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WO2023011431A1 - Anticorps anti-cd16 et son utilisation - Google Patents

Anticorps anti-cd16 et son utilisation Download PDF

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Publication number
WO2023011431A1
WO2023011431A1 PCT/CN2022/109574 CN2022109574W WO2023011431A1 WO 2023011431 A1 WO2023011431 A1 WO 2023011431A1 CN 2022109574 W CN2022109574 W CN 2022109574W WO 2023011431 A1 WO2023011431 A1 WO 2023011431A1
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seq
antibody
antigen
binding
bdd20
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Chinese (zh)
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王琼
刘雷
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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    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention relates to the field of biomedicine, in particular to CD16 antibody and its application.
  • NK cells Natural killer cells
  • NK cells are a component of the innate immune system, accounting for approximately 5-15% of circulating lymphocytes. Unlike B cells and T cells, NK cells do not express somatically rearranged antigen receptors, but instead express a series of activating and inhibitory receptors, the integration of activating and inhibitory signals from the interaction of ligands and different receptors And the balance determines the state of NK cell activation.
  • Activated NK cells kill target cells by similar means as cytotoxic T cells, namely by cytolytic granules containing perforin and granzymes and by the death receptor pathway.
  • Activated NK cells also secrete inflammatory cytokines such as IFN- ⁇ and chemokines that promote the recruitment of other inflammatory cells to target tissues.
  • NK cells do not require antigen priming and recognize targets by activating receptors in the absence of MHC recognition.
  • MHC I Class I HLA
  • the human IgG Fc receptor CD16 (Fc ⁇ RIII) consists of two subtypes (CD16a/Fc ⁇ RIIIa and CD16b/Fc ⁇ RIIIb), encoded by two highly homologous genes.
  • CD16b (Fc ⁇ RIIIb), mainly expressed on neutrophils, is a GPI-anchored glycoprotein that lacks the intracellular signal transduction domain.
  • CD16b There are gene polymorphisms in CD16b, which can produce three allotypes, namely NA1, NA2 and SH.
  • CD16a is a low-affinity receptor for human IgG Fc, a single transmembrane protein that is involved in antibody-dependent cellular cytotoxicity (ADCC) and triggers specific lysis of target cells by natural killer (NK) cells.
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCC is one of the dominant mechanisms of cytotoxicity by Fc ⁇ R-expressing effector cells to clear tumor cells.
  • a single nucleotide substitution (SNP) from G to T at position 559 of the CD16a gene in the cDNA of the CD16a gene resulted in two distinct Fc ⁇ RIIIa allotypes: at position 158 of the amino acid sequence, one encoded as valine Acid (V), the other encoded as phenylalanine (F).
  • V valine Acid
  • F phenylalanine
  • the presence of valine (V/V or V/F) enhances the binding affinity of NK cells for IgG1 or IgG3 antibodies compared to homozygous phenylalanine genotypes (F/F), resulting in higher NK Levels of cell-mediated ADCC.
  • NK cell-mediated ADCC is one of the mechanisms of anticancer effects of commonly used antibodies such as rituximab, trastuzumab, and cetuximab.
  • the ADCC function of NK cells has received high attention in antibody immunotherapy.
  • Bispecific antibodies that simultaneously recruit the ADCC receptor CD16a and recognize target antigens have been developed.
  • the development of therapeutic antibodies targeting CD16 has broad prospects.
  • CD16a and CD16b are highly similar in the extracellular domain, there are large differences in their functions, it is of great significance to increase the affinity of antibodies to CD16a and/or reduce their affinity to CD16b.
  • the presence of immunoglobulin affects the binding activity of antibodies to CD16, it is also necessary to screen for antibodies that better maintain CD16 binding activity in the presence of immunoglobulins. In view of this, the present invention is proposed.
  • the present disclosure provides an antibody or antigen-binding fragment specifically binding to CD16, a multispecific antigen-binding molecule, a nucleic acid fragment, a vector (vector), a host cell, an immune effector cell, a preparation method, a pharmaceutical composition, a pharmaceutical use, and cancer or Treatment of tumors, infectious diseases or autoimmune diseases.
