a CD3ε polypeptide.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar to or equivalent to those described herein can be used to implement the present invention, and 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, shall prevail.In addition, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0019] 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 become apparent from the description and drawings, and from the claims. [Brief description of the drawings]

[0020] [Figure 1A] 1A-1C: Diagram showing the proposed selective TRBC1 depletion strategy. (FIG. 1A) Healthy human T cells contain two TRBC families: TRBC1+ and TRBC2+. Similarly, malignant clonal T cells are TRBC1+. TRBC1-TRBC1 bispecific antibodies link healthy TRBC1+ T cells to malignant TRBC1+ cells and also link healthy TRBC1+ T cells to other healthy TRBC1+ T cells, resulting in selective killing of malignant and healthy TRBC1+ populations while sparing healthy TRBC2+ T cells. [Figure 1B] Figures 1A-1C: Diagram showing the proposed selective TRBC1 depletion strategy. (Figure 1B) After treatment with TRBC1-TRBC1 bispecific antibody, residual malignant TRBC1+ T cells may persist, while healthy TRBC1+ effector cells may be depleted. The "mop-up" strategy involves subsequent TRBC1-CD3 bispecific antibody treatment to redirect healthy TRBC2+ effector T cells to kill residual malignant TRBC1+ T cells. [Figure 1C] 1A-1C: Diagram showing the proposed selective TRBC1 depletion strategy. (FIG. 1C) After treatment with TRBC1-TRBC1 bispecific antibody, residual malignant TRBC1+ T cells may persist, while healthy TRBC1+ effector cells may be depleted. The second "mop-up" strategy involves subsequent treatment with TRBC1-antibody drug conjugate (ADC) molecules, where an ADC targeting TRBC1 can bind to and kill residual malignant TRBC1+ T cells, while healthy TRBC2+ cells persist. [Diagram 2]Figures 2A-2C: Structures of anti-TRBC antibodies. (Figures 2A-2B) Structures of bispecific antibodies. (Figure 2A) An exemplary TRBC1-CD3 bispecific antibody composed of an αTRBC1 scFv linked to an αCD3 scFv. (Figure 2B) An exemplary TRBC1-TRBC1 bispecific antibody composed of an αTRBC1 scFv linked to a second αTRBC1 scFv. (Figure 2C) An exemplary αTRBC1-ADC. [Diagram 3] Figure 3: Diagram showing the arrangement of the variable light (VL) chain in green, the variable heavy (VH) chain in orange, the short peptide linker (G4S), the long peptide linker ((G4S)3), and the polyhistidine tail (H) in yellow in the four TRBC1-TRBC1 bispecific antibody constructs. scDb: single chain diabody. BITE: bispecific T cell engager. [Figure 4] Figures 4A-4B: Expression of the four TRBC1-TRBC1 bispecific antibody constructs. The four TRBC1-TRBC1 bispecific antibodies were purified and analyzed using unstained polyacrylamide gel electrophoresis (Figure 4A) or Western blot with rabbit anti-6xHis antibody and anti-rabbit antibody conjugated to HRP (Figure 4B). [Diagram 5] 5A-5B: Binding of TRBC1-TRBC1 bispecific antibodies. (FIG. 5A) Histograms showing binding of four TRBC1-TRBC1 bispecific antibodies (#1, #2, #3, and #4) to Jurkat cells (expressing TRBC1), HPB-ALL cells (expressing TRBC2), Jurkat cells with a T cell receptor knockout (TCR-KO), and healthy human T cells (ABO4 donor, expressing both TRBC1 and TRBC2). (FIG. 5B) Histograms showing binding of four TRBC1-TRBC1 bispecific antibodies in combination with TRBC1-CD3 bispecific antibody (αC1-CD3) to Jurkat cells, HPB-ALL cells, Jurkat TCR-KO cells, and AB04 healthy human T cells. [Figure 6]Figure 6: TRBC1-TRBC1 bispecific antibody and TRBC-CD3 bispecific antibody induce T cell interferon gamma (IFNγ) release against T cell cancer cell lines in vitro. 5x104 normal human T cells (from human donor AB04) were incubated with 5x104 indicated target T cell cancer cell lines (Jurkat cells or HPB-ALL cells) for 17 hours in the presence of 5ng / mL or 50ng / mL TRBC1-TRBC1#3 (anti-C1#3) or TRBC1-TRBC1#4 (anti-C1#4) or TRBC1-CD3 (anti-C1-CD3). T cell cytokine release was then assessed by IFNγ ELISA. [Figure 7] Figure 7: 5x104 normal human T cells were incubated with bispecific antibodies TRBC1-TRBC1#3 or TRBC1-TRBC1#4 or TRBC-CD3 (50ng / ml) for 17 hours. 10μL of precision counting beads were added and flow cytometry was used to assess the number of TRBC1-PE- and GFP-expressing cells. 500 beads were collected for each condition. Numbers next to the dot plots indicate the number of viable cells. [Figure 8] Figure 8: 5x104 normal human T cells were incubated with 5x104 wild type Jurkat-GFP cells for 17 hours in the presence of bispecific antibodies TRBC1-TRBC1#3 or TRBC1-TRBC1#4 or TRBC-CD3 (50ng / ml). 10μL of precision counting beads were added and the number of TRBC1-PE and GFP expressing cells was assessed using flow cytometry. 500 beads were collected for each condition. Numbers next to the dot plots indicate the number of viable cells. [Figure 9]Figure 9: 5x104 normal human T cells were incubated with 5x104 wild type HPB-ALL-GFP cells for 17 hours in the presence of bispecific antibodies TRBC1-TRBC1#3, TRBC1-TRBC1#4, or TRBC-CD3 (50ng / ml). 10μL of precision counting beads were added and the number of TRBC1-PE- and GFP-expressing cells was assessed using flow cytometry. 500 beads were collected for each condition. Numbers next to the dot plots indicate the number of viable cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description The present specification provides methods and materials for treating clonal T cell proliferation (e.g., pathogenic clonal T cell proliferation, e.g., T cell cancer). In some cases, the present specification provides bispecific molecules that can be used to treat T cell cancer. For example, the present specification provides bispecific molecules comprising at least two antigen binding domains, where the first antigen binding domain (e.g., the first scFv) and the second antigen binding domain (e.g., the second scFv) can each bind to a TRBC polypeptide. For example, the present specification provides bispecific molecules comprising at least two antigen binding domains, where the first antigen binding domain (e.g., the first scFv) can bind to a TRBC polypeptide and the second antigen binding domain (e.g., the second scFv) can bind to a T cell co-receptor polypeptide (e.g., a CD3 polypeptide). The present specification also provides methods for treating T cell cancer. For example, one or more bispecific molecules provided herein (e.g., a composition containing one or more bispecific molecules provided herein) can be administered to a mammal to treat the mammal with T cell cancer. In some cases, one or more bispecific molecules provided herein (e.g., one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and optionally a bispecific molecule comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may be administered to a mammal to activate T cells in the mammal to target (e.g., target and destroy) T cells expressing a TRBC polypeptide that can be targeted by the bispecific molecule.For example, one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain, each capable of binding to a TRBC polypeptide, can be administered to a mammal (e.g., a human) to activate T cells to target (e.g., target and destroy) T cells expressing a TRBC polypeptide (e.g., cancerous T cells), and optionally thereafter, a bispecific molecule comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T cell co-receptor polypeptide (e.g., a CD3 polypeptide) can be administered to the mammal to activate T cells to target (e.g., target and destroy) remaining T cells expressing a TRBC polypeptide (e.g., cancerous T cells).

