WO2023179716A1 - 一种抗cd3抗体、其制备方法及应用 - Google Patents

一种抗cd3抗体、其制备方法及应用 Download PDF

Info

Publication number
WO2023179716A1
WO2023179716A1 PCT/CN2023/083410 CN2023083410W WO2023179716A1 WO 2023179716 A1 WO2023179716 A1 WO 2023179716A1 CN 2023083410 W CN2023083410 W CN 2023083410W WO 2023179716 A1 WO2023179716 A1 WO 2023179716A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
antibody
seq
sequence shown
Prior art date
Application number
PCT/CN2023/083410
Other languages
English (en)
French (fr)
Inventor
杨欣秀
夏凯文
曹晓丹
邓俗俊
Original Assignee
上海济煜医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海济煜医药科技有限公司 filed Critical 上海济煜医药科技有限公司
Publication of WO2023179716A1 publication Critical patent/WO2023179716A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention relates to the field of antibodies, specifically to an anti-CD3 antibody, its preparation method and application.
  • CD3 has four subunits, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ , which form three dimers: ⁇ , ⁇ and ⁇ respectively. They form a TCR-CD3 complex with TCR- ⁇ . There is an ITAM region (immunoreceptor tyrosine) at the intracellular end. Acid-activated motif), expressed on T cells. After the antigen-MHC complex binds to the TCR, it can activate T cells through CD3-mediated signaling and kill tumor cells or cells infected with foreign substances.
  • CD3 antibodies recognize all T cells and react with 70%-80% of human peripheral blood lymphocytes and 65%-85% of thymocytes.
  • T cells activated by CD3 antibodies are directed to the surrounding tumor cells. The two cells contact and form synapses, triggering the activation of the T cell receptor (TCR) signaling pathway. Granzyme expression and release then cause perforation of the tumor cell membrane, leading to the latter's death. Lysis and apoptosis.
  • the activation of the TCR signaling pathway also causes the expression and release of a series of cytokines. For example, the release of IL-2 feedback stimulates the proliferation of T cells and amplifies the immune killing effect.
  • TCR T cell receptor
  • Jurkat cells are human leukemic T lymphocytes that express the CD3/TCR complex.
  • the engineered cell line developed from them and overexpresses the reporter gene element can confirm whether Jurkat cells can be activated by CD3 by detecting the activation degree of the luciferase enzyme signal downstream of the reporter gene. Antibody activation.
  • the existing technology lacks anti-CD3 antibodies that can cross-react with monkey CD3, and the development convenience of antibodies that can only bind to human CD3 is low; at the same time, there is a lack of anti-CD3 antibodies with diverse binding activities and PBMC-activating activities, which is not conducive to anti-CD3 dual antibodies. Further research and development. In the current technology, although most antibodies bind to human CD3, they do not bind to primate monkey CD3, so primates cannot be used to evaluate the safety of antibody drugs.
  • the present invention provides an anti-CD3 antibody, a preparation method thereof, Pharmaceutical compositions containing the same, detection reagents containing the same, kits containing the same and applications thereof.
  • the anti-CD3 antibody of the present invention has high binding activity to human CD3, can directly activate signaling pathways downstream of CD3, activate T cells, has binding activity to monkey PBMC, and has cross-reactivity with monkey CD3;
  • Anti-CD3 antibodies can be used as an alternative to CD3-terminal antibodies of the CD3-TAA (tumor associated antigen) type bisAb.
  • the anti-CD3 antibody includes a light chain variable region (VL) and a heavy chain variable region (VH).
  • the light chain variable region includes LCDR1, LCDR2 and LCDR3, the LCDR1 includes the amino acid sequence shown in SEQ ID NO:1, 4 or 5, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:2, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:3
  • the amino acid sequence shown; the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, the HCDR1 includes the amino acid sequence shown in SEQ ID NO:6, and the HCDR2 includes the amino acid sequence shown in SEQ ID NO:7 Sequence, the HCDR3 includes the amino acid sequence shown in SEQ ID NO:8.
  • the CDR sequence is shown in Table 1 below:
  • the VL comprises the amino acid sequence shown in any one of SEQ ID NO: 10-15.
  • the VH comprises the amino acid sequence shown in any one of SEQ ID NO: 17-19.
  • the VL includes the amino acid sequence shown in SEQ ID NO: 10, and the VH includes the amino acid sequence shown in SEQ ID NO: 17; or, the VL includes the amino acid sequence shown in SEQ ID NO: 17
  • CDRs of antibodies can be defined in a variety of ways, such as Kabat's definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5th ed., U.S. National Institute of Health Research, Bethesda, MD (1991)) and Chothia definition rules based on the position of structural loop regions (see J Mol Biol 273:927-48, 1997).
  • the anti-CD3 antibody meets at least one of the following two items:
  • the anti-CD3 antibody is a full-length antibody, Fab, Fab', F(ab') 2 or Fv, and the Fv is preferably scFv;
  • the anti-CD3 antibody is a monoclonal antibody or a polyclonal antibody prepared from the anti-CD3 antibody described in item (1).
  • the anti-CD3 antibody is a full-length antibody, and the full-length antibody includes a light chain and a heavy chain;
  • the light chain includes a light chain constant region (CL), and the antibody light chain
  • the chain constant region is preferably a human antibody light chain constant region or a murine antibody light chain constant region, and the human antibody light chain constant region is more preferably a kappa subtype light chain constant region;
  • the heavy chain includes a heavy chain constant region (CH), the antibody heavy chain constant region is preferably a human or murine antibody heavy chain constant region, more preferably a heavy chain constant region of hIgG1, hIgG2, hIgG3 or hIgG4 subtype, further preferably a heavy chain of hIgG1 subtype constant region.
  • the human antibody light chain constant region includes the amino acid sequence shown in SEQ ID NO:20
  • the human antibody heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:21 The amino acid sequence shown.
  • the light chain includes the amino acid sequence shown in SEQ ID NO:23-28, and the heavy chain includes the amino acid sequence shown in any one of SEQ ID NO:30-32 .
  • the light chain includes the amino acid sequence shown in SEQ ID NO:23, and the heavy chain includes the amino acid sequence shown in SEQ ID NO:30; or, the light chain includes the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in SEQ ID NO: 27, and the heavy chain includes the amino acid sequence shown in SEQ ID NO: 32; or the light chain includes the amino acid sequence shown in SEQ ID NO: 27, and the heavy chain Comprises the amino acid sequence shown in SEQ ID NO:31; Or, the light chain includes the amino acid sequence shown in SEQ ID NO:26, and the heavy chain includes the amino acid sequence shown in SEQ ID NO:32; Or, the light chain includes the amino acid sequence shown in SEQ ID NO:26, and the heavy chain includes the amino acid sequence shown in SEQ ID NO:31; or, the light chain includes the amino acid sequence shown in SEQ ID NO:24 The amino acid sequence shown in SEQ ID NO:32, and the heavy chain includes the amino acid sequence shown in SEQ ID NO:32; or, the light chain includes the amino acid sequence shown
  • the amino acid sequence shown in SEQ ID NO:25, and the heavy chain includes the amino acid sequence shown in SEQ ID NO:32; or the light chain includes the amino acid sequence shown in SEQ ID NO:28, and the The heavy chain includes the amino acid sequence shown in SEQ ID NO:31.
  • the antibody or antigen-binding fragment thereof of the present invention contains 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the antibody or antigen-binding fragment thereof. or higher homology.
  • homology can be determined by sequence comparison and/or alignment using methods known in the art. For example, the sequence alignment algorithm BLAST or manual alignment can determine sequence identity or homology.
  • a certain aspect of the invention also provides an isolated nucleic acid encoding an anti-CD3 antibody according to the invention.
  • a certain aspect of the present invention also provides a recombinant expression vector comprising the isolated nucleic acid according to the present invention.
  • the recombinant expression vector includes a eukaryotic cell expression vector and/or a prokaryotic cell expression vector. More preferably, the eukaryotic cell expression vector is pcDNA3.4.
  • a certain aspect of the present invention also provides a transformant comprising the recombinant expression vector according to the present invention.
  • the host cell of the transformant is a prokaryotic cell and/or a eukaryotic cell
  • the prokaryotic cell is preferably an E.coli cell
  • the eukaryotic cell is preferably an Expi293F cell or a CHO cell.
  • a certain aspect of the present invention also provides a method for preparing the anti-CD3 antibody according to the present invention, which includes the following steps: cultivating the transformant according to the present invention, and obtaining the anti-CD3 antibody from the culture.
  • a certain aspect of the present invention also provides a pharmaceutical composition, which includes the anti-CD3 antibody according to the present invention, and a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable adjuvant.
  • a certain aspect of the present invention also provides a detection reagent, which contains the anti-CD3 antibody according to the present invention.
  • the detection reagent is in liquid dosage form, gas dosage form, solid dosage form or semi-solid dosage form.
  • the detection reagent further includes a secondary antibody, CD3 or a derivative thereof, such as an anti-human IgG antibody coupled to horseradish peroxidase and an anti-human IgG antibody coupled to biotin.
  • a certain aspect of the present invention also provides a pharmaceutical kit, which includes pharmaceutical kit A.
  • the pharmaceutical kit A contains the anti-CD3 antibody according to the present invention, the pharmaceutical composition according to the present invention and the pharmaceutical composition according to the present invention. one or more of the above detection reagents.
  • the kit also includes kit B, which contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations or Various.
  • kit B which contains other anti-tumor antibodies or pharmaceutical compositions containing the other anti-tumor antibodies, and/or consists of hormone preparations, targeted small molecule preparations or Various.
  • a certain aspect of the present invention also provides the anti-CD3 antibody according to the present invention, the pharmaceutical composition according to the present invention, the detection reagent according to the present invention and/or the pharmaceutical kit according to the present invention. Diagnose, prevent and/or treat CD3 Application in medicines for related diseases.
  • the CD3-related disease is a CD3-related tumor.
  • a certain aspect of the present invention also provides a method for detecting CD3 in a sample, which includes the step of contacting the sample with the anti-CD3 antibody according to the present invention or the detection reagent according to the present invention.
  • the method is for non-diagnostic and/or therapeutic purposes.
  • the sample is, for example, a blood sample (eg, whole blood sample and serum sample), a CD3-containing reagent, a CD3-containing antibody drug conjugate, or a CD3-containing chimeric antigen receptor cell.
  • the invention also provides an antibody combination, which includes the anti-CD3 antibody according to the invention.
  • the antibodies of the present invention can be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA technology, phage display technology, synthesis technology, or combinations of these technologies, or other techniques known in the art.
  • antibody molecules refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen-binding site that immunospecifically binds an antigen.
  • antibody encompasses not only intact antibody molecules, but also fragments of said antibodies and variants (including derivatives) of said antibodies and antibody fragments.
  • antibody molecule includes, for example, but is not limited to, single chain Fv (scFv), Fab fragment, Fab' fragment, F(ab') 2 , disulfide-linked Fv (sdFv), Fv, and intact Antibodies or full-length antibodies.
  • single chain Fv or “scFv” refers to a polypeptide comprising the VL domain of an antibody linked to the VH domain of the antibody.
  • Antibodies that immunospecifically bind CD3 can cross-react with other antigens. Preferably, the antibody that immunospecifically binds CD3 does not cross-react with other antigens.
  • Antibodies that immunospecifically bind CD3 can be identified, for example, by immunoassays or other methods known to those skilled in the art.
  • “Intact antibody” or “full-length antibody” refers to a protein containing two heavy chains (H) and two light chains (L) linked to each other by disulfide bonds, the protein containing: ( 1) in the case of a heavy chain, comprising a heavy chain variable region (herein abbreviated as "VH”) and a heavy chain constant region containing three domains CH1, CH2, CH3; and (2) in the case of a light chain, comprising The light chain variable region (herein abbreviated as "VL”) and the light chain constant region contain one domain CL.
  • VH and VL can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • VH and VL contain binding domains that interact with antigens.
  • the constant region of an antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and components of the classical complement system.
