Bi-specific antibodies for use in producing armed immune cells

Abstract

To develop efficient cancer therapy that specifically targets cancer cells without affecting normal cells or tissues.SOLUTION: Bispecific antibodies capable of binding to CD3 and a tumor associated antigen, immune cells armed with such bispecific antibodies, and therapeutic uses thereof in cancer treatment, as well as methods for producing immune cells armed with the bispecific antibodies.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 993,080, filed March 23, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Cancer is a disease characterized by abnormal cells that divide uncontrollably and have the ability to invade and destroy normal tissues and / or organs of a subject. Cancer is the second leading cause of death worldwide, responsible for an estimated 9.6 million deaths in 2018, with the most common cancers including lung cancer (approximately 2.09 million), breast cancer (approximately 2.09 million), colorectal cancer (approximately 1.8 million), prostate cancer (approximately 1.28 million), skin cancer (approximately 1.04 million), and stomach cancer (approximately 1.03 million).

[0003] Cancer treatments may vary depending on the type and stage of cancer. Traditional treatments for cancer include surgery, radiation therapy, and chemotherapy. Such treatments usually cause various complications or side effects, such as infection, blood clots, bleeding, nausea and vomiting, diarrhea, nerve or muscle damage, incontinence, and sexual and fertility problems. Immunotherapy offers an alternative strategy for cancer treatment, aiming to specifically stimulate a subject's immune response against cancer cells, for example, by blocking immune checkpoints or by enhancing the ability of immune cells (e.g., T cells or B cells) to target and destroy cancer cells. Serious adverse effects associated with overstimulation or nonspecific toxicity by immunotherapy drugs, including neurotoxicity, cytokine release syndrome (CRS), allergies, organ inflammation, and autoimmune disorders, have been reported in cancer patients.

[0004] Therefore, it is of great importance to develop efficient cancer treatments that specifically target cancer cells without affecting normal cells and / or tissues. Summary of the Invention

[0005] The present disclosure is based on the development of bispecific antibodies (BsAbs) that can bind to CD3 (e.g., human CD3) and tumor-associated antigens (TAAs). Such BsAbs can attach to the surface of CD3-positive immune cells via binding of the anti-CD3 moiety within the BsAb to cell surface CD3, generating armed immune cells.

[0006] Thus, in some aspects, the present disclosure features a bispecific antibody that includes (a) a first antigen-binding fragment that binds to human CD3 and (b) a second antigen-binding fragment that binds to a tumor-associated antigen (TAA). The first antigen-binding fragment comprises (i) a first heavy chain variable region (V H ), and (ii) a first heavy chain comprising a first light chain variable region (V L In some embodiments, the first light chain comprises a first V H comprises the same heavy chain complementarity determining regions (CDRs) as the first reference antibody. H contains five or fewer amino acid mutations in the CDRs compared to the first reference antibody. Alternatively, or in addition, the first V L may comprise the same light chain CDRs as the first reference antibody. L may contain five or fewer amino acid mutations in the CDRs compared to the first reference antibody. In some examples, the first reference antibody is CTA.02. In some examples, the first reference antibody is CTA.03. In other examples, the first reference antibody is CTA.04. In yet other examples, the first reference antibody is CTA.05. Structural information for these exemplary reference antibodies is provided in Table 1 below. In some examples, the first heavy chain and the first light chain have the same V H and V L Includes:

[0007] The second antigen-binding fragment comprises (i) a second heavy chain variable region (V H ), and (ii) a second heavy chain comprising a second light chain variable region (V L). The second antigen-binding fragment binds to a TAA. Examples include CD20, CD19, EGFR, HER2, PSMA, CEA, EpCAM, FAP, PD-L1, CD38, CD33, cMET, CD47, TRAIL-R2, mesothelin, or GD2. In some examples, the second V H comprises the same heavy chain complementarity determining regions (CDRs) as the second reference antibody. Alternatively, the second V H may contain no more than five amino acid mutations in the CDRs compared to the second reference antibody. L In other embodiments, the second V may comprise the same light chain CDRs. L may contain five or fewer amino acid mutations in the CDRs compared to the second reference antibody. In some examples, the second reference antibody is CTAT.01, CTAT.02, CTAT.03, CTAT.04, CTAT.05, CTAT.06, CTAT.07, CTAT.08, CTAT.09, CTAT.10, CTAT.11, CTAT.12, CTAT.13, CTAT.14, CTAT.15, or CTAT.16. See Table 2 below. In some examples, the second antigen-binding fragment has the same V as the second reference antibody. H and the same V L Includes:

[0008] In some embodiments, the first antigen-binding fragment is a Fab fragment and the second antigen-binding fragment is a single-chain variable fragment (scFv). In some examples, the Fab fragment is a first V H and a first heavy chain comprising a CH1 fragment, and a first V L and a first light chain comprising a light chain constant region. In specific examples, the Fab fragment may comprise a first heavy chain and a first light chain comprising the amino acid sequences of (a) SEQ ID NO: 10 and SEQ ID NO: 11, (b) SEQ ID NO: 23 and SEQ ID NO: 24, 25, or 228, (c) SEQ ID NO: 35 and SEQ ID NO: 36, or (d) SEQ ID NO: 46 and SEQ ID NO: 47. In some examples, the second antigen-binding fragment scFv comprises the amino acid sequence of any one of SEQ ID NOs: 254 to 271.

[0009] In some examples, the scFv is linked to a CH1 fragment, and w is optionally linked via a peptide linker. Alternatively, the scFv is optionally linked to a light chain constant region via a peptide linker. For example, a bispecific antibody can include a first polypeptide comprising a first light chain and a second polypeptide comprising, from N- to C-terminus, a first heavy chain, a peptide linker, and an scFv. Examples include any one of SEQ ID NOs: 229-248. Such a bispecific antibody can include a second polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 24, 25, and 228. See Table 3 below.

[0010] In other examples, the first antigen-binding fragment is a single-chain variable fragment (scFv) and the second antigen-binding fragment is a Fab fragment. The scFv may comprise the amino acid sequence of any one of SEQ ID NOs: 250-253. In some examples, the Fab fragment comprises the second V H and a second heavy chain comprising a CH1 fragment, and a second V L and a second light chain comprising a light chain constant region. In certain examples, the Fab fragment comprises a first heavy chain and a first light chain comprising the amino acid sequences of (1) SEQ ID NO:57 and SEQ ID NO:58, (2) SEQ ID NO:72 and SEQ ID NO:73, (3) SEQ ID NO:83 and SEQ ID NO:84, (4) SEQ ID NO:94 and SEQ ID NO:95, (5) SEQ ID NO:105 and SEQ ID NO:106, (6) SEQ ID NO:116 and SEQ ID NO:117, (7) SEQ ID NO:127 and SEQ ID NO:128, (8) SEQ ID NO:138 and SEQ ID NO:139, (9) SEQ ID NO:149 and SEQ ID NO:150, (10) SEQ ID NO:160 and SEQ ID NO:161, (11) SEQ ID NO:171 and SEQ ID NO:172, (12) SEQ ID NO:182 and SEQ ID NO:183, (13) SEQ ID NO:193 and SEQ ID NO:194, (14) SEQ ID NO:204 and SEQ ID NO:205, (15) SEQ ID NO:215 and SEQ ID NO:216, or (16) SEQ ID NO:226 and SEQ ID NO:227.

[0011] In some examples, the scFv is optionally linked to the CH1 fragment via a peptide linker. Alternatively, the scFv is optionally linked to the light chain constant region via a peptide linker. Any of the peptide linkers may be at least 5 amino acids in length.

[0012] In yet other examples, both the first antigen-binding fragment and the second antigen-binding fragment are scFv antibodies. In some examples, a bispecific antibody comprises a polypeptide comprising two scFv antibodies.

[0013] In another aspect, the present disclosure provides armed immune cells comprising an immune cell expressing surface CD3 and any of the bispecific antibodies disclosed herein (e.g., those exemplified in Tables 1-3). The armed immune cell displays the bispecific antibody on its surface via interaction between the first antigen-binding fragment of the bispecific antibody and CD3 expressed by the immune cell. In some embodiments, The immune cells are T cells, B cells, monocytes, macrophages, or a combination thereof. In some examples, the T cells can be CD4+ T cells, CD8+ T cells, regulatory T cells, or natural killer T cells. In some examples, the immune cells are human immune cells, e.g., immune cells derived from a human donor.

[0014] Additionally, provided herein are methods for producing the armed immune cells disclosed herein. The methods may include culturing a cell population comprising immune cells in the presence of a bispecific antibody disclosed herein to allow binding of the bispecific antibody to the immune cells, thereby producing armed immune cells. Armed immune cells produced by any of the methods disclosed herein are also within the scope of the present disclosure.

[0015] In some embodiments, the cell population comprises T cells, B cells, monocytes, macrophages, or a combination thereof. In some examples, the cell population comprises peripheral blood mononuclear cells (PBMCs) or immune cells derived in vitro from stem cells. The stem cells may be hematopoietic stem cells, umbilical cord blood stem cells, or induced pluripotent stem (iPS) cells.

[0016] In some embodiments, the culturing step is carried out in a culture medium that optionally contains cytokines, including interleukin 2 (IL-2), interleukin 7 (IL-7), transforming growth factor-β (TGF-β), or combinations thereof.

[0017] Further provided herein are methods for treating cancer, comprising administering an effective amount of any population of armed immune cells disclosed herein to a subject in need of cancer treatment. The subject has or is suspected of having a cancer that is positive for a TAA to which the second antigen-binding fragment of the bispecific antibody binds. In some embodiments, the subject is a human cancer patient. In some embodiments, the armed immune cells are autologous to the subject. Alternatively, the armed immune cells are allogeneic to the subject. Exemplary cancers include, but are not limited to, melanoma, esophageal cancer, gastric cancer, brain tumor, small cell lung cancer, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, thyroid cancer, testicular cancer, head and neck squamous cell carcinoma, leukemia, lymphoma, and myeloma.

[0018] In another aspect, the disclosure features a nucleic acid or set of nucleic acids (two nucleic acid molecules) that encode or collectively encode any of the bispecific antibodies disclosed herein. In some examples, the nucleic acid or set of nucleic acids is a vector or set of vectors, e.g., expression vectors. Host cells (e.g., bacterial cells, yeast cells, or mammalian cells) containing any of the nucleic acids or set of nucleic acids disclosed herein are also within the scope of the disclosure.

[0019] Additionally, the disclosure features a method for producing a bispecific antibody, including (i) culturing a host cell disclosed herein under conditions that allow for expression of the bispecific antibody, and (ii) harvesting the bispecific antibody.

[0020] Also included within the scope of the present disclosure is the use of any of the armed immune cells disclosed herein for use in cancer therapy or for the manufacture of a medicament for use in treating a targeted cancer.

[0021] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as the appended claims. [Brief explanation of the drawings]

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood in conjunction with the detailed description of specific embodiments presented herein.

