WO2025209593A1 - Activateur de lymphocytes t à anticorps multi-spécifiques - Google Patents

Activateur de lymphocytes t à anticorps multi-spécifiques

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Publication number
WO2025209593A1
WO2025209593A1 PCT/CN2025/087399 CN2025087399W WO2025209593A1 WO 2025209593 A1 WO2025209593 A1 WO 2025209593A1 CN 2025087399 W CN2025087399 W CN 2025087399W WO 2025209593 A1 WO2025209593 A1 WO 2025209593A1
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WIPO (PCT)
Prior art keywords
antibody
antigen
trop2
cancer
binding fragment
Prior art date
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PCT/CN2025/087399
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English (en)
Chinese (zh)
Inventor
陈川
朱祯平
彭健
苏晨鹏
田亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helixon Biotechnology Suzhou Co Ltd
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Helixon Biotechnology Suzhou Co Ltd
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Publication of WO2025209593A1 publication Critical patent/WO2025209593A1/fr
Pending legal-status Critical Current
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present disclosure generally relates to multispecific T cell engagers. Specifically, the present disclosure relates to multispecific antibodies and antigen-binding fragments directed against CD3 and/or CD28.
  • Multispecific antibodies are engineered antibodies that can simultaneously bind to different epitopes of one or more antigens.
  • multispecific antibodies that target T cell surface antigens (such as CD3, CD28) and another target antigen (such as a tumor antigen) are used as T cell engagers to recruit and activate T cells in a specific area.
  • CD3 is a conserved component of the T cell receptor (TCR) complex that has signal transduction capabilities.
  • TCR T cell receptor
  • pMHC major histocompatibility complex
  • pMHC major histocompatibility complex
  • CD3-binding antibody By binding a CD3-binding antibody to the CD3 complex, it is possible to bypass the restrictions of the pMHC and stimulate immunity (for example, activating the proliferation of cytotoxic lymphocytes (CTLs)).
  • CTLs cytotoxic lymphocytes
  • T cell activation and proliferation can also be enhanced by specific binding to co-stimulatory receptors (such as CD28 or 4-1BB).
  • co-stimulatory receptors such as CD28 or 4-1BB
  • Multispecific antibodies containing T cell co-stimulatory receptor targeting domains can regulate and redirect immune activation, such as multispecific antibodies that connect CD3 and co-stimulatory receptors (such as CD28 or 4-1BB), or multispecific antibodies that simultaneously target co-stimulatory receptors (such as CD28 or 4-1BB) and TAAs.
  • Tumor antigens include tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAAs are present in both cancer cells and normal somatic cells, but are overexpressed on the surface of cancer cells. Therefore, appropriate targets can be selected based on the type of cancer, using multispecific antibodies that simultaneously target cancer antigens and T cells to treat cancer.
  • TAAs tumor-associated antigens
  • TSAs tumor-specific antigens
  • CD3 antibodies may mediate overactivation of T cells, leading to adverse reactions such as cytokine storms. Furthermore, expressing multispecific CD3 antibodies at high levels often presents a technical challenge. Therefore, there is a need to establish a multispecific antibody T cell engager platform to develop more effective multispecific antibodies with enhanced therapeutic efficacy and expression levels.
  • an antibody means one antibody or more than one antibody.
  • the present disclosure provides a multispecific binding antibody or antigen-binding fragment thereof, an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof, a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and uses thereof.
  • CD3-VL and CD3-VH wherein the CD3-VL comprises SEQ ID NO: 133 and the CD3-VH comprises SEQ ID NO: 134;
  • X H1 is Q
  • X H2 is T
  • X H3 is N
  • X H4 is G
  • X H5 is G
  • X H6 is G
  • X H7 is N
  • X H8 is V
  • X H9 is G
  • X H10 is D
  • X H11 is S
  • X H13 is W.
  • the CD3 binding domain is a fragment antigen binding domain (Fab).
  • the TROP2 binding domain comprises TROP2-LCDR1, TROP2-LCDR2, TROP2-LCDR3, TROP2-HCDR1, TROP2-HCDR2 and TROP2-HCDR3, wherein the TROP2-LCDR1 comprises SEQ ID NO: 217, the TROP2-LCDR2 comprises SEQ ID NO: 218, the TROP2-LCDR3 comprises SEQ ID NO: 219 or 221, the TROP2-HCDR1 comprises SEQ ID NO: 214, the TROP2-HCDR2 comprises SEQ ID NO: 215 and the TROP2-HCDR3 comprises SEQ ID NO: 216 or 220.
  • the TROP2 binding domain comprises a combination of 6 CDRs as shown in Table 13.
  • the VL and VH of the TROP2 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 200 and 201, or SEQ ID NO: 204 and 205.
  • the VL and VH of the TROP2 binding domain respectively comprise the following sequence pairs: SEQ ID NO: 204 and 205.
  • the TROP2 binding domain is in Fab or IgG form.
  • the TROP2 binding domain comprises the sequence shown in Table 11.
  • the antibody or antigen-binding fragment thereof has the structure shown in FIG11 .
  • the antibody or antigen-binding fragment thereof comprises three polypeptides:
  • the antibody or antigen-binding fragment thereof comprises the sequence shown in Table 15.
  • a third polypeptide comprising (a) a VH of a TROP2 binding domain, (b) a heavy chain constant region, (c) the CD3 binding domain, and (d) a heavy chain constant region, wherein the CD3 binding domain is a scFv;
  • the antibody or antigen-binding fragment thereof comprises the sequence shown in Table 15.
  • the present disclosure provides an antibody or antigen-binding fragment thereof comprising:
  • CD28-VL and CD28-VH wherein the CD28-VL comprises the light chain variable region sequence shown in Table 7-8, and the CD28-VH comprises the heavy chain variable region sequence shown in Table 7-8.
  • the present disclosure provides a TROP2 antibody or an antigen-binding fragment thereof, wherein the TROP2 antibody comprises:
  • the present disclosure provides a TROP2 antibody or an antigen-binding fragment thereof, wherein the TROP2 antibody comprises the paired VH and VL shown in Table 11.
  • the antibody or antigen-binding fragment thereof is linked to one or more conjugate moieties.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.
  • the present disclosure provides an isolated polynucleotide encoding the aforementioned antibody or antigen-binding fragment thereof.
  • the present disclosure provides a method for producing an antibody or an antigen-binding fragment thereof, comprising culturing the aforementioned host cell under conditions where the antibody or antigen-binding fragment thereof is expressed, and recovering the antibody or antigen-binding fragment thereof.
  • the present disclosure provides a multispecific antibody or antigen-binding fragment thereof, comprising:
  • a CD3 binding domain comprising:
  • the CD3 binding domain comprises a combination of 6 CDRs as shown in Table 6.
  • the CD28 binding domain comprises a combination of the six CDR sequences shown in Table 10.
  • the target antigen is a cancer-associated antigen.
  • the antibody or antigen-binding fragment thereof comprises a TROP2 binding domain, wherein the TROP2 binding domain comprises:
  • TROP2-VL and TROP2-VH wherein the TROP2-VL comprises the light chain variable region sequence shown in Table 11, and the TROP2-VH comprises the heavy chain variable region sequence shown in Table 11.
  • the CD3 binding domain, CD28 binding domain, or target antigen binding domain is a fragment antigen binding domain (Fab).
  • the CD3 binding domain, CD28 binding domain, or target antigen binding domain is a variable region domain (Fv).
  • the CD3 binding domain, CD3 binding domain, or target antigen binding domain is a single chain variable domain (scFv).
  • the aforementioned antibody or antigen-binding fragment thereof has the structure shown in FIG11 .
  • the present disclosure provides a method for treating or ameliorating a disease that benefits from T lymphocyte killing and clearance or a disease associated with tumor-associated antigens in a subject, comprising administering to the subject a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment thereof, or the aforementioned pharmaceutical composition.
  • the disease is cancer or a disease of the immune system.
  • the cancer is selected from adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, neural or neuroendocrine tumors, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioblastoma multiforme, metastatic castration-resistant pulmonary neuroendocrine tumor, neuroblastoma, metastatic carcinoma, diffuse intrinsic pontine glioma, peritoneal cancer, central nervous
  • the immune system disease is selected from Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, reactive arthritis, undifferentiated spondyloarthropathy, juvenile spondyloarthropathy, Behcet's disease, enthesitis, ulcerative colitis, Crohn's disease, irritable bowel syndrome, inflammatory bowel disease, fibromyalgia, chronic fatigue syndrome, pain conditions associated with systemic inflammatory diseases, systemic lupus erythematosus, Sjogren's syndrome, rheumatoid arthritis, juvenile rheumatoid arthritis, juvenile-onset diabetes mellitus (also known as type 1 diabetes), Wegener's granulomatosis, polymyositis, dermatomyositis, inclusion body myositis, multiple endocrine failure, Schmidt's syndrome.
  • Guillain-Barre syndrome chronic
  • autoimmune uveitis Addison's disease, Grave's disease, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus hepatitis, atherosclerosis, multiple sclerosis, amyotrophic lateral sclerosis, hypoparathyroidism, Dressler's syndrome, myasthenia gravis, Eaton-Lambert syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia, scleroderma, progressive systemic sclerosis, CREST syndrome (calcification, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), adult-onset diabetes mellitus (also known as type 2 diabetes), mixed connective tissue disease tissue disease, poly(
  • the additional therapeutic agent directly acts on CD3, CD28, tumor-associated antigens or variants thereof, such as a monospecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a bispecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a multispecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a fusion protein targeting CD3, CD28, tumor-associated antigens or variants thereof, an ADC targeting CD3, CD28, tumor-associated antigens or variants thereof, or a cytokine targeting CD3, CD28, tumor-associated antigens or variants thereof.
  • a monospecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof such as a monospecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a bispecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a multispecific antibody targeting CD3, CD28, tumor-associated antigens or variants thereof, a fusion protein targeting
  • the one or more additional therapeutic agents are administered concurrently or sequentially with the antibody or antigen-binding fragment thereof.
  • FIG1 shows a schematic diagram of the structure of an antibody-antigen complex.
  • FIG2A shows the results of light chain mutation energy analysis of antibody CDR regions (Chothia encoding).
  • FIG2B shows the results of heavy chain mutation energy analysis of antibody CDR regions (Chothia encoding).
  • FIG9 shows the results of determining the kinetic characteristic parameters of the binding of anti-CD3 specific antibodies to CD3ed-HIS.
  • FIG17A to FIG17C show the detection results of kinetic characteristic parameters of the binding of double antibodies and triple antibodies to TROP2, CD28-HIS, and CD3ed-HIS.
  • FIG19 shows the results of ELISA assays of the affinity of anti-TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies for TROP2.
  • FIG20 shows the results of ELISA assays of the affinity of anti-TROP2 ⁇ CD3 dual antibodies and TROP2 ⁇ CD3 ⁇ CD28 triple antibodies to CD28-HIS.
  • FIG23 shows the results of FACS measurement of the binding affinities of anti-TROP2 ⁇ CD3 dual antibodies and TROP2 ⁇ CD3 ⁇ CD28 triple antibodies to 293-TCR target cells.
  • Figures 42 to 51 show the SDS-PAGE test results of the stability of TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody after repeated freeze-thaw cycles, wherein Figures 42 to 43 represent TPt0019, Figures 44 to 45 represent TPt0025, Figures 46 to 47 represent TPt0042, Figures 48 to 49 represent TPt0043, and Figures 50 to 51 represent TPb0059.
  • Figures 52-61 show the results of TDCC killing activity assays of TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies against 293-huTROP2 ( Figures 52A and 52B), BxPC3 ( Figures 53A and 53B), MDA-MB-468 ( Figures 54A to 54E), NCI-N87 ( Figures 55A and 55B), MDA-MB-231 ( Figure 56), DLD-1 ( Figures 57A to 57D), COLO205 ( Figures 58A and 58B), SW403 ( Figure 59), T84 ( Figure 60) and HEK293 ( Figure 61) tumor cells.
  • Figures 69-73 show the results of measuring the release levels of cytokines IL-2, IFN-r, IL6 and TNFa by anti-TROP2 ⁇ CD3 dual antibody and TROP2 ⁇ CD3 ⁇ CD28 triple antibody in the TDCC killing effect of 293-huTROP2 ( Figures 69A and 69B), BxPC3 ( Figure 70), MDA-MB-468 ( Figures 71A to 71E), DLD-1 ( Figure 72), and HEK293 ( Figure 73).
  • Figures 74 to 85 show the results of the killing effect test of TPt0042 and TPb0043 on TROP2 high-expressing, medium-expressing and low-expressing cells in the TDCC experiment.
  • Figures 86 and 87 show the results of the TDCC killing activity assay of TROP2 ⁇ CD3 ⁇ CD28 triple antibody weakened by CD28 antibody against BxPC3 and HEK293 cells.
  • Figures 89 and 90 show the results of TDCC killing activity assays against BxPC3 and SW403 tumor cells using TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 triple antibody attenuated by CD28 and TROP2 antibodies.
  • Figures 91-93 show the results of TDCC killing activity assays of TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 triple antibodies attenuated by CD28 and TROP2 antibodies against BxPC3 ( Figure 91), SW403 ( Figure 92) and Colo205 ( Figure 93) tumor cells at 24h, 48h and 72h.
  • Figures 94-96 show the results of measuring the release levels of cytokines IFNr, TNFa, IL-2 and IL10 during the TDCC killing effect of TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 triple antibody weakened by CD28 and TROP2 antibodies on BxPC3, SW403 and Colo205 tumor cells at 24h, 48h and 72h.
