WO2024017326A1 - Nanocorps anti-gprc5d et son utilisation - Google Patents

Nanocorps anti-gprc5d et son utilisation Download PDF

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WO2024017326A1
WO2024017326A1 PCT/CN2023/108360 CN2023108360W WO2024017326A1 WO 2024017326 A1 WO2024017326 A1 WO 2024017326A1 CN 2023108360 W CN2023108360 W CN 2023108360W WO 2024017326 A1 WO2024017326 A1 WO 2024017326A1
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antibody
antigen
amino acid
seq
gprc5d
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PCT/CN2023/108360
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English (en)
Chinese (zh)
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成广存
付雅媛
李玲
王义芳
曹卓晓
唐任宏
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山东先声生物制药有限公司
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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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas

Definitions

  • the present application relates to the field of antibodies, specifically to anti-GPRC5D Nanobodies.
  • Nanobodies generally have a long CDR3, which can form a stable large convex ring structure and can bind to some hidden epitopes. They are especially suitable for targets where antibodies are difficult to obtain, such as GPCRs, ion channels, and enzyme activity centers.
  • VHH nanobodies have small molecular weight, are easy to express in vitro, have good solubility, and have weak immunogenicity. They can pass through some protective barriers in the body, such as the blood-brain barrier, and enter the disease site to play a role.
  • MM Multiple myeloma
  • MM is a plasma cell malignant tumor characterized by the unrestricted proliferation of plasma cells in the bone marrow like tumor cells, accompanied by the secretion of monoclonal immunoglobulins, resulting in multiple lysates.
  • a series of clinical manifestations include bone damage, hypercalcemia, anemia, kidney damage, and recurrent infections.
  • Hematopoietic stem cell transplantation and combined therapy with immunomodulators and protease inhibitors can significantly improve the prognosis.
  • CAR T therapy has made breakthrough progress in multiple myeloma.
  • CAR T targeting B cell maturation antigen (BCMA) has shown positive clinical results in the treatment of multiple myeloma.
  • GPRC5D is an orphan receptor of G protein-coupled receptors (GPCRs). It is subtype D of the C5 family of G protein-coupled receptors and is a 7-transmembrane protein. GPRC5D has three exons, of which the amino-terminal domain is shorter, and its ligands and signaling mechanisms have not yet been clearly defined. GPRC5D is highly conserved among different species, with 92% homology between human and cynomolgus monkey GPRC5D. GPRC5D is highly expressed in multiple myeloma cells, but hardly expressed in normal tissues, and is limited to the hair follicle area. Since hair follicles are immune-privileged sites, based on this characteristic, GPRC5D has become an ideal target for the treatment of multiple myeloma (MM).
  • GPCRs G protein-coupled receptors
  • GPRC5D is mainly expressed in malignant plasma cells of MM patients. It has better specificity than BCMA. At the same time, the two expressions are independent. It can be targeted alone or dual-targeted to develop therapeutic drugs.
  • BCMA-targeted immunotherapy there are many studies on BCMA-targeted immunotherapy, but there is a lack of effective treatments for BCMA-negative or BCMA-low-expressing MM patients and patients with disease relapse due to antigen immune escape after BCMA-targeted therapy. treatment approach. Therefore, the development of therapeutic drugs targeting GPRC5D is expected to be the key to solving this problem.
  • the present disclosure provides a Nanobody that can specifically bind to G protein-coupled receptor C5 family subtype D (GPRC5D), Compared with full-length antibodies, it can better bind to difficult-to-bind antigens such as GPRC5D. At the same time, it provides new treatment methods for patients with no or low expression of BCMA and poor prognosis after BCMA target treatment.
  • GPRC5D G protein-coupled receptor C5 family subtype D
  • the present disclosure also provides antibodies that specifically bind GPRC5D or antigen-binding fragments thereof, multispecific antigen-binding molecules, nucleic acid fragments, vectors, host cells, preparation methods, pharmaceutical compositions, pharmaceutical uses, and tumors or cancers (e.g., B lymphoma or multiple myeloma).
  • the disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to human or monkey GPRC5D, the antibody or an antigen-binding fragment thereof comprising CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO: HCDR1, HCDR2 and HCDR3 of the VHH domain shown in any one of the sequences 10 to 13, 50 to 75, 76 to 77 and 96 to 99.
  • the present disclosure also provides a multispecific antigen-binding molecule, which comprises the aforementioned antibody or antigen-binding fragment thereof, and an antigen-binding molecule that binds to other antigens other than GPRC5D, or that binds to A GPRC5D epitope different from the aforementioned antibody or antigen-binding fragment thereof.
  • the present disclosure also provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment thereof or the aforementioned multispecific antigen-binding molecule.
  • the present disclosure also provides a vector comprising the aforementioned nucleic acid fragment.
  • the present disclosure also provides a host cell comprising the aforementioned nucleic acid fragment or the aforementioned vector.
  • the present disclosure also provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof or the aforementioned multi-specific antigen-binding molecule.
  • the method includes culturing the aforementioned host cell and isolating the antibody expressed by the host cell. Or its antigen-binding fragment or the aforementioned multi-specific antigen-binding molecule.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multi-specific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned carrier or the aforementioned carrier according to the aforementioned
  • the product is prepared by the method.
  • the present disclosure also provides a method for treating tumors or cancer, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned Nucleic acid fragments, the aforementioned vectors, products prepared according to the aforementioned methods or the aforementioned pharmaceutical compositions.
  • the present disclosure also provides the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned vector, the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition. Use in the preparation of drugs for treating tumors or cancer.
  • the present disclosure also provides the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned vector, the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition. , used to treat tumors or cancer.
  • the present disclosure also provides a kit comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned vector, according to the aforementioned The product obtained by the method or the aforementioned pharmaceutical composition is prepared.
  • the present disclosure also provides a method for detecting GPRC5D expression in a biological sample, the method comprising The biological sample is brought into contact with the antibody or antigen-binding fragment thereof under conditions capable of forming a complex between the aforementioned antibody or its antigen-binding fragment and GPRC5D.
  • the present disclosure also provides the use of the aforementioned antibody or antigen-binding fragment thereof in preparing a GPRC5D detection reagent.
  • the antibodies provided by the present disclosure have excellent binding ability to the GPRC5D antigen of humans and monkeys, and the development of drugs and immunotherapy based on the antibodies of the present disclosure is useful for improving BCMA target treatment of relapsed or BCMA-negative patients.
