T cell binding proteins

Abstract

The present disclosure generally relates to binding proteins that include an antigen-binding site, a T cell receptor-binding site, and a T cell costimulatory molecule-binding site. The present disclosure also provides compositions that include such binding proteins and nucleic acid molecules that encode such binding proteins. The present disclosure further relates to methods of treating disorders or conditions using such binding proteins.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 329,583, filed April 11, 2022, the contents of which are incorporated herein by reference.

[0002] The present disclosure generally relates to binding proteins that include an antigen-binding site, a T cell receptor-binding site, and a T cell costimulatory molecule-binding site. The present disclosure also provides compositions that include such binding proteins and nucleic acid molecules that encode such binding proteins. The present disclosure further relates to methods of treating disorders or conditions using such binding proteins.

[0003] Sequence Listing A computer readable form of the sequence listing has been submitted with this application by electronic submission and is incorporated by reference in its entirety into this application. The sequence listing is contained in the file named "21-0750-WO.xml", created on March 14, 2023, and is 71,648 bytes in size. [Background technology]

[0004] Recruiting T cell cytotoxic activity to destroy tumor cells is a valuable but complex treatment strategy for cancer. The development of CD3-based bispecific T cell engagers (TCEs) as cancer therapeutics has been underway for the past 30 years. TCEs simultaneously bind tumor-associated antigens (TAAs) and cluster of differentiation antigen 3 (CD3) on T cells to form a T cell receptor (TCR)-independent artificial immune synapse, which circumvents human leukocyte antigen (HLA) restriction and induces T cell activation and cytolysis of tumor cells.

[0005] The first generation of TCEs are simple bispecific T cell engagers (BiTEs), which are composed of two tandem single-chain variable fragments (scFvs) containing a strong CD3-binding arm and a TAA-binding domain. To date, there is only one Food and Drug Administration-approved BiTE, blinatumomab, which targets CD3 (using the Orthoclone OKT3 antibody) and cluster of differentiation 19 (CD19). The strong in vitro cytolytic activity observed during the development of BiTEs has generated excitement based on their potential use to treat cancer. However, the unexpected high cytokine release syndrome (CRS) observed in the clinic has somewhat dampened the excitement (Non-Patent Document 1). Another observed disadvantage of the BiTE format is that it exhibits a very short half-life and is poorly manufacturable (Non-Patent Document 2).

[0006] The second generation of TCEs contains a fragment crystallizable (Fc) domain, which can be modified to confer half-life extension and mutations to eliminate Fc receptor (FcR) binding, demonstrating improved ease of manufacture (Non-Patent Document 3). Nevertheless, these molecules still contain high-affinity CD3 binding domains that lead to neurotoxicity and induction of CRS in the clinic. More recent efforts have focused on developing CD3 binding domains with reduced affinity in the hope of significantly lowering associated cytokine release while maintaining potent T cell activation (Non-Patent Document 4).

[0007] Importantly, both CD3-based BiTEs and novel immunoglobulin G (IgG) format TCEs have been shown to bind and activate both cluster of differentiation 4 (CD4) and cluster of differentiation 8 (CD8) T cells, potentially engaging undesirable T cells, such as regulatory T cells (Tregs), and potentially reducing the cytolytic activity of CD8 T cells (5).

[0008] While T cell engager molecules show promise, therapeutic approaches have faced challenges to date. There is a need in the art for improved T cell binding proteins with increased activity and reduced off-target effects. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Teachey et al., “Cytokine release syndrome after blinatumomab treatment related to abnormal macrophage activation and ameliorated with cytokine-directed therapy,” Blood 121:5154-57 (2013) [Non-Patent Document 2] Ellerman, “Bispecific T cell engagers:Towards understanding variables influencing the in vitro potency and tumor selectivity and their modulation to enhance their efficacy and safety,”Methods 154:102-17(2019) [Non-Patent Document 3] Vafa et al., “Perspective:Designing T cell Engagers With Better Therapeutic Windows,”Front Oncol.10:446(2020) [Non-Patent Document 4] Trinklein et al., “Efficient tumor killing and minimal cytokine release with novel T cell agonist bispecific antibodies,” MAbs 11:639-52 (2019) [Non-Patent Document 5] Duell et al., “Frequency of regulatory T cells determines the outcome of the T cell-engaging antibody blinatumomab in patients with B-precursor ALL,” Leukemia 31:2181-90(2017) Summary of the Invention [Means for solving the problem]

[0010] The present disclosure provides a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the first and second polypeptide chains have the formula: L -C L and the third polypeptide chain has a structure represented by the formula: H1 -C H1- V H2 -Fc; the fourth polypeptide chain has a structure represented by the formula: V H1 -C H1 -V H3 -Fc; V L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is a heavy chain variable domain that specifically binds to the T cell receptor; V H3 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule; Fc is a C H2 and C H3 An immunoglobulin heavy chain constant domain.

[0011] Also provided herein is a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, the two polypeptide chains having the formula: L -C L and the two polypeptide chains have a structure represented by the formula: H1 -C H1- V H2 -Fc-II2 having the structure represented by: V L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is the heavy chain variable domain that specifically binds to the T cell receptor; Fc is the C H2 and C H3 is an immunoglobulin heavy chain constant domain; II1 and II2 are each, independently, a heavy chain variable domain that specifically binds to a T cell costimulatory molecule or are absent; and at least one of II1 and II2 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule.

[0012] Also provided herein is a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, the two polypeptide chains having the formula: L -C L -II2; and the two polypeptide chains have the structure represented by the formula: V H1 -C H1- V H2 -Fc having the structure represented by: V L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; Lis an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is the heavy chain variable domain that specifically binds to the T cell receptor; Fc is the C H2 and C H3 is an immunoglobulin heavy chain constant domain; II1 and II2 are each, independently, a heavy chain variable domain that specifically binds to a T cell costimulatory molecule or are absent; and at least one of II1 and II2 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule.

[0013] Also provided herein is a binding protein comprising three polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the first polypeptide chain has the formula: L -C L and the second polypeptide chain has a structure represented by the formula: V H1 -C H1- V H2 -Fc; the third polypeptide chain has a structure represented by the formula: V H1 -V H3 -Fc having the structure represented by: V L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is a heavy chain variable domain that specifically binds to the T cell receptor; V H3 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule; Fc is a C H2 and C H3 An immunoglobulin heavy chain constant domain.

[0014] Also provided herein is a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, the polypeptide chains being: (a) amino acids of the sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; (b) amino acids of the sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; (c) amino acids of the sequences SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:10; (d) amino acids of the sequences SEQ ID NO:1, SEQ ID NO:11, and SEQ ID NO:12; (e) amino acids of the sequences SEQ ID NO:1, SEQ ID NO:13, and SEQ ID NO:14; (f) amino acids of the sequences SEQ ID NO:1, SEQ ID NO:15, and SEQ ID NO:16; and amino acids of the sequences SEQ ID NO:19; (g) amino acids of the sequences SEQ ID NO:1, SEQ ID NO:22, and SEQ ID NO:23; (h) amino acids of the sequences SEQ ID NO:29 and SEQ ID NO:30; (i) amino acids of the sequences SEQ ID NO:31 and SEQ ID NO:32; (j) amino acids of the sequences SEQ ID NO:33 and SEQ ID NO:34; (k) amino acids of the sequences SEQ ID NO:35 and SEQ ID NO:36; (l) amino acids of the sequences SEQ ID NO:37 and SEQ ID NO:38; (m) amino acids of the sequences SEQ ID NO:39 and SEQ ID NO:40; (n) amino acids of the sequences SEQ ID NO:41 and SEQ ID NO:42; or (o) amino acids of the sequences SEQ ID NO:45 and SEQ ID NO:54.

[0015] Also provided herein is a binding protein comprising three polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains comprise (p) amino acids of the sequences of SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, or (q) amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:11, and SEQ ID NO:12.

[0016] The accompanying drawings are included to provide a further understanding of the disclosed methods and compositions, and are incorporated in and constitute a part of this disclosure. The drawings illustrate one or more aspects of the disclosure and, together with the description, serve to explain the principles and operation of the disclosure. [Brief description of the drawings]