  • Figures 97 and 98 show the anti-tumor efficacy test results of the anti-TROP2 ⁇ CD3 dual antibody in the BxPC3 mouse transplanted tumor model.
  • Figures 101 and 102 show the anti-tumor efficacy test results of the anti-TROP2 ⁇ CD3 dual antibody in the MDA-MB-231 mouse transplanted tumor model.
  • Figures 103A and 103B show the anti-tumor efficacy test results of the anti-TROP2 ⁇ CD3 dual antibody in the NCI-H292 mouse transplanted tumor model.
  • Figures 107A and 107B show the anti-tumor efficacy test results of anti-TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 triple antibody with weakened CD28 and TROP2 antibodies in the BxPC3 mouse transplanted tumor model.
  • antibody includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, single domain antibody, multispecific antibody, or bispecific antibody that binds to a specific antigen.
  • a natural, intact IgG antibody comprises two heavy (H) chains and two light (L) chains.
  • Mammalian heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , each consisting of a variable region ( VH ) and a first constant region, a second constant region, and a third constant region ( CH1 , CH2 , CH3 , respectively); mammalian light chains are classified as ⁇ or ⁇ , each consisting of a variable region ( VL ) and a constant region.
  • Antibodies are "Y"-shaped, with the stem of the Y consisting of the second constant region and the third constant region of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises the variable region and the first constant region of a single heavy chain, bound to the variable region and constant region of a single light chain.
  • variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable region of each chain typically contains three highly variable loops, termed complementarity determining regions (CDRs) (the light chain CDRs include LCDR1, LCDR2, LCDR3 and the heavy chain CDRs include HCDR1, HCDR2, HCDR3).
  • CDRs complementarity determining regions
  • the CDR boundaries of the antibodies and antigen binding domains disclosed herein can be defined or identified by Kabat, IMGT, AbM, Chothia, or Al-Lazikani conventions (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J. Mol.
  • the constant regions of the heavy and light chains do not participate in antigen binding, but exhibit various effector functions.
  • Antibodies are divided into multiple classes based on the amino acid sequence of their heavy chain constant regions.
  • the five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ heavy chains, respectively.
  • major antibody classes are divided into subclasses, such as IgG1 ( ⁇ 1 heavy chain), IgG2 ( ⁇ 2 heavy chain), IgG3 ( ⁇ 3 heavy chain), IgG4 ( ⁇ 4 heavy chain), IgA1 ( ⁇ 1 heavy chain), or IgA2 ( ⁇ 2 heavy chain).
  • antibody may also encompass single-domain antibodies, such as heavy-chain antibodies.
  • Heavy-chain antibodies or “HCAbs” refer to antibodies that contain two VH domains but no light chains (Riechmann L. and Muyldermans S., J Immunol Methods Dec 10;231(1-2):25-38 (1999); Muyldermans S., J Biotechnol. Jun;74(4):277-302 (2001); WO94/04678; WO94/25591; U.S. Patent No. 6,005,079).
  • Heavy-chain antibodies are originally derived from the Camelidae family (camels, dromedaries, and llamas).
  • variable domain of a heavy-chain antibody represents the smallest known antigen-binding unit produced by the adaptive immune response (Koch-Nolte F. et al. FASEB J. Nov;21(13):3490-8. Epub 2007 Jun 15 (2007)).
  • target antigen binding domain refers to an antigen binding domain that targets a target antigen.
  • the target antigen binding domain of the antibody or antigen binding fragment thereof provided herein may be a tumor antigen binding domain.
  • the target antigen includes a tumor surface antigen.
  • tumor surface antigen refers to an antigen that is primarily presented by tumor cells to distinguish it from non-malignant tissue, and is preferably located on the cell membrane of a tumor cell.
  • Tumor surface antigens can be in various forms, such as polypeptides (specifically glycosylated proteins) or polypeptides, glycosylation patterns, glycolipids (e.g., gangliosides, such as GM2), or even changes in the composition of lipids of the cell membrane, which may be characteristics of cancer cells.
  • Tumor surface antigens can be antigens that specifically express on cancer cells that trigger an immune response; and/or bind to T cell receptors (e.g., when presented by MHC molecules) or bind to antibodies.
  • tumor surface antigens trigger a humoral response (e.g., comprising the production of antigen-specific antibodies).
  • tumor surface antigens trigger a cellular response (e.g., involving T cells whose receptors specifically interact with tumor surface antigens).
  • the tumor surface antigen binds to the antibody and may or may not induce a specific physiological response in the organism.
  • multispecific antibody refers to an antibody or antigen-binding fragment thereof that comprises at least two different antigen-binding domains.
  • the at least two different antigen-binding domains target different antigens, or different epitopes of the same antigen.
  • a multispecific antibody can be configured as needed. In certain embodiments, the configuration of a multispecific antibody is shown in Figure 11.
  • antigen binding fragment refers to an antibody fragment formed by a part of an antibody including one or more CDRs or any other antibody fragment that binds to an antigen but does not include a complete native antibody structure.
  • antigen binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (divalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and divalent domain antibodies.
  • an antigen binding fragment can bind to the same antigen as the antigen bound by the parent antibody.
  • an antigen binding fragment can include one or more CDRs from a specific human antibody that is transplanted to the framework region from one or more different human antibodies. Further and detailed forms of antigen-binding fragments are described in Spiess et al., 2015 (supra) and Brinkman et al., Monoclonal Antibodies (mAbs), 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.
  • antigen binding domain refers to an antibody fragment formed by a portion of an antibody including one or more CDRs or any other antibody fragment that binds to an antigen but does not include a complete native antibody structure.
  • antigen binding fragments include, but are not limited to, bifunctional antibodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide-stabilized bifunctional antibodies (ds bifunctional antibodies), single-chain antibody molecules (scFv), scFv dimers (divalent bifunctional antibodies), bispecific antibodies, multispecific antibodies, camelized single domain antibodies, nanobodies, domain antibodies, and divalent domain antibodies.
  • an antigen binding domain may include one or more CDRs from a specific human antibody that is transplanted to a framework region from one or more different human antibodies. Further and detailed forms of antigen binding domains are described in Spiess et al., 2015 (supra) and Brinkman et al., Monoclonal Antibodies (mAbs), 9(2), pp. 182-212 (2017), which are incorporated herein by reference in their entirety.
  • the term "antigen” refers to a compound, composition, peptide, polypeptide, protein, or substance that can stimulate the production of antibodies or immune cells (e.g., T cells or myeloid cells) in a cell culture or animal, including compositions that are added to a cell culture (e.g., a hybridoma), injected or absorbed into an animal, or expressed on the surface of a cell (e.g., a composition comprising a cancer-specific protein).
  • Antigens react with products of specific humoral or cellular immunity (e.g., antibodies).
  • Fab with respect to antibodies refers to that portion of an antibody consisting of a single light chain (both variable and constant regions) bound to the variable and first constant regions of a single heavy chain by disulfide bonds.
  • F(ab) 2 refers to a dimer of Fab.
  • Fab refers to the Fab fragment including a portion of the hinge region.
  • F(ab') 2 refers to a dimer of Fab'.
  • fragment difficult in the context of antibodies refers to the amino-terminal half of a heavy chain fragment that can combine with a light chain to form a Fab.
  • an Fd fragment can consist of the VH and CH1 domains.
  • Fv with respect to antibodies refers to the smallest fragment of an antibody that carries a complete antigen-binding site.
  • An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.
  • Many Fv designs have been proposed, including dsFv, in which the association between the two domains is enhanced by an introduced disulfide bond, and scFv, which can be formed by joining the two domains together as a single polypeptide using a peptide linker.
  • Fv constructs containing the variable domains of an immunoglobulin heavy or light chain associated with the variable and constant domains of the corresponding immunoglobulin heavy or light chain have also been produced.
  • Fvs have also been multimerized to form bi- and tri-functional antibodies (Maynard et al., Annu Rev Biomed Eng 2 339-376 (2000)).
  • a “single-chain Fv antibody” or “scFv” refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region, which are linked to each other directly or through a peptide linker sequence (Huston JS et al., Proc. Natl. Acad. Sci. USA, 85:5879 (1988)). ScFv can also be used as a building block for developing multimeric structures (dimer: "diabody”; trimer: “tribody”; tetramer: “tetrabody”).
  • Diabodies or “dAbs” include small antibody fragments with two antigen-binding sites, wherein the fragments include a VH domain connected to a VL domain in the same polypeptide chain ( VH - VL or VL- VH ) (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA Jul 15;90(14):6444-8 (1993); EP404097; WO93 / 11161).
  • the antigen-binding sites can target the same or different antigens (or epitopes).
  • a "bispecific dsdiabody” is a bifunctional antibody that targets two different antigens (or epitopes).
  • dsFv refers to a disulfide-stabilized Fv fragment in which the variable region of a single light chain is connected to the variable region of a single heavy chain by a disulfide bond.
  • “(dsFv) 2 " or “(dsFv-dsFv')” comprises three peptide chains: two VH portions are connected by a peptide linker (e.g., a long flexible linker), and are each bound to two VL portions by a disulfide bridge.
  • dsFv-dsFv' has bispecificity, wherein each pair of heavy and light chains paired by disulfide bonds has a different antigenic specificity.
  • the term “valence” refers to the presence of a specified number of antigen binding sites in a given molecule.
  • the term “monovalent” refers to an antibody or antigen binding fragment having only one single antigen binding site; and the term “multivalent” refers to an antibody or antigen binding fragment having multiple (i.e., more than one) antigen binding sites.
  • the terms “bivalent,” “tetravalent,” and “hexavalent” refer to the presence of two binding sites, four binding sites, and six binding sites in an antigen binding molecule, respectively.
  • the antibody or its antigen binding fragment is bivalent.
  • Domain antibodies or “single domain antibodies” or “sdAbs” refer to antibody fragments that contain only the variable region of a heavy chain or a variable region of a light chain.
  • two or more VH domains are covalently joined by a peptide linker to create a bivalent or multivalent domain antibody.
  • the two VH domains of a bivalent domain antibody can target the same or different antigens.
  • Fc with respect to an antibody refers to the portion of the antibody consisting of the second and third constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain via disulfide bonds.
  • the Fc portion of an antibody is responsible for various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and phagocytosis.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • phagocytosis phagocytosis
  • chimeric means an antibody or antigen-binding domain having a portion of a heavy chain and/or light chain derived from a species and the remainder of the heavy chain and/or light chain derived from another different species.
  • a chimeric antibody may include a constant region derived from people and a variable region derived from a non-human animal (e.g., derived from a mouse).
  • a chimeric antibody may include a FR region derived from people and a CDR region derived from a non-human animal (e.g., derived from a mouse).
  • the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.
  • humanized means an antibody or antigen-binding domain that includes CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable).
  • operably linked refers to the juxtaposition of two or more biological sequences of interest, with or without a spacer, linker, or intervening sequence, in a manner that places the biological sequences of interest in a relationship that allows them to function in their intended manner.
  • the term means that the polypeptide sequences are linked in a manner that allows the linked product to have the intended biological function.
  • an antibody variable region can be operably linked to a constant region to provide a stable product with antigen binding activity.
  • an antigen binding domain can be operably linked to another antigen binding domain with an intervening sequence therebetween, and such intervening sequence can be a spacer or can include a much longer sequence, such as the constant region of an antibody.
  • the term can also be applied to polynucleotides.
  • a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it means that the polynucleotide sequences are linked in a manner that allows for regulated expression of the polypeptide from the polynucleotide.
  • fusion when applied to amino acid sequences (e.g., peptides, polypeptides, or proteins) refers to the combination of two or more amino acid sequences into a single amino acid sequence, for example, by chemical bonding or recombinant means.
  • a fused amino acid sequence can be produced by genetic recombination of two encoding polynucleotide sequences and can be expressed by introducing a construct containing the recombinant polynucleotide into a host cell.
  • CD3 refers to cluster of differentiation 3, a protein complex and T cell co-receptor involved in activating cytotoxic T cells and T helper cells.
  • the CD3 complex comprises a CD3 ⁇ chain, a CD3 ⁇ chain, two CD3 ⁇ chains, and a CD3- ⁇ (zeta) chain.
  • Human, mouse, and cynomolgus monkey CD3 amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot.
  • the term CD3 includes full-length wild-type CD3 and proteins thereof containing mutations (e.g., point mutations), fragments, insertions, deletions, and splice variants.
  • the human CD3 protein comprises the amino acid sequence shown in Table 1.
  • CD28 refers to cluster of differentiation 28, which is expressed on T cells and is used to provide co-stimulatory signals in the T cell activation and survival pathway. Through the joint action of CD28 and the T cell receptor, T cells can be stimulated and effectively activated, thereby producing a large amount of cytokines (such as IL-6).
  • cytokines such as IL-6
  • the amino acid and nucleic acid sequences of CD28 of humans, mice and crab-eating monkeys can be found in public databases such as GenBank, UniProt and Swiss-Prot.
  • the term CD28 includes full-length wild-type CD28 and proteins thereof containing mutations (e.g., point mutations), fragments, insertions, deletions and splice variants.
  • the human CD28 protein comprises the amino acid sequence shown in Table 1.
  • TROP2 also known as “TACSTD2” or “EGP-1” refers to tumor-associated calcium signal sensor 2 or epidermal glycoprotein-1.
