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WO2024152963A1 - Anticorps anti-cd3 humanisé et son utilisation - Google Patents

Anticorps anti-cd3 humanisé et son utilisation Download PDF

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Publication number
WO2024152963A1
WO2024152963A1 PCT/CN2024/071578 CN2024071578W WO2024152963A1 WO 2024152963 A1 WO2024152963 A1 WO 2024152963A1 CN 2024071578 W CN2024071578 W CN 2024071578W WO 2024152963 A1 WO2024152963 A1 WO 2024152963A1
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Prior art keywords
variable region
chain variable
seq
antibody
antigen
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PCT/CN2024/071578
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English (en)
Chinese (zh)
Inventor
徐汶新
郑欢
丁列明
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Betta Pharmaceuticals Co Ltd
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Betta Pharmaceuticals 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to the field of biomedicine, and in particular to anti-CD3 humanized antibodies and applications thereof.
  • the CD3 protein complex is composed of 6 peptide chains with non-covalent bonds. It is a transmembrane protein widely distributed on the surface of mature T cells. It contains 3 pairs of dimers ( ⁇ , ⁇ , ⁇ ) and is composed of four constant chains: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • the CD3 protein complex can bind to the T cell receptor (TCR) on T cells to form a receptor complex to activate T cells.
  • TCR T cell receptor
  • the N-terminal extracellular domain and transmembrane domain of the CD3 protein complex are highly consistent in all T cells.
  • the N-terminal extracellular domain is composed of two heterodimeric domains CD3 ⁇ and CD3 ⁇ , and the transmembrane domain is composed of a single homodimer CD3 ⁇ .
  • CD3 antibodies recognize all T cells, and they react with 70% to 80% of human peripheral blood lymphocytes and 65% to 85% of thymocytes. After being activated by CD3 antibodies, T cells are directed to the vicinity of tumor cells, and the two cells contact and form synapses, triggering the activation of the T cell receptor (TCR) signaling pathway, the expression and release of granzymes, and then causing tumor cell membrane perforation, leading to the latter's lysis and apoptosis. The activation of the TCR signaling pathway also causes the expression and release of a series of cytokines, such as the release of IL-2, which stimulates the proliferation of T cells and amplifies the immune killing effect.
  • TCR T cell receptor
  • cytokines such as the release of IL-2
  • CD3-binding antibody molecules are now known, especially bispecific antibody molecules containing CD3 binding specificity.
  • CD3 antibodies come from hybridoma platform mouse antibodies in the 1980s, such as OKT3, UCHT1, L2K and SP34.
  • the species cross-reactivity of CD3 monoclonal antibodies is extremely critical for the development of CD3 bispecific antibodies.
  • anti-CD3 antibodies for the CD3 protein complex is a key factor in the success of CD3-related antibodies.
  • anti-CD3 antibodies with too high affinity will lead to non-specific activation of T cells, thereby producing unnecessary cytokine release syndrome.
  • they will preferentially target peripheral T cells in the body and act less on tumor cells, resulting in decreased efficacy.
  • anti-CD3 antibodies with too low affinity are not enough to activate T cells so that T cells can exert their killing function.
  • anti-CD3 antibodies with different binding affinities and thus different T cell activation abilities, which can be used to meet unmet clinical needs, and can also be used to develop multi-specific antibodies that meet different tumor-associated antigens, such as bispecific antibodies or trispecific antibodies.
  • the present invention provides an antibody or antigen binding fragment that specifically recognizes and binds to CD3.
  • the provided antibody reduces the immunogenicity of sp34 by humanizing mouse sp34, and has specific recognition and binding activity with CD3.
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides an antibody or antigen-binding fragment that specifically recognizes and binds to CD3, selected from: a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:4; or a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:5; or a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:6; or a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:7; or a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:8; or a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:4; or a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:5; or The heavy chain variable region shown
  • a second aspect of the present invention provides an antibody or antigen-binding fragment that specifically recognizes and binds to CD3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the sequence shown in SEQ ID NO: 1, 2 or 3, and the light chain variable region is selected from the sequence shown in SEQ ID NO: 4, 5, 6, 7 or 8.
  • the third aspect of the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment according to any one of the first and second aspects of the present invention.
  • the fourth aspect of the present invention provides a construct comprising the polynucleotide described in the third aspect of the present invention.