  • the survival rate and prognosis of myeloma patients have high application value.
  • Figure 1A-1C shows the FACS detection of the expression levels of 293T-human GPRC5D, CHO-K1-human GPRC5D and 293T-cyno GPRC5D cell lines.
  • Figure 2 shows the FACS detection of the binding reaction between Lab01-huFc, Lab02-huFc, Lab03-huFc and Lab04-huFc recombinant antibodies and human multiple myeloma cells NCI-H929.
  • Figure 3A shows the FACS detection of the binding reaction of Lab01-huFc, Lab02-huFc, Lab03-huFc and Lab04-huFc recombinant antibodies with overexpressed cells 239T-human GPRC5D.
  • Figure 3B shows the FACS detection of the binding reaction between Lab01-huFc, Lab02-huFc, Lab03-huFc and Lab04-huFc recombinant antibodies and overexpressed cells 239T-cyno GPRC5D.
  • Figure 3C shows the FACS detection of the binding reaction of Lab01-huFc, Lab02-huFc, Lab03-huFc and Lab04-huFc recombinant antibodies with negative control cell 239T.
  • Figures 4A-4D are respectively ELISA detection of the binding reactions of HAB01, HAB02, HAB03 and HAB04 humanized antibodies with human GPRC5D protein.
  • Figures 5A-5D are respectively FACS detection of the binding reactions of HAB01, HAB02, HAB03 and HAB04 humanized antibodies with human multiple myeloma cells NCI-H929.
  • Figures 6A-6D are respectively FACS detection of the binding reactions of HAB01, HAB02, HAB03 and HAB04 humanized antibodies with human multiple myeloma cells molp-8.
  • Figures 7A-7D are respectively FACS detection of the binding reactions of HAB01, HAB02, HAB03 and HAB04 humanized antibodies with human multiple myeloma cells RPMI-8226.
  • Figure 8 shows the FACS detection of the binding reaction of recombinant antibodies and humanized antibodies with overexpressed cells CHOK1-human GPRC5D.
  • Figure 9 shows the FACS detection of the binding reaction of recombinant antibodies and humanized antibodies with human multiple myeloma cells NCI-H929.
  • Figure 10 shows the FACS detection of the binding reaction of recombinant antibodies and humanized antibodies with human multiple myeloma cells molp-8.
  • Figure 11 shows the FACS detection of the binding reaction of recombinant antibodies and humanized antibodies with human multiple myeloma cells RPMI-8226.
  • compositions including A and B should be understood as the following technical solution: a composition composed of A and B, as well as a composition containing other components in addition to A and B, all fall into the category Within the scope of the aforementioned "a composition”.
  • GPRC5D refers to G protein-coupled receptor C5 family subtype D, which is an orphan receptor and is a 7-transmembrane protein with no known ligand. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while its expression in normal tissues is limited to the hair follicle area. Studies have shown that 65% of multiple myeloma patients have GPRC5D exceeding the 50% expression threshold. With this feature, GPRC5D has become a potential target for the treatment of MM.
  • KD equilibrium dissociation constant
  • high affinity generally refers to having a KD of about 10 -6 M or lower, 10 -7 M or lower, about 10 -8 M or lower, or about 10 -9 M or lower.
  • the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis determination.
  • antigen-binding molecule refers to a molecule that specifically binds an antigen.
  • antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetic 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 mimetic includes but is not limited to affibody, affitin, affilin, and designed ankyrin repeat proteins. (DARPin), aptamer or Kunitz type domain peptide.
  • antibody is used herein in its broadest sense and refers to an antibody that contains sufficient sequence from the variable domain of an immunoglobulin heavy chain and/or sufficient sequence from the variable domain of an immunoglobulin light chain to be capable of specifically binding to an antigen. Polypeptide or combination of peptides. "Antibody” as used herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity. “Antibody” as used herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives.
  • CDRs complementarity determining regions
  • Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, for example, 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 intact antibodies and any antigen-binding fragments (i.e., “antigen-binding portions”) or single chains thereof.
  • Antibody refers to a glycoprotein, or an antigen-binding portion thereof, containing at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (herein abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • each VH and VL They are composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody can mediate 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 in this article can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain respectively. chain, gamma chain, alpha chain and epsilon chain.
  • Ig immunoglobulins
  • the same type of Ig can be divided into different subclasses based on differences in 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, and IgG4.
  • IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have either a kappa chain or a lambda chain.
  • Antibodies as used herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpacas.
  • Heavy-chain antibodies produced by camelids such as Vicugna pacos and immunoglobulin neoantigen receptors (IgNAR) found in cartilaginous fishes such as sharks.
  • antibody herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from the group consisting of primates, mammals, rodents and vertebrates, such as camelids, large animals, etc. Alpaca, guanaco, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (eg shark).
  • heavy chain antibody refers to an antibody that lacks the light chain of a conventional antibody.
  • 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.
  • anobody in this article refers to the natural heavy chain antibody lacking the light chain that exists in camels and other bodies. Cloning its variable region can obtain a single domain antibody consisting only of the heavy chain variable region, also known as VHH ( Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Variable domain of heavy chain of heavy chain antibody
  • VHH domain refers to the variable region of a cloned heavy chain antibody, which is constructed only by A single domain antibody consisting of a heavy chain variable region, which is the smallest fully functional antigen-binding fragment.
  • a heavy chain antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1)
  • CH1 light chain and heavy chain constant region 1
  • multispecificity refers to the ability of an antibody or antigen-binding fragment thereof to bind, for example, to different antigens or to at least two different epitopes on the same antigen.
  • terms such as “bispecific,””trispecific,””tetraspecific,” etc. refers to the number of different epitopes that an antibody can bind to.
  • conventional monospecific IgG-type antibodies have two identical antigen-binding sites (paratopes) and therefore can only bind to the same epitope (rather than to different epitopes).
  • multispecific antibodies have at least two different types of paratopes/binding sites and therefore can bind to 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").
  • price the actual number of CDRs/binding sites in a single antibody molecule.
  • a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes.
  • a multispecific antibody contains at least two (different) complementarity determining regions/binding sites.
  • the term “multispecific antibody” refers to an antibody that has more than one paratope and the ability to bind two or more different epitopes.
  • multispecific antibody includes in particular bispecific antibodies as defined above, but generally also includes proteins, such as antibodies, scaffolds that specifically bind three or more different epitopes, i.e. having three or more different epitopes. Antibodies with more than three paratopes/binding sites.