  • the present disclosure provides an antibody or antigen-binding fragment that specifically binds to CD16, comprising a combination of light chain CDRs and a combination of heavy chain CDRs:
  • the light chain CDRs combination comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 are selected from the sequences shown in the following table:
  • the heavy chain CDRs combination comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 are selected from the sequences shown in the following table:
  • an antibody or antigen-binding fragment of the invention comprises a combination of light chain CDRs and heavy chain CDRs selected from the group consisting of: VL2+VH1, VL3+VH1, VL4+VH1, VL5+VH1, VL6+VH1, VL7+VH1, VL8+VH1, VL9+VH1, VL10+VH1, VL11+VH1, VL12+VH1, VL13+VH1, VL14+VH1, VL15+VH1, VL1+VH2, VL1+VH3, VL1+VH4, VL1+VH5, VL1+ VH6, VL1+VH7, VL1+VH8, VL1+VH9, VL16+VH10, VL17+VH10, VL18+VH10, VL19+VH10, VL16+VH4, VL17+VH4, VL18+VH4, and VLL18+VH4,
  • said antibody or antigen-binding fragment comprises:
  • said antibody or antigen-binding fragment comprises:
  • the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, chemotherapeutics or immunomodulators, and the tracer selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the therapeutic agent is selected from radioisotopes, chemotherapeutics or immunomodulators
  • the tracer selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers.
  • the antibody or antigen-binding fragment is selected from one or more of F(ab) 2 , Fab', Fab, Fv, scFv, bispecific antibody and Nanobody; preferably, the The antibody or antigen-binding fragment is a scFv, more preferably, from the N-terminus to the C-terminus, the scFv includes a light chain variable region-Linker-heavy chain variable region, and the linker is preferably (G2S) 7 .
  • the antibody or antigen-binding fragment is also linked with other functional molecules, preferably, the other functional molecules can be selected from one or more of the following: signal peptides, protein tags, cell factors, angiogenesis inhibitors, or immune checkpoint inhibitors.
  • the cytokine can be IL2, IL-6, IL-12, IL-15, IL-21, IFN or TNF ⁇ ;
  • the angiogenesis inhibitor can be endostatin;
  • the The immune checkpoint inhibitor can be SIRP ⁇ .
  • the present disclosure also provides a multispecific antigen-binding molecule, which comprises the aforementioned antibody or antigen-binding fragment, and an antigen-binding molecule that binds to an antigen other than CD16, or binds to the aforementioned Different CD16 epitopes of antibodies or antigen-binding fragments; optionally, other antigens other than CD16 can be selected from: CD137, CD258, PD-1, PD-L1, 4-1BB, CD40, CD64, EGFR, VEGF, CD3 (preferably CD3 ⁇ ), HER2, HER1, HER3, IGF-1R, phosphatidylserine (Phosphatidylserine, PS), C-Met, BCMA, HSA, GPRC5D, MSLN or blood-brain barrier receptor;
  • said other antigen-binding molecule is an antibody or an antigen-binding fragment
  • said multispecific antigen binding molecule may be bispecific, trispecific or tetraspecific;
  • the multispecific antigen binding molecule may be bivalent, tetravalent or hexavalent.
  • the present disclosure also provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment or multispecific antigen-binding molecule.
  • the present disclosure also provides a vector, the vector comprising the aforementioned nucleic acid fragment.
  • the present disclosure also provides a host cell comprising the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
  • the present disclosure also provides a method for preparing the aforementioned antibody or antigen-binding fragment or multispecific antigen-binding molecule, the method comprising culturing the aforementioned cell, and isolating the antibody, antigen-binding fragment, or multispecific antigen-binding molecule expressed by the cell. Specific antigen-binding molecules.
  • the present disclosure also provides a pharmaceutical composition, which comprises the aforementioned antibody or antigen-binding fragment, multispecific antigen-binding molecule, nucleic acid fragment, carrier, or product prepared according to the aforementioned method;
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional antineoplastic agent.
  • the present disclosure also provides a method for treating tumors or cancers, inflammatory diseases or allergic diseases, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment, multispecific antigen Binding molecules, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods; preferably, the tumor or cancer is selected from non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, minimal residual disease, metastases.
  • the present disclosure also provides the aforementioned antibodies or antigen-binding fragments, multispecific antigen-binding molecules, nucleic acid fragments, vectors, products or pharmaceutical compositions prepared according to the aforementioned methods in the preparation and treatment of tumors or cancers, inflammatory diseases or Use in allergy medicine; preferably, the tumor or cancer is selected from non-Hodgkin's lymphoma, chronic lymphocytic leukemia, Hodgkin's disease, minimal residual disease, metastases.