  • TROP2 is associated with the occurrence and progression of cancer. It can interact with key molecular signaling pathways and play a role in tumor progression. TROP2 has been found to be abnormally overexpressed in some solid cancers, such as colorectal cancer, renal cancer, lung cancer and breast cancer. The amino acid and nucleic acid sequences of TROP2 in humans, mice and crab-eating macaques can be found in public databases such as GenBank, UniProt and Swiss-Prot.
  • TROP2 includes full-length wild-type TROP2 and proteins thereof containing mutations (e.g., point mutations), fragments, insertions, deletions and splice variants.
  • the human TROP2 protein comprises the amino acid sequence shown in Table 1.
  • GUI2C refers to guanylyl cyclase C, which is a type I transmembrane protein expressed by intestinal epithelial cells from the duodenum to the rectum. Importantly, the expression of GUCY2C remains unchanged at all stages of tumor transformation, from precancerous polyps to distal colorectal cancer metastasis. Many physiological processes, including intestinal cell proliferation, differentiation and metabolism, are regulated by GUCY2C signals, so it is a potential ideal target antigen for colorectal cancer immunotherapy.
  • the amino acid and nucleic acid sequences of GUCY2C in humans, mice and crab-eating macaques can be found in public databases such as GenBank, UniProt and Swiss-Prot.
  • GUCY2C includes full-length wild-type GUCY2C and proteins thereof comprising mutations (e.g., point mutations), fragments, insertions, deletions and splice variants.
  • the human GUCY2C protein comprises the amino acid sequence shown in Table 1.
  • the term “specific binding” or “specifically binds” refers to a non-random binding reaction between two molecules, such as an antibody or its antigen-binding domain and an antigen.
  • the antibody molecules or antigen-binding domains provided herein specifically bind to human CD3, human CD28 and/or tumor-associated antigens with a binding affinity ( KD ) of ⁇ 10-6 M (e.g., ⁇ 5 ⁇ 10-7 M, ⁇ 2 ⁇ 10-7 M, ⁇ 10-7 M, ⁇ 5 ⁇ 10-8 M, ⁇ 2 ⁇ 10-8 M, ⁇ 10-8 M, ⁇ 5 ⁇ 10-9 M, ⁇ 4 ⁇ 10-9 M).
  • KD refers to the ratio of the dissociation rate to the association rate ( koff / kon ), which can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance, microthermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). In certain embodiments, KD values can be suitably determined using flow cytometry.
  • epitope refers to a specific group of atoms or amino acids on the antigen to which an antibody is bound.
  • An epitope can be formed by continuous amino acids (also referred to as linear or sequential epitopes) or by non-continuous amino acids juxtaposed by the tertiary folding of a protein (also referred to as configurational or conformational epitopes).
  • Epitopes formed by continuous amino acids are typically arranged linearly along the primary amino acid residues on a protein, and small segments of continuous amino acids can be digested from antigen binding to major histocompatibility complex (MHC) molecules or retained when exposed to denaturing solvents, while epitopes formed by tertiary folding are typically lost when treated with denaturing solvents.
  • MHC major histocompatibility complex
  • an epitope typically includes at least 3 and more commonly at least 5, about 7, or about 8-10 amino acids. If two antibodies exhibit competitive binding for an antigen, they can bind to the same or closely related epitopes within the antigen.
  • an antibody or antigen-binding domain blocks at least 85% or at least 90% or at least 95% binding of a reference antibody to an antigen
  • the antibody or antigen-binding domain can be considered to bind to the same/closely related epitope as the reference antibody.
  • amino acid refers to an organic compound containing amino ( -NH2 ) and carboxyl (-COOH) functional groups and side chains unique to each amino acid.
  • Amino acid names are also represented in this disclosure by standard single-letter or three-letter codes, which are summarized below.
  • Constant substitutions with respect to amino acid sequences refer to substitutions of amino acid residues with different side chains having similar physicochemical properties. For example, conservative substitutions can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between residues with acidic side chains (e.g., Asp, Glu), between amino acids with basic side chains (e.g., His, Lys, and Arg), or between residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.
  • conservative substitutions generally do not cause significant changes in the conformational structure of the protein, and therefore the biological activity of the protein can be retained.
  • the term "subject” or “individual” or “animal” or “patient” refers to a human or non-human animal, including a mammal or primate, for whom diagnosis, prognosis, alleviation, prevention and/or treatment of a disease or condition is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, pigs, cattle, bears, and the like.
  • vector refers to a polynucleotide encoding a protein that can be operably inserted therein to cause the expression of the protein.
  • a vector can be used for transforming, transducing, or transfecting a host cell so that the genetic elements it carries are expressed in the host cell.
  • vectors include plasmids, phagemids, cosmids, and artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC), etc.), bacteriophages (such as lambda phage or M13 phage, etc.), and animal viruses.
  • the classification of animal viruses used as vectors includes retroviruses (including slow viruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).
  • Vectors can contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, vectors can contain an origin of replication. Vectors can also include materials that assist in entering cells, including but not limited to viral particles, liposomes, or protein coatings.
  • the vector can be an expression vector or a cloning vector.
  • host cell refers to a cell into which an exogenous polynucleotide and/or vector has been introduced.
  • cancer interchangeably with “tumor” refers to any medical condition characterized by malignant cell growth or neoplasm, abnormal proliferation, infiltration or metastasis, and includes solid tumors and non-solid cancers (malignant blood tumors) such as leukemia.
  • solid tumor refers to a solid mass of neoplastic and/or malignant cells.
  • a cancer or tumor includes hematological malignancies, oral cancer (e.g., lip cancer, tongue cancer or pharyngeal cancer), digestive organ cancer (e.g., esophageal cancer, gastric cancer, small intestine cancer, colon cancer, large intestine cancer or rectal cancer), peritoneal cancer, liver cancer and bile duct cancer, pancreatic cancer, respiratory system cancer such as laryngeal cancer or lung cancer (small cell and non-small cell), bone cancer, connective tissue cancer, skin cancer (e.g., melanoma), breast cancer, reproductive organ cancer (fallopian tube cancer, uterine cancer, cervical cancer, testicular cancer, ovarian cancer or prostate cancer), urinary tract cancer (e.g., bladder cancer or kidney cancer), brain cancer and endocrine gland cancer such as thyroid cancer.
  • oral cancer e.g., lip cancer, tongue cancer or pharyngeal cancer
  • digestive organ cancer e.g., esophageal cancer, gastric cancer, small
  • the cancer is selected from ovarian cancer, breast cancer, head and neck cancer, kidney cancer, bladder cancer, hepatocellular carcinoma and colorectal cancer. In certain embodiments, the cancer is selected from lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma and B cell lymphoma.
  • pharmaceutically acceptable means that the specified carrier, vehicle, diluent, excipient and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
  • the present disclosure provides multispecific antibodies or antigen-binding fragments thereof that bind to T cell surface antigens (e.g., CD3, CD28) and another target antigen (e.g., tumor antigen), which can serve as T cell engagers in immunotherapy to recruit and activate T cells in designated areas.
  • T cell surface antigens e.g., CD3, CD28
  • tumor antigen e.g., tumor antigen
  • the multispecific antibodies or antigen-binding fragments thereof provided herein comprise a CD3 binding domain and a target antigen binding domain. In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein comprise a CD28 binding domain and a target antigen binding domain. In certain embodiments, the multispecific antibodies or antigen-binding fragments thereof provided herein comprise a CD3 binding domain, a CD28 binding domain, and a target antigen binding domain.
  • T cell surface antigens and target antigens involved in the present disclosure are well known in the art.
  • Table 1 provides the sequences of exemplary T cell surface antigens and target antigens.
  • the present invention provides a series of anti-CD3 antibodies engineered based on the tidutamab antibody using an AI structure prediction model and an empirical force field energy function.
  • the variable region amino acid sequences of these anti-CD3 antibodies are shown in Table 2.
  • Table 2 Amino acid sequences of variable regions of energetically advantageous anti-CD3 antibodies based on tidutamab (CDRs classified according to IMGT rules are underlined)
  • anti-CD3 antibodies engineered based on the huSP34 antibody using an AI structure prediction model and empirical force field energy functions.
  • the variable region amino acid sequences of these anti-CD3 antibodies are shown in Table 3.
  • the present disclosure provides a series of optimized CD3 binding domains based on the AI structure prediction model and the empirical force field energy function, and the amino acid sequences of the variable regions thereof are shown in Table 4.
  • the optimized CD3 binding domains provided herein comprise a VL and a VH having the following general formula:
  • the present disclosure provides a series of optimized CD28 antibodies based on the AI structure prediction model and the empirical force field energy function, and the amino acid sequences of their variable regions are shown in Table 7.
  • TROP2 binding domains optimized based on the huE11 antibody using an AI structure prediction model and empirical force field energy functions.
  • the variable region amino acid sequences of these TROP2 binding domains are shown in Table 11.
  • TROP2 ⁇ CD3 bispecific antibodies designed based on huSP34, CD3-002IgG, CD3-002scFv, huE11, and TP-023IgG.
  • TROP2 ⁇ CD3 bispecific antibodies TPb0043 and TPt0042 constructed based on CD3-002 scFv and TP-023 IgG, with bispecific antibody amino acid sequences shown in Table 15 and configurations shown in Figure 11.
  • H44/L100 of the CD3-002 scFv are mutated to Cysteine to form an intramolecular disulfide bond to stabilize the antibody.
  • CD3xCD28 bispecific antibodies or TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies were designed, as shown in Figure 11.
  • the key antibody sequences are shown in Tables 11, 31, and 39.
  • CDRs are responsible for antigen binding, however, it has been found that not all 6 CDRs are essential or unchangeable. In other words, one or more CDRs provided herein for use in a CD3 binding domain can be replaced or changed or modified while still substantially retaining specific binding affinity for CD3.
  • the antibody or its Fab provided herein is humanized.
  • Humanized antigen-binding domains are desirable in terms of their immunogenicity in reducing people.
  • Humanized antigen-binding domains are chimeric in their variable regions because non-human CDR sequences are transplanted to people or substantially people's FR sequences.
  • the humanization of antigen-binding domains can be carried out substantially by replacing the corresponding people's CDR genes in human immunoglobulin genes with non-human (such as mouse) CDR genes (see, for example, Jones et al. (1986), ⁇ Nature > 321:522-525; Riechmann et al. (1988), ⁇ Nature > 332:323-327; Verhoeyen et al. (1988), ⁇ Science > 239:1534-1536).
  • Suitable human heavy chain and light chain variable domains can be selected using methods known in the art to achieve this purpose.
  • "best fit" method can be used, wherein for the database screening of known human variable domain sequences or BLAST non-human (for example, rodent) antibody variable domain sequences, and identify the human sequence closest to the non-human query sequence and use it as the people's support for transplanting non-human CDR sequences (see, for example, Sims et al., (1993) "Journal of Immunology (J.Immunol.)” 151:2296; Chothia et al. (1987) "Journal of Molecular Biology” 196:901).
  • the framework derived from the consensus sequence of all human antibodies can be used for the transplantation of non-human CDR (see, for example, Carter et al. (1992) “Proceedings of the National Academy of Sciences of the United States of America", 89:4285; Presta et al. (1993) “Journal of Immunology", 151:2623).
  • Various techniques can be used to produce such antigen-binding fragments.
  • Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression in host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), and screening from phage display libraries as discussed above (e.g., for ScFv).
  • Other techniques for producing antibody fragments will be apparent to the skilled practitioner.
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.
  • the antimicrobial agent used as a carrier can be added to the pharmaceutical composition in the multidose container, and the antimicrobial agent includes phenol or cresol, mercurials, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride.
  • Suitable excipients can include, for example, water, saline, dextran, glycerol or ethanol.
  • the unit dose parenteral formulation is packaged in an ampoule, a vial, or a syringe with a needle.As is known and practiced in the art, all preparations for parenteral administration should be sterile and pyrogen-free.
  • Reconstitution of the lyophilized powder with water for injection provides a formulation for parenteral administration.
  • sterile and/or pyrogen-free water or other suitable liquid carrier is added to the lyophilized powder. The exact amount depends on the selected therapy to be administered and can be determined empirically.
  • Host cell (containing a vector containing a polynucleotide)
  • the present disclosure provides isolated polynucleotides encoding the antibodies or antigen-binding fragments thereof provided herein.
  • polynucleotide sequences also implicitly encompass conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly specified.
  • degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acids Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • drosophilarum ATCC 36,906), K. thermotolerans, and K. marxianus (ATCC 16,045). arxianus); Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, such as A. nidulans and A. niger.
  • Suitable host cells for expressing the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • a variety of baculovirus strains and variants and corresponding permissive insect host cells have been identified, such as from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori.
  • viruses for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.
  • vertebrate cells are of greatest interest, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure.
  • useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., PNAS 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod.
  • monkey kidney cells (CV1 ATCC CCL 70); African Green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).
  • the host cell is a 293F cell.
  • antibody or its Fab that this paper provides can produce by homologous recombination known in the art.
  • Host cells for producing antibodies or Fabs thereof provided herein can be cultured in a variety of culture media.
  • Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing host cells.
  • any of these culture media can be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM drugs), trace elements (defined as inorganic compounds whose final concentrations are generally in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those skilled in the art.
  • the culture conditions (such as temperature, pH, etc.) are those previously used with the selected host cell for expression and will be apparent to those of ordinary skill in the art.