  • valency herein refers to the presence of a specified number of binding sites in the antibody/antigen binding molecule. Therefore, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites respectively in the antibody/antigen binding molecule. existence of points.
  • Fully-length antibody “intact antibody” and “intact antibody” are used interchangeably herein and refer to having a structure that is substantially similar to the structure of a native antibody.
  • Antigen-binding fragment and “antibody fragment” herein are used interchangeably. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have Ability to bind antigen.
  • antigen-binding fragments or “antibody fragments” herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single domains Antibody.
  • chimeric antibody refers to an antibody having variable sequences of immunoglobulins derived from one source organism (e.g. rat, mouse, rabbit or alpaca) and derived from a different organism (e.g. human) immunoglobulin constant region.
  • source organism e.g. rat, mouse, rabbit or alpaca
  • a different organism e.g. human immunoglobulin constant region.
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology with that of a human antibody.
  • CDR region of a humanized antibody comes 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) comes from a human source.
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.
  • Fully human antibody refers to an antibody having variable regions in which both FRs and CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are 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 refers to the region in the heavy or light chain of an antibody involved in allowing the antibody to bind to the antigen.
  • "Heavy chain variable region” is used interchangeably with “VH” and “HCVR”
  • "light chain variable region” is used interchangeably.
  • Area is used interchangeably with “VL” and “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three a hypervariable region (HVR). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • CDR complementarity determining region
  • HVR hypervariable regions
  • FR framework region
  • amino acid positions representing the hypervariable regions of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT) but not considered to be within a different set of criteria (such as KABAT or IMGT). One or more of these locations may also be found in extended hypervariable zones.
  • the present disclosure includes antibodies comprising modifications in these hybrid hypervariable positions.
  • the heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region can be abbreviated as LCDR.
  • the variable domains of native heavy and light chains each contain four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops that connect the sheet structure , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held closely together by the FR region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, MD. 1987; which is incorporated herein by reference).
  • CDR in this article 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.
  • the tool websites used include but are not limited to AbRSA website (http://cao.labshare .cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign .cgi#results).
  • CDRs herein include overlaps and subsets of differently defined amino acid residues.
  • Kabat numbering system 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 classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system in this article 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 refers to the carboxyl-terminal portion of the antibody heavy chain that is not directly involved in binding of the antibody to the antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's Variable domains have more conserved amino acid sequences.
  • the “heavy chain constant region” may be selected from the group consisting of 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 includes only "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 When an antibody is used, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from Fc or CH3 domains.
  • light chain constant region refers to the carboxyl-terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen.
  • the light chain constant region may 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.
  • the human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxy terminus of the heavy chain.
  • antibodies produced by the host cell may undergo post-translational cleavage, excluding one or more, particularly one or two amino acids, 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 comprise a full-length heavy chain, or it may comprise a cleaved variant of a full-length heavy chain.
  • This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may be present or absent.
  • the IgG Fc region contains IgG CH2 and IgG CH3 domains, and optionally may also contain a complete or partial hinge region, but not a CH1 domain.
  • the "CH2 domain" of the human IgG Fc region generally extends from amino acid residues at approximately position 231 to amino acid residues at approximately position 340. In one embodiment, the carbohydrate chain is attached to the CH2 domain.
  • the CH2 domains herein may be native sequence CH2 domains or variant CH2 domains.
  • “CH3 domain” includes that portion of the Fc region that is C-terminal to the CH2 domain (i.e., from amino acid residues at about position 341 to about amino acid residues at position 447 of IgG).
  • a CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., having "knobs” introduced in one strand thereof and a corresponding "cavity” introduced in the other strand thereof "Hole”, CH3 domain; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). As described herein, such variant CH3 domains can be used to promote heterodimerization of two different antibody heavy chains.
  • the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also known as the EU index, such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, Described in National Institutes of Health, Bethesda, MD, 1991.
  • Fc variant herein refers to changes in the structure or function of the Fc caused by the presence of one or more amino acid substitutions, insertions or deletions at appropriate sites on the Fc.
  • Interaction between Fc variants refers to the interaction between Fc variants designed by mutation, which can form space filling effects, electrostatic guidance, hydrogen bonding interactions, hydrophobic interactions, etc. Interactions between Fc variants contribute to the formation of stable heterodimeric proteins.
  • a preferred mutation design is a "Knob-into-Hole" form of mutation design.
  • Fc variants have been widely used in the field to prepare bispecific antibodies or heterodimeric Fc fusion protein forms.
  • Representative ones include the "Knob-into-Hole” form proposed by Cater et al.; Amgen technicians used electrostatic steering (Electrostatic Steering) to form an Fc-containing heterodimer form (US 20100286374 A1); Jonathan H.
  • heterodimer form formed by IgG/Ig chain exchange proposed by others; the bispecific molecule formed by Genmab's DuoBody platform technology; Xencor's technicians combined structural calculations and Fc amino acid mutations to synthesize different effects to form heterodimeric protein forms (mAbs 3: 6, 546-557; November/December 2011); Suzhou Alphamab’s Fc transformation method based on charge network (CN201110459100.7) obtains heterodimeric proteins form; and other genetic engineering methods based on Fc amino acid changes or functional modification methods to achieve the formation of heterodimer functional proteins.
  • the Knob/Hole structure on the Fc variant fragment described in the present disclosure refers to the mutation of each of the two Fc fragments, and after the mutation, they can be combined in the form of "Knob-into-Hole". It is preferable to use the "knob-into-hole" model of Cater et al. to carry out site mutation transformation on the Fc region, so that the resulting first Fc variant and the second Fc variant can be in the form of "knob-into-hole" Combine together to form heterodimers. Selection of specific immunoglobulin Fc regions from specific immunoglobulin classes and subclasses is within the purview of those skilled in the art.
  • Preferred human antibodies The Fc region of IgG1, IgG2, IgG3, and IgG4, and more preferably the Fc region of human antibody IgG1. Randomly select one of the first Fc variant or the second Fc variant to make a knob mutation, and the other to make a hole mutation.
  • amino acids 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 conserved amino acid residues, and the substitution of amino acid residues within the group belongs to the substitution of conservative amino acids:
  • identity can be calculated by aligning the sequences for optimal comparison purposes (e.g., for the purpose of determining the percent "identity" of two amino acid sequences or two nucleic acid sequences).
  • the alignment may 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.