[0017] [Figure 1]Figure 1 illustrates a representative binding protein format of the present disclosure that includes two binding sites for tumor-associated antigens (TAA), one binding site for the T cell receptor (TCR) on a T cell, and one binding site for a T cell costimulatory molecule. Upon cleavage by a tumor-specific protease at the sites shown, the Fc domain is released to activate the T cell by TCR binding and activation of the costimulatory receptor. [Figure 2A-2B] Figures 2A-2B show the structures of the binding proteins evaluated in different in vitro assays. Figure 2A illustrates the MCAZ 6.9 binding protein, which contains a protease cleavage site. Figure 2B illustrates the MCAZ 6.10 binding protein, which contains a protease cleavage site. [Figure 3A-3B] Figures 3A-3C show line graphs depicting the evaluation of MCAZ 6.9 and MCAZ 6.10 binding protein in vitro cytolysis against CD20+ B cell lines. The EC50 for both MCAZ 6.9 and MCAZ 6.10 is shown for each cell line. Figure 3A illustrates the cytolysis evaluation using Daudi cells and purified pan T cells. Figure 3B illustrates the cytolysis evaluation using Ramos cells and purified pan T cells. Figure 3C illustrates the cytolysis evaluation using Raji cells and PBMCs. [Figure 3C] Same as above. [Figure 4A-4B] Figures 4A-4B show line graphs showing the percentage of surface CD25+ cells using Daudi cells as a means of analyzing CD4 and CD8 T cell activation profiles. Figure 4A shows results with MCAZ 6.9 binding protein. Figure 4B shows results with MCAZ 6.10 binding protein. [Figure 5A-5B] Figures 5A-5B show line graphs showing the percentage of surface CD25+ cells using Ramos cells. CD25+ assessment is used as a means to analyze CD4 and CD8 T cell activation profiles. Figure 5A shows results with MCAZ 6.9 binding protein. Figure 5B shows results with MCAZ 6.10 binding protein. [Figure 6]FIG. 6 shows a line graph depicting the percentage of surface CD25+ cells using Raji cells as a means of analyzing CD4 and CD8 T cell activation profiles. [Figure 7A-7C] Figures 7A-7C show in vitro cell lysis and T cell activation profiles induced by CD20 T cell MCAZ 88 binding proteins. Figure 7A shows a representative structure of MCAZ 88 binding proteins. Figure 7B shows MCAZ 88-induced cell lysis assessed against the Raji cell line (CD20+, 85371 antigens per cell). Figure 7C shows CD4 and CD8 T cell activation profiles assessed by measuring the % of surface CD25+ cells. [Figure 8A] FIG. 8A is a schematic diagram of MCAZ 7.5 and MCAZ 89. [Fig. 8B-8C] Figures 8B-8C show bar graphs illustrating non-specific CD4 and CD8 T cell activation in solution as assessed by flow cytometry detecting CD25+ status. Binding proteins, shown from top to bottom in the legend, are shown from left to right along the x-axis. Figure 8B shows non-specific CD8 activation. Figure 8C shows non-specific CD4 activation. [Figure 9A-9B] 9A-9B show bar graphs illustrating non-specific CD8 and CD4 T cell activation in plate-adsorbed antibodies assessed by flow cytometry detecting surface expression of CD25+. FIG. 9A shows non-specific CD8 activation. Binding proteins shown from top to bottom in the legend are shown from left to right along the x-axis. FIG. 9B shows non-specific CD4 activation. Binding proteins shown from top to bottom in the legend are shown from left to right along the x-axis. [Figure 10A-10C]Figures 10A-10E show in vitro cell lysis and T cell activation profiles induced by uncleaved binding proteins. Figure 10A shows the structure of MCAZ 7.1 binding protein. Figure 10B shows the structure of MCAZ 10.3 binding protein. Figure 10C shows that cell lysis was evaluated against CD20+ B cell lines Toledo, Oci-LY18, and SU-DHL5 (expressing 12420, 20244, and 27152 CD20 antigens per cell, respectively). Figure 10D shows MCAZ 7.1 cell lysis EC50 values ​​per cell line. Figure 10E shows CD4 and CD8 T cell activation assessed as % CD25 expressing cells. [Fig. 10D-10E] Same as above. [Figure 11A-11B]Figures 11A-11H show in vitro cell lysis, specific and non-specific T cell activation profiles induced by binding proteins with modified linker length. Figure 11A shows the structure of MCAZ 7.7 binding protein containing an additional linker between the CD20 and TCR binding domains. Figure 11B shows that cell lysis against the CD20+ Raji B cell line (expressing 85000 CD20 antigens per cell) was evaluated. Figure 11C shows CD4 and CD8 T cell activation profiles evaluated as % CD25 expressing cells. Figure 11D shows that binding proteins were plate bound and 1.5e5 purified T cells were added in the absence of tumor cells and associated proteases. Non-specific CD8 and CD4 T cell activation levels were evaluated by flow cytometry as % CD69+ / CD25+ surface expression. Figure 11E shows the structure of MCAZ 10.1 binding protein with more rigid CD8 arms due to removal of TGGS (SEQ ID NO: 46) linker and HA tag in addition to removal of truncated linker. Figure 11F shows that cytolysis against OCI-Ly18 B cell line was evaluated. B cell line was CTV stained and incubated with PBMC at E:T ratio of 5:1 for 3 days. Cytolysis % was measured by flow cytometry. Figure 11G shows that CD8 and CD4 T cell activation profile was evaluated as % CD25 cell expression. Figure 11H shows that CD8 and CD4 T cell activation profile was evaluated as % CD25 cell expression. [Fig. 11C-11D] Same as above. [Fig. 11E-11F] Same as above. [Fig. 11G-11H] Same as above. [Figures 12A-12C]Figures 12A-12E show the evaluation of the effect of CD8 positioning on binding proteins. Figure 12A shows the structure of the MCAZ 8.71 binding protein. Figure 12B shows the structure of the MCAZ 8.81 binding protein. Figure 12C shows that cytolysis was evaluated against the CD20+B Raji cell line (expressing 85000 CD20 antigens per cell). Figure 12D shows the cytolysis EC50 values ​​per cell line. Figure 12E shows the CD4 and CD8 T cell activation profile evaluated as % CD25 expressing cells. [Fig. 12D-12E] Same as above. [Figures 13A-13C] Figures 13A-13E show the evaluation of TCR VHH and CD8 VHH bispecific binding proteins. Figure 13A shows the structure of MCAZ 8.69 binding protein. Figure 13B shows the structure of MCAZ 8.70 binding protein. Figure 13C shows the cell lysis evaluated against the CD20+B Raji cell line (expressing 85000 CD20 antigens per cell). Figure 13D shows the cell lysis EC50 values ​​per cell line. Figure 13E shows the CD4 and CD8 T cell activation profile evaluated as % CD25 expressing cells. [Fig. 13D-13E] Same as above. [Figure 14A-14B] FIG. 14A shows the non-specific T cell activation evaluation for various binding proteins. Various concentrations of binding proteins were plate-bound and 1.5e5 purified T cells were added in the absence of tumor cells and associated proteases. After 48 hours of incubation, the non-specific CD8 T cell activation level was evaluated by flow cytometry as CD69+ / CD25+ surface expression %. FIG. 14B shows the non-specific T cell activation evaluation for various binding proteins. Various concentrations of binding proteins were plate-bound and 1.5e5 purified T cells were added in the absence of tumor cells and associated proteases. After 48 hours of incubation, the CD4 T cell activation level was evaluated by flow cytometry as CD69+ / CD25+ surface expression %. [Figure 15A] FIG. 15A is a schematic diagram of MCAZ 8.71 binding proteins with different Fc regions. [Fig. 15B-15D]Figure 15B shows the cytolytic activity of the binding proteins against the OCI-Ly18 B cell line at 72 hours by PBMCs at an E:T ratio of 5:1. Figure 15C shows the activation profile of CD8 and CD4 T cells assessed by measuring the % of surface CD25+ T cells. Figure 15D shows the activation profile of CD8 and CD4 T cells assessed by measuring the % of surface CD25+ T cells. [Figures 16A-16C] Figures 16A-16C show the cytolytic activity of MCAZ 7.1 variants (TENG0093) with modified linkers. Specifically, the variants contained similar linkers on the CD8 and TCR VHH arms shown in Figure 16A. The variants had different profiles from the broad CD3xCD20 bivalent engager. Figure 16A shows the structure of modified MCAZ 7.1 with different linkers and the broad CD3xCD20 bivalent engager used as a comparator. Figure 16B shows the cytolytic activity of MCAZ 7.1 variants and CD3xCD20 bivalent engager against the OCI-Ly18 B cell line. Figure 16C shows the CD4 and CD8 T cell activation profile assessed at 72 hours as surface expression of CD25. [Figure 17A-17B]Figures 17A-17F show that MCAZ 7.1 showed strong binding to CD8 T cells and induced selective binding of CD20+ tumor cells to CD8 T cells compared to a broad range of CD3xCD20 bivalent engagers. Figure 17A shows the MCAZ 7.1 binding profile for a CD20+ tumor B cell line (OCI-Ly-18). Figure 17B shows the MCAZ 7.1 binding profile for purified CD4 T cells from PBMCs from a healthy donor. Figure 17C shows the MCAZ 7.1 binding profile for purified CD8 T cells from PBMCs from a healthy donor. Figure 17D shows the expansion of CD3xCD20 bivalent engagers binding to CD8 T cells. Figure 17E shows the percentage of CD8B cell conjugates and CD4T cell conjugates assessed by flow cytometry after staining of CD4 and CD8 T cells after incubation with MCAZ 7.1. FIG. 17F shows the % of CD8 B cell conjugates and CD4 T cell conjugates assessed by flow cytometry after staining of CD4 and CD8 T cells following incubation with CD3×CD20 bivalent engagers. [Fig. 17C-17F] Same as above. [Fig. 18A-18F] Figures 18A-18F show that MCAZ7.1 selective engagement of CD8 T cells during cytolysis was associated with significantly lower cytokine release than broad CD3+ T cell engagement by CD3xCD20 bivalent engagers. MCAZ7.1 binding protein and CD3xCD20 bivalent engagers were incubated with OCI-Ly18 B cell line and PBMCs at an E:T ratio of 5:1 for 72 hours. Supernatants were collected and analyzed by multiplex assays to measure the concentrations of released proinflammatory cytokines: IL-6 (Figure 18A), TNF-a (Figure 18B), IL-10 (Figure 18C), IFN-g (Figure 18D), IL-2 (Figure 18E), and IL-17A (Figure 18F). [Figure 19A]Figures 19A-19B show that MCAZ 7.1 did not induce significant levels of T cell activation and cytokine release compared to CD3xCD20 bivalent engagers. Various concentrations of MCAZ 7.1 binding protein and CD3xCD20 bivalent engagers were plate bound and incubated with 1.5e5 PBMCs from healthy donors for 48 hours. Figure 19A shows CD4 and CD8 non-specific T cell activation profiles assessed by flow cytometry for CD25 / CD69+ surface expression levels. Figure 19B shows multiplex assays measuring the concentration of released proinflammatory cytokines for collected supernatants. [Figure 19B] Same as above. [Figure 20A-20B] Figures 20A-20B show that MCAZ 7.1 variant (TENG0093) binding protein and CD3xCD20 bivalent engager induced strong cytolytic activity in a 3D spheroid model. A GFP-expressing CD20+ B cell line (TMD8, 100000 CD20 / cell) was plated in low attachment plates to form 3D spheroids for 72 hours. Purified panT cells were then added at an E:T ratio of 15:1 and co-incubated with no engager, MCAZ 7.1 variant binding protein, or CD3xCD20 bivalent engager for 96 hours. Figure 20A shows representative images of spheroids with MCAZ 7.1 variant binding protein or CD3xCD20 bivalent engager in the absence of binding protein. FIG. 20B shows the mean % of GFP signal intensity at various concentrations of T cell engagers after 96 hours of incubation compared to no engager controls. [Figure 21] Figure 21 shows PK evaluation of MCAZ 7.1 binding protein after a single dose. PanT cell-humanized NSG mice were injected with 0.5 mg / kg of the indicated binding protein and systemic concentrations of the binding protein were measured at 1, 6, 24, 48, 72, and 168 hours. [Figure 22]Figure 22 shows the in vivo efficacy of MCAZ 7.1 binding protein in B cell lymphoma in a humanized NSG mouse model. NSG mice were implanted with panT cells 2 days before the start of the study. 5e6 OCI-Ly18 CD20+ tumor cell line was implanted sc on day 0 and animals were dosed ip with 1 mg / kg MCAZ7.1 binding protein or CD3xCD20 bivalent engager on day 2 and then weekly at the same concentrations as indicated by the grey arrows under the x-axis. [Fig. 23A-23D] Figures 23A-23F show in vivo cytokine release assessment of MCAZ 7.1 binding protein compared to CD3xCD20 bivalent engagers in a CRS mouse model. Mean + / - SD are plotted for IL-6 (Figure 23A), TNF-a (Figure 23B), IL-10 (Figure 23C), IFN-g (Figure 23D), IL-2 (Figure 23E), and IL-17A (Figure 23F). [Fig. 23E-23F] Same as above. [Fig. 24A-24D] Figures 24A-24H show binding protein in vitro activity for MCAZ 7.1 and MCAZ 7.1 variants on PBMCs from non-Hodgkin's lymphoma (NHL) donors. B cell death was evaluated on PBMCs from NHL (DLBCL) donors. MCAZ 7.1 variant binding protein was spiked into PBMCs at the indicated concentrations and 48 hours later, and B cell % was evaluated by flow cytometry for each patient (Figure 24A, Figure 24C, Figure 24E, Figure 24G). B cell cytolysis % was shown at various concentrations of the binding protein shown for three different DLBCL donors (Figure 24B, Figure 24D, Figure 24F, Figure 24H). CD4 and CD8 T cell activation profile was evaluated as % of CD25+ T cells. [Fig. 24E-24F] Same as above. [Fig. 24G-24H] Same as above. [Fig. 25A-25B] Figures 25A-25B show representative molecular formats of EGFR TITAN and a range of CD3 engagers used as comparators. [Figure 26A-26B]Figure 26A shows the binding profile of EGFR TITAN (MCAZ13.8) and a broad CD3 comparator to NCIH196. Figure 26B shows the binding profile of EGFR TITAN (MCAZ13.8) and a broad CD3 comparator to MDA-MB231. [Figure 27A-27B] Figures 27A-27B show in vitro cytotoxicity and T cell activation profiles for NCIH196-EGFR high. Figure 27A shows equivalent cytolytic activity of EGFR TITAN and a broad range of CD3 engagers used as comparators against the NCIH196 tumor cell line. Figure 27B shows a strongly biased CD8 T cell activation profile for EGFR TITAN (MCAZ13.8) highlighted by the % CD25 surface expression on T cells. [Fig. 28A-28E] Figures 28A-28E show that EGFR TITAN (MCAZ13.8) selective engagement of CD8 T cells during cytolysis is associated with significantly lower cytokine release than broad CD3+ T cell engagement with the CD3xEGFR bivalent comparator. [Figure 29A-29B] Figures 29A-29B show in vitro cytotoxicity and T cell activation bias for MDA-MB-231-EGFR high. Figure 29A shows equivalent cytolytic activity of EGFR TITAN on the MDA-MB-231 tumor cell line and a broad range of CD3 engagers used as comparators. Figure 29B shows a strongly biased CD8 T cell activation profile for EGFR TITAN (MCAZ13.8) highlighted by the % CD25 surface expression on T cells. [Fig. 30A-30E] Figures 30A-30E show that EGFR TITAN (MCAZ13.8) selective engagement of CD8 T cells during cytolysis is associated with significantly lower cytokine release than broad CD3+ T cell engagement with the CD3xEGFR bivalent comparator. [Figure 31A]Figures 31A-31B show T cell activation. Figure 31A shows that EGFR TITAN (MCAZ 13.8) binding protein does not activate T cells when plate-bound at various concentrations. In comparison, a broad CD3 engager used as a comparator induces significant T cell activation at the highest concentration used. Figure 31B shows that the absence of T cell activation induced by plate-bound EGFR TITAN (MCAZ 13.8) is associated with the absence of cytokine release. [Figure 31B] Same as above. [Fig. 32A-32C] Figures 32A-32C show that TENG0093 potently depletes B cells in fully humanized NSG mice. NSG-SGM3 mice were humanized with human stem cells (CD34+ cells). After 14 weeks, animals were treated IP with 20 mg / kg Fc block and 16 hours later with 1 mg / kg TENG0093 or CD3xCD20 bivalent engager. At day 6 post engager treatment, B cell depletion efficacy was assessed by flow cytometry with anti-CD19 surface staining. Both CD20 T cell engagers induced potent and nearly complete B cell depletion efficacy in blood (Figure 32A), spleen (Figure 32B), and bone marrow (Figure 32C) compared to the PBS-treated group (untreated group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present disclosure generally relates to binding proteins that include an antigen-binding site, a T cell receptor-binding site, and a T cell costimulatory molecule-binding site. The present disclosure also provides compositions that include such binding proteins and nucleic acid molecules that encode such binding proteins. The present disclosure further relates to methods of treating disorders or conditions using such binding proteins.

