WO2021223719A1 - Antibody directed against cd19 antibody, and preparation therefor and application thereof - Google Patents
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- WO2021223719A1 WO2021223719A1 PCT/CN2021/091955 CN2021091955W WO2021223719A1 WO 2021223719 A1 WO2021223719 A1 WO 2021223719A1 CN 2021091955 W CN2021091955 W CN 2021091955W WO 2021223719 A1 WO2021223719 A1 WO 2021223719A1
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- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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- A—HUMAN NECESSITIES
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- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
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- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- the present invention relates to the field of biomedicine, and more specifically to an anti-CD19 antibody and its preparation and application.
- Antibodies are protective proteins produced by the body under the stimulation of antigens, which are secreted by plasma cells into blood and other body fluids. Antibodies can specifically bind to antigens to neutralize toxins and prevent pathogens from invading. According to the specific binding characteristics of antibodies and antigens, antibody drugs against disease-specific biological targets can be developed for the treatment of diseases. Antibody drugs have been applied to the field of anti-tumor and autoimmune treatments. At the same time, they also play an increasingly important role in the field of anti-viral and bacterial infections, cardiovascular and cerebrovascular, diabetes and rare disease treatments. It is the compound growth rate of current biological drugs. The highest class of drugs.
- CD19 is a kind of cluster differentiation antigen and an important membrane antigen related to B cell proliferation, differentiation, activation and antibody production.
- CD19 is highly expressed on the surface of most B-cell malignant tumors, and T cells modified by chimeric antigen receptors (CAR) developed independently by multiple centers target B cells expressing CD19 to relapse and refractory Malignant tumors have achieved unprecedented success.
- Immunotherapy led by CAR-T has brought hope to countless patients to "cure cancer", but while CAR-T has outstanding effectiveness, it also has an urgent problem that needs to be solved, that is, serious side effects-cytokines. Storm (CRS).
- CRS chimeric antigen receptors
- Cytokine storm means that after CAR-T infusion is completed in the human body, T lymphocytes are activated and proliferate rapidly in the body, causing TNF- ⁇ , IFN- ⁇ , IL-1, IL-2, IL-4, IL -6, IL-8, IL-10, IL-12 and other cytokines are excessively released in a cascade. These cytokines mediate a variety of immune responses, causing clinical manifestations such as high fever, hypotension, myalgia, coagulopathy, dyspnea, end organ disorders, etc., and may cause serious permanent damage or failure to human tissues and organs. , And even lead to death. In short, cytokine storm is a severe non-specific inflammatory response caused by the explosive secretion of large amounts of cytokines by immune cells in the body during CAR-T treatment.
- CAR-T cells can proliferate rapidly and release a large amount of cytokines, thereby killing target cells through cytokines. Therefore, cytokine storm cannot be regarded as a side effect of CAR-T, and it is also an effective clinical manifestation of CAR-T in vivo.
- CAR-T clinical treatment the generation of cytokine storm cannot be avoided, and we can only rely on routine symptomatic treatment such as close observation and active response.
- the field urgently needs to develop drugs and methods that can cope with the cytokine storm after CAR-T treatment.
- the purpose of the present invention is to provide an anti-CD19 antibody and its preparation and application.
- the heavy chain variable region includes the following three complementarity determining region CDRs:
- any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
- the heavy chain variable region further includes a human FR region or a murine FR region.
- the heavy chain variable region CDRs are shown in positions 50-54, 69-85, and 118-130 of the amino acid sequence shown in SEQ ID NO: 19.
- the heavy chain variable region CDRs are shown in positions 50-54, 69-85, and 118-123 of the amino acid sequence shown in SEQ ID NO:21.
- the heavy chain variable region CDRs are shown at positions 50-54, 69-85, and 118-129 of the amino acid sequence shown in SEQ ID NO: 23.
- amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, 27, 29, 31, 33.
- a heavy chain of an antibody having the heavy chain variable region of the first aspect of the present invention.
- the heavy chain of the antibody also includes a heavy chain constant region.
- the heavy chain constant region is of human, murine or rabbit origin.
- amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, 21 or 23 (the heavy chain constant region is of human origin).
- a light chain variable region of an antibody includes the following three complementarity determining region CDRs:
- any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
- the light chain variable region further includes a human FR region or a murine FR region.
- the light chain variable region CDRs are shown at positions 46-56, 72-78, and 114-119 of the amino acid sequence shown in SEQ ID NO: 20.
- the light chain variable region CDRs are shown at positions 46-61, 77-83, and 119-124 of the amino acid sequence shown in SEQ ID NO: 22.
- the CDR of the light chain variable region is shown at positions 46-60, 76-82, and 115-123 of the amino acid sequence shown in SEQ ID NO: 24.
- amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26, 28, 30, 32, and 34.
- a light chain of an antibody having the light chain variable region of the third aspect of the present invention.
- the light chain of the antibody also includes a light chain constant region.
- the light chain constant region is of human, murine or rabbit origin.
- amino acid sequence of the light chain is shown in SEQ ID NO: 20, 22 or 24 (the light chain constant region is of human origin).
- an antibody having:
- the antibody has: the heavy chain according to the second aspect of the present invention; and/or the light chain according to the fourth aspect of the present invention.
- the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
- the CDR region of the humanized antibody contains 1, 2, or 3 amino acid changes.
- the animal is a non-human mammal, preferably a rat, a sheep, or a rabbit.
- the antibody is a double-chain antibody or a single-chain antibody.
- the antibody is a monoclonal antibody.
- the antibody is a partially or fully humanized monoclonal antibody.
- the antibody is an antibody of the same type as IgG1.
- the number of added, deleted, modified and/or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, more preferably 10%.
- the number of added, deleted, modified and/or substituted amino acids is 1-7, preferably 1-3, and more preferably one.
- the added, deleted, modified and/or substituted at least one amino acid sequence is an amino acid sequence with at least 80% homology.
- the derivative sequence after addition, deletion, modification and/or substitution of at least one amino acid has the function of inhibiting the activity of the CD19 antibody.
- the affinity EC50 of the derived sequence of the anti-CD19 antibody to the CD19 antibody is 0.2-0.3 nM, preferably 0.01-10 nM, more preferably 0.001-100 nM.
- the anti-CD19 antibody antibody can bind to CD19-targeting CAR and CAR-T cells.
- the anti-CD19 antibody antibody can inhibit the function of CAR and CAR-T cells that target CD19.
- the CD19 antibody includes FMC63 and humanized FMC63.
- the CD19 antibody includes FMC63 and its derivative sequence
- the derivative sequence is a sequence obtained by adding, deleting, modifying and/or substituting at least one amino acid on the basis of the amino acid sequence of FMC63 .
- the FMC63-derived sequence retains binding affinity to CD19.
- the CD19 antibody includes a CD19 double-chain antibody or a CD19 single-chain antibody.
- the sixth aspect of the present invention provides a recombinant protein, the recombinant protein having:
- the tag sequence includes a 6His tag.
- the recombinant protein includes a fusion protein.
- the recombinant protein is a monomer, dimer, or multimer.
- the seventh aspect of the present invention provides a CAR construct.
- the scFv segment of the antigen-binding domain of the CAR construct is a binding region that specifically binds to the CD19 antibody, and the scFv has the characteristics of the first The variable region of the heavy chain as described in the aspect and the variable region of the light chain as described in the third aspect of the present invention.
- the eighth aspect of the present invention provides a recombinant immune cell that expresses an exogenous CAR construct as described in the seventh aspect of the present invention.
- the ninth aspect of the present invention provides an antibody-drug conjugate, said antibody-drug conjugate containing:
- An antibody portion which is selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, and the light chain according to the third aspect of the present invention Variable region, light chain according to the fourth aspect of the present invention, or antibody according to the fifth aspect of the present invention, or a combination thereof;
- a coupling portion coupled to the antibody portion, and the coupling portion is selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
- the antibody portion and the coupling portion are coupled through a chemical bond or linker.
- the tenth aspect of the present invention provides a use of an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, The light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the first aspect of the present invention.
- the detection reagent, detection plate or kit is used for:
- the detection reagent, detection plate or kit is used to detect immune cells expressing a CAR targeting CD19.
- the immune cells are selected from the group consisting of NK cells, T cells, and B cells.
- the immune cells are derived from human or non-human mammals (such as mice).
- the detection reagent, detection plate or kit is used for ELISA detection, FACS detection, electrochemiluminescence detection, and enzyme-linked immunoassay.
- mAb06 26E7D2
- mAb023 75H6C7
- mAb020 52H8C4 has the best affinity in FACS detection.
- the detection reagent is a positive antibody for ADA detection, a standard product.
- the drug is used to eliminate CD19 antibodies or immune cells expressing CARs targeting CD19.
- the drug is used to block the CD19 antibody or the immune cell function of expressing CAR that targets CD19.
- mAb019, mAb020, and mAb021 have the best blocking effects.
- the drug assists in the treatment of CD19-related diseases.
- the CD19-related disease is selected from the following group: cancer, autoimmune disease, metabolic-related disease, infectious disease, or a combination thereof.
- the cancer includes solid tumors and blood cancers.
- the cancer is a tumor with high CD19 expression.
- the tumor with high CD19 expression refers to the ratio of the CD19 transcript and/or protein level L1 in the tumor tissue to the transcript and/or protein level L0 in the normal tissue, L1/L0 ⁇ 2 , Preferably ⁇ 3.
- the metabolic-related diseases include diabetes, food-borne obesity, and fatty inflammation.
- infectious diseases include: bacterial and viral infections.
- the eleventh aspect of the present invention provides a pharmaceutical composition, which contains:
- An active ingredient which is selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, and the light chain according to the third aspect of the present invention Variable region, light chain according to the fourth aspect of the present invention, or antibody according to the fifth aspect of the present invention, recombinant protein according to the sixth aspect of the present invention, immune cell according to the eighth aspect of the present invention, The antibody drug conjugate according to the ninth aspect, or a combination thereof; and
- the pharmaceutical composition is a liquid preparation.
- the pharmaceutical composition is an injection.
- the twelfth aspect of the present invention provides a polynucleotide which encodes a polypeptide selected from the following group:
- the thirteenth aspect of the present invention provides a vector containing the polynucleotide according to the twelfth aspect of the present invention.
- the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
- the fourteenth aspect of the present invention provides a genetically engineered host cell, the host cell contains the vector according to the thirteenth aspect of the present invention or the genome integrates the polynucleus according to the twelfth aspect of the present invention. Glycidic acid.
- the fifteenth aspect of the present invention provides a method for detecting CD19 antibody in a sample (including diagnostic or non-diagnostic) in vitro, the method comprising the steps:
- the sixteenth aspect of the present invention provides a test board, the test board comprising: a substrate (support plate) and a test strip, the test strip containing the antibody according to the fifth aspect of the present invention or the present invention
- the immunoconjugate of the ninth aspect is not limited to: a substrate (support plate) and a test strip, the test strip containing the antibody according to the fifth aspect of the present invention or the present invention.
- the seventeenth aspect of the present invention provides a kit including:
- a first container containing the antibody according to the fifth aspect of the present invention and/or
- a second container which contains a secondary antibody against the antibody of the fifth aspect of the present invention
- the kit contains the detection plate according to the sixteenth aspect of the present invention.
- the eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
- the nineteenth aspect of the present invention provides a method for eliminating CD19 antibodies or immune cells expressing CARs targeting CD19, the method comprising: administering the antibodies described in the fifth aspect of the present invention, the An antibody-drug conjugate of an antibody, or a CAR-T cell expressing the antibody, or a combination thereof.
- the method further includes: administering other drugs or treatment methods to the subject in need for combined therapy.
- the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapeutics, and tumor radiotherapy.
- a method for preparing a chimeric antibody including the steps:
- the nucleotide sequence of the heavy chain variable region of the first aspect of the present invention and/or the light chain variable region of the third aspect of the present invention is cloned into an expression vector containing the nucleotide sequence of a human antibody constant region Afterwards, the human-mouse chimeric antibody was expressed by transfecting animal cells.
- a method for preparing a humanized antibody including the steps:
- the nucleotide sequence of the CDR region in the heavy chain variable region of the first aspect of the present invention and/or the light chain variable region of the third aspect of the present invention is implanted into a nucleoside containing the FR region of a human antibody After the acid sequence template is cloned into an expression vector containing the constant region of a human antibody, the humanized antibody is expressed by transfecting animal cells.
- a bispecific antibody comprising: the antibody of the fifth aspect of the present invention and a second antibody, and the second antibody is selected from the group consisting of Group: CD3, CD47, PD-1, PD-L1 antibodies, and anti-BCMA, CD20, CD22, CD33, CD123 anti-antibodies.
- Figure 1 shows the ELISA test results of the sera of immunized mice binding to FMC63.
- the four-digit number 7267-7274 indicates the mouse number
- TB3 indicates that the mouse has been immunized 3 times in total.
- Figure 2 shows the affinity (EC50) of anti-FMC63 antibodies detected by ELISA.
- FIG. 3 shows the affinity of anti-FMC63 antibodies detected by FACS.
- Figure 4 shows the test results of the anti-FMC63 antibody combination applied to the anti-antibody method.
- Figure 5 shows that the anti-FMC63 antibody can block the binding of CD19CAR-T cells to the CD19 target, where the abscissa represents the mAb number.
- FIG. 6 shows that anti-FMC63 antibodies can promote the proliferation of CD19CAR-T cells.
- Figure 7 shows that anti-FMC63 antibodies can promote cytokine release from CD19CAR-T cells.
- FIG. 8 shows that anti-FMC63 antibodies can activate CD19CAR-T cells.
- Figure 9 shows the CAR-positive rate of CAR-T cells targeting FMC63.
- Figure 10 shows that CAR-T cells targeting FMC63 can kill cells expressing the target antigen.
- Figure 11 shows that CAR-T cells targeting FMC63 release cytokines under the stimulation of cells expressing the target antigen.
- Figure 12 shows that CAR-T cells targeting FMC63 can be activated by cells expressing the target antigen.
- NT in the figure represents untreated T cells.
- the inventors unexpectedly discovered a humanized anti-CD19 antibody for the first time.
- the present invention uses a synthetic CD19 antibody (scFv) to immunize mice, take mouse spleens to prepare hybridoma cells, screen to obtain murine anti-CD19 antibody antibodies, and obtain humanized antibodies by replacing the human Fc segment.
- Anti-CD19 antibody The humanized anti-CD19 antibody can be used to detect CD19 antibody, prepare antibody drugs, and prepare CAR-T cells targeting CD19 antibody.
- VH refers to the variable region of the heavy chain
- VL refers to the variable region of the light chain
- VH-CDR1 refers to CDR1 of the heavy chain variable region
- VH-CDR2 refers to CDR2 of the heavy chain variable region
- VH-CDR3 refers to CDR3 of the heavy chain variable region.
- VL-CDR1 refers to CDR1 of the light chain variable region
- VL-CDR2 refers to CDR2 of the light chain variable region
- VL-CDR3 refers to CDR3 of the light chain variable region.
- FMC63 is a murine anti-CD19 IgG2a antibody.
- CAR-T cell drugs designed using FMC63 have been successfully marketed, and have good curative effects in the treatment of B-ALL and NHL.
- the amino acid sequence of FMC63 scFv is as follows:
- antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions.
- VH variable region
- Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain .
- Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.
- variable means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
- CDR complementarity determining regions
- FR framework region
- the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure.
- the CDRs in each chain are closely held together by the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain. Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
- immunoglobulins can be classified into one of two distinct categories (called kappa and lambda) based on the amino acid sequence of their constant regions. According to the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be divided into different types. There are mainly five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.
- the heavy chain constant regions corresponding to different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
- variable regions which are divided into 4 framework regions (FR), 4
- FR framework regions
- the amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the ⁇ sheets formed by the FRs in between are close to each other in space structure, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody.
- the amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
- the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
- antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art.
- Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombination techniques, and they are all useful antibodies.
- a chimeric antibody is a molecule in which different parts are derived from different animal species, for example, a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent Nos. 4,816,567 and U.S. Patent 4,816,397, which is hereby incorporated by reference in its entirety).