  • the fifth aspect of the present invention provides a host cell, wherein the host cell comprises the construct described in the fourth aspect of the present invention.
  • the sixth aspect of the present invention provides a multispecific antibody, comprising a first antigen binding portion and a second antigen binding portion, the first antigen binding portion specifically binds to a first epitope, the second antigen binding portion specifically binds to a second epitope, the first antigen binding portion is the antibody or antigen binding fragment described in the first aspect or the second aspect, and the second antigen binding portion is fused to the first antigen binding portion.
  • the seventh aspect of the present invention provides an antibody conjugate, comprising the antibody or antigen-binding fragment of the first aspect or the second aspect or the multispecific antibody of the sixth aspect, and a functional small molecule connected to the antibody or antigen-binding fragment or the multispecific antibody.
  • the eighth aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the first aspect or the second aspect or the multispecific antibody of the sixth aspect or the antibody conjugate of the seventh aspect, and a pharmaceutically acceptable carrier.
  • the ninth aspect of the present invention provides a kit comprising the antibody or antigen-binding fragment of the first aspect or the second aspect, or the multispecific antibody of the sixth aspect, or the antibody conjugate of the seventh aspect.
  • the tenth aspect of the present invention provides a method for producing an antibody or an antigen-binding fragment, wherein the antibody or antigen-binding fragment is the antibody or antigen-binding fragment described in the first aspect or the second aspect, and the method comprises: culturing the host cell described in the fifth aspect, and collecting the antibody or antigen-binding fragment from the culture.
  • the eleventh aspect of the present invention provides a method for preventing and/or treating a disease, comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment of the first or second aspect, or the multispecific antibody of the sixth aspect, or the antibody conjugate of the seventh aspect, or the pharmaceutical composition of the eighth aspect.
  • the twelfth aspect of the present invention provides a use of an antibody or antigen-binding fragment in the preparation of a drug or a kit or a multispecific antibody or an antibody conjugate.
  • FIG. 1 is an enzyme-linked immunosorbent assay (ELISA) test result of a humanized antibody provided in Example 2 of the present invention.
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 2 is the ELISA test result of the humanized antibody provided in Example 2 of the present invention.
  • FIG. 3 is a flow cytometry fluorescence sorting (FACs) test result of the humanized antibody and 293T-CD3 cells provided in Example 3 of the present invention.
  • FACs flow cytometry fluorescence sorting
  • FIG. 4 is a FACs detection result of the humanized antibody and 293T-CD3 cells provided in Example 3 of the present invention.
  • FIG. 5 is the FACs detection result of the humanized antibody and Jurkat cells provided in Example 3 of the present invention.
  • FIG. 6 is a FACS test result of humanized antibodies and Jurkat cells provided in Example 3 of the present invention.
  • CD3 herein refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR) and which is composed of a homodimer or heterodimer formed by two of the following four receptor chains: CD3-, CD3-.
  • antibodies that bind to CD3 or “anti-CD3 antibodies” or “anti-CD3 specific antibodies” used herein include antibodies and antigen-binding fragments thereof that specifically recognize or bind to a single CD3 subunit, as well as antibodies and antigen-binding fragments thereof that specifically recognize and bind to a dimeric complex of two CD3 subunits.
  • the antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3, and/or CD3 expressed on the cell surface.
  • Soluble CD3 includes natural CD3 protein and recombinant CD3 protein variants.
  • the term "effective amount” herein refers to the amount of a drug, preparation or active ingredient that can exhibit a detectable therapeutic effect or inhibitory effect.
  • the detectable therapeutic effect or inhibitory effect mentioned can be detected by any detection method known in the art.
  • first, second, and third are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated, nor should they be used to indicate a sequence of precedence.
  • first antigen-binding portion and the second antigen-binding portion are not It indicates the order of precedence and is not used to represent importance, but only to distinguish.
  • antibody is used in the broadest sense to refer to a protein or polypeptide comprising an antigen binding site, an antigen binding portion or an antigen binding fragment, covering natural antibodies and artificial antibodies of various structures, including but not limited to complete antibody forms or antigen binding fragments of antibodies.
  • each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (also referred to as a heavy chain constant domain, abbreviated as CH).
  • the heavy chain constant region includes a heavy chain constant domain CH1, a heavy chain constant domain CH2, and a heavy chain constant domain CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (also referred to as a light chain constant domain, abbreviated as CL).