  • the antibody or antigen-binding fragment thereof can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris of the host cells or lysed fragments can be removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of Escherichia coli.
  • the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes.
  • Cell debris can be removed by centrifugation.
  • the supernatant from such expression systems is typically first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit.
  • Protease inhibitors such as PMSF can be included in any of the preceding steps to inhibit proteolysis, and antibiotics can be included to prevent the growth of adventitious contaminants.
  • Antibodies or antigen-binding fragments thereof produced by the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being a preferred purification technique.
  • the protein A fixed on the solid phase is used for the immunoaffinity purification of antibodies or their antigen-binding fragments.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in antibodies or their antigen-binding fragments.
  • Protein A can be used for purifying antibodies based on people's ⁇ 1, ⁇ 2 or ⁇ 4 heavy chains (Lindmark et al., "Journal of Immunological Methods” 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and people's ⁇ 3 (Guss et al., "Journal of the European Molecular Biology Association (EMBO J.)" 5:1567 1575 (1986)).
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are also available. Compared with the flow rate and processing time that can be achieved with agarose, mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rate and shorter processing time. Where the antibody or antigen-binding fragment thereof comprises a CH3 domain, Bakerbond ABX TM resin (JT Baker, Phillipsburg, NJ) can be used for purification.
  • the mixture comprising the antibody molecule of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 and 4.5, preferably at low salt concentration (e.g., about 0-0.25 M salt).
  • the antibody or antigen-binding fragment thereof or the pharmaceutical composition is administered intravenously, intraarterially, intratumorally, intramuscularly or subcutaneously.
  • the methods provided herein further comprise administering to the subject one or more additional therapeutic agents, wherein the additional therapeutic agent is selected from a chemotherapeutic agent, an anticancer drug, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, a cytokine, an anti-infective agent, or an anti-inflammatory agent.
  • the additional therapeutic agent is selected from a chemotherapeutic agent, an anticancer drug, a radiotherapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, an antiviral agent, an antibiotic, an analgesic agent, an antioxidant, a metal chelator, a cytokine, an anti-infective agent, or an anti-inflammatory agent.
  • the subject has been diagnosed with or is at risk for a disease, disorder, or condition selected from the group consisting of cancer (e.g., solid tumors, hematological malignancies), inflammatory diseases, infectious diseases (e.g., chronic infections), autoimmune diseases (e.g., multiple sclerosis), neurological diseases, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction, and arthritis.
  • cancer e.g., solid tumors, hematological malignancies
  • infectious diseases e.g., chronic infections
  • autoimmune diseases e.g., multiple sclerosis
  • neurological diseases e.g., brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, transplant dysfunction, and arthritis.
  • the subject has been diagnosed with or is at risk for one or more solid tumors.
  • the condition or illness that can be treated by the methods provided herein can be an immune-related disease or illness, tumor and cancer, autoimmune disease or infectious disease.
  • the immune-related disease or illness is selected from the group consisting of: systemic lupus erythematosus, acute respiratory distress syndrome (ARDS), vasculitis, myasthenia gravis, idiopathic pulmonary fibrosis, Crohn's disease, asthma, rheumatoid arthritis, graft-versus-host disease, spondyloarthropathy (e.g., ankylosing spondylitis, psoriatic arthritis, isolated acute enteropathic arthritis associated with inflammatory bowel disease, reactive arthritis, Behcet's syndrome, undifferentiated spondyloarthropathy, anterior uveitis and juvenile idiopathic arthritis), multiple sclerosis, endometriosis, glomerulonephritis, sepsis, diabetes, acute coronary syndrome, ischemia
  • ARDS acute respiratory
  • the condition or illness that can be treated by the method provided herein include tumors and cancers.
  • the condition or illness that can be treated by the method provided herein include solid tumors and hematologic malignancies.
  • cancers and tumors include non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer, leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematologic malignancies, such as classical Hodgkin's lymphoma (CHL), primary longitudinal myeloma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematologic malignancies
  • CHL Hodg
  • the disease is cancer.
  • the cancer is selected from adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioalveolar lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, and vaginal cancer.
  • the cancer is a TROP2-positive cancer. In some embodiments, the cancer is a TROP2-positive cancer and a target antigen-positive cancer. In some embodiments, the subject to be treated has been identified as suffering from a TROP2-positive cancer, or a TROP2-positive cancer and a target antigen-positive cancer.
  • a "TROP2-positive” cancer refers to a cancer characterized by expressing TROP2 in cancer cells or expressing TROP2 in cancer cells at a level significantly higher than that expected from normal cells.
  • a "target antigen-positive” cancer refers to a cancer characterized by expressing a target antigen in cancer cells or expressing a target antigen in cancer cells at a level significantly higher than that expected from normal cells.
  • the cancer is a GUCY2C-positive cancer. In some embodiments, the cancer is a GUCY2C-positive cancer and a target antigen-positive cancer. In some embodiments, the subject to be treated has been identified as having a GUCY2C-positive cancer, or a GUCY2C-positive cancer and a target antigen-positive cancer.
  • a "GUCY2C-positive” cancer refers to a cancer characterized by expressing GUCY2C in cancer cells or expressing GUCY2C in cancer cells at a level significantly higher than that expected in normal cells.
  • a "target antigen-positive” cancer refers to a cancer characterized by expressing a target antigen in cancer cells or expressing a target antigen in cancer cells at a level significantly higher than that expected in normal cells.
  • the presence and/or amount of the target antigen in the biological sample of interest can be determined in a test biological sample from a subject using various suitable methods.
  • the test biological sample can be exposed to an anti-target antigen antibody or its antigen-binding fragment, which binds to and detects the expressed target antigen protein.
  • methods such as qPCR, reverse transcriptase PCR, microarrays, SAGE, FISH, etc. can also be used to detect the target antigen protein at the nucleic acid expression level.
  • the test sample is derived from cancer cells or tissues or tumor-infiltrating immune cells.
  • the presence or upregulation level of the target antigen protein in the test biological sample indicates the possibility of a response.
  • upregulation refers to an overall increase in the expression level of the target antigen protein in the test sample compared to the reference expression level of the target antigen by no less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more.
  • Reference level can be the level of target antigen expression found in normal cells of the same tissue type, optionally normalized relative to the expression level of another gene (e.g., housekeeping gene). Alternatively, reference level can be the level of target antigen expression found in healthy subjects.
  • Reference sample can be a control sample obtained from healthy or non-diseased individuals, or a healthy or non-diseased sample obtained from the same individual from which the test sample is obtained.
  • the reference sample can be a non-diseased sample adjacent to or near the test sample (e.g., tumor).
  • the test and/or reference are tested and/or determined substantially simultaneously with the test being paid attention to.
  • reference is a historical reference optionally embodied in a tangible medium. Typically, as will be appreciated by those skilled in the art, determine or characterize a reference under conditions or environments comparable to the conditions or environments in the assessment.
  • the tumor or cancer is selected from the group consisting of adrenal cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, stomach cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, non-small cell lung cancer, bronchioloalveolar cell lung cancer, mesothelioma, head and neck cancer, squamous cell carcinoma, melanoma, oral cancer, ovarian cancer, cervical cancer, penile cancer, prostate cancer, pancreatic cancer, skin cancer, sarcoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, neural or neuroendocrine tumors, small cell lung cancer (SCLC), large cell neuroendocrine carcinoma (LCNEC), gastrointestinal neuroendocrine tumor (GI-NEC), small cell bladder cancer (SCBC), glioblastoma multiforme, metastatic castration-resistant neuroendocrine tumor, neuroblastoma, central nervous system dysregulation, metastatic carcinoma, diffuse intrinsic pontine gliom
  • SCLC
  • the condition or disorder treatable by the methods provided herein comprises an autoimmune disease.
  • Autoimmune diseases include, but are not limited to, acquired immunodeficiency syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diabetes, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, sprue sprue-dermatitis herpetiformis; chronic fatigue immune dysfunction syndrome (CFS); CFIDS, chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, juvenile dermatomyositis, discoid
  • the conditions or disorders treatable by the methods provided herein include infectious diseases.
  • infectious diseases include, for example, chronic viral infections, e.g., fungal infections, parasitic/protozoal infections, or chronic viral infections, e.g., malaria, coccidioidomycosis immitis, histoplasmosis, onychomycosis, aspergillosis, blastomycosis, candidiasis albicans, paracoccidiomycosis, microsporidiosis, Acanthamoeba keratitis, amoebiasis, ascariasis, babesiosis, esiosis), Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enter
  • an antibody or antigen-binding fragment thereof as provided herein will depend on various factors known in the art, such as body weight, age, past medical history, current medications, the health status of the subject and the potential for cross-reactions, allergies, sensitivities and adverse side effects, as well as the route of administration and the extent of disease progression. As indicated by these and other circumstances or requirements, one of ordinary skill in the art (e.g., a physician or veterinarian) may proportionally reduce or increase the dosage.
  • antibody or its Fab as provided herein can be used with about 0.01mg/kg to the treatment effective dose of about 100mg/kg.
  • antibody or its Fab is used with about 50mg/kg or dosage still less, and in certain embodiments in these embodiments, dosage is 10mg/kg or still less, 5mg/kg or still less, 3mg/kg or still less, 1mg/kg or still less, 0.5mg/kg or still less or 0.1mg/kg or still less.
  • administration dosage can change in therapeutic process.For example, in certain embodiments, initial administration dosage can be higher than administration dosage subsequently.In certain embodiments, administration dosage can change according to experimenter's reaction in therapeutic process.
  • Dosage regimens may be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single dose may be administered, or several divided doses may be administered over time.
  • the antibodies or antigen-binding fragments thereof provided herein can be administered by any route known in the art, e.g., parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal or topical) routes.
  • parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection
  • non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal or topical routes.
  • the antibodies or antigen-binding fragments thereof disclosed herein can be administered alone or in combination with one or more additional therapeutic modalities or agents.
  • the antibodies or antigen-binding fragments thereof disclosed herein can be administered in combination with another therapeutic agent, such as a chemotherapeutic agent or an anticancer drug.
  • an antibody or antigen-binding fragment thereof as disclosed herein, administered in combination with one or more additional therapeutic agents can be administered concurrently with the one or more additional therapeutic agents, and in some of these embodiments, the antibody or antigen-binding fragment thereof and the additional therapeutic agent can be administered as part of the same pharmaceutical composition.
  • an antibody or antigen-binding fragment thereof administered "in combination" with another therapeutic agent need not be administered concurrently with the agent or in the same composition as the agent.
  • An antibody or antigen-binding fragment thereof administered before or after another agent is considered to be administered "in combination" with the agent, as the phrase is used herein, even if the antibody or antigen-binding fragment thereof and the second agent are administered by different routes.
  • the additional therapeutic agent administered in combination with the antibodies or antigen-binding fragments thereof disclosed herein is administered according to the schedule listed in the product information sheet of the additional therapeutic agent or according to the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)) or regimens well known in the art.
  • the multispecific molecules provided herein include an antigen binding domain provided herein and a target antigen binding domain provided herein. In certain embodiments, the multispecific molecules provided herein are designed as shown in FIG. 11 .
  • the multispecific molecules provided herein are capable of specifically binding to one, two, or three of the cell surface markers of human CD3, TROP2, and CD28.
  • the multispecific molecules provided herein retain specific binding affinity for one, two, or three of human CD3, TROP2, and CD28, and in certain embodiments, are at least comparable to or even better than the parent antibody in these aspects.
  • the binding of a multispecific molecule can be expressed as a "half maximal effective concentration" (EC50 ) value, which refers to the concentration of the antibody at which 50% of the maximal effect (e.g., binding or inhibition, etc.) of the antibody is observed.
  • EC50 values can be measured by methods known in the art, such as sandwich assays, such as ELISA, Western blots, flow cytometry assays, and other binding assays.
  • the binding affinity of the antigen-binding domains provided herein can also be expressed by a K value, which represents the ratio of the dissociation rate to the association rate when the binding between the antigen and the antigen-binding molecule reaches equilibrium (k off / kon ).
  • K D Antigen binding affinity
  • K D can be suitably determined using suitable methods known in the art (including, for example, flow cytometry).
  • the multispecific molecules provided herein specifically bind to human CD3, TROP2, or CD28 with a binding affinity ( KD ) as measured by an Octet assay.
  • the multispecific molecules provided herein specifically bind to human CD3, TROP2, or CD28 with a binding affinity ( KD ) as measured by an ELISA assay.
  • the multispecific molecules provided herein specifically bind to human CD3, TROP2, or CD28 with a binding affinity ( KD ) as measured by FACS assay.
  • the multispecific molecules provided herein also encompass various variants thereof.
  • Such variants retain the specific binding affinity of their parent antibody to CD3, TROP2 or CD28, but have one or more desirable properties conferred by the modification or substitution.
  • target residues e.g., charged residues such as Arg, Asp, His, Lys, and Glu
  • modified antibodies are generated and screened for properties of interest. If the substitution at a particular amino acid position shows a functional change of interest, the position can be identified as a potential residue for modification or substitution.
  • Potential residues can be further evaluated by replacing them with different types of residues (e.g., cysteine residues, positively charged residues, etc.).
  • the CD3 binding domain, CD28 binding domain, and/or TROP2 binding domain provided herein comprise one or more amino acid residue substitutions in one or more CDR sequences and/or one or more FR sequences and/or one or more variable region sequences.
  • the variant comprises no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions in a total of CDR sequences and/or FR sequences and/or one or more variable region sequences.
