WO2024087521A1 - Anti-cd3 antibody and application thereof - Google Patents
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- WO2024087521A1 WO2024087521A1 PCT/CN2023/086578 CN2023086578W WO2024087521A1 WO 2024087521 A1 WO2024087521 A1 WO 2024087521A1 CN 2023086578 W CN2023086578 W CN 2023086578W WO 2024087521 A1 WO2024087521 A1 WO 2024087521A1
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Classifications
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
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- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Definitions
- the present invention relates to the field of biomedicine, and in particular to an anti-CD3 antibody and application thereof.
- T lymphocytes play a very important role in the adaptive immune response and the precise regulation of the immune response.
- the T cell receptor (TCR) on the surface of T cells exists in the form of heterodimers of ⁇ and ⁇ or ⁇ and ⁇ . After receiving the antigen peptide signal from the antigen presenting cell, the TCR cannot directly mediate the intracellular signal, but needs to form a complex with the CD3 molecule. Not only ⁇ and ⁇ TCRs form a complex with CD3, but ⁇ and ⁇ TCRs also form a complex with CD3. The loss of TCR or CD3 genes can lead to the disappearance of the TCR-CD3 complex from the surface of T cells.
- the CD3 molecule is composed of 3 pairs of peptide chains formed by 5 peptide chains.
- CD3 90% of CD3 is composed of ⁇ , ⁇ and ⁇ , while the other 10% of CD3 is composed of ⁇ , ⁇ and ⁇ .
- the immunoreceptor tyrosine activation motif in the intracellular region of each CD3 peptide chain is phosphorylated by the tyrosine protein kinase p56 in the T cell, recruits and binds to tyrosine kinases such as ZAP-70, and participates in mediating TCR activation signals.
- bispecific antibodies based on T cell redirection
- the target of CD3 is widely selected.
- CD3-related bispecific antibodies can mediate T cell activation by bypassing TCR and peptide-major histocompatibility complex (pMHC), but the molecular process in the synapse is very similar to the interaction of classical TCR-pMHC.
- the activity of bispecific antibodies is affected by CD3 affinity.
- CD3 high-affinity bispecific antibodies have better promoting killing effects in in vitro experiments, but there is a higher risk of release factor syndrome in vivo.
- studies have found that very low-affinity CD3 antibody sequences can also effectively stimulate T cell activation after constructing bispecific antibodies.
- the bispecific antibody will prompt T cells to selectively locate to the tumor rather than in the peripheral circulation, avoiding systemic activation.
- CD3 antibodies are species-specific. For example, clones such as UCHT-1, OKT3, and L2K react only with chimpanzee CD3 but not with other primates such as cynomolgus monkeys and rhesus monkeys CD3 or mouse CD3. Since new drug development must undergo rigorous non-clinical trials before clinical research can be conducted, it is necessary to find relevant species for preclinical safety evaluation. The use of chimpanzees for drug safety testing is highly restricted. Among the currently known CD3 antibodies with human-monkey cross-reactivity, the affinity of the SP34 antibody decreases by an order of magnitude after forming a single-chain antibody.
- the inventors of the present application have successfully screened a mouse anti-CD3 monoclonal antibody that has high binding activity to human or monkey CD3 protein.
- the inventors partially humanized the constant region of the obtained mouse antibody and retained the CDR of the mouse anti-CD3 monoclonal antibody to obtain a chimeric antibody.
- the framework region in the light chain variable region or the heavy chain variable region of the chimeric antibody was humanized to obtain a fully humanized anti-CD3 antibody.
- the chimeric antibody and humanized antibody can not only specifically target and bind to human CD3 protein and monkey CD3 protein, but also have the characteristics of low immunogenicity, and can effectively treat and/or prevent CD3-mediated related diseases, such as autoimmune diseases.
- the bispecific antibody (bispecific antibody) or multispecific antibody (multispecific antibody) prepared using the anti-CD3 antibody can also specifically target and bind to human CD3 protein and monkey CD3 protein, and generally, based on the specificity of the bispecific antibody or multispecific antibody, the bispecific antibody or multispecific antibody can target T cells to other antigens, eliminate cells producing such antigens through T cell-mediated cell killing, thereby treating various diseases, such as tumors.
- the present invention proposes an antibody or antigen-binding fragment.
- it comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity therewith: heavy chain variable region CDR sequence: SEQ ID NO: 1-3, light chain variable region CDR sequence: SEQ ID NO: 4-6.
- the antibody or antigen-binding fragment according to the embodiment of the present invention can bind to human or monkey CD3 protein and effectively treat or prevent CD3-mediated related diseases.
- the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features:
- the antibody or antigen-binding fragment comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, 2 and 3, or an amino acid sequence with at least 95% identity to SEQ ID NO: 1, 2 and 3; and/or light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 4, 5 and 6, or an amino acid sequence with at least 95% identity to 4, 5 and 6.
- the antibody or antigen-binding fragment comprises: a heavy chain variable region CDR1 sequence as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, the heavy chain variable region CDR2 as shown in SEQ ID NO: 3, the light chain variable region CDR1 as shown in SEQ ID NO: 4, the light chain variable region CDR2 as shown in SEQ ID NO: 5, and the light chain variable region CDR3 as shown in SEQ ID NO: 6.
- the antibody or antigen-binding fragment comprises: at least one of a heavy chain FR region and a light chain FR region.
- At least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a human antibody, a primate antibody, a murine antibody, or a mutant thereof.
- the antibody or antigen-binding fragment comprises: at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NO: 7 to 10, respectively; or at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NO: 15 to 18, respectively.
- the antibody or antigen-binding fragment comprises: at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NO: 11 to 14, respectively; or at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NO: 19 to 22, respectively.
- the antibody or antigen-binding fragment comprises: at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO: 7 to 10, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO: 11 to 14, respectively; or at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO: 15 to 18, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO: 19 to 22, respectively.
- the antibody or antigen-binding fragment includes: a heavy chain variable region as shown in SEQ ID NO: 23 or SEQ ID NO: 27; and/or a light chain variable region as shown in SEQ ID NO: 24 or SEQ ID NO: 28.
- the antibody or antigen-binding fragment includes: 1) a heavy chain variable region as shown in SEQ ID NO:23, and a light chain variable region as shown in SEQ ID NO:24; or 2) a heavy chain variable region as shown in SEQ ID NO:27, and a light chain variable region as shown in SEQ ID NO:28.
- the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody and a murine antibody or a mutant thereof.
- the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.
- the light chain constant region and the heavy chain constant region are both derived from a murine IgG1 antibody or a mutant thereof or a human IgG1 antibody or a mutant thereof.
- the antibody has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 29 or 33 and/or a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 30 or 34.
- the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence shown in any one of SEQ ID NO: 35, 37 and 39, and a light chain having an amino acid sequence shown in any one of SEQ ID NO: 36, 38 and 40.
- the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in SEQ ID NO:35 and a light chain with an amino acid sequence shown in SEQ ID NO:36; the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in SEQ ID NO:37 and a light chain with an amino acid sequence shown in SEQ ID NO:38; or the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in SEQ ID NO:39 and a light chain with an amino acid sequence shown in SEQ ID NO:40.
- the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody.
- the monoclonal antibody includes at least one of a full-length antibody, Fv, a single-chain antibody, Fab, a single-domain antibody and a minimum recognition unit.
- the present invention proposes a bispecific antibody.
- it includes a first binding region, the first binding region comprises the antibody or antigen-binding fragment described in the first aspect; and a second binding region, the second binding region has BCMA or B7H6 binding activity.
- the bispecific antibody according to an embodiment of the present invention can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and can be applied to scientific research, or effectively treat or prevent CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- the binding activity of the second binding region is not particularly limited and may have other binding activities, as long as the bispecific antibody has the antibody or antigen-binding fragment described in the first aspect, and the antibody or antigen-binding fragment and the second binding region can both effectively function.
- the antibodies or antigen-binding fragments described in the present application can also be used to prepare more specific antibodies, such as trispecific, tetraspecific, and pentaspecific. Based on the multispecificity of the antibody, the antibody or antigen-binding fragment of the present invention can target T cells to other antigens and eliminate cells that produce such antigens through T cell-mediated cell killing.
- the above-mentioned bispecific antibody may further include at least one of the following additional technical features:
- the bispecific antibody comprises a symmetric bispecific antibody or an asymmetric bispecific antibody.
- the dual antibody is a symmetrical dual antibody.
- the antibody or antigen-binding fragment is an anti-CD3 single-chain antibody.
- the second binding region includes at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody and a minimum recognition unit having BCMA or B7H6 binding activity.
- the specific composition of the second binding region is not particularly limited, as long as it has BCMA or B7H6 binding activity, and can be a complete full-length antibody or an antibody fragment (antigen binding fragment).
- the second binding region comprises an anti-BCMA single-chain antibody or an anti-B7H6 single-chain antibody.
- the anti-CD3 single-chain antibody comprises an anti-CD3 antibody light chain variable region and an anti-CD3 antibody heavy chain variable region
- the anti-CD3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or 27
- the anti-CD3 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 24 or 28.
- the anti-CD3 single-chain antibody further includes a connecting peptide 1, wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the heavy chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the light chain variable region of the anti-CD3 antibody; or the N-terminus of the connecting peptide 1 is connected to the C-terminus of the light chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the heavy chain variable region of the anti-CD3 antibody.
- the anti-BCMA single-chain antibody comprises an anti-BCMA antibody light chain variable region and an anti-BCMA antibody heavy chain variable region
- the anti-BCMA antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:42
- the anti-BCMA antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:61.
- the anti-BCMA single-chain antibody further includes a connecting peptide 2, wherein the N-terminus of the connecting peptide 2 is connected to the C-terminus of the anti-BCMA antibody heavy chain variable region, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the anti-BCMA antibody light chain variable region; or the N-terminus of the connecting peptide 2 is connected to the C-terminus of the anti-BCMA antibody light chain variable region, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the anti-BCMA antibody heavy chain variable region.
- the anti-B7H6 single-chain antibody comprises an anti-B7H6 antibody light chain variable region and an anti-B7H6 antibody heavy chain variable region
- the anti-B7H6 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:63
- the anti-B7H6 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:62.
- the anti-B7H6 single-chain antibody further includes a connecting peptide 3, wherein the N-terminus of the connecting peptide 3 is connected to the C-terminus of the heavy chain variable region of the anti-B7H6 antibody, and the C-terminus of the connecting peptide 3 is connected to the N-terminus of the light chain variable region of the anti-B7H6 antibody; or the N-terminus of the connecting peptide 3 is connected to the C-terminus of the light chain variable region of the anti-B7H6 antibody, and the C-terminus of the connecting peptide 3 is connected to the N-terminus of the heavy chain variable region of the anti-B7H6 antibody.
- the connecting peptide 1 has an amino acid sequence (GGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
- At least one of the connecting peptide 2 and the connecting peptide 3 has an amino acid sequence (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
- connecting peptides 1, 2, and 3 described herein are not particularly limited, and any conventional connecting peptides in the art can be used, such as conventional flexible amino acid fragments or rigid amino acid fragments.
- the connecting peptide 1 has the amino acid sequence shown in SEQ ID NO:48.
- the connecting peptides 2 and 3 have the amino acid sequence shown in SEQ ID NO:41.
- the anti-CD3 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:45.
- the anti-BCMA single-chain antibody has an amino acid sequence as shown in SEQ ID NO:46.
- the anti-B7H6 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:47.
- the first antigen binding region further includes a first Fc peptide segment, and the N-terminus of the first Fc peptide segment is connected to the C-terminus of the antibody or antigen binding fragment.
- the second antigen binding region further includes a second Fc peptide segment, and the N-terminus of the second Fc peptide segment is connected to the C-terminus of the anti-BCMA single-chain antibody or the anti-B7H6 single-chain antibody.
- the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO:49.
- the second Fc peptide segment has the amino acid sequence shown in SEQ ID NO:50.
- the first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.
- the amino acid sequences of the first Fc peptide segment and the second Fc peptide segment may be the same or different, for example, when they have the same amino acid sequence, the first Fc peptide segment and the second Fc peptide segment may be connected via a disulfide bond.
- the first antigen binding region has the amino acid sequence shown in SEQ ID NO:51
- the second antigen binding region has the amino acid sequence shown in SEQ ID NO:52 or 53.
- nucleic acid encoding the antibody or antigen-binding fragment thereof or the bispecific antibody of the present invention is within the scope of the present invention. Based on its amino acid sequence, a person skilled in the art can easily obtain the corresponding nucleic acid sequence.
- the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment or the bispecific antibody described in the first aspect.
- the antibody or antigen-binding fragment encoded by the nucleic acid molecule can bind to human or monkey CD3 protein, effectively treat or prevent CD3-mediated related diseases
- the bispecific antibody encoded by the nucleic acid molecule can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, effectively treat or prevent CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- the above nucleic acid molecule may further include at least one of the following additional technical features:
- the nucleic acid molecule is DNA.
- nucleic acids mentioned in the specification and claims of the present invention those skilled in the art should understand that they actually include any one or both of the complementary double strands.
- the nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
- the present invention proposes an expression vector, the expression vector carries the aforementioned nucleic acid molecule.
- the expression vector may include an optional control sequence, the control sequence is operably connected to the nucleic acid molecule.
- the control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host.
- the expression vector proposed in the embodiment of the present invention can efficiently express the antibody or antigen-binding fragment in a large amount in a suitable host cell.
- operably connected means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended function of regulating the transcription and translation of the exogenous gene.
- control elements in the vector such as transcription control sequences and translation control sequences, etc.
- the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules.
- these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself.
- nucleic acid molecules used to encode antibodies or antigen-binding fragments can be inserted into different vectors separately and independently, and it is common to insert them into the same vector.
- Commonly used vectors can be, for example, plasmids, bacteriophages, etc.
- Plasmid-X plasmid Plasmid-X plasmid.
- the present invention proposes a method for preparing the above-mentioned antibody or antigen-binding fragment or bispecific antibody, comprising: introducing the above-mentioned expression vector into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment or bispecific antibody.
- the method proposed according to some specific embodiments of the present invention can effectively obtain the antibody or antigen-binding fragment or bispecific antibody in large quantities in vitro.
- the method for preparing the above-mentioned antibody or antigen-binding fragment or bispecific antibody may also include at least one of the following additional technical features:
- the cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used.
- the cell is a eukaryotic cell.
- the eukaryotic cell is a mammalian cell.
- the expression efficiency of the recombinant antibody is higher.
- the present invention provides a recombinant cell, wherein the recombinant cell contains the aforementioned nucleic acid, or expression vector, or is capable of expressing the aforementioned antibody or antigen-binding fragment or the bispecific antibody.
- the recombinant cell is obtained by transfecting or transforming the expression vector.
- the recombinant cell can efficiently and massively express the above-mentioned antibody or antigen-binding fragment or the bispecific antibody under appropriate conditions.
- the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages.
- the prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.
- the eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.
- the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.
- suitable conditions refer to conditions suitable for the expression of the antibody or antigen-binding fragment or double antibody described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the antibody or antigen-binding fragment or double antibody include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions” are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody or antigen-binding fragment or double antibody according to the specific environment of the laboratory.
- the present invention proposes an immunoconjugate, comprising the above-mentioned antibody or antigen-binding fragment or the bispecific antibody, and a therapeutic agent.
- the antibody or antigen-binding fragment of the embodiment of the present invention can effectively bind to CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or bind to human or monkey CD3 protein and B7H6 protein, effectively treating or preventing CD3 and BCMA, or CD3 and B7H6-mediated related diseases, therefore, the immunoconjugate containing the antibody or antigen-binding fragment can also bind to human or monkey CD3 protein, and the immunoconjugate containing the bispecific antibody can also bind to human or monkey CD3 protein and BCMA protein, or bind to human or monkey CD3 protein and B7H6 protein, and the immunoconjugate has good effects in preventing and/or treating CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases
- the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment, bispecific antibody, nucleic acid molecule, expression vector
- the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein
- the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and effectively inhibit the proliferation of tumor cells.
- the composition containing the above substances can also effectively bind to human or monkey CD3 protein, or to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and has good effects in preventing and/or treating CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- the type of the composition is not particularly limited and can be a food composition or a pharmaceutical composition.
- compositions of the invention may also be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent.
- the composition may also contain a chemotherapeutic agent.
- the antibodies or antigen-binding fragments thereof, or immunoconjugates of the invention may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit CD3 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or agents that interfere with CD3 upstream or downstream signal transduction.
- composition includes combinations separated in time and/or space, as long as they can work together to achieve the purpose of the present invention.
- the components contained in the composition can be administered to the subject as a whole, or separately.
- each component can be administered to the subject simultaneously or sequentially.
- the present invention proposes a drug, including the above-mentioned antibody or antigen binding fragment, bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or composition.
- the antibody or antigen binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein.
- a drug containing an effective amount of the antibody or antigen binding fragment or the active ingredient of the bispecific antibody or a series of substances thereof can also effectively bind to human or monkey CD3 protein, or to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and has good effects in preventing and/or treating CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- the above-mentioned medicine may further include at least one of the following additional technical features:
- the drug may further include a pharmaceutically acceptable carrier.
- the term "effective amount” or “effective dose” refers to an amount that can produce a function or activity on humans and/or animals and can be accepted by humans and/or animals.
- the effective amount of the antibody or antigen-binding fragment or the bispecific antibody of the present invention may vary with the mode of administration and the severity of the disease to be treated.
- the selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials).
- the factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.
- pharmaceutically acceptable ingredients are suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit/risk ratio.
- pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
- the medicine of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier.
- Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
- the drug preparation should match the mode of administration, wherein the mode of administration can be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration or intraperitoneal administration, and the dosage form of the drug of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent.
- it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
- the drug is preferably manufactured under sterile conditions.
- the antibody or antigen-binding fragment or bispecific antibody can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
- anti-CD3 monoclonal antibody or bispecific antibody herein can also be prepared as a part of a kit or other diagnostic reagent as required.
- the present invention proposes a kit, the kit containing the above-mentioned antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid molecule, expression vector or recombinant cell.
- the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein
- the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein.
- the kit containing the antibody or antigen-binding fragment can effectively detect human or monkey CD3 protein qualitatively or quantitatively
- the kit containing the bispecific antibody can effectively detect human or monkey CD3 protein and BCMA protein, or human or monkey CD3 protein and B7H6 protein qualitatively or quantitatively.
- the kit provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, etc., which involve the use of human or monkey CD3 and antibody specific binding properties for detection.
- kits may contain any one or more of the following: antagonists, anti-CD3 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc.
- Anti-CD3 antibodies can be used in different types of diagnostic tests, such as in vitro or in vivo detection of various diseases or the presence of drugs, toxins or other proteins.
- the serum or blood of a subject can be tested to test for related diseases.
- related diseases may include CD3-related diseases, such as cancer, wherein the cancer includes multiple myeloma, lymphoma, hemangioma, gastric cancer, At least one of liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- CD3-related diseases such as cancer, wherein the cancer includes multiple myeloma, lymphoma, hemangioma, gastric cancer, At least one of liver cancer, lung cancer, breast cancer, colorectal cancer, nasoph
- the antibodies or antigen-binding fragments provided herein can also be used for radioimmunoassay and radioimmunotherapy of the above-mentioned diseases.
- the dual antibodies are also applicable and will not be repeated here.
- the kit may also include conventional reagents for detecting CD3, or CD3 and BCMA, or CD3 and B7H6, such as coating fluid, etc.
- the present invention proposes the use of the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell or composition in the preparation of a drug, and the drug is used to prevent and/or treat CD3-mediated related diseases.
- the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, and therefore, a drug containing an effective amount of the antibody or antigen-binding fragment or a series of substances thereof can also effectively bind to human or monkey CD3 protein, and has a good effect of preventing and/or treating CD3-mediated related diseases.
- the above-mentioned use of preparing a medicine may also include at least one of the following additional technical features:
- the CD3-mediated related diseases include autoimmune diseases.
- the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- the present invention proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of drugs, which are used to prevent and/or treat CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- the bispecific antibodies of some specific embodiments of the present invention can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, therefore, drugs containing an effective amount of the active ingredients of the bispecific antibodies or a series of substances thereof can also effectively bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and have good effects in preventing and/or treating CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- the above-mentioned use of preparing a medicine may also include at least one of the following additional technical features:
- the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- the present invention proposes the use of the above-mentioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector or recombinant cell in preparing a kit for detecting CD3.
- the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein and block the binding of the CD3 protein to its receptor. Therefore, the antibody or antigen-binding fragment can be used to prepare a kit for detecting CD3 protein, and the kit can effectively perform qualitative or quantitative detection of human or monkey CD3 protein.
- the present invention proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors or recombinant cells in the preparation of a kit for detecting CD3 and/or BCMA, or CD3 and/or B7H6.
- the bispecific antibodies of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, therefore, the bispecific antibodies can be used to prepare a kit for detecting CD3 and/or BCMA, or CD3 and/or B7H6, and the kit can effectively perform qualitative or quantitative detection of human or monkey CD3 and/or BCMA, or CD3 and/or B7H6.
- the present invention proposes a method for treating or preventing CD3, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- the method comprises administering to a subject at least one of the following: 1) the antibody or antigen binding fragment described above; 2) the bispecific antibody described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; 5) the recombinant cell described above; 6) the composition described above; and 7) the drug described above.
- the bispecific antibody can effectively bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and the antibody or antigen binding fragment can bind to human or monkey CD3 protein, and can effectively treat or prevent CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, such as autoimmune diseases or cancer. Therefore, the method according to an embodiment of the present invention can effectively treat or prevent CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, preferably autoimmune diseases or cancer.
- the above method for treating or preventing a disease may further include at least one of the following additional technical features:
- the CD3-mediated related diseases include autoimmune diseases.
- the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic hemoglobin Patients with this disorder may also be diagnosed with leukoencephalitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, autoimmune thyroid disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.
- the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- the present invention proposes a method for diagnosing CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases.
- it includes using at least one of the following to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested: 1) the antibody or antigen binding fragment described above; 2) the bispecific antibody described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; and 5) the recombinant cell described above, based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is determined.
- the antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, or antibodies or antigen-binding fragments expressed by recombinant cells proposed in the present application can effectively bind to human or monkey CD3 protein, and the bispecific antibodies, or nucleic acid molecules, expression vectors, or bispecific antibodies expressed by recombinant cells can effectively bind to CD3, or CD3 and/or BCMA, or CD3 and/or B7H6. Therefore, the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample derived from the test individual, and can effectively diagnose related diseases caused by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- the above method for diagnosing a disease may further include at least one of the following additional technical features:
- the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is not less than the minimum standard for disease, which indicates that the test sample is derived from a patient suffering from a disease related to CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- the value of the minimum standard can be determined by comparative analysis and verification of the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a large number of individuals suffering from the disease related to CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 and a large number of healthy individuals.
- the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
- the CD3-mediated related diseases include autoimmune diseases.
- the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- the present invention proposes a method for staging CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases.
- it includes using at least one of the following to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested: 1) the antibody or antigen binding fragment described above; 2) the bispecific antibody described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; and 5) the recombinant cell described above, based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is determined.
- the bispecific antibodies proposed in the present application, or the bispecific antibodies expressed by nucleic acid molecules, expression vectors, and recombinant cells can effectively bind to human or monkey CD3 and/or BCMA, or CD3 and/or B7H6.
- the antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, and antibodies or antigen-binding fragments expressed by recombinant cells can effectively bind to human or monkey CD3 proteins.
- the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample from the subject, and evaluate the stage of related diseases caused by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 based on the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- the above method for staging a disease may further include at least one of the following additional technical features:
- the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is not lower than the standard level of stage IV tumor, which indicates that the test sample is derived from a patient with stage IV tumor.
- the content of CD3 and/or B7H6 being between the standard levels for tumor stage IV and stage III disease is an indication that the sample to be tested is derived from a patient with a tumor stage III; the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is between the standard levels for tumor stage III and stage II disease, is an indication that the sample to be tested is derived from a patient with a tumor stage II; the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is between the standard levels for tumor stage I and stage II disease, is an indication that the sample to be tested is derived from a patient with a tumor stage I.
- the levels of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 when the tumor is at stage I, stage II, stage III, or stage IV vary depending on the type of tumor, and to determine the stage of the tumor, it is only necessary to compare the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample with the standard level of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 at the corresponding tumor stage, or to compare the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample with the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a sample from an individual or group of individuals with a known stage of the disease.
- the values of the standard levels of tumor stages I, II, III, and IV can be determined by comparative analysis and verification of the differences in the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a large number of test samples from individuals suffering from the angiogenesis-related diseases and a large number of healthy individuals.