  • the CD16 antibody provided by the present disclosure has different specificities for CD16a and CD16b, and the antibody has at least one of the following advantages: (1) In the presence or absence of immunoglobulin, it can bind to CD16a (158F) and/or increased affinity for CD16a (158V); (2) decreased affinity for CD16b (NA1) and/or CD16b (NA2) in the presence or absence of immunoglobulins. Therefore, the antibody provided by the present disclosure can better distinguish the two subtypes of CD16, and then provide a better tumor immunotherapy method targeting the CD16 target.
  • CD16 refers to receptor type III for the Fc portion of immunoglobulin G (Fc ⁇ RIII), a group of differentiated molecules found on the surface of natural killer cells, neutrophils, monocytes, and macrophages.
  • CD16 has been identified as Fc receptors Fc ⁇ RIIIa (CD16a) and Fc ⁇ RIIIb (CD16b), involved in signal transduction.
  • Human Fc ⁇ RIIIA (CD16a) is a low-affinity receptor that can bind to IgG Fc expressed on human CD56 low-expressing natural killer (NK) cells, monocyte subsets, dendritic cells and rare T cells.
  • CD16b Human Fc ⁇ RIIIB (CD16b), encoded by different genes and mainly expressed in neutrophils, is a GPI-anchored glycoprotein that lacks the intracellular signal transduction domain.
  • CD16a is a type I membrane glycoprotein with a single transmembrane (TM) domain and a short cytoplasmic tail, whose expression on the cell surface depends on binding to the signaling molecules CD247 (TCR ⁇ ) and/or Fc- ⁇ RI- ⁇ , once they interact, they induce a series of signal transductions, leading to cytokine release and cell killing activity.
  • KD equilibrium dissociation constant
  • high affinity generally refers to having about 10 -6 M or lower, 10 -7 M or lower, about 10 -8 M or lower, about 10 -9 M or lower or about 10-10 M or lower KD.
  • 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 includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chains thereof.
  • Antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminal to the carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that can interact with antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • 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.
  • IgM, IgD, IgG, IgA, and IgE immunoglobulins
  • their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • 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 antibodies that do not comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca ( Heavy-chain antibodies (HCAbs) produced by camelids such as Vicugna pacos) and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptors Ig new antigen receptors, IgNAR
  • 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, large sheep Camels, protozoans, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
  • heavy chain antibody herein refers to an antibody that lacks the light chains of conventional antibodies.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • nanobody in this paper refers to the natural heavy chain antibody lacking the light chain in camels, etc., and its variable region can be cloned to obtain a single domain antibody consisting of only the variable region of the heavy chain, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • VHH domain and “nanobody” and “single domain antibody” (sdAb) herein have the same meaning and are used interchangeably, referring to the variable region of a cloned heavy chain antibody, constructed A single domain antibody consisting of only one heavy chain variable region, which is the smallest fully functional antigen-binding fragment.
  • the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • multispecific herein refers to the ability of an antibody or antigen-binding fragment to bind, for example, different antigens or at least two different epitopes on the same antigen.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody can bind.
  • conventional monospecific IgG-type antibodies have two identical antigen-binding sites (paratopes) and thus can only bind the same epitope (rather than bind different epitopes).
  • multispecific antibodies have at least two different types of paratopes/binding sites and thus can bind at least two different epitopes.
  • complementarity determining region refers to the antigen binding site of an antibody.
  • a single “specificity” may refer to one, two, three or more than three identical CDRs in a single antibody (the actual number of CDRs/binding sites in a single antibody molecule is referred to as " price").
  • a single native IgG antibody is monospecific and bivalent because it has two identical paratopes.
  • a multispecific antibody comprises at least two (different) complementarity determining regions/binding sites.
  • the term “multispecific antibody” refers to an antibody that has more than one paratope and has the ability to bind two or more different epitopes.
  • multispecific antibody includes in particular bispecific antibodies as defined above, but generally also proteins, e.g. antibodies, scaffolds which specifically bind three or more than three different epitopes, i.e. having three or more Antibodies with more than three paratopes/binding sites.
  • 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
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • exemplary, "antigen-binding fragment” or “antibody fragment” herein includes, but is not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabody and single domain Antibody.
