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WO2023088295A1 - Anticorps multi-spécifique et utilisation pharmaceutique associée - Google Patents

Anticorps multi-spécifique et utilisation pharmaceutique associée Download PDF

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Publication number
WO2023088295A1
WO2023088295A1 PCT/CN2022/132228 CN2022132228W WO2023088295A1 WO 2023088295 A1 WO2023088295 A1 WO 2023088295A1 CN 2022132228 W CN2022132228 W CN 2022132228W WO 2023088295 A1 WO2023088295 A1 WO 2023088295A1
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Prior art keywords
antibody
cell
binding
multispecific antibody
seq
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English (en)
Chinese (zh)
Inventor
杨翠青
邵小慧
张韫
付雅媛
曹卓晓
唐任宏
任晋生
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Jiangsu Simcere Pharmaceutical Co Ltd
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Jiangsu Simcere Pharmaceutical Co Ltd
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Priority to CN202280088186.3A priority Critical patent/CN118556077A/zh
Publication of WO2023088295A1 publication Critical patent/WO2023088295A1/fr
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present invention relates to the field of biomedicine, in particular to a multispecific antibody and its medicinal use.
  • the disclosure of the present invention provides a multispecific antibody, a nucleic acid fragment encoding the multispecific antibody, a vector, a cell, a composition, a preparation method, a pharmaceutical use and a treatment method thereof.
  • the present invention provides a multispecific antibody comprising at least three parts: (A) a target antigen binding part, (B) a half-life extending part, and (C) a T cell engaging part;
  • Target antigen-binding portion preferably a target antigen-binding antibody or a target antigen-binding ligand; the target antigen-binding antibody may be selected from any antigen-binding fragment;
  • (B) half-life extension moiety preferably anti-HSA antibody
  • T cell engaging portion wherein the T cell engaging portion is preferably an anti-CD3 antibody or an antigen-binding fragment;
  • the antigen-binding fragment is preferably Fd, Fv, scFv, diabody or single domain antibody (VHH);
  • the fragments of the three parts (A), (B) and (C) can be connected through a linker, or can be directly connected without a linker.
  • said multispecific antibody wherein the (A) target antigen binding portion or (C) T cell engaging portion can be repeated or comprise multiple portions;
  • the multispecific antibody comprises two target antigen-binding portions, A1 and A2, and A1 and A2 may be the same or different; in some embodiments, A1 and A2 bind different antigen targets; in some embodiments In the scheme, A1 and A2 bind different epitopes of the same antigen target;
  • portion (C) is a scFv that binds CD3; still in some embodiments, portion (C) is a scFv that binds human CD3.
  • the structural order of the multispecific antibody from the N-terminal to the C-terminal is:
  • said multispecific antibody (C) T cell engaging portion comprises complementarity determining regions (CDRs) selected from the following antibodies: OKT3, TRX4, MGA031, Nuvion, SP34, X35, VIT3, BMA030, CLB -T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2 -8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, S004-2-03, S004-2-06, S004-2-08, S004-2 -10, S004-2-18, 6-35.22-hu, 1-22.6-1-hu, 7-35.6-hu, and 6-44.5-hu;
  • CDRs complementarity determining regions
  • the (C)T cell engaging moiety comprises heavy chain CDRs and/or light chain CDRs selected from:
  • the heavy chain CDR1 is shown in SEQ ID NO.34, 39, 42, 47;
  • the heavy chain CDR2 is shown in SEQ ID NO.35, 40, 43, 45, 48;
  • the heavy chain CDR3 is shown in SEQ ID NO.36, 37, 38, 41, 44, 46, 49;
  • the light chain CDR1 is shown in SEQ ID NO.50, 55, 58, 61, 64;
  • the light chain CDR2 is shown in SEQ ID NO.51, 56, 59, 62, 65;
  • the light chain CDR3 is shown in SEQ ID NO.52, 53, 54, 57, 60, 63, 66;
  • the (C) T cell engaging portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% of the above heavy chain CDRs and/or light chain CDRs % identity sequence.
  • the target antigen of the multispecific antibody (A) part can be selected from the following group: CD19, BCMA, HER2, EGFR, VEGF, MSLN, CD33, CD70, CD5, CD20, CD40, CD47, CD38, CD137, TNF-alpha, HER3, CD27, EphA2, EpCAM, MUC1, MUC17, CEA, Claudin18.2, folate receptor, Claudin6, WT1, NY-ESO-1, MAGE3, ASGPR1, CDH16, GPRC5D, DLL3, ROR1, or GUCY2C;
  • the multispecific antibody comprises two target antigen-binding portions, A1 and A2, and A1 and A2 respectively bind to different epitopes of MSLN; for example, MLSN-R1 epitope or MSLN-R3 epitope.
  • the multispecific antibody comprises two target antigen-binding portions, A1 and A2, and A1 and A2 bind target antigens CD70 and CD33, respectively.
  • the target antigen binding portion comprises complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO.18-28, 103-106;
  • the (A) target antigen binding portion comprises heavy chain CDRs and/or light chain CDRs selected from:
  • the heavy chain CDR1 is shown in SEQ ID NO.76, 80, 89, 92, 110, 113, 116;
  • the heavy chain CDR2 is shown in SEQ ID NO.77, 79, 81, 90, 93, 111, 114, 117;
  • the heavy chain CDR3 is shown in SEQ ID NO.78, 82, 91, 94, 112, 115, 118;
  • the light chain CDR1 is shown in SEQ ID NO.83, 86, and 107;
  • the light chain CDR2 is shown in SEQ ID NO.84, 87, 108;
  • the light chain CDR3 is shown in SEQ ID NO.85, 88, 109;
  • the (A) target antigen binding portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% of the above heavy chain CDRs and/or light chain CDRs % identity sequence.
  • the multispecific antibody (B) part comprises complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO.15-17;
  • said portion (B) comprises CDRs selected from the group consisting of:
  • CDR1 is shown in SEQ ID NO.67, 70, 73;
  • CDR3 is shown in SEQ ID NO.69, 72, 75;
  • said portion (B) comprises sequences that are 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% identical to the above-mentioned CDRs.
  • the multispecific antibody uses a linker to connect the fragments of the three parts (A), (B), and (C); each of the linkers is independently selected from: (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, (GGGGS)n or (GGGGS)n(GGGS)n, where n is 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10;
  • the linker is GGGGS (Linker1), GGGGSGGGS (Linker2), GGGGSGGGGS (Linker3), GGGGSGGGGSGGGGS (Linker4), or GGGGSGGGGSGGGGSGGGGS (Linker5).