- the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
- the CD3-mediated related diseases include autoimmune diseases.
- the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- the present invention proposes a method for evaluating the prognosis of related diseases mediated by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- it includes using at least one of the following to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested: 1) the aforementioned double antibody; 2) the aforementioned antibody or antigen binding fragment; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; and 5) the aforementioned recombinant cell, based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, determine the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested.
- CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 has an important influence on cancer.
- the prognosis of such diseases can be effectively evaluated by monitoring the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in their tissues or excretions, such as peripheral blood, urine, etc.
- the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the subject before and after treatment can be compared, or the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the subject after treatment can be compared with the CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 levels in normal individuals or diseased individuals, etc.
- the bispecific antibodies proposed in the present application, or the bispecific antibodies expressed by nucleic acid molecules, expression vectors or recombinant cells can effectively bind to CD3 and/or BCMA, or CD3 and/or B7H6.
- the antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, antibodies or antigen-binding fragments expressed by recombinant cells can effectively bind to human or monkey CD3. Therefore, the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample from the subject, and evaluate the prognosis of related diseases caused by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 based on the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- the above method for evaluating disease prognosis may further include at least one of the following additional technical features:
- the sample to be tested is derived from a patient suffering from a CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related disease before or after treatment.
- the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
- the prognostic effect of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases is determined based on the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample of a patient suffering from CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases before or after the treatment.
- a decrease in the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a test sample of a patient suffering from a CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related disease after treatment is an indication of a good prognosis for the patient.
- the CD3-mediated related diseases include autoimmune diseases.
- the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- the present invention proposes the use of the aforementioned bispecific antibodies, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions or drugs in the treatment or prevention of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases.
- the bispecific antibodies have high binding activity with CD3 and/or BCMA, or CD3 and/or B7H6, and the antibodies or antigen-binding fragments can effectively bind to human or monkey CD3, and can effectively treat or prevent CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases.
- the above-mentioned use may further include at least one of the following additional technical features:
- the CD3-mediated related diseases include autoimmune diseases.
- the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- the present invention proposes the use of the aforementioned bispecific antibodies, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors or recombinant cells in diagnosing CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, staging CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, or evaluating the prognosis of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases.
- the bispecific antibodies proposed in the present application or the bispecific antibodies expressed by nucleic acid molecules, expression vectors or recombinant cells can effectively bind to CD3 and/or BCMA, or CD3 and/or B7H6, and the antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, antibodies or antigen-binding fragments expressed by recombinant cells can effectively bind to human or monkey CD3.
- the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample derived from the subject, and can effectively diagnose, stage and evaluate the prognosis of related diseases mediated by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- the above-mentioned use may further include at least one of the following additional technical features:
- the CD3-mediated related diseases include autoimmune diseases.
- the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- the "subject” or “individual” referred to in the present invention generally refers to a mammal, such as a primate and/or a rodent, in particular a human, a monkey or a mouse.
- the mouse anti-CD3 monoclonal antibody obtained in the present invention has higher CD3 protein binding activity than the existing CD3 monoclonal antibody, and the CD3 protein includes human CD3 protein and monkey CD3 protein.
- the humanized antibody obtained by humanizing the mouse anti-CD3 monoclonal antibody also has higher CD3 protein binding activity than the existing CD3 monoclonal antibody, the CD3 protein includes human CD3 protein and monkey CD3 protein, and the humanized antibody has lower immunogenicity and higher safety, The effect is longer lasting.
- FIG1A is a graph showing the ELISA test results of different concentrations of mouse CD3 antibody (m47B5) binding to human CD3EG protein according to an embodiment of the present invention
- FIG1B is a diagram showing the ELISA test results of different concentrations of mouse CD3 antibodies binding to human CD3ED protein according to an embodiment of the present invention.
- FIG1C is a diagram showing the ELISA test results of different concentrations of mouse CD3 antibodies binding to monkey CD3EG protein according to an embodiment of the present invention.
- FIG1D is a graph showing the ELISA test results of different concentrations of mouse CD3 antibodies binding to monkey CD3ED protein according to an embodiment of the present invention.
- FIG2A is a graph showing the detection results of different concentrations of human-mouse chimeric CD3 antibody ch47B5 binding to human PBMC cells according to an embodiment of the present invention
- 2B is a graph showing the detection results of different concentrations of human-mouse chimeric CD3 antibody ch47B5 binding to monkey PBMC cells according to an embodiment of the present invention
- FIG3A is a graph showing the detection results of humanized CD3 antibodies of different concentrations binding to human CD3 protein according to an embodiment of the present invention.
- FIG3B is a graph showing the detection results of humanized CD3 antibodies of different concentrations binding to monkey CD3 protein according to an embodiment of the present invention.
- FIG4A is a graph showing the detection results of CD3 ⁇ BCMA bispecific antibodies of different concentrations binding to human CD3 protein according to an embodiment of the present invention.
- FIG4B is a graph showing the detection results of CD3 ⁇ BCMA bispecific antibodies of different concentrations binding to human BCMA protein according to an embodiment of the present invention.
- FIG5 is a graph showing the killing test results of NCI-H929 cells by CD3 ⁇ BCMA bispecific antibodies of different concentrations according to an embodiment of the present invention.
- FIG6A is a graph showing the detection results of CD3 ⁇ B7H6 bispecific antibodies of different concentrations binding to human CD3 protein according to an embodiment of the present invention.
- FIG6B is a graph showing the detection results of different concentrations of CD3 ⁇ BCMA bispecific antibodies binding to human B7H6 protein according to an embodiment of the present invention.
- FIG. 7 is a graph showing the killing test results of HCT-15 cells by CD3 ⁇ BCMA bispecific antibodies of different concentrations according to an embodiment of the present invention.
- first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality” is two or more.
- any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values.
- the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.
- the antibody or antigen-binding fragment of the present invention is usually prepared by a biosynthetic method.
- a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include but are not limited to: PCR, DNA artificial synthesis, etc. For specific methods, please refer to J. Sambrook, "Molecular Cloning Laboratory Guide”.
- the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR. Wherein, the antibody or antigen fragment is numbered and defined using the Kabat numbering system.
- the term "monoclonal antibody” refers to an antibody that has a single antigen binding site.
- double antibody refers to an antibody having two different antigen binding sites.
- mutant or “variant” may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.
- CDR complementarity determining region
- CDR sequence refers to the amino acid sequence in an antibody that is responsible for antigen binding, for example, generally including: amino acid residues around 23-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region, and around 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Health Service, National Institutes of Health, Bethesda, MD.
- hypovariable loops e.g., amino acid residues around 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
- the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol.
- GAP Genetics Computing Group
- those skilled in the art can replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region.
- the variant sequence of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% consistency (or homology) with the reference sequence.
- the sequence consistency of the present invention can be measured using sequence analysis software.
- the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN.
- the amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.
- the monoclonal antibody of the present invention can be a full-length antibody or can only contain its functional fragments, or can be modified to affect function.
- the present invention includes anti-CD3 antibodies with modified glycosylation patterns. In some applications, it can be useful to modify to remove undesirable glycosylation sites, or antibodies in which fucose moieties are not present on the oligosaccharide chains to, for example, enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modification can be performed to alter complement-dependent cytotoxicity (CDC).
- the “full-length antibody” refers to a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).
- the term "functional fragment” as used herein refers in particular to antibody fragments such as CDR-grafted antibodies, Fab, F(ab')2, Fv or scFv, nanobodies, or any fragments that should be able to increase half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol (“PEGylation, PEGylation”) (called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG PEGylated fragments) (“PEG” is polyethylene glycol), or by incorporation into liposomes, wherein the fragment has CD3 binding activity.
- poly(alkylene) glycols such as polyethylene glycol (“PEGylation, PEGylation")
- PEG
- the functional fragment will be composed of or contain a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1/100 of the affinity of the antibody from which it is derived, and at least 1/10 in a more preferred manner.
- Such functional fragments will contain a minimum of 3 amino acids, and preferably 5, 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.
- antigen-binding fragment used generally refers to an antigen-binding antibody fragment, which may include a portion of a complete antibody, generally an antigen-binding region or a variable region, such as, for example, CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.
- CDR transplanted antibody refers to transplanting the CDR of a monoclonal antibody of one species to the variable region of an antibody of another species.
- the CDR of a mouse monoclonal antibody can be transplanted to the variable region of a human antibody to replace the human antibody CDR, so that the human antibody obtains the antigen binding specificity of the mouse monoclonal antibody while reducing its heterology.
- Fab antibody generally refers to an antibody containing only Fab molecules, which are composed of VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.
- F(ab') 2 antibody or “F(ab') 2” has two antigen-binding F(ab) portions linked together by disulfide bonds.
- the term “nanoantibody” single domain antibody or VHH antibody
- VHH antibody single domain antibody
- a “heavy chain antibody” i.e., “antibody lacking light chains”
- VH heavy chain variable region
- CH2 and CH3 regions which specifically bind to the antigen through the heavy chain variable region.
- Fv antibody generally refers to an antibody consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.
- single-chain antibody or “scFv” refers to a fragment consisting of the variable regions of the heavy and light chains of an antibody connected by a short peptide.
- the "knob into hole structure” refers to a knob (knob) and a hole (hole) mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate the heavy chain to bite and form a heterodimer.
- it is achieved by mutating the amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole”; T366W, S354C mutations in the other chain, i.e., "knob”).
- the amino acid numbering here is based on the Kabat numbering.
- T366S means that the T amino acid at position 366 according to the Kabat numbering system is replaced by the S amino acid.
- anti-human CD3 monoclonal antibodies were produced by immunizing mice.
- the experimental mice were C57BL/6, female, 6 weeks old (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), and the immunization antigens were human CD3E&D, human CD3E&G, monkey CD3E&D, and monkey CD3E&G extracellular segment protein (ACRO).
- the complete adjuvant (Sigma, F5881) was mixed with the antigen and then intraperitoneally immunized. Each mouse was intraperitoneally injected with 100 ⁇ g.
- the Ribi adjuvant system (Sigma, S6322) was mixed with the antigen for subsequent immunization.
- immunization was performed every 2 weeks, for a total of 3 immunizations.
- the serum of the immunized mice was taken for antibody titer ELISA detection, and the operation method was the conventional method in the art.
- mice with high antibody titer in serum were selected for spleen cell fusion.
- the selected mice were sprint immunized 72 hours before fusion, and a mixture of Ribi adjuvant system and the above antigens was intraperitoneally injected.
- the spleen lymphocytes were fused with myeloma cells Sp2/0 cells (ATCC, CRL-8287) using an optimized PEG-mediated fusion step to obtain hybridoma cells.
- the fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI), distributed in 96-well cell culture plates, and incubated at 37°C and 5% CO 2.
- HAT complete medium was added on the 5th day after fusion, 50 ⁇ L/well. On the 7th to 8th day after fusion, according to the cell growth density, the medium was completely replaced, and the culture medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), 200 ⁇ L/well.
- HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
- the ELISA experiment was used to detect the binding properties of the mouse CD3 antibody m47B5 obtained in Example 1.
- Human CD3ED protein ACRO biosystems, CDD-H52W1
- human CD3EG protein ACRO biosystems, CDG-H52W5
- monkey CD3ED protein ACRO biosystems, CDD-C52W4
- monkey CD3EG protein ACRO biosystems, CDG-H52W6
- coating buffer 35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6
- the plate was washed three times with PBST (0.05% Tween 20-PBS, pH 7.2).
- 300 ⁇ L of blocking buffer 1% BSA, 0.05% Tween20-PBS, pH 7.2
- mouse antibody m47B5 was added to each well and incubate at room temperature for 1 hour.
- wash 3 times with PBST Add 100 ⁇ L of HRP-sheep anti-mouse IgG secondary antibody (boster, item number BA1051) diluted with blocking buffer to each well and incubate at room temperature for 1 hour.
- Figure 1A shows that the mouse m47B5 antibody of the present invention can bind to human CD3EG
- Figure 1B shows that the mouse m47B5 antibody of the present invention can also bind to human CD3ED
- Figure 1C shows that the mouse m47B5 antibody of the present invention can bind to monkey CD3EG
- Figure 1D shows that the mouse m47B5 antibody of the present invention can bind to monkey CD3ED.
- the total number of candidate hybridoma cells of the m47B5 antibody screened in implementation 1 and 2 was cultured to 10 6 , and the cells were collected by centrifugation at 800 rpm for 10 minutes, and the total RNA was extracted using a Trizol kit (Invitrogen); the total RNA was used as a template to synthesize a cDNA library (Invitrogen), and the cDNA was used as a template to PCR amplify the nucleic acid sequence of the variable region of the m47B5 antibody corresponding to the hybridoma cells.
- the primer sequence used in the PCR amplification reaction is complementary to the first framework region or signal peptide region and constant region of the antibody variable region (for specific sequences, refer to Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156).
- PCR amplification was performed in a 50 ⁇ L reaction system, and 2 ⁇ L of cDNA, 5 ⁇ L of 10 ⁇ PCR buffer, 2 ⁇ L of upstream and downstream primers (5 ⁇ M), 2 ⁇ L of dNTP, 1 ⁇ L of Taq enzyme (Takara, Ex Taq), and 38 ⁇ L of H 2 O were added respectively; pre-denaturation at 95°C for 5 min, and temperature cycle was entered for PCR amplification.
- the reaction conditions were: denaturation at 94°C for 30S, annealing at 58°C for 45S, extension at 72°C for 50S, a total of 32 cycles, and then extension at 72°C for 7 min.
- the sequences of the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) of mouse monoclonal antibody m47B5 were obtained.
- the mouse monoclonal antibody m47B5 was used to construct a human-mouse chimeric antibody (ch47B5), wherein the heavy chain constant region of the chimeric antibody used was the heavy chain constant region of human IgG1.
- PBMC peripheral blood mononuclear cells
- monkey PBMC cells were diluted to 2 ⁇ 10 6 cells/mL with PBS, added to a 1.5mL EP tube at a volume of 100 ⁇ L/tube, and 10 ⁇ L/tube of goat serum was added thereto, and the cells were blocked at 4°C for 30min.
- the above-mentioned human-mouse chimeric antibody ch47B5 was added with gradient concentrations (5-fold dilution, the highest final concentration was 10 ⁇ g/mL), and incubated at 4°C for 30min. 1mL PBS was added to the EP tube, centrifuged at 4°C, 3500rpm ⁇ 5min, the supernatant was discarded, and then washed with PBS.
- the heavy chain or light chain variable region germline genes with high homology to the mouse monoclonal antibody m47B5 were selected as templates, and the CDRs of the mouse monoclonal antibody were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
- the amino acid residues were determined and annotated by the Kabat numbering system.
- the heavy chain variable region sequence of the obtained humanized CD3 antibody h47B5 is shown in SEQ ID NO: 27, and the light chain variable region sequence is shown in SEQ ID NO: 28.
- the nucleotide sequence encoding the heavy chain of the humanized antibody (SEQ ID NO: 43) and the nucleotide sequence encoding the light chain of the humanized antibody (SEQ ID NO: 44) were recombined into the pTT5 plasmid by molecular biology technology.
- the pTT5 vector carrying the humanized anti-human CD3 antibody h47B5 light chain and heavy chain was used to transiently transfect ExpiCHO-S cells (Gibco, catalog number A29127) to prepare antibodies.
- ExpiCHO-S cells The cell density of ExpiCHO-S cells was adjusted to (3-4) ⁇ 10 6 /mL and cultured overnight at 37°C, 8% CO 2 , and 120 rpm shaking.
- the cells grow to 7 ⁇ 10 6 ⁇ 1 ⁇ 10 7 /mL and the viability is greater than 95%, they are ready for transfection.
- Use fresh preheated ExpiCHO medium Gibco, Catalog No.
- A2910002 to dilute the cells to 6 ⁇ 10 6 /mL, and transfect the above pTT5 plasmid carrying the light and heavy chains of the humanized anti-human CD3 antibody h47B5 and ExpiFectamine CHO transfection reagent (Gibco, Catalog No. A29129) into ExpiCHO-S cells at a molar ratio of 2:1. Culture at 37°C, 8% CO 2 , 120rpm shaking. 18-22h after transfection, mix ExpiFectamine CHO Enhancer and ExpiCHO Feed and immediately add the transfected cells, mix, and culture at 32°C, 5% CO 2 , 120rpm shaking.
- the chromatography column was equilibrated with a pH 7.2 phosphate buffer, the supernatant was passed through the affinity chromatography column, eluted with an elution buffer (100 mM citric acid, pH 2.7), and finally concentrated and replaced with a PBS buffer.
- the purified antibody was identified by SDS-PAGE to have a purity of more than 95%, and finally a humanized m47B5 antibody (heavy chain as shown in SEQ ID NO: 39, light chain as shown in SEQ ID NO: 40) was obtained.
- the affinity of the humanized h47B5 antibody to the human CD3 antigen was tested by BIACORE. The results are shown in Table 2. The obtained humanized antibody has a good affinity to the CD3 antigen.
- Human CD3 antigen ACRO biosystems, CDD-H52W1
- monkey CD3 antigen ACRO biosystems, CDD-C52W4
- coating buffer 35mM NaHCO 3 , 15mM Na 2 CO 3 , pH 9.6
- 100 ⁇ L was added to each well of the ELISA plate and incubated at 4°C overnight. After that, it was washed 3 times with PBST (0.05% Tween 20-PBS, pH7.2).
- FIGS. 3A and 3B show that the humanized h47B5 antibody of the present invention can bind to human and monkey CD3 antigens.
- Examples 1-6 Based on the experimental results of Examples 1-6, the inventors designed a bispecific antibody CD3 ⁇ BCMA, and tested the binding activity and cell killing ability of the bispecific antibody.
- the antibody contains two monovalent units, one of which is in the form of anti-CD3 scFv-Fc, and its heavy chain and light chain variable region amino acid sequences are from the sequence of the humanized antibody h47B5 obtained in Example 6 of the present invention, and the other monovalent unit is in the form of anti-BCMA scFv-Fc (sequence from US009273141B2), and the bispecific antibody is named CD3 ⁇ BCMA.
- the bispecific antibody contains two polypeptide chains, namely: a heavy chain containing a CD3 single-chain antibody scFv (SEQ ID NO: 51), and a heavy chain containing a BCMA single-chain antibody scFv (SEQ ID NO: 52), wherein the heavy chain constant regions of the two chains are from human antibody IgG1. Since the molecule has a special asymmetric structure, in order to reduce the generation of homodimers, different amino acid mutations are introduced in the constant regions of the two chains to form a knob-in-hole structure. At the same time, in order to prevent cross-linking activation caused by Fc ⁇ receptors, (L234A/L235A) mutations are also introduced in the heavy chain constant region.
- the method for preparing the bispecific antibody refers to the method described in Example 6.2, and the bispecific antibody is prepared by combining the plasmid ratio (1:1 or other ratio) and undergoing one-step affinity purification, wherein the relevant sequence of the bispecific antibody CD3 ⁇ BCMA is shown in Table 3.
- Human BCMA (ACRO biosystems, BCA-H522y) or human CD3 antigen (ACRO biosystems, CDD-H52W1) was diluted to 2 ⁇ g/mL with coating buffer (35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6), and 100 ⁇ L was added to each well of the ELISA plate and incubated at 4°C overnight. Afterwards, the plate was washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 ⁇ L of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2 h. The plate was then washed three times with PBST.
- coating buffer 35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6
- NCI-H929 cells were labeled with CellTrace Violet (thermo, C34557) and the cell density was adjusted to 1 ⁇ 10 5 /mL.
- the cell density of PBMC was adjusted to 1 ⁇ 10 6 /mL, and PBMC and labeled NCI-H929 cells were mixed at a ratio of 1:1 and added to a 96-well round bottom plate, and then the above-mentioned bispecific antibodies or human IgG antibodies (control group) with gradient concentrations were added and mixed evenly. After incubation at 37°C for 24 hours, the cells were transferred to a flow tube through a 200-mesh nylon mesh, and the cells were tested 10 minutes after adding 7AAD.
- the killing efficiency was characterized by the ratio of CTV+7AAD+ cells.
- the results in Figure 5 show that as the concentration of CD3 ⁇ BCMA bispecific antibodies increases, the killing efficiency of PBMC against NCI-H929 multiple myeloma cells gradually increases.
- Examples 1-6 Based on the experimental results of Examples 1-6, the inventors designed a bispecific antibody CD3 ⁇ B7H6, and tested the binding activity and cell killing ability of the bispecific antibody.
- the antibody contains two monovalent units, one of which is in the form of anti-CD3scFv-Fc, in which the light chain and heavy chain variable regions in CD3scFv are from the humanized sequence of Example 6 of the present invention, and the other monovalent unit is in the form of anti-B7H6scFv-Fc (the B7H6scFv sequence is from CN114395045A).
- This bispecific antibody is named CD3 ⁇ B7H6.
- the bispecific antibody contains two polypeptide chains, namely: a heavy chain containing a CD3 single-chain antibody scFv (SEQ ID NO: 51) and a heavy chain containing a B7H6 single-chain antibody scFv (SEQ ID NO: 53).
- the method for preparing the bispecific antibody of CD3 ⁇ B7H6 refers to Example 6.2, and the CD3 ⁇ B7H6 bispecific antibody is prepared by combining the plasmid ratio (1:1 or other ratios) and undergoing one-step affinity purification.
- the relevant sequence of the bispecific antibody CD3 ⁇ B7H6 is shown in Table 4.
- Human B7H6 (ACRO biosystems, B76-H52H8) or human CD3 antigen (ACRO biosystems, CDD-H52W) was diluted to 2 ⁇ g/mL with coating buffer (35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6), and 100 ⁇ L was added to each well of the ELISA plate and incubated at 4°C overnight. Afterwards, the plate was washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 ⁇ L of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2 h.
- coating buffer 35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6
- PBST 0.05% Tween 20-PBS, pH 7.2
- 300 ⁇ L of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2)
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Abstract
An anti-CD3 antibody and an application thereof. The antibody comprises CDR sequences selected from at least one of the following or amino acid sequences at least 95% identical thereto: heavy chain variable region CDR sequences: SEQ ID NOs: 1-3; and light chain variable region CDR sequences: SEQ ID NOs: 4-6.
Description
本发明涉及生物医药领域，具体地，涉及一种抗CD3的抗体及应用。The present invention relates to the field of biomedicine, and in particular to an anti-CD3 antibody and application thereof.
T淋巴细胞在适应性免疫应答和免疫应答的精确调控过程中起着非常重要的作用。T细胞表面T细胞受体(TCR)以α和β或者γ和δ的异二聚体形式存在。T细胞在接受抗原递呈细胞的抗原肽信号后，TCR并不能直接介导细胞内信号，它需要和CD3分子形成复合物。不仅α和βTCR与CD3形成复合物，γ和δTCR也与CD3形成复合物。TCR或CD3基因的缺失均可导致TCR-CD3复合物从T细胞表面消失。CD3分子是由5条肽链形成的3对肽链组合而成，90％的CD3是由γε、δε和ζη的方式组合，而另外10％的CD3由γε、δε和ζζ的方式组合。CD3各条肽链胞内区的免疫受体酪氨酸活化基序，经由T细胞内酪氨酸蛋白激酶p56磷酸化，募集和结合ZAP-70等酪氨酸激酶后，参与介导TCR活化信号。T lymphocytes play a very important role in the adaptive immune response and the precise regulation of the immune response. The T cell receptor (TCR) on the surface of T cells exists in the form of heterodimers of α and β or γ and δ. After receiving the antigen peptide signal from the antigen presenting cell, the TCR cannot directly mediate the intracellular signal, but needs to form a complex with the CD3 molecule. Not only α and β TCRs form a complex with CD3, but γ and δ TCRs also form a complex with CD3. The loss of TCR or CD3 genes can lead to the disappearance of the TCR-CD3 complex from the surface of T cells. The CD3 molecule is composed of 3 pairs of peptide chains formed by 5 peptide chains. 90% of CD3 is composed of γε, δε and ζη, while the other 10% of CD3 is composed of γε, δε and ζζ. The immunoreceptor tyrosine activation motif in the intracellular region of each CD3 peptide chain is phosphorylated by the tyrosine protein kinase p56 in the T cell, recruits and binds to tyrosine kinases such as ZAP-70, and participates in mediating TCR activation signals.