[0022] Any suitable mammal (e.g., a mammal with clonal T cell proliferation, e.g., T cell cancer) can be treated as described herein. For example, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, and rats can be treated as described herein. In some cases, a human with T cell cancer can be administered one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain, each of which can bind to a TRBC polypeptide, and optionally a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide).

[0023] The materials and methods described herein can be used to treat mammals (e.g., humans) with any type of T cell cancer. In some cases, the T cell cancer treated as described herein can include one or more solid tumors. In some cases, the T cell cancer treated as described herein can be a hematological cancer. In some cases, the T cell cancer treated as described herein can be a primary cancer. In some cases, the T cell cancer treated as described herein can be a recurrent cancer. In some cases, the T cell cancer treated as described herein can be a metastatic cancer. In some cases, the T cell cancer treated as described herein can be a refractory cancer. In some cases, the T cell cancer treated as described herein can be a non-Hodgkin's lymphoma. Examples of T cell cancers that may be treated as described herein include, but are not limited to, acute lymphoblastic leukemia (ALL), peripheral T cell lymphoma (PTCL), angioimmunoblastic T cell lymphoma (AITL), T cell prolymphocytic leukemia (T-PLL), adult T cell leukemia / lymphoma (ATLL), enteropathy-associated T cell lymphoma (EATL), monomorphic epitheliotropic intestinal T cell lymphoma (MEITL), follicular T cell lymphoma (FTCL), nodal peripheral T cell lymphoma (nodal PTCL), cutaneous T cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL), T cell large granular lymphocytic leukemia (T-LGL), extranodal NK / T cell lymphoma (NKTL), and hepatosplenic T cell lymphoma.

[0024] In some cases, the materials and methods provided herein can be used to reduce or eliminate the number of cancer cells present in a mammal (e.g., a human) with a T-cell cancer. For example, a mammal in need thereof (e.g., a mammal with a T-cell cancer) can be administered one or more bispecific molecules provided herein (e.g., one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and optionally one or more bispecific molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T-cell co-receptor polypeptide, such as a CD3 polypeptide) to reduce or eliminate the number of cancer cells present in the mammal's body. For example, the materials and methods described herein can be used to reduce the number of cancer cells present in a mammal with a cancer by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. For example, the materials and methods described herein can be used to reduce the size (e.g., volume) of one or more tumors present in a mammalian body having cancer, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. In some cases, the number of cancer cells present in the mammalian body being treated can be monitored. Any suitable method can be used to determine whether the number of cancer cells present in the mammalian body is reduced. For example, imaging techniques can be used to assess the number of cancer cells present in the mammalian body.

[0025] In some cases, the materials and methods provided herein can be used to improve the survival of a mammal (e.g., a human) with a T-cell cancer. For example, a mammal in need thereof (e.g., a mammal with a T-cell cancer) can be administered one or more bispecific molecules provided herein (e.g., one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and optionally one or more bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T-cell co-receptor polypeptide, such as a CD3 polypeptide) to improve the survival of the mammal. For example, the materials and methods described herein can be used to improve the survival of a mammal with cancer, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. For example, the materials and methods described herein can be used to improve survival of a mammal with cancer, for example, by 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).

[0026] In some cases, when a mammal in need thereof (e.g., a mammal having a T-cell cancer) is administered one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and optionally a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T-cell co-receptor polypeptide, such as a CD3 polypeptide), a majority of normal T cells in the mammal's body (e.g., a sufficient number of normal T cells to maintain adequate T-cell immunity) can be preserved. For example, the materials and methods described herein can be used to treat a mammal having a T-cell cancer described herein while preserving, for example, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or more of normal (e.g., non-cancerous) T cells in the mammal's body. In some cases, when a mammal is administered one or more bispecific molecules provided herein, about 20% to about 75% (e.g., about 20% to about 65%, about 20% to about 55%, about 20% to about 45%, about 25% to about 75%, about 35% to about 75%, about 45% to about 75%, about 55% to about 75%, about 65% to about 75%, about 35% to about 65%, about 45% to about 55%, about 30% to about 50%, about 40% to about 60%, or about 50% to about 70%) of normal (e.g., non-cancerous) T cells in the mammal can be preserved.

[0027] In some cases, the methods described herein may also include identifying a mammal as having T cell cancer. Examples of methods for identifying a mammal as having T cell cancer include, but are not limited to, physical examination, clinical examination (e.g., blood and / or urine), biopsy, imaging examination (e.g., X-ray, PET / CT, MRI, and / or ultrasound), nuclear medicine scan (e.g., bone scan), endoscopy, and / or genetic testing. After being identified as having T cell cancer, the mammal may be administered or instructed to self-administer one or more bispecific molecules provided herein (e.g., one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain each capable of binding to a TRBC polypeptide, and optionally one or more bispecific molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide).

[0028] Any suitable bispecific molecule can be administered to a mammal (e.g., a human) as described herein. In some cases, the molecules provided herein can include at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) antigen binding domains. For example, a bispecific molecule can include at least two antigen binding domains, where a first antigen binding domain (e.g., a first scFv) and a second antigen binding domain (e.g., a second scFv) can each bind to a TRBC polypeptide. In some cases, the first antigen binding domain and the second antigen binding domain, each capable of binding to a TRBC polypeptide, can each bind to the same epitope on a TRBC polypeptide. In some cases, the first antigen binding domain and the second antigen binding domain, each capable of binding to a TRBC polypeptide, can have different affinities for a TRBC polypeptide. For example, a bispecific molecule can include at least two antigen binding domains, in which a first antigen binding domain (e.g., a first scFv) can bind to a TRBC polypeptide with a lower affinity than a second antigen binding domain can bind to the same TRBC polypeptide. In some cases, the first antigen binding domain and the second antigen binding domain, each capable of binding to a TRBC polypeptide, can bind to different epitopes on the TRBC polypeptide. For example, a bispecific molecule can include at least two antigen binding domains, in which a first antigen binding domain (e.g., a first scFv) can bind to a TRBC polypeptide and a second antigen binding domain can bind to a T cell co-receptor polypeptide (e.g., a CD3 polypeptide).Examples of molecules (e.g., bispecific molecules) that may include at least two antigen-binding domains, where a first antigen-binding domain (e.g., a first scFv) and a second antigen-binding domain (e.g., a second scFv) are each capable of binding to a TRBC polypeptide, include, but are not limited to, single-chain diabodies (scDbs), bispecific T cell engagers (BITEs), dual affinity retargeting molecules (DARTs), bivalent scFv-Fcs, and trivalent scFv-Fcs.

[0029] The antigen-binding domain of the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be any suitable type of antigen-binding domain. In some cases, the antigen-binding domain that can be used in the bispecific molecules provided herein can include a variable region (VL) of an immunoglobulin light chain and a variable region (VH) of an immunoglobulin heavy chain. For example, the antigen-binding domain that can be used in the bispecific molecules provided herein can include a first complementarity determining region (CDR) (VL) derived from an immunoglobulin light chain. L CDR1) and the second CDR (V L CDR2) and the third CDR (V L CDR3) and the first CDR (V H CDR1) and the second CDR (V H CDR2) and the third CDR (V HThe antigen-binding domain may comprise a single chain variable fragment (scFv), an antigen-binding fragment (Fab), an F(ab')2 fragment, and a biologically active fragment thereof (e.g., a fragment that retains the ability to bind to a target molecule, e.g., a TRBC polypeptide or a T cell co-receptor polypeptide, e.g., a CD3 polypeptide). In some cases, the antigen-binding domain that may be used as an antigen-binding domain in the bispecific molecules provided herein may be an scFv. In some cases, the two antigen-binding domains of the bispecific molecules provided herein may be the same type of antigen-binding domain. For example, each of the two antigen-binding domains of the bispecific molecules provided herein may be an scFv. In some cases, the two antigen-binding domains of the bispecific molecules provided herein may be different types of antigen-binding domains.