  • the structural order of the multispecific antibody from the N-terminus to the C-terminus is:
  • the antibody or antigen-binding fragment used in the multispecific antibody is:
  • the multispecific antibody comprises the sequence shown in SEQ ID NO.97, 98, 99, 119, 120, 121, or 122, or has 99%, 98%, 97%, 96 %, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identical sequences.
  • the present invention provides an isolated nucleic acid fragment encoding the multispecific antibody described in the first aspect of the present invention.
  • the present invention provides a vector comprising the nucleic acid fragment described in the second aspect of the present invention.
  • the present invention provides a host cell comprising the vector described in the third aspect of the present invention; in some embodiments, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterium (Escherichia coli) , fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • a prokaryotic cell such as a bacterium (Escherichia coli) , fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the present invention provides a method for preparing the multispecific antibody of the present invention, characterized in that the method includes culturing the cell described in the fourth aspect, and isolating the multispecific antibody expressed by the cell.
  • the present invention also provides a pharmaceutical composition, the pharmaceutical composition comprising the multispecific antibody described in the first aspect of the present invention, or the nucleic acid fragment described in the second aspect of the present invention, or the third aspect of the present invention
  • the carrier described in the aspect; or the product prepared by the method described in the fourth aspect of the present invention; optionally, the pharmaceutical composition also includes a pharmaceutically acceptable carrier (carrier), diluent or adjuvant; optionally , the pharmaceutical composition further comprises an additional antineoplastic agent.
  • the present invention provides a method for preventing and/or treating proliferative diseases, tumor diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergies, parasitic reactions, transplants
  • a method for host-versus-graft disease or host-versus-graft disease comprising administering an effective amount of the multispecific antibody described in the first aspect of the present invention, or the nucleic acid fragment described in the second aspect of the present invention, or the present invention to a patient in need thereof.
  • the tumor disease is a solid tumor expressing MSLN, CD70 and/or CD33 or a hematological tumor expressing MSLN, CD70 and/or CD33; in some embodiments, the tumor disease is mesothelioma , lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer, pleural cancer, cholangiocarcinoma, cervical cancer, gastric cancer, leukemia, histiocytic lymphoma, or renal cancer; in some embodiments, the neoplastic disease is epithelioid Malignant pleural mesothelioma, lung adenocarcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, monocytic leukemia, histiocytic lymphoma, clear cell adenocarcinoma of the kidney, T lymphocytic leukemia, or acute myeloid leukemia.
  • the present invention provides the multispecific antibody described in the first aspect, or the nucleic acid fragment described in the second aspect of the present invention, or the vector described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention
  • the diseases include proliferative diseases, tumor diseases, inflammatory diseases, immune disorders, autoimmune immune disease, infectious disease, viral disease, allergy, parasitic reaction, graft-versus-host disease or host-versus-graft disease;
  • the tumor disease is a solid tumor expressing MSLN, CD70 and/or CD33 or a hematological tumor expressing MSLN, CD70 and/or CD33; in some embodiments, the tumor disease is mesothelioma , lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer, pleural cancer, cholangiocarcinoma, cervical cancer, gastric cancer, leukemia, histiocytic lymphoma, or renal cancer; in some embodiments, the neoplastic disease is epithelioid Malignant pleural mesothelioma, lung adenocarcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, monocytic leukemia, histiocytic lymphoma, clear cell adenocarcinoma of the kidney, T lymphocytic leukemia, or acute myeloid leukemia.
  • compositions including A and B should be understood as the following technical scheme: a composition composed of A and B, and a composition containing other components in addition to A and B, all fall into Into the scope of the aforementioned "a composition”.
  • KD equilibrium dissociation constant
  • high affinity usually refers to having about 10-7 M or lower, about 10-8 M or lower, about 1 ⁇ 10-9 M or lower, about 1 ⁇ 10-10 M or lower, KD of 1 ⁇ 10 -11 M or lower or 1 ⁇ 10 -12 M or lower.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
  • antigen binding molecule is used herein in the broadest sense to refer to a molecule that specifically binds an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimic refers to an organic compound or binding domain that can specifically bind to an antigen, but has nothing to do with the structure of an antibody.
  • antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical "four-chain antibody”, which belongs to the immunoglobulins composed of two heavy chains (HC) and two light chains (LC); In the N-terminal to C-terminal direction, it consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain a heavy chain constant region CH2 domain
  • CH3 domain heavy chain constant region
  • the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain
  • the light chain is
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • IgA2 Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibody herein also includes “antigen-binding fragment” or “antibody fragment”, and “antigen-binding fragment” and “antibody fragment” are used interchangeably herein. Partial or partial variants that possess the ability to bind antigen. Including but not limited to Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabody and single domain antibody.
  • Papain digestion of intact antibodies yields two identical antigen-binding fragments, termed "Fab” fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is a Fab' fragment in which the cysteine residue of the constant domain bears a free thiol group. Pepsin treatment yields an F(ab')2 fragment with two antigen-combining sites (two Fab fragments) and part of the Fc region.
  • Fd refers to an antibody consisting of VH and CH1 domains.
  • Fv refers to an antibody fragment consisting of a single-armed VL and VH domain.
  • the Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (such as the Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although perhaps with a lower affinity than the full binding site.
  • scFv single-chain variable fragment
  • scFv single-chain variable fragment
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immunol.31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.
  • diabody herein, whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thus forcing the domains to The complementary domains of the other chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • single domain antibody single domain antibody, sdAb
  • VHH single domain antibody
  • nanobody the variable region of the heavy chain of a cloned antibody, constructed by only one A single domain antibody composed of the heavy chain variable region, which is the smallest fully functional antigen-binding fragment.
  • CH1 light chain and heavy chain constant region 1
  • Single domain antibodies can be derived from camelid heavy chain antibodies or cartilaginous fish IgNARs.
  • Antibody herein also includes antibodies that do not comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca (The heavy-chain antibodies (HCAbs) produced by Vicugna pacos) and the new immunoglobulin antigen receptor (Ig new antigen receptor, IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs heavy-chain antibodies
  • Ig new antigen receptor Ig new antigen receptor
  • an “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).
  • Antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibody, chimeric antibody, humanized antibody or fully human antibody.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variants (such as containing naturally occurring mutations or arising during the manufacture of a formulation, such variants typically appear as In addition to being present in small amounts), the individual antibodies comprising the population are identical and/or bind the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), in monoclonal antibody preparations each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies can be produced by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology and the use of transgenic animals containing all or part of the human immunoglobulin loci methods and other methods known in the art.
  • natural antibody herein refers to antibodies produced and paired by the immune system of a multicellular organism.
  • engineered antibody herein refers to a non-natural antibody obtained through genetic engineering, antibody engineering and other techniques.
  • engineered antibody includes humanized antibodies, small molecule antibodies (such as scFv, etc.), specific antibodies, etc.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
  • multispecific antibody herein refers to an antibody having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen. bit binding.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • full-length antibody intact antibody
  • intact antibody intact antibody
  • naked antibody refers to an antibody that is not conjugated to a therapeutic agent or tracer
  • conjugated antibody refers to an antibody conjugated to a therapeutic agent or tracer
  • Chimeric antibody herein refers to an antibody whose light chain and/or heavy chain are partly derived from an antibody (which may be derived from a specific species or belong to a specific class or subclass of antibodies). class), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the Binding activity to target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).
  • a primary antibody e.g., a murine antibody
  • the light chain constant region is from a second antibody (eg, a human antibody).
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region in the heavy or light chain of an antibody that is involved in making the antibody bind to an antigen
  • “heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • “light chain variable region” can be used interchangeably with “VL” and “LCVR”.
  • the variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). It can form a precise complementarity with the antigen epitope, so it is also called complementarity determining region.
  • the CDR of the variable region of the heavy chain can be abbreviated as HCDR
  • the CDR of the variable region of the light chain can be abbreviated as LCDR.
  • framework region or “FR region” are used interchangeably and refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs.
  • CDRs For further descriptions of CDRs, refer to Kabat et al., J.Biol.Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); People such as Chothia, J.Mol.Biol.196:901-917 (1987); People such as Al-Lazikani B., J.Mol.Biol., 273:927-948 (1997); People such as MacCallum, J.Mol .Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P.
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol. 27:55-77, 2003.
  • IMGT ImMunoGeneTics information system
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • the "heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • Heavy chain constant region includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • a typical "heavy chain constant region segment" can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxy-terminal part of the antibody obtained by papain hydrolysis of the whole antibody, which typically includes the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region can comprise or excluding Lys447.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the parameter set (and one that should be used unless otherwise stated) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, eg to identify other family member sequences or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • CD3 cluster of differentiation 3
  • the term "CD3" herein refers to a cluster of differentiation 3 protein derived from any vertebrate source, including mammals, such as primates (such as humans, monkeys) and rodents (such as mice and rats). mouse).
  • the CD3 molecule is a multiprotein complex of six chains, including: a CD3 ⁇ chain, a CD3 ⁇ chain, a homodimer of two CD3 ⁇ chains, and a CD3 ⁇ chain, where the CD3 ⁇ chain is the intracellular tail of the CD3 molecule, and
  • the CD3 gamma, CD3 delta and CD3 epsilon chains all contain an extracellular domain (ECD) expressed on the surface of T cells.
  • Exemplary sequences of human CD3 include human CD3 ⁇ protein (NCBI Ref Seq No. NP_000724 or NCBI: AAH49847.1), human CD3 ⁇ protein (NCBI Ref Seq No. NP_000723) and human CD3 ⁇ protein (NCBI Ref Seq No. NP_000064).
  • Exemplary sequences of non-human CD3 include Macaca fascicularis (monkey) CD3 ⁇ protein (NCBI Ref Seq No. NP_001270544), cynomolgus monkey (Macaca fascicularis) (monkey) CD3 ⁇ protein (NCBI Ref SeqNo.
  • NP_001274617 Crab monkey (Macaca fascicularis) (monkey) CD3 ⁇ protein (NCBI Ref Seq No.NP_001270839); mouse CD3 ⁇ protein (NCBI Ref Seq No.NP_031674), mouse CD3 ⁇ protein (NCBI Ref SeqNo.NP_038515), mouse CD3 ⁇ protein ( NCBI Ref Seq No.AAA37400); Rattus norvegicus (rat) CD3 ⁇ protein (NCBI Ref Seq No.NP_001101610), Rattus norvegicus (rat) CD3 ⁇ protein (NCBI Ref Seq No.NP_037301), Rattus norvegicus (rat ) CD3 ⁇ protein (NCBI Ref Seq No.NP_001071114).
  • CD3 ⁇ is intended to encompass any form of CD3 ⁇ subunit, for example, 1) a native unprocessed CD3 ⁇ molecule, a "full length” CD3 ⁇ chain or a naturally occurring CD3 ⁇ variant, including for example splice variants or alleles Variant; 2) any form of CD3 ⁇ produced by processing in the cell; or 3) full length, fragment (e.g. truncated form, ectodomain/transmembrane domain) or modification of the CD3 ⁇ subunit produced by recombinant means (e.g., mutated, glycosylated/pegylated, histidine-tagged/immunofluorescent fused forms).
  • recombinant means e.g., mutated, glycosylated/pegylated, histidine-tagged/immunofluorescent fused forms.
  • HSA human serum albumin, having a molecular weight of 67 kD. There are 17 disulfide bonds inside the HSA protein, which makes the whole protein have good stability. HSA has the advantages of prolonging the half-life and promoting the drug to pass through the blood-brain barrier. It is not easy to pass through the glomerulus, and the half-life in plasma is as long as 2 Zhou, which is widely distributed in the body and has no immunogenicity, is an ideal protein drug carrier.
  • CD70 herein, also known as “TNFSF7” or “CD27L”, is a member of the TNF ligand family and is a ligand of CD27 (also known as TNFRSF27).
  • CD70 herein includes mature or immature full-length wild-type CD70 protein or its mutants (such as point mutations, insertion mutations or deletion mutations), splice variants (splice variants), orthologs (Orthologs) and the aforementioned Fragment of CD70.
  • the "CD70” herein may be derived from humans, primates such as monkeys (eg rhesus monkeys, cynomolgus monkeys) and rodents such as mice and rats. Exemplarily, the amino acid sequence of human CD70 can be found in UniProt number: P32970, and the amino acid sequence of rhesus monkey CD70 can be found in UniProt number: F7GPA5.
  • CD33 herein is the smallest member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family.