- Humanized antibodies refer to antibody molecules derived from non-human species, with one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see U.S. Patent 5,585,089, This article is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.
- CDRs complementarity determining regions
- the antibody may be monospecific, bispecific, trispecific, or more multispecific.
- the antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
- conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
- substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Preferred substitution Ala(
- the antibody is an anti-CD19 antibody, and the CD19 antibody includes FMC63 and its derivative sequences.
- the antibody of the present invention includes a heavy chain containing a heavy chain variable region (VH) amino acid sequence and a light chain containing a heavy chain variable region (VH) amino acid sequence, and the light chain containing a light chain variable region (VL) amino acid sequence.
- the heavy chain variable region (VH) has a complementarity determining region CDR selected from the following group:
- the light chain variable region (VL) has a complementarity determining region CDR selected from the following group:
- any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
- the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology or sequence identity of at least 80%, preferably at least 85%, more preferably The ground is at least 90%, and most preferably at least 95% of the amino acid sequence.
- the preferred method for determining identity is to obtain the largest match between the tested sequences.
- the method for determining identity is compiled in a publicly available computer program.
- Preferred computer program methods for determining the identity between two sequences include but are not limited to: GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990).
- the public can obtain the BLASTX program from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990).
- the well-known Smith Waterman algorithm can also be used to determine identity.
- the antibody of the present invention may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.
- the animal is preferably a mammal, such as a mouse.
- the antibody derivatives of the present invention may be single-chain antibodies and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other antibody derivatives known in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
- the antibodies of the present invention may be chimeric antibodies, humanized antibodies, CDR grafted and/or modified antibodies that target CD19 antibodies.
- the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 19, 21 or 23.
- the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 20, 22 or 24.
- the antibody of the present invention may be an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody fragment, scFv), a single domain antibody (single domain antibody, sdAb), and One or more of single-domain antibodies (Signle-domain antibodies), and monoclonal antibodies or polyclonal antibodies made from the above antibodies.
- the monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
- the antibody full-length protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region.
- the heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a fully human antibody full-length protein.
- the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.
- the single-chain antibody is a conventional single-chain antibody in the field, which includes a heavy chain variable region, a light chain variable region and short peptides of 15-20 amino acids.
- the antigen-antibody binding domain protein fragments are conventional antigen-antibody binding domain protein fragments in the art, which include the light chain variable region, the light chain constant region and the Fd segment of the heavy chain constant region.
- the antigen-antibody binding domain protein fragments are Fab and F(ab').
- the single domain antibody is a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
- the single-domain antibody is a conventional single-domain antibody in the art, which only includes the variable region of the heavy chain.
- the preparation method of the recombinant protein is a conventional preparation method in the art.
- the preparation method is preferably: isolated from an expression transformant that recombinantly expresses the protein or obtained by artificially synthesizing a protein sequence.
- Said method of separating and obtaining from an expression transformant that recombinantly expresses the protein is preferably as follows: cloning a nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector, and transforming the obtained recombinant vector into a transformant to obtain recombinant expression
- the transformant can be isolated and purified to obtain the recombinant protein by culturing the obtained recombinant expression transformant.
- the present invention also provides a nucleic acid that encodes the heavy chain variable region or the light chain variable region of the aforementioned antibody or recombinant protein or anti-CD19 antibody.
- the preparation method of the nucleic acid is a conventional preparation method in the art, preferably, it includes the following steps: obtain the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtain the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-sequence synthesis .
- the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately substituted, deleted, altered, inserted or added to provide a polynucleotide homolog.
- the polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
- the present invention also provides a recombinant expression vector containing the nucleic acid.
- the recombinant expression vector can be obtained by conventional methods in the art, that is, the nucleic acid molecule of the present invention is connected to various expression vectors to be constructed.
- the expression vector is a variety of conventional vectors in the field, as long as it can hold the aforementioned nucleic acid molecule.
- Said vectors preferably include: various plasmids, cosmids, phage or virus vectors and the like.
- the present invention also provides a recombinant expression transformant comprising the above-mentioned recombinant expression vector.
- the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the recombinant expression vector is transformed into a host cell.
- the host cell is a variety of conventional host cells in the art, as long as the recombinant expression vector can replicate itself stably and the nucleic acid carried can be effectively expressed.
- the host cell is E. coli TG1 or E. coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody).
- the aforementioned recombinant expression plasmid is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention.
- the transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
- sequence of the DNA molecule of the antibody or its fragment of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening.
- the coding sequences of the light chain and the heavy chain can also be fused together to form a single chain antibody.
- the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
- artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
- the DNA sequence encoding the antibody (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
- the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
- the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
- Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
- the transformed host cell is cultured under conditions suitable for expression of the antibody of the present invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc.
- the antibody of the present invention is purified by conventional separation and purification means well-known to the person.
- the obtained monoclonal antibody can be identified by conventional means.
- the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
- RIA radioimmunoassay
- ELISA enzyme-linked immunosorbent assay
- the binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
- the antibody of the present invention can be expressed in the cell, on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
- conventional renaturation treatment treatment with protein precipitation agent (salting out method)
- centrifugation osmotic sterilization
- ultrasonic treatment ultracentrifugation
- molecular sieve chromatography gel filtration
- adsorption layer Analysis ion exchange chromatography
- ADC Antibody-drug conjugate
- the present invention also provides antibody-drug conjugate (ADC) based on the antibody of the present invention.
- ADC antibody-drug conjugate
- the antibody-conjugated drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and preferably is chemically coupled.
- the effector molecule is preferably a drug with therapeutic activity.
- the effector molecule may be one or more of toxic protein, chemotherapeutic drug, small molecule drug or radionuclide.
- the antibody of the present invention and the effector molecule may be coupled through a coupling agent.
- the coupling agent may be any one or more of non-selective coupling agents, coupling agents using carboxyl groups, peptide chains, and coupling agents using disulfide bonds.
- the non-selective coupling agent refers to a compound that makes the effector molecule and the antibody form a covalent bond, such as glutaraldehyde.
- the coupling agent using carboxyl groups can be any one or more of cis-aconitic acid anhydride coupling agents (such as cis-aconitic acid anhydride) and acyl hydrazone coupling agents (coupling sites are acyl hydrazones).
- Certain residues on the antibody are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes) , Stabilizers (such as glycol polymers) and therapeutic agents.
- the antibody can be conjugated to the functional agent to form an antibody-functional agent conjugate.
- Functional agents e.g. drugs, detection reagents, stabilizers
- the functional agent may be directly or indirectly linked to the antibody through a linker.
- Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs).
- ADC antibody-drug conjugates
- the ADC contains a linker between the drug and the antibody.
- the linker can be a degradable or a non-degradable linker.
- Degradable linkers are typically easily degraded in the intracellular environment, for example, the linker is degraded at the target site, so that the drug is released from the antibody.
- Suitable degradable linkers include, for example, enzymatically degraded linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, for example, which can be degraded by glucuronide.
- Enzymatically degraded glucuronide-containing linker may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine.
- suitable degradable linkers include, for example, pH-sensitive linkers (for example, linkers that are hydrolyzed at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (for example, disulfide bond linkers).
- Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by a protease.
- the antibody-drug conjugate ADC is represented by the following molecular formula:
- Ab is an antibody
- D is a drug
- CAR-T the full name is Chimeric Antigen Receptor T-Cell, refers to the chimeric antigen receptor T cell, among which the chimeric antigen receptor (CAR) is the core component of CAR-T, which gives T cells recognition in an HLA-independent manner The ability of tumor antigens, which allows CAR-modified T cells to recognize a wider range of targets than the natural T cell surface receptor TCR.
- the basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFv segment of the monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region and an intracellular signal Area.
- TAA tumor-associated antigen
- the design of CARs has gone through the following process:
- the first-generation CAR has only one intracellular signal component CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , And cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical effects.
- the second-generation CARs introduce a costimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function has been greatly improved, which further strengthens the persistence of CAR-T cells and the effect on tumor cells. The lethality.
- some new immunostimulatory molecules such as CD27 and CD134 are connected in series to develop into the third-generation and fourth-generation CARs.
- the antibody or ADC of the present invention can be used in detection applications, for example, to detect samples, thereby providing diagnostic information.
- the samples (samples) used include cells, tissue samples and biopsy specimens.
- the term "biopsy” used in the present invention shall include all kinds of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, excision samples of tumors, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
- the samples used in the present invention include fixed or preserved cell or tissue samples.
- the present invention also provides a kit containing the antibody (or a fragment thereof) of the present invention.
- the kit further includes a container, instructions for use, buffer, and the like.
- the antibody of the present invention can be immobilized on a detection plate.
- the invention also provides a composition.
- the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment or fusion protein or ADC or corresponding immune cell, and a pharmaceutically acceptable carrier.
- these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can be The nature of the formulated substance and the condition to be treated vary.
- the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
- the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration.
- the injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection.
- the pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably a tablet, capsule, or granule. , Injection or infusion, etc.
- the antibody of the present invention can also be used for cell therapy by expressing the nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
- CAR-T chimeric antigen receptor T cell immunotherapy
- the pharmaceutical composition of the present invention can be directly used in CAR-T cells that bind CD19 antibodies or target CD19, and thus can be used to assist in the treatment of tumors and other diseases.
- the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutical Acceptable carrier or excipient.
- a pharmaceutical Acceptable carrier or excipient include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
- the pharmaceutical preparation should match the mode of administration.
- the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
- the dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day.
- the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers.
- the pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient.
- the pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01-99.99% of the aforementioned protein and 0.01-99.99% of a pharmaceutical carrier, and the percentage is a mass percentage of the pharmaceutical composition.
- the administration amount of the pharmaceutical composition is an effective amount
- the effective amount is an amount that can alleviate or delay the progression of a disease, degenerative or traumatic condition.
- the effective amount can be determined on an individual basis and will be partly based on consideration of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the aforementioned factors such as individual basis and using no more than conventional experiments.
- a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases not more than about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight.
- the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
- the antibody of the present invention can be used to detect CD19 antibody, such as for ELISA detection, FACS detection and the like.
- the antibody of the present invention can block the function of CD19 antibody, such as FMC63.
- the antibody of the present invention binds to CAR-T targeting CD19, thereby blocking the function of CAR-T and inhibiting the occurrence and development of cytokine storm, neurotoxicity and other CAR-T-related toxic and side effects.
- FMC63 scFv CAR GMCSF signal peptide-FMC63 scFv-Flag-CD28 hinge-CD28 transmembrane region-CD28 costimulator region-CD3z
- FMC63 scFv CAR lentiviral vector Infect Jurkat cells, screen the monoclonal Jurkat-FMC63 scFv cells stably expressing FMC63 scFv CAR, and identify the monoclonal cells by flow cytometry.
- FMC63-scFv-mFc Integrate the nucleotide sequence of FMC63-scFv-mFc (a chain of FMC63 antibody) into the eukaryotic expression vector, and use PEI to transfer the expression vector carrying FMC63 nucleotides into 293 cells to express FMC63 protein, Purified by protein A column to obtain FMC63 protein (FMC63-scFv-mFc protein).
- mice were immunized according to the method shown in Table 1 below.
- mice were screened by ELISA/FACS.
- the mouse spleen lymphocytes were fused with Sp2/0-Ag14 cells to obtain hybridoma cells.
- the hybridoma cells were plated into a 96-well plate for growth, and the cells were taken 10-14 days later.
- Use ELISA/FACS to screen cells, dissociation rate constants, and select highly specific subclones for expansion culture and cryopreservation.
- the ELISA detection method is as follows: use 100ul 1ug/ml FMC63-scFv-mFc coated plate, incubate overnight at four degrees; wash three times with PBST, block with 1% BSA at 37°C for 1 hour; wash three times with PBST, add 50ul mAb anti-antibody ( 40ug/ml) and biotin-labeled FMC63-scFv-mFc; incubate at 37°C for 1 hour and wash three times with PBST; add 100ul secondary antibody to each well and incubate at 37°C for 1 hour; wash five times with PBST and add 100ul TMB to each well; Add 50ul 1M HCl to the well to stop the reaction; read the plate under the 450nm wavelength of the microplate reader;
- the FACS detection method is as follows: mix mAb with Jurkat-FMC63 scFv cells, incubate at room temperature for 30 minutes, wash once with DPBS, add anti-mouse Fc antibody to incubate at room temperature for 30 minutes, wash once with DPBS and perform flow-based detection.
- Antibody Clone number EC50(ug/ml)
- Antibody Clone number EC50(ug/ml) mAb01 4C5E2 0.03479 mAb013 9H2G1 0.03986 mAb02 8B3H4 0.02298 mAb014 20H5D10 0.0628 mAb03 14A10E7 0.8218 mAb015 22G8F7 53629 mAb04 17F10D3 0.02273 mAb016
- 40F12E5 0.04888 mAb05 26E2F11 0.0714 mAb017 35H8C4 0.05389 mAb06 26E7D2 0.0299 mAb018
- a competitive ELISA was used to detect antibody binding epitopes.
- Combinations of antibodies that bind different epitopes of FMC63 are selected and mixed at the same concentration as the positive product (PC), and the anti-antibody detection experiment is designed.
- the 23 antibodies shown in Table 2 were added to FMC63-CAR-T cells, and the density and number of FMC63-CAR-T cells were detected at different time points. The results are shown in Figures 6, 7, and 8. Anti-FMC63 antibodies can promote the proliferation, activation and cytokine release of FMC63-CAR-T cells.
- mAb019 and mAb020 can interfere with the binding of CD19 protein to Jurkat-FMC63 scFv cells, thereby blocking the binding of CAR to the target CD19.
- the Fc segments of the murine anti-FMC63 antibodies mAb02, mAb06 and mAb020 were replaced with human Fc to obtain human-mouse chimeric antibodies.
- the heavy chain amino acid sequences of the humanized mAb02, mAb06 and mAb020 antibodies are shown in SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, respectively, and the light chain amino acid sequences are shown in SEQ ID NO: 20, SEQ ID, respectively. NO: 22, SEQ ID NO: 24.
- the structure is signal peptide-anti-FMC63 scFv-CD8 hinge-CD8 transmembrane region-CD137 costimulatory region-CD3z, the amino acid sequence is as SEQ respectively ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49.
- Cell resuscitation Thaw PBMC in a 37°C water bath for about 2 minutes, transfer the cell suspension to 30 mL of pre-warmed CAR-T basal medium (X-vivo 15 with 1% HSA), mix well, and sample and count Centrifuge the remaining cells at 300g for 10 minutes.
- pre-warmed CAR-T basal medium X-vivo 15 with 1% HSA
- CAR-T basal medium After mixing, add appropriate amount of CAR-T basal medium to make the cell density reach 2 ⁇ 10 6 /mL, and add 1/1000 volume of IMC0003 . After mixing, take 2-3 mL of cell suspension in a 6-well plate as negative control cells, and the remaining cells are used for CAR-T cell preparation.
- CAR-T cells after pipetting and mixing, sample and count, take about 0.5 ⁇ 10 6 cells for positive rate detection, and add CART basal medium to continue culturing.
- the extracellular structure of CAR contains the amino acid sequence of DYKDDDDK, and anti-Flag antibody is used to detect the positive rate of CAR+.
- the results are shown in Fig. 9 that CAR was successfully expressed on T cells.
- CAR-T Incubate CAR-T with target cells (Hela-FMC63, K562-FMC63) according to an effective target ratio of 1:1; use real-time label-free dynamic cell analysis technology and flow cytometry to detect the activity of target cells to obtain CAR-T activation and Cytotoxic effect. At the same time, the cell supernatant was taken to detect the release of cytokines, including IL2 and IFN ⁇ .
- CAR-T targeting CD19 antibody can produce cytotoxic effect on target cell Hela-FMC63, as shown in Figure 10; CAR-T targeting CD19 antibody can be activated by target cell K562-FMC63 and release cytokines IFN ⁇ and IL2 , As shown in Figure 11 and Figure 12.