[0030] In some cases, the antigen-binding domain of the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may be a humanized antigen-binding domain.

[0031] The antigen-binding domain of the bispecific molecules provided herein that can bind to a TRBC polypeptide (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can bind to any suitable TRBC polypeptide. Examples of TRBC polypeptides that can be targeted by the antigen-binding domain of the bispecific molecules provided herein include, but are not limited to, TRBC1 polypeptide and TRBC2 polypeptide. In some cases, the antigen-binding domain capable of binding to a TRBC polypeptide is specific to that TRBC polypeptide. For example, an antigen-binding domain capable of binding to a TRBC polypeptide may have a concentration of about 0.01 nM to about 400 nM (e.g., about 0.01 nM to about 350 nM, about 0.01 nM to about 300 nM, about 0.01 nM to about 250 nM, about 0.01 nM to about 200 nM, about 0.01 nM to about 150 nM, about 0.01 nM to about 100 nM, about 0.01 nM to about 80 nM, about 0.01 nM to about 50 nM, about 0.01 nM to about 30 nM, about 0.01 nM to about 10 nM, about 0.01 nM to about 5 nM, about 0.01 nM to about 1 nM, about 0.01 nM to about 0.5 nM, about 0.4 nM 400 nM, about 1 nM to about 400 nM, about 5 nM to about 400 nM, about 10 nM to about 400 nM, about 50 nM to about 400 nM, about 100 nM to about 400 nM, about 200 nM to about 400 nM, about 300 nM to about 400 nM, about 0.05 nM to about 200 nM, about 0.1 nM to about 100 nM, about 0.03 nM to about 50 nM, about 0.04 nM to about 30 nM, about 0.05 nM to about 10 nM, about 0.1 nM to about 20 nM, about 0.2 nM to about 30 nM, about 0.3 nM to about 40 nM, about 0.4 nM to about 50 nM, or about 0.5 nM to about 100 nM) D) can bind to the TRBC polypeptide with an affinity having an affinity for the TRBC polypeptide. In some cases, an antigen-binding domain that specifically binds to a TRBC polypeptide does not bind (or does not substantially bind) to a different TRBC polypeptide.

[0032] In some cases, the antigen-binding domain that can be used in the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide such as a CD3 polypeptide) can bind to a TRBC1 polypeptide. For example, the antigen-binding domain capable of binding to a TRBC1 polypeptide can comprise each of the CDRs described below.

[0033] (Table 1) TIFF2024508749000001.tif53161

[0034] In some cases, the antigen-binding domains that can be used in the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may have one or more CDRs that are not 100% identical to the CDRs listed in Table 1 but retain the ability to bind to a TRBC1 polypeptide. For example, CDRs that include one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid substitutions with respect to the CDRs listed in Table 1 may be used in the antigen-binding domains that can be used in the bispecific molecules provided herein. Amino acid substitutions may be made in some cases by selecting substitutions that have a significantly different effect on maintaining (a) the structure of the peptide backbone in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at a particular site, or (c) the bulk of the side chain. For example, naturally occurring residues can be classified into the following groups based on the properties of their side chains: (1) hydrophobic amino acids (methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions.Non-limiting examples of conservative substitutions that may be made in the CDRs of the antigen-binding domains that may be used in the bispecific molecules provided herein include, but are not limited to, alanine to valine, arginine to lysine, asparagine to glutamine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to arginine, isoleucine to leucine, leucine to isoleucine, lysine to arginine, methionine to leucine, phenylalanine to leucine, proline to glycine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to phenylalanine, and / or valine to leucine substitutions.

[0035] In some cases, the antigen-binding domain capable of binding to a TRBC1 polypeptide comprises a V L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:2 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:3 L For example, an antigen-binding domain capable of binding to a TRBC1 polypeptide can include a light chain having an amino acid sequence as set forth in SEQ ID NO:7. For example, an antigen-binding domain capable of binding to a TRBC1 polypeptide can include a light chain having an amino acid sequence as set forth in SEQ ID NO:48. In some cases, an antigen-binding domain capable of binding to a TRBC1 polypeptide can include a light chain having an amino acid sequence as set forth in SEQ ID NO:4. H CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:5 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:6 HFor example, an antigen-binding domain capable of binding to a TRBC1 polypeptide can comprise a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:8. For example, an antigen-binding domain capable of binding to a TRBC1 polypeptide can comprise a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:49. In some cases, an antigen-binding domain capable of binding to a TRBC1 polypeptide can comprise a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:1. L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:2 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:3 L and a light chain having a V CDR3 and a V CDR4 comprising the amino acid sequence set forth in SEQ ID NO:4. H CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:5 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:6 H The antigen-binding domain capable of binding to a TRBC1 polypeptide may comprise a light chain comprising an amino acid sequence as set forth in SEQ ID NO:7, and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:8. For example, the antigen-binding domain capable of binding to a TRBC1 polypeptide may comprise a light chain comprising an amino acid sequence as set forth in SEQ ID NO:48, and a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO:49.

[0036] In some cases, the antigen-binding domain of a bispecific molecule provided herein capable of binding to a TRBC polypeptide (e.g., a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may be as described elsewhere (see, e.g., Maciocia et al., Nat. Med., 23:1416-1423 (2017); and U.S. Patent Application Publication No. 2017 / 0066827).

[0037] When the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell coreceptor polypeptide, such as a CD3 polypeptide) comprise an antigen-binding domain capable of binding to a T cell coreceptor polypeptide, the antigen-binding domain capable of binding to a T cell coreceptor polypeptide can bind to any suitable T cell coreceptor polypeptide. Examples of T cell coreceptor polypeptides that can be targeted by the second antigen-binding domain of the bispecific molecules provided herein include, but are not limited to, CD3 polypeptides, such as CD3γ polypeptides, CD3δ polypeptides, and CD3ε polypeptides.

[0038] In the case where the bispecific molecules provided herein comprise an antigen-binding domain capable of binding to a T cell co-receptor polypeptide, the bispecific molecule can bind to a CD3 polypeptide. For example, the antigen-binding domain capable of binding to a CD3 polypeptide can comprise one of each of the CDRs described below.

[0039] (Table 2) TIFF2024508749000002.tif180161

[0040] In some cases, the antigen-binding domains that can be used in the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may have one or more CDRs that are not 100% identical to the CDRs listed in Table 2 but retain the ability to bind to a TRBC1 polypeptide. For example, CDRs that include one or more (e.g., 1, 2, 3, 4, 5, or more) amino acid substitutions with respect to the CDRs listed in Table 2 may be used in the antigen-binding domains that can be used in the bispecific molecules provided herein. Amino acid substitutions may be made in some cases by selecting substitutions that have a significantly different effect on maintaining (a) the structure of the peptide backbone in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at a particular site, or (c) the bulk of the side chain. For example, naturally occurring residues can be classified into the following groups based on the properties of their side chains: (1) hydrophobic amino acids (methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions.Non-limiting examples of conservative substitutions that may be made in the CDRs of the antigen-binding domains that may be used in the bispecific molecules provided herein include, but are not limited to, alanine to valine, arginine to lysine, asparagine to glutamine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to arginine, isoleucine to leucine, leucine to isoleucine, lysine to arginine, methionine to leucine, phenylalanine to leucine, proline to glycine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to phenylalanine, and / or valine to leucine substitutions.