  • Siglec immunoglobulin-like lectin
  • the terminal amino acid consists of a conserved V-set immunoglobulin-like domain and a variable C2-set domain, in which V-set specifically recognizes and binds to sialic acid; the cytoplasmic tail
  • immunoreceptor tyrosine-based inhibitory motif immunoreceptor tyrosine-based inhibitory motif, ITIM
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • CD33 includes CD33 proteins of any human and non-human animal species, and specifically includes human CD33 as well as CD33 of non-human mammals.
  • MSLN Mesothelin
  • MSLN Mesothelin
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies of the invention in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g.
  • An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) unwanted physiological changes or pathological changes in the subject of treatment, such as cancer, autoimmune diseases and viral infections. progress.
  • Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject refers to an organism receiving treatment for a particular disease or condition as described herein.
  • subjects and patients include mammals, such as humans, primate (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • autoimmune disease refers to a condition in which cells, tissues and/or organs are damaged by a subject's immune response to its own cells, tissues and/or organs.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • 2A-2C ELISA detection of the binding reaction between MSLN multispecific antibody and albumin: 2A. HAS; 2B. CSA; 2C. MSA.
  • 3A-3B ELISA detection of the binding reaction of MSLN multispecific antibody to MSLN protein and CD3 protein simultaneously: 3A. Human CD3; 3B. Monkey CD3.
  • 4A-4E FACS detection of the binding reaction of MSLN multispecific antibodies to tumor cells: 4A.OVCAR3; 4B.Hela; 4C.Hs766T; 4D.NCI-H292; 4E.A431.
  • FIG. 6A FACS detection of the binding reaction of MSLN multispecific antibody to HEK293T-monkey MSLN cells
  • Fig. 6B FACS detection of the binding reaction of MSLN multispecific antibody to HEK293T cells.
  • FIG. 7A FACS detection of the binding reaction of MSLN multispecific antibody to human T cells
  • Fig. 7B FACS detection of the binding reaction of MSLN multispecific antibody to monkey T cells.
  • FIG. 8A ⁇ 8D MSLN multispecific antibody reporter assay: 8A.OVCAR3; 8B.Hela; 8C.Hs766T; 8D.NCI-H292.
  • Figure 9 ELISA detection of the effect of CA125 on the binding activity of MSLN multispecific antibodies.
  • FIG. 10 FACS detection of the effect of CA125 on the binding activity of MSLN multispecific antibodies.
  • FIGS 11A-11D Binding of bi-epitope MSLN multispecific antibodies.
  • FIG. 12A Endocytic activity of 100 nM MSLN multispecific antibody
  • FIG. 12B Endocytic activity of 10 nM MSLN multispecific antibody
  • FIG. 12C Endocytic activity of 1 nM MSLN multispecific antibody.
  • MSLN multispecific antibody mediates T cell killing activity of tumor cells in vitro and cytokine detection.
  • Multispecific antibody inhibits humanized ovarian cancer tumor growth curve
  • Figure 16 The tumor growth curve of the mixed subcutaneous model of ovarian cancer cell OVCAR-3 and PBMC inhibited by multispecific antibodies.
  • Figure 17 Multispecific antibody inhibition of humanized lung cancer tumor growth curve.
  • Figure 18 Comparison of pharmacokinetics of multispecific antibodies in cynomolgus monkeys.
  • Figure 19A Stability testing of multispecific antibodies in human serum.
  • Figure 19B Stability testing of multispecific antibodies in cynomolgus monkey serum.
  • Fig. 20A FACS detection of the binding reaction of CD33 ⁇ CD70 multispecific antibody to THP-1 cells.
  • Fig. 20B FACS detection of the binding reaction of CD33 ⁇ CD70 multispecific antibody to U937 cells.
  • Fig. 20C FACS detection of the binding reaction of CD33 ⁇ CD70 multispecific antibody to 786-O cells.
  • Fig. 20D FACS detection of the binding reaction of CD33 ⁇ CD70 multispecific antibody to Jurkat cells.
  • Figure 21 FACS detection of the binding reaction of CD33 ⁇ CD70 multispecific antibody to CHO-K1-human CD33 cells.
  • Figure 22 FACS detection of the binding reaction of CD33 ⁇ CD70 multispecific antibody to CHO-K1-human CD70 cells.
  • FIG. 23 Antibody killing activity experiment on tumor cells in different formats.
  • FIG. 24 Antibody killing activity experiment on monocytes in different formats.
  • the multispecific antibody of the present invention can be a tri-specific (tri-specific) or tetra-specific (tetra-specific) antibody, and the specificity can refer to binding to different antigenic targets, or can refer to binding to the same antigenic target. different epitopes. Please refer to Figures 1A-1B and Table 1 for the schematic structure of the multispecific antibody.
  • the multispecific antibody contains at least three parts: (A) target antigen binding part, (B) half-life extension part, and (C) T cell joint part.
  • the (A) target antigen-binding portion can be selected from a target antigen-binding antibody or a target antigen-binding ligand, and the target antigen-binding antibody can be selected from any antigen-binding fragment, preferably Fd, Fv, scFv, diabody or single Domain antibody (VHH).
  • the (B) half-life extending moiety is preferably an anti-HSA antibody.
  • the three parts (A), (B) and (C) of the multispecific antibody can be arranged or connected in various ways, as shown in Figure 1A, wherein (A) target antigen binding part or (C) T
  • the cell-engaging part may appear repeatedly or contain multiple parts (for example, a multispecific antibody contains two target antigen-binding parts, A1 and A2, and A1 and A2 may be the same or different).
  • the connection between the various parts of the multispecific antibody may be directly connected through a linker or not through a linker.
  • the nucleotide sequences containing the amino acid sequences encoding the multispecific antibodies were respectively constructed as recombinant plasmids.
  • the schematic structure of the multispecific antibody is shown in Fig. 1B. Plasmid construction and antibody expression and purification were completed by Taizhou Baiying Biotechnology Co., Ltd.
  • a tag His tag (HHHHHH) was added to the C-terminus of multispecific antibodies; The antibody sequence of His tag optimizes the method of antibody expression and purification.
  • the sequences of multispecific antibodies used in animal experiments are shown in Table 4, and there is no His tag at the C-terminus.