[0023] [Figure 1] Figures 1A-1N are schematic diagrams of exemplary bispecific antibody formats. Figures 1A-1D: Structure of an anti-CD3 Fab / anti-TAA scFv bispecific format. Figures 1E-1H: Structure of an anti-CD3 scFv / anti-TAA Fab bispecific format. Figures 1I-1L: Structure of an anti-CD3 scFv / anti-TAA scFv bispecific format. Figure 1M: Structure of an anti-CD3 knob / anti-TAA hole bispecific antibody format comprising a monovalent anti-CD3 antibody and a monovalent anti-TAA antibody. Figure 1N: Structure of an anti-CD3 knob / anti-TAA scFv hole antibody comprising a monovalent anti-CD3 antibody and a monovalent anti-TAA scFv-Fc fusion protein. [Figure 2]Figures 2A-2E contain schematic diagrams of DNA constructs for expressing the depicted recombinant bispecific antibodies. Figure 2A: An exemplary construct for expressing an anti-CD3 Fab / anti-TAA scFv bispecific antibody. Figure 2B: An exemplary construct for expressing an anti-CD3 scFv / anti-TAA Fab bispecific antibody. Figure 2C: An exemplary construct for expressing an anti-CD3 scFv / anti-TAA scFv bispecific antibody. Figure 2D: An exemplary construct for expressing an anti-CD3 knob / anti-TAA hole bispecific antibody. Figure 2E: An exemplary construct for expressing an anti-CD3 knob / anti-TAA scFv hole antibody. [Figure 3] FIG. 3 is a chart showing the binding affinity of exemplary bispecific antibodies to T cells as measured by flow cytometry. [Figure 4] FIG. 4 is a chart showing the cytotoxic effect of T cells armed with an exemplary bispecific antibody or activated by OKT3 antibody against HT-29 cancer cells. [Figure 5] FIG. 5 is a chart showing the time course of the levels of an exemplary bispecific antibody on the surface of T cells. [Figure 6] Figures 6A-6B include photographs showing the expression and assembly of an exemplary bispecific antibody as demonstrated by SDS-PAGE under non-reducing (Figure 6A) and reducing (Figure 6B) conditions. Estimated molecular weights: intact BsAb = 95 kDa, heavy chain = 60 kDa, and light chain = 25 kDa. Lane 1: protein marker, lane 2: CTA02Fab / CTAT02scFv, lane 3: CTA04Fab / CTAT02scFv, lane 4: CTA03Fab / CTAT02scFv, and lane 5: CTA05Fab / CTAT02scFv. [Figure 7]Figures 7A-7F contain photographs showing the expression and assembly of an exemplary anti-CD3 Fab / anti-tumor bispecific antibody as revealed via SDS-PAGE under reducing or non-reducing conditions. Estimated molecular weight: intact BsAb = 95 kDa. Figures 7A-7B: CTA03 Fab / anti-TAA scFv bispecific antibody under non-reducing conditions. Figures 7C-7D: CTA03 Fab / anti-TAA scFv bispecific antibody under reducing conditions. Figures 7E-7F: anti-CD3 scFv / CTAT03 Fab bispecific antibody under non-reducing and reducing conditions, respectively. [Figure 8] Figure 8 shows the binding activity of various bispecific antibodies on T cells and tumor cells. CD3+ T cells (Jurkat) and CD19+ B cell lymphoma (Raji) were incubated separately with exemplary bispecific antibodies CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, CTA04Fab / CTAT02scFv, and CTA05Fab / CTAT02scFv BsAb, followed by analysis with a FITC-conjugated goat anti-human IgG Fab antibody and flow cytometer. [Figure 9]Figures 9A-9L include diagrams showing the binding activity of exemplary anti-CD3 Fab / anti-tumor scFv bispecific antibodies exhibited on T cells and tumor cells. Figure 9A: CTA03Fab / CTAT02scFv binding to CD3+ T cells (Jurkat) and CD19+ B-cell lymphoma (Raji). Figure 9B: CTA03Fab / CTAT03scFv binding to CD3+ T cells (Jurkat) and EGFR+ triple-negative breast cancer (MDA-MB-231). Figure 9C: CTA03Fab / CTAT04scFv binding to CD3+ T cells (Jurkat) and HER2+ breast cancer (MCF7 / HER2). Figure 9D: CTA03Fab / CTAT05scFv binding to CD3+ T cells (Jurkat) and PSMA+ prostate cancer (LNCaP). Figure 9E: CTA03Fab / CTAT07scFv binding to CD3+ T cells (Jurkat) and EpCAM+ prostate cancer (LNCaP). Figure 9F: CTA03Fab / CTAT08scFv binding to CD3+ T cells (Jurkat) and FAP+ mouse fibroblast cells (3T3 / FAP). Figure 9G: CTA03Fab / CTAT09scFv binding to CD3+ T cells (Jurkat) and PDL1+ triple-negative breast cancer (MDA-MB-231). Figure 9H: CTA03Fab / CTAT10scFv binding to CD3+ T cells (Jurkat) and CD38+ B-cell lymphoma (Raji). Figure 9I: CTA03Fab / CTAT11scFv binding to CD3+ T cells (Jurkat) and CD33+ human acute myeloid leukemia (HL-60). Figure 9J: CTA03Fab / CTAT12scFv binding to CD3+ T cells (Jurkat) and EGFR+ human lung cancer (A549). Figure 9K: CTA03Fab / CTAT13scFv binding to CD3+ T cells (Jurkat) and CD47+ breast cancer (MCF7 / HER2). Figure 9L: BsAb CTA02scFv / CTAT03Fab. Binding of CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab to CD3+ T cells (Jurkat) (upper panel) and EGFR+ colon cancer (HT-29) (lower panel). Samples were analyzed with FITC-conjugated goat anti-human IgG Fab antibody and flow cytometry. [Figure 10]Figure 10 is a chart showing the retention ability of exemplary BsAbs on the T cell surface. Human T cells were incubated with variant anti-CD3Fab / anti-CD19scFv BsAbs bearing Fabs from four different anti-CD3 antibodies (CTA01Fab / CTAT02scFv, CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, and CTA05Fab / CTAT02scFv) for 1 hour, then cultured in medium for 5 minutes, 24, 48, and 72 hours. After culture, cells were stained with an FITC-conjugated goat anti-human IgG Fab antibody, and BsAb retention on the T cell surface was analyzed using flow cytometry. [Figure 11] Figures 11A and 11B contain diagrams showing the formation of T cells armed with exemplary BsAbs disclosed herein. Figure 11A: From left to right, OKT3, CTA01Fab / CTAT02scFv, and CTA02Fab / CTAT02scFv. Figure 11B: CTA03Fab / CTAT02scFv (left) and CTA05Fab / CTAT02scFv (right). PBMCs were cultured in the presence of OKT3 or various BsAbs. Cell cultures were then stained with FITC-conjugated CD8 antibody and PE-conjugated goat anti-human IgG Fab and then analyzed using flow cytometry. [Figure 12]Figures 12A-12D contain diagrams showing the formation of T cells armed with exemplary BsAbs as indicated. Figure 12A: OKT3, CTA03Fab / CTAT03scFv, and CTA03Fab / CTAT04scFv (top panel, left to right), and CTA03Fab / CTAT09scFv, CTA03Fab / CTAT010scFv, and CTA03Fab / CTAT11scFv (bottom panel, left to right). Figure 12B: CTA03Fab / CTAT05scFv, CTA03Fab / CTAT07scFv, and CTAFab / CTAT08scFv (top panel, left to right), and CTA03Fab / CTAT12scFv and CTA03Fab / CTAT13scFv (bottom, left to right). Figure 12C: OKT3, CTA01scFv / CTAT03Fab, and CTA02scFv / CTA03Fab (left to right). Figure 12D: CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab (left to right). Cell cultures were stained with anti-CD8 antibody and goat anti-human IgG Fab and then analyzed using flow cytometry. [Figure 13] Figures 13A-13B contain charts showing the cytotoxic activity of T cells induced by OKT3 antibody or armed with exemplary bispecific antibodies against tumor cells. Figure 13A: Anti-CD3 Fab / anti-CD19 scFv BsAb against B cell lymphoma. Figure 13B: Anti-CD3 scFv / CTAT03 Fab BsAb against HT29 cells. Cells were cultured with OKT3 or exemplary BsAb as indicated. Cell cultures were then incubated with CD19+ B cell lymphoma (Raji) at several effector cell:target cell ratios (3:1, 5:1, and 10:1) for 18 hours. Tumor cell death was determined using the CytoTox 96® non-radioactive cytotoxicity assay (Promega, G1780). [Figure 14]Figures 14A-14G include figures showing the in vitro cytotoxicity activity of anti-CD3 Fab / anti-EGFR scFv BsAb armed T cells against cancer cells. Figure 14A: CTA01 Fab / CTAT03 scFv or CTA03 Fab / CTAT03 scFv armed T cells against HT29 cells (EGFR+ colon cancer cells). Figure 14B: CTA01 Fab / CTAT03 scFv or CTA03 Fab / CTAT03 scFv armed T cells against HCT-116 cells (EGFR+ colon cancer cells). Figure 14C: Anti-CD3 Fab / anti-HER2 scFv (CTA03 Fab / CTAT04 scFv) armed T cells against HER2+ breast cancer cells (MCF-7 / HER2). Figure 14D: Anti-CD3 Fab / anti-PSMA scFv (CTA03 Fab / CTAT05 scFv) armed T cells against PSMA+ prostate cancer cells (LNCaP). Figure 14E: Anti-CD3 Fab / anti-EpCAM scFv (CTA03 Fab / CTAT07 scFv) armed T cells against EpCAM+ prostate cancer cells (LNCaP). Figures 14F-14G: Anti-CD3 Fab / anti-FAP scFv (CTA03 Fab / CTAT08 scFv) armed T cells against FAP- mouse fibroblast cells (3T3) (Figure 14F) or FAP+ mouse fibroblast cells (3T3 / FAP) (Figure 14G). OKT3-cultured T cells or armed T cells were co-cultured with cancer cells at several effector cell:target cell ratios (3:1, 5:1, and 10:1) for 18 hours. Tumor cell death was determined using the CytoTox 96® non-radioactive cytotoxicity assay (Promega, G1780). [Figure 15]Figures 15A-15E include charts showing the in vitro cytotoxic activity of exemplary anti-CD3Fab / anti-TAA scFv BsAbs against corresponding cancer cells. Figure 15A: Anti-CD3Fab / anti-PDL1 scFv (CTA03Fab / CTAT09scFv) armed T cells against triple-negative breast cancer cells (MDA-MB-231). Figure 15B: Anti-CD3Fab / anti-CD38 scFv (CTA03Fab / CTAT10scFv) armed T cells against CD38+ B-cell lymphoma cells (Raji). Figure 15C: Anti-CD3Fab / anti-CD33 scFv (CTA03Fab / CTAT11scFv) armed T cells against CD33+ human acute myeloid leukemia cells (HL-60). Figure 15D: Anti-CD3 Fab / anti-HGFR scFv (CTA03 Fab / CTAT12 scFv) armed T cells against HGFR+ human lung cancer cells (A549). Figure 15E: Anti-CD3 Fab / anti-CD47 scFv (CTA03 Fab / CTAT13 scFv) armed T cells against CD47+ breast cancer cells (MCF7 / HER2). OKT3-cultured T cells or armed T cells were co-cultured with cancer cells at several effector cell:target cell ratios (3:1, 5:1, and 10:1) for 18 hours. Tumor cell death was determined using the CytoTox 96® non-radioactive cytotoxicity assay (Promega, G1780). [Figure 16] Figures 16A-16C include diagrams showing the in vivo therapeutic efficacy of exemplary anti-CD3 Fab / anti-CD19 scFv armed T cells against lymphoma. SCID mice were inoculated intravenously with CD19+ B-cell lymphoma cells (2.5 x 106 cells / mouse). Three days later, T cells, CTA01 Fab / CTAT02 scFv armed T cells, and CTA03 Fab / CTAT02 scFv armed T cells were injected intravenously into the mice (5 x 106 cells / mouse, weekly for four injections). Figure 16A: Body weight. Figure 16B: Survival rate. Figure 16C: Incidence of hind limb paralysis. [Figure 17]Figures 17A-17B include diagrams showing the in vivo therapeutic efficacy of exemplary CTA03Fab / CTAT03scFv armed T cells and CTA03Fab / CTAT04scFv armed T cells against human triple-negative breast cancer. ASID mice were subcutaneously inoculated with clinical human breast tumor tissue. 19 days later, T cells, CTA03Fab / CTAT03scFv armed T cells, and CTA03Fab / CTAT04scFv armed T cells were injected intravenously into the mice (5 x 106 cells / mouse, weekly for 4 doses). Figure 17A: Body weight. Figure 17B: Tumor size. [Figure 18] Figures 18A-18D contain diagrams showing the formation of BsAb-armed NKT cells with various anti-CD3 / anti-TAA BsAbs. Figure 18A: OKT3 (left) and CTA03Fab / CTAT03scFv (right). Figure 18B: CTA03Fab / CTAT04scFv (left) and CTA03Fab / CTAT05scFv (right). Figure 18C: OKT3, CTA01scFv / CTAT03Fab, and CTA02scFv / CTAT03Fab (left to right). Figure 18D: CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab (left to right). NKT cells (CD8+CD25+) were cultured and differentiated in the presence of OKT3 antibody or BsAb as indicated. The cells were then stained with anti-CD8, anti-CD56, and FITC-conjugated anti-human IgG Fab antibodies, and the BsAbs on the cell surface were analyzed using flow cytometry. [Figure 19]Figures 19A-19C contain charts showing the binding activity and toxicity of the point mutant BsAb CTA03Fab / CTAT02scFv. Figure 19A: Binding activity to CD3+ T cells (Jurkat). Figure 19B: Binding activity to CD19+ B cell lymphoma (Raji). Figure 19C: Cytotoxicity of armed T cells compared to T cells cultured with OKT3. Cells were cultured with CTA03Fab / CTAT02scFv, CTA03-01Fab / CTAT02-01scFv, CTA03-01Fab / CTAT02-02scFv, CTA03-02Fab / CTAT02-02scFv, and CTA03-02Fab / CTAT02-01scFv BsAbs and then stained with a FITC-conjugated goat anti-human IgG Fab antibody. Fluorescent signals on the cell surface were detected using flow cytometry. For cytotoxicity analysis, T cells were cultured with OKT3 or various BsAbs to form armed T cells. The cells were then cocultured with CD19+ B-cell lymphoma (Raji) at several effector:target cell ratios (3:1, 5:1, and 10:1) for 18 hours. Tumor cell death was determined using the CytoTox 96® non-radioactive cytotoxicity assay (Promega, G1780). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure is based on the development of bispecific antibodies (BsAbs) capable of binding to CD3 (e.g., human CD3) and tumor-associated antigens (TAAs). Such BsAbs can attach to the surface of CD3-positive immune cells via binding of the anti-CD3 moiety in the BsAb to cell surface CD3, generating armed immune cells. As used herein, the term "armed immune cells" refers to immune cells that display the bispecific antibodies disclosed herein via binding of the anti-CD3 moiety in the bispecific antibody to cell surface CD3 molecules. Via the anti-TAA moiety in the bispecific antibody on the cell surface, armed immune cells can target diseased cells (e.g., cancer cells) that express TAA, thereby eliciting an immune response against the diseased cells.

[0025] The BsAbs disclosed herein inhibit CD3 + Immune cells and TAAs + High binding activity to both cancer cells and CD3 + It is reported herein that BsAbs exhibit high retention levels on immune cells. Immune cells armed with the BsAbs disclosed herein exhibited high cytotoxicity against cancer cells expressing the corresponding TAA both in vitro and in vivo. Therefore, the BsAbs and armed immune cells disclosed herein are expected to have high anti-cancer effects.