  • chimeric antibody refers to an antibody that has variable sequences derived from immunoglobulins of one source organism (such as rat, mouse, rabbit or alpaca) and derived from a different organism (such as human ) of the immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719):1202-1207. Transfectomas Provide Novel Chimeric Antibodies; Gillies et al., J Immunol Methods. 1989 Dec 20; 125(1-2):191-202; above incorporated by reference Incorporated into this article. See also patent US5807715A.
  • 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 or 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 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.
  • variable domains hypervariable regions
  • FR framework regions
  • amino acid positions representing the hypervariable regions of an antibody may vary according to the context and various definitions known in the art. Some positions within variable domains can be considered heterozygous hypervariable positions because these positions can be considered within hypervariable regions under one set of criteria (such as IMGT or KABAT) but under a different set of criteria (such as KABAT or IMGT) outside the hypervariable region. One or more of these positions may also be found in extended hypervariable regions.
  • the invention includes antibodies comprising modifications in these hybrid hypervariable positions.
  • the heavy chain variable region CDR may be abbreviated as HCDR and the light chain variable region may be abbreviated as LCDR.
  • the variable domains of the native heavy and light chains each comprise four framework regions predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held tightly together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference).
  • 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).
  • 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” may be selected from a CH1 domain, a hinge region, a CH2 domain, a 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.
  • typical "heavy chain constant region fragments" can be selected from 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 region is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • antibodies produced by host cells may undergo post-translational cleavage whereby one or more, especially one or two amino acids are excised from the C-terminus of the heavy chain.
  • an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include cleavage variants of the full-length heavy chain.
  • the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index).
  • the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
  • the IgG Fc region includes IgG CH2 and IgG CH3 domains, optionally, on this basis, it may also include a complete or partial hinge region, but does not include a CH1 domain.
  • the "CH2 domain" of a human IgG Fc region generally extends from an amino acid residue at about position 231 to an amino acid residue at about position 340.
  • the carbohydrate chain is attached to the CH2 domain.
  • the CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain.
  • a "CH3 domain” comprises the stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
  • the CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g.
  • the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service , National Institutes of Health, Bethesda, MD, 1991 described.
  • Fc variant herein refers to changes in the structure or function of Fc caused by one or more amino acid substitutions, insertions or deletion mutations at appropriate positions on the Fc.
  • Interaction between Fc variants refers to the space-filling effect, electrostatic guidance, hydrogen bond interaction, hydrophobic interaction, etc. between Fc variants designed by mutation. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins.
  • a preferred mutation design is a "Knob-into-Hole” style mutation design.
  • Fc variants have been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion proteins.
  • the representative one is the "Knob-into-Hole" form proposed by Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996); Heterodimer form of Fc (US 20100286374 A1); Heterodimer form of IgG/Ig chain exchange proposed by Jonathan H. Davis et al.
  • the Knob/Hole structure on the Fc variant fragments of the present invention means that the two Fc fragments are mutated respectively, and can be combined in a "Knob-into-Hole" form after the mutations. It is preferred to use the "knob-into-hole" model of Cater et al. to carry out site mutation modification on the Fc region, so that the obtained first Fc variant and the second Fc variant can be in the form of "knob-into-hole” Combine together to form heterodimers.
  • the selection of particular immunoglobulin Fc regions from particular immunoglobulin classes and subclasses is within the purview of those skilled in the art.
  • the Fc region of human antibody IgG1, IgG2, IgG3, IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred.
  • One of the first Fc variant or the second Fc variant is randomly selected for knob mutation and the other for hole mutation.
  • 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 algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using a Blossum62 matrix or a PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package (available at www.gcg.com), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. It is also possible to use a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4, using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11-17 ) to determine the percent identity between two amino acid sequences or nucleotide sequences.
  • nucleic acid sequences 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.
  • 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. Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP2101823B1).
  • isolated nucleic acid 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.
  • the pharmaceutical composition further comprises additional anti-tumor agents, such as chemotherapeutic agents, immune checkpoint inhibitors and the like.
  • the term "pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial, antifungal), isotonic agents, absorption delaying agents, Agents, salts, preservatives, drug stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc., and combinations thereof, are known to those skilled in the art (see For example, Remington's Pharmaceutical Sciences, 18th ed. Mack Printing Company, 1990, pp. 1289-1329). Except in cases of incompatibility with the active ingredient, any conventional carrier is contemplated for use in therapeutic or pharmaceutical compositions.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesired physiological changes or lesions, such as cancers and tumors, in the subject being treated.