  • VH and VL can be further divided into complementary determining regions (also referred to as hypervariable regions or hypervariable regions, abbreviated as CDR or HVR), with conserved framework regions (FR) inserted therebetween.
  • CDR complementary determining regions
  • FR conserved framework regions
  • Each VH and VL comprises three CDRs and four FRs, arranged in the following sequence from the amino terminus (N terminus) to the carboxyl terminus (C terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2 and HCDR3, respectively, starting from the amino terminus
  • the CDRs of the light chain variable region are referred to as LCDR1, LCDR2 and LCDR3, respectively, starting from the amino terminus.
  • Antigen binding fragment refers to a portion of a full-length antibody that exhibits specific binding to an antigen.
  • the hypervariable or hypervariable regions of the heavy and light chains of an intact antibody exhibit specific binding to an antigen.
  • antigen binding fragments or antigen binding portions include, but are not limited to, Fab, Fab', F(ab') 2 , bispecific Fab' and Fv fragments, linear antibodies, single-chain antibodies, single-domain antibodies, and the like. Papain digestion of an intact antibody produces two identical antigen binding fragments, called Fab fragments, each of which contains a heavy and light chain variable region and a light chain constant domain and a heavy chain constant domain CH1.
  • the Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain constant domain CH1, including one or more cysteines from the hinge region of the antibody. Pepsin digestion of an intact antibody yields a F(ab')2 fragment.
  • the F(ab')2 fragment has two antigen binding F(ab) portions linked together by a disulfide bond, and the F(ab')2 fragment is a bivalent antibody.
  • Single-chain antibodies are fusion proteins formed by connecting the variable regions of the heavy and light chains of antibodies through a flexible short peptide consisting of about 10-25 amino acids.
  • Single-domain antibodies are antibody fragments composed of the variable regions of a single monomer. Since single-domain antibodies are usually derived from the variable regions of the heavy chains of camelid antibodies or shark antibodies, they are often called nanobodies.
  • affinity or “binding affinity” mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and/or stability of the binding sites between an antigen and an antibody or an antigen-binding fragment.
  • Specific recognition means to distinguish it from non-specific interactions, and this specific binding can be measured by some methods commonly used in the art.
  • the ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, cells carrying antigens can be detected by flow cytometry, and the competitive binding between the antibody to be tested and the labeled antibody can be detected by measuring the positive rate index of the cells.
  • ELISA enzyme-linked immunosorbent assay
  • the antibody provided has an EC50 value of ⁇ 50nM, ⁇ 20nM, ⁇ 10nM, ⁇ 9nM, ⁇ 8nM, ⁇ 7nM, ⁇ 6nM, ⁇ 5nM, ⁇ 4nM, ⁇ 3nM, ⁇ 2nM, ⁇ 1nM, ⁇ 0.9nM, ⁇ 0.8nM, ⁇ 0.7nM, ⁇ 0.6nM, ⁇ 0.5nM, ⁇ 0.4nM, ⁇ 0.3nM, ⁇ 0.2nM, ⁇ 0.1nM, ⁇ 0.05nM, ⁇ 0.01nM, ⁇ 0.005nM, ⁇ 0.001nM.
  • the binding activity of the antibody to the antigen can also be determined by surface isoparticle resonance technology (SPR) or biofilm interferometry (BLI).
  • SPR surface isoparticle resonance technology
  • BBI biofilm interferometry
  • the ELISA activity of the humanized antibody provided is comparable to the sp34 activity, and the FACs binding activity is slightly weaker than the sp34 activity. Therefore, humanized antibodies will show higher safety.
  • the antibodies or antigen binding fragments, or multispecific antibodies, or polynucleotides mentioned herein are generally separable or recombinant.
  • “Separable” means capable of identifying and separating and/or recovering from cells or cell cultures expressing a polypeptide or protein. Typically, an isolated polypeptide will be prepared by at least one purification step.
  • isolated antibody means substantially free of different antigens.
  • Recombinant means that antibodies can be produced in exogenous host cells using genetic recombination techniques.
  • Humanized antibodies are generally based on the antigen-binding portion derived from a non-human species and the portion based on the human immunoglobulin molecule.
  • the entire antibody except for the CDR is encoded by a human-derived polynucleotide, which retains the antigen binding activity while reducing the immunogenicity.
  • back mutations can also be performed on this basis to maintain the antigen binding activity.