在基于T细胞再导向的双特异性抗体中，CD3这个靶点被广泛选择。与免疫卡控点阻断抗体不同的是，CD3相关的双特异性抗体可以越过TCR和肽-主要组织相容性复合物(pMHC)来介导T细胞活化，但在突触中的分子发挥作用过程与经典TCR-pMHC的相互作用很相似。双特异性抗体的活性受CD3亲和力的影响，CD3高亲和力双特异性抗体在体外实验中有着更好的促杀伤效果，但是在体内存在较高的释放因子综合征风险。同时有研究发现非常低亲和力的CD3抗体序列在构建双特异性抗体后也可以有效地刺激T细胞活化。当CD3抗体亲和力在合适范围且肿瘤靶点相关抗体是高亲和力时，双特异性抗体会促使T细胞选择性地定位到肿瘤局部而不是在外周循环，避免全身活化。Among bispecific antibodies based on T cell redirection, the target of CD3 is widely selected. Unlike immune checkpoint blocking antibodies, CD3-related bispecific antibodies can mediate T cell activation by bypassing TCR and peptide-major histocompatibility complex (pMHC), but the molecular process in the synapse is very similar to the interaction of classical TCR-pMHC. The activity of bispecific antibodies is affected by CD3 affinity. CD3 high-affinity bispecific antibodies have better promoting killing effects in in vitro experiments, but there is a higher risk of release factor syndrome in vivo. At the same time, studies have found that very low-affinity CD3 antibody sequences can also effectively stimulate T cell activation after constructing bispecific antibodies. When the CD3 antibody affinity is in the appropriate range and the tumor target-related antibody is high-affinity, the bispecific antibody will prompt T cells to selectively locate to the tumor rather than in the peripheral circulation, avoiding systemic activation.
已知的很多CD3抗体为物种特异性的，例如UCHT-1、OKT3和L2K等克隆都只与黑猩猩CD3反应但不与其他灵长类如食蟹猴和恒河猴CD3或鼠CD3反应。由于新药开发必需经过严格的非临床试验才能进行临床研究，因此临床前的安全性评价找到相关种属十分必要。而使用黑猩猩进行药物安全性测试是被高度限制的。目前已知的具有人猴交叉的CD3抗体中，SP34抗体在构成单链抗体后亲和力会呈现数量级下降。Many known CD3 antibodies are species-specific. For example, clones such as UCHT-1, OKT3, and L2K react only with chimpanzee CD3 but not with other primates such as cynomolgus monkeys and rhesus monkeys CD3 or mouse CD3. Since new drug development must undergo rigorous non-clinical trials before clinical research can be conducted, it is necessary to find relevant species for preclinical safety evaluation. The use of chimpanzees for drug safety testing is highly restricted. Among the currently known CD3 antibodies with human-monkey cross-reactivity, the affinity of the SP34 antibody decreases by an order of magnitude after forming a single-chain antibody.
因此，开发出与其他非人灵长类有特异性交叉的CD3抗体具有较大的实际应用价值。Therefore, developing CD3 antibodies with cross-specificity with other non-human primates has great practical application value.
发明内容Summary of the invention
本申请旨在至少在一定程度上解决现有技术中存在的技术问题之一：The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent:
本申请的发明人成功地筛选到一种鼠源抗CD3单克隆抗体，该单克隆抗体与人或猴CD3蛋白具有较高的结合活性，此外，发明人将获得的鼠源抗体的恒定区进行部分人源化，保留鼠源抗CD3单克隆抗体的CDR，以获得嵌合抗体，更进一步地，将所述嵌合抗体的轻链可变区或重链可变区中的框架区进行人源化，得到抗CD3的完全人源化抗体，所述嵌合抗体和人源化抗体不仅能够特异性的靶向结合人CD3蛋白和猴CD3蛋白，而且具有免疫原性低的特点，能够有效治疗和/或预防CD3介导的相关疾病，如自身免疫病。The inventors of the present application have successfully screened a mouse anti-CD3 monoclonal antibody that has high binding activity to human or monkey CD3 protein. In addition, the inventors partially humanized the constant region of the obtained mouse antibody and retained the CDR of the mouse anti-CD3 monoclonal antibody to obtain a chimeric antibody. Furthermore, the framework region in the light chain variable region or the heavy chain variable region of the chimeric antibody was humanized to obtain a fully humanized anti-CD3 antibody. The chimeric antibody and humanized antibody can not only specifically target and bind to human CD3 protein and monkey CD3 protein, but also have the characteristics of low immunogenicity, and can effectively treat and/or prevent CD3-mediated related diseases, such as autoimmune diseases.
利用所述抗CD3抗体制备的双特异性抗体(双抗)或者多特异性抗体(多抗)同样能够特异性的靶向结合人CD3蛋白和猴CD3蛋白，且通常地，基于双抗或者多抗体的特异性，所述双抗或者多抗体能够把T细胞靶向到其它抗原，通过T细胞介导的细胞杀伤作用来消除产生这种抗原的细胞，从而治疗多种疾病，如肿瘤。The bispecific antibody (bispecific antibody) or multispecific antibody (multispecific antibody) prepared using the anti-CD3 antibody can also specifically target and bind to human CD3 protein and monkey CD3 protein, and generally, based on the specificity of the bispecific antibody or multispecific antibody, the bispecific antibody or multispecific antibody can target T cells to other antigens, eliminate cells producing such antigens through T cell-mediated cell killing, thereby treating various diseases, such as tumors.
因此，在本发明的第一方面，本发明提出了一种抗体或抗原结合片段。根据本发明的实施例，包括选自下列至少之一的CDR序列或与其具有至少95％同一性的氨基酸序列：重链可变区CDR序列：SEQ ID NO:1～3，轻链可变区CDR序列：SEQ ID NO:4～6。根据本发明实施例的抗体或抗原结合片段能够与人或猴CD3蛋白进行结合，有效治疗或预防CD3介导的相关疾病。Therefore, in the first aspect of the present invention, the present invention proposes an antibody or antigen-binding fragment. According to an embodiment of the present invention, it comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity therewith: heavy chain variable region CDR sequence: SEQ ID NO: 1-3, light chain variable region CDR sequence: SEQ ID NO: 4-6. The antibody or antigen-binding fragment according to the embodiment of the present invention can bind to human or monkey CD3 protein and effectively treat or prevent CD3-mediated related diseases.
根据本发明的实施例，上述抗体或抗原结合片段还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above-mentioned antibody or antigen-binding fragment may further include at least one of the following additional technical features:
根据本发明的实施例，所述抗体或抗原结合片段包括：分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少95％同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；和/或分别如SEQ ID NO:4、5和6或者与4、5和6具有至少95％同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, 2 and 3, or an amino acid sequence with at least 95% identity to SEQ ID NO: 1, 2 and 3; and/or light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 4, 5 and 6, or an amino acid sequence with at least 95% identity to 4, 5 and 6.
根据本发明的实施例，所述抗体或抗原结合片段包括：如SEQ ID NO:1所示的重链可变区CDR1序列，如SEQ ID NO:
2所示的重链可变区CDR2，如SEQ ID NO:3所示的重链可变区CDR3，SEQ ID NO:4所示的轻链可变区CDR1，SEQ ID NO:5所示的轻链可变区CDR2，SEQ ID NO:6所示的轻链可变区CDR3。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: a heavy chain variable region CDR1 sequence as shown in SEQ ID NO: 1, as shown in SEQ ID NO: 2, the heavy chain variable region CDR2 as shown in SEQ ID NO: 3, the light chain variable region CDR1 as shown in SEQ ID NO: 4, the light chain variable region CDR2 as shown in SEQ ID NO: 5, and the light chain variable region CDR3 as shown in SEQ ID NO: 6.
根据本发明的实施例，所述抗体或抗原结合片段包括：重链FR区和轻链FR区中的至少之一。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of a heavy chain FR region and a light chain FR region.
根据本发明的实施例，所述重链FR区和轻链FR区的至少之一的至少一部分来自于人源抗体、灵长目源抗体和鼠源抗体或其突变体中的至少之一。According to an embodiment of the present invention, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a human antibody, a primate antibody, a murine antibody, or a mutant thereof.
根据本发明的实施例，所述抗体或抗原结合片段包括：分别如SEQ ID NO:7～10所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一；或分别如SEQ ID NO:15～18所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NO: 7 to 10, respectively; or at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NO: 15 to 18, respectively.
根据本发明的实施例，所述抗体或抗原结合片段包括：分别如SEQ ID NO:11～14所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一；或分别如SEQ ID NO:19～22所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NO: 11 to 14, respectively; or at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NO: 19 to 22, respectively.
根据本发明的实施例，所述抗体或抗原结合片段包括：分别如SEQ ID NO:7～10所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一；分别如SEQ ID NO:11～14所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一；或者分别如SEQ ID NO:15～18所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一；分别如SEQ ID NO:19～22所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO: 7 to 10, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO: 11 to 14, respectively; or at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO: 15 to 18, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO: 19 to 22, respectively.
根据本发明的实施例，所述抗体或抗原结合片段包括：如SEQ ID NO:23或SEQ ID NO:27所示的重链可变区；和/或如SEQ ID NO:24或SEQ ID NO:28所示的轻链可变区。According to an embodiment of the present invention, the antibody or antigen-binding fragment includes: a heavy chain variable region as shown in SEQ ID NO: 23 or SEQ ID NO: 27; and/or a light chain variable region as shown in SEQ ID NO: 24 or SEQ ID NO: 28.
根据本发明的实施例，所述抗体或抗原结合片段包括：1)如SEQ ID NO:23所示的重链可变区，和如SEQ ID NO:24所示的轻链可变区；或2)如SEQ ID NO:27所示的重链可变区，和如SEQ ID NO:28所示的轻链可变区。According to an embodiment of the present invention, the antibody or antigen-binding fragment includes: 1) a heavy chain variable region as shown in SEQ ID NO:23, and a light chain variable region as shown in SEQ ID NO:24; or 2) a heavy chain variable region as shown in SEQ ID NO:27, and a light chain variable region as shown in SEQ ID NO:28.
根据本发明的实施例，所述抗体或抗原结合片段含有重链恒定区和轻链恒定区的至少之一，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于人源抗体、灵长目源抗体和鼠源抗体或其突变体中的至少之一。According to an embodiment of the present invention, the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody and a murine antibody or a mutant thereof.
根据本发明的实施例，所述轻链恒定区和重链恒定区均来自于鼠源IgG抗体或其突变体或人源IgG抗体或其突变体。According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.
根据本发明的实施例，所述轻链恒定区和重链恒定区均来自于鼠源IgG1抗体或其突变体或人源IgG1抗体或其突变体。According to an embodiment of the present invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG1 antibody or a mutant thereof or a human IgG1 antibody or a mutant thereof.
根据本发明的实施例，所述抗体具有如SEQ ID NO:29或33所示氨基酸序列的重链恒定区和/或如SEQ ID NO:30或34所示氨基酸序列的轻链恒定区。According to an embodiment of the present invention, the antibody has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 29 or 33 and/or a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 30 or 34.
根据本发明的实施例，所述抗体或抗原结合片段具有SEQ ID NO:35、37和39任一项所示氨基酸序列的重链和具有SEQ ID NO:36、38和40任一项所示氨基酸序列的轻链。According to an embodiment of the present invention, the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence shown in any one of SEQ ID NO: 35, 37 and 39, and a light chain having an amino acid sequence shown in any one of SEQ ID NO: 36, 38 and 40.
根据本发明的实施例，所述抗体或抗原结合片段具有SEQ ID NO:35所示氨基酸序列的重链和SEQ ID NO:36所示氨基酸序列的轻链；所述抗体或抗原结合片段具有SEQ ID NO:37所示氨基酸序列的重链和SEQ ID NO:38所示氨基酸序列的轻链；或所述抗体或抗原结合片段具有SEQ ID NO:39所示氨基酸序列的重链和SEQ ID NO:40所示氨基酸序列的轻链。According to an embodiment of the present invention, the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in SEQ ID NO:35 and a light chain with an amino acid sequence shown in SEQ ID NO:36; the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in SEQ ID NO:37 and a light chain with an amino acid sequence shown in SEQ ID NO:38; or the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in SEQ ID NO:39 and a light chain with an amino acid sequence shown in SEQ ID NO:40.
根据本发明的实施例，所述抗体或抗原结合片段包括单抗或多抗。According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody.
根据本发明的实施例，所述单抗包括全长抗体、Fv、单链抗体、Fab、单域抗体以及最小识别单位中的至少之一。According to an embodiment of the present invention, the monoclonal antibody includes at least one of a full-length antibody, Fv, a single-chain antibody, Fab, a single-domain antibody and a minimum recognition unit.
在本发明的第二方面，本发明提出了一种双抗。根据本发明的实施例，包括第一结合区，所述第一结合区包含第一方面所述的抗体或抗原结合片段；和第二结合区，所述第二结合区具有BCMA或B7H6结合活性。根据本发明实施例的双抗能够与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，可以将其应用于科学研究，或有效治疗或预防CD3和BCMA，或者CD3和B7H6介导的相关疾病。In the second aspect of the present invention, the present invention proposes a bispecific antibody. According to an embodiment of the present invention, it includes a first binding region, the first binding region comprises the antibody or antigen-binding fragment described in the first aspect; and a second binding region, the second binding region has BCMA or B7H6 binding activity. The bispecific antibody according to an embodiment of the present invention can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and can be applied to scientific research, or effectively treat or prevent CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
本领域技术人员可以理解，所述第二结合区的结合活性并不受特别限制，也可以具有其它结合活性，只要所述双抗具有第一方面所述的抗体或抗原结合片段，且所述抗体或抗原结合片段和所述第二结合区均能够有效发挥功能即可。此外，也可以利用本申请所述的抗体或抗原结合片段制备更多特异性抗体，如三特异性、四特异性、五特异性，基于所述抗体的多特异性，本发明的抗体或抗原结合片段能够把T细胞靶向到其它抗原，通过T细胞介导的细胞杀伤作用来消除产生这种抗原的细胞。Those skilled in the art will appreciate that the binding activity of the second binding region is not particularly limited and may have other binding activities, as long as the bispecific antibody has the antibody or antigen-binding fragment described in the first aspect, and the antibody or antigen-binding fragment and the second binding region can both effectively function. In addition, the antibodies or antigen-binding fragments described in the present application can also be used to prepare more specific antibodies, such as trispecific, tetraspecific, and pentaspecific. Based on the multispecificity of the antibody, the antibody or antigen-binding fragment of the present invention can target T cells to other antigens and eliminate cells that produce such antigens through T cell-mediated cell killing.
根据本发明的实施例，上述双抗还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above-mentioned bispecific antibody may further include at least one of the following additional technical features:
根据本发明的实施例，所述双抗包括对称双抗或不对称双抗。According to an embodiment of the present invention, the bispecific antibody comprises a symmetric bispecific antibody or an asymmetric bispecific antibody.
根据本发明的实施例，所述双抗为对称双抗。According to an embodiment of the present invention, the dual antibody is a symmetrical dual antibody.
根据本发明的实施例，所述抗体或抗原结合片段为抗CD3单链抗体。
According to an embodiment of the present invention, the antibody or antigen-binding fragment is an anti-CD3 single-chain antibody.
根据本发明的实施例，所述第二结合区包括具有BCMA或B7H6结合活性的全长抗体、Fv、单链抗体、Fab、单域抗体以及最小识别单位中的至少之一。本领域技术人员可以理解，所述第二结合区的具体组成不受特别限制，只要具有BCMA或B7H6结合活性即可，可以是完整的全长抗体，也可以是抗体片段(抗原结合片段)。According to an embodiment of the present invention, the second binding region includes at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody and a minimum recognition unit having BCMA or B7H6 binding activity. It can be understood by those skilled in the art that the specific composition of the second binding region is not particularly limited, as long as it has BCMA or B7H6 binding activity, and can be a complete full-length antibody or an antibody fragment (antigen binding fragment).
根据本发明的实施例，所述第二结合区包括抗BCMA单链抗体或抗B7H6单链抗体。According to an embodiment of the present invention, the second binding region comprises an anti-BCMA single-chain antibody or an anti-B7H6 single-chain antibody.
根据本发明的实施例，所述抗CD3单链抗体包括抗CD3抗体轻链可变区和抗CD3抗体重链可变区，所述抗CD3抗体重链可变区具有SEQ ID NO:23或27所示的氨基酸序列，所述抗CD3抗体轻链可变区具有如SEQ ID NO:24或28所示的氨基酸序列。According to an embodiment of the present invention, the anti-CD3 single-chain antibody comprises an anti-CD3 antibody light chain variable region and an anti-CD3 antibody heavy chain variable region, the anti-CD3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or 27, and the anti-CD3 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 24 or 28.
根据本发明的实施例，所述抗CD3单链抗体进一步包括连接肽1，其中，所述连接肽1的N端与所述抗CD3抗体重链可变区的C端相连，所述连接肽1的C端与所述抗CD3抗体轻链可变区的N端相连；或所述连接肽1的N端与所述抗CD3抗体轻链可变区的C端相连，所述连接肽1的C端与所述抗CD3抗体重链可变区的N端相连。According to an embodiment of the present invention, the anti-CD3 single-chain antibody further includes a connecting peptide 1, wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the heavy chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the light chain variable region of the anti-CD3 antibody; or the N-terminus of the connecting peptide 1 is connected to the C-terminus of the light chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the heavy chain variable region of the anti-CD3 antibody.
根据本发明的实施例，所述抗BCMA单链抗体包括抗BCMA抗体轻链可变区和抗BCMA抗体重链可变区，所述抗BCMA抗体轻链可变区具有SEQ ID NO:42所示的氨基酸序列，所述抗BCMA抗体重链可变区具有如SEQ ID NO:61所示的氨基酸序列。According to an embodiment of the present invention, the anti-BCMA single-chain antibody comprises an anti-BCMA antibody light chain variable region and an anti-BCMA antibody heavy chain variable region, the anti-BCMA antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:42, and the anti-BCMA antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:61.
根据本发明的实施例，所述抗BCMA单链抗体进一步包括连接肽2，其中，所述连接肽2的N端与所述抗BCMA抗体重链可变区的C端相连，所述连接肽2的C端与所述抗BCMA抗体轻链可变区的N端相连；或所述连接肽2的N端与所述抗BCMA抗体轻链可变区的C端相连，所述连接肽2的C端与所述抗BCMA抗体重链可变区的N端相连。According to an embodiment of the present invention, the anti-BCMA single-chain antibody further includes a connecting peptide 2, wherein the N-terminus of the connecting peptide 2 is connected to the C-terminus of the anti-BCMA antibody heavy chain variable region, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the anti-BCMA antibody light chain variable region; or the N-terminus of the connecting peptide 2 is connected to the C-terminus of the anti-BCMA antibody light chain variable region, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the anti-BCMA antibody heavy chain variable region.
根据本发明的实施例，所述抗B7H6单链抗体包括抗B7H6抗体轻链可变区和抗B7H6抗体重链可变区，所述抗B7H6抗体重链可变区具有SEQ ID NO:63所示的氨基酸序列，所述抗B7H6抗体轻链可变区具有如SEQ ID NO:62所示的氨基酸序列。According to an embodiment of the present invention, the anti-B7H6 single-chain antibody comprises an anti-B7H6 antibody light chain variable region and an anti-B7H6 antibody heavy chain variable region, the anti-B7H6 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:63, and the anti-B7H6 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:62.
根据本发明的实施例，所述抗B7H6单链抗体进一步包括连接肽3，其中，所述连接肽3的N端与所述抗B7H6抗体重链可变区的C端相连，所述连接肽3的C端与所述抗B7H6抗体轻链可变区的N端相连；或所述连接肽3的N端与所述抗B7H6抗体轻链可变区的C端相连，所述连接肽3的C端与所述抗B7H6抗体重链可变区的N端相连。According to an embodiment of the present invention, the anti-B7H6 single-chain antibody further includes a connecting peptide 3, wherein the N-terminus of the connecting peptide 3 is connected to the C-terminus of the heavy chain variable region of the anti-B7H6 antibody, and the C-terminus of the connecting peptide 3 is connected to the N-terminus of the light chain variable region of the anti-B7H6 antibody; or the N-terminus of the connecting peptide 3 is connected to the C-terminus of the light chain variable region of the anti-B7H6 antibody, and the C-terminus of the connecting peptide 3 is connected to the N-terminus of the heavy chain variable region of the anti-B7H6 antibody.
根据本发明的实施例，所述连接肽1具有氨基酸序列(GGS)n，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。According to an embodiment of the present invention, the connecting peptide 1 has an amino acid sequence (GGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
根据本发明的实施例，所述连接肽2和连接肽3中的至少之一具有氨基酸序列(GGGGS)n，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。According to an embodiment of the present invention, at least one of the connecting peptide 2 and the connecting peptide 3 has an amino acid sequence (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
本领域技术人员可以理解，本文中所述的连接肽1、2、3不受特别限制，本领域常规连接肽均可使用，如常规的柔性氨基酸片段或刚性氨基酸片段。Those skilled in the art will appreciate that the connecting peptides 1, 2, and 3 described herein are not particularly limited, and any conventional connecting peptides in the art can be used, such as conventional flexible amino acid fragments or rigid amino acid fragments.
根据本发明的实施例，所述连接肽1具有SEQ ID NO:48所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptide 1 has the amino acid sequence shown in SEQ ID NO:48.
根据本发明的实施例，所述连接肽2和3具有SEQ ID NO:41所示的氨基酸序列。According to an embodiment of the present invention, the connecting peptides 2 and 3 have the amino acid sequence shown in SEQ ID NO:41.
根据本发明的实施例，所述抗CD3单链抗体具有如SEQ ID NO:45所示的氨基酸序列。According to an embodiment of the present invention, the anti-CD3 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:45.
根据本发明的实施例，所述抗BCMA单链抗体具有如SEQ ID NO:46所示的氨基酸序列。According to an embodiment of the present invention, the anti-BCMA single-chain antibody has an amino acid sequence as shown in SEQ ID NO:46.
根据本发明的实施例，所述抗B7H6单链抗体具有如SEQ ID NO:47所示的氨基酸序列。According to an embodiment of the present invention, the anti-B7H6 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:47.
根据本发明的实施例，所述第一抗原结合区进一步包括第一Fc肽段，所述第一Fc肽段的N端与所述抗体或抗原结合片段的C端相连。According to an embodiment of the present invention, the first antigen binding region further includes a first Fc peptide segment, and the N-terminus of the first Fc peptide segment is connected to the C-terminus of the antibody or antigen binding fragment.
根据本发明的实施例，所述第二抗原结合区进一步包括第二Fc肽段，所述第二Fc肽段的N端与所述抗BCMA单链抗体或所述抗B7H6单链抗体的C端相连。According to an embodiment of the present invention, the second antigen binding region further includes a second Fc peptide segment, and the N-terminus of the second Fc peptide segment is connected to the C-terminus of the anti-BCMA single-chain antibody or the anti-B7H6 single-chain antibody.
根据本发明的实施例，所述第一Fc肽段具有SEQ ID NO:49所示的氨基酸序列。According to an embodiment of the present invention, the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO:49.
根据本发明的实施例，所述第二Fc肽段具有SEQ ID NO:50所示的氨基酸序列。According to an embodiment of the present invention, the second Fc peptide segment has the amino acid sequence shown in SEQ ID NO:50.
根据本发明的实施例，所述第一Fc肽段和所述第二Fc肽段通过knob-into-hole结构进行连接。本领域技术人员可以理解，所述第一Fc肽段和所述第二Fc肽段的氨基酸序列可以相同也可以不同，例如，具有相同的氨基酸序列时，所述第一Fc肽段和所述第二Fc肽段可以通过二硫键进行连接。According to an embodiment of the present invention, the first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure. Those skilled in the art will appreciate that the amino acid sequences of the first Fc peptide segment and the second Fc peptide segment may be the same or different, for example, when they have the same amino acid sequence, the first Fc peptide segment and the second Fc peptide segment may be connected via a disulfide bond.
根据本发明的实施例，所述第一抗原结合区具有SEQ ID NO:51所示的氨基酸序列，所述第二抗原结合区具有SEQ ID NO:52或53所示的氨基酸序列。According to an embodiment of the present invention, the first antigen binding region has the amino acid sequence shown in SEQ ID NO:51, and the second antigen binding region has the amino acid sequence shown in SEQ ID NO:52 or 53.
编码本发明的抗体或其抗原结合片段或者所述双抗的核酸在本发明的范围内，根据其氨基酸序列，本领域技术人员能够很容易得到相应的核酸序列。
The nucleic acid encoding the antibody or antigen-binding fragment thereof or the bispecific antibody of the present invention is within the scope of the present invention. Based on its amino acid sequence, a person skilled in the art can easily obtain the corresponding nucleic acid sequence.