  • 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 herein refers to an organism receiving treatment for a particular disease or condition as described herein.
  • a “subject” includes a mammal, such as a human, a primate (eg, monkey) or a non-primate mammal, 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 relieve 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.
  • 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.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • Figure 1 is the SDS of human CD16a(158F)-Fc, human CD16a(158V)-Fc, human CD16b(NA1)-Fc, human CD16b(NA2)-Fc, human CD16b(SH)-Fc and cynomolgus monkey CD16-Fc protein samples -PAGE reducing gum and non-reducing gum detection results.
  • Lanes 1 and 2 are the protein bands of human CD16a(158F)-Fc under reducing and non-reducing conditions respectively
  • lanes 3 and 4 are the protein bands of human CD16a(158V)-Fc under reducing and non-reducing conditions respectively
  • lane 5 and 6 are the protein bands of human CD16b(NA1)-Fc under reducing and non-reducing conditions, respectively
  • lanes 7 and 8 are the protein bands of human CD16b(NA2)-Fc under reducing and non-reducing conditions, respectively
  • lanes 9 and 10 The protein bands of human CD16b(SH)-Fc under reducing and non-reducing conditions
  • lanes 11 and 12 are the protein bands of cynomolgus monkey CD16-Fc under reducing and non-reducing conditions, respectively
  • lane M is the protein maker band .
  • FIG. 2A FACS detection results of FlpinCHO cells transfected with human CD16a (158F) protein
  • Fig. 2B FACS detection results of FlpinCHO cells transfected with human CD16a (158V) protein
  • Fig. 2C FACS detection results of FlpinCHO cells transfected with human CD16b (NA1) protein
  • Fig. 2D FACS detection results of FlpinCHO cells transfected with human CD16b (NA2) protein
  • Fig. 2E FACS detection results of FlpinCHO cells transfected with cynomolgus CD16 protein.
  • Figure 3A shows the binding of CD16scFv-his antibody to FlpinCHO-human CD16a (158F) cells
  • Figure 3B shows the binding of CD16scFv-his antibody to FlpinCHO-human CD16a (158V) cells.
  • Figure 4A shows the binding of CD16scFv-his antibody to FlpinCHO-human CD16a (158F) cells in the presence of 10mg/mL human immunoglobulin;
  • Figure 4B shows the binding of CD16scFv-his antibody to FlpinCHO-human Binding of CD16a(158V) cells.
  • Figure 5A is the FACS detection of the binding reaction of the antibody to FlpinCHO-human CD16b (NA1) cells
  • Figure 5B is the FACS detection of the binding reaction of the antibody to FlpinCHO-human CD16b (NA2) cells.
  • Figure 6 is the activation of Jurkat-NFAT cells detected by the luciferase reporter system
  • Figure 7A shows the binding of CD16scFv-his antibody to human NK cells in the presence of 10 mg/mL human immunoglobulin
  • Figure 7B shows the binding of CD16scFv-his antibody to human NK cells in the presence of 10 mg/mL human immunoglobulin.
  • Figure 8A shows the binding of CD16scFv-his combined mutant antibody to FlpinCHO-human CD16a (158F) cells
  • Figure 8B shows the binding of CD16scFv-his combined mutant antibody to FlpinCHO-human CD16a (158V) cells.
  • Figure 9A shows the binding of CD16scFv-his combined mutant antibody to FlpinCHO-human CD16a(158F) cells in the presence of human immunoglobulin
  • Figure 9B shows the binding of CD16scFv-his combined mutant antibody to FlpinCHO-human CD16a(158V) in the presence of human immunoglobulin Cell binding.
  • Figure 10A is the FACS detection of the binding reaction of the combined mutant antibody to FlpinCHO-human CD16b (NA1) cells
  • Figure 10B is the FACS detection of the binding reaction of the combined mutant antibody to FlpinCHO-human CD16b (NA2) cells.
  • Figure 11 shows the activation of Jurkat-NFAT cells detected by the luciferase reporter system.
  • the extracellular domain (ECD, extra-cellular domain) of human and cynomolgus monkey CD16 protein was connected with human IgG1Fc, (G 3 S) 2 linker and AVI tag sequentially (the amino acid sequence of each element and the composition of the tag antigen are shown in Table 1) to design the tag antigen.