[0019] The binding proteins disclosed herein selectively bind to CD8+ T cells. As a result of selective binding to CD8+ T cells, the binding proteins avoid engagement with tumor-promoting T cells, such as Treg, Th2, and Th17 cells, and avoid engagement with CD4+ T cells, which produce most of the cytokines that cause CD4+ release syndrome (CRS). By lowering the proportion of activated T cells, the binding proteins disclosed herein lower CRS. In addition, CD8+ T cells bound by the binding proteins disclosed herein induce a type of programmed cell death called pyroptosis, which is immunogenic. Pyroptotic cells can be addressed by antigen-presenting cells to drive further tumor-specific T cell responses.

[0020] It should be understood that the specific aspects of the present disclosure described herein are not limited to the specific aspects presented and may vary. It should also be understood that the terminology used herein is only for describing specific aspects and is not intended to be limiting unless otherwise defined herein. Furthermore, the specific aspects disclosed herein can be combined with other aspects disclosed herein without limitation, as recognized by those skilled in the art.

[0021] Unless otherwise indicated, or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​herein expressed as ranges can assume any specific value or subrange within the ranges set forth in various embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless otherwise clearly indicated from the content.

[0022] Throughout this disclosure, unless the context specifically indicates otherwise, the terms "comprise" and "include" and variations thereof (e.g., "comprises," "comprising," "includes," and "including") will be understood to indicate the inclusion of a stated component, feature, element, or step, or group of components, features, elements, or steps, but not the exclusion of any other components, features, elements, steps, or group of components, features, elements, or steps. The terms "comprising," "consisting essentially of," and "consisting of" may all be substituted with either of the other two terms while retaining their ordinary meaning.

[0023] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0024] The percentages disclosed herein can vary in amounts of ±10, 20, or 30% from the disclosed values ​​and remain within the contemplated range of the disclosure.

[0025] Ranges and amounts used herein may be expressed as "about" a particular value or range. About also includes the exact amount. For example, "about 5%" means "about 5%" and also means "5%". The term "about" may also refer to ±10% of a given value or range of values. Thus, about 5% also means, for example, 4.5% to 5.5%.

[0026] As used herein, the terms "or" and "and / or" are used to describe multiple elements in combination with each other or exclusively with each other. For example, "x, y, and / or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z."

[0027] As utilized in accordance with this disclosure, unless otherwise indicated, all scientific and technical terms shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0028] The term "binding protein" as used herein refers to a non-naturally occurring (or recombinant) molecule comprising multiple polypeptide chains that form at least one antigen binding site.

[0029] A "recombinant" molecule is one prepared, expressed, produced, or isolated by means of recombinant DNA technology.

[0030] The term "antibody" as used herein refers to a protein capable of recognizing and specifically binding to an antigen. Normal or conventional mammalian antibodies are composed of a tetramer, which is typically composed of two identical pairs of polypeptide chains, each pair consisting of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light chain and each heavy chain typically contains a variable domain of about 100-110 or more amino acids that are typically responsible for antigen recognition. The variable domains may be subject to further protein engineering to humanize framework regions if the antibody is derived from a non-human source. The carboxyl-terminal portion of each chain typically defines a constant domain that is responsible for effector functions. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide comprises a variable domain (V H ), three constant domains (C H1 , C H2 , and C H3 ), and C H1 and C. H2 and the hinge region between V H The domain is located at the amino terminus of the polypeptide and isH3 The domains are located at the carboxyl terminus, and full-length light chain immunoglobulin polypeptides contain a variable domain (V L ) and the constant domain (C L ), including V L The domain is located at the amino terminus of the polypeptide and is L The domain is present at the carboxyl terminus.