因此，在本发明的第三方面，本发明提供了一种核酸分子，所述核酸分子编码第一方面所述的抗体或抗原结合片段或所述双抗。根据本发明一些具体实施方式中的核酸分子编码的抗体或抗原结合片段能够与人或猴CD3蛋白进行结合，有效治疗或预防CD3介导的相关疾病，所述核酸分子编码的双抗能够与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，有效治疗或预防CD3和BCMA，或者CD3和B7H6介导的相关疾病。Therefore, in the third aspect of the present invention, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment or the bispecific antibody described in the first aspect. According to some specific embodiments of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule can bind to human or monkey CD3 protein, effectively treat or prevent CD3-mediated related diseases, and the bispecific antibody encoded by the nucleic acid molecule can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, effectively treat or prevent CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
根据本发明的实施例，上述核酸分子还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above nucleic acid molecule may further include at least one of the following additional technical features:
根据本发明的实施例，所述核酸分子为DNA。According to an embodiment of the present invention, the nucleic acid molecule is DNA.
需要说明的是，对于本发明说明书和权利要求书中所提及的核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。It should be noted that, for the nucleic acids mentioned in the specification and claims of the present invention, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, in the specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
在本发明的第四方面，本发明提出了一种表达载体，所述表达载体携带前面所述的核酸分子。所述表达载体可包括可选的控制序列，所述控制序列与所述核酸分子进行可操作地连接。其中，所述控制序列为可指导所述核酸分子在宿主中表达的一个或多个控制序列。本发明实施例所提出的表达载体可在适合的宿主细胞中高效大量表达所述抗体或抗原结合片段。In a fourth aspect of the present invention, the present invention proposes an expression vector, the expression vector carries the aforementioned nucleic acid molecule. The expression vector may include an optional control sequence, the control sequence is operably connected to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiment of the present invention can efficiently express the antibody or antigen-binding fragment in a large amount in a suitable host cell.
本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。在将上述核酸分子连接到载体上时，可以将核酸分子与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。本领域技术人员可以理解，用来编码抗体或抗原结合片段的核酸分子，可以分别独立的插入到不同的载体上，常见的是插入到同一载体上。常用的载体例如可以为质粒、噬菌体等等。例如Plasmid-X质粒。Herein, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended function of regulating the transcription and translation of the exogenous gene. When the above-mentioned nucleic acid molecules are connected to the vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. It can be understood by those skilled in the art that the nucleic acid molecules used to encode antibodies or antigen-binding fragments can be inserted into different vectors separately and independently, and it is common to insert them into the same vector. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. For example, Plasmid-X plasmid.
在本发明的第五方面，本发明提出了一种制备前面所述的抗体或抗原结合片段或双抗的方法，包括：将前面所述的表达载体引入到细胞中；将所述细胞在适于蛋白表达和分泌的条件下进行培养，以便获得所述抗体或抗原结合片段或双抗。根据本发明一些具体实施方式所提出的方法可以有效体外大量获得所述抗体或抗原结合片段或所述双抗。In the fifth aspect of the present invention, the present invention proposes a method for preparing the above-mentioned antibody or antigen-binding fragment or bispecific antibody, comprising: introducing the above-mentioned expression vector into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment or bispecific antibody. The method proposed according to some specific embodiments of the present invention can effectively obtain the antibody or antigen-binding fragment or bispecific antibody in large quantities in vitro.
根据本发明的一些具体实施方式，上述制备前面所述的抗体或抗原结合片段或双抗的方法还可以包括下列附加技术特征中的至少之一：According to some specific embodiments of the present invention, the method for preparing the above-mentioned antibody or antigen-binding fragment or bispecific antibody may also include at least one of the following additional technical features:
根据本发明的一些具体实施方式，所述细胞不受特别限制，原核细胞或真核细胞均可使用。According to some specific embodiments of the present invention, the cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used.
根据本发明的一些具体实施方式，所述细胞为真核细胞。According to some specific embodiments of the present invention, the cell is a eukaryotic cell.
根据本发明的一些具体实施方式，所述真核细胞为哺乳动物细胞。根据本发明的一些具体实施例，当所述细胞为真核细胞，如哺乳动物细胞时所述重组抗体的表达效率较高。According to some specific embodiments of the present invention, the eukaryotic cell is a mammalian cell. According to some specific embodiments of the present invention, when the cell is a eukaryotic cell, such as a mammalian cell, the expression efficiency of the recombinant antibody is higher.
在本发明的第六方面，本发明提出了一种重组细胞，所述重组细胞前面所述的核酸，或表达载体，或能够表达前面所述的抗体或抗原结合片段或所述双抗。所述重组细胞是通过转染或者转化所述表达载体获得的。根据本发明的一些具体实施方式，所述重组细胞在合适条件下可高效并大量表达上述抗体或抗原结合片段或所述双抗。In a sixth aspect of the present invention, the present invention provides a recombinant cell, wherein the recombinant cell contains the aforementioned nucleic acid, or expression vector, or is capable of expressing the aforementioned antibody or antigen-binding fragment or the bispecific antibody. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently and massively express the above-mentioned antibody or antigen-binding fragment or the bispecific antibody under appropriate conditions.
需要注意的是，本发明所述重组细胞不受特别限制，可以为原核细胞、真核细胞或噬菌体。所述原核细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。所述真核细胞包括巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，草地粘虫等昆虫细胞，烟草等植物细胞，BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施例中，本发明所述重组细胞优选为哺乳动物细胞，包括BHK细胞、CHO细胞、NSO细胞或COS细胞，且不包括动物生殖细胞、受精卵或胚胎干细胞。It should be noted that the recombinant cells of the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.
需要说明的是，本申请说明书中所述的“适合条件”，是指适合本申请所述抗体或抗原结合片段或双抗表达的条件。本领域技术人员容易理解的是，适合抗体或抗原结合片段或双抗表达的条件包括但不限于合适的转化或转染方式、合适的转化或转条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述抗体或抗原结合片段或双抗表达的条件。It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of the antibody or antigen-binding fragment or double antibody described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the antibody or antigen-binding fragment or double antibody include but are not limited to suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the antibody or antigen-binding fragment or double antibody according to the specific environment of the laboratory.
在本发明的第七方面，本发明提出了一种免疫缀合物，包括前面所述的抗体或抗原结合片段或所述双抗，和治疗剂。如前所述，本发明实施例的抗体或抗原结合片段能够有效CD3蛋白进行结合，所述双抗能够与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，有效治疗或预防CD3和BCMA，或者CD3和B7H6介导的相关疾病，因此，包含所述抗体或抗原结合片段的免疫缀合物同样能够与人或猴的CD3蛋白进行结合，包含所述双抗的免疫缀合物同样能够与人或猴CD3蛋白和BCMA蛋白，或者与人或猴CD3蛋白和B7H6蛋白进行结合，所述免疫缀合物具有良好的预防和/或治疗CD3介导的疾病，或者CD3和BCMA，或者CD3和B7H6介导的相关疾病效果。In the seventh aspect of the present invention, the present invention proposes an immunoconjugate, comprising the above-mentioned antibody or antigen-binding fragment or the bispecific antibody, and a therapeutic agent. As mentioned above, the antibody or antigen-binding fragment of the embodiment of the present invention can effectively bind to CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or bind to human or monkey CD3 protein and B7H6 protein, effectively treating or preventing CD3 and BCMA, or CD3 and B7H6-mediated related diseases, therefore, the immunoconjugate containing the antibody or antigen-binding fragment can also bind to human or monkey CD3 protein, and the immunoconjugate containing the bispecific antibody can also bind to human or monkey CD3 protein and BCMA protein, or bind to human or monkey CD3 protein and B7H6 protein, and the immunoconjugate has good effects in preventing and/or treating CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
在本发明的第八方面，本发明提出了一种组合物，包括前面所述的抗体或抗原结合片段、双抗、核酸分子、表达载
体或重组细胞。如前所述，本发明一些具体实施方式的所述抗体或抗原结合片段能够有效与人或猴CD3蛋白进行结合，所述双抗能够与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，并有效抑制肿瘤细胞的增殖，因此，包含上述物质的组合物同样可以有效与人或猴CD3蛋白，或者与人或猴CD3蛋白和BCMA蛋白，或者与人或猴CD3蛋白和B7H6蛋白进行结合，具有良好的预防和/或治疗CD3介导的疾病，或者CD3和BCMA，或者CD3和B7H6介导的相关疾病效果，其中，所述组合物的种类不受特别限制，可以为食品组合物或药物组合物。In the eighth aspect of the present invention, the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment, bispecific antibody, nucleic acid molecule, expression vector As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and effectively inhibit the proliferation of tumor cells. Therefore, the composition containing the above substances can also effectively bind to human or monkey CD3 protein, or to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and has good effects in preventing and/or treating CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases. The type of the composition is not particularly limited and can be a food composition or a pharmaceutical composition.
本发明的组合物也可以相互组合、或与一种或多种其它的治疗化合物组合给药，例如，与化疗剂组合给药。因此，所述组合物还可以含有化疗剂。本发明的抗体或其抗原结合片段、或免疫缀合物还可以与第二治疗剂组合，所述第二治疗剂的示例性试剂包括但不限于抑制CD3活性的其他试剂(包括其他抗体或其抗原结合片段、肽抑制剂、小分子拮抗剂等)和/或干扰CD3上游或下游信号转导的试剂。The compositions of the invention may also be administered in combination with each other or with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Thus, the composition may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the invention may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit CD3 activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or agents that interfere with CD3 upstream or downstream signal transduction.
需要注意的是，所述组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述组合物中所含的成分可以以整体施用于受试者，或者分开施用于受试者。当所述组合物中所含的成分分开地施用于受试者时，各个成分可以同时或依次施用于受试者。It should be noted that the composition includes combinations separated in time and/or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole, or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.
在本发明的第九方面，本发明提出了一种药物，包括前面所述的抗体或抗原结合片段、双抗、核酸分子、表达载体、重组细胞或组合物。如前所述，本发明一些具体实施方式的所述抗体或抗原结合片段能够有效与人或猴CD3蛋白进行结合，所述双抗能够与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，因此，包含有效量的所述抗体或抗原结合片段或者所述双抗的活性成分或其一系列物质的药物同样可以有效与人或猴CD3蛋白进行结合，或者与人或猴CD3蛋白和BCMA蛋白，或者与人或猴CD3蛋白和B7H6蛋白进行结合，具有良好的预防和/或治疗CD3介导的疾病，或者CD3和BCMA，或者CD3和B7H6介导的相关疾病效果。In the ninth aspect of the present invention, the present invention proposes a drug, including the above-mentioned antibody or antigen binding fragment, bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or composition. As mentioned above, the antibody or antigen binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, and the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein. Therefore, a drug containing an effective amount of the antibody or antigen binding fragment or the active ingredient of the bispecific antibody or a series of substances thereof can also effectively bind to human or monkey CD3 protein, or to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and has good effects in preventing and/or treating CD3-mediated diseases, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
根据本发明的实施例，上述药物还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned medicine may further include at least one of the following additional technical features:
根据本发明的实施例，所述药物还可以包括药学上可接受的载体。According to an embodiment of the present invention, the drug may further include a pharmaceutically acceptable carrier.
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量。As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and/or animals and can be accepted by humans and/or animals.
本发明所述的抗体或抗原结合片段或所述双抗的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：所述的活性成分的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。The effective amount of the antibody or antigen-binding fragment or the bispecific antibody of the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be administered daily, or the dose may be reduced proportionally.
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.
本发明的药物含有安全有效量的本发明的活性成分以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，其中，给药的方式可以为口服给药、经鼻给药、皮内给药、皮下给药、肌内给药或静脉给药或腹腔内给药，本发明的药物的剂型为注射剂、口服制剂(片剂、胶囊、口服液)、透皮剂、缓释剂。例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物宜在无菌条件下制造。所述抗体或抗原结合片段或双抗可通过静脉输注或注射或肌肉内或皮下注射来施用。The medicine of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the drug preparation should match the mode of administration, wherein the mode of administration can be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration or intraperitoneal administration, and the dosage form of the drug of the present invention is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The drug is preferably manufactured under sterile conditions. The antibody or antigen-binding fragment or bispecific antibody can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
当然，本文中的抗CD3单抗或双抗还可以根据需要被制成试剂盒或者其他诊断性试剂的一部分。Of course, the anti-CD3 monoclonal antibody or bispecific antibody herein can also be prepared as a part of a kit or other diagnostic reagent as required.
在本发明的第十方面，本发明提出了一种试剂盒，所述试剂盒含有前面所述抗体或其抗原结合片段、双抗、核酸分子、表达载体或重组细胞。如前所述，本发明一些具体实施方式的抗体或抗原结合片段能够有效与人或猴CD3蛋白进行结合，所述双抗能够与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，因此，包含所述抗体或抗原结合片段的试剂盒能够有效的对人或猴的CD3蛋白进行定性或定量检测，包含所述双抗的试剂盒能够有效的对人或猴CD3蛋白和BCMA蛋白，或者人或猴CD3蛋白和B7H6蛋白进行定性或定量检测。应用本发明提供的试剂盒，例如可以用于免疫印迹、免疫沉淀等涉及到利用人或猴CD3和抗体特异性结合性能来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种：拮抗剂、抗CD3抗体或者药物参照材料；蛋白纯化柱；免疫球蛋白亲和纯化缓冲剂；细胞的测定稀释剂；说明书或者文献等。抗CD3抗体可被用于不同类型的诊断测试，例如可以在体外或者体内检测各种各样的疾病或者药物、毒素或者其他蛋白等的存在。例如可以通过对受试者的血清或者血液进行检测，用来测试相关疾病。这种相关疾病可包括CD3相关疾病，例如癌症，其中，所述癌症包括多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、
肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤中的至少一种。当然本文提供的抗体或抗原结合片段也可以用于上述疾病的放射免疫检测和放射免疫治疗等等。针对于上述应用场景，所述双抗同样适用，此处不再累述。In the tenth aspect of the present invention, the present invention proposes a kit, the kit containing the above-mentioned antibody or antigen-binding fragment thereof, bispecific antibody, nucleic acid molecule, expression vector or recombinant cell. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, the bispecific antibody can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein. Therefore, the kit containing the antibody or antigen-binding fragment can effectively detect human or monkey CD3 protein qualitatively or quantitatively, and the kit containing the bispecific antibody can effectively detect human or monkey CD3 protein and BCMA protein, or human or monkey CD3 protein and B7H6 protein qualitatively or quantitatively. The kit provided by the present invention can be used, for example, for immunoblotting, immunoprecipitation, etc., which involve the use of human or monkey CD3 and antibody specific binding properties for detection. These kits may contain any one or more of the following: antagonists, anti-CD3 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-CD3 antibodies can be used in different types of diagnostic tests, such as in vitro or in vivo detection of various diseases or the presence of drugs, toxins or other proteins. For example, the serum or blood of a subject can be tested to test for related diseases. Such related diseases may include CD3-related diseases, such as cancer, wherein the cancer includes multiple myeloma, lymphoma, hemangioma, gastric cancer, At least one of liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma. Of course, the antibodies or antigen-binding fragments provided herein can also be used for radioimmunoassay and radioimmunotherapy of the above-mentioned diseases. For the above-mentioned application scenarios, the dual antibodies are also applicable and will not be repeated here.
所述试剂盒还可以包括常规用于检测CD3，或者CD3和BCMA，或者CD3和B7H6的试剂，如包被液等。The kit may also include conventional reagents for detecting CD3, or CD3 and BCMA, or CD3 and B7H6, such as coating fluid, etc.
在本发明的第十一方面，本发明提出了前面所述的抗体或其抗原结合片段、核酸分子、表达载体、重组细胞或组合物在制备药物中的用途，所述药物用于预防和/或治疗CD3介导的相关疾病。如前所述，本发明一些具体实施方式的所述抗体或抗原结合片段能够有效与人或猴CD3蛋白进行结合，因此，包含有效量的所述抗体或抗原结合片段或其一系列物质的药物同样可以有效与人或猴CD3蛋白进行结合，具有良好的预防和/或治疗CD3介导的相关疾病效果。In the eleventh aspect of the present invention, the present invention proposes the use of the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant cell or composition in the preparation of a drug, and the drug is used to prevent and/or treat CD3-mediated related diseases. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein, and therefore, a drug containing an effective amount of the antibody or antigen-binding fragment or a series of substances thereof can also effectively bind to human or monkey CD3 protein, and has a good effect of preventing and/or treating CD3-mediated related diseases.
根据本发明的实施例，上述制备药物的用途还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above-mentioned use of preparing a medicine may also include at least one of the following additional technical features:
根据本发明的实施例，所述CD3介导的相关疾病包括自身免疫性疾病。According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.
根据本发明的实施例，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
在本发明的第十二方面，本发明提出了前面所述的双抗、核酸分子、表达载体、重组细胞或组合物在制备药物中的用途，所述药物用于预防和/或治疗CD3和BCMA，或者CD3和B7H6介导的相关疾病。如前所述，本发明一些具体实施方式的所述双抗能够与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，因此，包含有效量的所述双抗的活性成分或其一系列物质的药物同样可以有效与人或猴CD3蛋白和BCMA蛋白，或者与人或猴CD3蛋白和B7H6蛋白进行结合，具有良好的预防和/或治疗CD3和BCMA，或者CD3和B7H6介导的相关疾病效果。In the twelfth aspect of the present invention, the present invention proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of drugs, which are used to prevent and/or treat CD3 and BCMA, or CD3 and B7H6-mediated related diseases. As mentioned above, the bispecific antibodies of some specific embodiments of the present invention can bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, therefore, drugs containing an effective amount of the active ingredients of the bispecific antibodies or a series of substances thereof can also effectively bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and have good effects in preventing and/or treating CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
根据本发明的实施例，上述制备药物的用途还可以包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above-mentioned use of preparing a medicine may also include at least one of the following additional technical features:
根据本发明的实施例，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。According to an embodiment of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
根据本发明的实施例，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。According to an embodiment of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
在本发明的第十三方面，本发明提出了前面所述的抗体或抗原结合片段、核酸分子、表达载体或重组细胞在制备试剂盒中的用途，所述试剂盒用于检测CD3。如前所述，本发明一些具体实施方式的抗体或抗原结合片段能够有效与人或猴CD3蛋白进行结合，并阻断所述CD3蛋白与其受体进行结合，因此，所述抗体或抗原结合片段可以用于制备检测CD3蛋白的试剂盒，所述试剂盒能够有效的对人或猴的CD3蛋白进行定性或定量检测。In the thirteenth aspect of the present invention, the present invention proposes the use of the above-mentioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector or recombinant cell in preparing a kit for detecting CD3. As mentioned above, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein and block the binding of the CD3 protein to its receptor. Therefore, the antibody or antigen-binding fragment can be used to prepare a kit for detecting CD3 protein, and the kit can effectively perform qualitative or quantitative detection of human or monkey CD3 protein.
在本发明的第十四方面，本发明提出了前面所述的双抗、核酸分子、表达载体或重组细胞在制备试剂盒中的用途，所述试剂盒用于检测CD3和/或BCMA，或者，CD3和/或B7H6。如前所述，本发明一些具体实施方式的双抗能够有效与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，因此，所述双抗可以用于制备检测CD3和/或BCMA，或者，CD3和/或B7H6的试剂盒，所述试剂盒能够有效的对人或猴的CD3和/或BCMA，或者，CD3和/或B7H6进行定性或定量检测。In the fourteenth aspect of the present invention, the present invention proposes the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors or recombinant cells in the preparation of a kit for detecting CD3 and/or BCMA, or CD3 and/or B7H6. As mentioned above, the bispecific antibodies of some specific embodiments of the present invention can effectively bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, therefore, the bispecific antibodies can be used to prepare a kit for detecting CD3 and/or BCMA, or CD3 and/or B7H6, and the kit can effectively perform qualitative or quantitative detection of human or monkey CD3 and/or BCMA, or CD3 and/or B7H6.
在本发明的第十五方面，本发明提出了一种治疗或预防CD3，或者CD3和BCMA，或者CD3和B7H6介导的相关疾病的方法。根据本发明的实施例，所述方法包括向受试者施用以下中的至少之一：1)前面所述的抗体或抗原结合片段；2)前面所述的双抗；3)前面所述的核酸分子；4)前面所述的表达载体；5)前面所述的重组细胞；6)前面所述的组合物；和7)前面所述的药物。如前所述，所述双抗能够有效与人或猴CD3蛋白和BCMA蛋白进行结合，或者与人或猴CD3蛋白和B7H6蛋白进行结合，所述抗体或抗原结合片段能够与人或猴CD3蛋白进行结合，能够有效治疗或预防CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病，例如：自身免疫性疾病或者癌症，因此，根据本发明实施例的方法能够有效治疗或预防CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病，优选为自身免疫性疾病或者癌症。In the fifteenth aspect of the present invention, the present invention proposes a method for treating or preventing CD3, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases. According to an embodiment of the present invention, the method comprises administering to a subject at least one of the following: 1) the antibody or antigen binding fragment described above; 2) the bispecific antibody described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; 5) the recombinant cell described above; 6) the composition described above; and 7) the drug described above. As described above, the bispecific antibody can effectively bind to human or monkey CD3 protein and BCMA protein, or to human or monkey CD3 protein and B7H6 protein, and the antibody or antigen binding fragment can bind to human or monkey CD3 protein, and can effectively treat or prevent CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, such as autoimmune diseases or cancer. Therefore, the method according to an embodiment of the present invention can effectively treat or prevent CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, preferably autoimmune diseases or cancer.
根据本发明的实施例，上述治疗或预防疾病的方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above method for treating or preventing a disease may further include at least one of the following additional technical features:
根据本发明的实施例，所述CD3介导的相关疾病包括自身免疫性疾病。According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.
根据本发明的实施例，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血
管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic hemoglobin Patients with this disorder may also be diagnosed with leukoencephalitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, autoimmune thyroid disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, and acute idiopathic polyneuritis.
根据本发明的实施例，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。According to an embodiment of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
根据本发明的实施例，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。According to an embodiment of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
在本发明的第十六方面，本发明提出了一种诊断CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病的方法。根据本发明的实施例，包括使用下列中的至少之一对待测样品中的CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6进行检测：1)前面所述的抗体或抗原结合片段；2)前面所述的双抗；3)前面所述的核酸分子；4)前面所述的表达载体；和5)前面所述的重组细胞，基于所述CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6的检测结果，确定所述待测样品中CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6的含量。本申请提出的所述抗体或抗原结合片段，或核酸分子、表达载体、重组细胞表达的抗体或抗原结合片段均可以有效与人或猴CD3蛋白进行结合，所述双抗，或核酸分子、表达载体、重组细胞表达的双抗均可以有效与CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6进行结合，因此，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量，并可以对CD3，或者CD3和/或BCMA，或者CD3和/或B7H6引起的相关疾病进行有效诊断。In the sixteenth aspect of the present invention, the present invention proposes a method for diagnosing CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases. According to an embodiment of the present invention, it includes using at least one of the following to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested: 1) the antibody or antigen binding fragment described above; 2) the bispecific antibody described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; and 5) the recombinant cell described above, based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is determined. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, or antibodies or antigen-binding fragments expressed by recombinant cells proposed in the present application can effectively bind to human or monkey CD3 protein, and the bispecific antibodies, or nucleic acid molecules, expression vectors, or bispecific antibodies expressed by recombinant cells can effectively bind to CD3, or CD3 and/or BCMA, or CD3 and/or B7H6. Therefore, the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample derived from the test individual, and can effectively diagnose related diseases caused by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
根据本发明的实施例，上述诊断疾病的方法还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method for diagnosing a disease may further include at least one of the following additional technical features:
根据本发明的实施例，所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量不低于患病的最低标准是待测样品来源于患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6引起的相关疾病的患者的指示。所述最低标准的值可以通过对大量患有所述CD3，或者CD3和/或BCMA，或者CD3和/或B7H6引起的相关疾病的个体和大量健康个体的待测样本中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量进行差异比较分析、以及验证，而确定下来。According to an embodiment of the present invention, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is not less than the minimum standard for disease, which indicates that the test sample is derived from a patient suffering from a disease related to CD3, or CD3 and/or BCMA, or CD3 and/or B7H6. The value of the minimum standard can be determined by comparative analysis and verification of the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a large number of individuals suffering from the disease related to CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 and a large number of healthy individuals.
根据本发明的实施例，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
根据本发明的实施例，所述CD3介导的相关疾病包括自身免疫性疾病。According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.