  • Antibodies of the invention include, but are not limited to, monospecific, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies (including, for example, the invention anti-Id antibodies) and epitope-binding fragments of any of the above antibodies.
  • the immunoglobulin molecules of the invention may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of an immunoglobulin.
  • the antibody of the present invention can be a monoclonal antibody or a polyclonal antibody, and the monoclonal antibody is preferably a mouse anti-human monoclonal antibody.
  • the term "anti-CD3 antibody” means that the antibody can specifically bind to CD3.
  • Specific binding generally refers to an interaction that is measurable and reproducible. For example, in the binding between an antigen and an antibody, the antibody binds to the epitope through its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope.
  • an antibody that specifically binds a target (which may be an epitope) is an antibody that binds this target with higher affinity, avidity, more readily, and/or for a longer duration than it binds other targets.
  • An antibody is said to "specifically bind" an antigen when it binds to an epitope more readily through its antigen-binding domain than it would to a random, unrelated epitope.
  • CD3 refers to CD3 protein.
  • the CD3 protein has four subunits: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ , which form three dimers of ⁇ , ⁇ and ⁇ respectively, and form a TCR-CD3 complex with TCR- ⁇ .
  • ITAM region immunoglobulin binding protein
  • Receptor tyrosine activation motif expressed on T cells. After the antigen-MHC complex binds to the TCR, it can activate T cells through CD3-mediated signaling and kill tumor cells or cells infected with foreign substances.
  • variable region generally refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen.
  • the heavy and light chain variable regions may be referred to as “VH” and “VL” respectively.
  • VH and VL variable regions
  • These domains are typically the most variable parts of the antibody and contain the antigen-binding site.
  • HVR hypervariable regions
  • the decision regions (CDR) are LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 respectively.
  • the more highly conserved portions of the variable domains are called framework regions (FR).
  • variable domains of the natural heavy and light chains each contain four FR regions: HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4, most of which adopt a ⁇ -sheet configuration and pass through three CDR structural loop regions. connect.
  • the CDRs in each chain are held closely together by the FR region and, together with the CDRs from the other chain, form the antigen-binding site of the antibody.
  • CDRs of antibodies can be defined through a variety of methods, such as Kabat definition rules based on sequence variability, Chothia definition rules based on the position of the structural loop region, and concepts based on IMGT ontology (IMGT-ONTOLOGY).
  • the amino acid sequences of VL and VH of the anti-CD3 antibody are encoded according to Chothia coding rules
  • LCDR1-3 and HCDR1-3 of the anti-CD3 antibody are defined according to Chothia.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the antibodies in the population are identical except for the possible presence of small amounts of natural mutations. Monoclonal antibodies are usually highly specific for a single antigenic site.
  • the term "fully human antibody” or “human antibody” generally means that all parts of the antibody (including the variable region and constant region of the antibody) are encoded by genes of human origin. Fully human antibodies can greatly reduce the immune side reactions caused by heterologous antibodies to the human body. Methods for obtaining fully human antibodies in this field include phage display technology, transgenic mouse technology, ribosome display technology, etc.
  • isolated generally refers to being substantially free of components (eg, viruses, nucleic acids or proteins) that normally accompany or interact with it in its naturally occurring environment.
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogues of any length, isolated from their natural environment or artificially synthesized.
  • polynucleotide or “nucleic acid” are used interchangeably in the present invention to refer to a chain of nucleotides of any length, and includes DNA and RNA.
  • the nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate capable of being incorporated into the strand by DNA or RNA polymerase.
  • expression vector generally refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked and replicating itself in a suitable host.
  • Expression vectors include any genetic element, such as plasmids, transposons, artificial chromosomes, viruses, etc., that when combined with appropriate control elements is capable of self-replication and transfer of genetic sequences to or between hosts.
  • the expression vectors may include vectors primarily used for insertion of DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and vectors primarily used for expression of transcription and/or translation of DNA or RNA.
  • the expression vectors also include vectors having a variety of the above-mentioned functions.
  • the expression vector may also be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the expression vector can produce the desired expression product by culturing a host cell containing the expression vector.
  • expression vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA encapsulated in liposomes expression vectors and certain eukaryotic cells, such as producer cells.
  • host cell in the present invention may include cells into which exogenous nucleic acid has been introduced, including progeny of these cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cells and progeny derived therefrom, regardless of passage number.
  • the progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations.
  • the present invention includes mutant progeny that have the same function or biological activity as the cells screened or selected in the initially transformed cells.
  • K D is the dissociation rate constant (kdis, also known as “off-rate”). (koff)” or “kd”) to the binding rate constant (kon, also known as “binding rate (kon)” or “ka”).
  • Methods for determining association and dissociation rate constants are known in the art
  • Well-known methods include, but are not limited to, biofilm interference technology (BLI), radioimmunoassay (RIA), equilibrium dialysis, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (Co-IP), and Protein chip technology.
  • BBI biofilm interference technology
  • RIA radioimmunoassay
  • SPR surface plasmon resonance
  • FRET fluorescence resonance energy transfer
  • Co-IP co-immunoprecipitation
  • Protein chip technology Protein chip technology. The measured affinity for a particular protein-protein interaction can be different if measured under different conditions (eg, salt concentration, pH).
  • the "pharmaceutically acceptable adjuvant" described in the present invention includes, but is not limited to, one or a combination of surfactants, solution stabilizers, isotonic regulators or buffers.
  • surfactants include but are not limited to non-ionic surfactants such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or Tween 80); poloxamer (such as poloxamer 188); Triton; sodium lauryl sulfate (Tween 20 or Tween 80); SDS); sodium lauryl sulfate; tetradecyl or octadecyl sarcosine, etc., the amount added should minimize the tendency of granulation of the human Ang2-binding antibody or its antigen-binding fragment.
  • Solution stabilizers include, but are not limited to, sugars, such as reducing sugars or non-reducing sugars; amino acids, such as monosodium glutamate or histidine; alcohols, such as triols, propylene glycol or polyethylene glycol; solutions The stabilizer is added in an amount such that the final formulation remains stable for a period of time considered stable by those skilled in the art.
  • Isotonicity adjusting agents include, but are not limited to, sodium chloride, mannitol, or combinations thereof.
  • Buffers include, but are not limited to, Tris, histidine buffer, phosphate buffer, or combinations thereof.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the anti-CD3 antibodies provided by the invention also exhibit binding activity to monkey PBMC and have monkey CD3 cross-reactivity, which brings convenience to the safety research of the anti-research body and facilitates subsequent development; Moreover, the anti-CD3 antibody exhibits diverse binding and activating PBMC activities, which facilitates subsequent development and research of dual antibodies.
  • Figure 1 shows the binding curve of mouse monoclonal antibody and Jurkat cells.
  • Figure 2 shows the binding curve of mouse monoclonal antibody and monkey PBMC.
  • Figure 3 shows the results of hybridoma anti-CD3 antibody activating the Luciferase reporter gene in Jurkat NFAT-Luc cells.
  • Figure 4 shows the curve of hybridoma anti-CD3 antibody activating T cells and activating fresh CD4 + T cells to release IL-2.
  • Figure 5 is the binding curve of anti-CD3 chimeric antibody and Jurkat cells.
  • Figure 6 shows the results of anti-CD3 chimeric antibody activating the Luciferase reporter gene in Jurkat NFAT-Luc cells.
  • Figure 7 shows the curve of anti-CD3 chimeric antibody activating T cells and activating fresh CD4 + T cells to release IL-2.
  • Figures 8A and 8B are binding curves of humanized antibodies and Jurkat cells.
  • Figures 9A and 9B are binding curves of humanized antibodies and monkey PBMC.
  • Figures 10A and 10B show the results of humanized antibodies activating the Luciferase reporter gene in Jurkat NFAT-Luc cells.
  • Figures 11A and 11B show the curves of T cells activated by humanized antibodies and the release of IL-2 from fresh CD4 + T cells.
  • CD3 antigen including protein and cell lines overexpressing CD3 antigen
  • the fused cells are added to a 96-well cell culture plate and cultured in a CO 2 incubator for 9-10 days.
  • Flow cytometry was used to detect the binding activity of hybridoma supernatants in 96-well cell culture plates to Jurkat cells.
  • the cell clone corresponding to the positive hybridoma supernatant was transferred to a 24-well cell culture plate and continued to be cultured for 2-4 days.
  • the hybridoma supernatant in the 24-well cell culture plate was used to confirm the binding activity to Jurkat cells.
  • the monoclonal antibody light and heavy chain protein sequences are codon-optimized respectively, the codon-optimized DNA fragments are synthesized, and the synthesized gene fragments are cloned into the expression vector pcDNA3.4 (Life Technologies).
  • the double plasmids are co-transfected into Expi293F (ThermoFisher Scientific, A14527) or CHO-K1 cells (ECACC catalog no.85051005), and the antibody is transiently expressed according to the supplier's Expi293F or CHO-K1 expression system method.
  • the supernatant was centrifuged and purified through a proteinA column according to conventional methods.
  • FACS was used to identify the binding activity of mouse anti-human CD3 monoclonal antibody 10E1C11 to Jurkat cells and monkey PBMC cells.
  • Cultivate Jurkat cells (ATCC, TIB-152) in T175 cell culture flask to a density of 1E6/mL, take out the required number of cells, centrifuge at 300 ⁇ g for 5 minutes, discard the supernatant, and use FACS buffer (containing 2% FBS (PBS: ThermoFisher Scientific, 10099-141C; PBS: ThermoFisher Scientific, 10010023) was washed once, and then the cells were resuspended in FACS buffer to 2 ⁇ 10 cells per ml, and incubated at room temperature for 15 minutes.
  • FBS ThermoFisher Scientific, 10099-141C
  • PBS ThermoFisher Scientific, 10010023
  • the cell suspension was added into a U-shaped 96-well plate (Corning, 3798) at 50 ⁇ L/well. Dilute the monoclonal antibody in a gradient with FACS buffer, add the antibody dilution to the above 96-well plate, 50 ⁇ L/well, and incubate at 4°C for 1 hour. Centrifuge and discard the supernatant, wash the cells once with FACS buffer, add 100 ⁇ L of fluorescent (Alexa 488)-labeled secondary antibody (Thermo Fisher, A-11029) to each well, and incubate at 4°C for 1 hour.
  • fluorescent Alexa 488)-labeled secondary antibody
  • the binding curve of mouse monoclonal antibody 10E1C11 to Jurkat cells is shown in Figure 1.
  • the binding curve of mouse monoclonal antibody 10E1C11 to monkey PBMC is shown in Figure 2, showing that mouse monoclonal antibody 10E1C11 can bind to both monkey CD3 and human CD3. .
  • Each cycle includes the following steps: 1) regeneration of 10mM glycine solution at pH 1.7; 2) immersion in buffer for 60 seconds; 3) antibody solidified on the sensor for 35 seconds; 6) sensor immersion in buffer for 180 seconds; 7) antigen and antibody Binding, time 180s; 8) Dissociation of antigen and antibody, time 10 minutes; 9) Sensor regeneration.
  • Hybridoma anti-CD3 antibody activates Luciferase reporter gene in Jurkat NFAT-Luc cells
  • Jurkat NFAT-Luc cells are Jurkat cells containing a reporter gene system that contains NFAT elements to control the expression of its downstream luciferase gene. It has been confirmed that luciferase gene expression can be activated by CD3 antibodies.
  • Anti-CD3 antibody was coated on the plate, starting with an antibody coating concentration of 20 ⁇ g/mL, 2-fold gradient dilution, dilution to 11 points, and coating at 4°C overnight; the next day, Jurkat NFAT-Luc cells were collected, and the density was adjusted to 1 ⁇ 10 6 cells/mL (resuspend the cell pellet in 1640 (ThermoFisher Scientific, A10491) + 2% FBS), discard the anti-CD3 antibody, wash the plate twice with PBS, and add 100 ⁇ L Jurkat NFAT-Luc cells (1 ⁇ 10 5 cells/well), cultured at 37°C, 5% CO2 for 6 hours, pipet out 20 ⁇ L from each well into a white 96-well plate (Corning, 3917), and then add 50 ⁇ L Coelenterazine-utilizing luciferase detection medium (Invivogen, Cat. No.: rep- qlc1) to detect the luciferase activity in the sample.