[0031] Within full-length light and heavy chains, the variable and constant domains are typically linked by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit relatively conserved framework regions (FR) of identical overall structure linked by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both light and heavy chains typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0032] An "antigen-binding fragment thereof" refers to at least the smallest portion of an antibody that is capable of binding to a particular antigen to which the antibody is targeted, e.g., in the context of a typical antibody produced by a B cell, the heavy chain (V H ) variable domain and light chain (V L The term "antibody" refers to at least a portion of the complementarity determining region (CDR) of a variable domain of an antibody or antigen-binding fragment thereof. An antibody or antigen-binding fragment thereof may be a polyclonal antibody, a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, an epitope-binding fragment, such as Fab, Fab', and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), V L Domain or V H A fragment containing a domain alone or in combination with a portion of the opposing domain (e.g., the entire VL A domain and a portion V having one, two or three CDRs H domain), as well as fragments produced by a Fab expression library. scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0033] The term "native Fc" as used herein refers to a molecule, whether monomeric or multimeric, that comprises the sequence of a non-antigen-binding fragment resulting from the digestion of an antibody or generated by other means, and may contain a hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and may be any immunoglobulin. A native Fc molecule is composed of monomeric polypeptides, which may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent associations. The number of intramolecular disulfide bonds between monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is a disulfide-linked dimer resulting from papain digestion of IgG. The term "native Fc" as used herein is a collective term for monomeric, dimeric, and multimeric forms.

[0034] The term "Fc variant" as used herein refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor FcRn (neonatal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" can include a molecule or sequence that has been humanized from a non-human native Fc. Additionally, a native Fc includes regions that can be removed or mutated to generate an Fc variant that alters specific residues that provide structural features or biological activities not required for the binding proteins of the present disclosure. Thus, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues or in which one or more Fc portions or residues have been modified that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0035] The term "Fc" as used herein encompasses native Fc and Fc variants as defined above. As with Fc variants and native Fc molecules, the term "Fc" includes monomeric or multimeric forms of the molecule, whether digested from a full-length antibody or produced by other means.

[0036] Binding proteins encompassed by the present disclosure can be of or derived from any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0037] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule that can be recognized and bound by the antigen-binding portion of the binding protein of the present disclosure. The target antigen can be used to generate antibodies in an animal that can bind to an epitope of that antigen. A target antigen can have one or more epitopes. For each target antigen recognized by the antigen-binding portion of the binding protein, it can compete with an intact antibody that recognizes the target antigen.

[0038] The term "antigen-binding site" as used herein refers to a site occurring on the surface of a binding protein of the present disclosure, to which an antigen or an epitope on an antigen binds.

[0039] The term "linker" as used herein refers to one or more amino acid residues inserted between the domains of the binding protein of the present disclosure. For example, a linker can be inserted between the domains at the sequence level. The exact location of the domain transition can be determined by locating a peptide stretch that does not form a secondary structural element, such as a beta sheet or an alpha helix, as demonstrated by experimental data or as can be envisaged by modelling or secondary structure prediction techniques. Since natural linkers are often found between immunoglobulin domains, a linker may or may not be required depending on where the end and start residues of the protein fusion are selected.

[0040] The term "polynucleotide" as used herein includes single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). The term "nucleic acid" includes any type of nucleic acid, such as DNA or RNA.

[0041] The term "vector" as used herein may refer to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may contain a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. Certain types of vectors contemplated herein may be combined with or incorporated into viruses to facilitate cell transformation.

[0042] As used herein, the terms "treat", "treatment", or "treatment of" refer to alleviating disease pathology, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treating can refer to the ability of a therapy to alleviate the signs, signs, or causes of a disease when administered to a subject. Treating can also refer to the alleviation or reduction of at least one clinical symptom, and / or inhibiting or slowing the progression of a symptom, and / or preventing or delaying the onset of a disease or condition.

[0043] As used herein, the term "administration" or "administering" refers to providing, contacting, and / or delivering a binding protein by any route appropriate to achieve a desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.

[0044] As used herein, the terms "subject," "individual," or "patient" refer to any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0045] As used herein, an "effective amount" or "therapeutically effective amount" of an administered therapeutic agent, e.g., a binding protein, is an amount sufficient to carry out a specifically stated or intended purpose, such as treating cancer or the treatment of cancer. An "effective amount" can be determined based on routine experimentation for the stated purpose.

[0046] The term "pharmaceutical composition" as used herein refers to a compound or composition that can induce a desired therapeutic effect when properly administered to a subject. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of a binding protein of the present disclosure. As used herein, the term "pharmaceutical acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing the delivery of one or more binding proteins of the present disclosure.

[0047] In certain embodiments, the binding proteins disclosed herein may be formulated as pharmaceutical compositions with pharma- ceutically acceptable carriers, excipients, or stabilizers. In certain embodiments, such pharmaceutical compositions are suitable for administration to humans or non-human animals via any one or more routes of administration using methods known in the art. The term "pharmaceutically acceptable carrier" refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations may routinely contain salts, buffers, preservatives, compatible carriers, and optional other therapeutic agents. Such pharma- ceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that may be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients, such as serum albumin, gelatin, casein, salt-forming counterions, such as sodium, and the like. These and additional known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art and are, for example, as listed in "Remington: The Science & Practice of Pharmacy," 21st ed., Lippincott Williams & Wilkins, (2005) and "Physician's Desk Reference," 60th ed., Medical Economics, Montvale, NJ (2005). Pharmaceutically acceptable carriers suitable for the desired or required mode of administration, solubility, and / or stability can be selected.

[0048] In some embodiments, provided herein is a binding protein comprising two tumor-associated antigen (TAA) binding sites. In some embodiments, the tumor-associated antigen (TAA) is cluster of differentiation 20 (CD20). CD20 binds to Ca ++CD20 is a transmembrane protein involved in channeling, B cell activation, and proliferation. CD20 is a membrane-embedded surface molecule that plays a role in the development and differentiation of B cells into plasma cells. In some embodiments, the binding protein comprises a fragment of rituximab (see, e.g., U.S. Pat. No. 5,736,137).

[0049] In some embodiments, provided herein is a binding protein that comprises one T cell receptor (TCR) binding site. TCR comprises a heterodimer that comprises highly variable alpha (α) and beta (β) chains. The multicomponent complex of TCR contains the CD3 co-receptor, which plays an important role in activating T cells.

[0050] In some embodiments, provided herein is a binding protein comprising one T cell costimulatory molecule binding site. The costimulatory molecule comprises a costimulatory domain that can enhance or modulate the response of immune effector cells. The costimulatory domain can comprise, for example, a sequence from one or more of CD3 zeta (or CD3z), CD28, CD137 (4-1BB), OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. In some embodiments, the T cell costimulatory molecule is CD8. In some embodiments, the T cell costimulatory molecule is CD137 (4-1BB).

[0051] Some embodiments described herein provide a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the first and second polypeptide chains have the formula: L -C L and the third polypeptide chain has a structure represented by the formula: H1 -C H1- V H2 -Fc; the fourth polypeptide chain has a structure represented by the formula: V H1 -C H1 -V H3 -Fc; V Lis an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is a heavy chain variable domain that specifically binds to the T cell receptor; V H3 is the heavy chain variable domain that specifically binds to a T cell costimulatory molecule; Fc is the immunoglobulin hinge region, as well as C H2 and H3 An immunoglobulin heavy chain constant domain.

[0052] Some embodiments described herein provide a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the two polypeptide chains have the formula: L -C L and the two polypeptide chains have a structure represented by the formula: H1 -C H1- V H2 -Fc-II2 having the structure represented by: V L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is the heavy chain variable domain that specifically binds to the T cell receptor; Fc is the immunoglobulin hinge region, as well as C H2 and C H3is an immunoglobulin heavy chain constant domain; II1 and II2 are each, independently, a heavy chain variable domain that specifically binds to a T cell costimulatory molecule or are absent; and at least one of II1 and II2 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule.

[0053] Some embodiments described herein provide a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the two polypeptide chains have the formula: L -C L II2; the two polypeptide chains have the structure represented by the formula: V H1 -C H1- V H2 -Fc having the structure represented by: V L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is the heavy chain variable domain that specifically binds to the T cell receptor; Fc is the immunoglobulin hinge region, as well as C H2 and C H3 is an immunoglobulin heavy chain constant domain; II1 and II2 are each, independently, a heavy chain variable domain that specifically binds to a T cell costimulatory molecule or are absent; and at least one of II1 and II2 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule.

[0054] Some embodiments described herein provide a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the two polypeptide chains have the formula: L -C L and one polypeptide chain has a structure represented by the formula: VH1 -C H1- Fc; one polypeptide chain has the structure represented by the formula: H2 -V H3- Fc: L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule; V H3 is the heavy chain variable domain that specifically binds to the T cell receptor binding site; Fc is the immunoglobulin hinge region, as well as C H2 and C H3 An immunoglobulin heavy chain constant domain.

[0055] Some embodiments described herein provide a binding protein comprising three polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the first polypeptide chain has the formula: L -C L and the second polypeptide chain has a structure represented by the formula: V H1 -C H1- V H2 -Fc; the third polypeptide chain has a structure represented by the formula: V H1 -V H3 -Fc having the structure represented by: V L is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; L is an immunoglobulin light chain constant domain that specifically binds to a tumor-associated antigen; H1 is an immunoglobulin CH1 heavy chain constant domain that specifically binds to a tumor-associated antigen; H2is a heavy chain variable domain that specifically binds to the T cell receptor; V H3 is a heavy chain variable domain that specifically binds to a T cell costimulatory molecule; Fc is a C H2 and C H3 An immunoglobulin heavy chain constant domain.