根据本发明的实施例，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
根据本发明的实施例，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。According to an embodiment of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
根据本发明的实施例，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。According to an embodiment of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
在本发明的第十七方面，本发明提出了一种对CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病进行分期的方法。根据本发明的实施例，包括使用下列中的至少之一对待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6进行检测：1)前面所述的抗体或抗原结合片段；2)前面所述的双抗；3)前面所述的核酸分子；4)前面所述的表达载体；和5)前面所述的重组细胞，基于所述CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的检测结果，确定所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量。本申请提出的所述双抗，或核酸分子、表达载体、重组细胞表达的双抗均可以有效与人或猴的CD3和/或BCMA，或者CD3和/或B7H6进行结合，所述抗体或抗原结合片段，或核酸分子、表达载体、重组细胞表达的抗体或抗原结合片段均可以有效与人或猴的CD3蛋白进行结合，因此，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6含量，并基于所述CD3，或者CD3和/或BCMA，或者CD3和/或B7H6含量对CD3，或者CD3和/或BCMA，或者CD3和/或B7H6引起的相关疾病所处的时期进行评估。In the seventeenth aspect of the present invention, the present invention proposes a method for staging CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases. According to an embodiment of the present invention, it includes using at least one of the following to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested: 1) the antibody or antigen binding fragment described above; 2) the bispecific antibody described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; and 5) the recombinant cell described above, based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is determined. The bispecific antibodies proposed in the present application, or the bispecific antibodies expressed by nucleic acid molecules, expression vectors, and recombinant cells can effectively bind to human or monkey CD3 and/or BCMA, or CD3 and/or B7H6. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, and antibodies or antigen-binding fragments expressed by recombinant cells can effectively bind to human or monkey CD3 proteins. Therefore, the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample from the subject, and evaluate the stage of related diseases caused by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 based on the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
根据本发明的实施例，上述对疾病进行分期的方法还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method for staging a disease may further include at least one of the following additional technical features:
根据本发明的实施例，所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量不低于肿瘤IV期患病的标准水平是待测样品来源于患有肿瘤IV期的患者的指示，所述待测样品中CD3，或者CD3和/或BCMA，或者
CD3和/或B7H6的含量位于肿瘤IV期和III期患病的标准水平之间是待测样品来源于患有肿瘤III期的患者的指示；所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量处于肿瘤III期和II期患病的标准水平之间是待测样品来源于患有肿瘤II期的患者的指示；所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量处于I期和II期患病的标准水平之间是待测样品来源于患有肿瘤I期的患者的指示。本领域技术人员可以理解，所述的肿瘤I期、II期、III期、IV期患病时CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的水平根据肿瘤种类的不同而变化，判断肿瘤所述时期，只要将所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量与对应的该肿瘤阶段CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的标准水平进行对比即可知晓，或将所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量与已知患病时期的个体或群体来源的样品CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量进行对比。所述肿瘤I期、II期、III期、IV期标准水平的值可以通过对大量所述患有血管生成引起的相关疾病的个体和大量健康个体的待测样本中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量的差异进行比较分析、以及验证，而确定下来。According to an embodiment of the present invention, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is not lower than the standard level of stage IV tumor, which indicates that the test sample is derived from a patient with stage IV tumor. The content of CD3 and/or B7H6 being between the standard levels for tumor stage IV and stage III disease is an indication that the sample to be tested is derived from a patient with a tumor stage III; the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is between the standard levels for tumor stage III and stage II disease, is an indication that the sample to be tested is derived from a patient with a tumor stage II; the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested is between the standard levels for tumor stage I and stage II disease, is an indication that the sample to be tested is derived from a patient with a tumor stage I. Those skilled in the art will appreciate that the levels of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 when the tumor is at stage I, stage II, stage III, or stage IV vary depending on the type of tumor, and to determine the stage of the tumor, it is only necessary to compare the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample with the standard level of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 at the corresponding tumor stage, or to compare the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample with the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a sample from an individual or group of individuals with a known stage of the disease. The values of the standard levels of tumor stages I, II, III, and IV can be determined by comparative analysis and verification of the differences in the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a large number of test samples from individuals suffering from the angiogenesis-related diseases and a large number of healthy individuals.
根据本发明的实施例，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
根据本发明的实施例，所述CD3介导的相关疾病包括自身免疫性疾病。According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.
根据本发明的实施例，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
根据本发明的实施例，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。According to an embodiment of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
根据本发明的实施例，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。According to an embodiment of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
在本发明的第十八方面，本发明提出了一种评估CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病预后的方法。根据本发明的实施例，包括使用下列中的至少之一对待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6进行检测：1)前面所述的双抗；2)前面所述的抗体或抗原结合片段；3)前面所述的核酸分子；4)前面所述的表达载体；和5)前面所述的重组细胞，基于所述CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的检测结果，确定所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量。如前所述，CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量对癌症具有重要影响，患有相关疾病个体进行治疗后，通过监测其组织或排泄物，如外周血、尿液等中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量可以有效评估该类疾病的预后，例如，将治疗前后的受试者体内CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量进行比较，或将治疗后的受试者体内CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量与正常个体或患病个体的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6水平进行比较等方式，本申请提出的所述双抗，或核酸分子、表达载体或重组细胞表达的双抗均可以有效与CD3和/或BCMA，或者CD3和/或B7H6进行结合，所述抗体或抗原结合片段，或核酸分子、表达载体、重组细胞表达的抗体或抗原结合片段均可以有效与人或猴CD3进行结合，因此，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量，并基于所述CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量对CD3，或者CD3和/或BCMA，或者CD3和/或B7H6引起的相关疾病的预后进行评估。In the eighteenth aspect of the present invention, the present invention proposes a method for evaluating the prognosis of related diseases mediated by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6. According to an embodiment of the present invention, it includes using at least one of the following to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested: 1) the aforementioned double antibody; 2) the aforementioned antibody or antigen binding fragment; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; and 5) the aforementioned recombinant cell, based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, determine the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested. As mentioned above, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 has an important influence on cancer. After treatment of individuals with related diseases, the prognosis of such diseases can be effectively evaluated by monitoring the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in their tissues or excretions, such as peripheral blood, urine, etc. For example, the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the subject before and after treatment can be compared, or the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the subject after treatment can be compared with the CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 levels in normal individuals or diseased individuals, etc. The bispecific antibodies proposed in the present application, or the bispecific antibodies expressed by nucleic acid molecules, expression vectors or recombinant cells can effectively bind to CD3 and/or BCMA, or CD3 and/or B7H6. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, antibodies or antigen-binding fragments expressed by recombinant cells can effectively bind to human or monkey CD3. Therefore, the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample from the subject, and evaluate the prognosis of related diseases caused by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 based on the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
根据本发明的实施例，上述评估疾病预后的方法还可以进一步包括下列附加技术特征中的至少之一：According to an embodiment of the present invention, the above method for evaluating disease prognosis may further include at least one of the following additional technical features:
根据本发明的实施例，所述待测样品来源于治疗前或治疗后的患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的患者。According to an embodiment of the present invention, the sample to be tested is derived from a patient suffering from a CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related disease before or after treatment.
根据本发明的实施例，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。According to an embodiment of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cell, blood, serum, plasma, feces and urine.
根据本发明的实施例，基于所述治疗前或治疗后的患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的患者的待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量，确定CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的预后效果。According to an embodiment of the present invention, the prognostic effect of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases is determined based on the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample of a patient suffering from CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases before or after the treatment.
根据本发明的实施例，治疗后的患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的患者的待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量下降是患者预后良好的指示。According to an embodiment of the present invention, a decrease in the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a test sample of a patient suffering from a CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related disease after treatment is an indication of a good prognosis for the patient.
根据本发明的实施例，所述CD3介导的相关疾病包括自身免疫性疾病。According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.
根据本发明的实施例，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。
According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
根据本发明的实施例，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。According to an embodiment of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
根据本发明的实施例，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。According to an embodiment of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
在本发明的第十九方面，本发明提出了本发明提出了前面所述的双抗，抗体或抗原结合片段，核酸分子，表达载体、重组细胞，组合物或药物在治疗或预防CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病中的用途。如前所述，所述双抗与CD3和/或BCMA，或者，CD3和/或B7H6均具有较高的结合活性，所述抗体或抗原结合片段能够与人或猴CD3进行有效结合，能够有效治疗或预防CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病。In the nineteenth aspect of the present invention, the present invention proposes the use of the aforementioned bispecific antibodies, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions or drugs in the treatment or prevention of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases. As mentioned above, the bispecific antibodies have high binding activity with CD3 and/or BCMA, or CD3 and/or B7H6, and the antibodies or antigen-binding fragments can effectively bind to human or monkey CD3, and can effectively treat or prevent CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
根据本发明的实施例，所述CD3介导的相关疾病包括自身免疫性疾病。According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.
根据本发明的实施例，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
根据本发明的实施例，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。According to an embodiment of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
根据本发明的实施例，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。According to an embodiment of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
在本发明的第二十方面，本发明提出了前面所述的双抗，抗体或抗原结合片段，核酸分子，表达载体或重组细胞在诊断CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病、对CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病进行分期或评估CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病预后中的用途。如前所述，本申请提出的所述双抗，或核酸分子、表达载体或重组细胞表达的双抗均可以有效与CD3和/或BCMA，或者，CD3和/或B7H6进行结合，所述抗体或抗原结合片段，或核酸分子、表达载体、重组细胞表达的抗体或抗原结合片段均可以有效与人或猴CD3进行结合，因此，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6含量，并可以对CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病进行有效诊断、疾病分期和疾病预后评估。In the twentieth aspect of the present invention, the present invention proposes the use of the aforementioned bispecific antibodies, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors or recombinant cells in diagnosing CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, staging CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, or evaluating the prognosis of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases. As mentioned above, the bispecific antibodies proposed in the present application, or the bispecific antibodies expressed by nucleic acid molecules, expression vectors or recombinant cells can effectively bind to CD3 and/or BCMA, or CD3 and/or B7H6, and the antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, antibodies or antigen-binding fragments expressed by recombinant cells can effectively bind to human or monkey CD3. Therefore, the method described in the present application can effectively detect the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample derived from the subject, and can effectively diagnose, stage and evaluate the prognosis of related diseases mediated by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
根据本发明的实施例，上述用途还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
根据本发明的实施例，所述CD3介导的相关疾病包括自身免疫性疾病。According to an embodiment of the present invention, the CD3-mediated related diseases include autoimmune diseases.
根据本发明的实施例，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。According to an embodiment of the present invention, the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
根据本发明的实施例，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。According to an embodiment of the present invention, the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
根据本发明的实施例，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。According to an embodiment of the present invention, the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
本发明中涉及的“受试者”或“个体”一般指哺乳动物，如灵长类动物和/或啮齿类动物，特别是人、猴或鼠。The "subject" or "individual" referred to in the present invention generally refers to a mammal, such as a primate and/or a rodent, in particular a human, a monkey or a mouse.
本发明的有益效果：Beneficial effects of the present invention:
1)本发明获得的所述鼠源的抗CD3的单克隆抗体较现有CD3单克隆抗体具有更高的CD3蛋白结合活性，且所述CD3蛋白包括人CD3蛋白和猴CD3蛋白。1) The mouse anti-CD3 monoclonal antibody obtained in the present invention has higher CD3 protein binding activity than the existing CD3 monoclonal antibody, and the CD3 protein includes human CD3 protein and monkey CD3 protein.
2)将所述鼠源抗CD3单克隆抗体进行人源化后获得的所述人源化抗体同样较现有CD3单克隆抗体具有更高的CD3蛋白结合活性，所述CD3蛋白包括人CD3蛋白和猴CD3蛋白，且所述人源化抗体具有较低的免疫原性，安全性更高，
效力更加持久。2) The humanized antibody obtained by humanizing the mouse anti-CD3 monoclonal antibody also has higher CD3 protein binding activity than the existing CD3 monoclonal antibody, the CD3 protein includes human CD3 protein and monkey CD3 protein, and the humanized antibody has lower immunogenicity and higher safety, The effect is longer lasting.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:
图1A为根据本发明实施例的不同浓度的鼠源CD3抗体(m47B5)结合人CD3EG蛋白的ELISA检测结果图；FIG1A is a graph showing the ELISA test results of different concentrations of mouse CD3 antibody (m47B5) binding to human CD3EG protein according to an embodiment of the present invention;
图1B为根据本发明实施例的不同浓度的鼠源CD3抗体结合人CD3ED蛋白的ELISA检测结果图；FIG1B is a diagram showing the ELISA test results of different concentrations of mouse CD3 antibodies binding to human CD3ED protein according to an embodiment of the present invention;
图1C为根据本发明实施例的不同浓度的鼠源CD3抗体结合猴CD3EG蛋白的ELISA检测结果图；FIG1C is a diagram showing the ELISA test results of different concentrations of mouse CD3 antibodies binding to monkey CD3EG protein according to an embodiment of the present invention;
图1D为根据本发明实施例的不同浓度的鼠源CD3抗体结合猴CD3ED蛋白的ELISA检测结果图；FIG1D is a graph showing the ELISA test results of different concentrations of mouse CD3 antibodies binding to monkey CD3ED protein according to an embodiment of the present invention;
图2A为根据本发明实施例的不同浓度的人-鼠嵌合CD3抗体ch47B5结合人PBMC细胞的检测结果图；FIG2A is a graph showing the detection results of different concentrations of human-mouse chimeric CD3 antibody ch47B5 binding to human PBMC cells according to an embodiment of the present invention;
图2B为根据本发明实施例的不同浓度的人-鼠嵌合CD3抗体ch47B5结合猴PBMC细胞的检测结果图；2B is a graph showing the detection results of different concentrations of human-mouse chimeric CD3 antibody ch47B5 binding to monkey PBMC cells according to an embodiment of the present invention;
图3A为根据本发明实施例的不同浓度的人源化CD3抗体结合人CD3蛋白的检测结果图；FIG3A is a graph showing the detection results of humanized CD3 antibodies of different concentrations binding to human CD3 protein according to an embodiment of the present invention;
图3B为根据本发明实施例的不同浓度的人源化CD3抗体结合猴CD3蛋白的检测结果图；FIG3B is a graph showing the detection results of humanized CD3 antibodies of different concentrations binding to monkey CD3 protein according to an embodiment of the present invention;
图4A为根据本发明实施例的不同浓度的CD3×BCMA双特异性抗体结合人CD3蛋白的检测结果图；FIG4A is a graph showing the detection results of CD3×BCMA bispecific antibodies of different concentrations binding to human CD3 protein according to an embodiment of the present invention;
图4B为根据本发明实施例的不同浓度的CD3×BCMA双特异性抗体结合人BCMA蛋白的检测结果图；FIG4B is a graph showing the detection results of CD3×BCMA bispecific antibodies of different concentrations binding to human BCMA protein according to an embodiment of the present invention;
图5为根据本发明实施例的不同浓度的CD3×BCMA双特异性抗体对NCI-H929细胞的杀伤检测结果图；FIG5 is a graph showing the killing test results of NCI-H929 cells by CD3×BCMA bispecific antibodies of different concentrations according to an embodiment of the present invention;
图6A为根据本发明实施例的不同浓度的CD3×B7H6双特异性抗体结合人CD3蛋白的检测结果图；FIG6A is a graph showing the detection results of CD3×B7H6 bispecific antibodies of different concentrations binding to human CD3 protein according to an embodiment of the present invention;
图6B为根据本发明实施例的不同浓度的CD3×BCMA双特异性抗体结合人B7H6蛋白的检测结果图；以及FIG6B is a graph showing the detection results of different concentrations of CD3×BCMA bispecific antibodies binding to human B7H6 protein according to an embodiment of the present invention; and
图7为根据本发明实施例的不同浓度的CD3×BCMA双特异性抗体对HCT-15细胞的杀伤检测结果图。FIG. 7 is a graph showing the killing test results of HCT-15 cells by CD3×BCMA bispecific antibodies of different concentrations according to an embodiment of the present invention.
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
本发明所述的抗体或抗原结合片段通常由生物合成的方法制备。根据本发明所述的核苷酸序列，本领域技术人员可方便地用各种已知方法制得本发明的编码核酸。这些方法例如但不限于：PCR，DNA人工合成等，具体的方法可参见J.萨姆布鲁克，《分子克隆实验指南》。作为本发明的一种实施方式，可通过分段合成核苷酸序列再进行重叠延伸PCR的方法来构建本发明的编码核酸序列。其中，所述抗体或抗原片段是采用Kabat编号系统进行编号和定义的。The antibody or antigen-binding fragment of the present invention is usually prepared by a biosynthetic method. According to the nucleotide sequence of the present invention, a person skilled in the art can easily prepare the encoding nucleic acid of the present invention by various known methods. These methods include but are not limited to: PCR, DNA artificial synthesis, etc. For specific methods, please refer to J. Sambrook, "Molecular Cloning Laboratory Guide". As an embodiment of the present invention, the encoding nucleic acid sequence of the present invention can be constructed by synthesizing the nucleotide sequence in segments and then performing overlap extension PCR. Wherein, the antibody or antigen fragment is numbered and defined using the Kabat numbering system.
本文中，所述“单抗”是指具有单一抗原结合位点的抗体。As used herein, the term "monoclonal antibody" refers to an antibody that has a single antigen binding site.
本文中，所述“双抗”是指具有两个不同抗原结合位点的抗体。Herein, the term "double antibody" refers to an antibody having two different antigen binding sites.
在本文中，术语“突变体”或“变体”可以指包含对任何天然存在的或工程化的分子进行包含一个或多个核苷酸或氨基酸突变获得的分子。As used herein, the term "mutant" or "variant" may refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.
术语“互补决定区”或“CDR”或“CDR序列”是指抗体中负责抗原结合的氨基酸序列，例如，通常包括：轻链可变区中23-34(L1)、50-56(L2)和89-97(L3)附近，和重链可变区中31-35B(H1)、50-65(H2)和95-102(H3)附近的氨基酸残基(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD.(1991))；和/或来自“高变环”(例如，轻链可变区中26-32(LI)、50-52(L2)和91-96(L3)，和重链可变区中26-32(H1)、53-55(H2)和96-101(H3)附近的氨基酸残基(Chothia和Lesk J.Mol.Biol.196：901-917(1987))。The term "complementarity determining region" or "CDR" or "CDR sequence" refers to the amino acid sequence in an antibody that is responsible for antigen binding, for example, generally including: amino acid residues around 23-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region, and around 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Health Service, National Institutes of Health, Bethesda, MD. (1991)); and/or from “hypervariable loops” (e.g., amino acid residues around 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
在本文中，术语“同一性”用于描述相对于参考序列的氨基酸序列或核酸序列时，采用通过常规的方法进行确定两个氨基酸序列或核酸序列之间的相同氨基酸或核苷酸的百分比，例如参见，Ausubel等，编著(1995)，Current Protocols in
Molecular Biology，第19章(Greene Publishing and Wiley-Interscience，New York)；和ALIGN程序(Dayhoff(1978)，Atlas of Protein Sequence and Structure 5：Suppl.3(National Biomedical Research Foundation，Washington，D.C.)。关于比对序列和测定序列同一性有很多算法，包括，Needleman等(1970)J.Mol.Biol.48：443的同源性比对算法；Smith等(1981)Adv.Appl.Math.2：482的局部同源性算法；Pearson等(1988)Proc.Natl.Acad.Sci.85：2444的相似性搜索方法；Smith-Waterman算法(Meth.Mol.Biol.70：173-187(1997)；和BLASTP，BLASTN，和BLASTX算法(参见Altschul等(1990)J.Mol.Biol.215：403-410)。利用这些算法的计算机程序也是可获得的，并且包括但不限于：ALIGN或Megalign(DNASTAR)软件，或者WU-BLAST-2(Altschul等，Meth.Enzym.，266：460-480(1996))；或者GAP，BESTFIT，BLAST Altschul等，上文，FASTA，和TFASTA，在Genetics Computing Group(GCG)包，8版，Madison，Wisconsin，USA中可获得；和Intelligenetics，Mountain View，California提供的PC/Gene程序中的CLUSTAL。As used herein, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 102: 1444). .70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC/Gene program provided by Intelligenetics, Mountain View, California.
在不实质性影响抗体活性(保留至少95％的活性)的前提下，本领域技术人员可以对本发明的序列替换、添加和/或缺失一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述抗体或其功能性片段之序列的变体。它们都被视为包括在本发明保护的范围内。如在可变区将具有类似性质的氨基酸进行替换。本发明所述变体序列可以与参比序列具有至少80％、85％、90％、95％、96％、97％、98％或99％的一致性(或同源性)。本发明所述的序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST，尤其是BLASTP或TBLASTN。本发明述及的氨基酸序列均按照N端至C端的方式示出。Under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art can replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present invention to obtain variants of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% consistency (or homology) with the reference sequence. The sequence consistency of the present invention can be measured using sequence analysis software. For example, the computer program BLAST with default parameters is used, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are all shown in an N-terminal to C-terminal manner.
如前所述，本发明的单抗可以是全长抗体或可仅包含其功能性片段，或可以被修饰以影响功能。本发明包括具有修饰的糖基化模式的抗CD3抗体。在一些应用中，进行修饰以除去不期望的糖基化位点可以是有用的，或在寡糖链上不存在岩藻糖部分以例如增强抗体依赖性细胞毒性(ADCC)功能的抗体。在另一些应用中，可进行半乳糖基化修饰以改变补体依赖性细胞毒性(CDC)。As previously mentioned, the monoclonal antibody of the present invention can be a full-length antibody or can only contain its functional fragments, or can be modified to affect function. The present invention includes anti-CD3 antibodies with modified glycosylation patterns. In some applications, it can be useful to modify to remove undesirable glycosylation sites, or antibodies in which fucose moieties are not present on the oligosaccharide chains to, for example, enhance antibody-dependent cellular cytotoxicity (ADCC) function. In other applications, galactosylation modification can be performed to alter complement-dependent cytotoxicity (CDC).
在本文中，所述“全长抗体”是由两条相同的轻链和两条相同的重链通过链间二硫键连接而成的四肽链结构，如免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)或免疫球蛋白E(IgE)。As used herein, the “full-length antibody” refers to a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE).
本文所使用的术语“功能性片段”尤其是指抗体片段如CDR移植抗体、Fab、F(ab’)2、Fv或scFv，纳米抗体、或者通过化学修饰或通过掺入脂质体中应能够增加半寿期的任何片段，所述化学修饰例如添加聚(亚烷基)二醇，如聚乙二醇(“聚乙二醇化，PEG化”)(被称为Fv-PEG、scFv-PEG、Fab-PEG、F(ab’)2-PEG或Fab’-PEG的聚乙二醇化片段)(“PEG”为聚乙二醇)，所述片段具有CD3结合活性。优选地，所述功能性片段将由其来源抗体的重链可变区或轻链可变区的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，对于CD3，优选至少等于其来源抗体亲和力的1/100，在更优选方式中至少等于1/10。这种功能片段将包含最少3个氨基酸，优选其来源的抗体序列的5、10、15、25、50和100个连续氨基酸。The term "functional fragment" as used herein refers in particular to antibody fragments such as CDR-grafted antibodies, Fab, F(ab')2, Fv or scFv, nanobodies, or any fragments that should be able to increase half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation, PEGylation") (called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG PEGylated fragments) ("PEG" is polyethylene glycol), or by incorporation into liposomes, wherein the fragment has CD3 binding activity. Preferably, the functional fragment will be composed of or contain a partial sequence of the heavy chain variable region or light chain variable region of the antibody from which it is derived, and the partial sequence is sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1/100 of the affinity of the antibody from which it is derived, and at least 1/10 in a more preferred manner. Such functional fragments will contain a minimum of 3 amino acids, and preferably 5, 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.
在本发明中，在未作相反说明的情况下，使用的术语“抗原结合片段”通常是指抗原结合性抗体片段，可以包括完整抗体的一部分，一般是抗原结合区或可变区，示例性的，如包括CDR移植抗体、Fab、Fab’、F(ab’)2、Fv或scFv，纳米抗体等。In the present invention, unless otherwise specified, the term "antigen-binding fragment" used generally refers to an antigen-binding antibody fragment, which may include a portion of a complete antibody, generally an antigen-binding region or a variable region, such as, for example, CDR-grafted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.
在本文中，术语“CDR移植抗体”是指将一个物种单抗的CDR移植至另一物种抗体可变区。例如，可将鼠源单抗的CDR移植至人源抗体可变区，以便替代人源抗体CDR，使人源抗体获得鼠源单抗的抗原结合特异性，同时减少其异源性。In this article, the term "CDR transplanted antibody" refers to transplanting the CDR of a monoclonal antibody of one species to the variable region of an antibody of another species. For example, the CDR of a mouse monoclonal antibody can be transplanted to the variable region of a human antibody to replace the human antibody CDR, so that the human antibody obtains the antigen binding specificity of the mouse monoclonal antibody while reducing its heterology.