[0026] Accordingly, provided herein are bispecific antibodies capable of binding to CD3 and TAAs, armed immune cells that present such antibodies, methods of using bispecific antibodies to produce armed immune cells, and methods of using armed immune cells to treat cancer.

[0027] I. Bispecific antibodies that bind to CD3 and tumor-associated antigens In some aspects, the present disclosure provides bispecific antibodies capable of binding to CD3 (e.g., CD3+ cells) and tumor-associated antigens (TAA) (e.g., cancer cells expressing a TAA on their cell surface). Antibodies (used interchangeably in the plural) are immunoglobulin molecules that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, polypeptides, etc., via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The bispecific antibodies disclosed herein comprise two antigen-binding moieties, one of which binds to CD3, such as human CD3, and the other of which binds to a tumor-associated antigen, such as those disclosed herein.

[0028] A typical antibody molecule typically contains a heavy chain variable region (V H ) and the light chain variable region (V L ) included. V H and V LThe regions can be further subdivided into regions of hypervariability, also known as "complementarity-determining regions" ("CDRs"), flanked by more conserved regions known as "framework regions" ("FRs"). H and V L is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely defined using methods known in the art, for example, by the Kabat definition, Chothia definition, AbM definition, and / or contact definition, which are well known in the art. See, e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242; Chothia et al. (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al. (1997) J. Mol. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.

[0029] In some embodiments, the antibody portions disclosed herein contain the same heavy and / or light chain complementarity determining regions (CDRs) or the same V as the reference antibody. H and / or V L They may share a chain. H and / or V LTwo antibodies that have CDRs mean that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the contact approach, or the IMGT approach, as known in the art. See, e.g., bioinf.org.uk / abs / ). Such anti-CD19 antibodies have the same V H , the same V L , or both.

[0030] In some embodiments, antibody portions disclosed herein may share a certain level of sequence identity compared to a reference sequence. The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993, modified as described in Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0031] In some embodiments, the antibody portions disclosed herein may have one or more amino acid mutations compared to a reference antibody. The amino acid residue mutations disclosed in the present disclosure (e.g., in framework regions and / or CDRs) may be conservative amino acid residue substitutions. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those found in references compiling such methods (e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M.A.usubel, et al., eds., John Wiley & Sons, Inc., New York). Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0032] A. Bispecific antibodies The bispecific antibodies disclosed herein comprise a CD3-binding portion (an anti-CD3 portion) and a TAA-binding portion (an anti-TAA portion).

[0033] (i)CD3 binding part The anti-CD3 portion of any of the bispecific antibodies disclosed herein comprises an antigen-binding fragment specific for a CD3 molecule, e.g., human CD3. In some embodiments, the anti-CD3 portion comprises a heavy chain variable region (V H ) and the light chain variable region (V L). In some examples, the anti-CD3 portion can be derived from a reference anti-CD3 antibody. Exemplary reference anti-CD3 antibodies include CTA.02, CTA.03, CTA.04, or CTA.05. Structural information for these reference anti-CD3 antibodies is provided in Table 1 below (heavy and light chain complementarity determining regions (CDRs) based on the Kabat scheme are in bold and underlined). [Table 1] JPEG2025161873000003.jpg250170JPEG2025161873000004.jpg247170JPEG2025161873000005.jpg136170

[0034] An anti-CD3 binding moiety (and anti-TAA binding moiety disclosed below) derived from a reference antibody refers to a binding moiety that has substantially similar structural and functional characteristics to the reference antibody. Structurally, a binding moiety may contain the same heavy and / or light chain complementarity determining regions, or the same V complementarity determining regions, as the reference antibody. H and / or V L Alternatively, the binding moiety may have only a limited number of amino acid variations in one or more of the framework regions and / or one or more of the CDRs without significantly affecting its binding affinity and specificity compared to a reference antibody.

[0035] In some embodiments, an anti-CD3 binding moiety may comprise the same heavy chain CDRs as in antibody CTA.02, provided above in Table 1. Alternatively, or in addition, an anti-CD3 binding moiety may have the same light chain CDRs as in antibody CTA.02, also provided above in Table 1. Such an anti-CD3 binding moiety may have the same V H Chain and / or V LAlternatively, the anti-CD3 binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTA.02. For example, the anti-CD3 binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework region in CTA.02.

[0036] In some embodiments, the anti-CD3 portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTA.02. For example, the anti-CD3 portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTA.02. H Alternatively, or in addition, the anti-CD3 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L The antibody may comprise light chain CDRs that, individually or collectively, share at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity with the corresponding CDR of a reference antibody (e.g., an anti-CD3 reference antibody provided in Table 1 above, or any of the anti-TAA reference antibodies disclosed below). "Collectively" refers to the sequence identity of the three Vs of the antibody. H or V L The CDR combinations are identical to the corresponding three V H or V L It means that the CDR combinations share the sequence identity shown.

[0037] In some examples, the anti-CD3 portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTA.02. In some examples, the anti-CD3 portion can include a heavy chain CDR3 that is the same as the heavy chain CDR3 of CTA.02 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0038] In some embodiments, an anti-CD3 binding moiety may comprise the same heavy chain CDRs as in antibody CTA.03, provided above in Table 1. Alternatively, or in addition, an anti-CD3 binding moiety may have the same light chain CDRs as in antibody CTA.03, also provided above in Table 1. Such an anti-CD3 binding moiety may have the same V H Chain and / or V L Alternatively, the anti-CD3 binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTA.03. For example, the anti-CD3 binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework region in CTA.03. In one specific example, the anti-CD3 moiety disclosed herein comprises a mutation, e.g., an amino acid residue substitution (e.g., G58A), at position G58 of the VL chain relative to CTA.03. See, e.g., CTA.03 VL-01 in Table 1 above.

[0039] In some embodiments, the anti-CD3 portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTA.03. For example, the anti-CD3 portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTA.03. H Alternatively, or in addition, the anti-CD3 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. LIt may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0040] In some examples, the anti-CD3 moiety can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTA.03. In some examples, the anti-CD3 moiety can include a heavy chain CDR3 that is the same as that of CTA.03 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs. In specific examples, the anti-CD3 moiety disclosed herein can include an amino acid residue substitution, such as a mutation at position D57 of the VL chain compared to that of CTA.03, e.g., D57E. See, e.g., CTA.03 VL-02 in Table 1.

[0041] In some examples, an anti-CD3 binding moiety may comprise the same heavy chain CDRs as in antibody CTA.04, provided above in Table 1. Alternatively, or in addition, an anti-CD3 binding moiety may have the same light chain CDRs as in antibody CTA.04, also provided above in Table 1. Such an anti-CD3 binding moiety may have the same V H Chain and / or V L Alternatively, the anti-CD3 binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTA.04. For example, the anti-CD3 binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework region in CTA.04.

[0042] In some embodiments, the anti-CD3 portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTA.04. For example, the anti-CD3 portion may contain the V HAlternatively, or in addition, the anti-CD3 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0043] In some examples, the anti-CD3 portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTA.04. In some examples, the anti-CD3 portion can include a heavy chain CDR3 that is the same as the heavy chain CDR3 of CTA.04 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0044] In some examples, an anti-CD3 binding moiety may comprise the same heavy chain CDRs as in antibody CTA.05, provided above in Table 1. Alternatively, or in addition, an anti-CD3 binding moiety may have the same light chain CDRs as in antibody CTA.05, also provided above in Table 1. Such an anti-CD3 binding moiety may have the same V H Chain and / or V L Alternatively, the anti-CD3 binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTA.05. For example, the anti-CD3 binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework region in CTA.05.

[0045] In some embodiments, the anti-CD3 portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTA.05. For example, the anti-CD3 portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTA.05. HAlternatively, or in addition, the anti-CD3 antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0046] In some examples, the anti-CD3 portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTA.05. In some examples, the anti-CD3 portion can include a heavy chain CDR3 that is the same as the heavy chain CDR3 of CTA.05 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0047] (ii) TAA binding part In addition to the anti-CD3 binding portion, any of the bispecific antibodies disclosed herein further comprises a second binding portion specific for a tumor-associated antigen. The term "tumor-associated antigen" (TAA) is well understood in the art and refers to a molecule that is differentially expressed on and / or in cancer cells compared to non-cancerous cells of the same cell type. Non-limiting examples of TAAs include CD5, CD19, CD20, CD22, CD23, CD25, CD27, CD30, CD33, CD34, CD37, CD38, CD40, CD43, CD44v6, CD47, CD50, CD52, CD56, CD63, CD72a, CD74, CD78, CD79a, CD79b, CD86, CD134, CD137, CD138, CD248, CD319, αvβ3, α5β1, human epidermal growth factor receptor (EGFR or HER1). , HER2, HER3, HER4, vascular endothelial growth factor receptor 1 (VEGFR-1), VEGFR-2, VEGFR-3, TRAIL-R2, carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA), mucin 1 (MUC1), MUC2, MUC3, MUC4, MUC5, MUC7, ganglioside GD2, ganglioside GD3, ganglioside GM2, carbonic anhydrase IX (CAIX), sonic hedgehog (SHH), black Chromosome chondroitin sulfate proteoglycan (MCSP), chondroitin sulfate proteoglycan 4 (CSPG4), six-transmembrane epithelial antigen of the prostate (STEAP), A33 antigen, desmogroin-2 (Dsg2), Dsg3, Dsg4, E-cadherin neoepitope, fetal nicotinic acetylcholine receptor (fnAChR), Muellerian inhibitory substance type II receptor (MISIIR), tumor-associated antigen L6 (TAL6), Thomsen-Friedenreich (TF) antigen, EPHA1, EPHA2, EPHA3, EPHA4, EPHA7, EPHA8, EPHA10, EPHB4, cancer testis antigen (CTA), NY-BR1, tumor-associated glycoprotein 72 (TAG-72), alpha-fetoprotein (AFP), brothers of regulator of imbrinogen-activated receptors (BORIS), B-cell activating factor (BAFF), extradomain-B fibronectin (EDB-FN), glycoprotein A33 (GPA33), tenascin-C (TNC), melanoma-associated antigen (MAGE),Examples of such proteins include GAGE, BAGE, prostate stem cell antigen (PSCA), mesothelin, mucin-associated Tn, sialyl Tn, globoH, stage-specific embryonic antigen-4 (SSEA-4), epithelial cell adhesion molecule (EpCAM), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death 1 (PD-1), programmed cell death 1 ligand 1 (PD-L1), prostate-specific membrane antigen (PSMA), fibroblast activation protein (FAP), vascular cell adhesion protein 1 (VCAM-1), insulin-like growth factor receptor (IGFR), and hepatocyte growth factor receptor (HGFR).

[0048] In some embodiments, the anti-TAA binding moiety comprises a heavy chain variable region (V H ) and the light chain variable region (V L). In some examples, the anti-TAA binding moiety is specific for CD20 (e.g., human CD20). In some examples, the anti-TAA binding moiety is specific for CD19 (e.g., human CD19). In some examples, the anti-TAA binding moiety is specific for EGFR (e.g., human EGFR). In some examples, the anti-TAA binding moiety is specific for HER2 (e.g., human HER2). In some examples, the anti-TAA binding moiety is specific for PSMA (e.g., human PSMA). In some examples, the anti-TAA binding moiety is specific for CEA (e.g., human CEA). In some examples, the anti-TAA binding moiety is specific for EpCAM (e.g., human EpCAM). In some examples, the anti-TAA binding moiety is specific for FAP (e.g., human FAP). In some examples, the anti-TAA binding moiety is specific for PDL1 (e.g., human PDL1). In some examples, the anti-TAA binding moiety is specific for CD38 (e.g., human CD38). In some examples, the anti-TAA binding moiety is specific for CD33 (e.g., human CD33). In some examples, the anti-TAA binding moiety is specific for HGFR(cMET) (e.g., human cMET). In some examples, the anti-TAA binding moiety is specific for CD47 (e.g., human CD47). In some examples, the anti-TAA binding moiety is specific for TRAIL-R2 (e.g., human TRAIL-R2). In some examples, the anti-TAA binding moiety is specific for mesothelin (e.g., human mesothelin). In some examples, the anti-TAA binding moiety is specific for GD2 (e.g., human GD2).

[0049] In some examples, the anti-TAA portion may be derived from a reference anti-TAA antibody. Exemplary reference anti-TAA antibodies include CTAT.01 to CTAT.16. Structural information for these reference anti-CD3 antibodies is provided in Table 2 below (heavy and light chain complementarity determining regions (CDRs) based on the Kabat scheme are in bold and underlined). [Table 2] JPEG2025161873000007.jpg199170JPEG2025161873000008.jpg225170JPEG2025161873000009.jpg222170JPEG202 5161873000010.jpg230170JPEG2025161873000011.jpg212170JPEG2025161873000012.jpg228170JPEG20251618730 00013.jpg224170JPEG2025161873000014.jpg222170JPEG2025161873000015.jpg223170JPEG2025161873000016.j pg229170JPEG2025161873000017.jpg222170JPEG2025161873000018.jpg225170JPEG2025161873000019.jpg190170

[0050] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.01, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.01, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or V L Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.01. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) in one or more framework regions compared to the corresponding framework region in CTAT.01.

[0051] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to that of CTAT.01. For example, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to that of CTAT.01. HAlternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0052] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.01. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.01 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0053] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.02, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.02, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or V L Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.02. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) in one or more framework regions compared to the corresponding framework region in CTAT.02.