  • the present invention provides an antibody or antigen-binding fragment that specifically recognizes and binds to CD3, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the sequence shown in SEQ ID NO: 1, 2 or 3, and the light chain variable region is selected from the sequence shown in SEQ ID NO: 4, 5, 6, 7 or 8.
  • the antibody or antigen-binding fragment that specifically recognizes and binds to CD3 is obtained by humanizing sp34.
  • the heavy chain variable region sequence of antibody Sp34 is shown in SEQ ID NO: 9, and the light chain variable region sequence is shown in SEQ ID NO: 10.
  • the present invention provides an antibody or antigen-binding fragment that specifically recognizes and binds to CD3, selected from: the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:4; or the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:5; or the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:6; or the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:7; or the heavy chain variable region shown in SEQ ID NO:1 and the light chain variable region shown in SEQ ID NO:8; or the heavy chain variable region shown in SEQ ID NO:2 and the light chain variable region shown in SEQ ID NO:4; or the heavy chain variable region shown in SEQ ID NO:2 and the light chain variable region shown in SEQ ID NO:5 ; or the heavy chain variable region shown in SEQ ID NO:2 and the heavy chain variable region shown in SEQ
  • the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody, a monovalent antibody, a multivalent antibody, a complete antibody, Fab, Fd, scFv, F(ab') 2 , Fab', Fv.
  • the antibody or antigen-binding fragment is IgG1, IgG2, IgG3, IgG4, or a variant thereof.
  • the antibody or antigen-binding fragment further includes Fc or mutation.
  • the Fc mentioned can be derived from natural Fc or modified Fc.
  • the natural Fc can be of any origin, including but not limited to mouse, primate, human, preferably human Fc region.
  • the human Fc region mentioned can be obtained from a public database.
  • the antibody provided is hu01, which has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:4.
  • the antibody provided is hu02, which has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:5.
  • the antibody provided is hu03, which has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:6.
  • the antibody provided is hu04, which has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:7.
  • the antibody provided is hu05, which has a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:8.
  • the antibody provided is hu06, which has a heavy chain variable region shown in SEQ ID NO:2 and a light chain variable region shown in SEQ ID NO:4.
  • the monoclonal antibody provided is hu07, which has a heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region shown in SEQ ID NO: 5.
  • the monoclonal antibody provided is hu08, which has a heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region shown in SEQ ID NO: 6.
  • the monoclonal antibody provided is hu09, which has a heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region shown in SEQ ID NO: 7.
  • the monoclonal antibody provided is hu10, which has a heavy chain variable region shown in SEQ ID NO: 2 and a light chain variable region shown in SEQ ID NO: 8.
  • the monoclonal antibody provided is hu11, which has a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 4.
  • the monoclonal antibody provided is hu12, which has a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 5.
  • the monoclonal antibody provided is hu13, which has a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 6.
  • the monoclonal antibody provided is hu14, which has a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 7.
  • the monoclonal antibody provided is hu15, which has a heavy chain variable region shown in SEQ ID NO: 3 and a light chain variable region shown in SEQ ID NO: 8.
  • the present invention also provides a polynucleotide encoding an antibody or an antigen-binding fragment.
  • the polynucleotide mentioned is separable, including but not limited to DNA, RNA or cDNA, etc. Conventional methods in the art can be used to obtain isolated polynucleotide sequences for encoding antibodies or antigen-binding fragments.
  • the polynucleotide mentioned encodes one amino acid according to three codons, encoding the heavy chain variable region shown in SEQ ID NO: 1, 2 or 3.
  • the polynucleotide mentioned encodes one amino acid according to three codons, encoding the light chain variable region shown in SEQ ID NO: 4, 5, 6, 7 or 8.
  • the antibodies mentioned can be truncated or increased in individual amino acids.
  • the corresponding polynucleotides provided can delete or add multiple nucleotides as needed.
  • the polynucleotide sequence encoding the antibody or antigen-binding fragment can be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system.
  • the present invention also provides a construct comprising the polynucleotide described above.
  • a variety of methods commonly used in the art can be used to obtain the construct, including in vitro recombinant DNA technology, DNA Synthesis technology, in vivo recombination technology, etc., for example, a polynucleotide can be inserted into the multiple cloning site of an expression vector to form a construct.
  • the construct can contain a variety of operating factors such as a promoter, a terminator, a marker gene, etc. as needed, and these operating factors are operably connected to the polynucleotide.