在本文中，术语“Fab抗体”或“Fab”通常是指仅含Fab分子的抗体，其由重链的VH和CH1以及完整的轻链构成，轻链和重链之间通过一个二硫键连接。As used herein, the term "Fab antibody" or "Fab" generally refers to an antibody containing only Fab molecules, which are composed of VH and CH1 of the heavy chain and a complete light chain, with the light chain and the heavy chain connected by a disulfide bond.
在本文中，术语“F(ab’)2抗体”或“F(ab’)2”具有通过二硫键连接在一起的两个抗原结合F(ab)部分。As used herein, the term "F(ab') 2 antibody" or "F(ab') 2 " has two antigen-binding F(ab) portions linked together by disulfide bonds.
在本文中，术语“纳米抗体”(单域抗体或VHH抗体)，其最初被描述为“重链抗体”(即“缺乏轻链的抗体”)的抗原结合免疫球蛋白(可变)域(Hamers-Casterman C,AtarhouchT,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.:“Naturallyoccurring antibodies devoid of light chains”；Nature 363,446-448(1993))，只包含重链可变区(VH)和常规的CH2与CH3区，其通过重链可变区与抗原特异性结合。In this article, the term "nanoantibody" (single domain antibody or VHH antibody), which was originally described as the antigen-binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e., "antibody lacking light chains") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)), only contains the heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to the antigen through the heavy chain variable region.
在本文中，术语“Fv抗体”通常是指仅由轻链可变区(VL)和重链可变区(VH)通过非共价键连接而成的抗体，是抗体分了保留完整抗原结合部位的最小功能片段。As used herein, the term "Fv antibody" generally refers to an antibody consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds, and is the smallest functional fragment of an antibody that retains a complete antigen binding site.
在本文中，术语“单链抗体”或“scFv”是由抗体重链可变区和轻链可变区通过短肽连接而成的片段。As used herein, the term "single-chain antibody" or "scFv" refers to a fragment consisting of the variable regions of the heavy and light chains of an antibody connected by a short peptide.
在本文中，“knob into hole结构”为在抗体重链恒定区的CH3区形成钮(Knob)扣(hole)突变，便于重链咬合，形成异二聚体，例如，本申请中，通过突变人IgG1重链恒定区CH3结构域中氨基酸(一条链中为T366S、L368A、Y407V、Y349C突变，即“hole”；另一链中为T366W、S354C突变，即“knob”)实现。其中，此处的氨基酸编号为根据Kabat编
号系统进行编号，例如，所述“T366S”是指按照Kabat编号系统编号第366位的T氨基酸被S氨基酸替代。In this article, the "knob into hole structure" refers to a knob (knob) and a hole (hole) mutation formed in the CH3 region of the antibody heavy chain constant region to facilitate the heavy chain to bite and form a heterodimer. For example, in this application, it is achieved by mutating the amino acids in the CH3 domain of the human IgG1 heavy chain constant region (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole"; T366W, S354C mutations in the other chain, i.e., "knob"). The amino acid numbering here is based on the Kabat numbering. For example, "T366S" means that the T amino acid at position 366 according to the Kabat numbering system is replaced by the S amino acid.
本发明所涉及的序列说明详见表1。The sequence descriptions involved in the present invention are shown in Table 1.
表1：
Table 1:
Table 1:
以下将通过实施例对本发明进行详细描述。实施例或测试例中，未注明具体条件的实验方法的，均按照常规条件进行。The present invention will be described in detail below through examples. In the examples or test examples, experimental methods without specific conditions are all carried out under conventional conditions.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.
实施例1抗人CD3杂交瘤单克隆抗体的制备Example 1 Preparation of anti-human CD3 hybridoma monoclonal antibodies
本发明中，抗人CD3单克隆抗体通过免疫小鼠产生。实验用C57BL/6小鼠，雌性，6周龄(江苏集萃药康生物科技有限公司)，免疫抗原为人CD3E&D，人CD3E&G，猴CD3E&D，猴CD3E&G胞外段蛋白(ACRO)，首次免疫用弗
氏完全佐剂(Sigma，F5881)与抗原混合乳化后腹腔免疫，每只小鼠腹腔注射100μg，后续免疫用Ribi佐剂系统(Sigma，S6322)与抗原混合后腹腔免疫，其中，首免后每隔2周免疫一次，共免疫3次。取免疫后的小鼠血清进行抗体滴度ELISA检测，其操作方法为本领域常规方法。In the present invention, anti-human CD3 monoclonal antibodies were produced by immunizing mice. The experimental mice were C57BL/6, female, 6 weeks old (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), and the immunization antigens were human CD3E&D, human CD3E&G, monkey CD3E&D, and monkey CD3E&G extracellular segment protein (ACRO). After emulsification, the complete adjuvant (Sigma, F5881) was mixed with the antigen and then intraperitoneally immunized. Each mouse was intraperitoneally injected with 100 μg. The Ribi adjuvant system (Sigma, S6322) was mixed with the antigen for subsequent immunization. After the first immunization, immunization was performed every 2 weeks, for a total of 3 immunizations. The serum of the immunized mice was taken for antibody titer ELISA detection, and the operation method was the conventional method in the art.
根据抗体滴度的检测结果，选择血清中抗体滴度高小鼠进行脾细胞融合，融合前72小时冲刺免疫所选小鼠，使用Ribi佐剂系统和上述抗原的混合物腹腔注射。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(ATCC，CRL-8287)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中，于37℃、5％CO2孵育。融合后的第5天加入HAT完全培养基，50μL/孔。融合后第7天～8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，200μL/孔。According to the test results of antibody titer, mice with high antibody titer in serum were selected for spleen cell fusion. The selected mice were sprint immunized 72 hours before fusion, and a mixture of Ribi adjuvant system and the above antigens was intraperitoneally injected. The spleen lymphocytes were fused with myeloma cells Sp2/0 cells (ATCC, CRL-8287) using an optimized PEG-mediated fusion step to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1×HAT and 1×OPI), distributed in 96-well cell culture plates, and incubated at 37°C and 5% CO 2. HAT complete medium was added on the 5th day after fusion, 50μL/well. On the 7th to 8th day after fusion, according to the cell growth density, the medium was completely replaced, and the culture medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1×HT and 1×OPI), 200μL/well.
融合后第10-11天，根据细胞生长密度，进行流式细胞术结合检测。阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选为阳性的进行保种，并进行第二次亚克隆。第二次亚克隆为阳性的进行保种和蛋白表达。其中，亚克隆和保种操作均为本领域常规技术操作，通过无血清细胞培养法进一步制备抗体，通过protein G亲和层析纯化鼠源抗体，用于后续功能活性检测。On the 10th to 11th day after fusion, flow cytometry binding detection was performed according to the cell growth density. The positive wells were replaced with liquid and expanded to 24-well plates in time according to the cell density. The cell lines transferred into the 24-well plates were retested for seed preservation and the first subcloning. The first subclone screening was positive for seed preservation and the second subcloning was performed. The second subclone was positive for seed preservation and protein expression. Among them, subcloning and seed preservation operations are routine technical operations in this field. Antibodies are further prepared by serum-free cell culture method, and mouse antibodies are purified by protein G affinity chromatography for subsequent functional activity detection.
实施例2鼠源CD3抗体ELISA结合实验Example 2 Mouse CD3 antibody ELISA binding experiment
ELISA实验用于检测实施例1获得的鼠源CD3抗体m47B5的结合特性。将人CD3ED蛋白(ACRO biosystems，CDD-H52W1)，人CD3EG蛋白(ACRO biosystems，CDG-H52W5)，猴CD3ED蛋白(ACRO biosystems，CDD-C52W4)，猴CD3EG蛋白(ACRO biosystems，CDG-H52W6)分别用包被缓冲液(35mM NaHCO3，15mM Na2CO3,pH 9.6)稀释至2μg/mL，向酶联板中每孔加入100μL，4℃过夜。之后用PBST清洗3遍(0.05％Tween 20-PBS，pH7.2)。向板中加入300μL封闭缓冲液(1％BSA,0.05％Tween20-PBS,pH 7.2)，室温静置2h。再用PBST清洗3遍。每孔加入鼠源抗体m47B5，室温孵育1小时。再用PBST清洗3遍。每孔加入100μL用封闭缓冲液稀释的HRP-羊抗鼠IgG二抗(boster，货号BA1051)，室温孵育1小时。用PBST清洗3遍，每孔加入TMB，室温避光反应2-5分钟，每孔再用2M硫酸终止反应，最后用酶标仪读取OD450数值。其中，图1A表明本发明的鼠源m47B5抗体可以结合人CD3EG，图1B表明本发明的鼠源m47B5抗体也可以结合人CD3ED，图1C表明本发明的鼠源m47B5抗体可以结合猴CD3EG，图1D表明本发明的鼠源m47B5抗体可以结合猴CD3ED。The ELISA experiment was used to detect the binding properties of the mouse CD3 antibody m47B5 obtained in Example 1. Human CD3ED protein (ACRO biosystems, CDD-H52W1), human CD3EG protein (ACRO biosystems, CDG-H52W5), monkey CD3ED protein (ACRO biosystems, CDD-C52W4), and monkey CD3EG protein (ACRO biosystems, CDG-H52W6) were diluted to 2 μg/mL with coating buffer (35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6), and 100 μL was added to each well of the enzyme-linked plate and incubated at 4°C overnight. After that, the plate was washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2 hours. Then wash 3 times with PBST. Add mouse antibody m47B5 to each well and incubate at room temperature for 1 hour. Then wash 3 times with PBST. Add 100 μL of HRP-sheep anti-mouse IgG secondary antibody (boster, item number BA1051) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash 3 times with PBST, add TMB to each well, react at room temperature in the dark for 2-5 minutes, and then terminate the reaction with 2M sulfuric acid in each well, and finally read the OD450 value with an enzyme reader. Among them, Figure 1A shows that the mouse m47B5 antibody of the present invention can bind to human CD3EG, Figure 1B shows that the mouse m47B5 antibody of the present invention can also bind to human CD3ED, Figure 1C shows that the mouse m47B5 antibody of the present invention can bind to monkey CD3EG, and Figure 1D shows that the mouse m47B5 antibody of the present invention can bind to monkey CD3ED.
实施例3杂交瘤细胞测序Example 3 Hybridoma Cell Sequencing
将实施1和2筛选到的m47B5抗体候选杂交瘤细胞总数量培养到106，800rpm离心10分钟收集细胞，并以Trizol试剂盒(Invitrogen)提取总RNA；以总RNA为模板，逆转录合成cDNA文库(Invitrogen)，又以cDNA为模板PCR扩增杂交瘤细胞的所对应的m47B5抗体可变区核酸序列。PCR扩增反应中所使用的引物序列与抗体可变区第一框架区或信号肽区和恒定区互补(具体序列参考Larrick，J.W.，et al.，(1990)Scand.J.Immunol.，32，121 128和Coloma，J.J.et al.，(1991)BioTechniques，11，152 156)。PCR扩增在50μL反应体系中进行，分别加入cDNA 2μL，10×PCR缓冲液5μL，上游及下游引物2μL(5μM)，dNTP 2μL，Taq酶1μL(Takara，Ex Taq)，H2O 38μL；95℃预变性5min，进入温度循环，进行PCR扩增。反应条件为：94℃变性30S，58℃退火45S，72℃延伸50S，共32个循环，然后于72℃延伸7min。将扩增产物进行测序后，得到鼠单抗m47B5的重链可变区(SEQ ID NO:23)和轻链可变区(SEQ ID NO:24)序列。The total number of candidate hybridoma cells of the m47B5 antibody screened in implementation 1 and 2 was cultured to 10 6 , and the cells were collected by centrifugation at 800 rpm for 10 minutes, and the total RNA was extracted using a Trizol kit (Invitrogen); the total RNA was used as a template to synthesize a cDNA library (Invitrogen), and the cDNA was used as a template to PCR amplify the nucleic acid sequence of the variable region of the m47B5 antibody corresponding to the hybridoma cells. The primer sequence used in the PCR amplification reaction is complementary to the first framework region or signal peptide region and constant region of the antibody variable region (for specific sequences, refer to Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). PCR amplification was performed in a 50 μL reaction system, and 2 μL of cDNA, 5 μL of 10×PCR buffer, 2 μL of upstream and downstream primers (5 μM), 2 μL of dNTP, 1 μL of Taq enzyme (Takara, Ex Taq), and 38 μL of H 2 O were added respectively; pre-denaturation at 95°C for 5 min, and temperature cycle was entered for PCR amplification. The reaction conditions were: denaturation at 94°C for 30S, annealing at 58°C for 45S, extension at 72°C for 50S, a total of 32 cycles, and then extension at 72°C for 7 min. After sequencing the amplified products, the sequences of the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) of mouse monoclonal antibody m47B5 were obtained.
实施例4嵌合CD3抗体流式细胞术结合实验Example 4 Chimeric CD3 Antibody Flow Cytometry Binding Experiment
利用上述鼠单抗m47B5构建人--鼠嵌合抗体(ch47B5)，其中，所述嵌合抗体中的重链恒定区使用人IgG1的重链恒定区。The mouse monoclonal antibody m47B5 was used to construct a human-mouse chimeric antibody (ch47B5), wherein the heavy chain constant region of the chimeric antibody used was the heavy chain constant region of human IgG1.
用PBS将人外周血单个核细胞(PBMC)或者猴PBMC细胞稀释为2×106个/mL，以100μL/管的体积加于1.5mL EP管中，向其中加入10μL/管山羊血清，于4℃封闭30min。加梯度浓度(5倍稀释，终浓度最高10μg/mL)的上述人鼠嵌合抗体ch47B5，于4℃孵育30min。向EP管中加入1mL PBS，于4℃，3500rpm×5min离心，弃尽上清，再用PBS洗一遍。离心后弃尽上清，用100μL/管PBS重悬细胞，向其中加入0.1μL/管Alexa 647标记的大鼠抗人抗体二抗(biolegend)，4℃避光孵育30min。孵育结束后，用PBS洗两遍，离心后弃尽上清。用200μL/管PBS重悬细胞，然后用流式细胞仪进行检测，其中，以BT062抗体为对照抗体。图2A和图2B结果表明嵌合47B5抗体可以结合人PBMC和猴PBMC，说明嵌合CD3抗体可以结合细胞表面的CD3蛋白。
Human peripheral blood mononuclear cells (PBMC) or monkey PBMC cells were diluted to 2×10 6 cells/mL with PBS, added to a 1.5mL EP tube at a volume of 100μL/tube, and 10μL/tube of goat serum was added thereto, and the cells were blocked at 4°C for 30min. The above-mentioned human-mouse chimeric antibody ch47B5 was added with gradient concentrations (5-fold dilution, the highest final concentration was 10μg/mL), and incubated at 4°C for 30min. 1mL PBS was added to the EP tube, centrifuged at 4°C, 3500rpm×5min, the supernatant was discarded, and then washed with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended with 100μL/tube PBS, and 0.1μL/tube of Alexa 647-labeled rat anti-human antibody secondary antibody (biolegend) was added thereto, and incubated at 4°C for 30min in the dark. After the incubation, the cells were washed twice with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended with 200 μL/tube PBS and then detected by flow cytometry, wherein BT062 antibody was used as a control antibody. The results of Figure 2A and Figure 2B show that the chimeric 47B5 antibody can bind to human PBMC and monkey PBMC, indicating that the chimeric CD3 antibody can bind to the CD3 protein on the cell surface.
实施例5抗人CD3单克隆抗体的人源化Example 5 Humanization of anti-human CD3 monoclonal antibody
5.1 CD3单克隆抗体可变区框架的选择与回复突变5.1 Selection and backmutation of the variable region framework of CD3 monoclonal antibody
在实施例4获得的嵌合抗体的基础上，通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与鼠源单克隆抗体m47B5同源性高的重链或轻链可变区种系基因作为模板，将鼠源单克隆抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号系统确定并注释。Based on the chimeric antibody obtained in Example 4, by comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the heavy chain or light chain variable region germline genes with high homology to the mouse monoclonal antibody m47B5 were selected as templates, and the CDRs of the mouse monoclonal antibody were transplanted into the corresponding human templates to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were determined and annotated by the Kabat numbering system.
为了保持CDR区构象，对于VL和VH结合界面上的残基，靠近CDR并且包埋于蛋白内部的残基，与CDR有直接相互作用的残基进行回复突变，以保证可变区的活性不会受影响。In order to maintain the conformation of the CDR region, back mutations were performed on the residues on the VL and VH binding interface, the residues close to the CDR and buried in the protein, and the residues that directly interacted with the CDR to ensure that the activity of the variable region would not be affected.
5.2 CD3人源化抗体的表达5.2 Expression of humanized CD3 antibody
对上述CD3单克隆抗体可变区框架进行选择与回复突变后，获得的人源化CD3抗体h47B5的重链可变区序列如SEQ ID NO:27所示，轻链可变区序列如SEQ ID NO:28所示。将带有编码人源化抗体重链的核苷酸序列(SEQ ID NO:43)和编码人源化抗体轻链的核苷酸序列(SEQ ID NO:44)通过分子生物学技术分别重组到pTT5质粒上。After selecting and backmutating the variable region framework of the above CD3 monoclonal antibody, the heavy chain variable region sequence of the obtained humanized CD3 antibody h47B5 is shown in SEQ ID NO: 27, and the light chain variable region sequence is shown in SEQ ID NO: 28. The nucleotide sequence encoding the heavy chain of the humanized antibody (SEQ ID NO: 43) and the nucleotide sequence encoding the light chain of the humanized antibody (SEQ ID NO: 44) were recombined into the pTT5 plasmid by molecular biology technology.
用上述携带有人源化抗人CD3抗体h47B5轻链和重链的pTT5载体瞬时转染ExpiCHO-S细胞(Gibco，货号A29127)来制备抗体。转染前一天，将ExpiCHO-S细胞调整细胞密度为(3～4)×106/mL，37℃，8％CO2，120rpm摇动培养过夜。转染当天，细胞生长到7×106～1×107/mL，存活率大于95％时准备转染，使用新鲜预热的ExpiCHO培养基(Gibco，货号A2910002)将细胞稀释到6×106/mL，将上述携带有人源化抗人CD3抗体h47B5轻链和重链的pTT5质粒和ExpiFectamine CHO转染试剂(Gibco，货号A29129)以2：1摩尔比转染到ExpiCHO-S细胞中，于37℃，8％CO2，120rpm摇动培养。转染后18-22h，将ExpiFectamine CHO Enhancer和ExpiCHO Feed混匀后立即加入转染后细胞，混匀，于32℃，5％CO2，120rpm摇动培养。转染后第5天，向细胞中再补加8mL ExpiCHO Feed，混匀后继续培养。每日观察细胞数和细胞活率变化，待细胞活率下降至80％以下或培养10～14天后离心收获细胞。表达的上清用0.22μm滤膜过滤，利用Mabselect prism A亲和层析柱(GE公司，货号17549854)从表达上清中捕获带有Fc结构域的抗体，用pH7.2的磷酸盐缓冲液平衡层析柱后，上清过亲和层析柱，用洗脱缓冲液(100mM柠檬酸，pH2.7)洗脱，最后用PBS缓冲液浓缩置换，纯化后的抗体通过SDS-PAGE鉴定纯度在95％以上，最终获得了人源化m47B5抗体(重链如SEQ ID NO:39所示，轻链如SEQ ID NO:40所示)。The pTT5 vector carrying the humanized anti-human CD3 antibody h47B5 light chain and heavy chain was used to transiently transfect ExpiCHO-S cells (Gibco, catalog number A29127) to prepare antibodies. One day before transfection, the cell density of ExpiCHO-S cells was adjusted to (3-4)×10 6 /mL and cultured overnight at 37°C, 8% CO 2 , and 120 rpm shaking. On the day of transfection, when the cells grow to 7×10 6 ～1×10 7 /mL and the viability is greater than 95%, they are ready for transfection. Use fresh preheated ExpiCHO medium (Gibco, Catalog No. A2910002) to dilute the cells to 6×10 6 /mL, and transfect the above pTT5 plasmid carrying the light and heavy chains of the humanized anti-human CD3 antibody h47B5 and ExpiFectamine CHO transfection reagent (Gibco, Catalog No. A29129) into ExpiCHO-S cells at a molar ratio of 2:1. Culture at 37°C, 8% CO 2 , 120rpm shaking. 18-22h after transfection, mix ExpiFectamine CHO Enhancer and ExpiCHO Feed and immediately add the transfected cells, mix, and culture at 32°C, 5% CO 2 , 120rpm shaking. On the 5th day after transfection, add 8mL of ExpiCHO Feed to the cells, mix and continue to culture. The cell number and cell viability were observed daily, and the cells were harvested by centrifugation after the cell viability dropped below 80% or after culturing for 10 to 14 days. The expressed supernatant was filtered with a 0.22 μm filter membrane, and the antibody with the Fc domain was captured from the expression supernatant using a Mabselect prism A affinity chromatography column (GE, Cat. No. 17549854). After the chromatography column was equilibrated with a pH 7.2 phosphate buffer, the supernatant was passed through the affinity chromatography column, eluted with an elution buffer (100 mM citric acid, pH 2.7), and finally concentrated and replaced with a PBS buffer. The purified antibody was identified by SDS-PAGE to have a purity of more than 95%, and finally a humanized m47B5 antibody (heavy chain as shown in SEQ ID NO: 39, light chain as shown in SEQ ID NO: 40) was obtained.
5.3人源化抗体h47B5亲和力验证5.3 Affinity verification of humanized antibody h47B5
用BIACORE测试人源化h47B5抗体与人CD3抗原的亲和力，得到的结果如表2所示，获得的人源化抗体与CD3抗原具有较好的亲和力。The affinity of the humanized h47B5 antibody to the human CD3 antigen was tested by BIACORE. The results are shown in Table 2. The obtained humanized antibody has a good affinity to the CD3 antigen.
表2
Table 2
Table 2
实施例6人源化抗体h47B5的ELISA结合实验Example 6 ELISA binding experiment of humanized antibody h47B5
ELISA实验用于检测人源化抗体h47B5的结合特性，其中，以野生型人源IgG1抗体为对照。将人CD3抗原(ACRO biosystems，CDD-H52W1)和猴CD3抗原(ACRO biosystems，CDD-C52W4)用包被缓冲液(35mM NaHCO3，15mM Na2CO3，pH 9.6)稀释至2μg/mL，向酶联板中每孔加入100μL，4℃过夜。之后用PBST清洗3遍(0.05％Tween 20-PBS，pH7.2)。向板中加入300μL封闭缓冲液(1％BSA,0.05％Tween20-PBS,pH 7.2)，室温静置2h。再用PBST清洗3遍。每孔加入梯度浓度的人源化抗人CD3抗体h47B5或人源IgG1抗体，室温孵育1小时。再用PBST清洗3遍。每孔加入100μL用封闭缓冲液稀释的HRP-山羊抗人IgG二抗(Jackson ImmunoResearch，109-036-170))，室温孵育1小时。用PBST清洗3遍，每孔加入TMB，室温避光反应2～5分钟，每孔再用2M硫酸终止反应，最后用酶标仪读取OD450数值。图3A和图3B表明本发明的人源化h47B5抗体可以结合人和猴的CD3抗原。ELISA experiments were used to detect the binding properties of humanized antibody h47B5, in which wild-type human IgG1 antibody was used as a control. Human CD3 antigen (ACRO biosystems, CDD-H52W1) and monkey CD3 antigen (ACRO biosystems, CDD-C52W4) were diluted to 2μg/mL with coating buffer (35mM NaHCO 3 , 15mM Na 2 CO 3 , pH 9.6), and 100μL was added to each well of the ELISA plate and incubated at 4°C overnight. After that, it was washed 3 times with PBST (0.05% Tween 20-PBS, pH7.2). 300μL of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2h. It was then washed 3 times with PBST. Gradient concentrations of humanized anti-human CD3 antibody h47B5 or human IgG1 antibody were added to each well and incubated at room temperature for 1 hour. Then wash with PBST for 3 times. Add 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash with PBST for 3 times, add TMB to each well, react at room temperature in the dark for 2 to 5 minutes, and then terminate the reaction with 2M sulfuric acid in each well. Finally, read the OD450 value with an enzyme marker. Figures 3A and 3B show that the humanized h47B5 antibody of the present invention can bind to human and monkey CD3 antigens.