  • H2M2690N is
  • Hybridoma anti-CD3 antibody activates fresh CD4 + T cells to release IL-2
  • Anti-CD3 antibody was plated, starting with an antibody coating concentration of 10 ⁇ g/mL, 5-fold gradient dilution, diluting 5 points, and coating at 4°C overnight; the next day, first collect fresh CD4 + T cells (ALLCELLS, PB009-2- C), adjust the density to 2 ⁇ 10 6 cells/mL (the cell pellet is resuspended in 1640+2% FBS), and then use the cell suspension to prepare anti-CD28 antibody (BD, Cat.
  • hybridoma anti-CD3 antibodies activating fresh CD4 + T cells to release IL-2 are shown in Figure 4, indicating that hybridoma anti-CD3 antibodies can activate CD4 + T cells to release IL-2.
  • the heavy chain variable region sequence of hybridoma anti-CD3 antibody 10E1C11 was fused to human IgG1 LALA D265S (SEQ ID NO:21), and the light chain variable region was fused to the human Kappa chain constant region (SEQ ID NO:20) (human constant The region sequence is shown in Table 6), and the CD3 chimeric antibody CAb10E1 was constructed.
  • FACS was used to identify the activity of anti-CD3 chimeric antibody CAb10E1 binding to Jurkat cells. Cultivate Jurkat cells in T175 cell culture flask to a density of 10 cells/mL, take out the required number of cells, centrifuge at 300 ⁇ g for 5 minutes, discard the supernatant, and wash with FACS buffer (PBS containing 2% FBS) Once, the cells were then resuspended in FACS buffer to 2 ⁇ 10 6 cells per ml. After incubation at room temperature for 15 minutes, the cell suspension was added to a U-shaped 96-well plate (Corning, 3798) at 50 ⁇ L/well.
  • FACS buffer PBS containing 2% FBS
  • mice were resuspended in 30 ⁇ L/well FACS buffer and the results were detected and analyzed using flow cytometry intellycite plus (Sartorius).
  • Mouse isotype control antibody purchased from Thermofisher, Cat. No. 02-6502
  • human isotype control antibody purchased from Solarbio, Cat. No. SP001.
  • Example 8 Anti-CD3 chimeric antibody activates Luciferase reporter gene in Jurkat NFAT-Luc cells
  • the activation activity of anti-CD3 chimeric antibodies was detected using reporter gene activation assay in Jurkat NFAT-Luc cells. Dilute the antibody to five concentrations, 5-fold gradient dilution, 100 ⁇ L/well, add it to a 96-well plate (Corning, 3599), and cover the plate at 4°C overnight. Collect Jurkat NFAT-Luc cells, resuspend the cells in RPMI 1640 (Thermo Fisher, A1049101) + 2% FBS medium, adjust the density to 1E6/mL; aspirate the humanized CD3 antibody in the 96-well plate, and wash with PBS for 2 After spreading, add 100 ⁇ L cells to each well (1E5 cells/well). Place the 96-well plate into the incubator.
  • Example 9 Anti-CD3 chimeric antibody activates fresh CD4 + T cells to release IL-2
  • Example 6 The same experimental method as in Example 6 was used to determine the function of the anti-CD3 chimeric antibody in activating fresh CD4 + T cells to release IL-2.
  • Mouse isotype control antibody purchased from Baiying, Cat. No. B115101.
  • the following optimized design of the light chain variable region is based on the human antibody germline gene sequence IGKV4-1*01, IGKV3-20 or IGKJ4*01 as the framework, and 002hzVK0 (10E1C11VK) as the light chain variable region sequence of the chimeric antibody for CDR Transplanted and revertant humanized sequences:
  • Optimized mutation sites S5T, S9D, A15L, K18R, V19A, M21I, S22N, S43P, T63S, N77S, V78L, V83L, L103V, S105I.
  • Optimized mutation sites S5T, S9D, A15L, K18R, V19A, M21I, S22N, N30aQ, S43P, T63S, N77S, V78L, V83L, L103V, S105I.
  • Optimized mutation sites D1E, M4L, S5T, S9G, S10T, A12S, V13L, A15P, K18R, V19A, M21L, N30aQ, S43A, K45R, V58I, T63S, N77R, V78L, Q79E, A80P, L83F, L103V, S105I .
  • Optimized mutation sites D1E, M4L, S5T, S9G, S10T, A12S, V13L, A15P, K18R, V19A, M21L, N30aS, S43A, K45R, V58I, T63S, N77R, V78L, Q79E, A80P, L83F, L103V, S105I .
  • Optimized mutation sites D1E, M4L, S5T, S9G, S10T, A12S, V13L, A15P, K18R, V19A, M21L, N30aS, S43A, K45R, V58I, T63S, N77R, V78L, Q79E, A80P, F83L, L103V, S105I .
  • the following optimized design of the heavy chain variable region is based on the human antibody germline gene sequences IGHV1-3*01 and IGHJ4*01 as the framework, and 002hzVH0 (10E1C11VH) as the heavy chain variable region sequence of the chimeric antibody for CDR transplantation and reverse mutation of humanized sequences.
  • Optimized mutation sites Q5V, L9A, L11V, V12K, A17S, M20V, T23K, A33Y, M34I, K38R, R40A, G44R, I48M, T57S, N60S, E61Q, K64Q, A65G, K66R, T67V, S68T, L69I, A71R ,K73T,S75A,L81E,N82aS,T83R,S87T,I89V,F91Y,A93V,T108L,L109V.
  • Optimized mutation sites Q5V, L9A, L11V, V12K, A17S, M20V, T23K, A33Y, M34I, K38R, R40A, G44R, I48M, T57S, N60S, E61Q, K64Q, A65G, K66R, T67V, S68T, L69I, R71A ,T73K,S75A,L81E,N82aS,T83R,S87T,I89V,F91Y,A93V,T108L,L109V.
  • the above humanized light chain variable region sequence is combined with the human Kappa chain constant region (SEQ ID NO: 20) to form a humanized antibody light chain, and the above humanized heavy chain variable region sequence is combined with human IgG1 LALA D265S (SEQ ID NO:21) combines humanized antibody heavy chains.
  • the constant region sequences are shown in Table 6.
  • the 002hzVH2 heavy chain variable region and the 002hzVK8 light chain variable region were combined with the human heavy chain constant region and the human light chain constant region respectively and then paired to form humanized antibody 002hz28; 002hzVH2 heavy chain variable region and light chain variable region 002hzVK9 respectively After being combined with the human heavy chain constant region and the human light chain constant region, the humanized antibody 002hz29 is formed; the 002hzVH2 heavy chain variable region and the 002hzVK9b light chain variable region are combined with the human heavy chain constant region and the human light chain constant region respectively.
  • Pair to form humanized antibody 002hz29b; 002hzVH3 heavy chain variable region and 002hzVK1b light chain variable region are combined with human heavy chain constant region and human light chain constant region respectively and then paired to form humanized antibody 002hz31b.
  • the 002hzVH3 heavy chain variable region and the 002hzVK2b light chain variable region were combined with the human heavy chain constant region and the human light chain constant region respectively and then paired to form humanized antibody 002hz32b.
  • the 002hzVH3 heavy chain variable region and the 002hzVK8 light chain variable region are combined with the human heavy chain constant region and the human light chain constant region respectively and then paired to form humanized antibody 002hz38; the 002hzVH3 heavy chain variable region and the 002hzVK9 light chain variable region are respectively It is combined with the human heavy chain constant region and the human light chain constant region to form humanized antibody 002hz39.
  • the full-length recombinant monoclonal antibody light and heavy chain protein sequences were codon-optimized respectively, the codon-optimized DNA fragment was synthesized, and the synthesized gene fragment was cloned into the expression vector pcDNA3.4 (Life Technologies).
  • Expression plasmid amplification and plasmid extraction (Qiagen, Plasmid Maxi Kit, Cat. No. 12362), double plasmids are co-transfected into Expi293F (ThermoFisher Scientific, A14527) or CHO-K1 cells (ECACC catalog no. 85051005), and the antibody is transiently transfected according to the supplier's Expi293F or CHO-K1 expression system method.
  • Expression the general process is as follows:
  • Cell recovery Use a 125 mL disposable sterile shake flask to thaw the frozen CHO cells in a 37°C water bath, and dilute the cells to 0.3 ⁇ 10 6 cells/mL, with a volume of 30 mL. 125rpm (19mm orbit), 8% CO 2 , 37°C, culture to about 4-6 ⁇ 10 6 cells/mL.
  • Transient transfection cells are passed to passage 3 or above, and the density is about 4-6 ⁇ 10 6 cells/mL one day before transfection. Dilute the cells to a density of 3-4 ⁇ 10 6 cells/mL, and culture overnight. Day 7-10 ⁇ 10 6 , dilute to 6 ⁇ 10 6 , with a transient volume of 100 mL (500mL shake flask) as an example: solution 1: dilute 100 ⁇ g plasmid with culture medium (4mL) and mix well; solution 2: dilute 320 ⁇ L transfection reagent with culture medium (3.68mL) and mix well; add solution 2 to solution 1 in, the total volume is 8 mL, mix gently and incubate at room temperature for 1-5 minutes, no more than 5 minutes, add the mixed transfection solution drop by drop to the cell solution, while shaking.
  • solution 1 dilute 100 ⁇ g plasmid with culture medium (4mL) and mix well
  • solution 2 dilute 320 ⁇ L transfection reagent with culture medium (3.68mL) and mix well
  • Protein expression (maximum titer): Add auxiliary materials Feed-16mL, enhancer-0.6mL, and culture at 32°C for 18-22 hours. On the fifth day, add auxiliary material Feed-16mL, 123 rpm, 5% CO 2 , culture at 32°C, and centrifuge to collect the supernatant on the 12th to 14th day.
  • Antibody purification Protein A affinity chromatography column purification: 1) Equilibrium chromatography column: 1 ⁇ PBS, flow rate 1mL/min, 20mL; 2) Sample loading: flow rate 1mL/min; 3) Impurity washing: 1 ⁇ PBS, flow rate 1mL/min, 20mL; 4) Elution: citric acid buffer (PH3.4), 1mL/min, collected in separate tubes, each tube is about 500 ⁇ L. Collect a total of 10 tubes, and use the NanoDrop instrument to read the absorbance value at 280nm; 5) Dialysis: suck the high-concentration protein into the dialysis bag and place it in a beaker with 1 ⁇ PBS for dialysis. 6) Collect antibodies for later use.
  • a CH1 sensor (Fortébio, Cat. No.: 18-5127) was used. The remaining experimental methods and steps were the same as in Example 4. The results of the two experiments are shown in Tables 9 and 10. Among them, OKT3 is the positive control (purchased from invitrogen, product number: 16-0037-85). SP34 positive control (prepared with reference to WO2015181098A1).
  • variable regions of SP34.1 and SP34 are the same, and the constant regions are human IgG1 L234A, L235A, and D265S; the variable regions of OKT3.1 and OKT3 are the same, and the constant regions are human IgG1 L234A, L235A, and D265S.
  • Example 12 Flow cytometry binding of humanized anti-CD3 antibody to Jurkat cells and monkey PBMC cells
  • FACS FACS was used to determine the activity of humanized anti-CD3 antibodies in binding to Jurkat cells. Cultivate Jurkat cells in T175 cell culture flask to a density of 1 ⁇ 10 6 cells/mL, take out the required number of cells, centrifuge at 300 ⁇ g for 5 minutes, discard the supernatant, and use FACS buffer (PBS containing 2% FBS) to ), wash once, and then resuspend the cells in FACS buffer to 2 ⁇ 10 6 cells per ml. After incubation at room temperature for 15 minutes, add the cell suspension to a U-shaped 96-well plate (Corning, 3798) at 50 ⁇ L/well. .
  • FACS buffer PBS containing 2% FBS
  • FACS buffer to dilute the humanized antibody in a gradient manner, add the antibody dilution to the above 96-well plate, 50 ⁇ L/well, and incubate at 4°C for 1 hour. Centrifuge and discard the supernatant, wash the cells once with FACS buffer, add 100 ⁇ L of fluorescent (Alexa 488)-labeled secondary antibody (Thermo Fisher, A-11013) to each well, and incubate at 4°C for 1 hour. Wash the cells once with FACS buffer, add 100 ⁇ L of fixative [4% (v/v) paraformaldehyde] to each well to resuspend the cells, and wash the cells twice with FACS buffer after 10 minutes.
  • the cells were resuspended in 30 ⁇ L/well FACS buffer and the results were detected and analyzed using flow cytometry intellycite plus (Sartorius).
  • Human isotype control antibody purchased from Solarbio, Cat. No. SP001. Take cryopreserved monkey PBMC and perform flow cytometry in the same way after resuscitation.
  • the binding curves of humanized antibodies and Jurkat cells are shown in Figures 8A to 8B, and the binding curves of humanized antibodies and monkey PBMC cells are shown in Figures 9A to 9B, indicating that the humanized antibodies 002hz39, 002hz29, 002hz38, 002hz28, 002hz31b, 002hz32b and 002hz29b can bind to Jurkat cells and monkey PBMC.
  • the activation activity of humanized CD3 antibodies was detected by reporter gene activation assay in Jurkat NFAT-Luc cells. Dilute the antibody to five concentrations, 5-fold gradient dilution, 100 ⁇ L/well, add it to a 96-well plate (Corning, 3599), and cover the plate at 4°C overnight. Collect Jurkat NFAT-Luc cells, resuspend the cells in RPMI 1640 (Thermo Fisher, A1049101) + 2% FBS medium, adjust the density to 1E6/mL; aspirate the humanized CD3 antibody in the 96-well plate, and wash with PBS for 2 After spreading, add 100 ⁇ L cells to each well (1E5 cells/well). Place the 96-well plate into the incubator.
  • the experimental results are shown in Figure 10A to Figure 10B.
  • the humanized antibody has the activity of activating the Luciferase reporter gene in Jurkat NFAT-Luc cells.
  • Example 14 Humanized anti-CD3 antibody activates fresh CD4 + T cells to release IL-2
  • Anti-CD3 antibody coating starting with an antibody coating concentration of 10 ⁇ g/mL, 5-fold gradient dilution, dilution to 5 points, coating overnight at 4°C; the next day, first collect fresh CD4 + T cells, and adjust the density to 2 ⁇ 10 6 cells/mL (resuspend the cell pellet in 1640+2% FBS), then use the cell suspension to prepare anti-CD28 antibody (BD, Cat.
  • SP34 positive control prepared with reference to WO2015181098A1.
  • the variable regions of SP34.1 and SP34 are the same, and the constant regions are human IgG1 L234A, L235A, and D265S; the variable regions of OKT3.1 and OKT3 are the same, and the constant regions are human IgG1 L234A, L235A, and D265S.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本发明公开了一种抗CD3抗体、其制备方法及应用。所述抗CD3抗体包括轻链可变区(VL)和重链可变区(VH)，所述轻链可变区包括LCDR1、LCDR2和LCDR3；所述重链可变区包括HCDR1、HCDR2和HCDR3。所述抗CD3抗体具有较高的与人CD3结合的活性，可直接激活CD3下游的信号通路，活化T细胞，且对猴PBMC有结合活性，具有和猴CD3交叉反应性；本发明的抗CD3抗体可作为CD3-TAA(肿瘤相关抗原)类型双抗的CD3端抗体的一个选择。