  • the promoter is usually used to provide a signal to start transcription.
  • the promoter can select lactose promoter (Lac), Trp promoter, Tac promoter, PL and PR promoters of bacteriophage as needed; the terminator provides a signal for transcription termination during the transcription process, and the marker gene on the construct is often used for screening.
  • lactose promoter Lac
  • Trp promoter Trp promoter
  • Tac promoter Tac promoter
  • PL PL
  • PR promoters of bacteriophage as needed
  • the terminator provides a signal for transcription termination during the transcription process
  • the marker gene on the construct is often used for screening.
  • the expression vector is not particularly limited, and can be some commercially available expression vectors, or it can be an expression vector artificially modified as needed, such as a plasmid, a bacteriophage, a virus, etc.
  • the virus can be a plant cell virus, a mammalian cell virus, etc.
  • the construct can express antibodies or proteins in vitro, and can also be transferred into cells to express antibodies or proteins.
  • the present invention also provides a host cell containing the above-mentioned polynucleotide or the above-mentioned construct.
  • Any cell suitable for expressing antibodies or proteins with polynucleotides or constructs can be used as a host cell.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a yeast cell, a mammalian cell, etc. Commonly used host cells can be yeast cells, CHO, HEK-293 cells, COS cells, insect cells of Drosophila S2 or Sf9.
  • Host cells containing polynucleotides or constructs can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, etc.
  • the present invention also provides a multispecific antibody, comprising a first antigen binding part and a second antigen binding part, the first antigen binding part specifically binds to a first epitope, the second antigen binding part specifically binds to a second epitope, the first antigen binding part is the above-mentioned antibody or antigen binding fragment, and the second antigen binding part is fused to the first antigen binding part.
  • multispecific antibody refers to an antigen-binding portion that specifically binds to epitopes of at least two different biological molecules or at least two different epitopes of the same biological molecule.
  • a multispecific antibody can be a bispecific antibody or a trispecific antibody.
  • Multi means two or more.
  • quantifiers are added in front of the antigen-binding portion to distinguish them. For example, they can be expressed as "first antigen-binding portion", “second antigen-binding portion", “third antigen-binding portion”, etc. as needed.
  • the order of binding to antigens in the listed multispecific antibodies is arbitrary.
  • the multispecific antibody mentioned comprises an antigen-binding portion that binds to different epitopes of at least two different biological molecules.
  • first antigen binding moiety and the second antigen binding moiety mentioned in the fusion may be directly connected (e.g., directly connected through their terminal amino acids), or indirectly connected through a linker, and other functional region sequences may be connected between the two, such as Fc region, CH1, CL sequence, etc.
  • first antigen binding moiety and second antigen binding moiety respectively refer to moieties that can bind to different epitopes of different target antigens, which may be complete antibodies or parts of complete antibodies, such as heavy chain variable regions, Fab, Fab', F(ab') 2 , single-chain antibodies (ScFv), or single-domain antibodies (sdAb), etc.
  • the "first antigen binding moiety” and the "second antigen binding moiety” specifically bind to different epitopes of different antigens, respectively.
  • linker and “connector fragment” have the same meaning.
  • the linker can be some oligopeptides or polypeptides, and can be any amino acid sequence that can provide flexibility.
  • Available linkers are linkers commonly used in the art, including but not limited to the following groups: GS, SG, GGS, GSG, SGG, GGG, GGGS, GGGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGGGGS, GGGGGGGSGGGGS, GGGGGGGGGGGGSGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGS, GGGGSGGGGSGGGGGGGS, GGGGSGGGGSGGGGGGGS, GGGGSGGGGSGGGGGGGS, GGGG
  • the second antigen binding portion is capable of specifically recognizing and binding to different target antigens, including but not limited to CD19, BCMA, CD20, CD22, CD123, EGFR, C-Met, CLDN18.2, EpCAM, MSLN, GPC2/3, 5T4, Trop2, Her2, Her3, Nectin-4, etc.
  • the present invention also provides an antibody conjugate, comprising the antibody or antigen-binding fragment or multispecific antibody, and a functional small molecule or protease connected to the antibody or antigen-binding fragment or multispecific antibody.