[0056] In some embodiments, the heavy chain variable domain that specifically binds to a T cell costimulatory molecule is an immunoglobulin heavy chain variable domain. In some embodiments, the heavy chain variable domain that specifically binds to a T cell costimulatory molecule is a single domain sequence. In certain embodiments, the heavy chain variable domain that specifically binds to a T cell costimulatory molecule is a nanobody. In certain embodiments, the heavy chain variable domain that specifically binds to a T cell costimulatory molecule is from a camelid. In certain embodiments, the heavy chain variable domain that specifically binds to a T cell costimulatory molecule is a single domain variable neoantigen receptor.

[0057] In some embodiments, the heavy chain variable domain that specifically binds to the T cell receptor binding site is an immunoglobulin heavy chain variable domain. In some embodiments, the heavy chain variable domain that specifically binds to the T cell receptor binding site is a single domain sequence. In certain embodiments, the heavy chain variable domain that specifically binds to the T cell receptor binding site is a nanobody. In certain embodiments, the heavy chain variable domain that specifically binds to the T cell receptor binding site is from a camelid. In certain embodiments, the heavy chain variable domain that specifically binds to the T cell receptor binding site is a single domain variable neoantigen receptor.

[0058] In some embodiments, the Fc of the binding protein is derived from an IgG antibody, such as IgG1, IgG2, IgG3, IgA1, and IgGA2.

[0059] In some embodiments, the binding protein comprises a linker. The identity and sequence of the amino acid residues in the linker can vary depending on the type of the second structural element required to be achieved. For example, glycine, serine, and alanine are best for the linker to have maximum flexibility. If a more rigid and extended linker is required, some combination of glycine, proline, threonine, and serine is useful. Any amino acid residue can be considered as a linker, optionally combined with one or more other amino acid residues to build a larger peptide linker depending on the desired properties, which may be the same or different from the first amino acid residue. In some embodiments, the binding protein comprises a C on a third polypeptide chain. H1 and V H2 a linker, L1, located between the V and the V on the third polypeptide chain; H2 and Fc, and L1 and L2 are each, independently, a linker or absent. H1 and V H3 a linker located between L and V on the fourth polypeptide chain; H3 and Fc, and L4, wherein L3 and L4 are each independently a linker or absent. In some embodiments, L1, L2, L3, and L4 are each independently a linker or absent. In some embodiments, the linker comprises the amino acid sequence TGGS (SEQ ID NO: 46). In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 47). In some embodiments, the linker comprises the amino acid sequence AAAYPYDVPDYGSGEGTSTGSGGSGGSGGA (SEQ ID NO: 48). In some embodiments, the linker further comprises a hemagglutinin tag.

[0060] In some embodiments, the binding protein comprises a C on the third polypeptide chain. H1 and V H2 The immunoglobulin hinge region, H1, located between the V on the third polypeptide chain H2and Fc, and H1 and H2 are each, independently, an immunoglobulin hinge region or absent. H1 and V H3 The immunoglobulin hinge region, located between H3 and V on the fourth polypeptide chain H3 and Fc, and H3 and H4 are each independently an immunoglobulin hinge region or absent. In some embodiments, H1, H2, H3, and H4 are each independently an immunoglobulin hinge region or absent.

[0061] In some embodiments, the binding protein has the formula: V L -C L and a first and second polypeptide chain having a structure represented by the formula: V H1 -C H1 -H1-L1-V H2 -H2-L2-Fc and a third polypeptide chain having a structure represented by the formula: V H1 -C H1 -H3-L3-V H3 -H4-L4-Fc The fourth polypeptide chain has a structure represented by:

[0062] In other embodiments, the binding protein has the formula: V L -C L A first polypeptide chain having a structure represented by the formula: V H1 -C H1 -H1-L1-V H2 -H2-L2-Fc and a second polypeptide chain having a structure represented by the formula: V H1 -H3-L3-V H3 -H4-L4-Fc and a third polypeptide chain having a structure represented by:

[0063] In other embodiments, the binding protein has the formula: II1-V L -C L -II2 and two polypeptide chains having a structure represented by the formula: V H1 -C H1- V H2 -Fc The polypeptide comprises two polypeptide chains having a structure represented by:

[0064] In other embodiments, the binding protein has the formula: V L -C L A first polypeptide chain having a structure represented by the formula: V H1 -C H1- V H2 -Fc and a second polypeptide chain having a structure represented by the formula: V H1 -V H3 -Fc and a third polypeptide chain having a structure represented by:

[0065] In some embodiments, the binding protein comprises four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, the polypeptide chains being (a) the amino acids of the sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; (b) the amino acids of the sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; (c) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:10; (d) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:11, and SEQ ID NO:12; (e) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:13, and SEQ ID NO:14; (f) the amino acids of the sequences SEQ ID NO:1 and SEQ ID NO:19; (g) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:22, and SEQ ID NO:23; (h) the amino acids of the sequences of SEQ ID NO: 29 and SEQ ID NO: 30; (i) the amino acids of the sequences SEQ ID NO: 31 and SEQ ID NO: 32; (j) the amino acids of the sequences SEQ ID NO: 33 and SEQ ID NO: 34; (k) the amino acids of the sequences SEQ ID NO: 35 and SEQ ID NO: 36; or (l) the amino acids of the sequences of SEQ ID NO: 37 and SEQ ID NO: 38 Includes.

[0066] In some embodiments, the binding protein comprises three or four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, the polypeptide chains being (a) the amino acids of the sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; (b) the amino acids of the sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; (c) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:9, and SEQ ID NO:10; (d) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:11, and SEQ ID NO:12; (e) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:13, and SEQ ID NO:14; (f) the amino acids of the sequences SEQ ID NO:1 and SEQ ID NO:19; (g) the amino acids of the sequences of SEQ ID NO:1, SEQ ID NO:22, and SEQ ID NO:23; (h) the amino acids of the sequences of SEQ ID NO: 29 and SEQ ID NO: 30; (i) the amino acids of the sequences SEQ ID NO: 31 and SEQ ID NO: 32; (j) the amino acids of the sequences SEQ ID NO: 33 and SEQ ID NO: 34; (k) amino acids of the sequences of SEQ ID NO: 35 and SEQ ID NO: 36; (l) the amino acids of the sequences SEQ ID NO: 37 and SEQ ID NO: 38; (m) the amino acids of the sequences of SEQ ID NO: 39 and SEQ ID NO: 40; (n) the amino acids of the sequences SEQ ID NO: 41 and SEQ ID NO: 42; or (o) the amino acids of the sequences of SEQ ID NO: 45 and SEQ ID NO: 54 Includes.

[0067] In some aspects, the binding protein comprises three polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, the polypeptide chains comprising amino acids of the sequences SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, or (q) amino acids of the sequences SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO: 12. In one embodiment, the binding protein comprises three polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, the polypeptide chains comprising amino acids of the sequences SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45.

[0068] In some embodiments, provided herein is a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains comprise an amino acid sequence having at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-56. In some embodiments, provided herein is a binding protein comprising four polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains comprise an amino acid sequence having at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-42. In some embodiments, provided herein is a binding protein comprising three polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains comprise an amino acid sequence having at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-56. In some embodiments, provided herein is a binding protein comprising three polypeptide chains forming two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains comprise an amino acid sequence having at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 1-42.

[0069] In certain aspects, provided herein are amino acid sequences with conservative variants in which up to 10, up to 8, up to 5, and up to 3 amino acids are substituted with amino acids that are similar or have similar properties compared to the amino acid sequences of the sequences disclosed herein.

[0070] The binding proteins of the disclosure can be prepared using domains or sequences obtained or derived from any human or non-human antibody, including, for example, human, murine, or humanized antibodies.

[0071] Some aspects of the present disclosure pertain to isolated nucleic acid sequences encoding the binding proteins described herein. The isolated nucleic acid sequences may be contained in a vector.

[0072] In some aspects, the methods disclosed herein relate to treating a subject for cancer by administering an effective amount of the binding protein. The disclosure also provides a therapeutically effective amount of the binding protein for use in treating cancer in a subject. In one embodiment, a method is provided for treating a subject for inflammatory disease by administering an effective amount of the binding protein. In one embodiment, a pharmaceutical composition comprising the binding protein or the binding protein and a pharma- ceutically acceptable carrier for use as a medicament is provided. In another embodiment, a pharmaceutical composition comprising the binding protein or the binding protein and a pharma- ceutically acceptable carrier for use in the treatment of cancer is provided. In another embodiment, a pharmaceutical composition comprising the binding protein or the binding protein and a pharma- ceutically acceptable carrier for use in the treatment of inflammatory disease is provided. In another embodiment, a use of the binding protein or the pharmaceutical composition comprising the binding protein and a pharma- ceutically acceptable carrier in the manufacture of a medicament for use in the treatment of inflammatory disease is provided. In a further embodiment, a use of the binding protein or the pharmaceutical composition comprising the binding protein and a pharma- ceutically acceptable carrier in the manufacture of a medicament for use in the treatment of cancer is provided.

[0073] In some embodiments, the cancer comprises B-cell malignancies, including chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma.