Description

一种抗CD3抗体、其制备方法及应用
本申请要求申请日为2022/3/23的中国专利申请2022102940270的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及抗体领域，具体涉及一种抗CD3抗体、其制备方法及应用。
背景技术
CD3有四个亚基CD3γ、CD3δ、CD3ε和CD3ζ，分别组成εγ、εδ和ζζ三种二聚体，与TCR-αβ形成TCR-CD3复合物，胞内端有ITAM区域(免疫受体酪氨酸激活基序)，表达在T细胞上，抗原-MHC复合物结合TCR后，可通过CD3介导的信号传导活化T细胞，杀伤肿瘤细胞或感染外来物质的细胞。
CD3抗体识别所有T细胞，它和70％-80％的人外周血淋巴细胞和65％-85％的胸腺细胞发生反应。CD3抗体激活后的T细胞定向至肿瘤细胞周围，两种细胞接触进而形成突触，触发T细胞受体(TCR)信号通路的激活，颗粒酶表达、释放进而引起肿瘤细胞膜穿孔，导致后者的溶胞和凋亡。TCR信号通路的激活同时引起一系列细胞因子的表达与释放，例如IL-2的释放反馈刺激T细胞的增殖，放大免疫杀伤效应。临床前研究数据证明，在靶细胞存在下，双特异性抗体分子可有效激活T细胞，并刺激其增殖，同时引起靶细胞的死亡。
Jurkat细胞是人白血病T淋巴细胞，表达CD3/TCR复合物，由其发展而成的过表达报告基因元件的工程细胞株可通过检测报告基因下游luciferase酶信号的激活程度来确认Jurkat细胞可否被CD3抗体激活。
现有技术中缺乏能与猴CD3交叉反应的抗CD3抗体，仅能结合人CD3的抗体的研发便利性较低；同时缺乏结合活性和活化PBMC活性多样的抗CD3抗体，不利于抗CD3双抗的进一步研发。现有技术中，大多数抗体虽然和人CD3结合，但和灵长类动物猴CD3不结合，无法使用灵长类动物来评估抗体药的安全性。
发明内容
针对现有技术中缺乏能与猴CD3交叉反应的抗CD3抗体，且现有抗CD3抗体结合活性和活化PBMC活性多样性低的缺陷，本发明提供了一种抗CD3抗体、其制备方法、 含其的药物组合物、含其的检测试剂、含其的套装药盒及其应用。本发明所述抗CD3抗体具有较高的与人CD3结合的活性，可直接激活CD3下游的信号通路，活化T细胞，且对猴PBMC有结合活性，具有和猴CD3交叉反应性；本发明的抗CD3抗体可作为CD3-TAA(肿瘤相关抗原)类型双抗的CD3端抗体的一个选择。
为解决上述技术问题，本发明提供了一种抗CD3抗体，所述抗CD3抗体包括轻链可变区(VL)和重链可变区(VH)，所述轻链可变区包括LCDR1、LCDR2和LCDR3，所述LCDR1包含如SEQ ID NO:1、4或5所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:2所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:3所示的氨基酸序列；所述重链可变区包括HCDR1、HCDR2和HCDR3，所述HCDR1包含如SEQ ID NO:6所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:8所示的氨基酸序列。CDR序列见下表1：
表1 CDR序列信息
在本发明一较佳实施方案中，所述VL包含如SEQ ID NO:10-15任一所示的氨基酸序列。
在本发明一较佳实施方案中，所述VH包含如SEQ ID NO:17-19任一所示的氨基酸序列。
在本发明一具体实施方案中，所述VL包含如SEQ ID NO:10所示的氨基酸序列，且所述VH包含如SEQ ID NO:17所示的氨基酸序列；或，所述VL包含如SEQ ID NO:14所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或，所述VL包含如SEQ ID NO:14所示的氨基酸序列，且所述VH包含如SEQ ID NO:18所示的氨基酸序列；或，所述VL包含如SEQ ID NO:13所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或，所述VL包含如SEQ ID NO:13所示的氨基酸序列，且所述VH包含如SEQ ID NO:18所示的氨基酸序列；或，所述VL包含如SEQ ID NO:11所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或， 所述VL包含如SEQ ID NO:12所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或，所述VL包含如SEQ ID NO:15所示的氨基酸序列，且所述VH包含如SEQ ID NO:18所示的氨基酸序列。
在本申请中，上述所列CDR的氨基酸序列均是按照Chothia定义规则所示出的(本发明的权利要求中也是按照Chothia定义规则所示出的序列)。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273:927-48,1997)。
较佳地，所述的抗CD3抗体满足以下二项中至少一项：
(1)所述抗CD3抗体为全长抗体、Fab、Fab’、F(ab’)2或Fv，所述Fv优选为scFv；
(2)所述抗CD3抗体为由第(1)项中所述抗CD3抗体制得的单克隆抗体或多克隆抗体。
在本发明一较佳实施方案中，所述的抗CD3抗体为全长抗体，所述全长抗体包括轻链和重链；所述轻链包括轻链恒定区(CL)，所述抗体轻链恒定区优选为人源抗体轻链恒定区或鼠源抗体轻链恒定区，所述人源抗体轻链恒定区更优选为κ亚型的轻链恒定区；所述重链包括重链恒定区(CH)，所述抗体重链恒定区优选为人源或鼠源抗体重链恒定区，更优选为hIgG1、hIgG2、hIgG3或hIgG4亚型的重链恒定区，进一步优选为hIgG1亚型的重链恒定区。
在本发明一更佳实施方案中，所述人源抗体轻链恒定区包括如SEQ ID NO:20所示的氨基酸序列，且所述人源抗体重链恒定区包括如SEQ ID NO:21所示的氨基酸序列。
在本发明一较佳实施方案中，所述轻链包括如SEQ ID NO:23-28所示的氨基酸序列，且所述重链包括如SEQ ID NO:30-32任一所示的氨基酸序列。
更佳地，所述轻链包括如SEQ ID NO:23所示的氨基酸序列，且所述重链包括如SEQ ID NO:30所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:27所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:27所示的氨基酸序列，且所述重链包括如SEQ ID NO:31所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:26所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:26所示的氨基酸序列，且所述重链包括如SEQ ID NO:31所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:24所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如 SEQ ID NO:25所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:28所示的氨基酸序列，且所述重链包括如SEQ ID NO:31所示的氨基酸序列。
在本发明的抗体或其抗原结合片段包含与所述抗体或其抗原结合片段具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的同源性。这种同源性可由本领域已知的方法进行序列比较和/或比对来确定。例如，序列比对算法BLAST或手动比对可以测定序列同一性或同源性。
本发明某一方面还提供了一种分离的核酸，其编码如本发明所述的抗CD3抗体。
本发明某一方面还提供了一种重组表达载体，其包含如本发明所述的分离的核酸。较佳地，所述重组表达载体包含真核细胞表达载体和/或原核细胞表达载体。更佳地，所述真核细胞表达载体为pcDNA3.4。
本发明某一方面还提供了一种转化体，其包含如本发明所述的重组表达载体。较佳地，所述转化体的宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选为E.coli细胞，所述真核细胞优选为Expi293F细胞或CHO细胞。
本发明某一方面还提供了一种如本发明所述的抗CD3抗体的制备方法，其包括以下步骤：培养如本发明所述的转化体，从培养物中获得所述抗CD3抗体。
本发明某一方面还提供了一种药物组合物，其包含如本发明所述的抗CD3抗体，以及药学上可接受的载体和/或药学上可接受的佐剂。
本发明某一方面还提供了一种检测试剂，其包含如本发明所述的抗CD3抗体。较佳地，所述检测试剂为液体剂型、气体剂型、固体剂型和半固体剂型。更佳地，所述检测试剂还包括二抗、CD3或其衍生物，所述二抗例如抗人IgG的抗体偶联辣根过氧化物酶和抗人IgG的抗体偶联生物素蛋白。
本发明某一方面还提供了一种套装药盒，其包含药盒A，所述药盒A含有如本发明所述的抗CD3抗体、如本发明所述的药物组合物和如本发明所述的检测试剂中的一种或多种。
较佳地，所述套装药盒还包括药盒B，所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
本发明某一方面还提供了如本发明所述的抗CD3抗体、如本发明所述的药物组合物、如本发明所述的检测试剂和/或如本发明所述的套装药盒在制备诊断、预防和/或治疗CD3 相关疾病的药物中的应用。较佳地，所述CD3相关疾病为CD3相关肿瘤。
本发明某一方面还提供了一种检测样品中CD3的方法，其包括使用如本发明所述的抗CD3抗体或如本发明所述的检测试剂与所述样品接触的步骤。较佳地，所述方法为非诊断和/或治疗目的。所述样品例如血液样本(例如全血样本和血清样本)、包含CD3的试剂、含CD3的抗体药物偶联物或含CD3的嵌合抗原受体细胞。
本发明还提供了一种抗体组合，其中包括如本发明所述的抗CD3抗体。
本发明的抗体可利用该领域广为周知的技术制备，例如杂交瘤方法、重组DNA技术、噬菌体展示技术、合成技术或该等技术的组合、或该领域己知的其它技术。
术语
除非另有定义，否则本文使用的所有技术和科学术语都具有与本发明所属技术领域普通技术人员通常所理解的相同的含义。
本发明中，术语“一种”和“所述”通常包括多个指示物。