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Abstract
An antibody targeting a CD19 antibody, and a preparation method therefor and a use thereof. Specifically, provided is a novel antibody targeting a CD19 antibody. Also provided is a method for preparing a monoclonal antibody. The monoclonal antibody can bind with high specificity to a CD19 antibody and block the function of the CD19 antibody.
Description
本发明涉及生物医药领域，更具体地涉及一种抗CD19抗体的抗体及其制备和应用。The present invention relates to the field of biomedicine, and more specifically to an anti-CD19 antibody and its preparation and application.
抗体是机体在抗原刺激下产生的具有保护作用的蛋白质，由浆细胞分泌到血液等体液中。抗体可以与抗原特异性结合，起到中和毒素、阻止病原体入侵等作用。根据抗体与抗原特异结合的特性，可以开发针对疾病特异性生物靶点的抗体药物用于治疗疾病。抗体药已经应用到抗肿瘤领域和自身免疫的治疗，同时在抗病毒和细菌感染、心脑血管、糖尿病以及罕见病治疗等领域也发挥越来越重要的作用，是当前生物药中复合增长率最高的一类药物。Antibodies are protective proteins produced by the body under the stimulation of antigens, which are secreted by plasma cells into blood and other body fluids. Antibodies can specifically bind to antigens to neutralize toxins and prevent pathogens from invading. According to the specific binding characteristics of antibodies and antigens, antibody drugs against disease-specific biological targets can be developed for the treatment of diseases. Antibody drugs have been applied to the field of anti-tumor and autoimmune treatments. At the same time, they also play an increasingly important role in the field of anti-viral and bacterial infections, cardiovascular and cerebrovascular, diabetes and rare disease treatments. It is the compound growth rate of current biological drugs. The highest class of drugs.
CD19是簇分化抗原的一种，是B细胞增殖、分化、活化及抗体产生有关的重要膜抗原。绝大多数的B细胞性恶性肿瘤表面都高表达CD19,多个中心独立开展的利用嵌合抗原受体(Chimeric Antigen Receptor，CAR)修饰的T细胞靶向表达CD19的B细胞复发、难治性恶性肿瘤取得了前所未有的成功。以CAR-T为首的免疫疗法给无数患者带来了“治愈癌症”的希望，但CAR-T在具有突出有效性的同时，也有着一个亟待解决的问题，那就是严重的副作用——细胞因子风暴(CRS)。CD19 is a kind of cluster differentiation antigen and an important membrane antigen related to B cell proliferation, differentiation, activation and antibody production. CD19 is highly expressed on the surface of most B-cell malignant tumors, and T cells modified by chimeric antigen receptors (CAR) developed independently by multiple centers target B cells expressing CD19 to relapse and refractory Malignant tumors have achieved unprecedented success. Immunotherapy led by CAR-T has brought hope to countless patients to "cure cancer", but while CAR-T has outstanding effectiveness, it also has an urgent problem that needs to be solved, that is, serious side effects-cytokines. Storm (CRS).
细胞因子风暴是指在对人体完成CAR-T输注后，T淋巴细胞在体内被激活并快速增殖，引起了TNF-α、IFN-γ、IL-1、IL-2、IL-4、IL-6、IL-8、IL-10、IL-12等细胞因子过度的级联释放。这些细胞因子会介导多种免疫反应，引起患者高烧、低血压、肌痛、凝血障碍、呼吸困难、终末器官障碍等临床表现，有可能对人体的组织器官造成严重的永久性损伤或衰竭，甚至导致死亡。简而言之，细胞因子风暴就是在CAR-T治疗过程中，体内免疫细胞爆发性分泌大量的细胞因子所造成的严重非特异性炎症反应。Cytokine storm means that after CAR-T infusion is completed in the human body, T lymphocytes are activated and proliferate rapidly in the body, causing TNF-α, IFN-γ, IL-1, IL-2, IL-4, IL -6, IL-8, IL-10, IL-12 and other cytokines are excessively released in a cascade. These cytokines mediate a variety of immune responses, causing clinical manifestations such as high fever, hypotension, myalgia, coagulopathy, dyspnea, end organ disorders, etc., and may cause serious permanent damage or failure to human tissues and organs. , And even lead to death. In short, cytokine storm is a severe non-specific inflammatory response caused by the explosive secretion of large amounts of cytokines by immune cells in the body during CAR-T treatment.
然而，在体内靶细胞(癌细胞)的刺激下，CAR-T细胞才会快速增殖、释放大量的细胞因子，从而通过细胞因子杀伤靶细胞。因此，细胞因子风暴不可片面的认定为CAR-T的副作用，也是CAR-T在体内有效的临床表现。目前，在CAR-T临床治疗中，尚无法避免细胞因子风暴的产生，只能依靠密切观察、积极应对等常规对症治疗手段。However, under the stimulation of target cells (cancer cells) in the body, CAR-T cells can proliferate rapidly and release a large amount of cytokines, thereby killing target cells through cytokines. Therefore, cytokine storm cannot be regarded as a side effect of CAR-T, and it is also an effective clinical manifestation of CAR-T in vivo. At present, in CAR-T clinical treatment, the generation of cytokine storm cannot be avoided, and we can only rely on routine symptomatic treatment such as close observation and active response.
综上，本领域迫切需要开发能够应对CAR-T治疗后的细胞因子风暴的药物和方法。In summary, the field urgently needs to develop drugs and methods that can cope with the cytokine storm after CAR-T treatment.
发明内容Summary of the invention
本发明的目的在于提供一种抗CD19抗体的抗体及其制备和应用。The purpose of the present invention is to provide an anti-CD19 antibody and its preparation and application.
在本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：In the first aspect of the present invention, a heavy chain variable region of an antibody is provided. The heavy chain variable region includes the following three complementarity determining region CDRs:
SEQ ID NO:1所示的CDR1，CDR1 shown in SEQ ID NO:1,
SEQ ID NO:2所示的CDR2，和CDR2 shown in SEQ ID NO: 2, and
SEQ ID NO:3所示的CDR3；CDR3 shown in SEQ ID NO: 3;
或者，or,
SEQ ID NO:4的CDR1，CDR1 of SEQ ID NO: 4,
SEQ ID NO:5所示的CDR2，和CDR2 shown in SEQ ID NO: 5, and
SEQ ID NO:6所示的CDR3，CDR3 shown in SEQ ID NO: 6,
或者，or,
SEQ ID NO:7所示的CDR1，CDR1 shown in SEQ ID NO: 7,
SEQ ID NO:8所示的CDR2，和CDR2 shown in SEQ ID NO: 8, and
SEQ ID NO:9所示的CDR3；CDR3 shown in SEQ ID NO: 9;
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CD19抗体的结合亲和力的衍生序列。Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
在另一优选例中，所述重链可变区还包括人源的FR区或鼠源的FR区。In another preferred example, the heavy chain variable region further includes a human FR region or a murine FR region.
在另一优选例中，所述重链可变区CDR如SEQ ID NO:19所示的氨基酸序列的第50-54、69-85、118-130位所示。In another preferred embodiment, the heavy chain variable region CDRs are shown in positions 50-54, 69-85, and 118-130 of the amino acid sequence shown in SEQ ID NO: 19.
在另一优选例中，所述重链可变区CDR如SEQ ID NO:21所示的氨基酸序列的第50–54、69-85、118-123位所示。In another preferred example, the heavy chain variable region CDRs are shown in positions 50-54, 69-85, and 118-123 of the amino acid sequence shown in SEQ ID NO:21.
在另一优选例中，所述重链可变区CDR如SEQ ID NO:23所示的氨基酸序列的第50-54、69-85、118-129位所示。In another preferred example, the heavy chain variable region CDRs are shown at positions 50-54, 69-85, and 118-129 of the amino acid sequence shown in SEQ ID NO: 23.
在另一优选例中，所述重链可变区的氨基酸序列SEQ ID NO:25、27、29、31、33所示。In another preferred embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 25, 27, 29, 31, 33.
在本发明的第二方面，提供了一种抗体的重链，所述的重链具有本发明第一方面所述的重链可变区。In the second aspect of the present invention, there is provided a heavy chain of an antibody, the heavy chain having the heavy chain variable region of the first aspect of the present invention.
在另一优选例中，所述的抗体的重链还包括重链恒定区。In another preferred embodiment, the heavy chain of the antibody also includes a heavy chain constant region.
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的。In another preferred embodiment, the heavy chain constant region is of human, murine or rabbit origin.
在另一优选例中，所述重链的氨基酸序列如SEQ ID NO:19、21或23所示 (重链恒定区为人源)。In another preferred embodiment, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19, 21 or 23 (the heavy chain constant region is of human origin).
在本发明的第三方面，提供了一种抗体的轻链可变区，所述轻链可变区包括以下三个互补决定区CDR：In the third aspect of the present invention, a light chain variable region of an antibody is provided. The light chain variable region includes the following three complementarity determining region CDRs:
SEQ ID NO:10所示的CDR1’，CDR1' shown in SEQ ID NO: 10,
SEQ ID NO:11所示的CDR2’，和CDR2' shown in SEQ ID NO: 11, and
SEQ ID NO:12所示的CDR3’；CDR3' shown in SEQ ID NO: 12;
或者，or,
SEQ ID NO:13所示的CDR1’，CDR1' shown in SEQ ID NO: 13,
SEQ ID NO:14所示的CDR2’，和CDR2' shown in SEQ ID NO: 14, and
SEQ ID NO:15所示的CDR3’；CDR3' shown in SEQ ID NO: 15;
或者，or,
SEQ ID NO:16所示的CDR1’，CDR1' shown in SEQ ID NO: 16,
SEQ ID NO:17所示的CDR2’，和CDR2' shown in SEQ ID NO: 17, and
SEQ ID NO:18所示的CDR3’；CDR3' shown in SEQ ID NO: 18;
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CD19抗体的结合亲和力的衍生序列。Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
在另一优选例中，所述轻链可变区还包括人源的FR区或鼠源的FR区。In another preferred example, the light chain variable region further includes a human FR region or a murine FR region.
在另一优选例中，所述轻链可变区CDR如SEQ ID NO:20所示的氨基酸序列的第46–56、72-78、114-119位所示。In another preferred example, the light chain variable region CDRs are shown at positions 46-56, 72-78, and 114-119 of the amino acid sequence shown in SEQ ID NO: 20.
在另一优选例中，所述轻链可变区CDR如SEQ ID NO:22所示的氨基酸序列的第46-61、77-83、119-124位所示。In another preferred embodiment, the light chain variable region CDRs are shown at positions 46-61, 77-83, and 119-124 of the amino acid sequence shown in SEQ ID NO: 22.
在另一优选例中，所述轻链可变区CDR如SEQ ID NO:24所示的氨基酸序列的第46-60、76-82、115-123位所示。In another preferred embodiment, the CDR of the light chain variable region is shown at positions 46-60, 76-82, and 115-123 of the amino acid sequence shown in SEQ ID NO: 24.
在另一优选例中，所述轻链可变区的氨基酸序列如SEQ ID NO:26、28、30、32、34所示。In another preferred embodiment, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 26, 28, 30, 32, and 34.
在本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有本发明第三方面所述的轻链可变区。In the fourth aspect of the present invention, there is provided a light chain of an antibody, the light chain having the light chain variable region of the third aspect of the present invention.
在另一优选例中，所述的抗体的轻链还包括轻链恒定区。In another preferred embodiment, the light chain of the antibody also includes a light chain constant region.
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的。In another preferred embodiment, the light chain constant region is of human, murine or rabbit origin.
在另一优选例中，所述轻链的氨基酸序列如SEQ ID NO:20、22或24所示(轻链恒定区为人源)。In another preferred embodiment, the amino acid sequence of the light chain is shown in SEQ ID NO: 20, 22 or 24 (the light chain constant region is of human origin).
在本发明的第五方面，提供了一种抗体，所述抗体具有：In the fifth aspect of the present invention, an antibody is provided, the antibody having:
(1)本发明第一方面所述的重链可变区；和/或(1) The heavy chain variable region of the first aspect of the present invention; and/or
(2)本发明第三方面所述的轻链可变区；(2) The light chain variable region of the third aspect of the present invention;
或者，所述抗体具有：本发明第二方面所述的重链；和/或本发明第四方面所述的轻链。Alternatively, the antibody has: the heavy chain according to the second aspect of the present invention; and/or the light chain according to the fourth aspect of the present invention.
在另一优选例中，所述抗体选自：动物源抗体、嵌合抗体、人源化抗体、或其组合。In another preferred embodiment, the antibody is selected from: animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.
在另一优选例中，所述人源化抗体的CDR区包含1、2、或3个氨基酸的变化。In another preferred embodiment, the CDR region of the humanized antibody contains 1, 2, or 3 amino acid changes.
在另一优选例中，所述的动物为非人哺乳动物，较佳地为鼠、羊、兔。In another preferred embodiment, the animal is a non-human mammal, preferably a rat, a sheep, or a rabbit.
在另一优选例中，所述的抗体为双链抗体、或单链抗体。In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.
在另一优选例中，所述的抗体为单克隆抗体。In another preferred embodiment, the antibody is a monoclonal antibody.
在另一优选例中，所述的抗体是部分或全人源化的单克隆抗体。In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.
在另一优选例中，所述的抗体为IgG1同种类型抗体。In another preferred embodiment, the antibody is an antibody of the same type as IgG1.
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量，不超过初始氨基酸序列总氨基酸数量的40％，较佳地为20％，更佳地为10％。In another preferred example, the number of added, deleted, modified and/or substituted amino acids does not exceed 40% of the total number of amino acids in the initial amino acid sequence, preferably 20%, more preferably 10%.
在另一优选例中，所述添加、缺失、修饰和/或取代的氨基酸数量为1-7个，较佳地为1-3个，更佳地为1个。In another preferred example, the number of added, deleted, modified and/or substituted amino acids is 1-7, preferably 1-3, and more preferably one.
在另一优选例中，所述经过添加、缺失、修饰和/或取代的至少一个氨基酸序列为同源性为至少80％的氨基酸序列。In another preferred example, the added, deleted, modified and/or substituted at least one amino acid sequence is an amino acid sequence with at least 80% homology.
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸的衍生序列具有抑制CD19抗体活性的功能。In another preferred embodiment, the derivative sequence after addition, deletion, modification and/or substitution of at least one amino acid has the function of inhibiting the activity of the CD19 antibody.
在另一优选例中，抗CD19抗体的抗体的衍生序列对CD19抗体的亲和力EC50为0.2-0.3nM，较佳地为0.01-10nM，更佳地为0.001-100nM。In another preferred embodiment, the affinity EC50 of the derived sequence of the anti-CD19 antibody to the CD19 antibody is 0.2-0.3 nM, preferably 0.01-10 nM, more preferably 0.001-100 nM.
在另一优选例中，抗CD19抗体的抗体可以与靶向CD19的CAR和CAR-T细胞结合。In another preferred embodiment, the anti-CD19 antibody antibody can bind to CD19-targeting CAR and CAR-T cells.
在另一优选例中，抗CD19抗体的抗体可以抑制靶向CD19的CAR和CAR-T细胞的功能。In another preferred embodiment, the anti-CD19 antibody antibody can inhibit the function of CAR and CAR-T cells that target CD19.
在另一优选例中，所述的CD19抗体包括FMC63、人源化FMC63。In another preferred embodiment, the CD19 antibody includes FMC63 and humanized FMC63.
在另一优选例中，所述的CD19抗体包括FMC63及其衍生序列，所述的衍生序列是在FMC63的氨基酸序列的基础上，经过添加、缺失、修饰和/或取代至少一个氨基酸获得的序列。In another preferred example, the CD19 antibody includes FMC63 and its derivative sequence, and the derivative sequence is a sequence obtained by adding, deleting, modifying and/or substituting at least one amino acid on the basis of the amino acid sequence of FMC63 .
在另一优选例中，所述的FMC63衍生序列保留对CD19的结合亲和力。In another preferred embodiment, the FMC63-derived sequence retains binding affinity to CD19.
在另一优选例中，所述的CD19抗体包括CD19双链抗体、或CD19单链抗体。In another preferred embodiment, the CD19 antibody includes a CD19 double-chain antibody or a CD19 single-chain antibody.