[0041] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a VHV domain comprising the amino acid sequence set forth in SEQ ID NO:9. L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:10 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:11 L For example, an antigen-binding domain capable of binding to a CD3 polypeptide can comprise a light chain comprising an amino acid sequence set forth in SEQ ID NO:15. For example, an antigen-binding domain capable of binding to a CD3 polypeptide can comprise a light chain comprising an amino acid sequence set forth in SEQ ID NO:17.

[0042] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a VHV domain comprising the amino acid sequence set forth in SEQ ID NO:19. L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:23 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:26 LFor example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a light chain comprising the amino acid sequence set forth in SEQ ID NO:36.

[0043] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a VHV domain comprising the amino acid sequence set forth in SEQ ID NO:20. L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:24 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:27 L For example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a light chain comprising an amino acid sequence set forth in SEQ ID NO:38.

[0044] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a V L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:25 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:28 L For example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a light chain comprising the amino acid sequence set forth in SEQ ID NO:40.

[0045] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a V L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:23 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:26 LFor example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a light chain comprising the amino acid sequence set forth in SEQ ID NO:42.

[0046] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a VHV domain comprising the amino acid sequence set forth in SEQ ID NO:12. H CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:13 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:14 H For example, an antigen-binding domain capable of binding to a CD3 polypeptide can comprise a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:16. For example, an antigen-binding domain capable of binding to a CD3 polypeptide can comprise a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:18.

[0047] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a VHV domain comprising the amino acid sequence set forth in SEQ ID NO:29. H CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:31 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:34 H For example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:37.

[0048] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:29. H CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:31 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:34 HFor example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:39.

[0049] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a VHV domain comprising the amino acid sequence set forth in SEQ ID NO:30. H CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:32 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:35 H For example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:41.

[0050] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises an amino acid sequence set forth in SEQ ID NO:29. H CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:33 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:34 H For example, an antigen-binding domain capable of binding to a CD3 polypeptide can include a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:43.

[0051] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a VHV domain comprising the amino acid sequence set forth in SEQ ID NO:9. L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:10 L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:11 L and a light chain having a V CDR3 and a V CDR4 comprising the amino acid sequence set forth in SEQ ID NO:12. HCDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:13 H CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:14 H The antigen-binding domain capable of binding to a CD3 polypeptide can comprise a light chain comprising an amino acid sequence set forth in SEQ ID NO:15 and a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:16. For example, the antigen-binding domain capable of binding to a CD3 polypeptide can comprise a light chain comprising an amino acid sequence set forth in SEQ ID NO:17 and a heavy chain comprising an amino acid sequence set forth in SEQ ID NO:18.

[0052] In some cases, an antigen binding domain capable of binding to a CD3 polypeptide can include a light chain having a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:26, and can include a heavy chain having a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:34. For example, an antigen binding domain capable of binding to a CD3 polypeptide can include a light chain comprising the amino acid sequence set forth in SEQ ID NO:42, and can include a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:43.

[0053] In some cases, an antigen binding domain capable of binding to a CD3 polypeptide can include a light chain having a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:20, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:27, and can include a heavy chain having a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:31, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:34. For example, an antigen binding domain capable of binding to a CD3 polypeptide can include a light chain comprising the amino acid sequence set forth in SEQ ID NO:38, and can include a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:39.

[0054] In some cases, an antigen binding domain capable of binding to a CD3 polypeptide can include a light chain having a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:21, a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO:25, and a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO:28, and can include a heavy chain having a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:30, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:32, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:35. For example, an antigen binding domain capable of binding to a CD3 polypeptide can include a light chain comprising the amino acid sequence set forth in SEQ ID NO:40, and can include a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:41.

[0055] In some cases, the antigen binding domain capable of binding to a CD3 polypeptide comprises a V L CDR1 and V comprising the amino acid sequence set forth in SEQ ID NO:23L CDR2 and V comprising the amino acid sequence set forth in SEQ ID NO:26 L and a heavy chain having a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO:29, a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO:33, and a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO:34. For example, an antigen-binding domain capable of binding to a CD3 polypeptide can comprise a light chain comprising the amino acid sequence set forth in SEQ ID NO:42, and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:43.

[0056] When the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell coreceptor polypeptide, such as a CD3 polypeptide) comprise an antigen-binding domain capable of binding to a T cell coreceptor polypeptide, the antigen-binding domain capable of binding to the T cell coreceptor polypeptide may be as described elsewhere (see, e.g., Zhu et al., Journal of Immunology, 1551903-1910 (1995); Junttila et al., Cancer Research, 74:5561-5571 (2014); and Rodrigues et al., Int. J. Cancer. Suppl., 7:45-50 (1992)).

[0057] In some cases, the first and second antigen-binding domains of the bispecific molecules provided herein (e.g., bispecific molecules comprising a first and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may be connected via a linker (e.g., a polypeptide linker). The linker may comprise any suitable number of amino acids. For example, the linker may comprise about 5 amino acids to about 20 amino acids (e.g., about 5 amino acids to about 20 amino acids, about 5 amino acids to about 17 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 12 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 8 amino acids, about 7 amino acids to about 20 amino acids, about 10 amino acids to about 20 amino acids, about 13 amino acids to about 20 amino acids, about 15 amino acids to about 18 amino acids, about 7 amino acids to about 18 amino acids, about 10 amino acids to about 15 amino acids, about 7 amino acids to about 12 amino acids, about 10 amino acids to about 16 amino acids, or about 12 amino acids to about 18 amino acids). In some cases, the linker can change the mobility of the bispecific molecule. In some cases, the linker can change the solubility of the bispecific molecule. The linker can comprise any suitable amino acid. In some cases, the linker can be a glycine-rich linker. In some cases, the linker can be a serine-rich and / or threonine-rich linker. The linker can connect the first and second antigen-binding domains of the bispecific molecules provided herein in any order. For example, the linker can connect the N-terminus of the first antigen-binding domain of the bispecific molecules provided herein to the C-terminus of the second antigen-binding domain of the bispecific molecules, or vice versa.Examples of linkers that can be used to connect the first and second antigen-binding domains of the bispecific molecules provided herein include, but are not limited to, the GGGGS linker (SEQ ID NO:44), the (GGGGS)3 linker (SEQ ID NO:45), and. TIFF2024508749000003.tif4128 is included. In some cases, the linkers described herein can also be used to connect the VH and VL of the antigen binding domains described herein.

[0058] In some cases, the bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can comprise one or more additional molecules (e.g., one or more additional polypeptides and / or one or more additional nanoparticles). In some cases, the additional molecules can alter (e.g., improve) the stability of the bispecific molecules. For example, the additional molecules can increase the half-life of the bispecific molecules provided herein (e.g., after administration to a mammal, e.g., a human, having a T cell cancer). In some cases, the additional molecules can be used to detect (e.g., visualize) the bispecific molecules (e.g., after administration to a mammal, e.g., a human, having a T cell cancer). In some cases, the additional molecules can be used to bind (e.g., isolate and / or purify) the bispecific molecules. When the additional molecule is a polypeptide, the polypeptide can include any suitable amino acid. When the additional molecule is a polypeptide, the polypeptide can include any suitable number of amino acids. For example, the additional polypeptide can include about 2 amino acids to about 10 amino acids (e.g., about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 4 amino acids to about 10 amino acids, about 6 amino acids to about 10 amino acids, or about 4 amino acids to about 8 amino acids). In some cases, the additional polypeptide can be a polyhistidine polypeptide (e.g., a polyhistidine tail or polyhistidine tag). The additional molecule can be at any suitable location within the bispecific molecule provided herein. For example, the additional molecule can be at the N-terminus of the bispecific molecule, the C-terminus of the bispecific molecule, or both the N-terminus and the C-terminus of the bispecific molecule.Examples of additional molecules that can be included in the bispecific molecules provided herein include, but are not limited to, binding domains that can target albumin, albumin polypeptides (or fragments thereof), fragment crystallizable (Fc) regions, and polyhistidine polypeptides, such as polyhistidine polypeptides that include the amino acid sequence HHHHHH (SEQ ID NO:47). In some cases, additional molecules that can be included in the bispecific molecules provided herein can be as described elsewhere (see, for example, Dave et al., MAbs, 8(7):1319-1335 (2016); Muller et al., J. Biol. Chem., 282(17):12650-12660 (2007); and Liu et al., Front. Immunol., 8:38 (2017)).