本文所述的“抗体分子”或“抗体”是指免疫球蛋白分子和免疫球蛋白分子的免疫活性部分，即含有免疫特异性结合抗原的抗原结合位点的分子。因此，术语“抗体”不仅涵盖完整抗体分子，还包括所述抗体的片段以及所述抗体和抗体片段的变体(包括衍生物)。在本文中所述术语“抗体分子”例如包括但非限于单链Fv(scFv)，Fab片段，Fab’片段，F(ab’)2，二硫键连接的Fv(sdFv)，Fv，及完整抗体或全长抗体。术语“单链Fv”或“scFv”是指一种多肽，其包含与抗体VH结构域连接的抗体的VL结构域。免疫特异性结合CD3的抗体可以与其它抗原发生交叉反应。优选地，免疫特异性结合CD3的抗体与其它抗原不发生交叉反应。免疫特异性结合CD3的抗体可以例如通过免疫测定或其它本领域技术人员已知的方法鉴别。“完整抗体”或“全长抗体”指包含两条重链(H)和两条轻链(L)的蛋白，所述重链和轻链通过二硫键相互连接，所述蛋白包含：(1)就重链而言，包含重链可变区(本文缩写为“VH”)和含有三个结构域CH1、CH2、CH3的重链恒定区；和(2)就轻链而言，包含轻链可变区(本文缩写为“VL”)和含有一个结构域CL的轻链恒定区。VH和VL可进一步再分为高变区，称为互补决定区(CDR)，CDR散布在被称为框架区(FR)的更加保守的区域中。每个VH和VL均由三个CDR和四个FR组成，它们从氨基端向羧基端以如下顺序排列：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。VH和VL含有可与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，该宿主组织或因子包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的成分。本发明的抗体包括但非限于单特异性、多特异性、人或嵌合抗体、单链抗体、Fab片段、F(ab′)片段、抗独特型(抗-Id)抗体(包括例如本发 明抗体的抗-Id抗体)和上述任何抗体的表位结合片段。本发明的免疫球蛋白分子可以是免疫球蛋白的任何类型(例如IgG、IgE、IgM、IgD、IgA和IgY)，类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类。本发明的抗体可为一种单克隆抗体或者多克隆抗体，所述的单克隆抗体优选鼠抗人单克隆抗体。
本发明中，术语“抗CD3抗体”表示所述抗体可与CD3特异性结合。“特异性结合”通常是指可测量且可再现的相互作用。例如抗原和抗体之间的结合，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更高的亲和力、亲合力、更容易和/或以更长的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。
本发明中，术语“CD3”为CD3蛋白。所述CD3蛋白有四个亚基：CD3γ、CD3δ、CD3ε和CD3ζ，分别组成εγ、εδ和ζζ三种二聚体，与TCR-αβ形成TCR-CD3复合物，胞内端有ITAM区域(免疫受体酪氨酸激活基序)，表达在T细胞上，抗原-MHC复合物结合TCR后，可通过CD3介导的信号传导活化T细胞，杀伤肿瘤细胞或感染外来物质的细胞。
本发明中，术语“可变区”(也称“可变结构域”)通常是指参与抗体与抗原结合的抗体重链或轻链的结构域。重链和轻链可变区可以分别称为“VH”和“VL”。这些结构域通常是抗体中最可变的部分并且含有抗原结合位点。然而，可变性并非均匀地分布在抗体的整个可变区中，它集中在VH和VL中的三个区段，所述三个区段被称为高变区(HVR)或被称为互补决定区(CDR)，分别为LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3。可变结构域中更高度保守的部分被称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区：HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3和LFR4，大部分采用β-折叠构型，通过三个CDR结构环区连接。每条链中的CDR通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。
在本领域中，可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则、基于结构环区域位置的Chothia定义规则和基于IMGT本体论(IMGT-ONTOLOGY)的概念。本发明中，抗CD3抗体的VL和VH的氨基酸序列按Chothia编码规则进行编码，所述抗CD3抗体的LCDR1-3和HCDR1-3按Chothia定义。
本发明中，术语“单克隆抗体”通常是指从一群基本上同质的抗体中获得的抗体，即集群中的抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。
在本发明中，术语“全人源抗体”或“人抗体”通常是指抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。全人源抗体可以大大减少异源抗体部分对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术等。
本发明中，术语“分离的”通常是指大体上不含其天然存在的环境中通常伴随或与之相互作用的组分(例如病毒、核酸或蛋白质)。本发明中，术语“分离的核酸分子”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
如本领域已知，在本发明中可交换使用的“多核苷酸”或“核酸”是指任何长度的核苷酸链，并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。
本发明中，术语“表达载体”通常是指能够转运与它连接的另一个核酸分子，并在合适的宿主中自我复制的核酸分子。表达载体包括任何遗传元件，例如质粒、转座子、人工染色体和病毒等，其在与适当的控制元件组合时能够进行自我复制并且将基因序列转移到宿主或宿主之间。所述表达载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述表达载体还包括具有多种上述功能的载体。所述表达载体还可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述表达载体的宿主细胞，所述表达载体可以产生期望的表达产物。表达载体的实例包括但不限于病毒载体、裸DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂相关的DNA或RNA表达载体、包囊化于脂质体中的DNA或RNA表达载体以及某些真核细胞，例如生产细胞。
在本发明中术语“宿主细胞”可包括已经引入外源性核酸的细胞，包括这些细胞的子代。宿主细胞包括“转化子”和“转化的细胞”，其包括原代转化细胞以及由此来源的子代，而不考虑传代次数。子代在核酸含量上与亲代细胞可能不完全相同，但可能含有突变。本发明包括与在初始转化的细胞中筛选或选择的细胞具有相同功能或生物学活性的突变子代。
在本发明中“KD”、“KD”或“KD”可互换使用，本发明中使用的“KD”是解离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。确定结合和解离速率常数的方法为本领域 熟知的，包括但不限于生物膜干涉技术(BLI)、放射免疫法(RIA)、平衡透析法、表面等离子共振(SPR)、荧光共振能量迁移(FRET)、免疫共沉淀(Co-IP)以及蛋白质芯片技术。如果在不同的条件(例如盐浓度、pH下测量)，则所测得的某种特定蛋白-蛋白相互作用的亲和力可不同。
本发明所述的“药学上可接受的佐剂”包括但不限于表面活性剂、溶液稳定剂、等渗调节剂或缓冲液中的一种或其组合。其中，表面活性剂包括但不限于非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或吐温80)；poloxamer(如poloxamer 188)；Triton；十二烷基硫酸钠(SDS)；月桂硫酸钠；十四烷基或十八烷基肌氨酸等，其加入量应使结合人Ang2的抗体或其抗原结合片段的颗粒化趋势最小。溶液稳定剂包括但不限于糖类，例如还原性糖或非还原性糖；氨基酸类，例如谷氨酸单钠或组氨酸；醇类，例如三元醇、丙二醇或聚乙二醇；溶液稳定剂的加入量应使最后形成的制剂在本领域技术人员认为达到稳定的时间内保持稳定的状态。等渗调节剂包括但不限于氯化钠、甘露醇或其组合。缓冲液包括但不限于Tris、组氨酸缓冲液、磷酸盐缓冲液或其组合。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明提供的抗CD3抗体除与人CD3的结合活性高外，均表现与猴PBMC的结合活性，具有猴CD3交叉反应性，为评估抗研究体的安全性研究带来便利，便于后续开发；且所述抗CD3抗体表现出多样性的结合和活化PBMC的活性，为后续双抗的开发和研究提供便利。
附图说明
图1为鼠单克隆抗体与Jurkat细胞的结合曲线。
图2为鼠单克隆抗体与猴PBMC的结合曲线。
图3为杂交瘤抗CD3抗体激活Jurkat NFAT-Luc细胞中Luciferase报告基因的结果。
图4为杂交瘤抗CD3抗体活化T细胞，激活新鲜CD4+T细胞释放IL-2曲线。
图5为抗CD3嵌合抗体与Jurkat细胞的结合曲线。
图6为抗CD3嵌合抗体激活Jurkat NFAT-Luc细胞中Luciferase报告基因的结果。
图7为抗CD3嵌合抗体活化T细胞，激活新鲜CD4+T细胞释放IL-2曲线。
图8A和8B为人源化抗体与Jurkat细胞的结合曲线。
图9A和9B为人源化抗体与猴PBMC的结合曲线。
图10A和10B为人源化抗体激活Jurkat NFAT-Luc细胞中Luciferase报告基因的结果。
图11A和11B为人源化抗体活化T细胞，激活新鲜CD4+T细胞释放IL-2曲线。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1杂交瘤技术筛选抗CD3抗体
用CD3抗原(包括蛋白和过表达CD3抗原的细胞株)分别免疫Balb/c、SD小鼠(购自北京维通利华)3-4次，3-5×106细胞或25-50μg蛋白/只。依照常规方法进行PEG融合或者电融合后，将融合后的细胞加入96孔细胞培养板内，置CO2培养箱内培养9-10天。用流式检测96孔细胞培养板中的杂交瘤上清对Jurkat细胞的结合活性。阳性的杂交瘤上清对应的细胞克隆转至24孔细胞培养板继续培养2-4天后，24孔细胞培养板中的杂交瘤上清用于确认与Jurkat细胞的结合活性。从24孔细胞培养板中挑取阳性细胞进行亚克隆及亚克隆筛选，直至得到稳定的可分泌结合Jurkat细胞的杂交瘤单克隆抗体细胞株若干。并采用常规无血清培养基进行50毫升小规模生产，常规protein A柱纯化得到纯化单克隆抗体做后续鉴定。
实施例2杂交瘤抗体测序和嵌合抗体表达
培养杂交瘤单克隆细胞株，离心收集5×106个杂交瘤细胞，常规Trizol法(Thermofisher，15596026)提取总RNA，逆转录反应后得到的cDNA通过末端转移酶进行加G反应，后用VH，VK引物和polyC引物扩增含可变区序列的DNA，做TA克隆，测序后得鼠源杂交瘤克隆10E1C11的可变区序列如下表2：
表2鼠源杂交瘤克隆10E1C11抗体的轻重链可变区序列