  • the CD3 binding domain comprises 1, 2, 3, 4, 5 or 6 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to 1, 2, 3, 4, 5 or 6 sequences selected from Table 6, while retaining binding affinity for CD3 at a similar or even higher level relative to its parent antibody.
  • the CD28 binding domain comprises 1, 2, 3, 4, 5, or 6 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to 1, 2, 3, 4, 5, or 6 sequences selected from Table 10, while retaining binding affinity for CD28 at a similar or even higher level relative to its parent antibody.
  • the TROP2 binding domain comprises 1, 2, 3, 4, 5 or 6 CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to 1, 2, 3, 4, 5 or 6 sequences selected from Table 13, and at the same time retains binding affinity for TROP2 at a similar or even higher level relative to its parent antibody.
  • the multispecific molecules provided herein also encompass glycosylation variants, which can be obtained to increase or decrease the extent of glycosylation of the antigen binding domain or activating receptor domain of the multispecific molecule.
  • the multispecific molecules provided herein can include one or more amino acid residues having side chains to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached.
  • Glycosylation of the antibody antigen-binding domain is typically N-linked or O-linked.
  • N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue (e.g., an asparagine residue in a tripeptide sequence such as asparagine-X-serine and asparagine-X-threonine), where X is any amino acid except proline.
  • O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly to serine or threonine.
  • Natural glycosylation sites can be conveniently removed, for example, by altering the amino acid sequence such that one of the tripeptide sequences (for N-linked glycosylation sites) or the serine or threonine residues (for O-linked glycosylation sites) present in the sequence is substituted.
  • New glycosylation sites can be generated in a similar manner by introducing such a tripeptide sequence or a serine or threonine residue.
  • Cysteine engineered variants can be used to conjugate, for example, cytotoxic compounds and/or imaging compounds, labels, or radioisotopes at the site of the engineered cysteine via, for example, maleimide or haloacetyl groups.
  • Methods for engineering antibody polypeptides to introduce free cysteine residues are known in the art, see, for example, WO2006/034488.
  • the multispecific molecules provided herein also encompass Fc variants comprising one or more amino acid residue modifications or substitutions at the Fc region and/or hinge region thereof, for example, to provide altered effector functions such as ADCC, ADCP, and CDC.
  • Methods for altering ADCC activity by antibody engineering have been described in the art, see, for example, Shields RL. et al., J Biol Chem. 2001. 276(9): 6591-604; Idusogie EE. et al., J Immunol. 2000. 164(8): 4178-84; Steurer W. et al., J Immunol. 1995, 155(3): 1165-74; Idusogie EE. et al., J Immunol.
  • the CDC activity of the antibodies provided herein can also be altered, for example, by improving or reducing C1q binding and/or CDC (see, e.g., WO 99/51642; Duncan and Winter, Nature, 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821); and WO 94/29351 for other examples of Fc region variants.
  • One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be replaced with a different amino acid residue to alter C1q binding and/or reduce or eliminate complement-dependent cytotoxicity (CDC) (see U.S. Patent No. 6,194,551 to Idusogie et al.).
  • One or more amino acid substitutions can also be introduced to alter the ability of the antibody to fix complement (see PCT Publication WO 94/29351 to Bodmer et al.).
  • antibody-dependent cellular phagocytosis and "ADCP” refer to a process by which antibody-coated cells or particles are fully or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of an immunoglobulin.
  • phagocytic immune cells e.g., macrophages, neutrophils, and dendritic cells
  • Methods for altering the ADCP activity of antibodies by antibody engineering are known in the art, see, for example, Kellner C et al., Transfus Med Hemother, (2017) 44:327-336 and Chung AW et al., AIDS, (2014) 28:2523-2530.
  • Fc variants are known in the art, see, for example, Wang et al., Protein Cell 2018, 9(1):63-73 and Kang et al., Exp&Mol., Med. (2019) 51:138, which are incorporated herein in their entirety.
  • the Fc variants provided herein have increased ADCC and/or increased affinity for Fc ⁇ receptors (e.g., Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and/or Fc ⁇ RIII (CD16)) relative to wild-type Fc (e.g., Fc of IgG1).
  • Fc ⁇ receptors e.g., Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and/or Fc ⁇ RIII (CD16)
  • wild-type Fc e.g., Fc of IgG1
  • the Fc variants comprise one or more amino acid substitutions at one or more of the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 246, 247, 248, 249, 252, 254, 255, 256, 258, 260, 262, 263, 264, 265, 267, 268, 269, 270, 272, 274, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 300, 301, 303, 304, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335 35, 337, 338, 339, 340, 345, 360, 373, 376, 378, 382, 388, 389, 396, 398, 414, 416, 419, 430, 433, 434
  • Fc variants with strongly enhanced binding to Fc ⁇ RIIIa include variants with S239D/I332E and S239D/I332E/A330L mutations (which showed the greatest increase in affinity for Fc ⁇ RIIIa, decreased binding to Fc ⁇ RIIb, and potent cytotoxic activity) and variants with L235V, F243L, R292P, Y300L, V305I, and P396L mutations (which exhibited enhanced Fc ⁇ RIIIa and concomitant enhanced ADCC activity).
  • S239D/I332E and S239D/I332E/A330L mutations which showed the greatest increase in affinity for Fc ⁇ RIIIa, decreased binding to Fc ⁇ RIIb, and potent cytotoxic activity
  • variants with L235V, F243L, R292P, Y300L, V305I, and P396L mutations which exhibited enhanced Fc ⁇ RIIIa and concomitant enhanced ADCC activity.
  • Modifications that increase binding to C1q can be introduced to enhance CDC activity.
  • exemplary modifications include K326 (e.g., K326W) and/or E333 modifications in IgG2, or S267E/H268F/S324T modifications, alone or in combination, in IgG1 (see Idusogie et al. (2001) J. Immunol. 166:2571; Moore et al. (2010) Mabs 2:181).
  • Other exemplary modifications include K326W/E333S, S267E/H268F/S324T, and E345R/E430G/S440Y.
  • the Fc variants provided herein have reduced effector function relative to wild-type Fc (e.g., Fc of IgG1) and comprise one or more amino acid substitutions at positions selected from the group consisting of: 220, 226, 229, 233, 234, 235, 236, 237, 238, 267, 268, 269, 270, 297, 309, 318, 320, 322, 325, 328, 329, 330, and 331 of the Fc region (see WO 2016/196228; Richards et al. (2008) Mol Cancer Therapeutics 7:2517; Moore et al.
  • substitutions that reduce effector function include, but are not limited to, 220S, 226S, 228P, 229S, 233P, 234V, 234G, 234A, 234F, 234A, 235A, 235G, 235E, 236E, 236R, 237A, 237K, 238S, 267R, 268A, 268Q, 269R, 297A, 297Q, 297G, 309L, 318A, 322A, 325L, 328R, 330S, 331S, or any combination thereof (see WO 2016/196228; and Strohl (2009), Biotechnol. Current Rev. 20:685-691).
  • the Fc variant comprises one or more amino acid substitutions that improve binding affinity to the neonatal Fc receptor (FcRn) at pH 6.0 while retaining minimal binding at pH 7.4.
  • FcRn neonatal Fc receptor
  • Such variants can have a prolonged pharmacokinetic half-life because the variant binds to FcRn at acidic pH, thereby protecting it from degradation in lysosomes and allowing it to be transported and released outside the cell.
  • Methods for engineering antibodies and antigen-binding fragments thereof to improve binding affinity to FcRn are well known in the art, see, for example, Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Vol.
  • the present disclosure also provides methods of treating a CD28-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.
  • the present disclosure also provides methods of treating a TROP2-associated disease, disorder, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a multispecific molecule provided herein.
  • Sequence stability was assessed for all SP34-based engineered anti-CD3 antibodies using an AI structure prediction model and empirical force field energy functions to generate stability scores.
  • a stable antibody structure was first determined using the sequence-to-structure AI structure prediction model. Using the AI-predicted antibody structure as the starting point for energy optimization, the energy was optimized within 10 iterations using a greedy algorithm within the force field. Finally, all values were averaged. The prediction results are shown in Table 16.
  • All anti-CD28 antibodies based on TGN1412 were re-optimized using the antibody AI structure prediction model and empirical force field energy function.
  • the public protein structure database RCSB PDB structure 1YJD was used as a sequence and structural template to perform back mutation energy analysis on all sequences modified based on the TGN1412 sequence.
  • the mutation energy was analyzed to obtain multiple humanized and monomer stability multi-optimized precursors and reselect the starting point for the downstream algorithm.
  • Anti-CD28 antibody energy calculation results see Table 17
  • optimized sequences selected based on energy analysis see Table 18
  • the original sequence Pr1E11-Chi was humanized using a CDR grafting approach.
  • the human germline sequences IGHV1-46 and IGKV4-1 which share the highest homology with the original heavy and light chains, respectively, were selected as target germline sequences.
  • the original amino acids in all CDR regions were retained, and some amino acids in the FR regions were backmutated to obtain three humanized sequences (see Table 20, sequences huE11, huE11-2, and huE11-3). Sequence huE11 was selected as the starting antibody, and the amino acid sequence of the huE11 variable region is shown in Tables 11 and 20.
  • TROP2 antibodies Based on AI prediction and calculation results, we selected a series of advantageous mutations to design candidate TROP2 antibodies.
  • the candidate antibodies are shown in Table 11 for the antibody variable region (VL and VH) sequences and Table 12 for the single-chain antibody (scFv) sequences.
  • scFv single-chain antibody
  • huRS7 derived from patent WO2003074566A3
  • the huRS7 sequence is shown in Tables 11 and 12.
  • the heavy chain and light chain DNA fragments of the anti-CD3 monoclonal antibody designed by AI were subcloned into the pcDNA3.4 vector respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.
  • CD3-002IgG, CD3-006IgG, CD3-007IgG, and CD3-008IgG exhibited excellent physical and chemical properties.
  • the anti-CD3 scFv-Fc DNA fragment designed by AI was cloned into the pcDNA3.4 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with pH 3.4 sodium acetate buffer and dialyzed to pH 7.4 PBS. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.
  • Table 22 shows the DLS and SEC data for the yield, purity, Jurkat cell binding, CD3ed-HIS binding (ELISA), Tm values, and accelerated stability (7 days incubation at 40°C) of different anti-CD3 scFv-Fc proteins after AI optimization.
  • ELISA ed-HIS binding
  • Tm time incubation at 40°C
  • CD3-002scFv, CD3-006scFv, and CD3-007scFv exhibited excellent physical and chemical properties.
  • the expression level of CD3-002scFv was significantly higher than that of CD3-006scFv and CD3-007scFv.
  • CD3-002IgG (sequence shown in Table 4) as an example, the optimized sequence has good physicochemical properties.
  • CD3-002 IgG was re-predicted and the intramolecular interactions at the mutation sites were examined.
  • 244G245G-309V forms two small polar interactions that stabilize the two G loops
  • 264P forms a pair of polar interactions with 282T
  • 323Y and 326L form a pair of polar interactions.
  • the heavy chain and light chain DNA fragments of the anti-CD28 monoclonal antibody designed by AI were subcloned into the pcDNA3.4 vector respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.
  • CD28-041IgG and CD28-065IgG have good various physical and chemical properties.
  • the anti-CD28 scFv-Fc DNA fragment designed by AI was cloned into the pcDNA3.4 vector, and the recombinant plasmid was extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.
  • Table 24 shows the yield, purity, Jurkat cell binding, CD28-HIS binding (ELISA, KD), and accelerated stability (7 days incubation at 40°C) of different AI-optimized CD28 scFv-Fc proteins using DLS and SEC.
  • CD28-041scFv exhibited favorable physicochemical properties.
  • CD28-065scFv was not tested.
  • the heavy chain and light chain DNA fragments of the anti-TROP2 monoclonal antibody designed by AI were subcloned into the pcDNA3.4 vector respectively, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.
  • TP-021IgG and TP-023IgG exhibited excellent physical and chemical properties.
  • the AI-designed anti-TROP2 scFv-Fc DNA fragments were subcloned into the pcDNA3.4 vector.
  • the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument.
  • CHO cells were co-transfected with recombinant plasmids encoding the heavy and light chains or scFvs of anti-CD3, CD28, and TROP2 antibodies and Fc. After 7 days of culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument. Expression results are shown in Table 28.
  • the purified proteins were analyzed by HPLC.
  • the HPLC-SEC chromatogram is shown in Figure 4A , demonstrating that the purity of the monoclonal antibody monomers exceeded 98%, the purity of the CD3-002scFv single-chain antibody monomers exceeded 90%, and the purity of the remaining single-chain antibody monomers exceeded 95%.
  • SDS-PAGE analysis results are shown in Figure 4B , showing that the reduced purity of all antibodies was greater than 95%.
  • M protein marker
  • R reduced SDS-PAGE
  • N-R non-reduced SDS-PAGE.
  • the theoretical molecular weights of each protein are shown in Table 28.
  • Anti-CD3, CD28, and TROP2 antibodies were placed in a 40°C incubator and incubated for 7 days before analysis by HPLC-SEC.
  • the HPLC-SEC analysis profile is shown in Figure 5. No aggregation peaks were observed for CD3-002IgG, CD3-006IgG, CD3-007IgG, TP-021IgG, TP-023IgG, CD3-002scFv, CD3-006scFv, and CD3-007scFv. However, significant precipitation was observed for huSP34, huRS7, huE11, and huRS7-scFv, which were not detected by SEC (ND).