[0059] In some cases, one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a mammal (e.g., a human). For example, one or more bispecific molecules provided herein can be formulated into a pharma- ceutically acceptable composition for administration to a mammal (e.g., a human) having a T cell cancer. In some cases, one or more bispecific molecules provided herein can be formulated with one or more pharma- ceutically acceptable carriers (additives), excipients, preservatives, stabilizers, and / or diluents.Examples of pharma- ceutically acceptable carriers, excipients, preservatives, stabilizers, and diluents that may be used in the compositions described herein include, but are not limited to, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses, e.g., microcrystalline cellulose and cellulose ethers, e.g., hydroxypropyl cellulose (HPC) and the cellulose ether hydroxypropylmethylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), cross-linked polyvinylpyrrolidone (crospovidone), carboxymethylcellulose, polyethylene-polyoxypropylene block polymers, and cross-linked sodium carboxymethylcellulose (croscarmellose sodium). )), titanium dioxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methylparaben and propylparaben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, zinc salts, and sodium chloride, e.g., bacteriostatic 0.9% sodium chloride), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, and lecithin.

[0060] Compositions (e.g., pharmaceutical compositions) containing one or more bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be formulated into any suitable dosage form. Exemplary dosage forms include, but are not limited to, solid or liquid forms, including pills, capsules, tablets, gels, liquids, suspensions, solutions (e.g., sterile solutions), sustained release formulations, and delayed release formulations.

[0061] Compositions (e.g., pharmaceutical compositions) containing one or more bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be designed for oral or parenteral (e.g., topical, subcutaneous, intravenous, intraperitoneal, intrathecal, and intracerebroventricular) administration. When administered orally, the composition can be in the form of a pill, tablet, or capsule. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that can include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules 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.

[0062] A composition (e.g., a pharmaceutical composition) containing one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may be administered locally or systemically. For example, a composition containing one or more bispecific molecules provided herein may be administered locally by sentinel lymph node injection into one or more lymph nodes in the body of a mammal (e.g., a human). For example, a composition containing one or more bispecific molecules provided herein may be administered locally by intravenous injection into a mammal (e.g., a human). For example, a composition containing one or more bispecific molecules provided herein may be administered locally by intraperitoneal injection into a mammal (e.g., a human).

[0063] An effective amount (e.g., an effective dose) of one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) may vary depending on the severity of the T cell cancer, the route of administration, the age and general health of the subject, the use of excipients, the possibility of simultaneous use with other therapeutic treatments, e.g., the use of other agents, and / or the judgment of the treating physician.

[0064] An effective amount of a composition (e.g., a pharmaceutical composition) containing one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be any amount that can treat a mammal (e.g., a human) with a T cell cancer with a reasonable therapeutic index and / or without causing significant toxicity to the mammal. An effective amount of one or more bispecific molecules provided herein can be any suitable amount. The effective amount can remain constant or can be adjusted as a sliding scale or a variable dose depending on the mammal's response to the treatment. Various factors can affect the actual effective amount used for a particular application. For example, the frequency of administration, the duration of treatment, the use of multiple treatment agents, the route of administration, and the severity of the condition (e.g., T cell cancer) may require an increase or decrease in the actual effective amount administered.

[0065] The frequency of administration of a composition (e.g., a pharmaceutical composition) containing one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be any frequency that can treat a mammal (e.g., a human) having a T cell cancer with a reasonable therapeutic index and / or without causing significant toxicity to the mammal. For example, the administration frequency can be about twice a day to about once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every two months, once every three months, or once every four months. In some cases, administration can be continuous administration (e.g., continuous infusion). The administration frequency can remain constant or can be variable during the treatment period. A course of treatment with a composition containing one or more bispecific molecules provided herein can include a drug holiday. As with the effective amount, various factors may affect the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple therapeutic agents, route of administration, and severity of the condition (e.g., T-cell cancer) may require increased or decreased frequency of administration.

[0066] The effective period for administering a composition (e.g., a pharmaceutical composition) containing one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, or a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be any period that treats a mammal (e.g., a human) with a T cell cancer with a reasonable therapeutic index and / or without causing significant toxicity to the mammal. For example, the effective period can vary from a few days to a few weeks, months, or years. In some cases, the effective period for treating a mammal can range from about one month to about 10 years. Several factors can affect the actual effective period used for a particular treatment. For example, the effective period can vary depending on the frequency of administration, the effective amount, the use of multiple therapeutic agents, the route of administration, and the severity of the condition (e.g., T cell cancer) being treated.

[0067] In some cases, the methods and materials described herein may include administering one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide. For example, a mammal in need thereof (e.g., a mammal having a T-cell cancer) may be administered one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, but not a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T-cell co-receptor polypeptide (e.g., a CD3 polypeptide).

[0068] In some cases, the methods and materials described herein may include administering both one or more bispecific molecules comprising a first and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and one or more bispecific molecules comprising a first and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and a T cell co-receptor polypeptide (e.g., CD3 polypeptide). For example, a mammal in need thereof (e.g., a mammal having a T cell cancer) may be administered one or more bispecific molecules comprising a first and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and then one or more bispecific molecules comprising a first and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and a T cell co-receptor polypeptide (e.g., CD3 polypeptide). In some cases, a method comprising administering both one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and a bispecific molecule comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T cell co-receptor polypeptide (e.g., a CD3 polypeptide), may comprise a drug holiday between the administration of the one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and the administration of the one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain capable of binding to a TRBC polypeptide and a T cell co-receptor polypeptide (e.g., a CD3 polypeptide).In some cases, one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide may be administered to a mammal having a T-cell cancer, and when / at the time of recurrence of the T-cell cancer observed in the mammal, one or more bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T-cell co-receptor polypeptide (e.g., CD3 polypeptide) may be administered to the same mammal. For example, one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide may be administered to a mammal having a T-cell cancer, and one or more bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T-cell co-receptor polypeptide (e.g., CD3 polypeptide) may be administered to the same mammal from shortly after recurrence of the T-cell cancer observed in the mammal to several years after recurrence of the T-cell cancer observed in the mammal. For example, administration of one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, to a mammal and administration of one or more bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide (e.g., a CD3 polypeptide) to the same mammal may be administered at an interval of about 30 days to about 10 years.

[0069] In some cases, one or more bispecific molecules provided herein (e.g., bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and optionally a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be used as the sole active agent to treat a mammal (e.g., a human) with a T cell cancer. For example, one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide can be used as the sole active agent to treat a mammal (e.g., a human) with a T cell cancer. For example, one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide (e.g., a CD3 polypeptide), may be used as the sole active agent to treat a mammal (e.g., a human) with T cell cancer.