单克隆抗体轻重链蛋白质序列分别进行密码子优化，基因合成密码子优化后DNA片段，克隆合成后基因片段到表达载体pcDNA3.4(Life Technologies)。表达质粒扩增和质粒抽提后双质粒共转Expi293F(ThermoFisher Scientific，A14527)或CHO-K1细胞(ECACC catalogue no.85051005)，根据供应商Expi293F或CHO-K1表达系统方法进行抗体瞬转表达，上清离心后经proteinA柱按常规方法纯化。
实施例3杂交瘤抗CD3抗体流式结合Jurkat细胞和猴PBMC细胞
用FACS鉴定鼠抗人CD3单克隆抗体10E1C11结合Jurkat细胞和猴PBMC细胞的活性。将Jurkat细胞(ATCC，TIB-152)在T175细胞培养瓶中培养至密度为1E6/mL，取出所需数量的细胞，300×g离心5分钟，弃上清，用FACS缓冲液(含有2％FBS的PBS)(FBS:ThermoFisher Scientific,10099-141C；PBS:ThermoFisher Scientific,10010023)洗涤1次，然后将细胞用FACS缓冲液重悬至每毫升2×106个细胞，室温孵育15分钟后将细胞悬液按50μL/孔加入到U型96孔板(Corning，3798)中。用FACS缓冲液将单克隆抗体按梯度稀释，将抗体稀释液加入上述96孔板中，50μL/孔，4℃孵育1小时。离心弃上清，用FACS缓冲液洗涤细胞1次，每孔加入100μL荧光(Alexa 488)标记的二抗(Thermo Fisher，A-11029)，4℃孵育1小时。用FACS缓冲液洗涤细胞1次，每孔加入100μL固定液[4％(v/v)多聚甲醛]重悬细胞，10分钟后用FACS缓冲液洗涤细胞2次。用30μL/孔FACS缓冲液重悬细胞后用流式细胞仪intellycite plus(Sartorius)检测和分析结果。猴PBMC(澳赛尔斯，CAT#:NHP-PB001F)取冻存细胞复苏后按流式结合方法操作。小鼠同型对照抗体(购自Thermofisher，货号02-6502)。SP34阳性对照参考WO2015181098A1制备。
鼠单克隆抗体10E1C11与Jurkat细胞的结合曲线如图1所示，鼠单克隆抗体10E1C11与猴PBMC的结合曲线如图2所示，显示鼠单克隆抗体10E1C11与猴CD3和人CD3均可结合细胞。
实施例4鼠源抗CD3抗体亲和力测定
(1)采用Octet RED96e(Fortébio)测定鼠源抗CD3抗体与人CD3E&CD3G，Fc(KACTUS，货号：CD3-HM257)的亲和力，抗原及抗体均用1×PBST(1×PBS：生工， B548117-0500；0.02％吐温20：sigma-aldrich，P1379)稀释，抗原使用浓度为50nM，抗体使用浓度为33.3nM。
(2)样品上机检测(Octet Data Acquisition 11.1.0.11)：首先，将样品加入96孔板(Greiner bio-one，655209)，体系为200μL/well。然后设置软件参数，板温设定为30℃，收集标准动力学信号的频率为5.0HZ。接着，用1×PBST预湿AMQ传感器(Fortébio，货号：18-5022)10分钟，然后上机检测。每个循环包含以下步骤：1)10mM pH1.7的甘氨酸溶液再生；2)浸入缓冲液60s；3)抗体固化在传感器上，时间为35s；6)传感器浸入缓冲液180s；7)抗原与抗体结合，时间180s；8)抗原抗体的解离，时间10分钟；9)传感器再生。
(3)数据分析
采用Fortebio的Data Analysis 12.0软件，对抗原-抗体以1:1的结合方式，测定结合速率(Ka)和解离速率(Kd)，以此计算抗体的平衡解离常数(KD)，结果如下表3：
表3鼠源抗CD3抗体与人CD3E&CD3G的亲和力
实施例5杂交瘤抗CD3抗体激活Jurkat NFAT-Luc细胞中Luciferase报告基因
Jurkat NFAT-Luc细胞(Invivogen,jktl-nfat)是含有NFAT元件控制其下游luciferase基因表达的报告基因系统的Jurkat细胞，被确认luciferase基因表达可被CD3抗体激活。
抗CD3抗体包板，抗体包板浓度20μg/mL起始，2倍梯度稀释，稀释11个点，4℃包板过夜；第二天，收集Jurkat NFAT-Luc细胞，密度调整至1×106细胞/mL(细胞沉淀用1640(ThermoFisher Scientific,A10491)+2％FBS重悬)，吸弃anti-CD3抗体，板用PBS洗2遍后每孔加入100μL Jurkat NFAT-Luc细胞(1×105细胞/孔)，37℃、5％CO2培养6小时后，每孔吸出20μL到白色的96孔板(Corning，3917)中，然后加入50μL Coelenterazine-utilizing luciferase detection medium(Invivogen，货号：rep-qlc1)来检测样品中荧光素酶的活性。H2M2690N为阳性对照(参考US9657102B2制备)。
结果如图3所示，从图中可以看出，与对照抗体H2M2690N和SP34相比，抗体10E1C11可激活Jurkat NFAT-Luc细胞中Luciferase报告基因。
实施例6杂交瘤抗CD3抗体激活新鲜CD4+T细胞释放IL-2
抗CD3抗体包板，抗体包板浓度10μg/mL起始，5倍梯度稀释，稀释5个点，4℃包板过夜；第二天，首先收集新鲜CD4+T细胞(ALLCELLS,PB009-2-C)，密度调整至2×106细胞/mL(细胞沉淀用1640+2％FBS重悬)，然后用细胞悬液配制抗CD28抗体(BD，货号：555725)浓度为4μg/mL，接着吸弃抗CD3抗体，板用PBS洗2遍后每孔加入50μL新鲜CD4+T细胞(1×105细胞/孔)和50μL抗CD28抗体(终浓度2μg/mL)，37℃、5％CO2培养3d，最后离心取细胞上清稀释3倍，严格按照IL-2ELISA kit(Abclonal，货号：RK04123)检测IL-2的含量。数据用GraphPad Prism8.0软件进行处理。从图中结果可以看出，抗体均有活性。小鼠同型对照抗体购自百英(货号B115101)。
杂交瘤抗CD3抗体激活新鲜CD4+T细胞释放IL-2的结果如图4所示，表明杂交瘤抗CD3抗体可激活CD4+T细胞释放IL-2。
实施例7抗CD3嵌合抗体流式结合Jurkat细胞
将杂交瘤抗CD3抗体10E1C11的重链可变区序列与人IgG1 LALA D265S(SEQ ID NO:21)融合，轻链可变区与人Kappa链恒定区(SEQ ID NO:20)融合(人恒定区序列见表6)，构建得到CD3嵌合抗体CAb10E1。
用FACS鉴定抗CD3嵌合抗体CAb10E1结合Jurkat细胞的活性。将Jurkat细胞在T175细胞培养瓶中培养至密度为106个/mL，取出所需数量的细胞，300×g离心5分钟，弃上清，用FACS缓冲液(含有2％FBS的PBS)洗涤1次，然后将细胞用FACS缓冲液重悬至每毫升2×106个细胞，室温孵育15分钟后将细胞悬液按50μL/孔加入到U型96孔板(Corning，3798)中。用FACS缓冲液将单克隆抗体按梯度稀释，将抗体稀释液加入上述96孔板中，50μL/孔，4℃孵育1小时。离心弃上清，用FACS缓冲液洗涤细胞1次，每孔加入100μL荧光(Alexa 488)标记的二抗(Thermo Fisher，A-11029)，4℃孵育1小时。用FACS缓冲液洗涤细胞1次，每孔加入100μL固定液[4％(v/v)多聚甲醛]重悬细胞，10分钟后用FACS缓冲液洗涤细胞2次。用30μL/孔FACS缓冲液重悬细胞后用流式细胞仪intellycite plus(Sartorius)检测和分析结果。小鼠同型对照抗体(购自Thermofisher，货号02-6502)；人源同型对照抗体(购自Solarbio，货号SP001)。
抗CD3嵌合抗体CAb10E1流式结合Jurkat细胞的实验结果如图5所示，表明嵌合抗体CAb10E1保留有母本杂交瘤抗体的与Jurkat细胞结合的活性。
实施例8抗CD3嵌合抗体激活Jurkat NFAT-Luc细胞中Luciferase报告基因
用Jurkat NFAT-Luc细胞的报告基因激活实验检测抗CD3嵌合抗体的激活活性。将抗体稀释五个浓度，5倍梯度稀释，100μL/孔，加入96孔板(Corning，3599)中，4℃包板过夜。收集Jurkat NFAT-Luc细胞，用RPMI 1640(Thermo Fisher，A1049101)+2％FBS培养基重悬细胞，密度调整至1E6/mL；吸弃96孔板中的人源化CD3抗体，用PBS洗2遍后每孔加入100μL细胞(1E5细胞/孔)。将96孔板放入培养箱中，6小时后，每孔吸出20μL细胞到白色的96孔酶标板(Corning，3917)中，然后每孔加入50μL检测液QUANTI-Luc(INVIVOGEN，rep-qlc2)，用多功能酶标仪检测样品中荧光素酶的活性。小鼠同型对照抗体(购自Thermofisher，货号02-6502)；人源同型对照抗体(购自Solarbio，货号SP001)。
抗CD3嵌合抗体激活Jurkat NFAT-Luc细胞中Luciferase报告基因的实验结果如图6所示，表明嵌合抗体CAb10E1保留有母本杂交瘤抗体的激活Jurkat NFAT-Luc细胞中Luciferase报告基因。
实施例9抗CD3嵌合抗体激活新鲜CD4+T细胞释放IL-2
采用同实施例6中相同的实验方法，测定抗CD3嵌合抗体激活新鲜CD4+T细胞释放IL-2的功能。小鼠同型对照抗体(购自百英，货号B115101)。
结果如图7所示，表明嵌合抗体CAb10E1可激活CD4+T细胞释放IL2。
实施例10抗CD3抗体的人源化和表达
1.通过序列比对，挑选同源性最高的人抗体胚系基因(Human Antibody Germline Gene，数据来源：IMGT)作为人源化设计框架(Greg Winter,1986；Riechmann,L.,Clark,M.,Waldmann,H.et al.1988)。轻链以IGKV4-1*01或IGKV3-20，IGKJ4*01为框架，重链以IGHV1-3*01，IGHJ4*01为框架。对抗体轻重链可变区进行Chothia编号(Chothia&Lesk,1987)，定义抗体CDR区：LCDR1(L24-L34)、LCDR2(L50-L56)、LCDR3(L89-L97)、HCDR1(H26-H32)、HCDR2(H52-H56)和HCDR3(H95-H97)，由此定义的CDR如表4所示：
表4 Chothia编号的鼠源抗体10E1C11的CDR序列
根据序列比对和可变区结构信息对抗体轻重链可变区的氨基酸进行人源化设计；
2.设计表达载体，基因合成，哺乳细胞表达纯化重组抗体，比较人源化抗体和嵌合抗体活性，理化性质的差异，进行1-2轮人源化优化；
3.人胚系抗体基因氨基酸序列信息：
IGKV4-1*01：
IGKV3-20：
IGHV1-3*01：
IGKJ4*01：
IGHJ4*01：
以下轻链可变区的优化设计是以人抗体胚系基因序列IGKV4-1*01、IGKV3-20或IGKJ4*01为框架，002hzVK0(10E1C11VK)为嵌合抗体的轻链可变区序列进行CDR移植和回复突变的人源化序列：
002hzVK0
轻链可变区序列(SEQ ID NO:10)：
002hzVK1b
轻链可变区序列(SEQ ID NO:11)：
优化突变位点：S5T,S9D,A15L,K18R,V19A,M21I,S22N,S43P,T63S,N77S,V78L,V83L,L103V,S105I。
002hzVK2b
轻链可变区序列(SEQ ID NO:12)：
优化突变位点：S5T,S9D,A15L,K18R,V19A,M21I,S22N,N30aQ,S43P,T63S,N77S,V78L,V83L,L103V,S105I。
002hzVK8
轻链可变区序列(SEQ ID NO:13):
优化突变位点：D1E,M4L,S5T,S9G,S10T,A12S,V13L,A15P,K18R,V19A,M21L,N30aQ,S43A,K45R,V58I,T63S,N77R,V78L,Q79E,A80P,L83F,L103V,S105I。
002hzVK9
轻链可变区序列(SEQ ID NO:14)：
优化突变位点：D1E,M4L,S5T,S9G,S10T,A12S,V13L,A15P,K18R,V19A,M21L,N30aS,S43A,K45R,V58I,T63S,N77R,V78L,Q79E,A80P,L83F,L103V,S105I。
002hzVK9b
轻链可变区序列(SEQ ID NO:15)：
优化突变位点：D1E,M4L,S5T,S9G,S10T,A12S,V13L,A15P,K18R,V19A,M21L,N30aS,S43A,K45R,V58I,T63S,N77R,V78L,Q79E,A80P,F83L,L103V,S105I。
以下重链可变区的优化设计是以人抗体胚系基因序列IGHV1-3*01和IGHJ4*01为框架，002hzVH0(10E1C11VH)为嵌合抗体的重链可变区序列进行CDR移植和回复突变的人源化序列。
002hzVH0
重链可变区序列(SEQ ID NO:17)：
002hzVH2
重链可变区序列(SEQ ID NO:18)：
优化突变位点：Q5V,L9A,L11V,V12K,A17S,M20V,T23K,A33Y,M34I,K38R,R40A,G44R,I48M,T57S,N60S,E61Q,K64Q,A65G,K66R,T67V,S68T,L69I,A71R,K73T,S75A,L81E,N82aS,T83R,S87T,I89V,F91Y,A93V,T108L,L109V。
002hzVH3
重链可变区序列(SEQ ID NO:19)：
优化突变位点：Q5V,L9A,L11V,V12K,A17S,M20V,T23K,A33Y,M34I,K38R,R40A,G44R,I48M,T57S,N60S,E61Q,K64Q,A65G,K66R,T67V,S68T,L69I,R71A,T73K,S75A,L81E,N82aS,T83R,S87T,I89V,F91Y,A93V,T108L,L109V。
上述所有轻重链可变区的CDR如下表5所示：
表5各抗体CDR序列及编号