[0074] In some embodiments, the methods disclosed herein relate to treating a subject for inflammatory disease involving B cells by administering an effective amount of the binding protein. The present disclosure also provides a therapeutically effective amount of the binding protein used to treat inflammatory disease involving B cells in a subject. In the method of treatment of the present disclosure, the binding protein can selectively activate a subset of T cells in the subject. The subset of T cells can be CD8+ T cells. The CD8+ T cells can be selectively activated compared to CD4 T cells. The selective activation of CD8+ T cells reduces the engagement of tumor-promoting T cells and CD4+ T cells that produce most of the cytokines that cause release syndrome (CRS). In addition, the selective engagement of CD8+ T cells induces pyroptosis.

[0075] The activation of T cells by the method of the present disclosure can be determined by measuring the percentage of surface interleukin-2 receptor alpha chain-positive (CD25+) T cells. The percentage of surface CD25+ T cells that are CD8 T cells can be higher than the percentage of surface CD25+ T cells that are CD4 T cells. In certain embodiments, the activation of T cells can be determined by the percentage of CD69+ / CD25+ T cells. In certain embodiments, the activation of T cells can be determined by measuring the level of cytokines released by activated T cells.

[0076] The methods of treatment of the present disclosure may result in reduced engagement of regulatory T cells (Treg), increased cytolytic activity, and / or reduced incidence of cytokine release syndrome (CRS) compared to that caused by bispecific T cell engagers (BiTEs) previously known in the art. The present disclosure also provides therapeutically effective amounts of binding proteins for use in reducing engagement of regulatory T cells (Treg), increasing cytolytic activity, and / or reducing incidence of cytokine release syndrome (CRS) compared to that caused by bispecific T cell engagers (BiTEs) previously known in the art.

[0077] In light of the present disclosure, the methods and compositions described herein can be adapted by one of skill in the art to meet desired needs.

[0078] In some embodiments, the inflammatory disease or inflammatory disorder involving B cells is selected from rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, pemphigus, lupus erythematosus profundus, scleroderma, discoid lupus erythematosus, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, and allergic contact dermatitis. EXAMPLES

[0079] The following examples illustrate specific embodiments of the present disclosure and various uses thereof, which are provided for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0080] Example 1: Binding Protein Structure and Cytolytic T Cell Activity A novel class of binding proteins engineered to improve safety and increase efficacy is described herein. Multiple binding protein formats were tested to determine the arrangement of the TCR binding domain, the arrangement of the T cell costimulatory domain, the linker size and cleavability, and the Fc portion to be used. Figure 1 shows representative structures for these binding proteins. As shown in Figures 2A and 2B, binding proteins MCAZ 6.9 and MCAZ 6.10 were generated to contain two CD20 binding sites, a CD8 costimulatory binding arm (Figure 2A), and a CD137 binding arm (Figure 2B), a TCR binding domain, and two protease cleavage sites, respectively. Similarly, MCAZ 7.1 was generated to contain two CD20 binding sites, a CD8 costimulatory binding arm, and a TCR binding domain, but did not contain two protease cleavage sites (Figure 10A).

[0081] MCAZ 6.9 and MCAZ 6.10 binding proteins were evaluated for in vitro cytolysis against the CD20+ B cell lines Daudi, Ramos, and Raji. B cells and purified pan T cells were incubated with the B cell lines at an E:T ratio of 5:1 for 4 days, and % cytolysis was measured by flow cytometry. Cytolysis for MCAZ 7.1 was evaluated against the CD20+ B cell lines Toledo, Oci-LY18, and SU-DHL5 (expressing 12420, 20244, and 27152 CD20 antigens per cell, respectively). B cell lines were CTV stained and incubated with PBMCs at an E:T ratio of 5:1 for 4 days. % cytolysis was measured by flow cytometry.

[0082] MCAZ 6.9, MCAZ 6.10, and MCAZ 7.1 binding proteins induced strong in vitro cytolytic activity (Figures 3A-3C, 10C, and 10D). Cytolysis was associated with strong T cell activation biased towards CD8 T cells for MCAZ 6.9 and MCAZ 7.1 (Figures 4A, 5A, 6, and 10E), and with a CD4 biased or equivalent CD4 / CD8 profile for MCAZ 6.10 (Figures 4B, 5B, and 6).

[0083] To determine the optimal arrangement of the TCR binding domain, MCAZ 88, MCAZ 7.5, and MCAZ 89 were each generated with a unique arrangement of the TCR binding domain or the absence of a TCR binding domain. MCAZ 88 contains two TCR binding domains at each end of the variable heavy domain (FIG. 7A), MCAZ 7.5 has a TCR binding domain placed within the hinge portion of the binding protein (FIG. 8A), and the MCAZ89 binding protein does not contain any TCR binding domains (FIG. 8A).

[0084] To test cytolysis for MCAZ 88, induced cytolysis was evaluated against the Raji cell line (CD20+, 85371 antigens per cell). B cells and PBMCs were incubated with the B cell line at an E:T ratio of 5:1 for 4 days, and % cytolysis was measured by flow cytometry. CD4 and CD8 T cell activation profiles were also evaluated for MCAZ88, MCAZ7.5, and MCAZ89 binding proteins by measuring surface CD25+ cell % and CD69+ / CD25+ surface expression %. The results demonstrated that the presence of two TCR binding domains in the absence of a T cell costimulatory domain was sufficient to induce cytolysis (Figure 7B). However, both the absence of the TCR binding domain and the placement of the TCR binding domain at the top of the binding protein resulted in non-specific T cell activation (Figure 7C, Figure 8B, Figure 8C, Figure 9). Non-specific T cell activation was strongly reduced only when the TCR binding domain was within the hinge region of the binding protein (FIGS. 8B, 8C).

[0085] To evaluate the effect of CD8 positioning on binding protein function, binding proteins MCAZ 8.71 and MCAZ 8.81 were each produced with two T cell costimulatory molecules in a unique arrangement on the binding protein (Figure 12A-B). Cytolysis was evaluated against the CD20+B Raji cell line (expressing 85000 CD20 antigens per cell). B cell lines were CTV stained and incubated with PBMCs at an E:T ratio of 5:1 for 4 days. The % cytolysis was measured by flow cytometry. CD4 and CD8 T cell activation profiles were evaluated as % CD25 expressing cells. Despite the different positions of the T cell costimulatory molecules on the binding protein or the presence of additional T cell costimulatory molecules, both MCAZ 8.71 and MCAZ 8.81 showed similar cytolysis (Figure 12C-D). Cytolysis was associated with strong T cell activation, biased towards CD8 T cells, for MCAZ 8.71 and MCAZ 8.81 (Figure 12E). These results demonstrate that the location of the costimulatory molecules was flexible within the binding protein. However, additional molecules did not increase the potency of the binding protein.

[0086] To test the positioning of the T cell costimulatory domain and the T cell binding domain with respect to each other, various orientations of the binding proteins were tested. MCAZ 10.3 was generated to contain both the CD8 domain and the TCR binding domain on the same polypeptide (Figure 10C). The T cell costimulatory domain and the T cell binding domain were also tested on separate binding proteins. Specifically, MCAZ 8.69 was generated as a binding protein containing only the T cell costimulatory molecule, and MCAZ 8.70 was generated as a binding protein containing only the TCR binding domain (Figure 13A). Cytolysis was assessed against CD20+B Raji, Toledo, Oci-LY18, and SU-DHL5 cell lines. B cell lines were CTV stained and incubated with PBMCs at an E:T ratio of 5:1 for 4 days. The % cytolysis was measured by flow cytometry. CD4 and CD8 T cell activation profiles were evaluated as % CD25 expressing cells. The results demonstrated the ideal location of the CD8 domain and the TCR binding domain, on separate arms but within the same binding protein, for cell lysis and T cell activation (Figures 10C-E and 13).

[0087] Modified linker lengths were tested to determine the optimal linker for binding protein stability and function. Compared to MCAZ 7.1, in which a linker (T) is located between the CD20 binding domain and the TCR binding domain, MCAZ 7.7 was generated with a longer modified linker (GGGGSGGGGS) located between the CD20 binding domain and the TCR binding domain (FIG. 11A). Compared to MCAZ 7.1, MCAZ 10.1 was generated with a more rigid CD8 arm. Specifically, the linker (TGGS (SEQ ID NO: 46)) located between the CD20 binding domain and the CD8 domain was removed, and the linker located between the CD8 domain and the Fc domain was switched from (AAAYPYDVPDYGSGEGTSTGSGGSGGSGGA (SEQ ID NO: 48)) in MCAZ 7.1 to (G) in MCAZ 10.1 (FIG. 11E). In addition, MCAZ 10.1 was generated in which the linker (T) located between the TCR binding domain and the Fc was deleted compared to MCAZ 7.1 with the linker (GEGTSTGSGGSGGSGGA (SEQ ID NO: 49)) and the linker located between the CD20 binding domain and the TCR binding domain was deleted compared to MCAZ 7.1 with the shortened linker (G). MCAZ 7.7 was evaluated for cytolysis against CD20+Raji B cell line (expressing 85000 CD20 antigens per cell). B cell lines were stained with CTV and incubated with PBMCs at an E:T ratio of 5:1 for 4 days. The cytolysis % was measured by flow cytometry. MCAZ 10.1 was evaluated for cytolysis against OCI-Ly18 B cell line. B cell lines were stained with CTV and incubated with PBMCs at an E:T ratio of 5:1 for 3 days. The cytolysis % was measured by flow cytometry. T cell activation profiles were assessed as % CD25 expressing cells. For MCAZ 7.7, non-specific and specific T cell activation was assessed by plate binding of binding proteins and adding 1.5e5 purified T cells in the absence of tumor cells and associated proteases. After 48 hours of incubation, non-specific CD8 and CD4 T cell activation levels were assessed by flow cytometry as % CD69+ / CD25+ surface expression.The longer modified linker in MCZA 7.7 increased the level of cell lysis compared to MCZA 7.1. However, the longer linker in MCZA 7.7 resulted in non-specific T cell activation. Removal of the linker in MCAZ 10.1 inhibited the function of the binding protein, resulting in minimal cell lysis and T cell activation.