实施例7 CD3×BCMA的双特异性抗体的制备及结合活性和细胞杀伤能力检测Example 7 Preparation of CD3×BCMA bispecific antibodies and detection of binding activity and cell killing ability
基于实施例1-6的实验结果，发明人设计了一种双特异性抗体CD3×BCMA，并对该双特异性抗体的结合活性和细胞杀伤能力进行检测。
Based on the experimental results of Examples 1-6, the inventors designed a bispecific antibody CD3×BCMA, and tested the binding activity and cell killing ability of the bispecific antibody.
7.1 CD3×BCMA的双特异性抗体的设计及制备7.1 Design and preparation of CD3×BCMA bispecific antibodies
该抗体含有两个单价单元，其中一个单价单元为抗CD3scFv-Fc形式，其重链和轻链可变区氨基酸序列来自本发明实施例6获得的人源化抗体h47B5的序列，另一个单价单元为抗BCMA的scFv-Fc形式(序列来自US009273141B2)，该双特异性抗体命名为CD3×BCMA。该双特异性抗体含有两条多肽链，分别是：含有CD3单链抗体scFv的重链(SEQ ID NO：51)，含有BCMA单链抗体scFv的重链(SEQ ID NO：52)，其中两条链的重链恒定区来自于人源抗体IgG1。由于该分子有特殊的非对称结构，为了减少同源二聚体的产生，所以在两条链的恒定区引入了不同的氨基酸突变，形成knob-in-hole结构。同时为了防止由Fcγ受体引起的交联活化，在重链恒定区也引入了(L234A/L235A)突变。The antibody contains two monovalent units, one of which is in the form of anti-CD3 scFv-Fc, and its heavy chain and light chain variable region amino acid sequences are from the sequence of the humanized antibody h47B5 obtained in Example 6 of the present invention, and the other monovalent unit is in the form of anti-BCMA scFv-Fc (sequence from US009273141B2), and the bispecific antibody is named CD3×BCMA. The bispecific antibody contains two polypeptide chains, namely: a heavy chain containing a CD3 single-chain antibody scFv (SEQ ID NO: 51), and a heavy chain containing a BCMA single-chain antibody scFv (SEQ ID NO: 52), wherein the heavy chain constant regions of the two chains are from human antibody IgG1. Since the molecule has a special asymmetric structure, in order to reduce the generation of homodimers, different amino acid mutations are introduced in the constant regions of the two chains to form a knob-in-hole structure. At the same time, in order to prevent cross-linking activation caused by Fcγ receptors, (L234A/L235A) mutations are also introduced in the heavy chain constant region.
双特异性抗体的制备方法参考实施例6.2所述方法，并结合质粒配比(1：1或其他比例)并经过一步亲和纯化制备所述双特异性抗体，其中，所述双特异性抗体CD3×BCMA的相关序列如表3所示。The method for preparing the bispecific antibody refers to the method described in Example 6.2, and the bispecific antibody is prepared by combining the plasmid ratio (1:1 or other ratio) and undergoing one-step affinity purification, wherein the relevant sequence of the bispecific antibody CD3×BCMA is shown in Table 3.
表3
table 3
table 3
7.2 CD3×BCMA双特异性抗体与抗原的结合活性测定(ELISA)7.2 CD3×BCMA bispecific antibody binding activity assay with antigen (ELISA)
将人BCMA(ACRO biosystems，BCA-H522y)或人CD3抗原(ACRO biosystems，CDD-H52W1)用包被缓冲液(35mM NaHCO3，15mM Na2CO3，pH 9.6)稀释至2μg/mL，向酶联板中每孔加入100μL，于4℃过夜。之后用PBST清洗3遍(0.05％Tween 20-PBS，pH7.2)。向板中加入300μL封闭缓冲液(1％BSA，0.05％Tween20-PBS，pH 7.2)，室温静置2h。再用PBST清洗3遍。每孔加入相应双特异性抗体，室温孵育1小时。再用PBST清洗沉淀3遍。每孔加入100μL用封闭缓冲液稀释的HRP-山羊抗人IgG二抗(Jackson ImmunoResearch，109-036-170)，于室温孵育1小时。用PBST清洗3遍，每孔加入TMB，室温避光反应2～5分钟，每孔再用2M硫酸终止反应，最后用酶标仪读取OD450数值。图4A和4B显示本发明CD3×BCMA可以结合CD3和BCMA这两种抗原。Human BCMA (ACRO biosystems, BCA-H522y) or human CD3 antigen (ACRO biosystems, CDD-H52W1) was diluted to 2 μg/mL with coating buffer (35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6), and 100 μL was added to each well of the ELISA plate and incubated at 4°C overnight. Afterwards, the plate was washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2 h. The plate was then washed three times with PBST. The corresponding bispecific antibody was added to each well and incubated at room temperature for 1 hour. The precipitate was then washed three times with PBST. 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170) diluted with blocking buffer was added to each well and incubated at room temperature for 1 hour. Washed 3 times with PBST, TMB was added to each well, reacted at room temperature in the dark for 2 to 5 minutes, and 2M sulfuric acid was used to terminate the reaction in each well. Finally, the OD450 value was read with an ELISA reader. Figures 4A and 4B show that CD3×BCMA of the present invention can bind to both CD3 and BCMA antigens.
7.3细胞杀伤实验7.3 Cell killing assay
将多发性骨髓瘤NCI-H929细胞标记CellTrace Violet(thermo，C34557)，调整细胞密度为1×105/mL。PBMC调整细胞密度1×106/mL，将PBMC和标记后的NCI-H929细胞以1：1混合后加入到96孔圆底板中，再加入梯度浓度的上述双特异性抗体或人IgG抗体(对照组)，混合均匀。于37℃孵育24h后过200目尼龙网转入流式管，加入7AAD后10min进行检测，利用CTV+7AAD+细胞比例表征杀伤效率。图5结果显示随着CD3×BCMA双特异性抗体浓度增加，PBMC对NCI-H929多发性骨髓瘤细胞的杀伤效率逐渐增加。Multiple myeloma NCI-H929 cells were labeled with CellTrace Violet (thermo, C34557) and the cell density was adjusted to 1×10 5 /mL. The cell density of PBMC was adjusted to 1×10 6 /mL, and PBMC and labeled NCI-H929 cells were mixed at a ratio of 1:1 and added to a 96-well round bottom plate, and then the above-mentioned bispecific antibodies or human IgG antibodies (control group) with gradient concentrations were added and mixed evenly. After incubation at 37°C for 24 hours, the cells were transferred to a flow tube through a 200-mesh nylon mesh, and the cells were tested 10 minutes after adding 7AAD. The killing efficiency was characterized by the ratio of CTV+7AAD+ cells. The results in Figure 5 show that as the concentration of CD3×BCMA bispecific antibodies increases, the killing efficiency of PBMC against NCI-H929 multiple myeloma cells gradually increases.
实施例8 CD3×B7H6的双特异性抗体的制备及结合活性和细胞杀伤能力检测Example 8 Preparation of CD3×B7H6 bispecific antibody and detection of binding activity and cell killing ability
基于实施例1-6的实验结果，发明人设计了一种双特异性抗体CD3×B7H6，并对该双特异性抗体的结合活性和细胞杀伤能力进行检测。Based on the experimental results of Examples 1-6, the inventors designed a bispecific antibody CD3×B7H6, and tested the binding activity and cell killing ability of the bispecific antibody.
8.1 CD3×B7H6的双特异性抗体的设计及制备8.1 Design and preparation of CD3×B7H6 bispecific antibodies
该抗体含有两个单价单元，其中一个单价单元为抗CD3scFv-Fc形式，CD3scFv中的轻链和重链可变区来自本发明实施例6人源化后序列，另一个单价单元为抗B7H6scFv-Fc形式(B7H6scFv序列来自CN114395045A)，这个双特异性抗体命名为CD3×B7H6。该双特异性抗体含有两条多肽链，分别是：含有CD3单链抗体scFv的重链(SEQ ID NO：51)，含有B7H6单链抗体scFv的重链(SEQ ID NO：53)。由于该分子有特殊的非对称结构，为了减少同源二聚体的产生，所以在两条链的恒定区引入了不同的氨基酸突变，形成knob-in-hole结构。同时为了防止由Fcγ受体引起的交联活化，在重链恒定区也引入了(L234A/L235A)突变。The antibody contains two monovalent units, one of which is in the form of anti-CD3scFv-Fc, in which the light chain and heavy chain variable regions in CD3scFv are from the humanized sequence of Example 6 of the present invention, and the other monovalent unit is in the form of anti-B7H6scFv-Fc (the B7H6scFv sequence is from CN114395045A). This bispecific antibody is named CD3×B7H6. The bispecific antibody contains two polypeptide chains, namely: a heavy chain containing a CD3 single-chain antibody scFv (SEQ ID NO: 51) and a heavy chain containing a B7H6 single-chain antibody scFv (SEQ ID NO: 53). Since the molecule has a special asymmetric structure, in order to reduce the generation of homodimers, different amino acid mutations are introduced into the constant regions of the two chains to form a knob-in-hole structure. At the same time, in order to prevent cross-linking activation caused by Fcγ receptors, (L234A/L235A) mutations are also introduced into the heavy chain constant region.
CD3×B7H6的双特异性抗体的制备方法参考实施例6.2，并结合质粒配比(1：1或其他比例)并经过一步亲和纯化制备CD3×B7H6双特异性抗体，所述双特异性抗体CD3×B7H6的相关序列如表4所示。The method for preparing the bispecific antibody of CD3×B7H6 refers to Example 6.2, and the CD3×B7H6 bispecific antibody is prepared by combining the plasmid ratio (1:1 or other ratios) and undergoing one-step affinity purification. The relevant sequence of the bispecific antibody CD3×B7H6 is shown in Table 4.
表4
Table 4
Table 4
8.2双特异性抗体与抗原的结合活性测定(ELISA)
8.2 Determination of the binding activity of bispecific antibodies to antigens (ELISA)
将人B7H6(ACRO biosystems，B76-H52H8)或人CD3抗原(ACRO biosystems，CDD-H52W)用包被缓冲液(35mM NaHCO3，15mM Na2CO3，pH 9.6)稀释至2μg/mL，向酶联板中每孔加入100μL，于4℃过夜。之后用PBST清洗3遍(0.05％Tween 20-PBS，pH7.2)。向板中加入300μL封闭缓冲液(1％BSA，0.05％Tween20-PBS，pH 7.2)，室温静置2h。再用PBST清洗3遍。每孔加入相应双特异性抗体，室温孵育1小时。再用PBST清洗3遍。每孔加入100μL用封闭缓冲液稀释的HRP-山羊抗人IgG二抗(Jackson ImmunoResearch，109-036-170)，于室温孵育1小时。孵育结束后，用PBST清洗沉淀3遍，每孔加入TMB，室温避光反应2～5分钟，每孔再用2M硫酸终止反应，最后用酶标仪读取OD450数值。图6A和6B显示，本发明的双特异性抗体CD3×B7H6可以有效结合CD3和B7H6这两种抗原。Human B7H6 (ACRO biosystems, B76-H52H8) or human CD3 antigen (ACRO biosystems, CDD-H52W) was diluted to 2 μg/mL with coating buffer (35 mM NaHCO 3 , 15 mM Na 2 CO 3 , pH 9.6), and 100 μL was added to each well of the ELISA plate and incubated at 4°C overnight. Afterwards, the plate was washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2 h. The plate was then washed three times with PBST. The corresponding bispecific antibody was added to each well and incubated at room temperature for 1 hour. The plate was then washed three times with PBST. 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170) diluted with blocking buffer was added to each well and incubated at room temperature for 1 hour. After the incubation, the precipitate was washed 3 times with PBST, TMB was added to each well, and the reaction was carried out at room temperature in the dark for 2 to 5 minutes. 2M sulfuric acid was used to terminate the reaction in each well, and the OD450 value was read with an ELISA reader. Figures 6A and 6B show that the bispecific antibody CD3×B7H6 of the present invention can effectively bind to the two antigens CD3 and B7H6.
8.3细胞杀伤实验8.3 Cell killing assay
将结直肠癌HCT-15细胞消化后计数，调整细胞密度为2×105/mL。打开RTCA仪器(安捷伦)，选择仪器类型DP，选择实验模式，填写细胞信息和药物信息。进入schedule设置实验步骤，将板子加入50μL新鲜培养基(89％RPMI 1640培养基+10％胎牛血清+1％青链霉素)后放入仪器中，关好，点击第一步开始。完成Done后取出板子，加入100μL细胞悬液，室温静置15-30min防止边缘效应，放入仪器，点击开始。待生长到对数期后，暂停，加入50μL PBMC(4×106/mL)，并加入梯度浓度的双特异性抗体CD3×B7H6，点击开始，一段时间后进行分析。图7结果显示，随着CD3×B7H6双特异性抗体浓度增加，PBMC对结直肠癌HCT-15细胞的杀伤效率逐渐增加，说明CD3×B7H6可以很好地促进PBMC特异性地杀伤B7H6+的肿瘤细胞。After digestion, count the colorectal cancer HCT-15 cells and adjust the cell density to 2×10 5 /mL. Turn on the RTCA instrument (Agilent), select the instrument type DP, select the experimental mode, and fill in the cell information and drug information. Enter the schedule to set the experimental steps, add 50μL of fresh culture medium (89% RPMI 1640 culture medium + 10% fetal bovine serum + 1% penicillin and streptomycin) to the plate and put it into the instrument, close it, and click the first step to start. After Done, take out the plate, add 100μL of cell suspension, let it stand at room temperature for 15-30min to prevent edge effects, put it into the instrument, and click Start. After growing to the logarithmic phase, pause, add 50μL PBMC (4×10 6 /mL), and add gradient concentrations of bispecific antibody CD3×B7H6, click Start, and analyze after a period of time. The results in Figure 7 show that as the concentration of CD3×B7H6 bispecific antibody increases, the killing efficiency of PBMC on colorectal cancer HCT-15 cells gradually increases, indicating that CD3×B7H6 can well promote PBMC to specifically kill B7H6 + tumor cells.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.
Claims (87)
- 一种抗体或抗原结合片段，其特征在于，包括选自下列至少之一的CDR序列或与其具有至少95％同一性的氨基酸序列：An antibody or antigen-binding fragment, characterized in that it comprises a CDR sequence selected from at least one of the following or an amino acid sequence having at least 95% identity thereto:重链可变区CDR序列：SEQ ID NO:1～3，Heavy chain variable region CDR sequence: SEQ ID NO: 1～3,轻链可变区CDR序列：SEQ ID NO:4～6。Light chain variable region CDR sequence: SEQ ID NO: 4～6.
- 根据权利要求1所述的抗体或抗原结合片段，其特征在于，包括：The antibody or antigen-binding fragment according to claim 1, comprising:分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少95％同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列；和/或The heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO: 1, 2 and 3, respectively, or amino acid sequences that are at least 95% identical to SEQ ID NO: 1, 2 and 3; and/or分别如SEQ ID NO:4、5和6或者与4、5和6具有至少95％同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列。The light chain variable region CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO: 4, 5, and 6, respectively, or amino acid sequences that are at least 95% identical to 4, 5, and 6.
- 根据权利要求2所述的抗体或抗原结合片段，其特征在于，包括：The antibody or antigen-binding fragment according to claim 2, characterized in that it comprises:如SEQ ID NO:1所示的重链可变区CDR1序列，如SEQ ID NO:2所示的重链可变区CDR2，如SEQ ID NO:3所示的重链可变区CDR3，SEQ ID NO:4所示的轻链可变区CDR1，SEQ ID NO:5所示的轻链可变区CDR2，SEQ ID NO:6所示的轻链可变区CDR3。The heavy chain variable region CDR1 sequence as shown in SEQ ID NO:1, the heavy chain variable region CDR2 as shown in SEQ ID NO:2, the heavy chain variable region CDR3 as shown in SEQ ID NO:3, the light chain variable region CDR1 as shown in SEQ ID NO:4, the light chain variable region CDR2 as shown in SEQ ID NO:5, and the light chain variable region CDR3 as shown in SEQ ID NO:6.
- 根据权利要求3所述的抗体或抗原结合片段，其特征在于，包括：重链FR区和轻链FR区中的至少之一。The antibody or antigen-binding fragment according to claim 3, characterized in that it comprises: at least one of a heavy chain FR region and a light chain FR region.
- 根据权利要求4所述的抗体或抗原结合片段，其特征在于，所述重链FR区和轻链FR区的至少之一的至少一部分来自于人源抗体、灵长目源抗体和鼠源抗体或其突变体中的至少之一。The antibody or antigen-binding fragment according to claim 4, characterized in that at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a human antibody, a primate antibody and a mouse antibody or a mutant thereof.
- 根据权利要求4所述的抗体或抗原结合片段，其特征在于，包括：The antibody or antigen-binding fragment according to claim 4, characterized in that it comprises:分别如SEQ ID NO:7～10所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一；或At least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NOs: 7 to 10, respectively; or分别如SEQ ID NO:15～18所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一。At least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NO:15 to 18, respectively.
- 根据权利要求4所述的抗体或抗原结合片段，其特征在于，包括：The antibody or antigen-binding fragment according to claim 4, characterized in that it comprises:分别如SEQ ID NO:11～14所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一；或At least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NOs: 11 to 14, respectively; or分别如SEQ ID NO:19～22所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一。At least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NO: 19 to 22, respectively.
- 根据权利要求5～7任一项所述的抗体或其抗原结合片段，其特征在于，包括：分别如SEQ ID NO:7～10所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一；分别如SEQ ID NO:11～14所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一；或者The antibody or antigen-binding fragment thereof according to any one of claims 5 to 7, characterized in that it comprises: at least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NOs: 7 to 10, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NOs: 11 to 14, respectively; or分别如SEQ ID NO:15～18所示的重链框架区HFR1、HFR2、HFR3和HFR4序列中的至少之一；分别如SEQ ID NO:19～22所示的轻链框架区LFR1、LFR2、LFR3和LFR4序列中的至少之一。At least one of the heavy chain framework region HFR1, HFR2, HFR3 and HFR4 sequences shown in SEQ ID NO: 15 to 18, respectively; at least one of the light chain framework region LFR1, LFR2, LFR3 and LFR4 sequences shown in SEQ ID NO: 19 to 22, respectively.
- 根据权利要求8所述的抗体或其抗原结合片段，其特征在于，包括：The antibody or antigen-binding fragment thereof according to claim 8, characterized in that it comprises:如SEQ ID NO:23或SEQ ID NO:27所示的重链可变区；和/或The heavy chain variable region as shown in SEQ ID NO:23 or SEQ ID NO:27; and/or如SEQ ID NO:24或SEQ ID NO:28所示的轻链可变区。The light chain variable region as shown in SEQ ID NO:24 or SEQ ID NO:28.
- 根据权利要求9所述的抗体或其抗原结合片段，其特征在于，包括：The antibody or antigen-binding fragment thereof according to claim 9, characterized in that it comprises:1)如SEQ ID NO:23所示的重链可变区，和如SEQ ID NO:24所示的轻链可变区；或1) the heavy chain variable region shown in SEQ ID NO:23, and the light chain variable region shown in SEQ ID NO:24; or2)如SEQ ID NO:27所示的重链可变区，和如SEQ ID NO:28所示的轻链可变区。2) The heavy chain variable region shown in SEQ ID NO:27, and the light chain variable region shown in SEQ ID NO:28.
- 根据权利要求1所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段含有重链恒定区和轻链恒定区的至少之一，所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于人源抗体、灵长目源抗体和鼠源抗体或其突变体中的至少之一。The antibody or antigen-binding fragment according to claim 1, characterized in that the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody and a mouse antibody or a mutant thereof.
- 根据权利要求11所述的抗体或抗原结合片段，其特征在于，所述轻链恒定区和重链恒定区均来自于鼠源IgG抗体或其突变体或人源IgG抗体或其突变体。The antibody or antigen-binding fragment according to claim 11, characterized in that the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.
- 根据权利要求11所述的抗体或抗原结合片段，其特征在于，所述轻链恒定区和重链恒定区均来自于鼠源IgG1抗体或其突变体或人源IgG1抗体或其突变体。The antibody or antigen-binding fragment according to claim 11, characterized in that the light chain constant region and the heavy chain constant region are both derived from a murine IgG1 antibody or a mutant thereof or a human IgG1 antibody or a mutant thereof.
- 根据权利要求11所述的抗体或抗原结合片段，其特征在于，所述抗体具有如SEQ ID NO:29或33所示氨基酸序列的重链恒定区和/或如SEQ ID NO:30或34所示氨基酸序列的轻链恒定区。The antibody or antigen-binding fragment according to claim 11, characterized in that the antibody has a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 29 or 33 and/or a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 30 or 34.
- 根据权利要求11所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段具有SEQ ID NO:35、37和39任一项所示氨基酸序列的重链和具有SEQ ID NO:36、38和40任一项所示氨基酸序列的轻链。The antibody or antigen-binding fragment according to claim 11, characterized in that the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence shown in any one of SEQ ID NO: 35, 37 and 39, and a light chain with an amino acid sequence shown in any one of SEQ ID NO: 36, 38 and 40.
- 根据权利要求11所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段具有SEQ ID NO:35所示 氨基酸序列的重链和SEQ ID NO:36所示氨基酸序列的轻链；The antibody or antigen-binding fragment according to claim 11, characterized in that the antibody or antigen-binding fragment has a residue shown in SEQ ID NO: 35. The heavy chain of the amino acid sequence shown in SEQ ID NO:36 and the light chain of the amino acid sequence shown in SEQ ID NO:37;所述抗体或抗原结合片段具有SEQ ID NO:37所示氨基酸序列的重链和SEQ ID NO:38所示氨基酸序列的轻链；或The antibody or antigen-binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO:37 and a light chain with the amino acid sequence shown in SEQ ID NO:38; or所述抗体或抗原结合片段具有SEQ ID NO:39所示氨基酸序列的重链和SEQ ID NO:40所示氨基酸序列的轻链。The antibody or antigen-binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO:39 and a light chain with the amino acid sequence shown in SEQ ID NO:40.
- 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体或抗原结合片段包括单抗或多抗。The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody.
- 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述单抗包括全长抗体、Fv、单链抗体、Fab、单域抗体以及最小识别单位中的至少之一。The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the monoclonal antibody comprises at least one of a full-length antibody, Fv, a single-chain antibody, Fab, a single-domain antibody and a minimum recognition unit.
- 一种双抗，其特征在于，包括：A dual antibody, characterized by comprising:第一结合区，所述第一结合区包含权利要求1～13任一项所述的抗体或抗原结合片段；和A first binding region, the first binding region comprising the antibody or antigen-binding fragment of any one of claims 1 to 13; and第二结合区，所述第二结合区具有BCMA或B7H6结合活性。The second binding region has BCMA or B7H6 binding activity.
- 根据权利要求19所述的双抗，其特征在于，所述双抗包括对称双抗或不对称双抗。The bispecific antibody according to claim 19 is characterized in that the bispecific antibody comprises a symmetric bispecific antibody or an asymmetric bispecific antibody.
- 根据权利要求19所述的双抗，其特征在于，所述双抗为非对称双抗。The bispecific antibody according to claim 19 is characterized in that the bispecific antibody is an asymmetric bispecific antibody.
- 根据权利要求19所述的双抗，其特征在于，所述抗体或抗原结合片段为抗CD3单链抗体。The bispecific antibody according to claim 19, characterized in that the antibody or antigen-binding fragment is an anti-CD3 single-chain antibody.
- 根据权利要求19所述的双抗，其特征在于，所述第二结合区包括具有BCMA或B7H6结合活性的全长抗体、Fv、单链抗体、Fab、单域抗体以及最小识别单位中的至少之一。The bispecific antibody according to claim 19, characterized in that the second binding region comprises at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody and minimum recognition unit having BCMA or B7H6 binding activity.
- 根据权利要求23所述的双抗，其特征在于，所述第二结合区包括抗BCMA单链抗体或抗B7H6单链抗体。The bispecific antibody according to claim 23, characterized in that the second binding region comprises an anti-BCMA single-chain antibody or an anti-B7H6 single-chain antibody.
- 根据权利要求22所述的双抗，其特征在于，所述抗CD3单链抗体包括抗CD3抗体轻链可变区和抗CD3抗体重链可变区，所述抗CD3抗体重链可变区具有SEQ ID NO:23或27所示的氨基酸序列，所述抗CD3抗体轻链可变区具有如SEQ ID NO:24或28所示的氨基酸序列。The bispecific antibody according to claim 22 is characterized in that the anti-CD3 single-chain antibody comprises an anti-CD3 antibody light chain variable region and an anti-CD3 antibody heavy chain variable region, the anti-CD3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 23 or 27, and the anti-CD3 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 24 or 28.