  • Example 16 FACS detection of the binding of huRS7, huE11, TP-021 IgG, and TP-023 IgG to 293-huTROP2 and 293-cyTROP2 target cells
  • the DNA fragments encoding the extracellular domain (ECD) of human and monkey TROP2 (huTROP2, cyTROP2 in Table 1) were cloned into the mDeZ-TM (the company's own vector, with a secretion signal peptide, a transmembrane sequence and a Zeocin resistance gene) vector and transiently transfected into Expi293 cells. After 24 hours, 700ug/mL Zeocin (final concentration) was added and screened for 7 days to obtain Expi293 cells (293-huTROP2, 293-cyTROP2) with high display of the extracellular domain of human and monkey TROP2 on the cell surface.
  • mDeZ-TM the company's own vector, with a secretion signal peptide, a transmembrane sequence and a Zeocin resistance gene
  • 293-huTROP2 and 293-cyTROP2 which highly display the extracellular domain of human and monkey TROP2 on their cell surfaces, were used as target cells, and Expi293 cells (293-Ctl) were used as a negative control.
  • the cells were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded.
  • the cells were resuspended in PBS, diluted to a density of 1 ⁇ 106 cells/mL, and 100 ⁇ L/well was added to a 96-well plate.
  • the monoclonal antibody was diluted to 200 nM, and 100 ⁇ L/well was added to a 96-well plate.
  • the cells were mixed evenly with 293-huTROP2, 293-cyTROP2, and Expi293 cells. The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound antibody. Then, 100 ⁇ L/well of goat anti-human IgG-PE (1:200 dilution) was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 300 g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 ⁇ l of PBS, and antibody binding to the cells was measured using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using GraphPad Prism software. As shown in Figure 6, huRS7, huE11, TP-021 IgG, and TP-023 IgG all bound to both huTROP2 and cyTROP2 displayed on the surface of 293 cells.
  • Example 18 ELISA to detect the affinity of anti-CD3 antibodies to CD3ed-HIS
  • Recombinant CD3ed-HIS protein was diluted to 2 ⁇ g/ml with PBS and added to an ELISA plate at 100 ⁇ l/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 ⁇ l/well and incubated at room temperature for 2 hours. The blocking solution was removed, and the plates were washed three times with 250 ⁇ l/well of 0.5 ⁇ PBST.
  • the test results are shown in Figure 8.
  • the EC50 (unit: nM) of CD3-002IgG, CD3-006IgG, CD3-007IgG, huSP34 and CD3-002scFv-Fc, CD3-006scFv-Fc, CD3-007scFv-Fc binding to CD3ed-HIS were 0.14, 0.16, 0.15, 0.17, 1.41, 1.36 and 1.00, respectively.
  • the Fortebio Octet R8 molecular interaction instrument was used, and The AHC2 Biosensor probe capture method was used to determine the kinetic parameters of binding between anti-CD3-specific antibodies and CD3ed-HIS antigens.
  • the AHC2 probe was activated by soaking it in 1 ⁇ PBS for 20 minutes.
  • CD3-specific antibodies CD3-002IgG, CD3-006IgG, CD3-007IgG, CD3-002scFv-Fc, CD3-006scFv-Fc, CD3-007scFv-Fc, and huSP34 were diluted to 30 ⁇ g/ml in 1 ⁇ PBS and the probe was soaked in PBS for 120 seconds to allow binding. The probe was then soaked in 1 ⁇ PBS for another 120 seconds.
  • the CD3ed-HIS antigen was diluted two-fold downwards in 1 ⁇ PBS to create three concentration gradients, and the probe was soaked in PBS for 180 seconds to measure the association rate. The probe was then soaked in 1 ⁇ PBS for 360 seconds to measure the dissociation rate.
  • the kinetic parameters for the binding of anti-CD3-specific antibodies CD3-002IgG, CD3-006IgG, CD3-007IgG, huSP34, CD3-002scFv-Fc, CD3-006scFv-Fc, CD3-007scFv-Fc, and huSP34-scFv-Fc to CD3ed-HIS are shown in Table 29, and the kinetic characteristic parameter detection results are shown in Figure 9. The results show that all antibodies have good affinity for CD3ed-HIS.
  • CD3-002IgG, CD3-002scFv-Fc, CD3-006IgG, CD3-006scFv-Fc, and huSP34-scFv-Fc have approximately 3- to 4-fold lower affinity.
  • CD3-007IgG and CD3-007scFv-Fc showed no significant difference in affinity compared with huSP34.
  • the Fortebio Octet R8 molecular interaction instrument was used, and The AHC2 Biosensors probe capture assay was used to determine the kinetic parameters of binding between anti-TROP2 antibodies and the TROP2-HIS antigen.
  • the AHC2 probe was activated by soaking it in 1 ⁇ PBS for 20 minutes.
  • TROP2-specific antibodies huRS7, huE11, TP-021 IgG, and TP-023 IgG were diluted to 30 ⁇ g/ml in 1 ⁇ PBS and the probe was soaked for 120 seconds to allow binding. The probe was then soaked in 1 ⁇ PBS for another 120 seconds.
  • the TROP2-HIS antigen was diluted two-fold downwards from 400 nM in 1 ⁇ PBS to create three concentration gradients. The probe was soaked for 120 seconds to measure the association rate of the antigen and antibody, and the dissociation rate of the antigen and antibody was measured by soaking the probe in 1 ⁇ PBS for 240 seconds.
  • TP-021IgG The kinetic parameters for the binding of anti-TROP2-specific antibodies huRS7, huE11, TP-021IgG, and TP-023IgG to TROP2-HIS are shown in Table 30, and the results of the kinetic characteristic parameter testing are shown in Figure 10.
  • the results showed that TP-021IgG, TP-023IgG, and huE11 all had good affinity for TROP2-HIS.
  • TP-023IgG had an approximately 2.5-fold higher affinity than huE11.
  • TP-021IgG showed no significant change in affinity compared to HuE11.
  • CD3, CD28, and TROP2 primary antibodies were selected as the basis for constructing TROP2 ⁇ CD3 bispecific and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies.
  • the configurations are shown in Figure 11.
  • the H44/L100 positions of the CD3 single-chain antibody (scFv) were mutated to Cysteine to form an intramolecular disulfide bond to stabilize the antibody.
  • Knob-into-Hole (KIH) technology was also used to achieve recombinant heavy chain heterodimers. The sequences are shown in Table 31.
  • Table 31 Amino acid sequences of candidate anti-TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies
  • the designed double-antibody and triple-antibody DNA fragments were cloned into the pcDNA3.4 vector, and the recombinant plasmids were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was filtered by high-speed centrifugation and vacuum filtration with a microporous filter membrane, and then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer at pH 3.4 and dialyzed to PBS at pH 7.4. The absorbance value at 280 nm was read using a NanoDrop instrument to detect the protein concentration.
  • the expressed bispecific and tertiary antibodies were tested for protein yield, purity, cell binding (Jurkat, 293-CD28, and 293-huTROP2), antigen binding ELISA (TROP2-HIS, CD3ed-HIS, and CD28-HIS), accelerated stability (aggregation assayed by DLS and SEC after incubation at 40°C for 7 days), and antigen binding affinity (KD value).
  • the data are shown in Table 32.
  • TPt0019, TPt0025, TPt0042, TPb043, and TPb059 exhibited favorable physical and chemical properties and were selected for further testing.
  • the recombinant plasmids against heavy chain and light chain were co-transfected into Expi293 cells. After 3 days of cell culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The culture medium was then analyzed using a Fortebio Octet R8 molecular interaction instrument and a The ProA Biosensors probe capture assay was used to determine the kinetic parameters of binding between the alanine-scanning TP-023 antibody and the huTROP2-HIS antigen. The ProA probe was activated by soaking it in 1 ⁇ PBS for 20 minutes. The probe was then soaked in the antibody expression supernatant for 120 seconds to allow binding.
  • the probe was then soaked in 1 ⁇ PBS for another 120 seconds.
  • the huTROP2-HIS antigen was diluted to 200 nM in 1 ⁇ PBS and the probe was soaked for 100 seconds to measure the binding rate.
  • the probe was then soaked in 1 ⁇ PBS for 200 seconds to measure the dissociation rate.
  • the affinity (KD) of the alanine scanning antibody to huTROP2-HIS is shown in Table 38.
  • the results of the kinetic characteristic parameter detection of the binding of the alanine scanning antibody to huTROP2-HIS are shown in Figure 13.
  • Table 39 Amino acid sequences of candidate anti-TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies with weakened affinity
  • Example 28 Complete structural analysis of anti-TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody
  • TROP2 ⁇ CD3 dual antibody and TROP2 ⁇ CD3 ⁇ CD28 triple antibody expression vectors were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was centrifuged at high speed and vacuum filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument. Expression results are shown in Table 40.
  • the purified proteins were analyzed by HPLC.
  • HPLC-SEC purity data for the anti-TROP2 ⁇ CD3 and TROP2 ⁇ CD3 ⁇ CD28 triple antibodies are shown in Table 40 and Figure 15 .
  • the purity of the monoclonal antibodies, purified using a Protein A affinity column, was >88%.
  • SDS-PAGE analysis results are shown in Figure 15 , demonstrating that the reduced purity of the antibodies was >95%.
  • M protein marker
  • R reduced SDS-PAGE
  • N-R non-reduced SDS-PAGE.
  • Table 40 Optimized expression and purification results of TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody
  • the affinity-weakened TROP2 ⁇ CD3 dual antibody and TROP2 ⁇ CD3 ⁇ CD28 triple antibody expression vectors were extracted and co-transfected into CHO cells. After 7 days of cell culture, the culture medium was centrifuged at high speed and vacuum-filtered through a microporous filter membrane. The sample was then loaded onto a Protein A affinity chromatography column. The protein was eluted with sodium acetate buffer (pH 3.4) and dialyzed into PBS (pH 7.4). Protein concentration was determined by measuring absorbance at 280 nm using a NanoDrop instrument. Expression results are shown in Table 41.
  • HPLC-SEC purity data for the anti-TROP2 ⁇ CD3 and TROP2 ⁇ CD3 ⁇ CD28 triple antibodies are shown in Table 41 and Figure 16.
  • SDS-PAGE analysis results are shown in Figure 16, demonstrating that the reduced purity of the antibodies was >95%.
  • M protein marker
  • R reduced SDS-PAGE
  • N-R non-reduced SDS-PAGE.
  • Table 41 Optimized expression and purification results of TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies with weakened affinity
  • Example 31 Fortibio determined the affinity of anti-TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody for the antigens TROP2-HIS, CD28-HIS, and CD3ed-HIS
  • the Fortebio Octet R8 molecular interaction instrument was used, and The AHC2 Biosensor probe capture assay measured the kinetic parameters of binding between anti-TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies and the antigens TROP2-HIS, CD28-HIS, and CD3ed-HIS.
  • the AHC2 probe was activated by soaking in 1 ⁇ PBS for 20 minutes. The antibodies were diluted to 30 ⁇ g/ml in 1 ⁇ PBS, and the probe was soaked in this solution for 120 seconds to allow binding. The probe was then soaked in 1 ⁇ PBS for another 120 seconds.
  • the kinetic parameters for the binding of the bispecific and tertiary antibodies to TROP2-HIS, CD28-HIS, and D3ed-HIS are shown in Table 42.
  • the kinetic characteristic parameters for the binding of the bispecific and tertiary antibodies to TROP2, CD28-HIS, and CD3ed-HIS are shown in Figure 17.
  • the results showed that TPb0043, TPb0059, TPt0019, TPt0025, and TPt0042 had good affinity for TROP2; TPb0059, TPt0019, and TPt0025 had good affinity for CD28-HIS.
  • TPb0043, TPt0019, TPt0025, and TPt0042 had weaker affinity for CD3ed-HIS, with KD values of 3.00x10-8 M, 1.17x10-8 M, 1.25x10-7 M, and 1.29x10-7 M, respectively.
  • Table 42 Kinetic characteristic parameters of the binding of TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies to TROP2-His, CD28-HIS and CD3ed-HIS
  • Example 32 Fortibio determines the affinity (combination) of TROP2-biotin, CD28-biotin, and CD3ed-biotin antigens against the TROP2 ⁇ CD3 bispecific antibody and the TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody
  • the Fortebio Octet R8 molecular interaction instrument was used, and The streptavidin (SA) probe capture assay was used to determine the kinetic parameters of binding of TROP2-biotin, CD28-biotin, and CD3ed-biotin antigens to TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 tertiary antibodies.
  • the SA probes were activated by soaking in 1 ⁇ PBS for 20 minutes.
  • TROP2-biotin, CD28-biotin, and CD3ed-biotin were diluted to 10 ⁇ g/ml in 1 ⁇ PBS and soaked for 120 seconds to allow binding. The probes were then soaked in 1 ⁇ PBS for another 120 seconds.
  • the bispecific and tertiary antibodies were diluted two-fold in 1 ⁇ PBS from 400 nM to a gradient of 4-7 concentrations.
  • the probes were soaked in 1 ⁇ PBS for 120 seconds to measure the association rate.
  • the probes were then soaked in 1 ⁇ PBS for 240 seconds to measure the dissociation rate.