  • the molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • the percent identity between two sequences varies as a function of the identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences. For example, using the Needlema and Wunsch algorithms already integrated into the GAP program of the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or the 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. As another example, using the GAP program in the GCG software package (available at www.gcg.com), use the NWSgapdna.CMP matrix with gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and the 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. You can also use the PAM120 weighted remainder table, the gap length penalty of 12, the gap penalty of 4, and the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0) to determine the relationship between two amino acid sequences or nucleotide sequences. percent identity.
  • nucleic acid sequences and protein sequences described in the present disclosure may further be used as "query sequences" to perform searches against public databases, for example, to identify other family member sequences or related sequences.
  • the NBLAST and XBLAST programs (version 2.0) can be used to perform such searches.
  • the default parameters of the corresponding programs eg, XBLAST and NBLAST
  • the default parameters of the corresponding programs eg, XBLAST and NBLAST
  • nucleic acid herein includes any compound and/or substance that contains a polymer of nucleotides.
  • Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), Composed of sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • a nucleic acid molecule is 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 as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA containing two A polymer that is a mixture 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 the antibodies of the present disclosure in vitro and/or in vivo, such as in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • 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 produce 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. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule but which are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is 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 refers to a cell into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the originally transformed cells are included herein.
  • pharmaceutical composition refers to a preparation that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain substances that are unacceptable to the subject administered the pharmaceutical composition. Toxicity of additional ingredients.
  • pharmaceutically acceptable carrier herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorbent agents, etc. Delay agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. and combinations thereof are known to those skilled in the art. Except where incompatible with the active ingredient, any conventional carrier is contemplated for use in the therapeutic or pharmaceutical compositions.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as cancer and tumors.
  • Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented.
  • slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • a “subject” refers to an organism undergoing treatment for a particular disease or condition as described herein.
  • a “subject” includes a mammal such as a human, a primate (eg, a monkey), or a non-primate mammal undergoing treatment for a disease or condition.
  • an effective amount refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, is effective in preventing or ameliorating the symptoms of a disease or the progression of a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • cancer refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers.
  • tumor or “tumor” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” as used herein are not mutually exclusive.
  • EC50 refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents 50% of the antibody concentration at which its maximum effect is observed and can be measured by methods known in the art.
  • the present disclosure provides a Nanobody that can specifically bind to G protein-coupled receptor C5 family subtype D (GPRC5D). Compared with a full-length antibody, it can better bind to difficult-to-bind antigens such as GPRC5D. At the same time, it provides new treatment methods for patients with no or low expression of BCMA and poor prognosis after BCMA target treatment.
  • GPRC5D G protein-coupled receptor C5 family subtype D
  • the present disclosure also provides antibodies that specifically bind GPRC5D or antigen-binding fragments thereof, multispecific antigen-binding molecules, nucleic acid fragments, vectors, host cells, preparation methods, pharmaceutical compositions, pharmaceutical uses, and tumors or cancers (e.g., B lymphoma or multiple myeloma).
  • the disclosure provides an antibody or an antigen-binding fragment thereof that specifically binds to human or monkey GPRC5D, the antibody or an antigen-binding fragment thereof comprising CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 respectively comprising SEQ ID NO: HCDR1, HCDR2 and HCDR3 of the VHH domain represented by any one of the sequences 10 to 13, 50 to 75, 76 to 77 and 96 to 99.
  • the HCDR1, HCDR2 and HCDR3 are determined according to the IMGT, Kabat or Chothia numbering system and are selected from any one of the following:
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 14, 15 and 16; the amino acid sequences shown in SEQ ID NO: 26, 27 and 28 ; Or the amino acid sequence shown in SEQ ID NO: 38, 39 and 28;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 17, 18 and 19; the amino acid sequences shown in SEQ ID NO: 29, 30 and 31 ; Or the amino acid sequence shown in SEQ ID NO: 40, 41 and 31;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 20, 21 and 22; the amino acid sequences shown in SEQ ID NO: 32, 33 and 34 ; Or the amino acid sequence shown in SEQ ID NO: 42, 43 and 34;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 23, 24 and 25; the amino acid sequences shown in SEQ ID NO: 35, 36 and 37 ; Or the amino acid sequence shown in SEQ ID NO: 44, 45 and 37;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 78, 79 and 80; the amino acid sequences shown in SEQ ID NO: 84, 85 and 86 ; Or the amino acid sequence shown in SEQ ID NO: 90, 91 and 86;
  • the HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 81, 82 and 83; the amino acid sequences shown in SEQ ID NO: 87, 88 and 89 ; Or the amino acid sequence shown in SEQ ID NO: 92, 93 and 89;
  • the CDR1, CDR2 and/or CDR3 comprise at least 80, 85%, 90%, 91%, 92%, 93%, 94% compared to the aforementioned HCDR1, HCDR2 and/or HCDR3. , 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.
  • the antibody or antigen-binding fragment thereof is a single domain antibody, and the single domain antibody includes the aforementioned CDR1, CDR2 and CDR3.
  • the single domain antibody comprises the amino acid sequence shown in any one of SEQ ID NOs: 10-13, 50-75, 76-77, and 96-99.
  • the single domain antibody includes at most 20, 19, and 18 sequences compared to the sequence shown in any one of SEQ ID NOs: 10-13, 50-75, 76-77, and 96-99. , 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
  • a mutated amino acid sequence the mutation is selected from insertion, deletion and/or substitution, and the substitution is preferably a conservative amino acid substitution.
  • the single domain antibody comprises at least 80%, 85%, 90% of the sequence shown in any one of SEQ ID NOs: 10-13, 50-75, 76-77 and 96-99. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.
  • the antibody comprises the FR region in the VHH domain shown in any one of SEQ ID NOs: 10-13, 50-75, 76-77, and 96-99.
  • the antibody comprises at most 15, 14 or more FR regions in the VHH domain shown in any one of SEQ ID NOs: 10-13, 50-75, 76-77 and 96-99. 1, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutated amino acid sequences, and the mutations are selected from Insertion, deletion and/or substitution, the substitution is preferably a substitution of conservative amino acids.
  • the antibody comprises at least 80%, 85% of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequences.
  • the antibody or antigen-binding fragment thereof is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof.
  • the antibody or antigen-binding fragment thereof includes or does not include an antibody heavy chain constant region; optionally, the antibody heavy chain constant region is selected from the group consisting of human, alpaca, mouse, rat, Rabbit or sheep; optionally, the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE or IgD; optionally, the IgG is selected from IgG1, IgG2, IgG3 or IgG4; optionally, the The heavy chain constant region is selected from the group consisting of Fc region, CH3 region or complete heavy chain constant region.