[0088] To test different uncleaved Fc regions, various binding proteins were generated, including IgG1 (MCAZ 11.1), IgG2 (MCAZ 11.2), IgG3 (MCAZ 11.3), IgG4 (MCAZ 11.5), mutated IgG1 (MCAZ 11.5), IgD (MCAZ 11.6), and IgA1 (MCAZ 11.7), as illustrated in FIG. 15A. The cytolytic activity of the binding proteins was tested against the OCI-Ly18 B cell line in PBMCs at a 5:1 E:T ratio at 72 hours. The CD8 and CD4 T cell activation profile was assessed by measuring the % of surface CD25+ T cells. The binding proteins with IgG Fc regions were the most effective in inducing cytolysis and T cell activation (FIGS. 15B-D).

[0089] Based on the experiments described above, it was found that the format described, for example, in MCAZ 7.1, provides optimal cell lysis and induction of T cell activation.

[0090] Example 2: In vitro testing of binding proteins compared to a broad range of CD3xCD20 bivalent engagers The activity of the novel binding proteins disclosed herein was compared to a broad range of CD20 bivalent engagers established to be effective in clinical treatments.

[0091] The activity of MCAZ 7.1 variants and a broad range of CD3xCD20 bivalent engagers was compared (Figure 16A). Specifically, the variants contained linkers between the CD20 binding domain and the CD8 domain, and between the CD20 binding domain and the TCR domain (TGGS (SEQ ID NO: 46)), as well as linkers between the CD8 domain, the Fc region, the TCR domain and the Fc domain (GGGGS (SEQ ID NO: 47)). The binding proteins and bivalent engagers were incubated with target CD20+ cells at an E:T ratio of 5:1 with PBMCs from healthy donors for 72 hours. The MCAZ7.1 variants induced similar Emax cytolysis (~pM EC50), but a significantly CD8-biased activation profile was evident (Figure 16B).

[0092] Binding of MCAZ 7.1 variants and CD3×CD20 bivalent engagers to CD8 T cells was compared. MCAZ7.1 variant binding profiles were evaluated on a CD20+ tumor B cell line (OCI-Ly-18) and on purified CD4 and CD8 T cells from PBMCs from healthy donors. To evaluate CD3×CD20 bivalent engager-induced B cell:T cell binding, OCI-Ly18 tumor cells were CTV stained and mixed with pan-T cells from healthy donors in a 1:1 ratio and incubated for 1 hour at room temperature. The percentage of CD8 B cell conjugates and CD4 T cell conjugates was then evaluated by flow cytometry after staining of CD4 and CD8 T cells after incubation with MCAZ7.1 variants and CD3×CD20 bivalent engagers. The results demonstrated that, compared to a broad range of therapeutically effective CD3 × CD20 bivalent engagers, the MCAZ7.1 variants exhibited strong binding to CD8 T cells and induced selective engagement of CD20+ tumor cells with CD8 T cells (Figures 17A-F).

[0093] A 3D spheroid model was used to determine the cytolytic activity of MCAZ 7.1 variants compared to CD3×CD20 bivalent engagers. A GFP-expressing CD20+ B cell line (TMD8, 100000 CD20 / cell) was plated in low attachment plates to form 3D spheroids for 72 hours. Purified panT cells were then added at an E:T ratio of 15:1 and co-incubated with no engager, TENG0093, or CD3×CD20 bivalent engagers for 96 hours. GFP+TMD8 cells were quantified using a Cellinsight CX7 HCS Platform imager. MCAZ 7.1 variants achieved potent CD20+ cell killing comparable to CD3×CD20 bivalent molecules (Figure 20B).

[0094] To evaluate the inflammatory cytokine release of MCAZ7.1 binding proteins compared to CD3×CD20 bivalent engagers, MCAZ7.1 and CD3×CD20 bivalent engagers were incubated with OCI-Ly18 B cell line and PBMCs at an E:T ratio of 5:1 for 72 hours. Supernatants were collected and analyzed by multiplex assays to measure the concentrations of released proinflammatory cytokines: IL-6 (FIG. 18A), TNF-a (FIG. 18B), IL-10 (FIG. 18C), IFN-g (FIG. 18D), IL-2 (FIG. 18E), and IL-17A (FIG. 18F). CD8-specific engagement of MCAZ7.1 achieved Emax death of CD20+ tumor cells similar to CD3×CD20 bivalent engagers, but cell lysis was associated with significantly lower proinflammatory cytokine release than CD3+ T cell engagement by CD3×CD20 bivalent engagers.

[0095] Example 3: In vivo efficacy in B cell lymphoma in a humanized NSG mouse model The pharmacokinetics of MCAZ 7.1 was evaluated after a single dose. Specifically, panT cell-humanized NSG mice were injected with 0.5 mg / kg T cell engager, and the systemic concentration of T cell engager was measured 1 hour, 6 hours, 24 hours, 48 ​​hours, 72 hours, and 168 hours later. The results demonstrate that the MCAZ 7.1 binding protein was stable in vivo (Figure 21).

[0096] To test the efficacy of MCAZ 7.1 binding proteins in tumor growth inhibition, NSG mice were implanted with panT cells 2 days before the start of the study. 5e6 OCI-Ly18 CD20+ tumor cell line was implanted sc on day 0 and animals were dosed ip with 1 mg / kg MCAZ7.1 or CD3xCD20 bivalent engager on day 2 and then weekly at the same concentrations as indicated by the grey arrows under the X-axis. MCAZ 7.1 and CD3xCD20 bivalent engager showed similar efficacy and complete tumor growth inhibition (Figure 22).

[0097] To evaluate the in vivo cytokine release of MCAZ 7.1 compared to CD3xCD20 bivalent engagers, NSG mice were irradiated (2.3 Gy) and treated with Fc block (400 mg / mouse ip) before transfer of 10e6 PBMC (ip) 48 hours later, and treated with 2 mg / kg (ip) for OKT3 antibody or 1 mg / kg (ip) of the indicated binding protein 24 hours later. Blood was collected 6 and 24 hours after injection for evaluation of cytokine concentrations by multiplex assay. In vivo cytokine release evaluation in a CRS (cytokine release syndrome) model confirmed that MCAZ 7.1 induced lower cytokine release compared to broad CD3+ T cell engagement induced by CD3xCD20 bivalent molecules (Figures 23A-F).

[0098] Example 4: Binding protein in vitro activity on PBMCs from non-Hodgkin's lymphoma donors To determine the efficacy of the binding proteins in B cell death and T cell activation, B cell death was evaluated on PBMCs from NHL (DLBCL) donors. MCAZ 7.1 variants were spiked into PBMCs at various concentrations and 48 hours later, and B cell % was evaluated by flow cytometry for each patient. Both CD8-specific T cell engagers, MCAZ 7.1 and MCAZ 7.1 variants, showed similar robust CD20+ B cell death on PBMCs from non-Hodgkin's lymphoma donors, and confirmed robust activation biased towards CD8 T cells (Figures 24A-F).

[0099] Based on the multiple binding protein formats tested, a binding protein with a novel conformation was identified using IgG Fc, which includes a TCR binding domain and a T cell costimulatory domain on separate arms located in the hinge region. This binding protein advantageously increased cytolytic activity both in vitro and in vivo during lytic activity in the absence of tumor target cells (non-specific T cell activation) and in the presence of tumor target cells, and reduced the incidence of cytokine release. In addition, the binding format disclosed herein achieves reduced CD4 engagement compared to a broad range of CD3 engagers, including (i) limiting Treg activation and proliferation, and limiting potential suppressive activity on CD8 cytolytic T cells, and (ii) reducing engagement of other CD4 T cell subsets (Th1, Th2, Th9, TH17), all of which have been shown to be involved in the drivers of cytokine release syndrome events.

[0100] The embodiments described herein as examples may suitably be practiced in the absence of any element or elements, or limitation or limitations not specifically disclosed herein. The terms and expressions used are used as terms of description and not as limitations, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the claimed embodiments. Thus, although the present description is specifically disclosed by embodiments, it should be understood that those skilled in the art may rely on optional features, modifications, and variations of the concepts disclosed herein, and such modifications and variations are considered to be within the scope of these embodiments as defined by the specification and the appended claims. Although some aspects of the present disclosure may be identified herein as particularly advantageous, it is intended that the present disclosure is not limited to these particular aspects of the disclosure.

[0101] A claim or specification including "or" between one or more members of a group is deemed to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise relevant to a given product or process, unless the contrary is stated or the context makes clear otherwise. The present disclosure includes aspects in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes aspects in which more than one or all of the members of the group are present in, used in, or otherwise relevant to a given product or process.

[0102] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the recited claims are introduced into another claim. For example, any claim that is dependent on another claim can be amended to include one or more limitations found in any other claim that is dependent on the same base claim. When elements are presented as a list, for example in Markush group format, each subgroup of elements is also disclosed, and any element can be removed from the group.

[0103] In general, when the disclosure or aspects of the disclosure are referred to as comprising certain elements and / or features, it is to be understood that the particular aspect of the disclosure or aspects of the disclosure consist or consist essentially of such elements and / or features, and for the sake of brevity, those aspects have not been specifically described in these terms herein.