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：The sixth aspect of the present invention provides a recombinant protein, the recombinant protein having:
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；以及(i) The heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, the light chain variable region according to the third aspect of the present invention, and the fourth aspect of the present invention The light chain of the aspect, or the antibody of the fifth aspect of the present invention; and
(ii)任选的协助表达和/或纯化的标签序列。(ii) Optional tag sequence to assist expression and/or purification.
在另一优选例中，所述的标签序列包括6His标签。In another preferred example, the tag sequence includes a 6His tag.
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.
本发明的第七方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合结构域的scFv段为特异性结合于CD19抗体的结合区，并且所述scFv具有如本发明第一方面所述的重链可变区和如本发明第三方面所述的轻链可变区。The seventh aspect of the present invention provides a CAR construct. The scFv segment of the antigen-binding domain of the CAR construct is a binding region that specifically binds to the CD19 antibody, and the scFv has the characteristics of the first The variable region of the heavy chain as described in the aspect and the variable region of the light chain as described in the third aspect of the present invention.
本发明的第八方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明第七方面所述的CAR构建物。The eighth aspect of the present invention provides a recombinant immune cell that expresses an exogenous CAR construct as described in the seventh aspect of the present invention.
本发明的第九方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：The ninth aspect of the present invention provides an antibody-drug conjugate, said antibody-drug conjugate containing:
(a)抗体部分，所述抗体部分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、或其组合；和(a) An antibody portion, which is selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, and the light chain according to the third aspect of the present invention Variable region, light chain according to the fourth aspect of the present invention, or antibody according to the fifth aspect of the present invention, or a combination thereof; and
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。(b) A coupling portion coupled to the antibody portion, and the coupling portion is selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。In another preferred embodiment, the antibody portion and the coupling portion are coupled through a chemical bond or linker.
本发明的第十方面，提供了一种活性成分的用途，所述活性成分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、本发明第六方面所述的重组蛋白、本发明第八方面所述的免疫细胞、本发明第九方面所述的抗体药物偶联物、或其组合，所述活性成分用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备预防和/或治疗CD19相关疾病的药物。The tenth aspect of the present invention provides a use of an active ingredient selected from the group consisting of the heavy chain variable region of the first aspect of the present invention, the heavy chain of the second aspect of the present invention, The light chain variable region according to the third aspect of the present invention, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, the first aspect of the present invention The immune cell according to the eighth aspect, the antibody drug conjugate according to the ninth aspect of the present invention, or a combination thereof, wherein the active ingredient is used for (a) preparing a detection reagent, a detection plate or a kit; and/or (b ) Preparation of drugs for the prevention and/or treatment of CD19-related diseases.
在另一优选例中，所述检测试剂、检测板或试剂盒用于：In another preferred embodiment, the detection reagent, detection plate or kit is used for:
(1)检测样品中的CD19抗体；和/或(1) Detect the CD19 antibody in the sample; and/or
(2)检测免疫细胞表面的CD19抗体；和/或(2) Detection of CD19 antibodies on the surface of immune cells; and/or
(3)检测表达CD19抗体的免疫细胞。(3) Detection of immune cells expressing CD19 antibody.
在另一优选例中，所述检测试剂、检测板或试剂盒用于检测表达靶向CD19的CAR的免疫细胞。In another preferred embodiment, the detection reagent, detection plate or kit is used to detect immune cells expressing a CAR targeting CD19.
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞、B细胞。In another preferred embodiment, the immune cells are selected from the group consisting of NK cells, T cells, and B cells.
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。In another preferred embodiment, the immune cells are derived from human or non-human mammals (such as mice).
在另一优选例中，所述检测试剂、检测板或试剂盒用于ELISA检测、FACS检测、电化学发光检测、酶联免疫检测。其中在ELISA检测中mAb06(26E7D2)、mAb023(75H6C7)亲和力最优，mAb020(52H8C4)在FACS检测中亲和力最优。In another preferred embodiment, the detection reagent, detection plate or kit is used for ELISA detection, FACS detection, electrochemiluminescence detection, and enzyme-linked immunoassay. Among them, mAb06 (26E7D2) and mAb023 (75H6C7) have the best affinity in ELISA detection, and mAb020 (52H8C4) has the best affinity in FACS detection.
在另一优选例中，所述检测试剂为ADA检测的阳性抗体，标准品。In another preferred embodiment, the detection reagent is a positive antibody for ADA detection, a standard product.
在另一优选例中，所述的药物用于清除CD19抗体或表达靶向CD19的CAR的免疫细胞。In another preferred embodiment, the drug is used to eliminate CD19 antibodies or immune cells expressing CARs targeting CD19.
在另一优选例中，所述的药物用于阻断CD19抗体或表达靶向CD19的CAR的免疫细胞功能。其中mAb019、mAb020、mAb021阻断效果最优。In another preferred example, the drug is used to block the CD19 antibody or the immune cell function of expressing CAR that targets CD19. Among them, mAb019, mAb020, and mAb021 have the best blocking effects.
在另一优选例中，所述的药物辅助治疗CD19相关疾病。In another preferred embodiment, the drug assists in the treatment of CD19-related diseases.
在另一优选例中，所述CD19相关疾病选自下组：癌症、自身免疫疾病、代谢相关疾病、感染疾病、或其组合。In another preferred embodiment, the CD19-related disease is selected from the following group: cancer, autoimmune disease, metabolic-related disease, infectious disease, or a combination thereof.
在另一优选例中，所述的癌症包括实体瘤、血液癌。In another preferred example, the cancer includes solid tumors and blood cancers.
在另一优选例中，所述的癌症为CD19高表达的肿瘤。In another preferred example, the cancer is a tumor with high CD19 expression.
在另一优选例中，所述的CD19高表达的肿瘤指肿瘤组织中CD19转录本和/或蛋白的水平L1与正常组织中转录本和/或蛋白的水平L0之比，L1/L0≥2，较佳地≥3。In another preferred example, the tumor with high CD19 expression refers to the ratio of the CD19 transcript and/or protein level L1 in the tumor tissue to the transcript and/or protein level L0 in the normal tissue, L1/L0≥2 , Preferably ≥3.
在另一优选例中，所述代谢相关疾病包括：糖尿病、食源性肥胖和脂肪炎症。In another preferred example, the metabolic-related diseases include diabetes, food-borne obesity, and fatty inflammation.
在另一优选例中，所述感染疾病包括：细菌和病毒感染。In another preferred example, the infectious diseases include: bacterial and viral infections.
本发明的第十一方面，提供了一种药物组合物，所述的药物组合物含有：The eleventh aspect of the present invention provides a pharmaceutical composition, which contains:
(i)活性成分，所述活性成分选自下组：本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体、本发明第六方面所述的重组蛋白、本发明第八方面所述的免疫细胞、本发明第九方面所述的抗体药物偶联物、或其组合；以及(i) An active ingredient, which is selected from the group consisting of the heavy chain variable region according to the first aspect of the present invention, the heavy chain according to the second aspect of the present invention, and the light chain according to the third aspect of the present invention Variable region, light chain according to the fourth aspect of the present invention, or antibody according to the fifth aspect of the present invention, recombinant protein according to the sixth aspect of the present invention, immune cell according to the eighth aspect of the present invention, The antibody drug conjugate according to the ninth aspect, or a combination thereof; and
(ii)药学上可接受的载体。(ii) A pharmaceutically acceptable carrier.
在另一优选例中，所述的药物组合物为液态制剂。In another preferred embodiment, the pharmaceutical composition is a liquid preparation.
在另一优选例中，所述的药物组合物为注射剂。In another preferred embodiment, the pharmaceutical composition is an injection.
本发明的第十二方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：The twelfth aspect of the present invention provides a polynucleotide which encodes a polypeptide selected from the following group:
(1)本发明第一方面所述的重链可变区、本发明第二方面所述的重链、本发明第三方面所述的轻链可变区、本发明第四方面所述的轻链、或本发明第五方面所述的抗体；或(1) The heavy chain variable region described in the first aspect of the present invention, the heavy chain described in the second aspect of the present invention, the light chain variable region described in the third aspect of the present invention, and the light chain variable region described in the fourth aspect of the present invention Light chain, or the antibody of the fifth aspect of the present invention; or
(2)本发明第六方面所述的重组蛋白；(2) The recombinant protein of the sixth aspect of the present invention;
(3)本发明第七方面所述的CAR构建物。(3) The CAR construct described in the seventh aspect of the present invention.
本发明的第十三方面，提供了一种载体，所述的载体含有本发明第十二方面所述的多核苷酸。The thirteenth aspect of the present invention provides a vector containing the polynucleotide according to the twelfth aspect of the present invention.
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。In another preferred example, the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
本发明的第十四方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有本发明第十三方面所述的载体或基因组中整合有本发明第十二方面所述的多核苷酸。The fourteenth aspect of the present invention provides a genetically engineered host cell, the host cell contains the vector according to the thirteenth aspect of the present invention or the genome integrates the polynucleus according to the twelfth aspect of the present invention. Glycidic acid.
本发明的第十五方面，提供了一种体外检测(包括诊断性或非诊断性)样品中CD19抗体的方法，所述方法包括步骤：The fifteenth aspect of the present invention provides a method for detecting CD19 antibody in a sample (including diagnostic or non-diagnostic) in vitro, the method comprising the steps:
(1)在体外，将所述样品与本发明第五方面所述的抗体接触；(1) In vitro, contacting the sample with the antibody of the fifth aspect of the present invention;
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD19抗体。(2) Detect whether an antigen-antibody complex is formed. The formation of a complex indicates the presence of CD19 antibody in the sample.
本发明的第十六方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有本发明第五方面所述的抗体或本发明第九方面所述的免疫偶联物。The sixteenth aspect of the present invention provides a test board, the test board comprising: a substrate (support plate) and a test strip, the test strip containing the antibody according to the fifth aspect of the present invention or the present invention The immunoconjugate of the ninth aspect.
本发明的第十七方面，提供了一种试剂盒，所述试剂盒中包括：The seventeenth aspect of the present invention provides a kit including:
(1)第一容器，所述第一容器中含有本发明第五方面所述的抗体；和/或(1) A first container containing the antibody according to the fifth aspect of the present invention; and/or
(2)第二容器，所述第二容器中含有抗本发明第五方面所述的抗体的二抗；(2) A second container, which contains a secondary antibody against the antibody of the fifth aspect of the present invention;
或者，所述试剂盒含有本发明第十六方面所述的检测板。Alternatively, the kit contains the detection plate according to the sixteenth aspect of the present invention.
本发明的第十八方面，提供了一种重组多肽的制备方法，所述方法包括：The eighteenth aspect of the present invention provides a method for preparing a recombinant polypeptide, the method comprising:
(a)在适合表达的条件下，培养本发明第十四方面所述的宿主细胞；(a) Culturing the host cell according to the fourteenth aspect of the present invention under conditions suitable for expression;
(b)从培养物中分离出重组多肽，所述的重组多肽是本发明第五方面所述 的抗体或本发明第六方面所述的重组蛋白。(b) Isolating a recombinant polypeptide from the culture, where the recombinant polypeptide is the antibody according to the fifth aspect of the present invention or the recombinant protein according to the sixth aspect of the present invention.
本发明的第十九方面，提供了一种清除CD19抗体或表达靶向CD19的CAR的免疫细胞的方法，所述方法包括：给需要的对象施用本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。The nineteenth aspect of the present invention provides a method for eliminating CD19 antibodies or immune cells expressing CARs targeting CD19, the method comprising: administering the antibodies described in the fifth aspect of the present invention, the An antibody-drug conjugate of an antibody, or a CAR-T cell expressing the antibody, or a combination thereof.
在另一优选例中，所述的方法还包括：给需要的对象施用其他药物或治疗方法进行联合治疗。In another preferred embodiment, the method further includes: administering other drugs or treatment methods to the subject in need for combined therapy.
在另一优选例中，所述的其他药物或治疗方法包括：抗肿瘤免疫治疗药物、肿瘤靶向药物、肿瘤化疗药物、肿瘤放射治疗。In another preferred example, the other drugs or treatment methods include: anti-tumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapeutics, and tumor radiotherapy.
在本发明的第二十方面，提供了一种制备嵌合抗体的方法，包括步骤：In the twentieth aspect of the present invention, a method for preparing a chimeric antibody is provided, including the steps:
将本发明第一方面所述的重链可变区和/或本发明第三方面所述的轻链可变区的核苷酸序列克隆入含有人抗体恒定区的核苷酸序列的表达载体后，通过转染动物细胞表达人-鼠嵌合抗体。The nucleotide sequence of the heavy chain variable region of the first aspect of the present invention and/or the light chain variable region of the third aspect of the present invention is cloned into an expression vector containing the nucleotide sequence of a human antibody constant region Afterwards, the human-mouse chimeric antibody was expressed by transfecting animal cells.
在本发明的第二十一方面，提供了一种制备人源化抗体的方法，包括步骤：In the twenty-first aspect of the present invention, a method for preparing a humanized antibody is provided, including the steps:
将本发明第一方面所述的重链可变区和/或本发明第三方面所述的轻链可变区中的CDR区的核苷酸序列植入含人源抗体FR区的核苷酸序列模板，再将其克隆入含有人抗体恒定区的表达载体后，通过转染动物细胞表达人源化抗体。The nucleotide sequence of the CDR region in the heavy chain variable region of the first aspect of the present invention and/or the light chain variable region of the third aspect of the present invention is implanted into a nucleoside containing the FR region of a human antibody After the acid sequence template is cloned into an expression vector containing the constant region of a human antibody, the humanized antibody is expressed by transfecting animal cells.
在本发明的第二十二方面，提供了一种双特异性抗体，所述的双特异性抗体包括：本发明第五方面所述抗体和第二抗体，所述的第二抗体选自下组：CD3、CD47、PD-1、PD-L1抗体，以及抗BCMA、CD20、CD22、CD33、CD123抗抗体。In the twenty-second aspect of the present invention, there is provided a bispecific antibody, the bispecific antibody comprising: the antibody of the fifth aspect of the present invention and a second antibody, and the second antibody is selected from the group consisting of Group: CD3, CD47, PD-1, PD-L1 antibodies, and anti-BCMA, CD20, CD22, CD33, CD123 anti-antibodies.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.
图1显示了免疫小鼠血清与FMC63结合的ELISA测试结果。图中，四位数字编号(7267-7274)表示小鼠编号，TB3表示小鼠共进行3次免疫，Figure 1 shows the ELISA test results of the sera of immunized mice binding to FMC63. In the figure, the four-digit number (7267-7274) indicates the mouse number, and TB3 indicates that the mouse has been immunized 3 times in total.
图2显示了ELISA检测的抗FMC63抗体的亲和力(EC50)。Figure 2 shows the affinity (EC50) of anti-FMC63 antibodies detected by ELISA.
图3显示了FACS检测的抗FMC63抗体的亲和力。Figure 3 shows the affinity of anti-FMC63 antibodies detected by FACS.
图4显示了抗FMC63抗体组合应用到抗抗体方法的检测结果。Figure 4 shows the test results of the anti-FMC63 antibody combination applied to the anti-antibody method.
图5显示抗FMC63抗体可以阻断CD19CAR-T细胞与CD19靶点的结合，其中，横坐标表示mAb编号。Figure 5 shows that the anti-FMC63 antibody can block the binding of CD19CAR-T cells to the CD19 target, where the abscissa represents the mAb number.
图6显示了抗FMC63抗体可以促进CD19CAR-T细胞的增殖。Figure 6 shows that anti-FMC63 antibodies can promote the proliferation of CD19CAR-T cells.
图7显示了抗FMC63抗体可以促进CD19CAR-T细胞的细胞因子释放。Figure 7 shows that anti-FMC63 antibodies can promote cytokine release from CD19CAR-T cells.
图8显示了抗FMC63抗体可以激活CD19CAR-T细胞。Figure 8 shows that anti-FMC63 antibodies can activate CD19CAR-T cells.
图9显示了靶向FMC63的CAR-T细胞的CAR阳性率。Figure 9 shows the CAR-positive rate of CAR-T cells targeting FMC63.