  • the functional small molecule mentioned can be a developed or undeveloped small molecule drug, which is chemically coupled with the antibody or antigen-binding fragment or multispecific antibody to enhance the activity of the drug. It can enhance the therapeutic effect of anti-tumor drugs and reduce adverse reactions. Small molecules such as toxins, chemotherapeutic drugs, and photosensitizers can also be coupled to the antibodies mentioned above through linkers.
  • antibody-Protac conjugates can also be obtained by coupling antibodies with protac molecules, such as connecting antibodies and protease targeting ligands (such as E3 ligase ligands) through linkers to shorten the distance between the target protein and the E3 ubiquitin ligase in the cell, and using the ubiquitin-proteasome pathway to specifically degrade the target protein.
  • protac molecules such as connecting antibodies and protease targeting ligands (such as E3 ligase ligands) through linkers to shorten the distance between the target protein and the E3 ubiquitin ligase in the cell, and using the ubiquitin-proteasome pathway to specifically degrade the target protein.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment or multispecific antibody or antibody conjugate, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier can be a pharmaceutically acceptable salt, such as an acid addition salt or a base addition salt (for example, as described in Berge, S.M.etal (1977) J.Pharma.Sci.66:1-19).
  • the pharmaceutically acceptable carrier mentioned is acceptable to the subject at the dose or concentration used.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers or salts, such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris; sugars, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc.
  • buffers or salts such as disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris
  • sugars such as trehalose, polysorbate, sucrose, mannitol
  • surfactants such as polysorbate
  • preservatives such as
  • Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtering with a sterile filter membrane. Those skilled in the art can select different pharmaceutically acceptable carriers for the pharmaceutical composition as needed to prepare different dosage forms, such as freeze-dried dosage forms, injections, etc.
  • the prepared different pharmaceutical dosage forms can be formulated into any suitable administration route for administration to the subject, including but not limited to intravenous, dermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, etc.
  • the pharmaceutical composition mentioned may further include other therapeutic agents.
  • the therapeutic agent mentioned is a substance that can exert a therapeutic effect.
  • anti-CD19 antibodies, anti-BCMA antibodies, anti-CD20 antibodies, anti-EGFR antibodies, anti-CLDN18.2 antibodies, anti-EpCAM antibodies or conjugates or derivatives of these antibodies, etc.
  • the present invention also provides a kit, which includes the above-mentioned antibody or antigen-binding fragment, or the above-mentioned multispecific antibody, or the above-mentioned antibody conjugate.
  • the kit may also include a container, a buffer reagent, and a control such as a positive control and a negative control as needed. Those skilled in the art can make corresponding selections as needed. Accordingly, the kit may also include instructions for use to facilitate the operation and use of those skilled in the art.
  • the present invention also provides a medicine box, which includes the above-mentioned antibody or antigen-binding fragment, or the above-mentioned multispecific antibody, or the above-mentioned antibody conjugate.
  • the present invention also provides a method for producing an antibody or an antigen-binding fragment, comprising: culturing the above host cell, and collecting the antibody or antigen-binding fragment from the culture.
  • the antibody or antigen-binding fragment collected from the culture can be purified to obtain a substantially pure product.
  • substantially pure means that the purity of the antibody or antigen-binding fragment is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or even greater than 99.5%, 99.6%, 99.7%, or 99.8%.
  • the present invention provides a method for preventing and/or treating a disease, comprising: administering an effective amount of an antibody or antigen-binding fragment, or a multispecific antibody, or an antibody conjugate or a pharmaceutical composition to a subject in need.
  • treatment means that it can lead to a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or a reduction in the pain caused by the disease.
  • the effective amount of antibodies used to "prevent” the disease will usually be lower than the effective amount of antibodies used to treat the disease.
  • Effective amount is an amount sufficient to achieve or at least partially achieve the desired effect.
  • the inhibition rate of cells in the subject's body reaches 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or even 90% or more or more than 95% compared to a subject not treated with the antibody.
  • the subject mentioned here can be an animal or a human. For example, it can be a mammal, including cattle, sheep, mice, horses, etc.
  • the antibodies or antigen-binding fragments thereof provided can be used to treat diseases including but not limited to cancer, inflammatory diseases, chronic infections, cardiovascular and cerebrovascular diseases, fibrosis, neurodegenerative diseases, immunomodulatory diseases, central nervous system diseases and diabetes.
  • diseases including but not limited to cancer, inflammatory diseases, chronic infections, cardiovascular and cerebrovascular diseases, fibrosis, neurodegenerative diseases, immunomodulatory diseases, central nervous system diseases and diabetes.