  • plasmid and transfection reagent (Thermofisher, product number: A29133) to OptiPRO SFM (Thermofisher, product number: 12309019) and mix well, then let it stand for 5 minutes, add it to ExpiCHO-S TM cells (manufacturer: Thermofisher, product number: A29127), put Into 5% CO 2 , 120 rpm, 37°C shaker culture. On the second day after transfection, feed was added, and the shaker temperature was adjusted to 32°C to continue culturing. On day 9 of transfection, the cell supernatant was collected.
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with PBS solution containing 5mM imidazole, wash 2-5 times the column volume. The replaced supernatant sample was loaded onto the column for binding, and the column was washed with PBS solution containing 5 mM imidazole until the A280 reading dropped to the baseline. Afterwards, the chromatographic column was washed with PBS+10mM imidazole to remove non-specifically bound impurity proteins, and the effluent was collected.
  • the target protein was then eluted with PBS solution containing 300 mM imidazole, and the elution peaks were collected. After concentration, the collected eluted product can be further purified by gel chromatography Superdex200 (GE), the mobile phase is PBS, the polymer and foreign protein peaks are removed, and the eluted peak of the target product is collected. The obtained proteins were electrophoresed and identified by SEC-HPLC and sorted for use.
  • GE gel chromatography Superdex200
  • the sequences of the positive control antibody and negative control antibody of Anti-MSLN come from the SEQ ID NO: 101 and SEQ ID NO: 99 of the patent US20180327508A1, named MB001 and MB060 respectively (see Table 4 for the sequence), all of which are trispecific antibodies with a structure
  • a schematic is shown in Figure 1B. Both the positive control antibody and the negative control antibody were constructed by Taizhou Baiying Biotechnology Co., Ltd. and the production and purification of antibodies were completed. For specific methods, see Example 1.2.
  • human serum albumin purchased from Chengdu Rongsheng, article number: S10940024
  • monkey serum albumin purchased from: Abcam, product number: ab184894
  • mouse serum albumin purchased from: Alpha Diagnist, product number: ALB14-N-10) was diluted with PBS to a final concentration of 4 ⁇ g/mL, and then added to a 96-well ELISA plate at 50 ⁇ l/well , sealed with a plastic film and incubated at 4°C overnight, washed the plate twice with PBST the next day, and added blocking solution [PBS+2% (w/w) BSA] to block at room temperature for 2 hours.
  • Enzyme-linked immunosorbent assay to detect the ability of multispecific antibodies to simultaneously bind to MSLN protein and CD3 protein
  • Human MSLN extramembrane protein (296-580, GenBank: AAH09272.1, His-tag, self-expression) was diluted with PBS to a final concentration of 2 ⁇ g/mL, then added to 96-well ELISA plate at 50 ⁇ l/well, covered with plastic film Seal and incubate overnight at 4°C, wash the plate twice with PBST the next day, add blocking solution [PBS+2% (w/w) BSA] to block at room temperature for 2 hours. Pour off the blocking solution, wash the plate twice with PBST, and add the test antibody or control antibody with an initial concentration of 100nM or 200nM and a 1:3 gradient dilution at 50 ⁇ l/well.
  • the human ovarian cancer cell line OVCAR3 (purchased from ATCC, product number: HTB-161) with high expression of MSLN, the human cervical cancer cell line Hela and the human pancreatic cancer cell line Hs766T (purchased from the Chinese Academy of Medical Sciences Foundation) were used in the experiment. Institute of Medicine Cell Resource Center), human lung cancer cell line NCI-H292 with low MSLN expression (purchased from ATCC, catalog number: CRL-1848), and human skin cancer cell line A431 that does not express human MSLN protein.
  • Expand the required cells to the logarithmic growth phase in a T175 cell culture flask remove the medium, wash twice with PBS buffer, digest the cells with trypsin, then stop the digestion with complete medium, and pipette the cells to single cell suspension. After counting the cells, centrifuge, wash the cell pellet twice with PBS, resuspend the cell pellet with FACS buffer (PBS+2% fetal calf serum) to 2 ⁇ 10 6 cells/mL, add 50 ⁇ l per well to 96-well FACS In the reaction plate, add the test antibody or control antibody (200nM as the initial concentration, 3-fold or 5-fold gradient dilution) at 50 ⁇ l/well, mix with the cell suspension, and incubate at 4°C for 1 hour.
  • FACS buffer PBS+2% fetal calf serum
  • HEK293T cell line HEK293T-hMSLN-R3 expressing human MSLN-R3 protein
  • PCT/CN2021/136419 coding the amino acid sequence of human MSLN-R3 (NCBI: Met487-Ser606 of AAH09272.1)
  • plasmid transfection 3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015
  • puromycin to selectively culture for 2 weeks, sorting positive monoclonal cells into 96-well plates and selecting some monoclonal wells for amplification.
  • the amplified clones were detected and analyzed by FACS flow cytometer with specific antibodies, and the cell lines with better growth and higher fluorescence intensity were selected to continue to be expanded and cultured and frozen in liquid nitrogen.
  • the detection shows that HEK293T-hMSLN-R3 after puromycin pressure selection has a relatively single positive peak).
  • the preparation of the detection cells and the antibody to be tested and the detection method refer to Example 2.3.
  • the analysis results are shown in Figure 5 and Table 11.
  • HEK293T cell line HEK293T-monkey MSLN expressing monkey MSLN protein please refer to PCT/CN2021/136419 (the nucleotide sequence encoding the full-length amino acid sequence of monkey MSLN (NCBI: XP_028696439.1) was cloned into the pcDNA3.1 vector And prepare plasmid.
  • HEK293T cell line (purchased from ATCC) is carried out plasmid transfection ( 3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015), using puromycin to selectively culture for 2 weeks, sorting positive monoclonal cells into 96-well plates and selecting some monoclonal wells for amplification.
  • the amplified clones were detected and analyzed by FACS flow cytometer with specific antibodies, and the cell lines with better growth and higher fluorescence intensity were selected to continue to be expanded and cultured and frozen in liquid nitrogen.
  • the detection showed that the HEK293T-monkey MSLN after puromycin pressure selection had a relatively single positive peak).
  • the preparation of the detection cells and the antibody to be tested and the detection method refer to Example 2.3.
  • the analysis results are shown in Figures 6A-6B and Table 12, wherein MB060 is a negative control. The results showed that all the Anti-MSLN multispecific antibodies had specific binding activity to the recombinant cells expressing
  • Human PBMC cells purchased from Aussells Biotechnology (Shanghai) Co., Ltd.