  • the heavy chain constant region is a human Fc region; preferably, the antibody or antigen-binding fragment thereof is a heavy chain antibody.
  • the antibody or antigen-binding fragment thereof is also coupled with a therapeutic agent or tracer; preferably, the therapeutic agent is selected from radioactive isotopes, chemotherapeutics, immunomodulators, or combinations thereof; preferably Preferably, the tracer is selected from radiological contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents, photosensitizers or combinations thereof.
  • the antibody or its antigen-binding fragment is also connected to other functional molecules.
  • the other functional molecules are selected from one or more of the following: signal peptide, protein tag, cell factors, angiogenesis inhibitors, or immune checkpoint inhibitors.
  • the present disclosure also provides a multispecific antigen-binding molecule, which comprises the aforementioned antibody or antigen-binding fragment thereof, and an antigen-binding molecule that binds to other antigens other than GPRC5D, or that binds to GPRC5D epitopes different from the aforementioned antibodies or antigen-binding fragments thereof; optionally, the other antigens other than GPRC5D are selected from: CD3 (preferably CD3 ⁇ ), CD16, CD137, CD258, PD-1, PD-L1, 4 -1BB, CD40, CD64, EGFR, VEGF, HER2, HER1, HER3, IGF-1R, phosphatidylserine (PS), C-Met, HSA, BCMA, MSLN, blood-brain barrier receptor, GPC3, PSMA, CD33, GD2, ROR1, ROR2, FR ⁇ or Gucy2C.
  • CD3 preferably CD3 ⁇
  • the antigen-binding molecule that binds to other antigens other than GPRC5D is an antibody or an antigen-binding fragment thereof.
  • the multispecific antigen-binding molecule may be bispecific, trispecific or tetraspecific.
  • the multispecific antigen-binding molecule may be bivalent, trivalent, tetravalent, pentavalent or hexavalent.
  • the present disclosure also provides an isolated nucleic acid fragment encoding the aforementioned antibody or antigen-binding fragment thereof or the aforementioned multispecific antigen-binding molecule.
  • the present disclosure also provides a vector comprising the aforementioned nucleic acid fragment.
  • the present disclosure also provides a host cell, which contains the aforementioned nucleic acid fragment or the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria such as Escherichia coli, fungi such as Yeast, insect cells or mammalian cells, such as CHO cell line or 293T cell line.
  • a host cell which contains the aforementioned nucleic acid fragment or the aforementioned vector; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria such as Escherichia coli, fungi such as Yeast, insect cells or mammalian cells, such as CHO cell line or 293T cell line.
  • the present disclosure also provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof or the aforementioned multi-specific antigen-binding molecule.
  • the method includes culturing the aforementioned host cell and isolating the antibody expressed by the host cell or Its antigen-binding fragment or the aforementioned multi-specific antigen-binding molecule.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multi-specific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned carrier or the aforementioned carrier according to the aforementioned
  • the product is prepared by a method; optionally, the pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent or auxiliary agent; optionally, the pharmaceutical composition also includes an additional anti-tumor agent agent.
  • the present disclosure also provides a method for treating tumors or cancer, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned Nucleic acid fragment, front
  • the above-mentioned vector, the product prepared according to the above-mentioned method or the above-mentioned pharmaceutical composition is preferably B-cell lymphoma; more preferably it is multiple myeloma (MM).
  • the present disclosure also provides the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned vector, the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition in Use in the preparation of drugs for treating tumors or cancer;
  • the tumor or cancer is preferably B-cell lymphoma; more preferably it is multiple myeloma (MM).
  • the present disclosure also provides the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned vector, the product prepared according to the aforementioned method, or the aforementioned pharmaceutical composition,
  • the tumor or cancer is preferably B-cell lymphoma; more preferably it is multiple myeloma (MM).
  • the present disclosure also provides a kit comprising the aforementioned antibody or antigen-binding fragment thereof, the aforementioned multispecific antigen-binding molecule, the aforementioned nucleic acid fragment, the aforementioned vector, according to the aforementioned The product obtained by the method or the aforementioned pharmaceutical composition is prepared.
  • the present disclosure also provides a method for detecting the expression of GPRC5D in a biological sample, which method includes causing the aforementioned antibody or antigen-binding fragment thereof to form a complex with GPRC5D under conditions.
  • a biological sample is contacted with said antibody or antigen-binding fragment thereof; preferably, said method further comprises detecting the formation of said complex indicating the presence or expression level of GPRC5D in the sample.
  • the present disclosure also provides the use of the aforementioned antibody or antigen-binding fragment thereof in preparing a GPRC5D detection reagent.
  • the control antibodies used in this example are all from published patent sequences.
  • the JNJ7564 and 5F11 antibody sequences are from published patents WO2020148677A1 and WO2019154890A1. Unless otherwise specified, the JNJ7564 and 5F11 control antibodies use human IgG1+ ⁇ subtype. Perform recombinant expression.
  • the Nanobodies and their humanized antibodies used in the examples were all recombinantly expressed in human Fc fusion form. The specific sequence information of the antibodies is shown in Table 1.
  • hinge region sequence in bold in the human IgG1Fc sequence contains the C220S mutation, which is underlined.
  • the obtained antibody sequences were cloned into the eukaryotic expression vector pTT5 (Ubao Biotechnology, VT2202), and transiently transfected into Expi293F cells (Gibco, A14527) through PEI (Polysciences, 24765-1). After 7 days of culture, the expressed antibodies were collected by high-speed centrifugation. of cell culture supernatant. Use 3-5 times the column volume of 0.1M NaOH to wash the Protein A (Borgron, AA0273) protein chromatography column, and then use 3-5 times the column volume of pure water. Use 3-5 times the column volume of 1 ⁇ PBS (pH7.4) buffer system as the balancing buffer to balance the chromatography column.
  • the cell supernatant is loaded and combined with a low flow rate, and the flow rate is controlled so that the retention time is about 1 minute or longer.
  • control antibodies were named JNJ7564-hlgG1 and 5F11-hlgG1.
  • the nucleotide sequence encoding the full-length amino acid sequence of human GPRC5D (UniProt: Q9NZD1, SEQ ID NO: 8) was cloned into the pLVX lentiviral vector (Ubao Biotechnology, VT1465), and the virus was prepared in HEK293T (purchased from ATCC) cells Particles.