  • the methods provided by the present invention can be used to treat CD3 target-related diseases.
  • the cancers mentioned include but are not limited to lymphoma (e.g.
  • myeloma such as multiple myeloma
  • leukemia such as acute lymphoblastic leukemia
  • non-small cell lung cancer small cell lung cancer, squamous cell carcinoma, glioma, colon cancer, kidney cancer,
  • the antibody or antigen-binding fragment mentioned above can also be combined with other treatment methods to treat diseases.
  • the other treatment methods mentioned above can be chemotherapy or radiotherapy.
  • chemotherapy or radiotherapy By combining chemotherapy or radiotherapy and other treatment methods clinically, the therapeutic effect of the drug can be further improved, and the treatment effect of the patient can be improved.
  • the present invention also provides the use of the antibody or antigen-binding fragment in preparing a drug or a kit or a multispecific antibody or antibody conjugate.
  • the drug is used to treat cancer.
  • the provided antibody or antigen-binding fragment can be used to prepare a drug for treating a variety of diseases. It can also be used to prepare a kit for use as an immunodiagnostic reagent.
  • Example 1 The mouse sp34 antibody was humanized. Specifically, humanized sequence templates were searched in the database based on the sp34 antibody sequence, and the importance of different amino acids was analyzed based on the 3D structure. If necessary, the key amino acids that affect the structure were reversely mutated to determine the corresponding humanized antibody.
  • the sequences with the highest similarity to the framework region (FR) were first selected for analysis.
  • V7-46*01 was selected as the template for humanized germline to design VL mutants
  • V3-72*01 was selected as the template for humanized germline to design VH mutants.
  • the humanized sequences formed are shown in SEQ ID NO: 2 ⁇ 3 (heavy chain variable region) and SEQ ID NO: 13 ⁇ 15 (light chain variable region).
  • the heavy chain variable region of one of the humanized antibodies is shown in SEQ ID NO: 2, and the light chain variable region is shown in SEQ ID NO: 13; the heavy chain variable region of one of the humanized antibodies is shown in SEQ ID NO: 2, and the light chain variable region is shown in SEQ ID NO: 14; the heavy chain variable region of one of the humanized antibodies is shown in SEQ ID NO: 2, and the light chain variable region is shown in SEQ ID NO: 15; the heavy chain variable region of one of the humanized antibodies
  • the activities of the humanized antibodies were verified as shown in SEQ ID NO:3, and the light chain variable region was shown in SEQ ID NO:13; the heavy chain variable region of one of the humanized antibodies was shown in SEQ ID NO:3, and the light chain variable region was shown in SEQ ID NO:14; the heavy chain variable region of one of the humanized antibodies was shown in SEQ
  • the humanized antibody was fused with the constant region sequence of human origin (the heavy chain constant domain sequence used is shown in SEQ ID NO:11, and the light chain constant domain sequence is shown in SEQ ID NO:12), constructed into the PCDNA3.1 mammalian expression vector, and constructed into the form of a chimeric antibody. Then, the different antibodies obtained were expressed and purified using a mammalian cell system to obtain different antibodies with a purity of at least 90%.
  • the expressed antibody sequences are hu01 to hu15, respectively. Among them, the antibody hu01, its heavy chain variable region is shown in SEQ ID NO:1, and the light chain variable region is shown in SEQ ID NO:4.
  • the antibody hu02, its heavy chain variable region is shown in SEQ ID
  • the heavy chain variable region is shown in SEQ ID NO:1, and the light chain variable region is shown in SEQ ID NO:5.
  • Antibody hu03, its heavy chain variable region is shown in SEQ ID NO:1, and the light chain variable region is shown in SEQ ID NO:6.
  • Antibody hu04, its heavy chain variable region is shown in SEQ ID NO:1, and the light chain variable region is shown in SEQ ID NO:7.
  • Antibody hu05, its heavy chain variable region is shown in SEQ ID NO:1, and the light chain variable region is shown in SEQ ID NO:8.
  • Antibody hu06 its heavy chain variable region is shown in SEQ ID NO:2, and the light chain variable region is shown in SEQ ID NO:4.
  • Antibody hu07 its heavy chain variable region is shown in SEQ ID NO:2, and the light chain variable region is shown in SEQ ID NO:5.