[0054] In one specific example, the anti-TAA moiety disclosed herein comprises a mutation, e.g., an amino acid residue substitution (e.g., G42A), at position G42 of the VL chain relative to CTAT.02. See, e.g., CTAT.02 VL-01 in Table 2 above. In another specific example, the anti-TAA moiety disclosed herein comprises a mutation, e.g., an amino acid residue substitution (e.g., D41E), at position D41 of the VL chain relative to CTAT.02. See, e.g., CTAT.02 VL-02 in Table 2 above.

[0055] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.02. For example, the anti-TAA portion may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0056] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.02. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.02 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0057] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.03, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.03, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moieties may comprise amino acid mutations in one or more of the framework regions compared to the corresponding framework regions in CTAT.03. For example, the anti-TAA binding moieties may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework regions in CTAT.03.

[0058] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.03. For example, the anti-TAA portion may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0059] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.03. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.03 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0060] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.04, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.04, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise amino acid mutations in one or more of the framework regions compared to the corresponding framework regions in CTAT.04. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework regions in CTAT.04.

[0061] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.04. For example, the anti-TAA moiety may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0062] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.04. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.04 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0063] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.05, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.05, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moieties may comprise amino acid mutations in one or more of the framework regions compared to the corresponding framework regions in CTAT.05. For example, the anti-TAA binding moieties may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework regions in CTAT.05.

[0064] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.05. For example, the anti-TAA portion may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0065] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.05. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.05 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0066] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.06, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.06, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moieties may comprise amino acid mutations in one or more of the framework regions compared to the corresponding framework regions in CTAT.06. For example, the anti-TAA binding moieties may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework regions in CTAT.06.

[0067] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to that of CTAT.06. For example, the anti-TAA portion may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0068] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.06. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.06 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0069] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.07, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.07, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moieties may comprise amino acid mutations in one or more of the framework regions compared to the corresponding framework regions in CTAT.07. For example, the anti-TAA binding moieties may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework regions in CTAT.07.

[0070] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.07. For example, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.07. H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0071] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.07. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.07 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0072] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.08, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.08, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V and V sequences as CTAT.08. H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise amino acid mutations in one or more of the framework regions compared to the corresponding framework regions in CTAT.08. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework regions in CTAT.08.

[0073] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to that of CTAT.08. For example, the anti-TAA moiety may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0074] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.08. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.08 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0075] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.09, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.09, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.09. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework region in CTAT.09.

[0076] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.09. For example, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.09. H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0077] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.09. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.09 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0078] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.10, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.10, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise amino acid mutations in one or more of the framework regions compared to the corresponding framework regions in CTAT.10. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework regions in CTAT.10.

[0079] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.10. For example, the anti-TAA portion may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0080] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.10. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.10 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0081] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.11, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.11, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.11. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) in one or more framework regions compared to the corresponding framework region in CTAT.11.

[0082] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.11. For example, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.11. H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0083] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.11. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.11 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0084] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.12, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.12, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.12. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework region in CTAT.12.

[0085] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.12. For example, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.12. H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0086] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.12. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.12 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0087] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.13, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.13, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.13. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) in one or more framework regions compared to the corresponding framework region in CTAT.13.

[0088] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.13. For example, the anti-TAA moiety may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0089] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.13. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.13 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0090] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.14, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.14, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.14. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more framework regions compared to the corresponding framework region in CTAT.14.

[0091] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.14. For example, the anti-TAA moiety may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0092] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.14. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.14 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0093] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.15, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.15, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.15. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) in one or more framework regions compared to the corresponding framework region in CTAT.15.

[0094] In some embodiments, the anti-TAA moiety may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.15. For example, the anti-TAA moiety may contain the V H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0095] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.15. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.15 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0096] In some examples, an anti-TAA binding moiety may comprise the same heavy chain CDRs as in antibody CTAT.16, provided above in Table 2. Alternatively, or in addition, an anti-TAA binding moiety may have the same light chain CDRs as in antibody CTAT.16, also provided above in Table 2. Such an anti-TAA binding moiety may have the same V and V sequences as CTAT.16. H Chain and / or VL Alternatively, the anti-TAA binding moiety may comprise an amino acid mutation in one or more of the framework regions compared to the corresponding framework region in CTAT.16. For example, the anti-TAA binding moiety may collectively comprise up to 15 amino acid mutations (e.g., up to 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation) in one or more framework regions compared to the corresponding framework region in CTAT.16.

[0097] In some embodiments, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.16. For example, the anti-TAA portion may contain a certain level of mutation in one or more of the CDRs compared to those of CTAT.16. H Alternatively, or in addition, the anti-TAA antibody may comprise heavy chain CDRs that, individually or collectively, are at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identical to the CDRs. L It may comprise light chain CDRs that individually or collectively have at least 80% (eg, 85%, 90%, 95%, or 98%) sequence identity compared to the CDRs.

[0098] In some examples, the anti-TAA portion can include up to 10 amino acid mutations (e.g., up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) in one or more of the heavy and light chain CDRs collectively compared to those in the CDRs of CTAT.16. In some examples, the anti-TAA portion can include a heavy chain CDR3 that is the same as that of CTAT.16 and can include one or more amino acid mutations in one or more of the other heavy and light chain CDRs.

[0099] (iii) Anti-CD3 / anti-TAA bispecific antibody The bispecific antibodies disclosed herein may be in any suitable format known in the art, such as those disclosed in Mol. Immunol. 67(2):95-106 (2015), the relevant disclosure of which is incorporated by reference for the subject matter and purposes referenced herein. Some examples are provided below. See also Figures 1A-1N.

[0100] In some embodiments, a bispecific antibody disclosed herein may comprise one antigen-binding portion in Fab format and the other antigen-binding portion in single-chain variable fragment (scFv) format. Such a bispecific antibody may comprise two polypeptides, one comprising the heavy or light chain of the Fab fragment linked to the scFv fragment, and the other comprising the light or heavy chain of the Fab fragment that is not linked to the scFv fragment.

[0101] In some examples, a Fab fragment comprises two polypeptide chains, one comprising a VH domain linked to a fragment of a heavy chain constant region (e.g., CH1) and the other comprising a VL domain linked to a light chain constant region. The heavy chain constant region fragment can be derived from any Ig subclass, e.g., IgG, IgA, IgE, IgD, or IgM. In some examples, the heavy chain constant region fragment is derived from an IgG molecule (e.g., a human IgG molecule). The light chain constant region can be a kappa or lambda chain (e.g., a human kappa or lambda chain). An scFv fragment comprises a VH domain and a VL domain linked by a peptide linker. See, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. In some examples, the scFv fragment has a VH-linker-VL orientation from N- to C-terminus. Alternatively, the scFv fragment has a VL-linker-VH orientation from N- to C-terminus. In bispecific antibodies, the scFv fragment can be linked to the heavy chain of a Fab fragment. Alternatively, the scFv can be linked to the light chain of a Fab fragment. See Figures 1A-1H.

[0102] In some examples, the bispecific antibodies disclosed herein can comprise an anti-CD3 binding portion in Fab format and an anti-TAA binding portion in scFv format. Exemplary diagrams are provided in Figures 1A-1D. The anti-CD3 Fab comprises a heavy chain VH-CH1 domain and a light chain VL-CK or VL-Cλ domain. The anti-TAA scFv comprises a VH domain and a VL domain. Figures 1A-1D. In some examples, the anti-CD3 Fab can be linked to the anti-TAA scFv via a peptide linker disposed between the CH1 domain of the anti-CD3 Fab heavy chain and the VH domain of the anti-tumor scFv. An exemplary diagram is provided in Figure 1A. In other examples, the CH1 domain of the anti-CD3 Fab heavy chain can be linked to the VL domain of the anti-tumor scFv, as shown in Figure 1B. In other examples, an anti-TAA scFv can be linked to the Cκ or Cλ domain of an anti-CD3 Fab light chain via the VL domain of the scFv (FIG. 1C) or via the VH domain of an anti-tumor scFv (FIG. 1D). Examples of anti-CD3 Fab heavy chains (VH-CH1) and light chains (VL-Ck), as well as examples of anti-TAA scFv fragments, are provided in Tables 1 and 2, respectively. Any combination thereof is within the scope of this disclosure.

[0103] In some examples, the bispecific antibodies disclosed herein can comprise an anti-TAA binding portion in Fab format and an anti-CD3 binding portion in scFv format. Exemplary diagrams are provided in Figures 1E-1H. The anti-TAA Fab comprises a heavy chain VH-CH1 domain and a light chain VL-CK or VL-Cλ domain. The anti-CD3 scFv comprises a VH domain and a VL domain. Figures 1E-1H. In some examples, the anti-TAA Fab can be linked to the anti-CD3 scFv via a peptide linker disposed between the CH1 domain of the anti-TAA Fab heavy chain and the VH domain of the anti-CD3 scFv. An exemplary diagram is provided in Figure 1E. In other examples, the CH1 domain of the anti-TAA Fab heavy chain can be linked to the VL domain of the anti-CD3 scFv, as shown in Figure 1F. In other examples, an anti-CD3 scFv can be linked to the Cκ or Cλ domain of an anti-TAA Fab light chain via the VL domain of the scFv (FIG. 1G) or via the VH domain of the anti-CD3 scFv (FIG. 1H). Examples of anti-TAA Fab heavy chains (VH-CH1) and light chains (VL-Ck), as well as examples of anti-CD3 scFv fragments, are provided in Tables 2 and 1, respectively. Any combination thereof is within the scope of this disclosure.

[0104] In some embodiments, the bispecific antibodies disclosed herein may comprise both antigen-binding portions in scFv format. An exemplary illustration is provided in Figures 1I-1L.

[0105] In some examples, the VH domain of an anti-CD3 scFv can be linked to the VH domain of an anti-TAA scFv via a peptide linker (FIG. 1I). In some examples, the VH domain of an anti-CD3 scFv can be linked to the VL domain of an anti-TAA scFv via a peptide linker (FIG. 1J). In some examples, the VL domain of an anti-CD3 scFv can be linked to the VH domain of an anti-TAA scFv via a peptide linker (FIG. 1K). In other examples, the VL domain of an anti-CD3 scFv can be linked to the VH domain of an anti-TAA scFv via a peptide linker (FIG. 1L). Exemplary anti-CD3 scFv fragments and exemplary anti-TAA scFv fragments are provided in Tables 1 and 2, respectively. Any combination thereof to construct a bispecific antibody is within the scope of this disclosure.

[0106] In yet other embodiments, the bispecific antibodies disclosed herein may comprise one or more Fc regions, optionally in a "knob-into-hole" configuration, in which knobs in the CH2 domain, CH3 domain, or both, of a first heavy chain are created by replacing certain amino acid side chains with alternatives, and holes in juxtaposed positions in the CH3 domain of a second heavy chain are created by replacing appropriate amino acid side chains with alternatives. Exemplary diagrams are provided in Figures 1M and 1N.

[0107] Typically, the terms "knob and a hole" or "knobs-into-holes" are used interchangeably herein. Knob-into-hole amino acid changes are a rational design strategy known in the art for heavy (H) chain heterodimerization in the production of bispecific IgG antibodies. Carter, J. Immunol. Methods, 248(1-2):7-15 (2001), the relevant disclosures of which are incorporated herein by reference for the purposes and subject matter referred to herein.

[0108] In one example, a "knob-into-hole" approach is provided, e.g., as described in Ridgway JBB et al., (1996) Protein Engineering, 9(7):617-21 and US Pat. No. 5,731,168, the relevant disclosures of each of which are incorporated herein by reference for the purposes and subject matter referenced herein. This approach has been shown to promote the formation of heterodimers of a first polypeptide and a second polypeptide chain and prevent the assembly of the corresponding homodimer. In one aspect, the knob is created by replacing a small amino side chain at the interface between the CH3 domains with a larger one, while the hole is constructed by replacing a large side chain with a smaller one. In a specific example, the "knob" mutation includes T366W, and the "hole" mutations include T366S, L368A, and Y407V (Atwell S et al., (1997) J. Mol. Biol. 270:26-35).

[0109] In some examples, a bispecific antibody may comprise an anti-CD3 binding moiety comprising a first VH-CH1-CH2-CH3 domain and a first VL-CK or VL-Cλ domain, and an anti-TAA binding moiety comprising a second VH-CH1-CH2-CH3 domain and a second VL-CK or VL-Cλ domain ( Figure 1M ). The CH2 and / or CH3 in the heavy chain of the anti-TAA binding moiety may comprise a knob / hole modification, allowing binding between the two heavy chains. In other cases, a bispecific antibody may comprise an anti-CD3 binding moiety comprising a first VH-CH1-CH2-CH3 domain and a first VL-CK or VL-Cλ domain, and an anti-TAA scFv linked to a second CH2-CH3 domain. The CH2 and / or CH3 of the anti-TAA binding site may contain knob / hole modifications, allowing binding between the two heavy chains, as well as the CH2 and / or CH3 of the anti-CD3 binding site heavy chain (Figure 1N). In this configuration, the format of the anti-CD3 binding site and the format of the anti-TAA binding site may be switched.