  • fetal calf serum containing 2 ⁇ g/ml puromycin was Selectively cultured in DMEM medium for 2 weeks, using human anti-human GPRC5D antibody (JNJ7564-hIgG1, prepared as described in Section 1.2) and goat anti-human IgG (H+L) antibody (Jackson, 109605088) for flow cytometry FACS CantoII (purchased from BD Biosciences) was used for detection, and cells with high expression levels and single peak shapes were amplified, and the amplified cells were retested by flow cytometry analysis.
  • human anti-human GPRC5D antibody JNJ7564-hIgG1, prepared as described in Section 1.2
  • goat anti-human IgG (H+L) antibody Jackson, 109605088
  • the obtained cell line was named 293T-human GPRC5D.
  • the expression of the cell line detected by FACS is shown in Figure 1A. CHOK1 cells were stably transfected using the same method, and the resulting cell line was named CHO-K1-human GPRC5D.
  • the expression of cell lines detected by FACS is shown in Figure 1B.
  • the nucleotide sequence encoding the full-length amino acid sequence of cyno GPRC5D NCBI: The cell line was named 293T-cyno GPRC5D, and the expression of the cell line detected by FACS is shown in Figure 1C.
  • Cyno GPRC5D full-length amino acid sequence (NCBI: XP_005570249.1, SEQ ID NO: 9):
  • the CHO-K1-human GPRC5D overexpression cell line was used to immunize four times, and the amount of cellular immunity each time With 2 ⁇ 10 7 cells per camel, the serum titer results of the sixth immunization are shown in Table 2.
  • the data in the table are OD450nm values.
  • SfiI purchased from NEB, R0123L
  • the ligation product was then electroporated into TG1 (Lucigen, 60502) competent cells, and a total of 8 times of electroporation transformation were performed. Immediately after electroporation, 1 mL of SOC medium was added to the electroporation cup for recovery and culture for 1 hour. A total of 8.4 ml of TG1 cell recovery product was obtained.
  • the TG1 bacterial liquid was serially diluted 10 4 and 10 5 times, and the number of transformants in the Nanobody library was determined. The remaining bacterial liquid was spread on eight 150 mm ampicillin-resistant plates. The sizes of the calculated storage capacities are 4.9 ⁇ 10 10 and 1.7 ⁇ 10 10 respectively. Sequencing of 96 samples showed that the insertion rate of the library was about 99% and there were no repetitive sequences, indicating that the phage library was successfully constructed.
  • Human GPRC5D overexpressing cells were used as antigens for phage library panning.
  • the specific panning strategy is shown in Table 3.
  • Human GPRC5D overexpressing cells (target cells) were used in panning rounds 1, 3, and 5.
  • negative cells 293T were used for negative selection to remove non-specific binding.
  • the specific operation process is as follows: The first round of phages that bind to the target cells are recovered and amplified overnight. In the second round, the selected phages from the first round are applied to background cells, unbound phages are recovered for the third round of panning, and the third round phages that bind to the target cells are recovered and amplified overnight, and the fourth round is carried out. Round negative round panning. To perform a final round, split the amplified phage from round 4 of negative selection into two panning samples, one for target cells and the other for background cell panning.
  • Gly-HCl was used for elution, and the phages selected in the 1st, 3rd, and 5th rounds were added to E. coli TG1 (Lucigen, 60502) in the logarithmic growth phase, and incubated at 37°C for 30 minutes to obtain a culture medium of TG1. Gradient dilute the culture solution of TG1, spread it on the plate, and culture it at 37°C overnight. The number of clones in the 293T-human GPRC5D cell group and the control group was calculated, and 48 clones were selected for sequencing.
  • the clones on the plate were washed and collected with 2YT medium (Sangon, A507019), inoculated into fresh medium, and cultured at 37°C to the logarithmic phase.
  • 2YT medium Wang, A507019
  • helper phage M13KO7 N0315S
  • the ratio of helper phage to E. coli TG1 is 20:1, mix well, and let stand at 37°C for 30 minutes.
  • incubate with shaking at 37°C for 30 minutes, 4000rpm Collect the cells after centrifugation for 10 minutes, add fresh 2YT medium containing ampicillin and kanamycin resistance, and culture with shaking at 30°C overnight.
  • NCI-H929 ATCC, CRL-9068
  • overexpression cell lines 293T-human GPRC5D and 293T-cyno GPRC5D were expanded and cultured in T-175 cell culture flasks to 90% confluence.
  • the overexpression cell line was drained of the culture medium, washed once with PBS buffer, and then treated with Trypsin-EDTA (Gibco, 25200072) and collected. After counting the cells, wash the cells twice with PBS phosphate buffer, dilute to 2 ⁇ 10 6 cells per ml, and add 50 ⁇ L per well to a 96-well FACS reaction plate.
  • Nanobody sequences were cloned into the eukaryotic expression vector pTT5 with hIgG1-Fc tag, transiently transfected into Expi293F cells (Gibco, A14527) via PEI, and cultured for 7 days.
  • the antibody-expressing cell culture supernatant was collected by high-speed centrifugation.
  • the antibodies were purified according to the purification method described in Example 1.2 to obtain corresponding recombinant Nanobodies, which were named Lab01-huFc, Lab02-huFc, Lab03-huFc and Lab04-huFc respectively.
  • Use the SEC-HPLC method to test the purity of the purified antibodies, and prepare them for use after passing the test.
  • NCI-H929 Multiple myeloma cells NCI-H929 were expanded and cultured in T-175 culture flasks to the logarithmic growth phase. After counting the cells, they were centrifuged and the cell pellet was resuspended in FACS buffer (PBS+2% fetal calf serum) to 2 ⁇ 10 6 cells/ml, add 50 ⁇ l per well to the 96-well FACS reaction plate, centrifuge and discard the supernatant, add 50 ⁇ l of the antibody sample to be tested (135 nM as the starting concentration, 5-fold gradient dilution) per well, and mix with the cells Mix well and incubate at 4°C for 1 hour.
  • FACS buffer PBS+2% fetal calf serum
  • the recombinant antibodies Lab01-huFc, Lab02-huFc, Lab03-huFc and Lab04-huFc can specifically bind to NCI-H929 cells.
  • 293T-human GPRC5D, 293T-cyno GPRC5D and 293T cells were expanded and cultured in T-175 culture flasks to the logarithmic growth phase.