[0104] [Table 1]

[0105] [Table 2]

[0106] [Table 3]

[0107] [Table 4]

[0108] [Table 5]

[0109] [Table 6]

[0110]

Table 7

[0111]

Table 8

[0112]

Table 9

[0113]

Table 10

[0114]

Table 11

[0115]

Table 12

[0116]

Table 13

[0117]

Table 14

[0118]

Table 15

[0119]

Table 16

[0120]

Table 17

[0121]

Table 18

[0122]

Table 19

[0123]

Table 20

Claims

1. A binding protein comprising four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the first and second polypeptide chains are of the formula: V Ｌ -C Ｌ It has a structure represented by; The third polypeptide chain is given by formula: V Ｈ１ -C Ｈ１－ V Ｈ２ -Fc It has a structure represented by; The fourth polypeptide chain is given by formula: V Ｈ１ -C Ｈ１ -V Ｈ３ -Fc It has a structure represented by; V Ｌ is an immunoglobulin light chain variable domain that specifically binds to a tumor-associated antigen; V Ｈ１ This is a variable domain of immunoglobulin heavy chains that specifically binds to tumor-associated antigens; C Ｌ This is the constant domain of the immunoglobulin light chain; C Ｈ１ This is the constant domain of the immunoglobulin CH1 heavy chain; V Ｈ２ This is a heavy chain variable domain that specifically binds to the T cell receptor (TCR); V Ｈ３ This is a heavy chain variable domain that specifically binds to T cell costimulatory molecules; Fc is C Ｈ２ and C Ｈ３ The constant domain of the immunoglobulin heavy chain, Binding protein.

2. C on the third polypeptide chain Ｈ１ and V Ｈ２ L is a linker located between them. １ and / or V on the third polypeptide chain Ｈ２ L is a linker located between and Fc. ２ The binding protein according to claim 1, further comprising:

3. C on the fourth polypeptide chain Ｈ１ and V Ｈ３ L is a linker located between them. ３ and / or V on the fourth polypeptide chain Ｈ３ L is a linker located between and Fc. ４ The binding protein according to claim 2, further comprising:

4. C on the third polypeptide chain Ｈ１ and V Ｈ２ The immunoglobulin hinge region located between is H １ and / or V on the third polypeptide chain Ｈ２ H is an immunoglobulin hinge region located between and the aforementioned Fc. ２ The binding protein according to claim 3, further comprising:

5. C on the fourth polypeptide chain Ｈ１ and V Ｈ３ The immunoglobulin hinge region located between is H ３ and / or V on the fourth polypeptide chain Ｈ３ H is an immunoglobulin hinge region located between and the aforementioned Fc. ４ The binding protein according to claim 4, further comprising:

6. The first and second polypeptide chains are of the formula: V Ｌ -C Ｌ It has a structure represented by, The third polypeptide chain is given by formula: V Ｈ１ -C Ｈ１ -H １ -L １ -V Ｈ２ -H ２ -L ２ -Fc It has a structure represented by, The fourth polypeptide chain is given by formula: V Ｈ１ -C Ｈ１ -H ３ -L ３ -V Ｈ３ -H ４ -L ４ The binding protein according to claim 5, having a structure represented by -Fc.

7. The binding protein according to any one of claims 1 to 6, wherein the Fc is derived from an IgG antibody.

8. The binding protein according to any one of claims 1 to 6, wherein the binding protein activates T cells only when it is bound to a tumor-associated antigen at one or both of the antigen-binding sites.

9. The binding protein according to any one of claims 1 to 6, wherein the tumor-associated antigen is CD20.

10. The binding protein according to any one of claims 1 to 6, wherein the T cell costimulatory molecule is CD8 or CD137.

11. A binding protein comprising four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the two polypeptide chains are of the formula: V Ｌ -C Ｌ It has a structure represented by, Two polypeptide chains, formula: II １ -V Ｈ１ -C Ｈ１－ V Ｈ２ -Fc-II ２ It has a structure represented by: V Ｌ This is a variable domain of the immunoglobulin light chain that specifically binds to tumor-associated antigens; V Ｈ１ This is a variable domain of immunoglobulin heavy chains that specifically binds to tumor-associated antigens; C Ｌ This is the constant domain of the immunoglobulin light chain; C Ｈ１ This is the constant domain of the immunoglobulin CH1 heavy chain; V Ｈ２ This is a heavy chain variable domain that specifically binds to the T cell receptor (TCR); Fc is C Ｈ２ and C Ｈ３ It is the constant domain of the immunoglobulin heavy chain; II １ and II ２ Each of these is either an independent heavy chain variable domain that specifically binds to a T cell costimulatory molecule, or it is absent; II １ and II ２ At least one of these is a heavy chain variable domain that specifically binds to T cell costimulatory molecules. Binding protein.

12. A binding protein comprising four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the two polypeptide chains are of the formula: II １ -V Ｌ -C Ｌ -II ２ It has a structure represented by; Two polypeptide chains, formula: V Ｈ１ -C Ｈ１－ V Ｈ２ -Fc It has a structure represented by: V Ｌ This is a variable domain of the immunoglobulin light chain that specifically binds to tumor-associated antigens; V Ｈ１ This is a variable domain of immunoglobulin heavy chains that specifically binds to tumor-associated antigens; C Ｌ This is the constant domain of the immunoglobulin light chain; C Ｈ１ This is the constant domain of the immunoglobulin CH1 heavy chain; V Ｈ２ This is a heavy chain variable domain that specifically binds to the T cell receptor (TCR); Fc is C Ｈ２ and C Ｈ３ It is the constant domain of the immunoglobulin heavy chain; II １ and II ２ Each of these is either an independent heavy chain variable domain that specifically binds to a T cell costimulatory molecule, or it is absent; II １ and II ２ At least one of these is a heavy chain variable domain that specifically binds to T cell costimulatory molecules. Binding protein.

13. A binding protein comprising three polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the first polypeptide chain has the formula: V Ｌ -C Ｌ It has a structure represented by, The second polypeptide chain is given by formula: V Ｈ１ -C Ｈ１－ V Ｈ２ -Fc It has a structure represented by, The third polypeptide chain is given by formula: V Ｈ１ -V Ｈ３ -Fc It has a structure represented by: V Ｌ This is a variable domain of the immunoglobulin light chain that specifically binds to tumor-associated antigens; V Ｈ１ is an immunoglobulin heavy chain variable domain that specifically binds to a tumor-associated antigen; C Ｌ This is the constant domain of the immunoglobulin light chain; C Ｈ１ This is the constant domain of the immunoglobulin CH1 heavy chain; V Ｈ２ This is a heavy chain variable domain that specifically binds to the T cell receptor (TCR); V Ｈ３ This is a heavy chain variable domain that specifically binds to T cell costimulatory molecules; Fc is C Ｈ２ and C Ｈ３ The constant domain of the immunoglobulin heavy chain, Binding protein.

14. The binding protein according to any one of claims 11 to 13, wherein the binding protein activates T cells only when it is bound to a tumor-associated antigen at one or both of the antigen-binding sites.

15. The binding protein according to any one of claims 11 to 13, wherein the tumor-associated antigen is CD20.

16. The binding protein according to any one of claims 11 to 13, wherein the T cell costimulatory molecule is CD8 or CD137.

17. The binding protein comprises four polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains are (a) Amino acids in the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; (b) Amino acids in the sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 4; (c) Amino acids in the sequences of SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 10; ； (d) Amino acids in the sequences of SEQ ID NO: 1, SEQ ID NO: 13, and SEQ ID NO: 14; (e) Amino acids in the sequences of SEQ ID NO: 1 and SEQ ID NO: 19; (f) Amino acids in the sequences of SEQ ID NO: 1, SEQ ID NO: 22, and SEQ ID NO: 23; (g) Amino acids in the sequences of SEQ ID NOs: 29 and 30; (h) Amino acids in the sequences of SEQ ID NOs: 31 and 32; (i) Amino acids in the sequences of SEQ ID NOs: 33 and 34; (j) Amino acids in the sequences of SEQ ID NOs: 35 and 36; (k) Amino acids in the sequences of SEQ ID NOs: 37 and 38; (l) Amino acids in the sequences of SEQ ID NO: 39 and SEQ ID NO: 40; (m) Amino acids in the sequence of SEQ ID NOs: 41 and 42; or (n) Amino acids in the sequences of SEQ ID NOs. 45 and 54 A binding protein containing this protein.

18. A binding protein comprising three polypeptide chains that form two tumor-associated antigen binding sites, a T cell receptor binding site, and a T cell costimulatory molecule binding site, wherein the polypeptide chains comprise amino acids in the sequences of SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, or (q) amino acids in the sequences of SEQ ID NO: 1, SEQ ID NO: 11, and SEQ ID NO:

12.

19. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 6, 11 to 13, 17, and 18, and a pharmaceutically acceptable carrier.

20. An isolated nucleic acid sequence encoding the binding protein according to claim 17 or 18.

21. A vector comprising the isolated nucleic acid sequence described in claim 20.

22. A pharmaceutical composition according to claim 19, used for the treatment of cancer.

23. A pharmaceutical composition according to claim 19, used for the treatment of inflammatory diseases.

24. The pharmaceutical composition according to claim 22, wherein the binding protein preferentially activates a subset of T cells in the target.

25. The pharmaceutical composition according to claim 24, wherein the subset of T cells is CD8 T cells.

26. The pharmaceutical composition according to claim 25, wherein the CD8 T cells are preferentially activated compared to CD4 T cells.

27. ​​The pharmaceutical composition according to claim 24, wherein the activation of T cells is determined by measuring the percentage of surface CD25+ T cells.

28. The pharmaceutical composition according to claim 27, wherein the percentage of surface CD25+ T cells, which are CD8 T cells, is higher than the percentage of surface CD25+ T cells, which are CD4 T cells.

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