[0110] The term "peptide linker" refers to a peptide containing natural or synthetic amino acid residues for connecting two polypeptides. For example, a peptide linker can be used to connect one VH domain and one VL domain to form a single-chain variable fragment (e.g., scFv); to connect one scFv and one Fab to form an scFv / Fab recombinant antibody; to connect two scFvs to form an scFv / scFv recombinant antibody; or to connect two monovalent antibodies (e.g., two monovalent IgGs), two monovalent antibody fragments (e.g., two monovalent scFv-Fc fusion proteins), or one monovalent antibody and one monovalent antibody fragment (e.g., one monovalent IgG and one monovalent scFv-Fc fusion protein) to form a bivalent antibody. Preferably, the peptide linker is a peptide having at least 5 amino acid residues in length, e.g., 5 to 100 amino acid residues in length, more preferably 10 to 30 amino acid residues in length. The peptide linker in the scFv is a peptide having a length of at least 5 amino acid residues, preferably 15 to 20 amino acid residues. n S) m where G=glycine, S=serine, and n and m are independently numbers 1 to 4. In one embodiment, the linker comprises the sequence (G2S)4. In another embodiment, the linker comprises the sequence (G4S)3.

[0111] The peptide linker for linking the first antibody fragment (i.e., anti-CD3 antibody fragment) and the second antibody fragment (i.e., anti-TAA antibody fragment) can be any peptide suitable for connecting two polypeptides. According to certain embodiments of the present disclosure, the peptide linker can be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, The peptide linker of the present recombinant antibody is a peptide having at least 5 amino acid residues in length, and having a length of 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acid residues in length. Preferably, the peptide linker of the present recombinant antibody consists of 10 to 30 glycine (G) and / or serine (S) residues.

[0112] In some embodiments, the bispecific antibodies described herein specifically bind to the corresponding target antigens (CD3 and TAA) or one or both of their epitopes. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, with longer duration, and / or with higher affinity with a particular target antigen compared to alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, avidity, more readily, and / or with longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (CD3 and / or TAA) or an antigen epitope therein is an antibody that binds to this target antigen with higher affinity, avidity, more readily, and / or with longer duration than it binds to other antigens or other epitopes within the same antigen. It is also understood that this definition, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (but can include) exclusive binding. In some examples, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes within the same antigen (i.e., with conventional methods, only baseline binding activity may be detected).

[0113] In some embodiments, the bispecific antibodies described herein have suitable binding affinity to one or both of the target antigens (e.g., CD3 and TAA) or their antigenic epitopes. As used herein, "binding affinity" refers to the apparent association constant or K A K A is the dissociation constant (K D The bispecific antibodies described herein have a binding affinity (K) for CD3 of at least 100 nM, 10 nM, 1 nM, 0.1 nM or less (e.g., less than 1 nM or less than 0.1 nM). DAlternatively, the bispecific antibodies described herein may have a binding affinity (K) for the TAA of at least 100 nM, 10 nM, 1 nM, 0.1 nM or less. D ).

[0114] Increased binding affinity corresponds to a decreased K D The higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a higher K for binding to the second antigen. A (or number K D ) for binding to the first antigen A (or a smaller number K D ). In such cases, the antibody has specificity for a first antigen (e.g., a first protein in a first conformation or a mimetic thereof) compared to a second antigen (e.g., the same first protein in a second conformation or a mimetic thereof, or a second protein). The difference in binding affinity (e.g., for specificity or other comparison) may be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 90, 100, 500, 1000, 10,000, or 10 5 In some embodiments, either the anti-CD3 and / or anti-TAA antibodies used to generate the bispecific antibody may be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

[0115] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). An exemplary condition for assessing binding affinity is HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20). Using these techniques, the concentration of bound binding protein can be measured as a function of target protein concentration. The concentration of bound binding protein ([bound]) is generally related to the concentration of free target protein ([free]) by the following formula: [bound] = [free] / (Kd + [free])

[0116] However, it is sufficient to obtain a quantitative measure of affinity, determined using methods such as, for example, ELISA or FACS analysis, and the K A and therefore may be used for comparison, such as determining whether a higher affinity is, for example, 2-fold higher, to obtain a qualitative measure of affinity or to obtain an inference of affinity by activity in a functional assay, e.g., in vitro or in vivo assay, since K A It is not necessary to make an accurate determination of the

[0117] Exemplary bispecific antibodies disclosed herein are provided below in Table 3 (using the anti-CD3 binding portion of CTA.03 as an example). Anti-CD3 binding portions from other anti-CD3 reference antibodies (e.g., CTA.02, CTA.04, and CTA.05) are also within the scope of this disclosure. [Table 3] JPEG2025161873000021.jpg200170JPEG2025161873000022.jpg230170JPEG2025161873000023.jpg225170JPEG2025161873000024.jpg54170

[0118] Also provided herein are pharmaceutical compositions comprising any of the bispecific antibodies disclosed herein (or armed immune cells also disclosed herein), further comprising a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient may be any inert substance combined with an active molecule (such as a bispecific antibody or armed immune cells) to prepare a suitable or convenient dosage form. Generally, a pharmaceutically acceptable excipient is non-toxic to recipients at the dosages and concentrations employed and is compatible with other components of the formulation, including the recombinant antibody. Examples of pharmaceutically acceptable excipients suitable for use in the present pharmaceutical compositions include, but are not limited to, water, phosphate buffer, acetate buffer, succinate buffer, citrate buffer, tris(hydroxymethyl)aminomethane (Tris) buffer, phosphate-buffered saline (PBS), Ringer's solution, lactated Ringer's solution, and combinations thereof. Optionally, the pharmaceutical composition may further comprise an agent for preserving and / or stabilizing the recombinant antibody, e.g., amino acid residues (such as histidine (H) or serine (S) residues), glucose, galactose, xylitol, sorbitol, mannitol, sucrose, trehalose, or an antioxidant. Other agents, such as antimicrobial agents, may be added to prevent spoilage during storage, i.e., to inhibit the growth of microorganisms such as yeast and mold.

[0119] B. Methods for Producing Bispecific Antibodies Any of the bispecific antibodies described herein can be produced by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, anti-CD3 and / or anti-TAA antibodies for use in producing bispecific antibodies can be produced by conventional hybridoma technology. Alternatively, anti-CD3 and / or anti-TAA antibodies can be identified from a suitable library (e.g., a human antibody library). In some examples, high-affinity fully human CD3 and / or TAA binders can be obtained from a human antibody library, for example, an affinity maturation library (e.g., with mutations in one or more of the CDR regions). There are several conventional methods known in the art for identifying and isolating antibodies capable of binding to the target antigens described herein, including phage display, yeast display, ribosome display, or mammalian display technologies.

[0120] In some embodiments, the bispecific antibodies disclosed herein may be produced by conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific for a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Once isolated, the DNA can be placed into one or more expression vectors, which are then transfected into host cells such as E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, resulting in the synthesis of the monoclonal antibody in the recombinant host cells. See, e.g., PCT Publication No. 87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0121] In some examples, nucleic acids encoding one or both chains of the bispecific antibodies described herein can be cloned into a single expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one example, the nucleotide sequences encoding the heavy and light chains are each operably linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter such that both the heavy and light chains are expressed from the same promoter. If desired, an internal ribosome entry site (IRES) can be inserted between the heavy chain-encoding sequence and the light chain-encoding sequence.

[0122] In some examples, the nucleotide sequences encoding the two chains of an antibody can be cloned into two vectors, which can be introduced into the same or different cells. If the two chains are expressed in different cells, they can be isolated from the host cells in which they are expressed, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to allow antibody formation.

[0123] Generally, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and be joined together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter depends on the type of host cell to be used in producing the antibody.

[0124] A variety of promoters can be used to express the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate-early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

[0125] Regulatable promoters can also be used, including those that use the lac repressor from E. coli as a transcriptional regulator to regulate transcription from mammalian cell promoters containing the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)] and those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad. Sci. USA 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16, or p65 using astroradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0126] Regulatable promoters containing repressors with operons can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional regulator to regulate transcription from mammalian cell promoters with lac operators [M. Brown et al., Cell, 49:603-612 (1987); Gossen and Bujard (1992); M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)]. The tetracycline repressor (tetR) was combined with a transcription activator (VP16) to create the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP16) along with a minimal promoter with tetO derived from the human cytomegalovirus (hCMV) major immediate-early promoter, creating a tetR-tet operator system for controlling gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. The tetracycline repressor (tetR) alone, but not tetR-mammalian cell transcription factor fusion derivatives, can function as a potent transregulator to control gene expression in mammalian cells when the tetracycline operator is appropriately positioned downstream of the TATA element of the CMVIE promoter (Yao et al., Human Gene Therapy, 10(16):1392-1399 (2003)). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which in some instances may be toxic to cells to achieve their regulatory effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0127] Additionally, vectors can contain some or all of the following: a selectable marker gene such as the neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate-early gene of human CMV for high-level transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; the SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

[0128] Examples of polyadenylation signals useful in practicing the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

[0129] Exemplary constructs for producing bispecific antibodies of various configurations disclosed herein are provided in Figures 2A-2E.

[0130] One or more vectors (e.g., expression vectors) containing a nucleic acid encoding any of the antibodies can be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under conditions suitable for expression of the antibody or any polypeptide chains thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or culture supernatant) via conventional methods, such as affinity purification. If desired, the antibody polypeptide chains can be incubated under suitable conditions for a suitable period of time to allow production of the antibody.

[0131] In some embodiments, methods for preparing the antibodies described herein involve recombinant expression vectors encoding both chains of the bispecific antibodies described herein. The recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions that allow for the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow for antibody formation.

[0132] In one example, two recombinant expression vectors are provided, each encoding one chain of the bispecific antibody disclosed herein. Both recombinant expression vectors can be introduced into suitable host cells (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow expression of the antibody polypeptide chains. When two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cell or from the culture medium and then incubated under suitable conditions that allow antibody formation. When two expression vectors are introduced into different host cells, each can be recovered from the corresponding host cell or from the corresponding culture medium. The two polypeptide chains can then be incubated under conditions that allow antibody formation.

[0133] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G coupled matrix.

[0134] Any of the nucleic acids encoding the bispecific antibodies described herein, vectors (e.g., expression vectors) containing such antibodies, and host cells containing the vectors are within the scope of this disclosure. Methods for producing such bispecific antibodies (e.g., using host cells via recombinant techniques) are also within the scope of this disclosure.

[0135] II. Armed immune cells In another aspect, provided herein are immune cells armed with any of the bispecific antibodies disclosed herein (e.g., those comprising Fab fragments and / or scFv chains provided in Tables 1 and 2, or the exemplary bispecific antibodies provided in Table 3 above). Bispecific antibodies can be presented on the surface of CD3+ immune cells via binding to cell surface CD3 molecules.

[0136] The immune cells may be any type of immune cell (e.g., human immune cell) that expresses surface CD3 or a mixture thereof. Examples include, but are not limited to, T cells, B cells, monocytes, and / or macrophages. In some examples, the T cells are conventional CD4+ and / or CD8+ T cells. In some examples, the T cells are regulatory T cells (Tregs). In other examples, the T cells are natural killer T cells (NKTs). The immune cells may be obtained from a donor, such as a body fluid donor (e.g., a healthy donor). Alternatively, the immune cells may be obtained from a cell line or differentiated from stem cells, such as hematopoietic stem cells, bone marrow cells, umbilical cord blood cells, or induced pluripotent stem cells.

[0137] Any of the armed immune cells can be produced by incubating suitable immune cells with any of the bispecific antibodies disclosed herein (e.g., those comprising Fab fragments and / or scFv chains provided in Tables 1 and 2, or the exemplary bispecific antibodies provided in Table 3 above) under suitable conditions for a suitable period of time. Unlike anti-CD3 antibodies alone (e.g., OKT3), incubation of the bispecific antibodies disclosed herein with immune cells results in the production of armed immune cells in which the bispecific antibody is displayed on the cell surface. The bispecific antibody can induce the proliferation and / or differentiation of immune cells, for example, by binding to CD3 molecules on T cells via its anti-CD3 binding portion, thereby inducing the differentiation of naive T cells into effector cells. The armed immune cells produced in this manner are capable of targeting cancer cells by recognizing TAA molecules expressed on cancer cells by the anti-TAA binding portion of the bispecific antibody displayed on the surface of the armed immune cells.

[0138] In some examples, the armed immune cells disclosed herein can be produced using peripheral blood mononuclear cells (PBMCs). For example, PBMCs can be isolated from a donor (e.g., a human donor) using conventional methods. Suitable methods for isolating PBMCs from a donor include, but are not limited to, density centrifugation (e.g., FICOLL® Paque), cell preparation tubes (CPT), and SEPMATE™ tubes. In some examples, PBMCs can be isolated from a whole blood sample obtained from a donor via density centrifugation according to the manufacturer's instructions. The isolated PBMCs can then be cultured with the bispecific antibody in a suitable cell culture medium for at least 7 days, e.g., 7, 8, 9, 10, 11, 12, 13, 14 days, or more, preferably at least 14 days. In some embodiments, CD3 T cells, such as T cells, can be cultured with the bispecific antibody. + The number of immune cells (e.g., CD4 / CD8 T cells and / or NKT cells) is expanded after 7 days of culture. In another embodiment, the culture is continued for 14 days, and the number of CD3 + The number of T cells increases threefold.

[0139] In other examples, immune cells derived from cell culture can be used to generate the armed immune cells disclosed herein. In vitro cultured immune cells can be derived from established cell lines. Alternatively, immune cells can be differentiated from suitable stem cells, such as hematopoietic stem cells, bone marrow cells, umbilical cord blood cells, or induced pluripotent stem cells, according to conventional methods.