[0070] In some cases, the methods and materials described herein can include one or more (e.g., 1, 2, 3, 4, 5, or more) additional therapeutic agents used to treat a mammal (e.g., a human) with a T-cell cancer. For example, a mammal in need thereof (e.g., a mammal with a T-cell cancer) can be administered one or more bispecific molecules provided herein (e.g., one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide, and optionally one or more bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T-cell co-receptor polypeptide, such as a CD3 polypeptide) in combination with one or more anti-cancer agents (e.g., chemotherapeutic agents). In some cases, the anti-cancer agent can be an alkylating agent. In some cases, the anti-cancer agent can be a platinum compound. In some cases, the anti-cancer agent can be a taxane. In some cases, the anti-cancer agent can be a luteinizing hormone-releasing hormone (LHRH) agonist. In some cases, the anti-cancer agent may be an anti-estrogen. In some cases, the anti-cancer agent may be an aromatase inhibitor. In some cases, the anti-cancer agent may be an angiogenesis inhibitor. In some cases, the anti-cancer agent may be a checkpoint inhibitor. In some cases, the anti-cancer agent may be an immunotherapeutic agent. In some cases, the anti-cancer agent may be a poly(ADP)-ribose polymerase (PARP) inhibitor. In some cases, the anti-cancer agent may be a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) inhibitor. In some cases, the anti-cancer agent may be an inhibitor of PD / PD-L1 signaling. In some cases, the anti-cancer agent may target one or more epigenetic changes (e.g., DNA methylation and histone modifications).Examples of anti-cancer drugs include, but are not limited to, vincristine, prednisone, dexamethasone, busulfan, cisplatin, carboplatin, paclitaxel, docetaxel, nab-paclitaxel, altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine. , goserelin, leuprolide, tamoxifen, letrozole, anastrozole, exemestane, bevacizumab, olaparib, rucaparib, niraparib, nivolumab, pembrolizumab, durvalumab, atezolizumab, radioisotopes, monomethyl auristatin E (MMAE; e.g., vedotin), calicheamicin, deruxtecan, DM1, and any combination thereof. In some cases, the anticancer agent can be as described elsewhere (see, e.g., Zhang et al., Clin. Epigenet., 12:169 (2020), e.g., Tables 3 and 5; and Ghione et al., Curr. Hematol. Malig. Rep., 13(6):494-506 (2018), e.g., Sections II, III, and Table 1). In some cases, one or more additional therapeutic agents may be administered together with one or more bispecific molecules provided herein (e.g., in a single composition). For example, the antigen-binding domain capable of binding to a TRBC polypeptide described herein may be conjugated to one or more anti-cancer agents (e.g., in the form of an ADC). In some cases, one or more additional therapeutic agents may be administered independently of one or more bispecific molecules provided herein. When one or more additional therapeutic agents are administered independently of one or more bispecific molecules provided herein, one or more bispecific molecules provided herein may be administered first and one or more additional therapeutic agents may be administered second, or vice versa.

[0071] In some cases, the methods and materials described herein may include administering both one or more bispecific molecules comprising a first and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and one or more molecules comprising the first antigen binding domain, each capable of binding to a TRBC polypeptide, conjugated to one or more anti-cancer drugs. For example, a mammal in need thereof (e.g., a mammal having a T-cell cancer) may be administered one or more bispecific molecules comprising a first and a second antigen binding domain, each capable of binding to a TRBC polypeptide, followed by one or more molecules comprising the first antigen binding domain, each capable of binding to a TRBC polypeptide, conjugated to one or more anti-cancer drugs. In some cases, the method comprising administering both one or more bispecific molecules comprising a first and a second antigen binding domain each capable of binding to a TRBC polypeptide, and one or more molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide conjugated to one or more anti-cancer drugs, can include a drug holiday between the administration of one or more bispecific molecules comprising a first and a second antigen binding domain each capable of binding to a TRBC polypeptide, and the administration of one or more molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide conjugated to one or more anti-cancer drugs. In some cases, one or more bispecific molecules comprising a first and a second antigen binding domain each capable of binding to a TRBC polypeptide can be administered to a mammal having a T-cell cancer, and when / at the time a recurrence of the T-cell cancer is observed in the mammal, one or more molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide conjugated to one or more anti-cancer drugs can be administered to the same mammal.For example, one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide may be administered to a mammal having a T-cell cancer, and one or more molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide conjugated to one or more anti-cancer drugs may be administered to the same mammal from shortly after recurrence of T-cell cancer is observed in the mammal to several years after recurrence of T-cell cancer is observed in the mammal. For example, the administration of one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain each capable of binding to a TRBC polypeptide to a mammal and the administration of one or more molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide conjugated to one or more anti-cancer drugs to the same mammal may be administered at an interval of about 30 days to about 10 years.

[0072] In some cases, one or more molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide conjugated to one or more anti-cancer drugs (e.g., TRBC-ADC) can be administered to a mammal (e.g., a human) having a T-cell cancer independently of one or more bispecific molecules provided herein (e.g., one or more bispecific molecules comprising a first antigen binding domain and a second antigen binding domain each capable of binding to a TRBC polypeptide, and optionally one or more bispecific molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T-cell co-receptor polypeptide such as a CD3 polypeptide). For example, one or more molecules comprising a first antigen binding domain capable of binding to a TRBC polypeptide conjugated to one or more anti-cancer drugs can be used as the only active agent to treat a mammal (e.g., a human) having a T-cell cancer.

[0073] In some cases, the methods and materials described herein may include one or more (e.g., one, two, three, four, five, or more) additional treatments (e.g., therapeutic interventions) that are effective in treating T-cell cancer. For example, a mammal in need thereof (e.g., a mammal having a T-cell cancer) may be administered one or more bispecific molecules provided herein (e.g., one or more bispecific molecules comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and optionally one or more bispecific molecules comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T-cell co-receptor polypeptide, such as a CD3 polypeptide) in combination with one or more therapeutic interventions. Examples of therapeutic interventions that may be used as described herein to treat T-cell cancer include, but are not limited to, cancer surgery, radiation therapy, blood transplants (e.g., autologous and allogeneic blood transplants), bone marrow transplants (e.g., autologous and allogeneic bone marrow transplants), and any combination thereof. In some cases, one or more additional treatments effective to treat T cell cancer may be administered simultaneously with administration of one or more bispecific molecules provided herein. In some cases, one or more additional treatments effective to treat T cell cancer may be administered prior to and / or after administration of one or more bispecific molecules provided herein.

[0074] In some cases, one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first and a second antigen binding domain, each capable of binding to a TRBC polypeptide, and optionally a bispecific molecule comprising a first antigen binding domain capable of binding to a TRBC polypeptide and a second antigen binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be used to treat a mammal having clonal T cell proliferation other than cancer (e.g., pathogenic clonal T cell proliferation). For example, a mammal having a disease, disorder, or condition other than a T cell cancer associated with clonal T cell proliferation can be administered one or more bispecific molecules provided herein. In some cases, the disease, disorder, or condition other than a T cell cancer associated with clonal T cell proliferation can be an autoimmune disease. In some cases, the disease, disorder, or condition other than a T cell cancer associated with clonal T cell proliferation can be associated with graft rejection. Examples of diseases, disorders, and conditions associated with clonal T cell expansion that can be targeted using one or more bispecific molecules provided herein include, but are not limited to, graft-versus-host disease (GVHD), celiac disease, Felty's syndrome, Sjogren's syndrome, scleroderma, eosinophilic fasciitis, sclerosing myxedema, myositis, multiple sclerosis, Rasmussen's encephalitis, autoimmune thyroid disease, neuromyelitis optica, aplastic anemia, paroxysmal nocturnal hemoglobinuria, Alzheimer's disease, narcolepsy, and aging. For example, one or more bispecific molecules provided herein (e.g., a bispecific molecule comprising a first antigen-binding domain and a second antigen-binding domain, each capable of binding to a TRBC polypeptide, and optionally a bispecific molecule comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and a second antigen-binding domain capable of binding to a T cell co-receptor polypeptide, such as a CD3 polypeptide) can be used to selectively deplete clonally expanded T cells while sparing a portion of healthy T cells (e.g., a sufficient number of healthy T cells to maintain adequate T cell immunity).