图10显示了靶向FMC63的CAR-T细胞可以杀伤表达靶抗原的细胞。Figure 10 shows that CAR-T cells targeting FMC63 can kill cells expressing the target antigen.
图11显示了靶向FMC63的CAR-T细胞在表达靶抗原的细胞刺激下细胞因子释放。Figure 11 shows that CAR-T cells targeting FMC63 release cytokines under the stimulation of cells expressing the target antigen.
图12显示了靶向FMC63的CAR-T细胞可以被表达靶抗原的细胞激活。Figure 12 shows that CAR-T cells targeting FMC63 can be activated by cells expressing the target antigen.
注，附图中NT表示未处理的T细胞。Note, NT in the figure represents untreated T cells.
本发明人经过广泛而深入地研究，首次意外地发现一种人源化抗CD19抗体的抗体。具体地，本发明通过使用合成的CD19抗体(scFv)免疫小鼠，取小鼠脾脏制备杂交瘤细胞，筛选得到鼠源抗CD19抗体的抗体，并经过替换人Fc段获得到人源化的抗CD19抗体的抗体。人源化的抗CD19抗体的抗体可以应用于检测CD19抗体、制备抗体药、制备靶向CD19抗体的CAR-T细胞。After extensive and in-depth research, the inventors unexpectedly discovered a humanized anti-CD19 antibody for the first time. Specifically, the present invention uses a synthetic CD19 antibody (scFv) to immunize mice, take mouse spleens to prepare hybridoma cells, screen to obtain murine anti-CD19 antibody antibodies, and obtain humanized antibodies by replacing the human Fc segment. Anti-CD19 antibody. The humanized anti-CD19 antibody can be used to detect CD19 antibody, prepare antibody drugs, and prepare CAR-T cells targeting CD19 antibody.
术语the term
本发明中，“VH”指重链可变区，“VL”指轻链可变区。“VH-CDR1”指重链可变区的CDR1；“VH-CDR2”指重链可变区的CDR2；“VH-CDR3”指重链可变区的CDR3。“VL-CDR1”指轻链可变区的CDR1；“VL-CDR2”指轻链可变区的CDR2；“VL-CDR3”指轻链可变区的CDR3。In the present invention, "VH" refers to the variable region of the heavy chain, and "VL" refers to the variable region of the light chain. "VH-CDR1" refers to CDR1 of the heavy chain variable region; "VH-CDR2" refers to CDR2 of the heavy chain variable region; "VH-CDR3" refers to CDR3 of the heavy chain variable region. "VL-CDR1" refers to CDR1 of the light chain variable region; "VL-CDR2" refers to CDR2 of the light chain variable region; "VL-CDR3" refers to CDR3 of the light chain variable region.
FMC63FMC63
FMC63是一种鼠源抗CD19的IgG2a抗体。利用FMC63设计的CAR-T细胞药物已经成功上市，在治疗B-ALL和NHL上有较好的疗效。FMC63 is a murine anti-CD19 IgG2a antibody. CAR-T cell drugs designed using FMC63 have been successfully marketed, and have good curative effects in the treatment of B-ALL and NHL.
FMC63 scFv氨基酸序列如下：The amino acid sequence of FMC63 scFv is as follows:
抗体Antibody
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约 150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain . Special amino acid residues form an interface between the variable regions of the light chain and the heavy chain.
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。As used herein, the term "variable" means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR). The variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a β-sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-sheet structure. The CDRs in each chain are closely held together by the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain. Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
脊椎动物抗体(免疫球蛋白)的“轻链”可根据其恒定区的氨基酸序列归为明显不同的两类(称为κ和λ)中的一类。根据其重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类。主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM，其中一些还可进一步分成亚类(同种型)，如IgG1、IgG2、IgG3、IgG4、IgA和IgA2。对应于不同类免疫球蛋白的重链恒定区分别称为α、δ、ε、γ、和μ。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct categories (called kappa and lambda) based on the amino acid sequence of their constant regions. According to the amino acid sequence of the constant region of their heavy chains, immunoglobulins can be divided into different types. There are mainly five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
一般，抗体的抗原结合特性可由位于重链和轻链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。Generally, the antigen-binding properties of antibodies can be described by 3 specific regions located in the variable regions of the heavy and light chains, called variable regions (CDR), which are divided into 4 framework regions (FR), 4 The amino acid sequence of FR is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the β sheets formed by the FRs in between are close to each other in space structure, and the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。The present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以通过标准的DNA重组技术获得，它们都是有用的抗体。嵌合抗体是一个分子，其中不同的部分来自不同的动物种，例如具有来自鼠的单克隆抗体的可变区，和来自人免疫球蛋白的恒定区的嵌合抗体(见例如美国 专利4,816,567和美国专利4,816,397,在此通过引用方式整体引入本文)。人源化的抗体是指来源于非人物种的抗体分子，具有一个或多个来源于非人物种的互补决定区(CDRs)和来源于人免疫球蛋白分子的框架区域(见美国专利5,585,089，在此通过引用方式整体引入本文)。这些嵌合和人源化的单克隆抗体可以采用本领域熟知的DNA重组技术制备。In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombination techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are derived from different animal species, for example, a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent Nos. 4,816,567 and U.S. Patent 4,816,397, which is hereby incorporated by reference in its entirety). Humanized antibodies refer to antibody molecules derived from non-human species, with one or more complementarity determining regions (CDRs) derived from non-human species and framework regions derived from human immunoglobulin molecules (see U.S. Patent 5,585,089, This article is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。In the present invention, the antibody may be monospecific, bispecific, trispecific, or more multispecific.
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。In the present invention, the antibody of the present invention also includes its conservative variants, which means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
表ATable A
| 最初的残基Initial residues | 代表性的取代Representative substitution | 优选的取代Preferred substitution |
| Ala(A)Ala(A) | Val；Leu；IleVal; Leu; Ile | ValVal |
| Arg(R)Arg(R) | Lys；Gln；AsnLys; Gln; Asn | LysLys |
| Asn(N)Asn(N) | Gln；His；Lys；ArgGln; His; Lys; Arg | GlnGln |
| Asp(D)Asp(D) | GluGlu | GluGlu |
| Cys(C)Cys(C) | SerSer | SerSer |
| Gln(Q)Gln(Q) | AsnAsn | AsnAsn |
| Glu(E)Glu(E) | AspAsp | AspAsp |
| Gly(G)Gly(G) | Pro；AlaPro; Ala | AlaAla |
| His(H)His(H) | Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg | ArgArg |
| Ile(I)Ile(I) | Leu；Val；Met；Ala；PheLeu; Val; Met; Ala; Phe | LeuLeu |
| Leu(L)Leu(L) | Ile；Val；Met；Ala；PheIle; Val; Met; Ala; Phe | IleIle |
| Lys(K)Lys(K) | Arg；Gln；AsnArg; Gln; Asn | ArgArg |
| Met(M)Met(M) | Leu；Phe；IleLeu; Phe; Ile | LeuLeu |
| Phe(F)Phe(F) | Leu；Val；Ile；Ala；TyrLeu; Val; Ile; Ala; Tyr | LeuLeu |
| Pro(P)Pro(P) | AlaAla | AlaAla |
| Ser(S)Ser(S) | ThrThr | ThrThr |
| Thr(T)Thr(T) | SerSer | SerSer |
| Trp(W)Trp(W) | Tyr；PheTyr; Phe | TyrTyr |
| Tyr(Y)Tyr(Y) | Trp；Phe；Thr；SerTrp; Phe; Thr; Ser | PhePhe |
| Val(V)Val(V) | Ile；Leu；Met；Phe；AlaIle; Leu; Met; Phe; Ala | LeuLeu |
抗CD19抗体的抗体Anti-CD19 antibody
本发明中，所述抗体为抗CD19抗体的抗体，所述的CD19抗体包括FMC63及其衍生序列。本发明的抗体包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。In the present invention, the antibody is an anti-CD19 antibody, and the CD19 antibody includes FMC63 and its derivative sequences. The antibody of the present invention includes a heavy chain containing a heavy chain variable region (VH) amino acid sequence and a light chain containing a heavy chain variable region (VH) amino acid sequence, and the light chain containing a light chain variable region (VL) amino acid sequence.
优选地，Preferably,
所述的重链可变区(VH)具有选自下组的互补决定区CDR：The heavy chain variable region (VH) has a complementarity determining region CDR selected from the following group:
SEQ ID NO:1所示的CDR1，CDR1 shown in SEQ ID NO:1,
SEQ ID NO:2所示的CDR2，和CDR2 shown in SEQ ID NO: 2, and
SEQ ID NO:3所示的CDR3；CDR3 shown in SEQ ID NO: 3;
或者，or,
SEQ ID NO:4的CDR1，CDR1 of SEQ ID NO: 4,
SEQ ID NO:5所示的CDR2，和CDR2 shown in SEQ ID NO: 5, and
SEQ ID NO:6所示的CDR3，CDR3 shown in SEQ ID NO: 6,
或者，or,
SEQ ID NO:7所示的CDR1，CDR1 shown in SEQ ID NO: 7,
SEQ ID NO:8所示的CDR2，和CDR2 shown in SEQ ID NO: 8, and
SEQ ID NO:9所示的CDR3；CDR3 shown in SEQ ID NO: 9;
所述的轻链可变区(VL)具有选自下组的互补决定区CDR：The light chain variable region (VL) has a complementarity determining region CDR selected from the following group:
SEQ ID NO:10所示的CDR1’，CDR1' shown in SEQ ID NO: 10,
SEQ ID NO:11所示的CDR2’，和CDR2' shown in SEQ ID NO: 11, and
SEQ ID NO:12所示的CDR3’；CDR3' shown in SEQ ID NO: 12;
或者，or,
SEQ ID NO:13所示的CDR1’，CDR1' shown in SEQ ID NO: 13,
SEQ ID NO:14所示的CDR2’，和CDR2' shown in SEQ ID NO: 14, and
SEQ ID NO:15所示的CDR3’；CDR3' shown in SEQ ID NO: 15;
或者，or,
SEQ ID NO:16所示的CDR1’，CDR1' shown in SEQ ID NO: 16,
SEQ ID NO:17所示的CDR2’，和CDR2' shown in SEQ ID NO: 17, and
SEQ ID NO:18所示的CDR3’；CDR3' shown in SEQ ID NO: 18;
其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CD19抗体的结合亲和力的衍生序列。Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性或序列相同性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。In another preferred example, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence preferably has a homology or sequence identity of at least 80%, preferably at least 85%, more preferably The ground is at least 90%, and most preferably at least 95% of the amino acid sequence.
本领域普通技术人员公知的测定序列同源性或相同性的方法包括但不限 于：计算机分子生物学(Computational Molecular Biology)，Lesk，A.M.编，牛津大学出版社，纽约，1988；生物计算：信息学和基因组项目(Biocomputing:Informatics and Genome Projects)，Smith，D.W.编，学术出版社，纽约，1993；序列数据的计算机分析(Computer Analysis of Sequence Data)，第一部分，Griffin，A.M.和Griffin，H.G.编，Humana Press，新泽西，1994；分子生物学中的序列分析(Sequence Analysis in Molecular Biology)，von Heinje，G.，学术出版社，1987和序列分析引物(Sequence Analysis Primer)，Gribskov，M.与Devereux，J.编M Stockton Press，纽约，1991和Carillo，H.与Lipman，D.，SIAM J.Applied Math.，48:1073(1988)。测定相同性的优选方法要在测试的序列之间得到最大的匹配。测定相同性的方法编译在公众可获得的计算机程序中。优选的测定两条序列之间相同性的计算机程序方法包括但不限于：GCG程序包(Devereux，J.等，1984)、BLASTP、BLASTN和FASTA(Altschul，S，F.等，1990)。公众可从NCBI和其它来源得到BLASTX程序(BLAST手册，Altschul，S.等，NCBI NLM NIH Bethesda，Md.20894；Altschul，S.等，1990)。熟知的Smith Waterman算法也可用于测定相同性。Methods of determining sequence homology or identity well-known to those of ordinary skill in the art include but are not limited to: Computational Molecular Biology, Lesk, AM edited, Oxford University Press, New York, 1988; Biocomputing: Information Biocomputing: Information and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG , Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux , J. Ed. M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the tested sequences. The method for determining identity is compiled in a publicly available computer program. Preferred computer program methods for determining the identity between two sequences include but are not limited to: GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The public can obtain the BLASTX program from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.
本发明的抗体可以是选自动物源抗体、嵌合抗体、人源化抗体，更优选为人源化抗体、人-动物嵌合抗体，更优选为全人源化抗体。The antibody of the present invention may be selected from animal-derived antibodies, chimeric antibodies, and humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and more preferably fully humanized antibodies.
其中，所述动物优选为哺乳动物，如鼠。Among them, the animal is preferably a mammal, such as a mouse.
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab’、(Fab’)2或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。The antibody derivatives of the present invention may be single-chain antibodies and/or antibody fragments, such as: Fab, Fab', (Fab')2 or other antibody derivatives known in the field, etc., as well as IgA, IgD, IgE , IgG and IgM antibodies or any one or more of other subtypes of antibodies.
本发明抗体可以是靶向CD19抗体的嵌合抗体、人源化抗体、CDR嫁接和/或修饰的抗体。The antibodies of the present invention may be chimeric antibodies, humanized antibodies, CDR grafted and/or modified antibodies that target CD19 antibodies.
在另一优选例中，所述抗体的重链可变区含有SEQ ID NO:19、21或23所示的氨基酸序列。In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 19, 21 or 23.
在另一优选例中，所述抗体的轻链可变区含有SEQ ID NO:20、22或24所示的氨基酸序列。In another preferred embodiment, the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO: 20, 22 or 24.
本发明抗体可以是抗体全长蛋白、抗原抗体结合域蛋白质片段、双特异性抗体、多特异性抗体、单链抗体(single chain antibody fragment，scFv)、单域抗体(single domain antibody，sdAb)和单区抗体(Signle-domain antibody)中的一种或多种，以及上述抗体所制得的单克隆抗体或多克隆抗体。所述单克隆抗体可以由多种途径和技术进行研制，包括杂交瘤技术、噬菌体展示技术、单淋巴细胞基因克隆技术等，主流是通过杂交瘤技术从野生型或转基因小鼠制备单克隆抗体。The antibody of the present invention may be an antibody full-length protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (single chain antibody fragment, scFv), a single domain antibody (single domain antibody, sdAb), and One or more of single-domain antibodies (Signle-domain antibodies), and monoclonal antibodies or polyclonal antibodies made from the above antibodies. The monoclonal antibody can be developed by a variety of approaches and technologies, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice through hybridoma technology.
所述的抗体全长蛋白为本领域常规的抗体全长蛋白，其包括重链可变区、 轻链可变区、重链恒定区和轻链恒定区。所述的蛋白质的重链可变区和轻链可变区与人源重链恒定区和人源轻链恒定区构成全人源抗体全长蛋白。较佳地，所述的抗体全长蛋白为IgG1、IgG2、IgG3或IgG4。The antibody full-length protein is a conventional antibody full-length protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, the human heavy chain constant region and the human light chain constant region constitute a fully human antibody full-length protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.
所述的单链抗体为本领域常规的单链抗体，其包括重链可变区、轻链可变区和15-20个氨基酸的短肽。The single-chain antibody is a conventional single-chain antibody in the field, which includes a heavy chain variable region, a light chain variable region and short peptides of 15-20 amino acids.
所述的抗原抗体结合域蛋白质片段为本领域常规的抗原抗体结合域蛋白质片段，其包括轻链可变区、轻链恒定区和重链恒定区的Fd段。较佳地，所述的抗原抗体结合域蛋白质片段为Fab和F(ab’)。The antigen-antibody binding domain protein fragments are conventional antigen-antibody binding domain protein fragments in the art, which include the light chain variable region, the light chain constant region and the Fd segment of the heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragments are Fab and F(ab').
所述的单域抗体为本领域常规的单域抗体，其包括重链可变区和重链恒定区。The single domain antibody is a conventional single domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.