[0140] A suitable amount of immune cells (e.g., 3 x 10 5 cells) may be cultured in a suitable cell culture medium in the presence of about 500 ng to about 3,000 ng (e.g., 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, or 3,000 ng) of the bispecific antibody under suitable conditions and for a suitable period of time to produce armed immune cells. The cell culture medium may also include one or more cytokines to maintain the growth of and / or stimulate the activation of immune cells, such as T cells. Examples include, but are not limited to, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-12, IL-18, IL-21, IL-23, IL-25, IL-27, IL-31, interferon-γ (IFN-γ), TGF-β, or a combination thereof. Additionally or alternatively, the medium may contain antibodies or carbohydrates for activation purposes, such as anti-CD28 antibodies or mannose.

[0141] In some examples, IL-2 is used in the culture medium to culture PBMCs, e.g., to induce cell proliferation of armed CD8 + In another example, IL-2 and IL-7 may be used in the culture medium for culturing PBMCs to produce armed CD4+ T cells. To produce armed Treg cells, IL-2, anti-CD28 antibody, and mannose may be used in the cell culture medium.

[0142] Armed immune cells produced by any of the methods disclosed herein are also within the scope of this disclosure.

[0143] III. Cancer treatment with armed immune cells In another aspect, the present disclosure provides methods for treating cancer using the armed immune cells disclosed herein. To practice the methods disclosed herein, an effective amount of armed immune cells or a pharmaceutical composition comprising such immune cells can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time. In some examples, the armed immune cells are autologous to the subject. In other cases, the armed immune cells are allogeneic to the subject.

[0144] The subject treated by the methods described herein may be a mammal, more preferably a human or non-human primate. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats. A human subject in need of treatment may be a human patient suffering from, at risk of, or suspected of suffering from a target disease / disorder characterized by tumor cells expressing the target TAA to which the bispecific antibody binds. Exemplary cancers include, but are not limited to, melanoma, esophageal cancer, gastric cancer, brain tumor, small cell lung cancer, non-small cell lung cancer, bladder cancer, breast cancer, pancreatic cancer, colon cancer, rectal cancer, colorectal cancer, kidney cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, thyroid cancer, testicular cancer, head and neck squamous cell carcinoma, leukemia, lymphoma, and myeloma.

[0145] The presence of specific tumor-associated antigens by specific types of cancer cells is known in the art. For example, B-cell malignancies are often associated with CD19+ (e.g., B-cell acute lymphoblastic leukemia) and / or CD20+ cancer cells (e.g., B-cell non-Hodgkin's lymphoma). EGFR is expressed on various types of cancer, such as lung cancer and colon cancer. HER2 is associated with, for example, breast cancer. PSMA is associated with, for example, prostate cancer. CEA is associated with various types of cancer, including colon cancer, rectal cancer, and pancreatic cancer. EpCAM, FAP, CD47, and TRAIL-R2 are associated with solid tumors. PDL1 is associated with various cancers, such as bladder cancer, non-small cell lung cancer, breast cancer, and small cell lung cancer. CD38 is associated with, for example, multiple myeloma. CD33 is associated with, for example, AML. cMET (HGFR) is associated with, for example, non-small cell lung cancer. Mesothelin is associated with mesothelioma. GD2 is associated with neuroblastoma. Therefore, it is within the knowledge of a physician to select a bispecific antibody disclosed herein with an anti-TAA binding moiety suitable for treating a particular type of cancer.

[0146] Subjects with target cancers can be identified by routine examination, for example, clinical tests, organ function tests, CT scans, or ultrasound. In some embodiments, subjects treated by the methods described herein may be human cancer patients who have undergone or are undergoing anti-cancer therapy, for example, chemotherapy, radiation therapy, immunotherapy, or surgery. Subjects suspected of having any of such target diseases / disorders may exhibit one or more symptoms of the disease / disorder. Subjects at risk for a disease / disorder may have one or more risk factors for the disease / disorder.

[0147] As used herein, "effective amount" refers to the amount of each active agent required to confer a therapeutic effect on a subject, either alone or in combination with one or more other active agents. Determining whether a certain amount of antibody achieves a therapeutic effect will be apparent to one of ordinary skill in the art. Effective amounts will vary, as recognized by those skilled in the art, depending on the specific condition being treated, the severity of the condition, individual patient parameters including age, physical condition, size, sex, and weight, duration of treatment, nature of concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of health care professionals. These factors are well known to those skilled in the art and can be addressed with no more than routine experimentation. It is generally preferred to use the maximum dose of each individual component or combination thereof, i.e., the highest safe dose according to sound medical judgment.

[0148] Empirical considerations such as half-life will generally contribute to determining the dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment and is generally, but not necessarily, based on the treatment and / or suppression and / or improvement and / or delay of the target disease / disorder. Alternatively, a sustained continuous release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0149] In one example, dosages of the antibodies described herein can be empirically determined in individuals given one or more doses of the antibody. The individual is given incremental doses of an agonist. Disease / disorder indicators can be followed to assess the effectiveness of the agonist.

[0150] The particular dosing regimen, i.e., dosage, timing, and repetition, will depend on the particular individual and their medical history, as well as the characteristics of the particular drug (such as the drug's half-life and other considerations well known in the art).

[0151] For purposes of the present disclosure, the appropriate dosage of armed immune cells as described herein depends on the particular bispecific antibody directed against the immune cells, the type of immune cells (or composition thereof) used, the type and severity of the disease / disorder, the patient's clinical history and response to the agonist, and the discretion of the attending physician. Typically, a clinician will administer armed immune cells until a dosage is reached that achieves the desired result. Methods for determining whether a dosage has produced the desired result will be apparent to those of skill in the art. Administration of one or more doses of armed immune cells can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of armed immune cells can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, for example, either before, during, or after the onset of the target disease or disorder.

[0152] In some embodiments, the amount of armed immune cells, such as armed T cells, administered to a subject is about 1 x 10 4 ~1×10 7 In certain embodiments, the amount of armed immune cells, such as armed T cells, in a subject can be about 1 x 10 cells / Kg body weight. 5 ~1×10 6 Cells / Kg body weight of the subject may be administered. The dose may be administered in a single dose, or alternatively, in multiple doses.

[0153] As used herein, the term "treating" refers to the application or administration to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder of a subject of a composition comprising one or more active agents for the purpose of treating, curing, alleviating, mitigating, altering, relieving, ameliorating, improving, or affecting the disorder, symptom of the disease, or predisposition to the disease or disorder.

[0154] Alleviating a target disease / disorder includes delaying the onset or progression of the disease, reducing the severity of the disease, or extending survival. Alleviating a disease or extending survival does not necessarily require a curative outcome. As used herein, "delaying" the onset of a target disease or disorder means delaying, preventing, slowing, delaying, stabilizing, and / or postponing the progression of the disease. This delay can be for various lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease or delaying the occurrence of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease within a given time frame and / or reduces the severity of symptoms within a given time frame compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

[0155] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of the disease. Disease onset can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes occurrence, recurrence, and onset. As used herein, "onset" or "occurrence" of a target disease or disorder includes initial onset and / or recurrence.

[0156] Depending on the type of cancer or site of the cancer being treated, armed immune cells or pharmaceutical compositions comprising such immune cells can be administered to a subject using conventional methods known to those skilled in the medical arts. In some instances, armed immune cells can be administered via intravenous infusion.

[0157] In some embodiments, the armed immune cells disclosed herein can be used in combination with another anti-cancer agent, e.g., a chemotherapeutic agent, an immunotherapeutic agent, or a combination thereof. For example, the armed immune cells disclosed herein can be used in combination with an immune checkpoint inhibitor, such as an anti-PD-1 antibody or an anti-PDL1 antibody. As used herein, the terms "combination," "combined," and related terms refer to the simultaneous or sequential administration of multiple therapeutic agents according to the present disclosure. For example, the armed immune cells disclosed herein can be administered simultaneously with another therapeutic agent, sequentially in separate unit dosage forms, or together in a single unit dosage form.

[0158] In one example, a method for treating a subject (e.g., a human cancer patient) having cancer cells that express a TAA using the armed immune cells disclosed herein may include the following steps: (a) isolating PBMCs from the patient, (b) culturing the PBMCs of step (a) with a bispecific antibody disclosed herein that comprises a binding moiety specific for a TAA to produce armed immune cells, such as armed T cells, and (c) administering an effective amount of the armed immune cells to the subject.

[0159] In step (a), PBMCs can be isolated from a subject. The subject can be any mammal, such as a human, mouse, rat, chimpanzee, rabbit, monkey, sheep, goat, cat, dog, horse, or pig. Preferably, the subject is a human. Suitable methods for isolating PBMCs from a subject include, but are not limited to, density centrifugation (e.g., FICOLL® Paque), cell preparation tubes (CPT), and SEPMATE™ tubes.

[0160] In step (b), the isolated PBMCs are cultured in a medium containing the recombinant antibody of the present invention for a sufficient period (e.g., at least 7 days) to produce TAA-specific T cells. The bispecific antibody can induce T cell activation by its anti-CD3 antibody fragment. The armed T cells thus produced have the bispecific antibody bound to their surface, and therefore can specifically target cancer cells via the anti-TAA antibody fragment of the bispecific antibody.

[0161] In step (c), the armed immune cells, such as armed T cells, produced in step (b) can be administered to a subject to treat cancer. The amount of T cells administered to a subject is about 1 x 10 4 ~1×10 7 In certain embodiments, the amount of T cells is about 1 x 10 cells / Kg body weight of a subject. 5 ~1×10 6 The cells / Kg body weight are administered to the subject. The dose can be administered in a single dose, or alternatively, in multiple doses.

[0162] Optionally, prior to step (c), the method may further isolate T cells from the product of step (b) by a method suitable for isolating or purifying immune cells, e.g., affinity columns, or magnetic beads. The efficacy of the treatment can be examined through routine practice.

[0163] IV. Cancer Treatment Kits The present disclosure also provides kits comprising any of the armed immune cells, such as armed T cells, or any of the bispecific antibodies disclosed herein. Such kits can be used to treat or alleviate the targeted cancers disclosed herein. Such kits can include one or more containers containing the armed immune cells or bispecific antibodies described herein.

[0164] In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering armed immune cells or using a bispecific antibody to generate armed immune cells to treat, delay the onset of, or alleviate a target disease as described herein. The kit can further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for the target disease.

[0165] Instructions for use of armed immune cells, such as armed T cells or bispecific antibodies, generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. Instructions provided with kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0166] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or ameliorating the disease, such as cancer or an immune disorder (e.g., an autoimmune disease). Instructions for practicing any of the methods described herein can be provided.

[0167] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar bags or plastic bags), etc. Also contemplated are packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), or injection devices such as minipumps. The kit may have a sterile access port (e.g., the container may be an infusion bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an infusion bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an armed immune cell or a bispecific antibody as described herein.

[0168] Kits may optionally provide additional components such as buffers and interpretive information. Typically, kits include a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the above-described kit.

[0169] Common techniques The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are described in the literature, e.g., Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press, Oligonucleotide Synthesis (MJ Gait, ed. 1984), Methods in Molecular Biology, Humana Press, Cell Biology: A Laboratory Notebook (JECellis, ed., 1989) Academic Press, Animal Cell Culture (RIFreshney, ed. 1987), Introuction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and DG Newell, eds. 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology(DMWeir and CCBlackwell, eds.): Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al. eds. 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994), Current Protocols in Immunology (JEColigan et al., eds.,1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988 - 1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J.D. Capra, eds. Harwood Academic Publishers, 1995), DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985), Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)), Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984)), Animal Cell Culture (R.I. Freshney, ed. (1986)), Immobilized Cells and Enzymes (lRL Press, (1986)), and B. Perbal, A practical Guide To Molecular Cloning (1984), are fully described in F.M. Ausubel et al. (eds.).

[0170] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purpose or subject matter referenced therein.

[0171] Example 1: Production of recombinant bispecific antibodies In this example, recombinant antibodies having the structures shown in Figures 1A-1L were prepared using the DNA constructs shown in Figures 2A-2E, respectively. In the case of anti-CD3 Fab / anti-TAA scFv bispecific antibodies, the constructs comprised, from N- to C-terminus, (a) an Igk leader sequence (LS), an anti-CD3 VL-CK domain or an anti-CD3 VL-Cλ domain, an internal ribosome entry site (IRES), an LS, an anti-CD3 VH-CH1 domain, a peptide linker, and an anti-TAA scFv (e.g., anti-EGFR scFv) (Figures 1A-1D) and the top two constructs in Figure 2A; or (b) an LS, an anti-CD3 VL-CK domain or an anti-CD3 VL-Cλ domain, a peptide linker, an anti-TAA scFv (e.g., anti-EGFR scFv), an IRES, an LS, and an anti-CD3 VH-CH1 domain (Figures 1B and 2A, the bottom two constructs).

[0172] In the case of anti-CD3 scFv / anti-TAA Fab, the constructs comprise, from N- to C-terminus, (a) an LS, an anti-TAA VL-CK domain (e.g., an anti-EGFR VL-CK domain), an IRES, an LS, an anti-TAA VH-CH1 domain (e.g., an anti-EGFR VH-CH1 domain), a peptide linker, and an anti-CD3 VH-VL domain or an anti-CD3 VL-VH domain (Figures 1E-1H and 2B, top two constructs); or (b) an LS, an anti-TAA VL-CK domain (e.g., an anti-EGFR VL-CK domain), a peptide linker, an anti-CD3 VH-VL domain or an anti-CD3 VL-VH domain, an IRES, an LS, and an anti-TAA VH-CH1 domain (e.g., an anti-EGFR VH-CH1 domain) (Figures 1F and 2B, bottom two constructs).