[0075] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0076] Example 1: Antibodies targeting the TCR β chain constant region for the treatment of T cell malignancies Antibodies against pan-B cell markers CD19 or CD20 have shown success in treating B cell malignancies. Such treatments result in the loss of healthy B cells, but this depletion is usually well tolerated in patients. Similar targeting of pan-T cell markers may help control T cell malignancies, but the associated depletion of healthy T cells results in severe and intolerable immunosuppression.

[0077] This example describes the generation and evaluation of BsAbs targeting TRBC for the treatment of T cell cancer. BsAbs targeting TRBC can selectively target and deplete cancerous T cells. Bispecific antibodies targeting TRBC1 and TRBC2 + TRBC1 while sparing healthy human T cells + T-cell cancer and TRBC1 + Healthy human T cells can be selectively depleted (Figure 1A). + Healthy T cells are sufficient to maintain a functional immune system.

[0078] TRBC1 + Generation of bispecific antibodies to selectively target malignant T cells A TRBC1-CD3 bispecific antibody was generated by linking anti-TRBC1 scFv with anti-CD3 scFv (Figure 2A). + The second set of TRBC1-TRBC1 bispecific antibodies was generated by linking two anti-TRBC1 scFvs (Figure 2B). The TRBC1-TRBC1 bispecific antibodies bind to TRBC1 +T cells are then treated with another TRBC1 + Only connects with T cells. TRBC1-TRBC1 bispecific antibodies were generated using four different formats (Figure 3). All bispecific antibodies were generated by HEK293F cell transfection and subsequently purified using nickel affinity chromatography. The expression of bispecific antibodies was detected by SDS-PAGE gel electrophoresis (Figure 4A, 4B).

[0079] ScFv sequence TIFF2024508749000004.tif23654

[0080] The TRBC1-TRBC1 bispecific antibody + Binds to Jurkat T cells and TRBC2 + No binding to HPB-ALL cells Human T-cell carcinoma-derived cell lines have rearranged TCRβ genes and express either TRBC1 or TRBC2. Jurkat cell lines derived from T-cell acute lymphoblastic leukemia (T-ALL) patients express TRBC1. HPB-ALL cell lines derived from different T-ALL patients express TRBC2. Incubation of the four TRBC1-TRBC1 bispecific antibodies with Jurkat and HPB-ALL cell lines showed that TRBC1-TRBC1#3 and TRBC1-TRBC1#4 bound to Jurkat cells but not to HPB-ALL cells (Figure 5A). Jurkat cells with TCR gene knockout (TCR-KO) were used as a negative control and showed no binding to any of the four TRBC1-TRBC1 bispecific antibodies. Thus, the TRBC1-TRBC1#3 and TRBC1-TRBC1#4 bispecific antibodies retained TRBC1 binding ability, whereas the TRBC1-TRBC1#1 and TRBC1-TRBC1#2 bispecific antibodies did not. Healthy polyclonal T cells obtained from a human donor (AB04) consisted of both TRBC1 and TRBC2 subsets. TRBC1-TRBC1#3 and TRBC1-TRBC1#4 were able to bind to subsets of healthy human T cells (Figure 5A). Binding of the TRBC1-CD3 bispecific antibody to Jurkat cells, HPB-ALL cells, and healthy human T cells obtained from donor AB04 was also tested. The TRBC1-CD3 bispecific antibody was able to bind to both Jurkat cells (using anti-TRBC1 scFv and anti-CD3 scFv) and HPB-ALL cells (using anti-CD3 scFv) (Figure 5B). TRBC1-CD3 did not bind to Jurkat TCR-KO cells because they lack both TRBC1 and CD3 on the cell surface. TRBC1-CD3 also showed binding to healthy human T cells obtained from donor AB04 with two different intensities (peak 1 and peak 2). This is likely because TRBC1-CD3 binds to TRBC1+ healthy human T cells using both TRBC1 and CD3 antigens, causing higher intensity peak 1 staining.TRBC1-CD3 bound to TRBC2+ healthy human T cells using only the CD3 antigen and therefore also gave rise to a lower intensity peak 2.

[0081] TRBC1-TRBC1 bispecific antibody + Activating healthy human T cells against T cell cancer cell lines Since bispecific antibodies TRBC1-TRBC1#3 and TRBC1-TRBC1#4 showed binding to Jurkat cells, the ability of these bispecific antibodies to induce healthy human T cell activation against T cell cancer cell lines was studied. To assess the activity of TRBC1-TRBC1 bispecific antibody and TRBC1-CD3 bispecific antibody against T cell malignancies, healthy human T cells were co-cultured with T cell cancer cell lines in the presence or absence of different antibodies. Bispecific antibodies TRBC1-TRBC1#3 and TRBC1-TRBC1#4 showed increased IFNγ production in the presence of Jurkat cells but not in the presence of HPB-ALL cells (Figure 6). In contrast, TRBC1-CD3 bispecific antibody induced IFNγ secretion in the presence of both Jurkat and HPB-ALL cells.

[0082] The TRBC1-TRBC1 bispecific antibody + Selectively kills T cells and TRBC2 + Inducing healthy human T cells to preserve T cells To assess the cytotoxicity of the bispecific antibodies against healthy human T cells, healthy human T cells (AB04) were cultured in the presence of bispecific antibodies TRBC1-TRBC1#3, TRBC1-TRBC1#4, or TRBC1-CD3. Exposure to TRBC1-TRBC1#3 and TRBC1-TRBC1#4 increased the cytotoxicity of TRBC2. + This resulted in the selective loss of healthy human TRBC1+ T cell subsets without affecting T cell subsets (Figure 7). Conversely, exposure to the TRBC1-CD3 bispecific antibody increased the number of TRBC1 + and TRBC2 + This results in depletion of both healthy human T cell subsets (Figure 7).+ To test the cytotoxicity of the bispecific antibodies against T cell cancer cell lines, healthy human T cells were co-cultured with GFP-expressing Jurkat cells. Jurkat cells and TRBC1 were co-cultured with the bispecific antibodies TRBC1-TRBC1#3 or TRBC1-TRBC1#4. + Depletion of healthy T cells was observed, whereas TRBC2 + Healthy human T cells were preserved (Figure 8). Similar co-cultures in the presence of TRBC1-CD3 showed that TRBC1 + and TRBC2 + The depletion of both healthy human T cells and Jurkat cells was shown (Figure 8). This indicates that the bispecific antibodies TRBC1-TRBC1#3 and TRBC1-TRBC1#4 inhibited the expression of TRBC2 + TRBC1 while sparing healthy human T cells + We demonstrated that TRBC1-CD3 bispecific antibody treatment can kill Jurkat cells. + When healthy T cells are present, TRBC1 + and TRBC2 + Along with the loss of both healthy T cells, TRBC1 + Resulting in eradication of Jurkat cells. TRBC2 + To test the cytotoxicity of the bispecific antibodies against T cell cancer cell lines, healthy human T cells were co-cultured with GFP-expressing HPB-ALL cells. Exposure to bispecific antibodies TRBC1-TRBC1#3 or TRBC1-TRBC1#4 failed to eradicate HPB-ALL cells. Similar co-culture in the presence of TRBC1-CD3 bispecific antibody showed depletion of HPB-ALL cells (Figure 9).