  • T Cell Activation/Expansion Kit human (purchased from Miltenyi, product number: 130-091-441) to complete the T cell activation. Isolation and in vitro activation amplification experiments. When the cells were cultured for 14 days, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS. SP34 antibody was used as the primary antibody, and Alexa647-labeled secondary antibody (purchased from Jackson Immuno, catalog number: 109-605-098) was used for FACS (FACS CantoTM, purchased from BD Company) detection. The results showed that human T cells had a higher CD3 protein expression.
  • Example 2.3 For the preparation and detection methods of the above detection cells and antibodies to be tested, refer to Example 2.3.
  • the analysis results are shown in FIGS. 7A to 7B and Table 13. The results showed that all Anti-MSLN multispecific antibodies had binding activity to human T cells expressing human CD3 protein, and had cross-binding activity to monkey T cells expressing monkey CD3 protein.
  • Multispecific antibodies can simultaneously bind Jurkat-NFAT-Luciferase-CD16a cells (purchased from: Promega, CD3 positive) and tumor cells expressing MSLN (OVCAR3, Hela, Hs766T, NCI-H292), thereby causing Jurkat-NFAT-Luciferase-
  • MSLN MSLN
  • OVCAR3, Hela, Hs766T, NCI-H292 tumor cells expressing MSLN
  • the NFAT-related signaling pathway in CD16a cells was activated, the expression level of luciferase increased, and the fluorescent signal could be detected after adding the substrate.
  • the intensity of the fluorescent signal can characterize the intensity of the activation of the signaling pathway.
  • Example 2.3 for the preparation process of tumor cells, adjust the cell density to 4 ⁇ 10 5 cells/ml, add 100 ⁇ l per well into a 96-well cell culture plate, and culture overnight in a cell incubator. Discard the cell culture medium, wash twice with PBS, add the antibody to be tested or control antibody (60nM as the initial concentration, 5-fold serial dilution, 10-12 dilution points) at 50 ⁇ l/well, and then add 50 ⁇ l/well Jurkat-NFAT-Luciferase-CD16a cells (at a density of 1 ⁇ 10 6 cells/ml) were mixed evenly and placed in a cell incubator for 6 hours.
  • CA125 has been confirmed to bind to MSLN in the R1 region of the distal end of the membrane. To confirm that antibodies against the R1 epitope can block the binding of CA125 to MSLN protein, both protein and cellular levels were evaluated.
  • the main experimental procedures of ELISA and FACS can refer to Examples 2.1 and 2.3, respectively.
  • ELISA and FACS primary antibody preparation instructions Add the antibody to be tested or the control antibody (400nM as the initial concentration, 3-fold serial dilution, 11 dilution points) at 50 ⁇ l/well, incubate at room temperature for 30 minutes, and then add 0.4 ⁇ g at 50 ⁇ l/well /ml CA125 solution (purchased from Baiying, product number: B475601), mix well and continue to incubate at room temperature for 1 hour.
  • the ELISA secondary antibody was horseradish peroxidase-coupled Streptavidin (purchased from Sigma, product number: S2438), and the FACS secondary antibody was APC-coupled Streptavidin (purchased from Biolegend, product number: 405243).
  • the main experimental process can refer to Example 2.3.
  • Primary antibody preparation instructions Add the antibody to be tested or control antibody (200nM as the initial concentration, 5-fold serial dilution, 8 dilution points) at 50 ⁇ l/well, then add 50 ⁇ l/well of 500nM monoclonal antibody solution, and mix well.
  • the analysis results are shown in FIGS. 11A to 11D and Table 20.
  • the results showed that the binding activity of MB001 and MB048 decreased in the presence of high-concentration NB149-95 monoclonal antibody, while MB065 (MSLN R1/R3) and MB069 (MSLN R1/R3) had no binding activity in the presence of different epitope monoclonal antibodies.
  • OVCAR3 cell preparation and detection methods refer to Example 2.3.
  • Instructions for primary antibody preparation and endocytosis conditions Add the antibody to be tested or the control antibody (200nM as the initial concentration, 10-fold serial dilution, 3 dilution points) at 50 ⁇ l/well, mix well and incubate at 4°C for 1 hour, wash with PBS Discard the supernatant after 3 times. Add 50 ⁇ l/well of FACS buffer, resuspend the cells, place one at 4°C for 1 hour, and the other at 37°C for 1 hour. After the supernatant was centrifuged, the diluted secondary antibody was added.
  • Endocytosis rate (4°C_MFI-37°C_MFI)/4°C_MFI*100%
  • Example 7 In vitro activation state detection of T lymphocytes mediated by multispecific antibodies
  • CD69 molecule is an early marker of T cell activation. T cells cultured in a resting state rarely express CD69 molecules. Once T lymphocytes are activated, the expression of CD69 is significantly up-regulated. It is a test to detect whether T lymphocytes are effectively induced and activated. of markers.
  • NCI-H292 cells were digested with trypsin to prepare a single cell suspension. Adjust the tumor cell density to 2 ⁇ 10 5 cells/ml and the T cell density to 1 ⁇ 10 6 cells/ml with complete medium. Take 50 ⁇ l of adjusted NCI-H292 cells and 50 ⁇ l of primary T cells and mix them evenly, and add them to each well of a flat-bottomed 96-well plate with a micropipette.
  • the volume of the cell mixture is 100 ⁇ l/well (tumor cells and The numbers of T cells were 1 ⁇ 10 4 and 5 ⁇ 10 4 respectively).
  • the antibodies were serially diluted with complete medium (the initial antibody concentration was 2nM, 6-fold dilution, 8 concentration gradients), and 100 ⁇ l of antibody dilutions of different concentrations were added to each well, so that the final volume of each well was 200 ⁇ l. Cultivate in vitro for 24 hours in an incubator.
  • MB001 has the strongest T cell activation activity due to the use of high-affinity CD3 antibodies, while MB048, MB065 and MB069 with low-affinity CD3 have weaker T cell activation activities; MB060 negative control has no T cells because it cannot bind to MSLN Activation activity.
  • OVCAR3 was human ovarian cancer cells with high expression of MSLN
  • NCI-H292 was human lung cancer cells with low expression of MSLN
  • A431 was human lung cancer cells without MSLN expression.
  • Skin squamous cell carcinoma cells were used as a negative control.
  • Primary T cells were isolated from PBMCs of healthy donors using a T cell isolation kit (purchased from STEMCELL technologies, product number 17951, refer to the kit instructions for usage) and cultured overnight.