[0173] In the case of anti-CD3 scFv / anti-TAA scFv, the construct comprises, from N- to C-terminus, LS, anti-TAA scFv (e.g., anti-EGFR scFv), a peptide linker, and anti-CD3 VH-VL domains or anti-CD3 VL-VH domains (Figures 1I-1L and 2C).

[0174] In the case of anti-CD3 knob / anti-TAA hole antibodies, the anti-CD3 knob construct contained, from N- to C-terminus, an LS, an anti-CD3 VL-Ck domain or an anti-CD3 VL-Cλ domain, an IRES, an LS, and an anti-CD3 VH-CH1 knob Fc, whereas the anti-tumor hole contained the following sequence: an LS, an anti-TAA VL-Ck (e.g., an anti-EGFR VL-Ck domain), an IRES, an LS, and an anti-TAA VH-CH1-hole Fc (e.g., an anti-EGFR VH-CH1-hole Fc) (Figure 2D).

[0175] In the case of anti-CD3 knob / anti-TAA scFv hole antibodies, the anti-CD3 knob construct contained the following sequence: LS, anti-CD3 VL-CK domain or anti-CD3 VL-Cλ domain, IRES, LS, and anti-CD3 VH-CH1-knob Fc, while the anti-tumor hole contained the following sequence: LS, anti-TAA scFv (e.g., anti-EGFR scFv), peptide linker, and hole Fc (Figure 2E).

[0176] Two recombinant antibodies, designated CTA02scFv / CTAT03Fab (formerly named anti-EGFR Fab / CAT.02 scFv) and CTA01scFv / CTAT03Fab (formerly named anti-EGFR Fab / aCD3 scFv, the structural information of which is provided in WO2018 / 177371, the relevant disclosure of which is incorporated by reference for the subject matter and purposes referenced herein), were accordingly prepared. Both CTA02scFv / CTAT03Fab and CTA01scFv / CTAT03Fab comprised anti-EGFR Fab and anti-CD3 scFv, and the VH-CH1 and VL-Ck domains of the anti-EGFR Fab had the amino acid sequences of SEQ ID NOs: 83 and 84, respectively. The anti-CD3 scFv of CTA02scFv / CTAT03Fab has the amino acid sequence of SEQ ID NO: 9, and the anti-CD3 scFv of CTA01scFv / CTAT03Fab is provided in WO2018 / 177371 (CTA01 is the same antibody named OKT3 in WO2018 / 177371).

[0177] Example 2: Effect of recombinant bispecific antibodies on T cells This example investigates the biological activity of the recombinant bispecific antibodies disclosed herein.

[0178] binding affinity The binding affinity of the recombinant antibodies CTA02scFv / CTAT03Fab and CTA01scFv / CTAT03Fab was examined by flow cytometry in this example. Both CTA02scFv / CTAT03Fab and CTA01scFv / CTAT03Fab were able to bind to CD3-positive T cells. The binding affinity of CTA02scFv / CTAT03Fab was higher than that of CTA01scFv / CTAT03Fab (Figure 3). The binding affinity results are provided in Table 4 below. [Table 4]

[0179] Cytotoxic effects In this example, the cytotoxic effects of recombinant antibodies CTA02scFv / CTAT03Fab or CTA01scFv / CTAT03Fab on cancer cells were evaluated. Approximately 2.5%, 18.2%, and 26.9% of HT-29 cells expressed CD3 activators activated by mouse OKT3. + / CD8 + Approximately 13.8%, 34.8%, and 65.7% of HT-29 cells were killed by T cells at effector-to-target cell ratios (E / T ratios) of 3:1, 5:1, and 10:1, respectively, and CD3 T cells were killed by CTA01scFv / CTAT03Fab-armed CD3 T cells. + / CD8 + It was found that approximately 28.1%, 44.4%, and 76.7% of HT-29 cells were killed by T cells armed with CTA02scFv / CTAT03Fab at the same E / T ratio (Figure 4 and Table 5). [Table 5]

[0180] The data showed that both of the tested bispecific antibodies exhibited higher cytotoxic effects against cancer cells compared to the OKT3 antibody (anti-CD3 antibody), and CTA02scFv / CTAT03Fab exhibited better cytotoxic effects.

[0181] Time effect on the amount of antibody remaining on the surface of T cells CTA02scFv / CTAT03Fab and CTA01scFv / CTAT03Fab were incubated with T cells in the presence of 20% FBS for 24 hours. The T cells were then analyzed by flow cytometry to assess the amount of antibody remaining on the surface of the T cells. The results of this assay show that the amount of antibody on the surface of T cells decreased over time (see Figure 5 and Table 6 below). Compared to CTA01scFv / CTAT03Fab, CTA02scFv / CTAT03Fab was less susceptible to degradation. Approximately 84.5% of CTA02scFv / CTAT03Fab remained on the T cell surface after 24 hours, while the level of CTA01scFv / CTAT03Fab remaining on the T cell surface decreased to approximately 40% after 24 hours. [Table 6]

[0182] In summary, the bispecific antibodies disclosed herein (exemplified by the CTA02scFv / CTAT03Fab antibody) exhibited higher binding affinity to T cells, higher cytotoxicity, and higher levels of T cell engagement over time compared to the CTA01scFv / CTAT03Fab control antibody.

[0183] Example 2: Construction of variant anti-CD3 / anti-tumor bispecific antibodies A panel of various anti-CD3 / anti-tumor-associated antigen (TAA) bispecific antibodies (BsAbs) was constructed by genetic engineering. These BsAbs were derived from four anti-CD3 antibodies and 16 anti-TAA antibodies (CD20 (CTAT01), CD19 (CTAT02), EGFR (CTAT03), HER2 (CTAT04), PSMA (CTAT05), CEA (CTAT06), EpCAM (CTAT07), FAP (CTAT08), PDL1 (CTAT09), CD38 (CTAT10), CD33 (CTAT11), HGFR (CTAT12), CD47 (CTAT13), TRAIL-R2 (CTAT14), mesothelin (CTAT15), and GD2 (CTAT16)). See Tables 1 and 2 above.

[0184] BsAbs were produced via recombinant technology in mammalian host cells, collected, and examined by SDS-PAGE under reducing and non-reducing conditions. Briefly, protein electrophoresis using 8% SDS-PAGE was performed under non-reducing and reducing conditions to analyze the structure and molecular weight of various BsAbs, including anti-CD3 and anti-TAA fragments.

[0185] Figures 6A-6B show the expression and assembly of BsAbs, each containing Fabs of four different anti-CD3 antibodies (CTA02, CTA03, CTA04, and CTA05, see Table 1 above) and an anti-CD19 scFv (CTAT02, see Table 2 above).

[0186] Figures 7A-7D show the expression and assembly of BsAbs, each containing an anti-CD3 Fab (CTA03, see Table 1 above) and scFvs of 16 different anti-tumor antibodies (CD20 (CTAT01), CD19 (CTAT02), EGFR (CTAT03), HER2 (CTAT04), PSMA (CTAT05), CEA (CTAT06), EpCAM (CTAT07), FAP (CTAT08), PDL1 (CTAT09), CD38 (CTAT10), CD33 (CTAT11), HGFR (CTAT12), CD47 (CTAT13), TRAIL-R2 (CTAT14), mesothelin (CTAT15), and GD2 (CTAT16), see Table 2 above). Figures 7E and 7F show the expression of BsAbs, each containing the scFv of one of the four anti-CD3 antibodies (CTA02 to CTA05) and the Fab fragment of anti-EGFR CTAT03.

[0187] Example 3: Anti-CD3 / anti-TAA BsAb binds to T cells and targets TAA-expressing tumor cells The binding activity of various anti-CD3 / anti-tumor BsAbs to T cells and tumor cells was analyzed using flow cytometry. CD3- and CD19-specific BsAbs (CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, CTA04Fab / CTAT02scFv, and CTA05Fab / CTAT02scFv) all bound to CD3. + T cells (Jurkat) and CD19 + T cells armed with such BsAb showed binding activity to B cell lymphoma (Raji), and CD19 + We demonstrated that CD19+ disease cells, such as B-cell lymphoma, can be targeted.

[0188] We also investigated the binding activity of BsAbs with binding fragments to other TAAs. As shown in Figures 9A to 10K, CTA03Fab / CTAT02scFv binds to CD3 + T cells (Jurkat) and CD19 + showed binding activity to B-cell lymphoma (Raji) ( Fig. 9A ); CTA03Fab / CTAT03scFv binds to CD3 + T cells (Jurkat) and EGFR + It showed binding activity to triple-negative breast cancer (MDA-MB-231) ( Figure 9B ); CTA03Fab / CTAT04scFv binds to CD3 + T cells (Jurkat) and HER2 + It showed binding activity to breast cancer (MCF7 / HER2) ( Figure 9C ); CTA03Fab / CTAT05scFv binds to CD3 + T cells (Jurkat) and PSMA + It showed binding activity to prostate cancer (LNCaP) ( Figure 9D ); CTA03Fab / CTAT07scFv binds to CD3 + T cells (Jurkat) and EpCAM + It showed binding activity to prostate cancer (LNCaP) ( Figure 9E ); CTA03Fab / CTAT08scFv binds to CD3+ T cells (Jurkat) and FAP + It showed binding activity to mouse fibroblast cells (3T3 / FAP) ( Figure 9F ); CTA03Fab / CTAT09scFv binds to CD3 + T cells (Jurkat) and PDL1 + It showed binding activity to triple-negative breast cancer (MDA-MB-231) ( Figure 9G ); CTA03Fab / CTAT10scFv binds to CD3 + T cells (Jurkat) and CD38 + showed binding activity to B-cell lymphoma (Raji) ( Figure 9H ); CTA03Fab / CTAT11scFv binds to CD3 + T cells (Jurkat) and CD33 + It showed binding activity to human acute myeloid leukemia (HL-60) ( Figure 9I ); CTA03Fab / CTAT12scFv binds to CD3 + T cells (Jurkat) and HGFR + It showed binding activity to human lung cancer (A549) (Figure 9J); and CTA03Fab / CTAT13scFv binds to CD3 + T cells (Jurkat) and CD47 + It showed binding activity to breast cancer (MCF7 / HER2) (Fig. 9K).

[0189] Furthermore, the binding activity of various anti-CD3 scFv / anti-tumor Fab BsAbs to T cells and tumor cells was analyzed using flow cytometry. Figure 9L shows the targeting ability of BsAbs consisting of scFvs from four different anti-CD3 antibodies (CTA02, CTA03, CTA04, and CTA05) and an anti-EGFR Fab (CTAT03) to CD3+ T cells (Jurkat) and EGFR+ colon cancer (HT-29). The results demonstrated that CTA02scFv / CTAT03Fab, CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab all possessed the targeting ability to CD3+ T cells (Jurkat) and EGFR+ colon cancer (HT-29).

[0190] Example 4: Retention ability of BsAb on the surface of T cells The retention time of BsAb on the T cell surface was analyzed using an in vitro incubation platform. Briefly, human T cells were incubated with various anti-CD3 Fab / anti-CD19 scFvs (CTA01 Fab / CTAT02 scFv, CTA02 Fab / CTAT02 scFv, CTA03 Fab / CTAT02 scFv, and CTA05 Fab / CTAT02 scFv) for 1 hour and then cultured in medium for 5 minutes, 24 hours, 48 ​​hours, and 72 hours. After culture, the remaining amount of BsAb on the T cell surface was detected using flow cytometry. After 72 hours, CTA01Fab / CTAT02scFv, CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, and CTA05Fab / CTAT02scFv were all detected on the T cell surface, with CTA03Fab / CTAT02scFv having the highest retention on the T cell surface (Figure 10).

[0191] Example 5: Preparation of armed immune cells with BsAb by one-step incubation Human peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in the presence of OKT3 antibody or in the presence of exemplary BsAbs disclosed herein (CTA01Fab / CTAT02scFv, CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, and CTA05Fab / CTAT02scFv are used as examples) and differentiated into T cells. All groups were cultured under the same conditions (in an incubator with 5% CO2 supply and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to measure the production of BsAb-armed T cells.

[0192] As shown in Figures 11A and 11B, the OKT3 anti-CD3 antibody induced differentiation of PBMCs only into normal T cells, but not into armed T cells. In contrast, the CTA01Fab / CTAT02scFv, CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, and CTA05Fab / CTAT02scFv BsAbs all successfully generated armed T cells.

[0193] In another experiment, PBMCs were cultured and differentiated into T cells with OKT3 or exemplary anti-CD3 Fab / anti-tumor scFv BsAbs (CTA03Fab / CTAT03scFv, CTA03Fab / CTAT04scFv, CTA03Fab / CTAT05scFv, CTA03Fab / CTAT07scFv, CTA03Fab / CTAT08scFv, CTA03Fab / CTAT9scFv, CTA03Fab / CTAT10scFv, CTA03Fab / CTAT11scFv, CTA03Fab / CTAT12scFv, and CTA03Fab / CTAT13scFv). All groups were cultured under the same conditions (in an incubator with 5% CO2 and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to determine whether BsAb-armed T cells were successfully generated. Figures 12A and 12B show that the OKT3 antibody led to the differentiation of PBMCs into normal T cells, but not to the production of armed T cells. In contrast, the CTA03Fab / CTAT03scFv, CTA03Fab / CTAT04scFv, CTA03Fab / CTAT05scFv, CTA03Fab / CTAT07scFv, CTA03Fab / CTAT08scFv, CTA03Fab / CTAT9scFv, CTA03Fab / CTAT10scFv, CTA03Fab / CTAT11scFv, CTA03Fab / CTAT12scFv, and CTA03Fab / CTAT13scFv BsAbs all led to the production of armed T cells.