[0083] Residual TRBC1 + Elimination of cancer cells TRBC1-TRBC1 bispecific antibody inhibits healthy TRBC1 by fratricide + This can result in the death of T cells. + Healthy T cells express TRBC1 +Before cancer cells are completely eliminated, they may fall to insufficient numbers to complete their task (Fig. 1B and Fig. 8). In such a scenario, a "mop-up" treatment with TRBC1-CD3 bispecific antibody may be used (Fig. 1B). + Since healthy T cells are absent or very scarce, T cell activation mediated by the anti-TRBC1 portion of the TRBC1-CD3 bispecific antibody is absent or minimal, and thus, TRBC2 + The T cell population remained largely intact, with residual TRBC1 + The remaining task of completely eliminating the cancer cells can be accomplished.

[0084] Treatment with toxin-conjugated TRBC1 scFv or TRBC1 antibody (TRBC1-ADC) inhibits TRBC1 without requiring an effector T cell population. + A second "mop-up" strategy may be provided to eliminate malignant cells (Figure 1C). This strategy also involves the elimination of healthy TRBC2 + T cell populations can remain intact, and healthy TRBC2 + T cell populations can maintain cell-mediated immunity.

[0085] summary Bispecific antibodies TRBC1-TRBC1#3 and TRBC1-TRBC1#4 bind to TRBC1 + Binds only to T cells and TRBC1 + Healthy TRBC1 for T-cell cancer + Activates human T cells and inhibits healthy TRBC2 + TRBC1 while preserving T cells + On the other hand, the TRBC1-CD3 bispecific antibody selectively depletes TRBC1 cells. + T cells and TRBC2 +T cells, resulting in the near-complete loss of all T cells. This demonstrates that the bispecific antibodies TRBC1-TRBC1#3 and TRBC1-TRBC1#4 are viable candidates for immunotherapy against T cell cancers. Furthermore, through fratricide, TRBC1-TRBC1 bispecific antibodies inhibited TRBC1 + When healthy T cells fall to insufficient numbers, residual TRBC1 + To completely eliminate cancer cells, TRBC2 + A "mop-up" strategy is proposed in which a TRBC1-CD3 bispecific antibody is used to recruit healthy T cells. Alternatively, a TRBC1-ADC molecule can be used to target residual TRBC1 + It may also be used to kill malignant cells.

[0086] Sequence Listing TIFF2024508749000005.tif186128TIFF2024508749000006.tif229159TIFF2024508749000007.tif223158TIFF2024508749000008.tif2163 9TIFF2024508749000009.tif22353TIFF2024508749000010.tif224159TIFF2024508749000011.tif208157TIFF2024508749000012.tif55158

[0087] Other Aspects Although the present invention has been described in conjunction with a detailed description thereof, it is to be understood that the above description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. a polypeptide comprising a first antigen-binding domain capable of binding to a T cell receptor beta chain constant region (TRBC) polypeptide; a polypeptide comprising a second antigen-binding domain capable of binding to the TRBC polypeptide; 2. A single chain bispecific molecule comprising:

2. The bispecific molecule of claim 1, wherein the polypeptide comprising a first antigen-binding domain capable of binding to a TRBC polypeptide and the polypeptide comprising a second antigen-binding domain capable of binding to a TRBC polypeptide are each independently selected from the group consisting of a single-chain variable fragment (scFv), an antigen-binding fragment (Fab), an F(ab')2 fragment, and biologically active fragments thereof.

3. The bispecific molecule of claim 1, wherein the binding affinity of the first antigen binding domain capable of binding to a TRBC polypeptide is lower than the binding affinity of the second antigen binding domain capable of binding to a TRBC polypeptide.

4. The bispecific molecule of any one of claims 1 to 3, wherein the TRBC polypeptide is a TRBC1 polypeptide or a TRBC2 polypeptide.

5. The bispecific molecule of claim 4, wherein the TRBC polypeptide is a TRBC1 polypeptide.

6. A first antigen-binding domain capable of binding to a TRBC1 polypeptide, or a second antigen-binding domain capable of binding to a TRBC1 polypeptide, V having the amino acid sequence set forth in SEQ ID NO:1 L CDR1 and V having the amino acid sequence set forth in SEQ ID NO:2 L CDR2 and V having the amino acid sequence set forth in SEQ ID NO:3 L a light chain comprising a CDR3 and a light chain comprising a V having the amino acid sequence set forth in SEQ ID NO:4 H CDR1 and V having the amino acid sequence set forth in SEQ ID NO:5 H CDR2 and V having the amino acid sequence set forth in SEQ ID NO:6 H Heavy chain comprising CDR3 and 6. The bispecific molecule of claim 5, comprising:

7. The bispecific molecule of claim 6, wherein the light chain comprises the amino acid sequence set forth in SEQ ID NO:7 and the heavy chain comprises the amino acid sequence set forth in SEQ ID NO:

8.

8. The bispecific molecule of claim 6, wherein said light chain comprises the amino acid sequence set forth in SEQ ID NO:48 and said heavy chain comprises the amino acid sequence set forth in SEQ ID NO:

49.

9. 9. The bispecific molecule of any one of claims 1 to 8, further comprising a molecule capable of improving the stability of said bispecific molecule.

10. A bispecific molecule described in any one of claims 1 to 9, comprising a single-chain diabody (scDb) format or a bispecific T cell engager (BITE) format.

11. The bispecific molecule described in claim 10, wherein the scDb format or BITE format includes a VL-VH-VL-VH format.

12. A pharmaceutical composition comprising a bispecific molecule according to any one of claims 1 to 11 for use in a method for treating a mammal having a T-cell cancer, the method comprising administering the bispecific molecule to the mammal.

13. 13. The pharmaceutical composition of claim 12, wherein the mammal is a human.

14. 14. The pharmaceutical composition of claim 12 or 13, wherein the T cell cancer is a clonal T cell cancer.

15. The T-cell cancers include acute lymphoblastic leukemia (ALL), peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma (AITL), T-cell prolymphocytic leukemia (T-PLL), adult T-cell leukemia / lymphoma (ATLL), enteropathy-associated T-cell lymphoma (EATL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), and follicular T-cell lymphoma.

15. The pharmaceutical composition according to any one of claims 12 to 14, selected from the group consisting of T-cell lymphoma (FTCL), nodal peripheral T-cell lymphoma (nodal PTCL), cutaneous T-cell lymphoma (CTCL), anaplastic large cell lymphoma (ALCL), T-cell large granular lymphocytic leukemia (T-LGL), extranodal NK / T-cell lymphoma (NKTL), and hepatosplenic T-cell lymphoma.

16. The pharmaceutical composition of any one of claims 12 to 15, wherein said cancer cells in said mammalian body are reduced by at least 50%.

17. The pharmaceutical composition of any one of claims 12 to 16, wherein the method is effective to improve survival of the mammal.

18. The method comprising, after administration of the bispecific molecule: a polypeptide comprising a third antigen-binding domain capable of binding to a TRBC polypeptide; a polypeptide comprising an antigen-binding domain capable of binding to a CD3 polypeptide; 18. The pharmaceutical composition of any one of claims 12-17, further comprising administering to the mammal a second bispecific molecule comprising:

19. The pharmaceutical composition of claim 18, wherein the CD3 polypeptide is selected from the group consisting of a CD3γ polypeptide, a CD3δ polypeptide, and a CD3ε polypeptide.

20. The method, after administration of the bispecific molecule, a polypeptide comprising a third antigen-binding domain capable of binding to a TRBC polypeptide; Anticancer drugs and The pharmaceutical composition of any one of claims 12 to 17, further comprising administering to the mammal a molecule comprising:

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