  • the medium was aspirated, washed twice with PBS buffer, digested with trypsin, and then used with complete medium. Terminate digestion and pipette cells to a single cell suspension. After counting the cells, centrifuge and resuspend the cell pellet in FACS buffer (PBS + 2% fetal calf serum) to 2 ⁇ 10 6 cells per ml. Then, perform FACS detection and data analysis according to the method of Example 3.2. The results are shown in Figure 3A-3C.
  • the recombinant antibodies Lab01-huFc, Lab02-huFc, Lab03-huFc and Lab04-huFc can effectively bind to human GPRC5D and cyno GPRC5D overexpressing recombinant cells, and the binding activities are better than the positive control antibodies. .
  • the recombinant antibody did not bind to 293T cells, showing good specificity.
  • the heavy chain variable region germline genes with high homology to nanobodies were selected as templates, and the nanobodies were The CDR sequences of the antibody based on the IMGT or Kabat naming method are transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the CDR sequence of the Lab01 antibody is defined based on IMGT rules, and its humanization templates are IGHV3-7*01 and IGHJ3*01.
  • the Lab01 antibody was renamed HAB01 during the humanization process, and was named HAB01-H after CDR transplantation.
  • the CDR sequence of the Lab02 antibody is defined based on IMGT rules, and its humanization templates are IGHV3-30*13 and IGHJ2*01.
  • the Lab02 antibody was renamed HAB02 during the humanization process, and was named HAB02-H after CDR transplantation.
  • the CDR sequence of Lab03 antibody is defined based on Kabat rules, and its humanization templates are IGHV3-7*01 and IGHJ3*01. Lab03 antibody was renamed HAB03 during the humanization process and named HAB03-H after CDR transplantation.
  • the CDR sequence of the Lab04 antibody is defined based on IMGT rules, and its humanization templates are IGHV3-7*01 and IGHJ3*01.
  • the Lab04 antibody was renamed HAB04 during the humanization process, and was named HAB04-H after CDR transplantation.
  • Humanized antibodies are obtained by transplanting the CDRs of antibodies HAB01, HAB02, HAB03 and HAB04 into their human templates respectively.
  • the amino acid sequence of its humanized template and the humanized antibody sequence after CDR transplantation are shown in Table 6.
  • Graft (IGHV3-7*01) represents the implantation of the Nanobody CDR into the human germline FR region sequence; S35A represents the mutation of S at position 35 of Graft (IGHV3-7*01) to A, and so on.
  • Graft(IGHV3-30*13) represents implanting the Nanobody CDR into the human germline FR region sequence; Q1E represents mutating Q at position 1 of Graft(IGHV3-30*13) to E, and so on.
  • the key amino acids in the FR region sequence of the HAB03 humanized antibody were back-mutation to the corresponding amino acids of the camel antibody to ensure the original affinity.
  • the specific details of the mutation points after the reverse mutation (the reverse mutation points are numbered in natural order) and the specific amino acids The sequences are shown in Table 13 and Table 14.
  • Graft(IGHV3-7*01) represents implanting the Nanobody CDR into the human germline FR region sequence; F27D represents mutating F at position 27 of Graft(IGHV3-7*01) to D, and so on.
  • Graft (IGHV3-7*01) represents the implantation of the Nanobody CDR into the human germline FR region sequence; S35G represents the mutation of S at position 35 of Graft (IGHV3-7*01) to G, and so on.
  • the humanized antibody variable region sequence gene was synthesized and cloned into the pTT5 vector with human hinge region and hIgG1-Fc constant region sequence to form the VHH-huFc (C220S) expression sequence, and a plasmid was prepared.
  • the antibody plasmid was transiently transfected into Expi293F cells via PEI. After culturing for 7 days, the supernatant was collected and the antibody was purified by Protein A according to the method in Example 1.2.
  • ELISA Enzyme-linked immunosorbent assay
  • Human GPRC5D protein (Kaika Biotech, GPR-HM05P) was diluted with PBS to a final concentration of 1 ⁇ g/mL, then 50 ⁇ l per well was added to a 96-well ELISA plate, and incubated at 4°C overnight. The next day, wash the plate twice with PBST, add blocking solution [PBS+2% (w/w) BSA] and block at room temperature for 2 hours. Pour off the blocking solution and add 5-fold gradient dilution of humanized recombinant antibody with a starting concentration of 135 nM, and 50 ⁇ l of positive and negative control antibodies per well. After incubation at 37°C for 1 hour, the plate was washed three times with PBST.
  • Example 3.2 For the preparation of detection cells and antibodies to be tested and the detection method, please refer to Example 3.2.
  • the test results are shown in Figures 5 to 7.
  • the humanized antibodies can all interact well with endogenous cells NCI-H929, molp-8 (Nanjing Kebai, CBP60562) and RPMI-8226 (ATCC, CCL-155). Binding activity, specific binding activity and numerical values are shown in Tables 23 to 34.
  • Human GPRC5D overexpression cells (293T-human GPRC5D) were used as antigens for phage library panning.
  • the specific panning strategy is as follows: collect the cells, wash the cells twice with 5% serum-PBS, add 100 ⁇ l/well of 4% paraformaldehyde, Fix at room temperature for 25-30min. Add the blocked phage dilution and incubate at 37°C for 1 hour; remove unbound phage and wash the cells 3 times with 0.1% PBST and 2 times with PBS; add 500 ⁇ L Gly-HCl eluent and incubate at 37°C for 8 minutes to elute specificity.
  • Bound phage transfer the eluate to a 1.5 mL sterile centrifuge tube and quickly neutralize it with 50 ⁇ L Tris-HCl neutralization buffer; collect the phage supernatant for the next round of biopanning. After multiple rounds of panning, positive phages are continuously enriched during the panning process in order to screen out Nanobodies with good specificity and high affinity.
  • 293T-human GPRC5D and 293T negative cells were seeded into a 96-well plate and cultured overnight; the next day, 100 ⁇ l of 4% paraformaldehyde was added to fix the cells. Wash 2 times with PBS, add 300ul 5% skim milk to each well, and block for 1 hour at 37°C; add 50 ⁇ L phage culture supernatant and 50 ⁇ L 5% skim milk to each well, incubate at 37°C for 1 hour; wash 5 times with PBST, add horseradish and rinse Oxidase-labeled anti-M13 antibody (diluted 1:10000 in PBS). Add TMB chromogenic solution to develop color, read the OD450nm absorbance value with a microplate reader, and select ELISA positive clones for the next step of FACS verification.