  • TROP2-Biotin had good affinity for both the bispecific and tertiary antibodies
  • CD28-HIS had good binding to TPb0059, TPt0019, and TPt0025
  • CD3ed-Biotin had moderate binding to TPb0043 and TPt0019
  • Table 43 Kinetic characteristic parameters of the binding of TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies to TROP2-biotin, CD28-biotin and CD3ed-biotin
  • Recombinant TROP2-HIS protein was diluted to 2 ⁇ g/ml with PBS and added to an ELISA plate at 100 ⁇ l/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 ⁇ l/well and incubated at room temperature for 2 hours. The blocking solution was removed, and the plate was washed three times with 250 ⁇ l/well of 0.5 ⁇ PBST. The antibody was then diluted to 100 nM in blocking solution, and a five-fold dilution was used to form 12 concentration gradients (maximum concentration 100 nM). 100 ⁇ l/well was added to the blocked ELISA plate and incubated at room temperature for 1 hour.
  • the plate was washed three times with PBST (removing any remaining droplets with absorbent paper), and HRP-labeled goat anti-human IgG antibody was added at 100 ⁇ l/well and incubated at room temperature for 45 minutes.
  • the plate was washed five times with 0.5 ⁇ PBST, TMB was added at 100 ⁇ l/well, and the plate was placed in the dark at room temperature for 5 minutes. Stop solution was added at 100 ⁇ l/well to stop the substrate color development reaction, and the OD value at 450 nm was read with a microplate reader. The data were analyzed with GraphPad, and graphs were drawn and EC50 was calculated.
  • the detection results are shown in Figure 19.
  • the EC50 (unit: nM) of anti-TROP2 ⁇ CD3 bispecific antibodies TPb0043 and TPb0059, TROP2 ⁇ CD3 ⁇ CD28 triple antibodies TPt0019, TPt0025, TPt0042 and positive control huRS7 and huE11 monoclonal antibodies binding to TROP2-HIS were 0.049, 0.043, 0.12, 0.15, 0.012, 0.022 and 0.027, respectively.
  • Recombinant CD28-HIS protein was diluted to 2 ⁇ g/ml with PBS and added to the ELISA plate at 100 ⁇ l/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight.
  • the coating solution was removed and blocking solution was added at 200 ⁇ l/well and incubated at room temperature for 2 hours.
  • the blocking solution was removed and the plates were washed three times with 250 ⁇ l/well of 0.5 ⁇ PBST.
  • the antibody was then diluted to 2 ⁇ M with blocking solution and diluted fivefold to form 12 concentration gradients (highest concentration 2 ⁇ M).
  • the plates were then added to the blocked ELISA plate at 100 ⁇ l/well and incubated at room temperature for 1 hour.
  • the plates were washed three times with 0.5 ⁇ PBST (removing any remaining droplets with absorbent paper) and HRP-labeled goat anti-human IgG antibody was added at 100 ⁇ l/well and incubated at room temperature for 45 minutes.
  • the plate was washed five times with 0.5 ⁇ PBST, TMB was added at 100 ⁇ l/well, and the plate was placed in the dark at room temperature for 5 minutes. Stop solution was added at 100 ⁇ l/well to stop the substrate color development reaction, and the OD value at 450 nm was read with a microplate reader.
  • the data were analyzed with GraphPad, and graphs were drawn and EC50 was calculated.
  • the test results are shown in FIG20 .
  • the EC50 (unit: nM) of the anti-TPb0059, TPt0019 and TPt0025 antibody molecules binding to CD28-HIS are 5.40, 2.68 and 1.03, respectively.
  • Recombinant CD3ed-HIS protein was diluted to 2 ⁇ g/ml in PBS and added to an ELISA plate at 100 ⁇ l/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 ⁇ l/well. The plate was incubated at room temperature for 2 hours. The blocking solution was removed, and the plate was washed three times with 250 ⁇ l/well of 0.5 ⁇ PBST. The antibody was then diluted to 2 ⁇ M in blocking solution. A five-fold dilution series (maximum concentration 2 ⁇ M) was created, and 12 concentrations were added to the blocked plate at 100 ⁇ l/well.
  • the plate was incubated at room temperature for 1 hour. The plate was washed three times with 0.5 ⁇ PBST (removing any remaining droplets with absorbent paper). HRP-conjugated goat anti-human IgG antibody was added at 100 ⁇ l/well and incubated at room temperature for 45 minutes. The plate was washed five times with 0.5 ⁇ PBST, TMB was added at 100 ⁇ l/well, and the plate was placed in the dark at room temperature for 5 minutes. Stop solution was added at 100 ⁇ l/well to stop the substrate color development reaction, and the OD value at 450 nm was read with a microplate reader. The data were analyzed with GraphPad, and graphs were drawn and EC50 was calculated.
  • the test results are shown in FIG21 .
  • the EC50 (unit: nM) of TPb0043, TPt0019, TPt0025, TPt0042 and the positive control huSP34 monoclonal antibody binding to CD3ed-HIS were 0.58, 0.79, 3.74, 7.00 and 0.035, respectively.
  • Example 36 FACS detection of the binding affinity of anti-TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody to target cells
  • the DNA fragments encoding human CD3 and 1G4 TCR were cloned into mDeZ-HIS (the company's own vector, with a secretion signal peptide and a Zeocin resistance gene) and mDeH-HIS (the company's own vector, with a secretion signal peptide and a Hygromycin resistance gene) vectors, respectively.
  • the dual plasmids were transiently transfected into Expi293 cells. After 24 hours, 700ug/mL Zeocin (final concentration) and 500ug/ml Hygromycin were added and screened for 7 days to obtain Expi293 cells (293-TCR) with high display of human TCR on the cell surface.
  • Expi293 cells which display a highly expressed TCR on their cell surface, were used as target cells (293-TCR).
  • Expi293 cells were used as negative cells. Wash the cells three times with PBS, centrifuging at 300g for 5 minutes each time, and discarding the supernatant. Resuspend the cells in PBS and dilute them to a density of 1 ⁇ 106 cells/mL. 100 ⁇ L/well of the cells were added to a 96-well plate. Antibody was diluted to 2 ⁇ M and serially diluted 5-fold over eight steps (maximum concentration: 1 ⁇ M). 100 ⁇ L/well of the antibody was added to a 96-well plate and mixed with the 293-huTROP2 cells.
  • Jurkat cells which display a high TCR on their cell surface, were used as target cells.
  • the cells were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded.
  • the cells were resuspended in PBS and diluted to a density of 1 ⁇ 106 cells/mL.
  • 100 ⁇ L/well of the solution was added to a 96-well plate.
  • the antibody was diluted to 1 ⁇ M and serially diluted 5-fold over eight steps (maximum concentration 500 nM).
  • 100 ⁇ L/well of the solution was added to a 96-well plate and mixed thoroughly with the Jurkat cells. The cells were incubated at 4°C for 30 minutes.
  • the cells were washed twice with PBS to remove unbound antibody. Then, 100 ⁇ L/well of goat anti-human IgG-PE was added and incubated at 4°C for 30 minutes. The cells were centrifuged at 300 g for 5 minutes and washed twice with PBS to remove unbound secondary antibody. Finally, the cells were resuspended in 200 ⁇ L of PBS, and antibody binding to the cells was detected using a Beckman Coulter CytoFLEX flow cytometer. The data were analyzed using GraphPad Prism software.
  • the experimental results are shown in FIG24 .
  • the EC50 (nM) of TPt0019, TPt0025, TPt0042 and TPb0043 binding to Jurkat cells were 6.15, 7.08, 445.6 and 35.8, respectively.
  • Example 39 FACS detection of the binding of TROP2 antibody-weakened anti-TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody to 293-huTROP2 target cells
  • 293-huTROP2 cells which display the extracellular domain of human TROP2 on their cell surface, were used as target cells.
  • the cells were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded.
  • the cells were resuspended in PBS and diluted to a density of 1 ⁇ 106 cells/mL. 100 ⁇ L/well of the solution was added to a 96-well plate.
  • Each antibody was serially diluted fivefold starting at 200 nM, and 100 ⁇ L/well of the solution was added to a 96-well plate and mixed evenly with the 293-huTROP2 cells.
  • the cells were incubated at 4°C for 30 minutes.
  • Example 40 FACS detection of the binding of anti-TROP2 ⁇ CD3 bispecific antibody and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibody weakened by TROP2 antibody to tumor target cells
  • BxPC3, SW403, and COLO 205 tumor cells were used as target cells. They were washed three times with PBS, centrifuged at 300 g for 5 minutes each time, and the supernatant discarded. The cells were resuspended in PBS and diluted to a density of 1 ⁇ 106 cells/mL. 100 ⁇ L/well of each solution was added to a 96-well plate. Each antibody was serially diluted fivefold starting at 800 nM, and 100 ⁇ L/well of each solution was added to the 96-well plate and mixed thoroughly. The cells were incubated at 4°C for 30 minutes. The cells were washed twice with PBS to remove unbound antibody.
  • the purity of TPt0019/TPt0042/TPt0025 in the histidine system (pH 5.5-6.5), the citric acid system (pH 5.0-5.5), and PBS at 4°C for 28 days was above 97%.
  • the purity of the PBS group decreased more rapidly than that of the other groups, but the overall value was above 95%.
  • the overall purity of TPb0043 samples decreased significantly after the solution change, except for the original PBS system, indicating poor stability.
  • the purity of TPb0059 decreased to below 90% after 28 days at 4°C, and there was no significant change in purity after 14 days at 40°C.
  • Table 48 show that after 28 days at 4°C, the average particle size and PI (dispersity index) of TPt0019 in each buffer system gradually increased.
  • the average particle size of TPt0042 in each buffer system did not increase significantly, with most PIs ⁇ 0.3.
  • TPt0025 had a PI ⁇ 0.3 in all buffer systems, indicating good particle size uniformity.
  • the average particle size of TPb0043 increased significantly, with two groups of PIs >0.3, indicating a polydisperse state with aggregates.
  • TPb0059 had a PI ⁇ 0.2, indicating good particle size uniformity. After 14 days at 40°C, TPb0043 and TPb0059 showed a clear tendency to aggregate.
  • Figures 27 to 41 show that after 7 and 14 days at 40°C, no obvious degradation bands were observed for TPt0019, TPt0025, TPt0042, TPb0043, and TPb0059 in various buffer systems.
  • Figures 42 to 51 show that after five freeze-thaw cycles, no obvious degradation bands were observed for TPt0019, TPt0025, TPt0042, TPb0043, and TPb0059.
  • SDS-PAGE revealed a band slightly larger than the monomer for purified TPt0025 and TPb0043, which was confirmed by mass spectrometry to be a hole-hole dimer.
  • Tpt0019 and TPt0042 performed better than TPb0043 in terms of purity, thermal stability, and colloidal stability.
  • Example 42 Study on the TDCC killing activity of anti-TROP2 ⁇ CD3 dual antibody and TROP2 ⁇ CD3 ⁇ CD28 triple antibody against BxPC3, MDA-MB-468, NCI-N87 and other tumor cells and negative HEK293 cells
  • TROP2 is expressed on the surface of tumor cells.
  • Anti-TROP2 ⁇ CD3 bispecific antibodies and TROP2 ⁇ CD3 ⁇ CD28 trispecific antibodies can exert strong T-cell dependent cellular cytotoxicity (TDCC), thereby specifically killing tumor cells.
  • TDCC T-cell dependent cellular cytotoxicity
  • This experiment used TROP2-high-expressing cells 293-huTROP2, BxPC3, MDA-MB-468, and NCI-N87; TROP2-intermediate-expressing MDA-MB-231; TROP2-low-expressing cells DLD-1, Colo-205, SW403, and T84; and negative cells HEK293 as target cells.
  • Tumor cells in the logarithmic growth phase were digested with trypsin (Source Culture, Cat#S310KJ) and resuspended in phenol red-free 1640 medium (Source Culture, Cat#L230KJ) supplemented with 2% FBS (Gibco, Cat#10091148). The cell density was adjusted to 2 ⁇ 105 cells/mL. Tumor cells were added to a 96-well U-bottom plate (NEST, Cat#701101) at a density of 1 ⁇ 104 cells/well, with 50 ⁇ L added to each well. The cells were allowed to adhere overnight. Test antibodies were then added at varying concentrations diluted in phenol red-free 1640 medium supplemented with 2% FBS.
  • the starting working concentration of the test antibody was 30 nM or 300 nM (3X working concentration), and a ten-fold serial dilution was performed, totaling 7-9 different concentrations, with 50 ⁇ L added to each well.
  • Effector Pan T cells were isolated from commercially purchased PBMCs (Sai Li Biotechnology, Donor ID#XW0801211W) using the EasySep TM Human T Cell Isolation Kit (Stemcell, Cat#17951) according to the kit's instructions. Cells were resuspended in phenol red-free 1640 medium supplemented with 2% FBS, and the cell concentration was adjusted to 1 ⁇ 106 cells/mL using an E:T ratio of 5:1. 5 ⁇ 104 cells/50 ⁇ L were added to each well.
  • Frozen PBMCs can be thawed one day in advance and cultured overnight in RPMI 1640 (Source Cell, Cat#L210KJ) supplemented with 10% FBS. DNase was added as appropriate to prevent DNA entanglement in dead cells. The 96-well U-bottom plate was then incubated in a 37°C, 5% CO2 incubator for 24 hours. Two hours before the end of the incubation period, 10 ⁇ Lysis buffer was added to the target cell-only wells and incubated for an additional hour. Centrifuge the culture plate at 300g for 5 minutes and transfer 50 ⁇ L of supernatant to a 96-well plate.