- 根据权利要求22所述的双抗，其特征在于，所述抗CD3单链抗体进一步包括连接肽1，其中，所述连接肽1的N端与所述抗CD3抗体重链可变区的C端相连，所述连接肽1的C端与所述抗CD3抗体轻链可变区的N端相连；或所述连接肽1的N端与所述抗CD3抗体轻链可变区的C端相连，所述连接肽1的C端与所述抗CD3抗体重链可变区的N端相连。The bispecific antibody according to claim 22 is characterized in that the anti-CD3 single-chain antibody further comprises a connecting peptide 1, wherein the N-terminus of the connecting peptide 1 is connected to the C-terminus of the heavy chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the light chain variable region of the anti-CD3 antibody; or the N-terminus of the connecting peptide 1 is connected to the C-terminus of the light chain variable region of the anti-CD3 antibody, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the heavy chain variable region of the anti-CD3 antibody.
- 根据权利要求24所述的双抗，其特征在于，所述抗BCMA单链抗体包括抗BCMA抗体轻链可变区和抗BCMA抗体重链可变区，所述抗BCMA抗体轻链可变区具有SEQ ID NO:42所示的氨基酸序列，所述抗BCMA抗体重链可变区具有如SEQ ID NO:61所示的氨基酸序列。The bispecific antibody according to claim 24 is characterized in that the anti-BCMA single-chain antibody comprises an anti-BCMA antibody light chain variable region and an anti-BCMA antibody heavy chain variable region, the anti-BCMA antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:42, and the anti-BCMA antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:61.
- 根据权利要求27所述的双抗，其特征在于，所述抗BCMA单链抗体进一步包括连接肽2，其中，所述连接肽2的N端与所述抗BCMA抗体重链可变区的C端相连，所述连接肽2的C端与所述抗BCMA抗体轻链可变区的N端相连；或所述连接肽2的N端与所述抗BCMA抗体轻链可变区的C端相连，所述连接肽2的C端与所述抗BCMA抗体重链可变区的N端相连。The bispecific antibody according to claim 27 is characterized in that the anti-BCMA single-chain antibody further comprises a connecting peptide 2, wherein the N-terminus of the connecting peptide 2 is connected to the C-terminus of the anti-BCMA antibody heavy chain variable region, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the anti-BCMA antibody light chain variable region; or the N-terminus of the connecting peptide 2 is connected to the C-terminus of the anti-BCMA antibody light chain variable region, and the C-terminus of the connecting peptide 2 is connected to the N-terminus of the anti-BCMA antibody heavy chain variable region.
- 根据权利要求24所述的双抗，其特征在于，所述抗B7H6单链抗体包括抗B7H6抗体轻链可变区和抗B7H6抗体重链可变区，所述抗B7H6抗体重链可变区具有SEQ ID NO:63所示的氨基酸序列，所述抗B7H6抗体轻链可变区具有如SEQ ID NO:62所示的氨基酸序列。The bispecific antibody according to claim 24 is characterized in that the anti-B7H6 single-chain antibody comprises an anti-B7H6 antibody light chain variable region and an anti-B7H6 antibody heavy chain variable region, the anti-B7H6 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:63, and the anti-B7H6 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:62.
- 根据权利要求29所述的双抗，其特征在于，所述抗B7H6单链抗体进一步包括连接肽3，其中，所述连接肽3的N端与所述抗B7H6抗体重链可变区的C端相连，所述连接肽3的C端与所述抗B7H6抗体轻链可变区的N端相连；或所述连接肽3的N端与所述抗B7H6抗体轻链可变区的C端相连，所述连接肽3的C端与所述抗B7H6抗体重链可变区的N端相连。The bispecific antibody according to claim 29 is characterized in that the anti-B7H6 single-chain antibody further comprises a connecting peptide 3, wherein the N-terminus of the connecting peptide 3 is connected to the C-terminus of the heavy chain variable region of the anti-B7H6 antibody, and the C-terminus of the connecting peptide 3 is connected to the N-terminus of the light chain variable region of the anti-B7H6 antibody; or the N-terminus of the connecting peptide 3 is connected to the C-terminus of the light chain variable region of the anti-B7H6 antibody, and the C-terminus of the connecting peptide 3 is connected to the N-terminus of the heavy chain variable region of the anti-B7H6 antibody.
- 根据权利要求26所述的双抗，其特征在于，所述连接肽1具有氨基酸序列(GGS)n，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。The bispecific antibody according to claim 26 is characterized in that the connecting peptide 1 has an amino acid sequence (GGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
- 根据权利要求28或30所述的双抗，其特征在于，所述连接肽2和连接肽3具有氨基酸序列(GGGGS)n，其中n为大于或等于1的整数，优选为1、2、3、4、5、6、7、8、9或10。The bispecific antibody according to claim 28 or 30 is characterized in that the connecting peptide 2 and the connecting peptide 3 have an amino acid sequence (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
- 根据权利要求31所述的双抗，其特征在于，所述连接肽1具有SEQ ID NO:48所示的氨基酸序列。The bispecific antibody according to claim 31 is characterized in that the connecting peptide 1 has the amino acid sequence shown in SEQ ID NO:48.
- 根据权利要求32所述的双抗，其特征在于，所述连接肽2和连接肽3具有SEQ ID NO:41所示的氨基酸序列。The bispecific antibody according to claim 32 is characterized in that the connecting peptide 2 and the connecting peptide 3 have the amino acid sequence shown in SEQ ID NO:41.
- 根据权利要求22所述的双抗，其特征在于，所述抗CD3单链抗体具有如SEQ ID NO:45所示的氨基酸序列。The bispecific antibody according to claim 22 is characterized in that the anti-CD3 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:45.
- 根据权利要求24所述的双抗，其特征在于，所述抗BCMA单链抗体具有如SEQ ID NO:46所示的氨基酸序列；或The bispecific antibody according to claim 24, characterized in that the anti-BCMA single-chain antibody has an amino acid sequence as shown in SEQ ID NO: 46; or所述抗B7H6单链抗体具有如SEQ ID NO:47所示的氨基酸序列。 The anti-B7H6 single-chain antibody has the amino acid sequence shown in SEQ ID NO:47.
- 根据权利要求19所述的双抗，其特征在于，所述第一抗原结合区进一步包括第一Fc肽段，所述第一Fc肽段的N端与所述抗体或抗原结合片段的C端相连。The bispecific antibody according to claim 19 is characterized in that the first antigen binding region further comprises a first Fc peptide segment, and the N-terminus of the first Fc peptide segment is connected to the C-terminus of the antibody or antigen binding fragment.
- 根据权利要求19所述的双抗，其特征在于，所述第二抗原结合区进一步包括第二Fc肽段，所述第二Fc肽段的N端与所述抗BCMA单链抗体或所述抗B7H6单链抗体的C端相连。The bispecific antibody according to claim 19, characterized in that the second antigen binding region further comprises a second Fc peptide segment, and the N-terminus of the second Fc peptide segment is connected to the C-terminus of the anti-BCMA single-chain antibody or the anti-B7H6 single-chain antibody.
- 根据权利要求37所述的双抗，其特征在于，所述第一Fc肽段具有SEQ ID NO:49所示的氨基酸序列。The bispecific antibody according to claim 37 is characterized in that the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO:49.
- 根据权利要求38所述的双抗，其特征在于，所述第二Fc肽段具有SEQ ID NO:50所示的氨基酸序列。The bispecific antibody according to claim 38 is characterized in that the second Fc peptide segment has the amino acid sequence shown in SEQ ID NO:50.
- 根据权利要求37或38所述的双抗，其特征在于，所述第一Fc肽段和所述第二Fc肽段通过knob-into-hole结构进行连接。The bispecific antibody according to claim 37 or 38, characterized in that the first Fc peptide segment and the second Fc peptide segment are connected via a knob-into-hole structure.
- 根据权利要求37或38所述的双抗，其特征在于，所述第一抗原结合区具有SEQ ID NO:51所示的氨基酸序列，所述第二抗原结合区具有SEQ ID NO:52或53所示的氨基酸序列。The bispecific antibody according to claim 37 or 38 is characterized in that the first antigen binding region has the amino acid sequence shown in SEQ ID NO:51, and the second antigen binding region has the amino acid sequence shown in SEQ ID NO:52 or 53.
- 一种核酸分子，其特征在于，所述核酸分子编码权利要求1～18任一项所述的抗体或抗原结合片段或权利要求19～42任一项所述的双抗。A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the antibody or antigen-binding fragment according to any one of claims 1 to 18 or the bispecific antibody according to any one of claims 19 to 42.
- 一种表达载体，其特征在于，携带权利要求43所述的核酸分子。An expression vector, characterized in that it carries the nucleic acid molecule described in claim 43.
- 一种重组细胞，其特征在于，携带权利要求43所述的核酸分子或权利要求44所述的表达载体或能够表达权利要求1～18任一项所述的抗体或抗原结合片段或权利要求19所述的双抗。A recombinant cell, characterized in that it carries the nucleic acid molecule of claim 43 or the expression vector of claim 44 or is capable of expressing the antibody or antigen-binding fragment of any one of claims 1 to 18 or the bispecific antibody of claim 19.
- 根据权利要求45所述的重组细胞，其特征在于，所述重组细胞是通过将权利要求44所述的表达载体导入至宿主细胞中而获得的。The recombinant cell according to claim 45 is characterized in that the recombinant cell is obtained by introducing the expression vector according to claim 44 into a host cell.
- 根据权利要求46所述的重组细胞，其特征在于，所述重组细胞为真核细胞。The recombinant cell according to claim 46, characterized in that the recombinant cell is a eukaryotic cell.
- 根据权利要求47所述的重组细胞，其特征在于，所述重组细胞为哺乳动物细胞。The recombinant cell according to claim 47, characterized in that the recombinant cell is a mammalian cell.
- 一种组合物，其特征在于，包括权利要求1～18任一项所述的抗体或抗原结合片段、权利要求19～42任一项所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞。A composition, characterized in that it comprises the antibody or antigen-binding fragment according to any one of claims 1 to 18, the bispecific antibody according to any one of claims 19 to 42, the nucleic acid molecule according to claim 43, the expression vector according to claim 44, or the recombinant cell according to any one of claims 45 to 48.
- 一种药物，其特征在于，包括权利要求1～18任一项所述的抗体或抗原结合片段、权利要求19～42任一项所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞或权利要求49所述的组合物。A drug, characterized in that it comprises the antibody or antigen-binding fragment according to any one of claims 1 to 18, the bispecific antibody according to any one of claims 19 to 42, the nucleic acid molecule according to claim 43, the expression vector according to claim 44, or the recombinant cell according to any one of claims 45 to 48, or the composition according to claim 49.
- 一种试剂盒，其特征在于，所述试剂盒含有包括权利要求1～18任一项所述的抗体或抗原结合片段、权利要求19～42任一项所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞。A kit, characterized in that the kit contains the antibody or antigen-binding fragment according to any one of claims 1 to 18, the bispecific antibody according to any one of claims 19 to 42, the nucleic acid molecule according to claim 43, the expression vector according to claim 44, or the recombinant cell according to any one of claims 45 to 48.
- 权利要求1～18任一项所述的抗体或抗原结合片段、权利要求43所述的核酸分子、权利要求44所述的表达载体、权利要求45～48任一项所述的重组细胞或权利要求49所述的组合物在制备药物中的用途，所述药物用于预防和/或治疗CD3介导的相关疾病。Use of the antibody or antigen-binding fragment of any one of claims 1 to 18, the nucleic acid molecule of claim 43, the expression vector of claim 44, the recombinant cell of any one of claims 45 to 48, or the composition of claim 49 in the preparation of a medicament for preventing and/or treating CD3-mediated related diseases.
- 根据权利要求52所述的用途，其特征在于，所述CD3介导的相关疾病包括自身免疫性疾病。The use according to claim 52 is characterized in that the CD3-mediated related diseases include autoimmune diseases.
- 根据权利要求53所述的用途，其特征在于，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。The use according to claim 53 is characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- 权利要求19～42任一项所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体、权利要求45～48任一项所述的重组细胞或权利要求49所述的组合物在制备药物中的用途，所述药物用于预防和/或治疗CD3和BCMA，或者CD3和B7H6介导的相关疾病。Use of the bispecific antibody according to any one of claims 19 to 42, the nucleic acid molecule according to claim 43, the expression vector according to claim 44, the recombinant cell according to any one of claims 45 to 48, or the composition according to claim 49 in the preparation of a drug for preventing and/or treating CD3 and BCMA, or CD3 and B7H6-mediated related diseases.
- 根据权利要求55所述的用途，其特征在于，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。The use according to claim 55 is characterized in that the related diseases mediated by CD3 and BCMA, or CD3 and B7H6 include cancer.
- 根据权利要求56所述的用途，其特征在于，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。The use according to claim 56 is characterized in that the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- 权利要求1～18任一项所述的抗体或抗原结合片段、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞在制备试剂盒中的用途，所述试剂盒用于检测CD3。Use of the antibody or antigen-binding fragment of any one of claims 1 to 18, the nucleic acid molecule of claim 43, the expression vector of claim 44, or the recombinant cell of any one of claims 45 to 48 in the preparation of a kit for detecting CD3.
- 权利要求19～42任一项所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞在制备试剂盒中的用途，所述试剂盒用于检测CD3和/或BCMA，或者，CD3和/或B7H6。 Use of the bispecific antibody according to any one of claims 19 to 42, the nucleic acid molecule according to claim 43, the expression vector according to claim 44 or the recombinant cell according to any one of claims 45 to 48 in the preparation of a kit for detecting CD3 and/or BCMA, or CD3 and/or B7H6.
- 一种治疗或预防CD3，或者CD3和BCMA，或者CD3和B7H6介导的相关疾病的方法，其特征在于，包括：向受试者施用权利要求1～18任一项所述的抗体或抗原结合片段、权利要求19～42所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体、权利要求45～48任一项所述的重组细胞、权利要求49所述的组合物或权利要求50所述的药物中的至少之一。A method for treating or preventing CD3, or CD3 and BCMA, or CD3 and B7H6-mediated related diseases, characterized in that it comprises: administering to a subject at least one of the antibody or antigen-binding fragment of any one of claims 1 to 18, the bispecific antibody of claims 19 to 42, the nucleic acid molecule of claim 43, the expression vector of claim 44, the recombinant cell of any one of claims 45 to 48, the composition of claim 49, or the drug of claim 50.
- 根据权利要求60所述的方法，其特征在于，所述CD3介导的相关疾病包括自身免疫性疾病。The method according to claim 60, characterized in that the CD3-mediated related diseases include autoimmune diseases.
- 根据权利要求61所述的方法，其特征在于，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。The method according to claim 61 is characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- 根据权利要求60所述的方法，其特征在于，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。The method according to claim 60, characterized in that the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- 根据权利要求63所述的方法，其特征在于，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。The method of claim 63, wherein the cancer comprises at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- 一种诊断CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6介导的相关疾病的方法，其特征在于，包括使用权利要求1～18任一项所述的抗体或抗原结合片段、权利要求19～42所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞中的至少之一对待测样品中的CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6进行检测，确定所述待测样品中CD3，或者CD3和/或BCMA，或者，CD3和/或B7H6的含量。A method for diagnosing CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases, characterized in that it comprises using at least one of the antibody or antigen-binding fragment described in any one of claims 1 to 18, the bispecific antibody described in claims 19 to 42, the nucleic acid molecule described in claim 43, the expression vector described in claim 44, or the recombinant cell described in any one of claims 45 to 48 to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a sample to be tested, and determining the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested.
- 根据权利要求65所述的方法，其特征在于，所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量不低于患病的最低标准是待测样品来源于患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6引起的相关疾病的患者的指示。The method according to claim 65 is characterized in that the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is not lower than the minimum standard for the disease, which is an indication that the test sample is derived from a patient suffering from a related disease caused by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- 根据权利要求65所述的方法，其特征在于，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。The method according to claim 65 is characterized in that the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces and urine.
- 根据权利要求65所述的方法，其特征在于，所述CD3介导的相关疾病包括自身免疫性疾病。The method according to claim 65, characterized in that the CD3-mediated related diseases include autoimmune diseases.
- 根据权利要求68所述的方法，其特征在于，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。The method according to claim 68, characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- 根据权利要求65所述的方法，其特征在于，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。The method according to claim 65, characterized in that the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- 根据权利要求70所述的方法，其特征在于，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。The method of claim 70, wherein the cancer comprises at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- 一种对CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病进行分期的方法，其特征在于，包括：使用权利要求1～18任一项所述的抗体或抗原结合片段、权利要求19～42所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞中的至少之一对待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6进行检测，基于所述CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的检测结果，确定所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量。A method for staging related diseases mediated by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, characterized in that it comprises: using at least one of the antibody or antigen-binding fragment described in any one of claims 1 to 18, the bispecific antibody described in claims 19 to 42, the nucleic acid molecule described in claim 43, the expression vector described in claim 44, or the recombinant cell described in any one of claims 45 to 48 to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a sample to be tested, and determining the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6.
- 根据权利要求72所述的方法，其特征在于，所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量不低于肿瘤IV期患病的标准水平是待测样品来源于患有肿瘤IV期的患者的指示；所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量位于肿瘤IV期和III期患病的标准水平之间是待测样品来源于患有肿瘤III期的患者的指示；所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量处于肿瘤III期和II期患病的标准水平之间是待测样品来源于患有肿瘤II期的患者的指示；所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量处于I期和II期患病的标准水平之间是待测样品来源于患有肿瘤I期的患者的指示。The method according to claim 72 is characterized in that the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is not lower than the standard level for tumor stage IV, which is an indication that the test sample is derived from a patient with tumor stage IV; the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is between the standard levels for tumor stage IV and stage III, which is an indication that the test sample is derived from a patient with tumor stage III; the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is between the standard levels for tumor stage III and stage II, which is an indication that the test sample is derived from a patient with tumor stage II; the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample is between the standard levels for tumor stage I and stage II, which is an indication that the test sample is derived from a patient with tumor stage I.
- 根据权利要求72所述的方法，其特征在于，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。The method according to claim 72 is characterized in that the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces and urine.
- 根据权利要求72所述的方法，其特征在于，所述CD3介导的相关疾病包括自身免疫性疾病。 The method according to claim 72 is characterized in that the CD3-mediated related diseases include autoimmune diseases.
- 根据权利要求75所述的方法，其特征在于，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。The method according to claim 75, characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- 根据权利要求72所述的方法，其特征在于，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。The method according to claim 72 is characterized in that the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- 根据权利要求77所述的方法，其特征在于，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。The method of claim 77, wherein the cancer comprises at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
- 一种评估CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病预后的方法，其特征在于，包括使用权利要求1～18任一项所述的抗体或抗原结合片段、权利要求19～42所述的双抗、权利要求43所述的核酸分子、权利要求44所述的表达载体或权利要求45～48任一项所述的重组细胞中的至少之一对待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6进行检测，基于所述CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的检测结果，确定所述待测样品中CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量。A method for evaluating the prognosis of related diseases mediated by CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, characterized in that it comprises using at least one of the antibody or antigen-binding fragment described in any one of claims 1 to 18, the bispecific antibody described in claims 19 to 42, the nucleic acid molecule described in claim 43, the expression vector described in claim 44, or the recombinant cell described in any one of claims 45 to 48 to detect CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in a sample to be tested, and based on the detection result of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6, determining the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the sample to be tested.
- 根据权利要求79所述的方法，其特征在于，所述待测样品来源于治疗前或治疗后的患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的患者。The method according to claim 79 is characterized in that the sample to be tested is derived from a patient suffering from CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases before or after treatment.
- 根据权利要求79所述的方法，其特征在于，所述待测样品包括下列中的至少之一：血液、唾液、汗液、组织、细胞、血液、血清、血浆、粪便和尿液。The method according to claim 79 is characterized in that the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces and urine.
- 根据权利要求79所述的方法，其特征在于，基于所述治疗前或治疗后的患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的患者的待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量，确定CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的预后效果。The method according to claim 79 is characterized in that the prognostic effect of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases is determined based on the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample of the patient suffering from CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related diseases before or after the treatment.
- 根据权利要求82所述的方法，其特征在于，治疗后的患有CD3，或者CD3和/或BCMA，或者CD3和/或B7H6介导的相关疾病的患者的待测样品中的CD3，或者CD3和/或BCMA，或者CD3和/或B7H6的含量下降是患者预后良好的指示。The method according to claim 82 is characterized in that a decrease in the content of CD3, or CD3 and/or BCMA, or CD3 and/or B7H6 in the test sample of a patient suffering from a CD3, or CD3 and/or BCMA, or CD3 and/or B7H6-mediated related disease after treatment is an indication of a good prognosis for the patient.
- 根据权利要求79所述的方法，其特征在于，所述CD3介导的相关疾病包括自身免疫性疾病。The method according to claim 79 is characterized in that the CD3-mediated related diseases include autoimmune diseases.
- 根据权利要求84所述的方法，其特征在于，所述自身免疫性疾病包括下列中的至少之一：系统性红斑狼疮、类风湿性关节炎、系统性血管炎、硬皮病、皮肌炎、自身免疫性溶血性贫血、甲状腺自身免疫病、溃疡性结肠炎、慢性淋巴细胞性甲状腺炎、甲状腺功能亢进、胰岛素依赖型糖尿病、重症肌无力、溃疡性结肠炎、恶性贫血伴慢性萎缩性胃炎、肺出血肾炎综合征、寻常天疱疮、类天疱疮、原发性胆汁性肝硬化、多发性脑脊髓硬化症和急性特发性多神经炎。The method according to claim 84 is characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis and acute idiopathic polyneuritis.
- 根据权利要求79所述的方法，其特征在于，所述CD3和BCMA，或者CD3和B7H6介导的相关疾病包括癌症。The method according to claim 79 is characterized in that the CD3 and BCMA, or CD3 and B7H6-mediated related diseases include cancer.
- 根据权利要求86所述的方法，其特征在于，所述癌症包括下列中的至少之一：多发性骨髓瘤、淋巴瘤、血管瘤、胃癌、肝癌、肺癌、乳腺癌、结直肠癌、鼻咽癌、膀胱癌、宫颈癌、前列腺癌、甲状腺癌、肾癌、食道癌、黑色素瘤、纤维肉瘤、星形细胞瘤、神经母细胞瘤和神经胶质瘤。 The method according to claim 86 is characterized in that the cancer includes at least one of the following: multiple myeloma, lymphoma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, astrocytoma, neuroblastoma and glioma.
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| CN114395044A (en) * | 2021-12-07 | 2022-04-26 | 合肥天港免疫药物有限公司 | Recombinant antibodies and uses thereof |
| CN114395047A (en) * | 2021-12-07 | 2022-04-26 | 合肥天港免疫药物有限公司 | Bispecific antibodies and uses thereof |
| WO2022135536A1 (en) * | 2020-12-25 | 2022-06-30 | 江苏先声药业有限公司 | Cd3 humanized antibody and application thereof |
| US20220204630A1 (en) * | 2019-05-30 | 2022-06-30 | Sunshine Lake Pharma Co., Ltd. | Anti-trka antibodies and uses thereof |
| WO2022170033A1 (en) * | 2021-02-04 | 2022-08-11 | The Trustees Of Dartmouth College | Engineered natural ligand-based car: directed evolution of the stress-receptor nkp30 |
| CN115611985A (en) * | 2022-10-27 | 2023-01-17 | 合肥天港免疫药物有限公司 | anti-CD 3 antibodies and uses thereof |
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| US20220204630A1 (en) * | 2019-05-30 | 2022-06-30 | Sunshine Lake Pharma Co., Ltd. | Anti-trka antibodies and uses thereof |
| WO2022135536A1 (en) * | 2020-12-25 | 2022-06-30 | 江苏先声药业有限公司 | Cd3 humanized antibody and application thereof |
| WO2022170033A1 (en) * | 2021-02-04 | 2022-08-11 | The Trustees Of Dartmouth College | Engineered natural ligand-based car: directed evolution of the stress-receptor nkp30 |
| CN114395044A (en) * | 2021-12-07 | 2022-04-26 | 合肥天港免疫药物有限公司 | Recombinant antibodies and uses thereof |
| CN114395047A (en) * | 2021-12-07 | 2022-04-26 | 合肥天港免疫药物有限公司 | Bispecific antibodies and uses thereof |
| CN115611985A (en) * | 2022-10-27 | 2023-01-17 | 合肥天港免疫药物有限公司 | anti-CD 3 antibodies and uses thereof |
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