  • Antibody hu08 its heavy chain variable region is shown in SEQ ID NO:2, and the light chain variable region is shown in SEQ ID NO:6.
  • Antibody hu09, its heavy chain variable region is shown in SEQ ID NO:2, and the light chain variable region is shown in SEQ ID NO:7.
  • Antibody hu10 its heavy chain variable region is shown in SEQ ID NO:2, and its light chain variable region is shown in SEQ ID NO:8.
  • Antibody hu14, its heavy chain variable region is shown in SEQ ID NO:3, and its light chain variable region is shown in SEQ ID NO:7.
  • Example 2 The activity of the humanized antibody prepared in Example 1 was detected by ELISA method.
  • the experimental process is as follows:
  • Example 3 The binding activity of the humanized antibody prepared in Example 1 was detected by FACS technology.
  • the experimental process is as follows:
  • 293T-CD3 cells or Jurkat cells were plated, washed with FACS buffer, and centrifuged at 1200 rpm for 3-5 min; then, different concentrations of diluted antibodies were added, incubated at 4°C for 0.5 h, centrifuged at 1200 rpm for 3-5 min, and washed with FACS buffer; then, goat anti-human IgG secondary antibody (manufacturer: Abcam, ab98596) was added at 100 uL/well, incubated at 4°C for 0.5 h; centrifuged at 1200 rpm for 3-5 min, washed with FACS buffer, and 50 uL of FACS buffer was added to resuspend the cells, and detected by flow cytometry.
  • FACS buffer 293T-CD3 cells or Jurkat cells were plated, washed with FACS buffer, and centrifuged at 1200 rpm for 3-5 min; then, different concentrations of diluted antibodies were added, incubated at 4°C for 0.5

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Abstract

L'invention concerne un anticorps anti-CD3 humanisé et son utilisation. L'anticorps selon l'invention a une région variable de chaîne lourde représentée par SEQ ID NO : 1, 2 ou 3 et une région variable de chaîne légère représentée par SEQ ID NO : 4,5,6, 7 ou 8. L'anticorps selon l'invention peut spécifiquement reconnaître et se lier à CD3.
PCT/CN2024/071578 2023-01-18 2024-01-10 Anticorps anti-cd3 humanisé et son utilisation Ceased WO2024152963A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1421459A (zh) * 2001-11-23 2003-06-04 上海中信国健药业有限公司 人源化抗cd3单克隆抗体
CN104822704A (zh) * 2012-06-14 2015-08-05 医疗生物科学有限公司 针对分化簇3(cd3)的人源化的抗体
CN107001468A (zh) * 2014-08-07 2017-08-01 艾芙美德有限公司 Cd3结合结构域
WO2019034580A1 (fr) * 2017-08-14 2019-02-21 Morphosys Ag Anticorps humanisés pour cd3
CN113461820A (zh) * 2021-09-03 2021-10-01 苏州近岸蛋白质科技股份有限公司 抗cd3人源化抗体
WO2023217072A1 (fr) * 2022-05-09 2023-11-16 杭州微诺迈博生物科技有限公司 Agent thérapeutique comprenant un anticorps multispécifique et son utilisation en thérapie tumorale

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CN1421459A (zh) * 2001-11-23 2003-06-04 上海中信国健药业有限公司 人源化抗cd3单克隆抗体
CN104822704A (zh) * 2012-06-14 2015-08-05 医疗生物科学有限公司 针对分化簇3(cd3)的人源化的抗体
CN107001468A (zh) * 2014-08-07 2017-08-01 艾芙美德有限公司 Cd3结合结构域
WO2019034580A1 (fr) * 2017-08-14 2019-02-21 Morphosys Ag Anticorps humanisés pour cd3
CN113461820A (zh) * 2021-09-03 2021-10-01 苏州近岸蛋白质科技股份有限公司 抗cd3人源化抗体
WO2023217072A1 (fr) * 2022-05-09 2023-11-16 杭州微诺迈博生物科技有限公司 Agent thérapeutique comprenant un anticorps multispécifique et son utilisation en thérapie tumorale

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S. PESSANO ET AL.: "The T3/T Cell Receptor Complex: The T3/T Cell Receptor Complex Antigenic Distinction between the Two 20-kd T3 (T3-delta and T3-epsilon) Subunits", THE EMBO JOURNAL, vol. 4, no. 2, 28 February 1985 (1985-02-28), XP055348187 *

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