  • Example 43 Study on the Activation Effect of Anti-TROP2 ⁇ CD3 Bispecific Antibodies and TROP2 ⁇ CD3 ⁇ CD28 Trispecific Antibodies on T Cells in the TDCC Killing Assay of Tumor Cells Such as BxPC3, MDA-MB-468, NCI-N87, and MDA-MB-231 and Negative Cells HEK293
  • the 96-well U-bottom plate (NEST, Cat#701101) was removed and centrifuged at 450 g for 5 minutes, and the supernatant was discarded. Then, a staining buffer (PBS + 2% FBS + 5mM EDTA) was used to prepare a staining solution containing Brilliant Violet 785 TM anti-human CD3 Antibody (BioLegend, Cat#344842), Brilliant Violet 605 TM anti-human CD4 Antibody (BioLegend, Cat#344646), Brilliant Violet 421 TM anti-human CD8 Antibody (BioLegend, Cat#344748), BD Pharmingen TM PE Mouse Anti-Human CD25 (BD, Cat#555432), and BD Pharmingen TM APC Mouse Anti-Human CD69 (BD, Cat#555533) and other flow cytometry antibody solutions (panel as shown in Table 49) were prepared. The cells were resuspended with the prepared flow cytometry
  • T cell activation in the TROP2-positive cell killing assay was TPt0019 ⁇ TPt0025>TPb0043+TPt0059>TPt0042 ⁇ TPb0043.
  • TROP2-negative HEK293 cells TPt0019>TPt0025 resulted in a small amount of non-specific T cell activation, while no T cell activation was detected with TPt0042, TPb0043, or TPb0043+TPt0059.
  • the 96-well U-bottom plate (NEST, Cat# 701101) was removed and centrifuged at 450 g for 5 minutes to collect the cell supernatant.
  • the release levels of various cytokines in the TDCC reaction supernatant were then measured according to the CBA reagent instructions.
  • the standard beads were transferred to a centrifuge tube and dissolved in diluent.
  • IL-2 BD TM Cytometric Bead Array (CBA) Human IL-2 Flex Set, BD, Cat#558270
  • TNF-a BD TM Cytometric Bead Array (CBA) Human TNF Flex Set, BD, Cat#560112
  • IFN- ⁇ BD TM Cytometric Bead Array
  • IL-6 Human IL-6 Flex Set, BD 558276
  • microspheres were vortexed thoroughly before mixing and then aliquoted into a 96-well plate, with 50 ⁇ l per well. Dilute the supernatant sample according to the experimental requirements and prepare the standard according to the kit instructions. Add the diluted standard and sample to a 96-well plate, mix thoroughly with the previously added microsphere mixture, and shake on a shaker at 500 rpm for 5 minutes. Incubate in the dark for 2 hours. Detection antibodies for IL-2, TNF- ⁇ , IFN- ⁇ , and IL-6 were mixed in a 1:1 ratio and aliquoted into a 96-well plate, 50 ⁇ l per well. Shake on a shaker at 500 rpm for 5 minutes, and incubate in the dark for 1 hour.
  • the starting working concentration of the test antibody was 300 nM (3X the working concentration), and a ten-fold serial dilution was performed, resulting in nine different concentrations. 50 ⁇ L was added to each well.
  • Effector Pan T cells were isolated from commercially purchased PBMCs (Sai Li Biotechnology, Donor ID#XW0801211W) using the EasySep TM Human T Cell Isolation Kit (Stemcell, Cat#17951) according to the kit's instructions. Cells were resuspended in phenol red-free 1640 medium supplemented with 2% FBS, and the cell concentration was adjusted to 1 ⁇ 106 mL using an E:T ratio of 5:1. 5 ⁇ 104 cells/50 ⁇ L were added to each well.
  • Table 51 Grouping and Dosage Regimen N Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 ⁇ L/g body weight.
  • TGI% The relative tumor inhibition rate
  • TGI% (1-T/C) ⁇ 100%.
  • mice with tumor volumes ranging from 192.34 to 399.81 mm3 were randomly divided into three groups of 5 mice each based on tumor volume and body weight for the regimen described in Table 55.
  • mice with tumor volumes ranging from 411.35 to 592.92 mm3 were randomly divided into three groups of 5 mice each based on tumor volume and body weight for the regimen described in Table 56. Dosing began on Day 0.
  • TV (length ⁇ width 2 )/2.
  • the experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202312280152.
  • MDA-MB-231 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS. 5 ⁇ 10 6 MDA-MB-231 cells (100 ⁇ L) of PBS were mixed with an equal volume of Matrigel and inoculated subcutaneously in the axilla of the right forelimb of mice in a 0.2 ml inoculation volume.
  • TGI% The relative tumor inhibition rate
  • TGI% (1-T/C) ⁇ 100%.
  • TGI% (1-T/C) ⁇ 100%.
  • NCI-H292 cells were routinely subcultured for subsequent in vivo experiments. Cells were harvested by centrifugation and resuspended in PBS. 5 ⁇ 10 6 NCI-H292 cells in 100 ⁇ L of PBS were mixed with an equal volume of Matrigel and inoculated subcutaneously in the axilla of the right forelimb of mice in a volume of 0.2 ml.
  • mice When tumors grew to an average size of approximately 100-200 mm3 , 15 tumor-bearing mice were randomly divided into three groups of five mice each based on tumor volume and body weight. Ten days before grouping, 1 ⁇ 107 PBMCs were injected via the tail vein. The day of grouping was designated Day 0, and dosing began. The groupings and dosing schedule are shown in Table 63.
  • mice The body weight and tumor volume of mice were measured twice a week, as shown in Figures 103A and 103B.
  • the relative tumor inhibition rate (TGI%) was calculated on Day 28 using the following formula:
  • TV (length ⁇ width 2 )/2.
  • the tumor inhibition rate results are shown in Table 64.
  • the TPt0042 and TPb0043 groups significantly inhibited tumor growth compared to the vehicle group, with tumor inhibition rates of 100.00% and 100.00%, respectively.
  • the body weight of the mice was also measured, and as shown in Figure 103B, no significant differences in mouse body weight were observed.
  • the experiment used SPF-grade female NCG mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), and the animal certificate number was NO.B202310070147.
  • each mouse was inoculated subcutaneously with 5 ⁇ 106 NCI-N87 cells on the right flank.
  • 5 ⁇ 106 PBMCs were injected via the tail vein. This grouping date was designated Day 0.
  • Dosing began on Day 1, the second day of grouping. The grouping and dosing schedule are shown in Table 65.
  • Tumor volume and mouse body weight were measured twice weekly. The results are shown in Figures 104A and 104B.
  • the relative tumor inhibition rate (TGI%) was calculated on Day 28 using the following formula:
  • TGI% (1-T/C) ⁇ 100%.
  • TV (length ⁇ width 2 )/2.
  • Table 65 Grouping and Dosage Regimen N Number of animals in each group. Dosage volume: The administration volume for animals was adjusted to 10 ⁇ L/g body weight.
  • the experiment used SPF-grade female Balb/c mice (18-25 g, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with the certificate number NO.A202312070173.
  • mice After the animals were released from quarantine, they were divided into two groups of three mice each. Each mouse was intravenously injected with 1 mg/kg TPt0042, and then blood was collected at the time points shown in Table 67.
  • Recombinant TROP2-HIS protein was diluted to 2 ⁇ g/ml in PBS and added to an ELISA plate at 100 ⁇ l/well (coated with an equal amount of BSA as a control) and incubated at 4°C overnight. The coating solution was removed, and blocking solution was added at 200 ⁇ l/well. The plates were incubated at room temperature for 2 hours. The blocking solution was removed, and the plates were washed three times with 250 ⁇ l/well of 0.5 ⁇ PBST. Serum samples were then diluted in blocking solution.
  • Three dilutions were set: 800x, 1600x, and 3200x at 5 min, 30 min, 1 hour, 3 hours, and 6 hours, D1, D2, D3, D4, and D5; and three dilutions were set: 200x, 400x, and 800x at D7, D14, and D25.
  • the positive control, TPt0042 was diluted two-fold starting at 1 nM to form a six-point concentration gradient (maximum concentration 1 nM). The plate was then added to the blocked ELISA plate at 100 ⁇ l/well and incubated at room temperature for 1 hour.
  • the plate was washed three times with 0.5 ⁇ PBST, and HRP-labeled goat anti-human IgG antibody was added at 100 ⁇ l/well and incubated at room temperature for 45 minutes.
  • the plate was washed five times with 0.5 ⁇ PBST, and TMB was added at 100 ⁇ l/well.
  • the plate was incubated at room temperature in the dark for 5 minutes.
  • the substrate color development reaction was terminated by adding 100 ⁇ l/well stop solution.
  • the OD value at 450 nm was read using a microplate reader.
  • the data were analyzed using GraphPad, and graphs were constructed and half-life (T1/2) was calculated.
  • the PK results are shown in Figure 105.
  • the half-life T1/2 of TPt0042 is 152.2h.
  • mice SPF-grade female N PG mice (18-25g, purchased from Beijing Weitongda Biotechnology Co., Ltd.). After the animals were out of quarantine, each animal was injected with 1 ⁇ 10 7 PBMC (Sai Li Bio, donor: XW0801187W) through the tail vein to construct a humanized animal model. Three days after PBMC inoculation, each mouse was subcutaneously inoculated with 5 ⁇ 10 6 BxPC3 cells on the right side. Seven days after tumor inoculation, the average tumor volume reached about 170 mm 3. 40 tumor-bearing mice were randomly divided into 8 groups based on tumor volume and body weight, with 5 mice in each group. The day of grouping was defined as Day 0. Drug administration began on the day of grouping. The grouping situation and dosing schedule are shown in Table 68.
  • Tumor volume and mouse body weight were measured twice weekly. The results are shown in Figures 106A and 106B.
  • the tumor growth inhibition rate (TGI%) was calculated using the following formula:
  • TGI% (1-T/C) ⁇ 100%.
  • mice in the TPt0051 0.03 mg/kg and TPt0052 0.03 mg/kg groups showed a downward trend, which may be related to the activation and expansion of T cells.
  • mice SPF-grade female N PG mice (18-25g, purchased from Beijing Weitongda Biotechnology Co., Ltd.). After the animals were released from quarantine, each animal was injected with 1 ⁇ 10 7 PBMC (Miaoshun Bio, donor: P123041110C) through the tail vein to construct a humanized animal model.
  • PBMC Human PBMC
  • PBMC Human PBMC
  • BxPC3 cells BxPC3 cells on the right side.
  • tumor inoculation the average tumor volume reached about 85 mm 3. 25 tumor-bearing mice were randomly divided into 5 groups based on tumor volume and body weight, with 5 mice in each group. The day of grouping was defined as Day 0. Drug administration began on the day of grouping. The grouping situation and dosing schedule are shown in Table 70.
  • Tumor volume and mouse body weight were measured twice weekly. The results are shown in Figures 107A and 107B.
  • the relative tumor inhibition rate (TGI%) was calculated using the Day 25 data as follows:
  • TGI% (1-T/C) ⁇ 100%.
  • TV (length ⁇ width 2 )/2.
  • mice in each group showed a downward trend, which may be related to GVHD after PBMC reconstitution.
  • mice SPF-grade female hTrop2/hCD3E humanized mice (18-25 g, purchased from Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.) were used. After the animals were released from quarantine, eight mice were randomly divided into four groups based on body weight, with two mice in each group. The day of grouping was defined as Day 0. Dosing began on the same day of grouping. The grouping and dosing schedule are shown in Table 72.
  • mice The weight and status of the mice were monitored regularly after administration, and the results are shown in Figure 108: at a dose of 0.1 mg/kg for antibody TPt0042, the weight of the mice showed a downward trend from Day 1 to Day 5, and then the weight recovered; at a dose of 1 mg/kg for antibody TPt0042, the weight of the mice showed a downward trend from Day 1 until all died on Day 4; at a dose of 1 mg/kg for antibody TPt0047, the weight of the mice showed a downward trend from Day 0 to Day 2, and then the weight recovered; at a dose of 10 mg/kg for antibody TPt0047, the weight of the mice showed a downward trend from Day 1 to Day 5, and then the weight recovered, which demonstrated that the mouse tolerance dose of TPt0047 antibody was higher than that of TPt0042 antibody.
  • a single male rhesus monkey received repeated intravenous injections of the TROP2xCD3 bispecific antibody TPt0047. Doses were administered four times on Days 1, 8, 15, and 22 (doses of 0.05 mg/kg, 0.15 mg/kg, 0.5 mg/kg, and 3 mg/kg, respectively). No abnormal clinical symptoms or weight changes were observed. However, increases in basophil percentage (BASO%), absolute eosinophil count (EO#), reticulocyte count (RET%/RET#), lactate dehydrogenase (LDH), and gamma-glutamyl transpeptidase (GGT) were observed.
  • BASO% absolute eosinophil count
  • RET%/RET# reticulocyte count
  • LH lactate dehydrogenase
  • GTT gamma-glutamyl transpeptidase

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Abstract

L'invention concerne un anticorps multi-spécifique ou un fragment de liaison à l'antigène associé, un polynucléotide isolé codant pour l'anticorps ou le fragment de liaison à l'antigène associé, une composition pharmaceutique comprenant l'anticorps ou le fragment de liaison à l'antigène associé, et son utilisation.
PCT/CN2025/087399 2024-04-06 2025-04-07 Activateur de lymphocytes t à anticorps multi-spécifiques Pending WO2025209593A1 (fr)

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