4.人源化抗体的重链轻链组合与抗体表达
将上述人源化的轻链可变区序列与人Kappa链恒定区(SEQ ID NO:20)组合成人源化抗体轻链，将上述人源化的重链可变区序列与人IgG1 LALA D265S(SEQ ID NO:21)组合成人源化抗体重链。恒定区序列如表6所示。
表6人轻链和重链恒定区
002hzVH2重链可变区和002hzVK8轻链可变区分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体002hz28；002hzVH2重链可变区和轻链可变区002hzVK9分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体002hz29；002hzVH2重链可变区和002hzVK9b轻链可变区分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体002hz29b；002hzVH3重链可变区和002hzVK1b轻链可变区分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体002hz31b。
002hzVH3重链可变区和002hzVK2b轻链可变区分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体002hz32b。
002hzVH3重链可变区和002hzVK8轻链可变区分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体002hz38；002hzVH3重链可变区和002hzVK9轻链可变区分别与人重链恒定区和人轻链恒定区组合后配对组成人源化抗体002hz39。
上述鼠源抗体、嵌合抗体和人源化抗体的轻重链全长列序列信息如下表7所示：
表7抗体轻重链


优化设计序列后，全长重组单克隆抗体轻重链蛋白质序列分别进行密码子优化，基因合成密码子优化后DNA片段，克隆合成后基因片段到表达载体pcDNA3.4(Life Technologies)。表达质粒扩增和质粒抽提(Qiagen,Plasmid Maxi Kit,Cat.No.12362)后双质粒共转Expi293F(ThermoFisher Scientific，A14527)或CHO-K1细胞(ECACC catalogue no.85051005)，根据供应商Expi293F或CHO-K1表达系统方法进行抗体瞬转表达，大致过程如下：
(一)细胞复苏：使用125mL一次性无菌摇瓶，将冻存的CHO细胞37℃水浴解冻，细胞稀释至0.3×106个/mL，体积30mL。125rpm(19mm轨道)，8％CO2，37℃培养至4-6×106个/mL左右。
(二)传代：细胞长至4-6×106个/mL左右，传代密度为0.2-0.3×106个/mL，培养3天。
(三)瞬转转染：细胞传至3代以上，转染前一天密度4-6×106个/mL左右，稀释细胞至密度3-4×106个/mL，过夜培养，第二天7-10×106，稀释至6×106，以瞬转体积100 mL(500mL摇瓶)为例：溶液1：用培养液(4mL)稀释100μg质粒，混匀；溶液2：用培养液(3.68mL)稀释320μL转染试剂，混匀；将溶液2加入溶液1中，总体积是8mL，轻柔混匀后室温孵育1-5分钟，不超过5分钟，将混合转染液逐滴加入细胞液中，边摇边加。
(四)蛋白表达(最大滴度)：18-22个小时加辅料Feed-16mL，增强剂Enhancer-0.6mL，32℃培养。第五天加辅料Feed-16mL，123rpm，5％CO2，32℃培养，待第12-14天离心收集上清。
(五)抗体纯化：Protein A亲和层析柱纯化：1)平衡层析柱：1×PBS，流速1mL/min，20mL；2)上样：流速1mL/min；3)洗杂：1×PBS，流速1mL/min，20mL；4)洗脱：柠檬酸缓冲液(PH3.4)，1mL/min，分管收集，每管约500μL。共收集10管，使用NanoDrop仪器读取280nm吸光度值；5)透析：将高浓度蛋白吸至透析袋放1×PBS的烧杯中透析。6)收集抗体备用。
抗体表达结果如表8所示，表明利用上述方法能表达出抗CD3抗体。
表8表达量数据
实施例11人源化抗CD3抗体的亲和力测定
测定人源化抗CD3抗体与人CD3E&CD3G，Fc的亲和力，使用CH1传感器(Fortébio，货号：18-5127)，其余实验方法及步骤同实施例4，两次实验结果如表9和表10。其中OKT3为阳性对照(购自invitrogen，货号为16-0037-85)。SP34阳性对照(参考WO2015181098A1制备)。SP34.1与SP34的可变区相同，恒定区为人IgG1 L234A、L235A和D265S；OKT3.1与OKT3的可变区相同，恒定区为人IgG1 L234A、L235A和D265S。
表9人源化抗CD3抗体与人CD3E&CD3G的亲和力

表10人源化抗CD3抗体与人CD3E&CD3G的亲和力
由表9和表10可知，人源化CD3抗体与嵌合抗体CAb10E1相比，其与人CD3E&CD3G，Fc的亲和力差异不明显，所以均进一步进行体外功能实验评估。
实施例12人源化抗CD3抗体的流式结合Jurkat细胞和猴PBMC细胞
用FACS鉴定人源化抗CD3抗体结合Jurkat细胞的活性。将Jurkat细胞在T175细胞培养瓶中培养至密度为1×106个/mL，取出所需数量的细胞，300×g离心5分钟，弃上清，用FACS缓冲液(含有2％FBS的PBS)洗涤1次，然后将细胞用FACS缓冲液重悬至每毫升2×106个细胞，室温孵育15分钟后将细胞悬液按50μL/孔加入到U型96孔板(Corning，3798)中。用FACS缓冲液将人源化抗体按梯度稀释，将抗体稀释液加入上述96孔板中，50μL/孔，4℃孵育1小时。离心弃上清，用FACS缓冲液洗涤细胞1次，每孔加入100μL荧光(Alexa 488)标记的二抗(Thermo Fisher，A-11013)，4℃孵育1小时。用FACS缓冲液洗涤细胞1次，每孔加入100μL固定液[4％(v/v)多聚甲醛]重悬细胞，10分钟后用FACS缓冲液洗涤细胞2次。用30μL/孔FACS缓冲液重悬细胞后用流式细胞仪intellycite plus(Sartorius)检测和分析结果。人源同型对照抗体(购自Solarbio，货号SP001)。取冻存猴PBMC，复苏后按相同方法做流式结合。
人源化抗体与Jurkat细胞的结合曲线如图8A至8B所示，人源化抗体与猴PBMC细胞的结合曲线如图9A至9B所示，表明人源化后的抗体002hz39、002hz29、002hz38、002hz28、002hz31b、002hz32b和002hz29b可与Jurkat细胞和猴PBMC的结合。
实施例13人源化抗体激活Jurkat NFAT-Luc细胞中Luciferase报告基因
用Jurkat NFAT-Luc细胞的报告基因激活实验检测人源化CD3抗体的激活活性。将抗体稀释五个浓度，5倍梯度稀释，100μL/孔，加入96孔板(Corning，3599)中，4℃包板过夜。收集Jurkat NFAT-Luc细胞，用RPMI 1640(Thermo Fisher，A1049101)+2％FBS培养基重悬细胞，密度调整至1E6/mL；吸弃96孔板中的人源化CD3抗体，用PBS洗2遍后每孔加入100μL细胞(1E5细胞/孔)。将96孔板放入培养箱中，6小时后，每孔吸 出20μL细胞到白色的96孔酶标板(Corning，3917)中，然后每孔加入50μL检测液QUANTI-Luc(INVIVOGEN，rep-qlc2)，用多功能酶标仪检测样品中荧光素酶的活性。小鼠同型对照抗体(购自Thermofisher，货号02-6502)，人源同型对照抗体(购自Solarbi，货号SP001)。
实验结果如图10A至图10B所示，人源化后的抗体具有激活Jurkat NFAT-Luc细胞中Luciferase报告基因的活性。
实施例14人源化抗CD3抗体激活新鲜CD4+T细胞释放IL-2
抗CD3抗体包板，抗体包板浓度10μg/mL起始，5倍梯度稀释，稀释5个点，4℃包板过夜；第二天，首先收集新鲜CD4+T细胞，密度调整至2×106细胞/mL(细胞沉淀用1640+2％FBS重悬)，然后用细胞悬液配制抗CD28抗体(BD，货号：555725)浓度为4μg/mL，接着吸弃抗CD3抗体，板用PBS洗2遍后每孔加入50μL新鲜CD4+T细胞(1×105细胞/孔)和50μL抗CD28抗体(终浓度2μg/mL)，37℃、5％CO2培养3d，最后离心取细胞上清稀释3倍，严格按照IL-2ELISA kit(Abclonal，货号：RK04123)检测IL-2的含量。其中OKT3为阳性对照(购自invitrogen，货号为16-0037-85)。SP34阳性对照(参考WO2015181098A1制备)。SP34.1与SP34的可变区相同，恒定区为人IgG1 L234A、L235A和D265S；OKT3.1与OKT3的可变区相同，恒定区为人IgG1 L234A、L235A和D265S。
实验结果如图11A至图11B所示，表明抗体均可激活新鲜CD4+T细胞释放IL-2。