  • OVCAR3, NCI-H292 or A431 cells were digested with trypsin to prepare a single cell suspension. Adjust the tumor cell density to 2 ⁇ 10 5 cells/ml and the T cell density to 1 ⁇ 10 6 cells/ml with complete medium. Take 50 ⁇ l of adjusted OVCAR3, NCI-H292 or A431 cells and 50 ⁇ l of primary T cells and mix them evenly (the numbers of tumor cells and T cells in each well are 1 ⁇ 10 4 and 5 ⁇ 10 4 ), and use a micropipette Add the pipette to each well of a flat-bottomed 96-well plate, and the volume of the cell mixture is 100 ⁇ l/well.
  • the antibodies were serially diluted with complete medium (the initial antibody concentration was 25nM, 6-fold dilution, 8 gradients), and 100 ⁇ l of antibody diluents of different concentrations were added to each well, so that the final volume of each well was 200 ⁇ l. After culturing for 24 hours, the supernatant was taken to detect the production of IFN ⁇ , TNF ⁇ and IL-6 cytokines in T cells by ELISA (the kit was purchased from BD, the article numbers were 555142, 555212 and 555220, respectively, and the method of use was referred to the kit instruction manual).
  • the CellTiter-Glo kit purchased from Promega, catalog number G9243, refer to the product manual for usage was used to detect cell viability.
  • MB069 shows the strongest activation of T cells and tumor cells in vitro.
  • T cell activation and in vitro tumor cell killing activity of MB048 and MB065 are comparable to MB001.
  • MB001, MB048, MB065 and MB069 have no killing effect on MSLN-negative A431 cells, indicating that the killing effect of CD3 multispecific antibody has good target antigen specificity, and will not produce non-specific killing on normal tissues that do not express the target antigen .
  • the release level of cytokines reflects the activity of multispecific antibodies, and generally positively correlates with the activation level of T cells, killing activity in vitro and antitumor activity in vivo. At the same time, the stronger the T cell activation activity of multispecific antibodies, the higher the risk of cytokine storm (CRS) after entering the human body.
  • CRS cytokine storm
  • Example 9 In vivo anti-tumor activity of multispecific antibody in human ovarian cancer OVCAR-3 (highly expressed MSLN) xenograft model
  • human PBMC cells are injected into immunodeficiency NPG mice (5-6w, female, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) by tail vein injection to obtain a human PBMC reconstruction model, and then in this animal model Established a human ovarian cancer OVCAR-3 xenograft model (xenograft model) with high MSLN expression and a human lung cancer NCI-H292 xenograft model with low MSLN expression, and validated multispecific antibodies in these two models antitumor activity in vivo.
  • Expand OVCAR-3 cells (American Type Culture Collection, ATCC) to the required number in T-300 cell culture flasks, and in its logarithmic growth phase (confluence is about 80%; the day before inoculation needs to be given Cells were replaced with fresh medium) to begin collection.
  • the medium in the cell culture flask was removed, washed twice with phosphate buffer saline (PBS, Thermo Fisher Scientific Co., Ltd., Hyclone, 29588138), and then an appropriate amount of 0.25% trypsin digestion solution (Thermo Fisher Co., Ltd., Gibco, #25200-072/219085), shake the bottom of the bottle, so that the trypsin digestion solution evenly covers the cell surface, digest at 37°C for 5min, add 20% fetal bovine serum (Fetal Bovine serum, FBS, Sai The complete medium of Mofei Shier Technology Co., Ltd., Gibco, #10099-141C/2261480CP) was used to stop the digestion reaction, and the cells adhered to the bottom of the bottle were gently blown away from the culture bottle, and the digested cell suspension was collected into a 50 mL centrifuge Tube, 350g, centrifuged for 5min, absorb an appropriate amount of serum-free RPMI-1640 medium (Thermo
  • the above cell suspension was mixed with Matrigel (Corning Biotechnology Co., Ltd., #356237) in equal proportions, and 200 ⁇ L of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.
  • PBMC peripheral blood mononuclear cells
  • RPMI-1640 without Serum culture medium
  • the PBMC cell concentration was adjusted to 2.5 ⁇ 10 7 cells/mL using RPMI-1640 serum-free medium, placed on ice, and transferred to the SPF animal room through the transfer window for inoculation modeling. 200 ⁇ L of the above cell suspension was inoculated into the tail vein of each mouse for immune reconstitution. After inoculation, the tumor growth was monitored.
  • the dosage regimen is shown in Table 23, wherein MB060 is a negative control, MB001 is a positive control, and MB048, MB065 and MB069 are antibodies to be tested.
  • Rat administration mass where m is the administration mass, n is the administration molarity, and M is the molecular weight of the drug, and the administration doses in this article are calculated according to this principle.
  • the treatment method of OVCAR-3 cells is the same as that in 9.1.
  • the treatment method of PBMC is the same as that in 9.1.
  • Example 10 In vivo anti-tumor activity of multispecific antibody in human PBMC reconstituted human lung cancer NCI-H292 (low expression of MSLN) xenograft model
  • the human lung cancer cell NCI-H292 with low expression of MSLN was selected to establish a mouse (NPG, 5-6w, female, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) in vivo model, and the multi-specificity was verified on this model. anti-tumor activity of antibodies in vivo.
  • Human lung cancer cells NCI-H292 (American Type Culture Collection, ATCC) were processed according to the method described in Example 9.1 (the complete medium containing 10% fetal bovine serum was terminated digestion), and finally according to the cell counting results, use Serum-free medium was used to adjust the cell density to 5 ⁇ 10 7 cells/mL, placed on ice, and transferred to the SPF animal room through the transfer window for inoculation modeling, and 200 ⁇ L of the above cell suspension was inoculated subcutaneously in the right axilla of each mouse.
  • Serum-free medium was used to adjust the cell density to 5 ⁇ 10 7 cells/mL, placed on ice, and transferred to the SPF animal room through the transfer window for inoculation modeling, and 200 ⁇ L of the above cell suspension was inoculated subcutaneously in the right axilla of each mouse.
  • the PBMCs were treated in the same way as in 9.1.
  • each mouse was inoculated with 200 ⁇ L of the above cell suspension in the tail vein for immune reconstitution. After inoculation, the tumor growth was monitored. When the overall tumor volume was around 100 mm 3 , blood was collected from the orbit to detect the reconstitution of the human immune system.