[0194] PBMCs were then cultured and differentiated into T cells with OKT3 or various anti-CD3 scFv / anti-tumor Fab BsAbs (CTA01scFv / CTAT03Fab, CTA02scFv / CTAT03Fab, CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab). All groups were cultured under the same conditions (in an incubator with 5% CO2 and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to determine whether BsAb-armed T cells were successfully generated. Figures 12C and 12D show that the conventional OKT3 method differentiated PBMCs into only normal T cells, but not armed T cells. However, the CTA01scFv / CTAT03Fab, CTA02scFv / CTAT03Fab, CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab BsAbs all successfully generated armed T cells.

[0195] Example 6: In vitro toxicity of BsAb-armed T cells against tumor cells CD19 of T cells armed with anti-CD3 / anti-CD19 BsAb + Cytotoxic activity against B-cell lymphoma (Raji) was investigated in this example. Exemplary anti-CD3 / anti-CD19 BsAbs disclosed herein include CTA01Fab / CTAT02scFv, CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, and CTA05Fab / CTAT02scFv. T cells cultured with OKT3 antibody were analyzed using an in vitro cytotoxicity assay. CD19 + No significant cytotoxicity was observed in T cells cultured with OKT3 against B-cell lymphoma (Raji). Differently, T cells cultured with CTA01Fab / CTAT02scFv, CTA02Fab / CTAT02scFv, CTA03Fab / CTAT02scFv, or CTA05Fab / CTAT02scFv expressed CD19 +They efficiently killed B-cell lymphoma (Raji). Of the BsAbs tested, CTA03Fab / CTAT02scFv-armed T cells had the best cytotoxic activity. Figure 13A.

[0196] Furthermore, T cells armed with anti-CD3 scFv / anti-EGFR Fab BsAb consisting of five different anti-CD3 scFvs (CTA01 scFv / CTAT03 Fab, CTA02 scFv / CTAT03 Fab, CTA03 scFv / CTAT03 Fab, CTA04 scFv / CTAT03 Fab, and CTA05 scFv / CTAT03 Fab) and T cells cultured with the conventional OKT3 antibody showed EGFR activity. + Colon cancer (HT-29) killing activity was analyzed using an in vitro cytotoxicity assay. Figure 13B shows that T cells incubated with OKT3 express EGFR + Although armed T cells cultured with CTA01scFv / CTAT03Fab, CTA02scFv / CTAT03Fab, CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab did not efficiently kill colon cancer (HT-29) cells, they were able to inhibit EGFR + Figure 13B shows that colon cancer (HT-29) was efficiently killed.

[0197] In a separate experiment, the tumor cell-killing activity of armed T cells generated with various anti-CD3 Fab / anti-tumor scFv BsAbs (including CTA03Fab / CTAT03scFv, CTA03Fab / CTAT04scFv, CTA03Fab / CTAT05scFv, CTA03Fab / CTAT07scFv, CTA03Fab / CTAT08scFv, CTA03Fab / CTAT9scFv, CTA03Fab / CTAT10scFv, CTA03Fab / CTAT11scFv, CTA03Fab / CTAT12scFv, and CTA03Fab / CTAT13scFv) was further analyzed. As shown in Figures 14A and 14B, CTA03Fab / CTAT03scFv-armed T cells inhibited the EGFR +CTA03Fab / CTAT04scFv-armed T cells efficiently killed colon cancer cells HT29 and HCT-116. Figure 14C shows that CTA03Fab / CTAT04scFv-armed T cells efficiently killed HER2 + Figure 14D shows that CTA03Fab / CTAT05scFv-armed T cells efficiently killed breast cancer (MCF7 / HER2). + Figure 14E shows that CTA03Fab / CTAT07scFv-armed T cells efficiently killed EpCAM-mediated prostate cancer (LNCaP). + Furthermore, Figures 14F-14G show that CTA03Fab / CTAT08scFv-armed T cells efficiently killed LNCaP cells. + It shows that mouse fibroblast cells (3T3 / FAP) were killed efficiently.

[0198] In addition, Figure 15A shows that CTA03Fab / CTAT09scFv-armed T cells inhibited PDL1 + Figure 15B shows that CTA03Fab / CTAT10scFv-armed T cells efficiently killed triple-negative breast cancer (MDA-MB-231). + Figure 15C shows that CTA03Fab / CTAT11scFv-armed T cells efficiently killed B-cell lymphoma (Raji). + Figure 15D shows that CTA03Fab / CTAT12scFv armed T cells efficiently killed human acute myeloid leukemia (HL-60) cells. + Figure 15E shows that CTA03Fab / CTAT13scFv-armed T cells efficiently killed human lung cancer (A549). + This shows that breast cancer (MCF7 / HER2) was killed efficiently.

[0199] Example 7: In vivo anti-cancer activity of CTA03Fab / CTAT02scFv-armed T cells The in vivo cancer inhibition of anti-CD3 Fab / anti-CD19 scFv (CTA01 Fab / CTAT02 scFv and CTA03 Fab / CTAT02 scFv) armed T cells was evaluated. CTA01 Fab / CTAT02 scFv armed T cells and CTA03 Fab / CTAT02 scFv armed T cells were intravenously injected into SCID mice bearing B cell lymphoma (Raji). Body weight, survival rate, and incidence of hind limb paralysis were recorded. Figures 16A-16C show that CTA03 Fab / CTAT02 scFv armed T cells had the best therapeutic efficacy for effectively inhibiting cancer.

[0200] Example 8: In vivo antitumor activity of CTA03Fab / CTAT03scFv-armed T cells and CTA03Fab / CTAT04scFv-armed T cells In vivo tumor inhibition of anti-CD3 Fab / anti-EGFR scFv (CTA03Fab / CTAT03scFv) armed T cells and anti-CD3 Fab / anti-HER2 scFv (CTA03Fab / CTAT04scFv) armed T cells was evaluated. CTA03Fab / CTAT03scFv armed T cells and CTA03Fab / CTAT04scFv armed T cells were intravenously injected into patient-derived xenograft (PDX) mouse models with human triple-negative breast cancer. Body weight and tumor size were recorded. Figures 17A-17B show that both CTA03Fab / CTAT03scFv armed T cells and CTA03Fab / CTAT04scFv armed T cells efficiently inhibited tumor growth of human triple-negative breast cancer.

[0201] Example 9: One-step incubation to generate armed NKT cells from PBMCs using BsAb Human peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured and NKT cells (CD8 + CD56 +) All groups were cultured in the same environment (in an incubator with 5% CO2 and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to determine whether BsAb-armed NKT cells were successfully generated. Figures 18A and 18B show that the OKT3 method induced PBMCs to differentiate only into normal NKT cells, but did not induce the formation of armed T cells. Differently, CTA03Fab / CTAT03scFv, CTA03Fab / CTAT04scFv, and CTA03Fab / CTAT05scFv BsAbs all successfully generated armed NKT cells.

[0202] In a similar assay, human peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured and NKT cells (CD8) were stimulated in the presence of OKT antibody or with CTA01scFv / CTAT03Fab, CTA02scFv / CTAT03Fab, CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab BsAbs. + CD56 + ) All groups were cultured in the same environment (in an incubator with 5% CO2 and a stable humidity level at 37°C). After 7 days, all groups were analyzed using flow cytometry to determine whether BsAb-armed NKT cells were successfully generated. Figure 34 shows that the conventional OKT3 method differentiated PBMCs only into normal NKT cells, but not into armed T cells. However, the CTA01scFv / CTAT03Fab, CTA02scFv / CTAT03Fab, CTA03scFv / CTAT03Fab, CTA04scFv / CTAT03Fab, and CTA05scFv / CTAT03Fab BsAbs all successfully generated armed NKT cells.

[0203] Example 10: Construction of CTA03Fab / CTAT02scFv BsAb with point mutations and Its characterization Point mutations were introduced into the CTA03Fab / CTAT02scFv BsAb by genetic engineering, resulting in the BsAbs CTA03-01Fab / CTAT02-01scFv (CTA03Fab(VLG58A) / CTAT02scFv(VLG42A)), CTA03-01Fab / CTAT02-02scFv (CTA03Fab(VLG58A) / CTAT02scFv(VLD41E)), CTA03-02Fab / CTAT02-02scFv (CTA03Fab(VLD57E) / CTAT02scFv(VLD41E)), and CTA03-02Fab / CTAT02-01scFv (CTA03Fab(VLD57E) / CTAT02scFv(VLG42A)). More specifically, point mutations G58A and D57E were introduced into the VL of CTA03, and G42A and D41E were introduced into the VL of CTAT02 (see Table 2 above).

[0204] CD3 + T cells (Jurkat) and CD19 + The binding ability of the BsAbs to B-cell lymphoma (Raji) was analyzed using flow cytometry. Figures 19A-19B show that CTA03-01Fab / CTAT02-01scFv, CTA03-01Fab / CTAT02-02scFv, CTA03-02Fab / CTAT02-02scFv, and CTA03-02Fab / CTAT02-01scFv BsAbs all bind to CD3 + T cells (Jurkat) and CD19 + It has been shown that the BsAb has the ability to target B-cell lymphoma (Raji). Additionally, the binding of the BsAb to the target cells is dose-dependent.

[0205] CD19 expression by armed T cells generated with point mutant BsAbs (CTA03-01Fab / CTAT02-01scFv, CTA03-01Fab / CTAT02-02scFv, CTA03-02Fab / CTAT02-02scFv, and CTA03-02Fab / CTAT02-01scFv) +The killing activity against B cell lymphoma was compared with that of the original CTA03Fab / CTAT02scFv BsAb-armed T cells. Figure 19C shows that T cells incubated with OKT3 express CD19 + On the other hand, armed T cells cultured with CTA03-01Fab / CTAT02-01scFv, CTA03-01Fab / CTAT02-02scFv, CTA03-02Fab / CTAT02-02scFv, or CTA03-02Fab / CTAT02-01scFv showed no cytotoxicity against B-cell lymphoma (Raji). + They efficiently killed B-cell lymphoma (Raji), and all had better cytotoxicity than the parental CTA03Fab / CTAT02scFv BsAb.

[0206] Other embodiments All of the features disclosed herein can be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0207] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to adapt the present invention to various uses and conditions without departing from the spirit and scope of the present invention. Accordingly, other embodiments are also within the scope of the claims.

[0208] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or obtaining the results and / or one or more advantages described herein, and each such variation and / or modification is deemed to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application or applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, the foregoing embodiments are presented by way of example only, and it should be understood that, within the scope of the appended claims and their equivalents, embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0209] All definitions defined and used herein should be understood to control for dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0210] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may include the entire document.

[0211] The open-ended articles "a" and "an" as used in this specification and claims should be understood to mean "at least one," unless expressly indicated to the contrary. The term "and / or," as used in the specification and claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements, whether related or unrelated to those elements specifically identified, may optionally be present other than the elements specifically identified by the "and / or" phrase. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," can refer in one embodiment to A only (optionally including elements other than B); in another embodiment to B only (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements); etc.

[0212] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be construed as inclusive, i.e., at least one of, but including more than one of, a number or list of elements, and optionally including additional, undisclosed items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0213] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within an accepted standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably even up to ±1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within two-fold, of a value. When particular values ​​are described in the present application and claims, unless otherwise specified, the term "about" is implicit and, in this context, means within an acceptable error range for the particular value. In some embodiments, the hinge domain is a hinge domain of a naturally occurring protein.

[0214] As used herein and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, may optionally be present, whether related to those specifically identified elements or not. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B," or equivalently, "at least one of A and / or B") can refer in one embodiment to at least one A (optionally including more than one A) with no B present (and optionally including elements other than B); in another embodiment to at least one B (optionally including more than one B) with no A present (and optionally including elements other than A); in yet another embodiment to at least one A (optionally including more than one A) and at least one B (and optionally including other elements); etc.

[0215] It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

Claims

[Claim 1] A bispecific antibody, (a) a first antigen-binding fragment that binds to human CD3, wherein the first antigen-binding fragment comprises a first heavy chain variable region (V Ｈ a first heavy chain comprising a first light chain variable region (V Ｌ ), wherein the first V Ｈ contains the same heavy chain complementarity determining regions (CDRs) or no more than five amino acid mutations compared to the first reference antibody, and said first V Ｌ a first antigen-binding fragment comprising the same light chain CDRs or no more than five amino acid mutations compared to the reference antibody, wherein the first reference antibody is CTA.02, CTA.03, CTA.04, or CTA.05; (b) a second antigen-binding fragment that binds to a tumor-associated antigen (TAA), wherein the second antigen-binding fragment comprises a second heavy chain variable region (V Ｈ a second heavy chain comprising a second light chain variable region (V Ｌ and a second antigen-binding fragment comprising a second light chain comprising:

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