  • Entrust General Biosystems (Anhui) Co., Ltd. to synthesize the nucleotide sequence corresponding to the amino acid sequence of the tag antigen and clone it into the pTT5 vector containing the signal peptide, and prepare the plasmid according to the established standard molecular biology method.
  • HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
  • PI Polysciences, Cat. No.: 24765-1
  • FreeStyle TM 293 Thermofisher scientific, Cat. No.: 12338018
  • Protein A packing material AT Protein A Diamond and chromatography column BXK16/26 are purchased from Bogeron, article number: AA0273 and B-1620 respectively), using PBS phosphate After washing with buffer (pH7.4), wash with 20mM PB, 1M NaCl (pH7.2), and finally use pH3.4 citrate buffer for elution, and collect the eluted protein A chromatography column.
  • Table 1 The sequence of each element of CD16-Fc tag antigen and the composition of tag antigen
  • the mouse antibody 3G8 that recognizes human CD16 was obtained by immunizing mice with PMN (Fleit HBet al., Proc Natl Acad Sci U S A.1982 May), which can recognize human CD16a (158F and 158V) and CD16b (NA1, NA2 and SH), and has cross-binding activity with cynomolgus monkey CD16.
  • the heavy chain variable region and light chain variable region sequences of 3G8 were obtained according to the patent WO2007009065A2. Taizhou Baiying Biotechnology Co., Ltd. cloned the heavy chain variable region sequence of 3G8 into a heavy chain containing signal peptide and murine antibody IgG1.
  • the light chain variable region sequence was cloned into the expression vector pcDNA3.4 containing the signal peptide and the light chain constant region of the mouse antibody IgG1 - From B1MLK (Taizhou Baiying Biotechnology Co., Ltd.), the sequence of 3G8-mIgG1 was obtained, and the sequence information is shown in Table 2.
  • the specific method is referring to Sambrook, J., Fritsch, EF, and Maniatis, T. (1989).
  • the expression vector was transiently transfected into HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the instructions of PEI (purchased from Polysciences, catalog number: 24765-1), and used FreeStyle TM 293 (Thermofisher scientific, catalog number: 12338018) were continuously cultured at 37°C for 5 days, and centrifuged to remove cell components to obtain a culture supernatant containing antibodies.
  • PEI purchased from Polysciences, catalog number: 24765-1
  • FreeStyle TM 293 Thermofisher scientific, catalog number: 12338018
  • the culture supernatant was loaded onto a protein A chromatography column (protein A filler AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borglon (article numbers: AA0273 and B-1620 respectively), washed with PBS phosphate buffer (pH7.4) and then washed with 20mM PB, 1M NaCl (pH7.2), and finally Use citrate buffer at pH 3.4 for elution, collect the Fc-labeled antibody eluted from the protein A chromatography column, neutralize with 1/10 volume of 1M Tris at pH 8.0, and use PBS at 4 Dialyze at °C overnight, and the dialyzed protein is sterile-filtered through a 0.22-micron filter membrane and stored at -80°C.
  • the full-length amino acid sequence encoding human CD16a (158F) (Uniprot ID: P08637), the full-length amino acid sequence of human CD16a (158V) (NCBI ID: AAH17865.1), the full-length amino acid sequence of human CD16b (NA1) (NCBI ID: AAA35881.1), the full-length amino acid sequence of human CD16b (NA2) (Uniprot ID: O75015) and the nucleotide sequence of the full-length amino acid sequence of cynomolgus monkey CD16 (NCBI ID: NP_001270121.1) were cloned into the pcDNA5-FRT vector (Thermofisher scientific, Cat.
  • the transfected FlipinCHO cells were placed in a 37°C, 5% (v/v) CO 2 incubator, and contained 800 ⁇ g/ml hygromycin and 10 Selective culture in F12 medium with % (v/v) fetal bovine serum, and after about 2 weeks, some cells were taken to detect the expression of cell surface antigens by flow cytometry, and the cell lines that recovered growth continued to be expanded and cultured in liquid nitrogen. Freeze.
  • the test results are shown in Table 3 and Figures 2A-2E.
  • the 3G8 antibody can interact with Flpin CHO-human CD16a (158F), Flpin CHO-human CD16a (158V), Flpin CHO-human CD16b (NA1), Flpin CHO-human CD16b ( NA2) combined with Flpin CHO-monkey CD16 cell lines, these cell lines have been able to stably express the corresponding CD16 antigen, which can be used for subsequent antibody screening and identification.