所述的单区抗体为本领域常规的单区抗体，其仅包括重链可变区。The single-domain antibody is a conventional single-domain antibody in the art, which only includes the variable region of the heavy chain.
其中，所述重组蛋白的制备方法为本领域常规的制备方法。所述制备方法较佳地为：从重组表达该蛋白质的表达转化体中分离获得或者通过人工合成蛋白质序列获得。所述的从重组表达该蛋白质的表达转化体中分离获得优选如下方法：将编码所述蛋白质并且带有点突变的核酸分子克隆到重组载体中，将所得重组载体转化到转化体中，得到重组表达转化体，通过培养所得重组表达转化体，即可分离纯化获得所述重组蛋白。Wherein, the preparation method of the recombinant protein is a conventional preparation method in the art. The preparation method is preferably: isolated from an expression transformant that recombinantly expresses the protein or obtained by artificially synthesizing a protein sequence. Said method of separating and obtaining from an expression transformant that recombinantly expresses the protein is preferably as follows: cloning a nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector, and transforming the obtained recombinant vector into a transformant to obtain recombinant expression The transformant can be isolated and purified to obtain the recombinant protein by culturing the obtained recombinant expression transformant.
核酸Nucleic Acid
本发明还提供一种核酸，其编码上述的抗体或重组蛋白或抗CD19抗体的抗体的重链可变区或轻链可变区。The present invention also provides a nucleic acid that encodes the heavy chain variable region or the light chain variable region of the aforementioned antibody or recombinant protein or anti-CD19 antibody.
所述核酸的制备方法为本领域常规的制备方法，较佳地，包括以下的步骤：通过基因克隆技术获得编码上述蛋白质的核酸分子，或者通过人工全序列合成的方法得到编码上述蛋白质的核酸分子。The preparation method of the nucleic acid is a conventional preparation method in the art, preferably, it includes the following steps: obtain the nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtain the nucleic acid molecule encoding the above-mentioned protein by the method of artificial full-sequence synthesis .
本领域技术人员知晓，编码上述蛋白质的氨基酸序列的碱基序列可以适当引入替换、缺失、改变、插入或增加来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对编码该蛋白序列基因的一个或多个碱基在保持抗体活性范围内进行替换、缺失或增加来制得。Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately substituted, deleted, altered, inserted or added to provide a polynucleotide homolog. The polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the scope of maintaining the activity of the antibody.
载体Carrier
本发明还提供一种包含所述核酸的重组表达载体。The present invention also provides a recombinant expression vector containing the nucleic acid.
其中所述重组表达载体可通过本领域常规方法获得，即：将本发明所述的核酸分子连接于各种表达载体上构建而成。所述的表达载体为本领域常规的各种载体，只要其能够容载前述核酸分子即可。所述载体较佳地包括：各种质粒、粘粒、噬菌体或病毒载体等。The recombinant expression vector can be obtained by conventional methods in the art, that is, the nucleic acid molecule of the present invention is connected to various expression vectors to be constructed. The expression vector is a variety of conventional vectors in the field, as long as it can hold the aforementioned nucleic acid molecule. Said vectors preferably include: various plasmids, cosmids, phage or virus vectors and the like.
本发明还提供一种包含上述重组表达载体的重组表达转化体。The present invention also provides a recombinant expression transformant comprising the above-mentioned recombinant expression vector.
其中，所述重组表达转化体的制备方法为本领域常规的制备方法，较佳地 为：将上述重组表达载体转化至宿主细胞中制得。所述的宿主细胞为本领域常规的各种宿主细胞，只要能满足使上述重组表达载体稳定地自行复制，且所携带所述的核酸可被有效表达即可。较佳地，所述宿主细胞为E.coli TG1或E.coli BL21细胞(表达单链抗体或Fab抗体)，或者HEK293或CHO细胞(表达全长IgG抗体)。将前述重组表达质粒转化至宿主细胞中，即可得本发明优选的重组表达转化体。其中所述转化方法为本领域常规转化方法，较佳地为化学转化法，热激法或电转法。Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the recombinant expression vector is transformed into a host cell. The host cell is a variety of conventional host cells in the art, as long as the recombinant expression vector can replicate itself stably and the nucleic acid carried can be effectively expressed. Preferably, the host cell is E. coli TG1 or E. coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody). The aforementioned recombinant expression plasmid is transformed into a host cell to obtain the preferred recombinant expression transformant of the present invention. The transformation method is a conventional transformation method in the field, preferably a chemical transformation method, a heat shock method or an electrotransformation method.
抗体的制备Antibody preparation
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。The sequence of the DNA molecule of the antibody or its fragment of the present invention can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain and the heavy chain can also be fused together to form a single chain antibody.
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。Once the relevant sequence is obtained, the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
目前，已经可以完全通过化学合成来得到编码所述的本发明的抗体(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。At present, the DNA sequence encoding the antibody (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。The present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。优选的动物细胞包括(但并不限于)：CHO-S、HEK-293细胞。The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。Generally, the transformed host cell is cultured under conditions suitable for expression of the antibody of the present invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc. The antibody of the present invention is purified by conventional separation and purification means well-known to the person.
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。单克隆抗体的结合亲和力例如可用Munson等,Anal.Biochem.,107:220(1980)的Scatchard分析来测定。The obtained monoclonal antibody can be identified by conventional means. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).
本发明的抗体可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的 蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超声处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。The antibody of the present invention can be expressed in the cell, on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
抗体-药物偶联物(ADC)Antibody-drug conjugate (ADC)
本发明还提供了基于本发明抗体的抗体偶联药物(antibody-drug conjugate，ADC)。The present invention also provides antibody-drug conjugate (ADC) based on the antibody of the present invention.
典型地，所述抗体偶联药物包括所述抗体、以及效应分子，所述抗体与所述效应分子偶联，并优选为化学偶联。其中，所述效应分子优选为具有治疗活性的药物。此外，所述效应分子可以是毒蛋白、化疗药物、小分子药物或放射性核素中的一种或多种。Typically, the antibody-conjugated drug includes the antibody and an effector molecule, and the antibody is coupled to the effector molecule, and preferably is chemically coupled. Among them, the effector molecule is preferably a drug with therapeutic activity. In addition, the effector molecule may be one or more of toxic protein, chemotherapeutic drug, small molecule drug or radionuclide.
本发明抗体与所述效应分子之间可以是通过偶联剂进行偶联。所述偶联剂的例子可以是非选择性偶联剂、利用羧基的偶联剂、肽链、利用二硫键的偶联剂中的任意一种或几种。所述非选择性偶联剂是指使效应分子和抗体形成共价键连接的化合物，如戊二醛等。所述利用羧基的偶联剂可以是顺乌头酸酐类偶联剂(如顺乌头酸酐)、酰基腙类偶联剂(偶联位点为酰基腙)中的任意一种或几种。The antibody of the present invention and the effector molecule may be coupled through a coupling agent. Examples of the coupling agent may be any one or more of non-selective coupling agents, coupling agents using carboxyl groups, peptide chains, and coupling agents using disulfide bonds. The non-selective coupling agent refers to a compound that makes the effector molecule and the antibody form a covalent bond, such as glutaraldehyde. The coupling agent using carboxyl groups can be any one or more of cis-aconitic acid anhydride coupling agents (such as cis-aconitic acid anhydride) and acyl hydrazone coupling agents (coupling sites are acyl hydrazones).
抗体上某些残基(如Cys或Lys等)用于与多种功能基团相连，其中包括成像试剂(例如发色基团和荧光基团)，诊断试剂(例如MRI对比剂和放射性同位素)，稳定剂(例如乙二醇聚合物)和治疗剂。抗体可以被偶联到功能剂以形成抗体-功能剂的偶联物。功能剂(例如药物，检测试剂，稳定剂)被偶联(共价连接)至抗体上。功能剂可以直接地、或者是通过接头间接地连接于抗体。Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging reagents (such as chromophores and fluorescent groups), diagnostic reagents (such as MRI contrast agents and radioisotopes) , Stabilizers (such as glycol polymers) and therapeutic agents. The antibody can be conjugated to the functional agent to form an antibody-functional agent conjugate. Functional agents (e.g. drugs, detection reagents, stabilizers) are coupled (covalently linked) to the antibody. The functional agent may be directly or indirectly linked to the antibody through a linker.
抗体可以偶联药物从而形成抗体药物偶联物(ADCs)。典型地，ADC包含位于药物和抗体之间的接头。接头可以是可降解的或者是不可降解的接头。可降解的接头典型地在细胞内环境下容易降解，例如在目标位点处接头发生降解，从而使药物从抗体上释放出来。合适的可降解的接头包括，例如酶降解的接头，其中包括可以被细胞内蛋白酶(例如溶酶体蛋白酶或者内体蛋白酶)降解的含有肽基的接头，或者糖接头例如，可以被葡糖苷酸酶降解的含葡糖苷酸的接头。肽基接头可以包括，例如二肽，例如缬氨酸-瓜氨酸，苯丙氨酸-赖氨酸或者缬氨酸-丙氨酸。其它合适的可降解的接头包括，例如，pH敏感接头(例如pH小于5.5时水解的接头，例如腙接头)和在还原条件下会降解的接头(例如二硫键接头)。不可降解的接头典型地在抗体被蛋白酶水解的条件下释放药物。Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, the ADC contains a linker between the drug and the antibody. The linker can be a degradable or a non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, for example, the linker is degraded at the target site, so that the drug is released from the antibody. Suitable degradable linkers include, for example, enzymatically degraded linkers, including peptidyl-containing linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, for example, which can be degraded by glucuronide. Enzymatically degraded glucuronide-containing linker. The peptidyl linker may include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (for example, linkers that are hydrolyzed at a pH of less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (for example, disulfide bond linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by a protease.
在一些实施方式中，抗体药物偶联物ADC如下分子式所示：In some embodiments, the antibody-drug conjugate ADC is represented by the following molecular formula:
其中：in:
Ab是抗体，Ab is an antibody,
LU是接头；LU is the connector;
D是药物；D is a drug;
而且下标p是选自1到8的值。And the subscript p is a value selected from 1 to 8.
嵌合抗原受体T细胞Chimeric antigen receptor T cell
CAR-T，全称是Chimeric Antigen Receptor T-Cell，指的是嵌合抗原受体T细胞，其中嵌合抗原受体(CAR)是CAR-T的核心部件，赋予T细胞HLA非依赖的方式识别肿瘤抗原的能力，这使得经过CAR改造的T细胞相较于天然T细胞表面受体TCR能够识别更广泛的目标。CAR的基础设计中包括一个肿瘤相关抗原(tumor-associated antigen，TAA)结合区(通常来源于单克隆抗体抗原结合区域的scFv段)，一个胞外铰链区，一个跨膜区和一个胞内信号区。CARs的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效。第二代CARs在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CARs相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CARs。CAR-T, the full name is Chimeric Antigen Receptor T-Cell, refers to the chimeric antigen receptor T cell, among which the chimeric antigen receptor (CAR) is the core component of CAR-T, which gives T cells recognition in an HLA-independent manner The ability of tumor antigens, which allows CAR-modified T cells to recognize a wider range of targets than the natural T cell surface receptor TCR. The basic design of CAR includes a tumor-associated antigen (TAA) binding region (usually derived from the scFv segment of the monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region and an intracellular signal Area. The design of CARs has gone through the following process: The first-generation CAR has only one intracellular signal component CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , And cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical effects. The second-generation CARs introduce a costimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function has been greatly improved, which further strengthens the persistence of CAR-T cells and the effect on tumor cells. The lethality. On the basis of the second-generation CARs, some new immunostimulatory molecules such as CD27 and CD134 are connected in series to develop into the third-generation and fourth-generation CARs.
检测用途和试剂盒Testing purposes and kits
本发明的抗体或其ADC可用于检测应用，例如用于检测样本，从而提供诊断信息。The antibody or ADC of the present invention can be used in detection applications, for example, to detect samples, thereby providing diagnostic information.
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如肿瘤的切除样本、通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。In the present invention, the samples (samples) used include cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention shall include all kinds of biopsy known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, excision samples of tumors, tissue samples prepared by endoscopic methods or organ puncture or needle biopsy.
本发明中使用的样本包括固定的或保存的细胞或组织样本。The samples used in the present invention include fixed or preserved cell or tissue samples.
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。The present invention also provides a kit containing the antibody (or a fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, and the like. In a preferred example, the antibody of the present invention can be immobilized on a detection plate.
药物组合物Pharmaceutical composition
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的免疫细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值 可随被配制物质的性质以及待治疗的病症而有所变化。The invention also provides a composition. In a preferred example, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or active fragment or fusion protein or ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can be The nature of the formulated substance and the condition to be treated vary.
配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。典型地，本发明所述的药物组合物的给药途径较佳地为注射给药或口服给药。所述注射给药较佳地包括静脉注射、肌肉注射、腹腔注射、皮内注射或皮下注射等途径。所述的药物组合物为本领域常规的各种剂型，较佳地为固体、半固体或液体的形式，可以为水溶液、非水溶液或混悬液，更佳地为片剂、胶囊、颗粒剂、注射剂或输注剂等。The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection administration preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is a variety of conventional dosage forms in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably a tablet, capsule, or granule. , Injection or infusion, etc.
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。The antibody of the present invention can also be used for cell therapy by expressing the nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
本发明的药物组合物可直接用于结合CD19抗体或靶向CD19的CAR-T细胞，因而可用于辅助治疗肿瘤等疾病。The pharmaceutical composition of the present invention can be directly used in CAR-T cells that bind CD19 antibodies or target CD19, and thus can be used to assist in the treatment of tumors and other diseases.
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。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。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，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。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody (or conjugate thereof) of the present invention and a pharmaceutical Acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the polypeptides of the present invention can also be used together with other therapeutic agents.
本发明中，较佳地，本发明所述的药物组合物还包括一种或多种药用载体。所述的药用载体为本领域常规药用载体，所述的药用载体可以为任意合适的生理学或药学上可接受的药物辅料。所述的药物辅料为本领域常规的药物辅料，较佳的包括药学上可接受的赋形剂、填充剂或稀释剂等。更佳地，所述的药物组合物包括0.01～99.99％的上述蛋白质和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。In the present invention, preferably, the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipients are conventional pharmaceutical excipients in the field, and preferably include pharmaceutically acceptable excipients, fillers or diluents. More preferably, the pharmaceutical composition includes 0.01-99.99% of the aforementioned protein and 0.01-99.99% of a pharmaceutical carrier, and the percentage is a mass percentage of the pharmaceutical composition.
本发明中，较佳地，所述的药物组合物的施用量为有效量，所述有效量为能够缓解或延迟疾病、退化性或损伤性病症进展的量。所述有效量可以以个体基础来测定，并将部分基于待治疗症状和所寻求结果的考虑。本领域技术人员可以通过使用个体基础等上述因素和使用不超过常规的实验来确定有效量。In the present invention, preferably, the administration amount of the pharmaceutical composition is an effective amount, and the effective amount is an amount that can alleviate or delay the progression of a disease, degenerative or traumatic condition. The effective amount can be determined on an individual basis and will be partly based on consideration of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the aforementioned factors such as individual basis and using no more than conventional experiments.
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When the pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases not more than about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
本发明的主要优点包括：The main advantages of the present invention include:
(a)本发明的抗体能够用于检测CD19抗体，如用于ELISA检测、FACS检测等。(a) The antibody of the present invention can be used to detect CD19 antibody, such as for ELISA detection, FACS detection and the like.
(b)本发明的抗体能够阻断CD19抗体，如FMC63的功能。(b) The antibody of the present invention can block the function of CD19 antibody, such as FMC63.
(c)本发明的抗体与靶向CD19的CAR-T结合，从而阻断CAR-T的功能，抑制细胞因子风暴、神经毒性和其它CAR-T相关毒副作用的发生发展。(c) The antibody of the present invention binds to CAR-T targeting CD19, thereby blocking the function of CAR-T and inhibiting the occurrence and development of cytokine storm, neurotoxicity and other CAR-T-related toxic and side effects.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.