  • the overexpressing cell line 293T-human GPRC5D was used to further verify the ELISA-positive clones. For specific methods, refer to Example 2.4. After multiple rounds of optimization and screening, a total of 2 positive clones capable of recognizing GPRC5D were obtained, named Lab05 and Lab06 respectively.
  • the CDRs of its sequences were analyzed using the IMGT, KABAT and Chothia numbering systems. The corresponding sequence information is shown in Table 36 and Table 37 below.
  • Table 36 shows the amino acid sequences of the two Nanobody molecules
  • Table 37 shows the two Nanobody molecules.
  • the heavy chain variable region germline genes with high homology to nanobodies were selected as templates and the nanobodies were The CDR sequences of the antibody based on the IMGT or KABAT naming method are transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the CDR sequence of the Lab05 antibody is defined based on IMGT rules, and its humanization templates are IGHV3-74*03 and IGHJ3*01.
  • the Lab05 antibody was renamed HAB05 during the humanization process, and was named HAB05.H after CDR transplantation.
  • the CDR sequence of the Lab06 antibody is defined based on Kabat rules, and its humanization templates are IGHV3-23*04 and IGHJ3*01.
  • the Lab06 antibody was renamed HAB06 during the humanization process, and was named HAB06.H after CDR transplantation.
  • the CDRs of antibodies HAB05 and HAB06 were respectively transplanted into their human templates to obtain humanized antibodies.
  • the amino acid sequence of its humanized template is shown in Table 38.
  • the key amino acids in the FR region sequence of the HAB05 humanized antibody were back mutated to the corresponding amino acids of the alpaca antibody to ensure the original affinity.
  • the details of the specific mutation points after the reverse mutation (the reverse mutation points are numbered in natural order) and the specific The amino acid sequences are shown in Table 39 and Table 40.
  • Graft(IGHV3-74*03) represents implanting the Nanobody CDR into the human germline FR region sequence; H35A represents mutating H at position 35 of Graft(IGHV3-74*03) to A, and so on.
  • Graft (IGHV3-23*04) represents the implantation of the Nanobody CDR into the human germline FR region sequence; V37F represents the mutation of V at position 37 of Graft (IGHV3-23*04) to F, and so on.
  • Example 8 Expression, purification and binding detection of chimeric and humanized Nanobodies
  • the screened Nanobody sequences, humanized sequences, and control antibody sequences were cloned into the eukaryotic expression vector pTT5 with hIgG1-Fc tag, transiently transfected into Expi293F cells (Gibco, A14527) with PEI, cultured for 7 days, and centrifuged at high speed. Collect the culture supernatant of cells expressing antibodies.
  • the antibody was purified according to the purification method described in Example 1.2 to obtain the corresponding recombinant Nanobody and humanized Nanobody.
  • the recombinant Nanobodies were named Lab05-huFc and Lab06-huFc.
  • the humanized Nanobodies are named HAB05-H2, HAB05-H3, HAB06-H1 and HAB06-H5 respectively.
  • the positive control sequence comes from patent WO2020/092854A2 and is named MCARH109-scFv-huFc. Its VH and VL sequences are shown in Table 45. Use SEC-HPLC to test the purity of the purified antibodies, and distribute them for use after passing the test.
  • Example 3.2 For the preparation of detection cells and antibodies to be tested and the detection method, please refer to Example 3.2. The test results are shown in Figures 9 to 11. Both recombinant antibodies and humanized antibodies have good binding activity to endogenous cells NCI-H929, molp-8 and RPMI-8226. The specific binding activities and values are as follows in Table 47 - Table 49.

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Abstract

La présente divulgation concerne un anticorps dirigé contre le sous-type D de la famille C5 du récepteur couplé à la protéine G (GPRC5D) et son utilisation. Plus particulièrement, la présente invention concerne un nanocorps ou un fragment de liaison à l'antigène de celui-ci qui se lie de manière spécifique à GPRC5D, un acide nucléique codant pour celui-ci, un vecteur d'expression et une cellule d'expression de celui-ci, son procédé de préparation, une composition pharmaceutique de celui-ci, et son utilisation dans la préparation d'une composition pharmaceutique pour le traitement de maladies, par exemple, son utilisation dans le traitement de tumeurs. Par comparaison avec des anticorps pleine longueur, le nanocorps selon la présente divulgation peut se lier de manière plus efficace à la protéine cible GPRC5D contre laquelle il est difficile de développer un anticorps. La présente divulgation a une grande importance pour le développement d'un médicament thérapeutique et d'un réactif de détection pour l'anticorps GPRC5D.
PCT/CN2023/108360 2022-07-21 2023-07-20 Nanocorps anti-gprc5d et son utilisation WO2024017326A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109715667A (zh) * 2016-07-20 2019-05-03 詹森药业有限公司 抗-gprc5d抗体、结合gprc5d和cd3的双特异性抗原结合分子及其用途
WO2020092854A2 (fr) * 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Récepteurs antigéniques chimériques spécifiques du gprc5d (élément d du groupe 5 de classe c des récepteurs couplés à la protéine g)
CN113429482A (zh) * 2014-12-05 2021-09-24 纪念斯隆-凯特琳癌症中心 靶向g-蛋白偶联受体的抗体和使用方法
WO2022148370A1 (fr) * 2021-01-05 2022-07-14 Lanova Medicines Development Co., Ltd. Anticorps monoclonaux anti-gprc5d et leurs utilisations
US20220259318A1 (en) * 2019-07-31 2022-08-18 Hoffmann-La Roche Inc. Antibodies binding to gprc5d

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113429482A (zh) * 2014-12-05 2021-09-24 纪念斯隆-凯特琳癌症中心 靶向g-蛋白偶联受体的抗体和使用方法
CN109715667A (zh) * 2016-07-20 2019-05-03 詹森药业有限公司 抗-gprc5d抗体、结合gprc5d和cd3的双特异性抗原结合分子及其用途
WO2020092854A2 (fr) * 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Récepteurs antigéniques chimériques spécifiques du gprc5d (élément d du groupe 5 de classe c des récepteurs couplés à la protéine g)
US20220259318A1 (en) * 2019-07-31 2022-08-18 Hoffmann-La Roche Inc. Antibodies binding to gprc5d
WO2022148370A1 (fr) * 2021-01-05 2022-07-14 Lanova Medicines Development Co., Ltd. Anticorps monoclonaux anti-gprc5d et leurs utilisations

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