  • the IgG subtype control in Table 3 and Figure 2 is the mouse IgG1 control.
  • the sequence of the BDD20-00 antibody comes from the patent WO2019175368A1. It is an antibody that recognizes human CD16 obtained by screening a fully human phage display library using human CD16-Fc protein. The amino acid sequences of its VL and VH are shown in SEQ ID NO: 12, SEQ ID NO: 13. Using MOE to predict the three-dimensional structure of BDD20-00 antibody, 22 antibodies were designed based on the structure prediction. The above 23 human CD16 antibodies, positive control 3G8 and negative control NC (Anti-Hel) were prepared from N-terminal to C-terminal according to the form of VL-(G 2 S) 7 -VH-his(his ⁇ 6) by Taizhou Baiying Biotechnology Co., Ltd.
  • the dialyzed protein was aseptically filtered through a 0.22-micron membrane filter and stored at -80°C.
  • Antibody VL and VH sequences are shown in Table 4; scFv-His antibody design is shown in Table 5; CD16 antibody CDRs defined according to the Kabat numbering system are shown in Table 6.
  • CD16 scFv-his antibody except for BDD20-02, BDD20-05, BDD20-06, BDD20-11, BDD20-12, BDD20-16, BDD20-20 and BDD20-22, all It can be combined with FlpinCHO-human CD16a cells.
  • Table 7 shows the maximum average fluorescence intensity and EC50 of scFv antibodies detected by FACS and FlpinCHO-CD16a cells.
  • the binding of the antibody to be tested to FlpinCHO-human CD16b cells was detected according to the method in Example 3.1.
  • the 3G8 antibody is a positive antibody binding to CD16b, and the binding activity of all the tested antibodies to FlpinCHO-human CD16b (NA1) and FlpinCHO-human CD16b (NA2) is weaker than that of the 3G8 antibody.
  • Luciferase reporter gene detects the function of human CD16 antibody
  • the Jurkat-NFAT luciferase reporter cell line stably expressing human CD16a (158V) (purchased from BPS Bioscience, Cat. No. 60541) was used to detect the signal pathway in the luciferase reporter cell line activated by the CD16 antibody, and the luciferase signal was detected according to Bright -Glo TM Luciferase Assay System kit (Promega, catalog number: E2620) operating instructions.
  • the experimental method is as follows: 2 ⁇ g/mL anti-his tag antibody (GenScript, product number: A00186) was coated overnight at 4°C to capture the antibody to be tested in the form of scFv-his, and the next day the antibody to be detected was coated with Dilute in RIPM 1640 medium with 5% FBS, the initial antibody concentration is 100nM, dilute 1:5, then add 50 ⁇ l/well to a 96-well flat-bottomed cell culture plate, pre-incubate at 37°C for 15 minutes, and collect in logarithmic For the luciferase reporter cell line in the growth phase, the cell concentration was adjusted to 8 ⁇ 10 5 /mL with medium containing 2% FBS (RPMI 1640, purchased from Gibco, catalog number 12633012), and the cells were added to the culture plate, 50 ⁇ l/well.
  • RPMI 1640 purchased from Gibco, catalog number 12633012
  • BDD20-07, BDD20-08, BDD20-10, BDD20-13, BDD20-14 and BDD20-17 antibodies After combined mutation of the mutation sites of BDD20-07, BDD20-08, BDD20-10, BDD20-13, BDD20-14 and BDD20-17 antibodies, a total of 8 antibodies from BDD20-23 to BDD20-30 were obtained.
  • the antibody was prepared according to the method of Example 2, the antibody sequence is shown in Table 12, the scFv design is shown in Table 13, and the CDR sequence is shown in Table 14:
  • Example 3.1 According to the method of Example 3.1, the binding of the combined mutant antibody to FlpinCHO-human CD16a (158F) and FlpinCHO-human CD16a (158V) was detected.
  • the results are shown in Table 15 and Figures 8A-8B. All combined antibodies can bind to FlpinCHO-human CD16a cell binding, the binding EC50 values of BDD20-23, BDD20-24, BDD20-27, BDD20-28, BDD20-29 and BDD20-30 to different CD16a mutants were all smaller than BDD20-00, BDD20-25, BDD20-26, The EC50 value of BDD20-30 binding to CD16a mutant was comparable to that of BD20-00.