除非另外说明，实施例中的所有载体、细胞、试剂等均为常规市售的。实施例中涉及的核苷酸序列(FMC63 scFv CAR、FMC63-scFv-mFc)均由第三方基因合成公司合成，Unless otherwise specified, all vectors, cells, reagents, etc. in the examples are conventionally commercially available. The nucleotide sequences (FMC63 scFv CAR, FMC63-scFv-mFc) involved in the examples were all synthesized by a third-party gene synthesis company.
实施例1 构建表达FMC63scFv CAR的单克隆细胞(Jurkat-FMC63 scFv)Example 1 Construction of monoclonal cells expressing FMC63scFv CAR (Jurkat-FMC63 scFv)
将FMC63 scFv CAR(GMCSF信号肽-FMC63 scFv-Flag-CD28铰链-CD28跨膜区-CD28共刺激区-CD3z)的核苷酸序列整合到慢病毒载体中，包被FMC63 scFv CAR慢病毒载体，感染Jurkat细胞，筛选得到稳定表达的FMC63 scFv CAR的单克隆Jurkat-FMC63 scFv细胞，使用流式细胞术鉴定单克隆细胞。Integrate the nucleotide sequence of FMC63 scFv CAR (GMCSF signal peptide-FMC63 scFv-Flag-CD28 hinge-CD28 transmembrane region-CD28 costimulator region-CD3z) into the lentiviral vector, and coat the FMC63 scFv CAR lentiviral vector, Infect Jurkat cells, screen the monoclonal Jurkat-FMC63 scFv cells stably expressing FMC63 scFv CAR, and identify the monoclonal cells by flow cytometry.
结果显示，成功获得表达FMC63scFv CAR的单克隆细胞(Jurkat-FMC63 scFv细胞)。The results showed that monoclonal cells (Jurkat-FMC63 scFv cells) expressing FMC63scFv CAR were successfully obtained.
实施例2 FMC63-scFv-mFc免疫小鼠Example 2 Immunization of mice with FMC63-scFv-mFc
将FMC63-scFv-mFc(FMC63抗体的一条链)的核苷酸序列整合到真核细胞表达载体中，使用PEI将携带FMC63核苷酸的表达载体转入293细胞中，使其表达FMC63蛋白，使用protein A柱纯化得到FMC63蛋白(FMC63-scFv-mFc蛋白)。Integrate the nucleotide sequence of FMC63-scFv-mFc (a chain of FMC63 antibody) into the eukaryotic expression vector, and use PEI to transfer the expression vector carrying FMC63 nucleotides into 293 cells to express FMC63 protein, Purified by protein A column to obtain FMC63 protein (FMC63-scFv-mFc protein).
按照下表1所示方法免疫小鼠。Mice were immunized according to the method shown in Table 1 below.
表1 小鼠免疫方法Table 1 Mouse Immunization Method
|
第0天 |
1×10 7的Jurkat-FMC63 scFv细胞IP注射免疫小鼠 IP injection of 1×10 7 Jurkat-FMC63 scFv cells to immunize mice |
| 第14天Day 14 | 1×10 7的Jurkat-FMC63 scFv细胞IP注射免疫小鼠 IP injection of 1×10 7 Jurkat-FMC63 scFv cells to immunize mice |
| 第35天Day 35 | 1×10 7的Jurkat-FMC63 scFv细胞IP注射免疫小鼠 IP injection of 1×10 7 Jurkat-FMC63 scFv cells to immunize mice |
| 第56天Day 56 | 25ug FMC63-scFv-mFc蛋白IP注射免疫小鼠25ug FMC63-scFv-mFc protein IP injection to immunize mice |
结果如图1所示，Jurkat-FMC63 scFv细胞免疫后的小鼠血清与FMC63-scFv-mFc有结合信号，说明小鼠免疫成功，可以继续进行筛选。The results are shown in Figure 1. The serum of mice immunized with Jurkat-FMC63 scFv cells has a binding signal with FMC63-scFv-mFc, indicating that the mice are successfully immunized and can continue to be screened.
实施例3 杂交瘤获取与筛选Example 3 Hybridoma acquisition and screening
通过ELISA/FACS筛选免疫有应答的小鼠，取小鼠脾脏淋巴细胞与Sp2/0-Ag14细胞融合获得杂交瘤细胞，将杂交瘤细胞铺板至96孔板中进行生长，10-14天后取上清用ELISA/FACS筛选细胞、解离速率常数，选择高特异的亚克隆进行扩大培养以及冻存。The immune-responsive mice were screened by ELISA/FACS. The mouse spleen lymphocytes were fused with Sp2/0-Ag14 cells to obtain hybridoma cells. The hybridoma cells were plated into a 96-well plate for growth, and the cells were taken 10-14 days later. Use ELISA/FACS to screen cells, dissociation rate constants, and select highly specific subclones for expansion culture and cryopreservation.
ELISA检测方法如下：使用100ul 1ug/ml FMC63-scFv-mFc包被板，四度孵育过夜；PBST洗三次，1％BSA于37℃封闭1小时；PBST洗三次，每孔加入50ul mAb抗抗体(40ug/ml)和生物素标记FMC63-scFv-mFc；于37℃孵育1小时，PBST洗三次；每孔加100ul二抗，37℃孵育1小时；PBST洗五次，每孔加100ul TMB；每孔加入50ul 1M的HCl终止反应；酶标仪450nm波长下读板；The ELISA detection method is as follows: use 100ul 1ug/ml FMC63-scFv-mFc coated plate, incubate overnight at four degrees; wash three times with PBST, block with 1% BSA at 37°C for 1 hour; wash three times with PBST, add 50ul mAb anti-antibody ( 40ug/ml) and biotin-labeled FMC63-scFv-mFc; incubate at 37°C for 1 hour and wash three times with PBST; add 100ul secondary antibody to each well and incubate at 37°C for 1 hour; wash five times with PBST and add 100ul TMB to each well; Add 50ul 1M HCl to the well to stop the reaction; read the plate under the 450nm wavelength of the microplate reader;
FACS检测方法如下：将mAb与Jurkat-FMC63 scFv细胞混合，室温孵育30分钟后DPBS洗一次，加抗鼠Fc抗体室温孵育30分钟，DPBS洗一次后流式上机检测。The FACS detection method is as follows: mix mAb with Jurkat-FMC63 scFv cells, incubate at room temperature for 30 minutes, wash once with DPBS, add anti-mouse Fc antibody to incubate at room temperature for 30 minutes, wash once with DPBS and perform flow-based detection.
根据初步的ELISA结果，D value>10，得到103个克隆进行二次筛选。使用ELISA和FACS方法对103个克隆进行进一步筛选，得到23个单克隆细胞表达结合FMC63的抗体(对应表2中的mAb01-mAb023)。According to the preliminary ELISA results, D value>10, 103 clones were obtained for secondary screening. 103 clones were further screened using ELISA and FACS methods, and 23 monoclonal cells were obtained to express antibodies that bind to FMC63 (corresponding to mAb01-mAb023 in Table 2).
ELISA和FACS检测结果如图2-3和表2-4所示。ELISA and FACS test results are shown in Figure 2-3 and Table 2-4.
结果显示，mAb06(26E7D2)、mAb023(75H6C7)在ELISA检测中效果最优，mAb020(52H8C4)在FACS中检测中效果最优。The results showed that mAb06 (26E7D2) and mAb023 (75H6C7) had the best effect in ELISA detection, and mAb020 (52H8C4) had the best effect in FACS detection.
表2 ELISA检测抗FMC63抗体的亲和力(EC50)数值Table 2 Affinity (EC50) values of anti-FMC63 antibodies detected by ELISA
| 抗体Antibody | 克隆号Clone number | EC50(ug/ml)EC50(ug/ml) | 抗体Antibody | 克隆号Clone number | EC50(ug/ml)EC50(ug/ml) |
| mAb01mAb01 | 4C5E24C5E2 | 0.034790.03479 | mAb013mAb013 | 9H2G19H2G1 | 0.039860.03986 |
| mAb02mAb02 | 8B3H48B3H4 | 0.022980.02298 | mAb014mAb014 | 20H5D1020H5D10 | 0.06280.0628 |
| mAb03mAb03 | 14A10E714A10E7 | 0.82180.8218 | mAb015mAb015 | 22G8F722G8F7 | 5362953629 |
| mAb04mAb04 | 17F10D317F10D3 | 0.022730.02273 | mAb016mAb016 | 40F12E540F12E5 | 0.048880.04888 |
| mAb05mAb05 | 26E2F1126E2F11 | 0.07140.0714 | mAb017mAb017 | 35H8C435H8C4 | 0.053890.05389 |
| mAb06mAb06 | 26E7D226E7D2 | 0.02990.0299 | mAb018mAb018 | 24E8E1124E8E11 | 0.086290.08629 |
| mAb07mAb07 | 27C5E1127C5E11 | 0.099050.09905 | mAb019mAb019 | 66B2A966B2A9 | 0.069810.06981 |
| mAb08mAb08 | 28B6F728B6F7 | 0.072260.07226 | mAb020mAb020 | 52H8C452H8C4 | 0.1130.113 |
| mAb09mAb09 | 28E12C328E12C3 | 0.056640.05664 | mAb021mAb021 | 47H1B347H1B3 | 0.048390.04839 |
| mAb010mAb010 | 35G7G635G7G6 | 0.0590.059 | mAb022mAb022 | 42C10G242C10G2 | 0.17160.1716 |
| mAb011mAb011 | 36A11E336A11E3 | 0.060010.06001 | mAb023mAb023 | 75H6C775H6C7 | 0.051150.05115 |
| mAb012mAb012 | 39D4C539D4C5 | 0.056070.05607 | To | To | To |
表3 FACS检测抗FMC63抗体的亲和力(EC50)数值Table 3 Affinity (EC50) values of anti-FMC63 antibodies detected by FACS
表4 ELISA和FACS筛选后的抗FMC63抗体Table 4 Anti-FMC63 antibodies screened by ELISA and FACS
实施例4 抗FMC63抗体表位检测Example 4 Anti-FMC63 antibody epitope detection
使用竞争性ELISA检测抗体结合表位。使用100ul 1ug/ml抗FMC63抗体包被板，四度孵育过夜；PBST洗三次，1％BSA于37℃封闭1小时；PBST洗三次，每孔加入50ul抗抗体(40ug/ml)和FMC63-scFv-mFc(EC80)；于37℃孵育1小时，PBST洗三次；每孔加100ul二抗，37℃孵育1小时；PBST洗五次，每孔加100ul TMB；每孔加入50ul 1M的HCl终止反应；酶标仪450nm波长下读板。A competitive ELISA was used to detect antibody binding epitopes. Use 100ul 1ug/ml anti-FMC63 antibody to coat the plate and incubate overnight for four degrees; wash three times with PBST and block with 1% BSA for 1 hour at 37°C; wash three times with PBST, add 50ul anti-antibody (40ug/ml) and FMC63-scFv to each well -mFc(EC80); incubate at 37°C for 1 hour, wash with PBST three times; add 100ul secondary antibody to each well, incubate for 1 hour at 37°C; wash five times with PBST, add 100ul TMB to each well; add 50ul 1M HCl to each well to stop the reaction ; Read the plate under the 450nm wavelength of the microplate reader.
将结合FMC63不同表位的抗体分类，结果如表5所示。The antibodies that bind to different epitopes of FMC63 were classified, and the results are shown in Table 5.
表5 抗FMC63抗体表位分类Table 5 Anti-FMC63 antibody epitope classification
实施例5 单克隆测序Example 5 Monoclonal sequencing
选取单克隆细胞mAb02(8B3H4)、mAb06(26E7D2)、mAb07(27C5E11)、mAb020(52H8C4)、mAb023(75H6C7)进行DNA测序，测序后得到的抗体重链可变区和轻链可变区的氨基酸序列如表6所示，表6还显示了抗体测序分析后得到的CDR区。Select monoclonal cells mAb02 (8B3H4), mAb06 (26E7D2), mAb07 (27C5E11), mAb020 (52H8C4), mAb023 (75H6C7) for DNA sequencing, and the amino acids of the heavy chain variable region and light chain variable region of the antibody obtained after sequencing The sequence is shown in Table 6, which also shows the CDR regions obtained after antibody sequencing analysis.
表6 mAb抗体CDR区序列和重链、轻链可变区序列Table 6 mAb antibody CDR region sequence and heavy chain, light chain variable region sequence
实施例7 抗FMC63抗体应用于抗抗体检测方法Example 7 Application of anti-FMC63 antibody to anti-antibody detection method
选用结合FMC63不同表位的抗体组合(mAb01,mAb02,mAb07,mAb10,mAb011,mAb021,mAb022,mAb023)等浓度混合后作为阳性品(PC)，设计抗抗体检测实验。向MSD板中加入300ul封闭液封闭2小时，把配置好的混合PC，一定浓度的biotin-FMC63-scFv与一定浓度的sulfo-tag-FMC63-scFv的混合液吸取100ul在另一块板中孵育1小时。把MSD板中的封闭液丢弃并洗板3次。把孵育的混合液以及每孔50ul的体积转移至MSD板中孵育1小时。把MSD板的混合液丢弃并洗板3次。加入150ul每孔2x read buffer读板。Combinations of antibodies that bind different epitopes of FMC63 (mAb01, mAb02, mAb07, mAb10, mAb011, mAb021, mAb022, mAb023) are selected and mixed at the same concentration as the positive product (PC), and the anti-antibody detection experiment is designed. Add 300ul of blocking solution to the MSD plate for 2 hours, then mix the configured mixed PC, a certain concentration of biotin-FMC63-scFv and a certain concentration of sulfo-tag-FMC63-scFv, draw 100ul and incubate in another plate 1 Hour. Discard the blocking solution in the MSD plate and wash the plate 3 times. Transfer the incubation mixture and the volume of 50ul per well to the MSD plate and incubate for 1 hour. Discard the MSD plate mixture and wash the plate 3 times. Add 150ul 2x read buffer per well to read the plate.
结果如图4和表7所示，MSD检测结果显示抗FMC63抗体可以与FMC63-scFv按照剂量依赖的方式结合，说明抗FMC63抗体可以应用到MSD方法中。The results are shown in Figure 4 and Table 7. The MSD test results showed that the anti-FMC63 antibody can bind to FMC63-scFv in a dose-dependent manner, indicating that the anti-FMC63 antibody can be applied to the MSD method.
表7 抗FMC63抗体组合应用到MSD方法检测原始数据Table 7 Application of anti-FMC63 antibody combination to MSD detection raw data
实施例8 抗体的功能检测Example 8 Functional detection of antibodies
将表2所示的23种抗体与生物素标记的CD19蛋白混合后，加入到Jurkat-FMC63 scFv细胞孵育1小时，DPBS洗一次，加入链霉亲和素-APC孵育30分钟，DPBS洗一次后流式上机检测CD19蛋白结合。After mixing 23 kinds of antibodies shown in Table 2 with biotin-labeled CD19 protein, add them to Jurkat-FMC63 scFv cells and incubate for 1 hour, wash once with DPBS, add streptavidin-APC and incubate for 30 minutes, wash once with DPBS Flow cytometric detection of CD19 protein binding.
将表2所示的23种抗体与FMC63-CAR-T细胞共孵育1小时，将孵育后的FMC63-CAR-T细胞添加到铺有Hela-CD19的RTCA板中，观察与抗体结合后的CAR-T对靶细胞的杀伤功能变化，从而筛选得到可以阻断FMC63-CAR-T功能的抗体。Incubate the 23 kinds of antibodies shown in Table 2 with FMC63-CAR-T cells for 1 hour, add the incubated FMC63-CAR-T cells to the RTCA plate covered with Hela-CD19, and observe the CAR after binding to the antibody -T changes in the killing function of target cells, so as to screen for antibodies that can block the function of FMC63-CAR-T.
将表2所示的23种抗体加入到FMC63-CAR-T细胞，在不同时间点检测FMC63-CAR-T细胞的密度和数量。结果如图6、7、8显示，抗FMC63抗体可以促进FMC63-CAR-T细胞的增殖、激活和细胞因子释放。The 23 antibodies shown in Table 2 were added to FMC63-CAR-T cells, and the density and number of FMC63-CAR-T cells were detected at different time points. The results are shown in Figures 6, 7, and 8. Anti-FMC63 antibodies can promote the proliferation, activation and cytokine release of FMC63-CAR-T cells.