  • the dosage regimen is shown in Table 25, wherein MB060 is a negative control, MB001 is a positive control, and MB048, MB065 and MB069 are antibodies to be tested.
  • Example 12 Stability experiment of multispecific antibody in human serum and cynomolgus monkey serum
  • the culture and treatment of PBMC cells are the same as in Example 7; the culture and treatment of Hela cells are the same as that of NCI-H292 cells in Example 7.
  • Antibody treatment is divided into two types: 1. The antibody is not pretreated; 2. The antibody is pre-incubated in human serum at 37°C for two days. Differently treated antibodies were serially diluted with complete medium (the initial antibody concentration was 25 nM, 6-fold dilution, 10 concentration gradients). 100 ⁇ l of antibody diluents of different concentrations were added to each well, so that the final volume of each well was 200 ⁇ l.
  • the in vitro cytotoxic activity test treatment is divided into two types: 1. The in vitro killing test is carried out in 10% FBS complete medium; 2. The in vitro killing test system is additionally added with 20% human serum.
  • the experimental method is the same as 12.1, only human serum is partially replaced with cynomolgus monkey serum. After culturing for 24 hours, the cell viability was measured with the CellTiter-Glo kit (purchased from Promega, refer to the product manual for the usage method), and the results are shown in FIG. 19B . After two days of incubation at 37°C in cynomolgus monkey serum, the activities of MB001 and MB048 decreased slightly, while those of MB065 and MB069 hardly decreased.
  • Tumor cells THP-1 purchased from the Cell Bank of Chinese Academy of Sciences, article number: TCHu 57
  • U937 purchasedd from ATCC, article number: CRL-1593.2
  • 786-O purchasedd from ATCC, article number: CRL-1932
  • Jurkat purchased from the Cell Bank of Chinese Academy of Sciences, article number: TCHU123. Expand the desired cells in T-175 cell culture flasks to the logarithmic growth phase, aspirate the medium, wash twice with PBS buffer, digest the cells with trypsin, then stop the digestion with complete medium, and blow the cells to a single cell suspension.
  • CHO-K1-human CD33 expressing human CD33 protein For the preparation method of the recombinant cell line CHO-K1-human CD33 expressing human CD33 protein, please refer to PCT/CN2022/075621 (the nucleotide sequence encoding the full-length amino acid sequence of human CD33 (NCBI: XP_011525834.1) was cloned into pcDNA3.1 Vector and plasmid preparation (completed by General Biosystems (Anhui) Co., Ltd.). Plasmid transfection was carried out on CHO-K1 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) ( 3000 Transfection Kit, purchased from Invitrogen, item number: L3000-015), and puromycin was used for pressure screening.
  • the recombinant cell line CHO-K1-human CD70 expressing human CD70 protein was purchased from Kangyuan Biotech (KC-1267) and the preparation and detection methods of the detection cells and antibodies to be tested refer to Example 13.1.
  • the analysis results are shown in Figure 22, where MB060 is the negative control, and BDD20-09-9# has only CD33 monoclonal antibody but no CD70 monoclonal antibody.
  • the results showed that the Anti-CD33 ⁇ CD70 multispecific antibody had binding activity to the recombinant cells expressing human CD70 protein, indicating that the combination of Anti-CD70 monoclonal antibody into multispecific antibody retained the characteristics of monoclonal antibody.
  • Example 14 Different formats of polyclonal antibodies mediate differences in the killing activity of tumor cells and normal cells expressing CD33
  • the AML3 cell line Molm13 with high expression of CD33 was added to PBMC of healthy donors to simulate the in vivo environment of AML patients, and to verify that different formats of polyclonal antibodies mediate tumor cells and normal cells expressing CD33 (mainly CD14+ monocytes in peripheral blood) Differences in killing activity.
  • Resuscitate frozen healthy donor PBMC cells label the revived PBMC cells with CellTrace TM Violet dye, use complete medium to adjust the Molm13 tumor cells to 2.5 ⁇ 10 6 cells/ml, and adjust the PBMC cell density to 1 ⁇ 10 6 pieces/ml.
  • the device was added to each well of a flat-bottomed 96-well plate, and the volume of the cell mixture was 100 ⁇ l/well.
  • the antibodies were serially diluted with complete medium (the initial antibody concentration was 5 ⁇ g/ml, 20-fold dilution, 8 gradients), and 100 ⁇ l of antibody diluents of different concentrations were added to each well, so that the final volume of each well was 200 ⁇ l.
  • Fc Receptor Blocking Solution purchased from Biolegend, Cat. Cat. No. 367110 staining, react in the dark for 30 minutes, centrifuge to remove the supernatant, wash twice with flow buffer, add PI (purchased from Thermo Fisher, Cat. No.

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Abstract

L'invention concerne un anticorps multi-spécifique et une utilisation pharmaceutique associée. L'anticorps multi-spécifique comprend au moins trois parties : (A) une partie de liaison à l'antigène cible, (B) une partie d'extension de demi-vie, et (C) une partie engageant les lymphocytes T. En se liant à deux cibles ou plus en même temps, l'anticorps multi-spécifique peut simultanément exercer de multiples fonctions, de façon à prévenir et/ou traiter des maladies prolifératives.
PCT/CN2022/132228 2021-11-17 2022-11-16 Anticorps multi-spécifique et utilisation pharmaceutique associée Ceased WO2023088295A1 (fr)

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CN110913904A (zh) * 2017-05-05 2020-03-24 美国安进公司 用于改善的储存和施用的包含双特异性抗体构建体的药物组合物
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CA2956471C (fr) * 2014-07-31 2024-09-10 Amgen Res Munich Gmbh Constructions optimisees d'anticorps monocatenaires, bispecifiques, specifiques d'especes croisees
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US5686578A (en) * 1994-08-05 1997-11-11 Immunomedics, Inc. Polyspecific immunoconjugates and antibody composites for targeting the multidrug resistant phenotype
WO2012175400A1 (fr) * 2011-06-23 2012-12-27 Ablynx Nv Protéines se liant à la sérumalbumine
CN112062853A (zh) * 2013-12-20 2020-12-11 豪夫迈·罗氏有限公司 双特异性her2抗体及使用方法
CN110198737A (zh) * 2016-11-23 2019-09-03 哈普恩治疗公司 靶向psma的三特异性蛋白质及使用方法
CN110913904A (zh) * 2017-05-05 2020-03-24 美国安进公司 用于改善的储存和施用的包含双特异性抗体构建体的药物组合物
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