  • Example 3.2 According to the method of Example 3.2, the influence of human immunoglobulin on the binding activity of CD16 antibody was detected, and the results are shown in Table 16 and Figures 9A-9B. All antibodies to be tested combined with FlpinCHO-human CD16a (158F) The binding activity to FlpinCHO-human CD16a(158V) cells is comparable to or higher than that of BDD20-00.
  • the binding of the antibody to be tested to FlpinCHO-human CD16b cells was detected according to the method in Example 3.2. As shown in Figure 10A- Figure 10B and Table 17, at 100nM and 20nM antibody concentrations, the maximum binding fluorescence intensity of all detection antibodies to FlpinCHO-human CD16b (NA1) FlpinCHO-human CD16b (NA2) was weaker than that of the BDD20-00 antibody.
  • Example 4.1 the activation activity of the CD16 combined mutant antibody on the Jurkat-NFAT luciferase reporter cell line stably expressing human CD16a (158V) was detected.
  • the results are shown in Table 18 and Figure 11. In the report assay, all antibodies could activate the downstream signaling pathway of Jurkat cells.
  • Human CD16-ECD-hFc protein was captured using a Protein A chip (GE Healthcare; 29-127-558).
  • Sample and running buffer was HBS-EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25°C.
  • the sample block was set to 16°C. Both were preconditioned with running buffer.
  • the antibody to be tested was first captured with a Protein A chip, and then a single concentration of anti-human CD16 scFv-his antibody was injected to record the binding and dissociation process of the antibody and the antigenic protein, and finally Glycine pH1.5 (Cytiva : BR-1003-54) to complete chip regeneration, wherein the mobile phase is HBS-EP+pH7.4 (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (Cytiva: BR100669), flow rate 30 ⁇ L/min, binding time 240 seconds, dissociation time 600 seconds, regeneration time 30 seconds, detection temperature 25°C.
  • HBS-EP+pH7.4 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20

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Abstract

La présente divulgation concerne un anticorps anti-CD16 et son utilisation. En particulier, la présente invention concerne un anticorps ou un fragment de liaison à l'antigène se liant de manière spécifique au CD16, un acide nucléique codant, un vecteur d'expression et une cellule d'expression associés, un procédé de préparation, une composition pharmaceutique et une utilisation de ceux-ci dans le traitement de maladies, par exemple, une utilisation dans le traitement de tumeurs. L'invention présente une grande importance pour le développement d'un médicament thérapeutique à base d'anticorps anti-CD16 et d'un réactif de détection.
PCT/CN2022/109574 2021-08-03 2022-08-02 Anticorps anti-cd16 et son utilisation Ceased WO2023011431A1 (fr)

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CN118878695A (zh) * 2024-07-30 2024-11-01 郑州市乘黄生物医药科技有限公司 结合cd16a和her3的双特异性抗体-nk细胞偶联物及其用途
WO2024230345A1 (fr) * 2023-05-10 2024-11-14 Suzhou Biomissile Pharmaceuticals Co., Ltd. Anticorps anti-cd16a et leurs utilisations
CN119080933A (zh) * 2024-09-12 2024-12-06 武汉海沙百得生物技术有限公司 靶向CD16b的抗体及其应用
WO2024251628A1 (fr) * 2023-06-06 2024-12-12 Molecular Partners Ag Protéines de liaison à cd16a recombinantes et leur utilisation
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WO2024230345A1 (fr) * 2023-05-10 2024-11-14 Suzhou Biomissile Pharmaceuticals Co., Ltd. Anticorps anti-cd16a et leurs utilisations
WO2024251628A1 (fr) * 2023-06-06 2024-12-12 Molecular Partners Ag Protéines de liaison à cd16a recombinantes et leur utilisation
WO2025140623A1 (fr) * 2023-12-27 2025-07-03 博生吉医药科技(苏州)有限公司 Nanocorps anti-cd16a, son procédé de préparation et son utilisation
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CN118878695B (zh) * 2024-07-30 2025-08-12 郑州市乘黄生物医药科技有限公司 结合cd16a和her3的双特异性抗体-nk细胞偶联物及其用途
CN119080933A (zh) * 2024-09-12 2024-12-06 武汉海沙百得生物技术有限公司 靶向CD16b的抗体及其应用

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