结果如图5所示，mAb019、mAb020可以干扰CD19蛋白与Jurkat-FMC63 scFv细胞的结合，从而阻断CAR与靶点CD19结合发挥作用。The results are shown in Figure 5, mAb019 and mAb020 can interfere with the binding of CD19 protein to Jurkat-FMC63 scFv cells, thereby blocking the binding of CAR to the target CD19.
实施例9 人源化抗FMC63抗体的制备Example 9 Preparation of humanized anti-FMC63 antibody
将鼠源抗FMC63抗体mAb02、mAb06和mAb020的Fc段更换为人源Fc，得到人鼠嵌合抗体。人源化的mAb02、mAb06和mAb020抗体的重链氨基酸序列分别如SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO:23，轻链的氨基酸序列分别如SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24。The Fc segments of the murine anti-FMC63 antibodies mAb02, mAb06 and mAb020 were replaced with human Fc to obtain human-mouse chimeric antibodies. The heavy chain amino acid sequences of the humanized mAb02, mAb06 and mAb020 antibodies are shown in SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 23, respectively, and the light chain amino acid sequences are shown in SEQ ID NO: 20, SEQ ID, respectively. NO: 22, SEQ ID NO: 24.
实施例10 靶向CD19抗体的CAR-T的制备Example 10 Preparation of CAR-T Targeting CD19 Antibody
利用抗FMC63抗体的抗体mAb02、mAb06、mAb07、mAb020和mAb023制备CAR-T，结构为信号肽-anti-FMC63 scFv-CD8铰链-CD8跨膜区-CD137共刺激区-CD3z,氨基酸序列分别如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49。Use anti-FMC63 antibodies mAb02, mAb06, mAb07, mAb020 and mAb023 to prepare CAR-T, the structure is signal peptide-anti-FMC63 scFv-CD8 hinge-CD8 transmembrane region-CD137 costimulatory region-CD3z, the amino acid sequence is as SEQ respectively ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49.
具体方法如下：The specific method is as follows:
细胞复苏：取PBMC于37℃水浴锅中解冻约2min，将细胞悬液转移至30mL已预热的CAR-T基础培养基(含1％HSA的X-vivo15)中，混匀后，取样计数，剩余细胞300g离心10分钟。Cell resuscitation: Thaw PBMC in a 37°C water bath for about 2 minutes, transfer the cell suspension to 30 mL of pre-warmed CAR-T basal medium (X-vivo 15 with 1% HSA), mix well, and sample and count Centrifuge the remaining cells at 300g for 10 minutes.
T细胞接种与活化T cell seeding and activation
室温下，将分选后的阳性细胞300g离心8min，弃上清，用适量CAR-T基础培 养基重悬细胞沉淀。按照CD3/CD28 dynabeads与细胞数量1:1加入适当体积CD3/CD28 dynabeads，补加等体积CAR-T基础培养基洗涤磁珠，置于DynaMag50上静置1min，弃上清，用1-5mL CAR-T基础培养基重悬磁珠，并转移至细胞悬液中，混匀后补加适量CAR-T基础培养基使细胞密度达到2×10
6/mL，并补加1/1000体积的IMC0003。混匀后，取2-3mL细胞悬液于6孔板中作为阴性对照细胞，剩余细胞用于CAR-T细胞制备。
Centrifuge the sorted positive cells at 300 g for 8 min at room temperature, discard the supernatant, and resuspend the cell pellet with an appropriate amount of CAR-T basal medium. Add an appropriate volume of CD3/CD28 dynabeads according to the number of CD3/CD28 dynabeads and the number of cells 1:1, add an equal volume of CAR-T basal medium to wash the magnetic beads, place them on DynaMag50 and let stand for 1 min, discard the supernatant, and use 1-5mL CAR -T basal medium resuspend the magnetic beads and transfer to the cell suspension. After mixing, add appropriate amount of CAR-T basal medium to make the cell density reach 2×10 6 /mL, and add 1/1000 volume of IMC0003 . After mixing, take 2-3 mL of cell suspension in a 6-well plate as negative control cells, and the remaining cells are used for CAR-T cell preparation.
T细胞病毒感染(Day1)T cell virus infection (Day1)
将细胞取出，混匀后取样计数，并根据细胞体积计算细胞量，收集细胞悬液300g离心8分钟。按照2×10
6/mL密度计算接种体积，并根据公式“慢病毒载体体积＝MOI*细胞量/生物滴度”计算慢病毒载体体积。弃上清，用适当CAR-T基础培养基(体积＝接种体积-慢病毒载体体积)重悬细胞沉淀，补加1/1000接种体积的IL2，并将复苏后病毒加入细胞悬液中，进行感染。
The cells were taken out, sampled and counted after mixing, and the cell volume was calculated according to the cell volume, and the cell suspension was collected and centrifuged at 300g for 8 minutes. Calculate the inoculation volume according to the density of 2×10 6 /mL, and calculate the volume of the lentiviral vector according to the formula "lentiviral vector volume = MOI * cell volume / biological titer". Discard the supernatant, resuspend the cell pellet with appropriate CAR-T basal medium (volume = inoculation volume-lentiviral vector volume), add 1/1000 inoculation volume of IL2, and add the resuscitated virus to the cell suspension. Infect.
CAR-T细胞扩增培养(Day8)CAR-T cell expansion culture (Day8)
CAR-T细胞，吹打混匀后，取样计数，取约0.5×10
6细胞进行阳性率检测，补加CART基础培养基继续培养。在CAR胞外结构中包含DYKDDDDK的氨基酸序列，使用抗Flag抗体检测CAR+阳性率。结果如图9显示，CAR成功表达在T细胞上。
CAR-T cells, after pipetting and mixing, sample and count, take about 0.5×10 6 cells for positive rate detection, and add CART basal medium to continue culturing. The extracellular structure of CAR contains the amino acid sequence of DYKDDDDK, and anti-Flag antibody is used to detect the positive rate of CAR+. The results are shown in Fig. 9 that CAR was successfully expressed on T cells.
CAR-T细胞毒性检测CAR-T cytotoxicity test
将CAR-T与靶细胞(Hela-FMC63、K562-FMC63)按照效靶比1:1孵育；使用实时无标记动态细胞分析技术、流式检测靶细胞的活性，得出CAR-T的激活和细胞毒作用效果。同时取细胞上清检测细胞因子释放，包括IL2、IFNγ。Incubate CAR-T with target cells (Hela-FMC63, K562-FMC63) according to an effective target ratio of 1:1; use real-time label-free dynamic cell analysis technology and flow cytometry to detect the activity of target cells to obtain CAR-T activation and Cytotoxic effect. At the same time, the cell supernatant was taken to detect the release of cytokines, including IL2 and IFNγ.
结果显示，靶向CD19抗体的CAR-T可以对靶细胞Hela-FMC63产生细胞毒作用，如图10；靶向CD19抗体的CAR-T可以被靶细胞K562-FMC63激活并且释放细胞因子IFNγ和IL2，如图11和图12。The results show that CAR-T targeting CD19 antibody can produce cytotoxic effect on target cell Hela-FMC63, as shown in Figure 10; CAR-T targeting CD19 antibody can be activated by target cell K562-FMC63 and release cytokines IFNγ and IL2 , As shown in Figure 11 and Figure 12.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
Claims (15)
- 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：A heavy chain variable region of an antibody, characterized in that the heavy chain variable region includes the following three complementarity determining region CDRs:SEQ ID NO:7所示的CDR1，CDR1 shown in SEQ ID NO: 7,SEQ ID NO:8所示的CDR2，和CDR2 shown in SEQ ID NO: 8, andSEQ ID NO:9所示的CDR3；CDR3 shown in SEQ ID NO: 9;或者，or,SEQ ID NO:4的CDR1，CDR1 of SEQ ID NO: 4,SEQ ID NO:5所示的CDR2，和CDR2 shown in SEQ ID NO: 5, andSEQ ID NO:6所示的CDR3，CDR3 shown in SEQ ID NO: 6,或者，or,SEQ ID NO:1所示的CDR1，CDR1 shown in SEQ ID NO:1,SEQ ID NO:2所示的CDR2，和CDR2 shown in SEQ ID NO: 2, andSEQ ID NO:3所示的CDR3；CDR3 shown in SEQ ID NO: 3;其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CD19抗体的结合亲和力的衍生序列。Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
- 一种抗体的重链，其特征在于，所述的重链具有如权利要求1所述的重链可变区。An antibody heavy chain, characterized in that the heavy chain has the heavy chain variable region of claim 1.
- 一种抗体的轻链可变区，其特征在于，所述的轻链可变区包括以下三个互补决定区CDR：A light chain variable region of an antibody, characterized in that the light chain variable region includes the following three complementarity determining region CDRs:SEQ ID NO:16所示的CDR1’，CDR1' shown in SEQ ID NO: 16,SEQ ID NO:17所示的CDR2’，和CDR2' shown in SEQ ID NO: 17, andSEQ ID NO:18所示的CDR3’；CDR3' shown in SEQ ID NO: 18;或者，or,SEQ ID NO:13所示的CDR1’，CDR1' shown in SEQ ID NO: 13,SEQ ID NO:14所示的CDR2’，和CDR2' shown in SEQ ID NO: 14, andSEQ ID NO:15所示的CDR3’；CDR3' shown in SEQ ID NO: 15;或者，or,SEQ ID NO:10所示的CDR1’，CDR1' shown in SEQ ID NO: 10,SEQ ID NO:11所示的CDR2’，和CDR2' shown in SEQ ID NO: 11, andSEQ ID NO:12所示的CDR3’；CDR3' shown in SEQ ID NO: 12;其中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留对CD19抗体的结合亲和力的衍生序列。Wherein, any one of the above amino acid sequences further includes a derivative sequence that is optionally added, deleted, modified and/or substituted for at least one amino acid and can retain the binding affinity for the CD19 antibody.
- 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求3所述的轻链可变区。An antibody light chain, characterized in that the light chain has the light chain variable region according to claim 3.
- 一种抗体，其特征在于，所述抗体具有：An antibody, characterized in that the antibody has:(1)如权利要求1所述的重链可变区；和/或(1) The heavy chain variable region of claim 1; and/or(2)如权利要求3所述的轻链可变区；(2) The light chain variable region of claim 3;或者，所述抗体具有：如权利要求2所述的重链；和/或如权利要求4所述的轻链。Alternatively, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.
- 一种重组蛋白，其特征在于，所述的重组蛋白具有：A recombinant protein, characterized in that the recombinant protein has:(i)如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体；以及(i) The heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, or The antibody of claim 5; and(ii)任选的协助表达和/或纯化的标签序列。(ii) Optional tag sequence to assist expression and/or purification.
- 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：An antibody-drug conjugate, characterized in that the antibody-drug conjugate contains:(a)抗体部分，所述抗体部分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、或如权利要求5所述的抗体、或其组合；和(a) An antibody portion, which is selected from the group consisting of the heavy chain variable region according to claim 1, the heavy chain according to claim 2, and the light chain variable region according to claim 3 , The light chain of claim 4, or the antibody of claim 5, or a combination thereof; and(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、酶、或其组合。(b) A coupling part coupled to the antibody part, and the coupling part is selected from the group consisting of detectable markers, drugs, toxins, cytokines, enzymes, or combinations thereof.
- 一种CAR构建物，其特征在于，所述的CAR构建物的抗原结合结构域包含如权利要求1所述的重链可变区和如权利要求3所述的轻链可变区，较佳地，所述嵌合抗原受体构成还包含：铰链区、跨膜区、共刺激信号区和CD3z。A CAR construct, characterized in that the antigen binding domain of the CAR construct comprises the heavy chain variable region according to claim 1 and the light chain variable region according to claim 3, preferably Specifically, the chimeric antigen receptor composition further comprises: a hinge region, a transmembrane region, a costimulatory signal region and CD3z.
- 一种工程化免疫细胞，其特征在于，所述的工程化免疫细胞表达外源的如权利要求8所述的CAR构建物。An engineered immune cell, characterized in that the engineered immune cell expresses an exogenous CAR construct according to claim 8.
- 一种活性成分的用途，其特征在于，所述活性成分选自下组：如权利要求 1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体、如权利要求7所述的抗体药物偶联物、如权利要求9所述的工程化免疫细胞、或其组合，其特征在于，所述活性成分用于(a)制备检测试剂、检测板或试剂盒；和/或(b)制备药物，和/或(c)用于刺激靶向CD19的CAR的免疫细胞扩增；和/或(d)用于分离纯化靶向CD19的CAR的免疫细胞；A use of an active ingredient, characterized in that the active ingredient is selected from the following group: the heavy chain variable region according to claim 1, the heavy chain according to claim 2, and the heavy chain according to claim 3. Light chain variable region, light chain as claimed in claim 4, antibody as claimed in claim 5, antibody-drug conjugate as claimed in claim 7, engineered immune cell as claimed in claim 9, Or a combination thereof, characterized in that the active ingredient is used to (a) prepare a detection reagent, a detection plate or a kit; and/or (b) prepare a drug, and/or (c) be used to stimulate a CAR targeting CD19 Immune cell expansion; and/or (d) Immune cells used to isolate and purify CD19-targeted CAR;
- 如权利要求10所述的用途，其特征在于，所述的药物用于中和CD19抗体或用于封闭在细胞上表达的靶向CD19的CAR。The use according to claim 10, wherein the drug is used to neutralize CD19 antibodies or to block CD19-targeting CAR expressed on cells.
- 如权利要求11所述的用途，其特征在于，所述细胞包括表达靶向CD19的CAR的肿瘤细胞或免疫细胞，较佳地为表达靶向CD19的CAR的肿瘤B细胞和表达靶向CD19的CAR的T细胞。The use according to claim 11, wherein the cells comprise tumor cells or immune cells expressing CARs targeting CD19, preferably tumor B cells expressing CARs targeting CD19 and tumor B cells expressing CARs targeting CD19 CAR T cells.
- 一种药物组合物，其特征在于，所述的药物组合物含有：A pharmaceutical composition, characterized in that the pharmaceutical composition contains:(i)活性成分，所述活性成分选自下组：如权利要求1所述的重链可变区、如权利要求2所述的重链、如权利要求3所述的轻链可变区、如权利要求4所述的轻链、如权利要求5所述的抗体、如权利要求7所述的抗体药物偶联物、如权利要求9所述的工程化免疫细胞、或其组合；以及(i) An active ingredient, which is selected from the group consisting of the heavy chain variable region according to claim 1, the heavy chain according to claim 2, and the light chain variable region according to claim 3 , The light chain of claim 4, the antibody of claim 5, the antibody-drug conjugate of claim 7, the engineered immune cell of claim 9, or a combination thereof; and(ii)药学上可接受的载体。(ii) A pharmaceutically acceptable carrier.
- 一种在体内中和CD19抗体或中和在细胞上表达的靶向CD19的CAR的方法，所述方法包括：给需要的对象施用如权利要求5所述的抗体、如权利要求7所述的抗体药物偶联物、如权利要求9所述的工程化免疫细胞、或其组合。A method for neutralizing a CD19 antibody or a CD19-targeting CAR expressed on a cell in vivo, the method comprising: administering the antibody according to claim 5, the antibody according to claim 7 to a subject in need An antibody drug conjugate, the engineered immune cell according to claim 9, or a combination thereof.
- 一种体外检测样品中CD19抗体或表达靶向CD19的CAR的细胞的方法，所述方法包括步骤：An in vitro method for detecting CD19 antibodies or cells expressing CARs targeting CD19 in a sample, the method comprising the steps:(1)在体外，将所述样品与权利要求5所述的抗体接触；(1) In vitro, contacting the sample with the antibody of claim 5;(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CD19抗体或表达靶向CD19的CAR的细胞。(2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD19 antibodies or cells expressing CARs targeting CD19 in the sample.
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Also Published As
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| TWI800824B (en) | 2023-05-01 |
| CN115515984A (en) | 2022-12-23 |
| TW202144435A (en) | 2021-12-01 |
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