Claims (14)

  1. 一种抗CD3抗体，所述抗CD3抗体包括轻链可变区(VL)和重链可变区(VH)，所述轻链可变区包括LCDR1、LCDR2和LCDR3，所述LCDR1包含如SEQ ID NO:1、4或5所示的氨基酸序列，所述LCDR2包含如SEQ ID NO:2所示的氨基酸序列，所述LCDR3包含如SEQ ID NO:3所示的氨基酸序列；所述重链可变区包括HCDR1、HCDR2和HCDR3，所述HCDR1包含如SEQ ID NO:6所示的氨基酸序列，所述HCDR2包含如SEQ ID NO:7所示的氨基酸序列，所述HCDR3包含如SEQ ID NO:8所示的氨基酸序列。
  2. 如权利要求1所述的抗CD3抗体，其特征在于，所述VL包含如SEQ ID NO:10-15任一所示的氨基酸序列；和/或，所述VH包含如SEQ ID NO:17-19任一所示的氨基酸序列；
    较佳地，所述VL包含如SEQ ID NO:10所示的氨基酸序列，且所述VH包含如SEQ ID NO:17所示的氨基酸序列；或，所述VL包含如SEQ ID NO:14所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或，所述VL包含如SEQ ID NO:14所示的氨基酸序列，且所述VH包含如SEQ ID NO:18所示的氨基酸序列；或，所述VL包含如SEQ ID NO:13所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或，所述VL包含如SEQ ID NO:13所示的氨基酸序列，且所述VH包含如SEQ ID NO:18所示的氨基酸序列；或，所述VL包含如SEQ ID NO:11所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或，所述VL包含如SEQ ID NO:12所示的氨基酸序列，且所述VH包含如SEQ ID NO:19所示的氨基酸序列；或，所述VL包含如SEQ ID NO:15所示的氨基酸序列，且所述VH包含如SEQ ID NO:18所示的氨基酸序列。
  3. 如权利要求1或2所述的抗CD3抗体，其特征在于，所述的抗CD3抗体满足以下二项中至少一项：
    (1)所述抗CD3抗体为全长抗体、Fab、Fab’、F(ab’)2或Fv，所述Fv优选为scFv；
    (2)所述抗CD3抗体为由第(1)项中所述抗CD3抗体制得的单克隆抗体或多克隆抗体。
  4. 如权利要求1-3任一项所述的抗CD3抗体，其特征在于，所述的抗CD3抗体为全长抗体，所述全长抗体包括轻链和重链；所述轻链包括轻链恒定区(CL)，所述轻链恒定区优选为人源抗体轻链恒定区或鼠源抗体轻链恒定区，所述人源抗体轻链恒定区更优 选为κ亚型的轻链恒定区；所述重链包括重链恒定区(CH)，所述重链恒定区优选为人源或鼠源抗体重链恒定区，更优选为hIgG1、hIgG2、hIgG3或hIgG4亚型的重链恒定区，进一步优选为hIgG1亚型的重链恒定区；
    较佳地，所述人源抗体轻链恒定区包括如SEQ ID NO:20所示的氨基酸序列，且所述人源抗体重链恒定区包括如SEQ ID NO:21所示的氨基酸序列。
  5. 如权利要求1-4任一项所述的抗CD3抗体，其特征在于，所述轻链包括如SEQ ID NO:23-28任一所示的氨基酸序列，且所述重链包括如SEQ ID NO:30-32任一所示的氨基酸序列；
    较佳地，所述轻链包括如SEQ ID NO:23所示的氨基酸序列，且所述重链包括如SEQ ID NO:30所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:27所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:27所示的氨基酸序列，且所述重链包括如SEQ ID NO:31所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:26所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:26所示的氨基酸序列，且所述重链包括如SEQ ID NO:31所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:24所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:25所示的氨基酸序列，且所述重链包括如SEQ ID NO:32所示的氨基酸序列；或，所述轻链包括如SEQ ID NO:28所示的氨基酸序列，且所述重链包括如SEQ ID NO:31所示的氨基酸序列。
  6. 一种分离的核酸，其编码如权利要求1-5任一项所述的抗CD3抗体。
  7. 一种重组表达载体，其包含如权利要求6所述的分离的核酸；
    较佳地，所述重组表达载体包含真核细胞表达载体和/或原核细胞表达载体；
    更佳地，所述真核细胞表达载体为pcDNA3.4。
  8. 一种转化体，其包含如权利要求7所述的重组表达载体；
    较佳地，所述转化体的宿主细胞为原核细胞和/或真核细胞，所述原核细胞优选为E.coli细胞，所述真核细胞优选为Expi293F细胞或CHO细胞。
  9. 一种如权利要求1-5任一项所述的抗CD3抗体的制备方法，其包括以下步骤：培养如权利要求8所述的转化体，从培养物中获得所述抗CD3抗体。
  10. 一种药物组合物，其包含如权利要求1-5任一项所述的抗CD3抗体，以及药学上可接受的载体和/或药学上可接受的佐剂。
  11. 一种检测试剂，其包含如权利要求1-5任一项所述的抗CD3抗体；
    较佳地，所述检测试剂为液体剂型、气体剂型、固体剂型和半固体剂型；
    更佳地，所述检测试剂还包括二抗、CD3或其衍生物，所述二抗例如抗人IgG的抗体偶联辣根过氧化物酶和抗人IgG的抗体偶联生物素蛋白。
  12. 一种套装药盒，其包含药盒A，所述药盒A含有如权利要求1-5任一项所述的抗CD3抗体、如权利要求10所述的药物组合物和如权利要求11所述的检测试剂中的一种或多种；
    较佳地，所述套装药盒还包括药盒B，所述药盒B含有其他抗肿瘤抗体或者包含所述其他抗肿瘤抗体的药物组合物，和/或由激素制剂、靶向小分子制剂、蛋白酶体抑制剂、成像剂、诊断剂、化疗剂、溶瘤药物、细胞毒性剂、细胞因子、共刺激分子的激活剂、抑制性分子的抑制剂以及疫苗组成的群组中的一种或多种。
  13. 如权利要求1-5任一项所述的抗CD3抗体、如权利要求10所述的药物组合物、如权利要求11所述的检测试剂和/或如权利要求12所述的套装药盒在制备诊断、预防和/或治疗CD3相关疾病的药物中的应用；
    较佳地，所述CD3相关疾病为CD3相关肿瘤。
  14. 一种检测样品中CD3的方法，其包括使用如权利要求1-5任一项所述的抗CD3抗体或如权利要求11所述的检测试剂与所述样品接触的步骤；
    较佳地，所述方法为非诊断和/或治疗目的；所述样品例如血液样本、包含CD3的试剂、含CD3的抗体药物偶联物或含CD3的嵌合抗原受体细胞。
PCT/CN2023/083410 2022-03-23 2023-03-23 一种抗cd3抗体、其制备方法及应用 WO2023179716A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210294027 2022-03-23
CN202210294027.0 2022-03-23

Publications (1)

Publication Number Publication Date
WO2023179716A1 true WO2023179716A1 (zh) 2023-09-28

Family

ID=88100102

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/083410 WO2023179716A1 (zh) 2022-03-23 2023-03-23 一种抗cd3抗体、其制备方法及应用

Country Status (2)

Country Link
TW (1) TW202346356A (zh)
WO (1) WO2023179716A1 (zh)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104640881A (zh) * 2012-09-21 2015-05-20 瑞泽恩制药公司 抗cd-3抗体,与cd3及cd20结合的双特异性抗原结合分子,及其用途

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104640881A (zh) * 2012-09-21 2015-05-20 瑞泽恩制药公司 抗cd-3抗体,与cd3及cd20结合的双特异性抗原结合分子,及其用途

Also Published As

Publication number Publication date
TW202346356A (zh) 2023-12-01

Similar Documents

Publication Publication Date Title
JP7317272B2 (ja) Ｔｉｇｉｔ抗体、その抗原結合断片及びその医療用途 本願は、２０１９年９月２９日に出願された出願番号ｃｎ２０１７１０９０８５６５．３に基づいたものであり、その優先権を主張する。その開示は、その全体が参照により本明細書に組み込まれる。
CN112625136B (zh) 针对冠状病毒具有中和活性的双特异性抗体及其用途
JP2023075294A (ja) 抗ｃｄ４７抗体及びその応用
WO2021058000A1 (zh) 抗人Claudin18.2抗体及其应用
CN111518214A (zh) 靶向cldn18.2的双特异性抗体及其制备方法和应用
WO2020168555A1 (zh) Cd3抗原结合片段及其应用
JP7457822B2 (ja) 抗ｃｄ３および抗ｃｄ１２３二重特異性抗体およびその使用
WO2023125888A1 (zh) 一种gprc5d抗体及其应用
WO2023025315A1 (zh) 抗b7-h3抗体、其制备方法及用途
CN115386006A (zh) 抗gprc5d抗体、其制备方法与用途
KR102249981B1 (ko) 단클론성 항-gpc-1 항체 및 이의 용도
CN115386007A (zh) 抗gprc5d抗体、其制备方法与用途
WO2021143914A1 (zh) 一种激活型抗ox40抗体、生产方法及应用
WO2023001155A1 (zh) 一种磷脂酰肌醇蛋白聚糖3抗体及其应用
WO2022100694A1 (zh) 抗体及其制备方法
TWI815136B (zh) 一種雙特異性抗體及其用途
WO2023179716A1 (zh) 一种抗cd3抗体、其制备方法及应用
JP2021501569A (ja) Ｐｄｌ１を標的とする抗体及びそれを用いる方法
WO2021244371A1 (zh) 一种抗pd-l1/vegf融合蛋白
CN117616048A (zh) Cd19抗体及其应用
CN116761889A (zh) 抗原结合蛋白及其用途
WO2024032664A1 (zh) 一种靶向pd-l1和vegf的抗体及其应用
WO2024012513A1 (en) Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
WO2024131846A1 (en) Antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
TWI845767B (zh) 抗人Claudin18.2抗體及其應用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23773962

Country of ref document: EP

Kind code of ref document: A1