WO2016112855A1 - 抗cd19单克隆抗体及其制备方法 - Google Patents
抗cd19单克隆抗体及其制备方法 Download PDFInfo
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- WO2016112855A1 WO2016112855A1 PCT/CN2016/070831 CN2016070831W WO2016112855A1 WO 2016112855 A1 WO2016112855 A1 WO 2016112855A1 CN 2016070831 W CN2016070831 W CN 2016070831W WO 2016112855 A1 WO2016112855 A1 WO 2016112855A1
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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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
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- the present invention belongs to the field of biomedicine, and in particular, the present invention relates to an anti-CD19 monoclonal antibody and a process for the preparation thereof.
- CD19 is expressed in both normal and malignant B lymphocytes and is considered to be the most reliable surface marker for a long period of coverage during B cell development.
- CD19 is expressed in germinal center B cells and follicular dendritic cells, mantle cells, and dendritic cells in the intercellular follicle T cell region.
- the CD19 molecule is mainly expressed in early B cells and is a transmembrane glycoprotein unique to the B cell line of about 95KDa.
- CD19 As a cell surface molecule that B cells express during their differentiation and proliferation.
- CD19 is generally considered to be a therapeutic target for the treatment of B cell disorders or diseases such as B cell malignancies, autoimmune diseases and transplant rejection, which is expressed on B cell line malignancies including, but not limited to, non-Hodge Golden lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.
- B cell malignancies such as B cell malignancies, autoimmune diseases and transplant rejection
- B cell line malignancies including, but not limited to, non-Hodge Golden lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.
- a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
- the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO: 2.
- a heavy chain of an antibody having the first aspect of the present invention
- the heavy chain variable region and the heavy chain constant region described in the aspects are provided.
- the heavy chain constant region is of human or murine origin.
- the light chain variable region has the amino acid sequence set forth in SEQ ID NO:4.
- a light chain of an antibody having the light chain variable region and the light chain constant region of the fourth aspect of the invention.
- the constant region of the light chain is of human or murine origin.
- an antibody having:
- the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.
- the antibody is an antibody specific for the anti-CD19 protein.
- the antibody comprises: a single chain antibody, a double chain antibody, a monoclonal antibody, a chimeric antibody (such as a human mouse chimeric antibody), a murine antibody, a humanized antibody, and a bispecific antibody. (BiTE) and chimeric antigen receptor antibody (CAR).
- a recombinant protein having:
- the tag sequence comprises a 6His tag.
- the recombinant protein specifically binds to the CD19 protein.
- the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the fourth aspect of the invention a light chain as described in the aspect, or an antibody according to the fifth aspect of the invention; or
- polynucleotide has the sequence set forth in SEQ ID NO.: 1, 3, 8, 9, 10, 14, 15, or 16.
- a vector comprising the polynucleotide of the seventh aspect of the invention is provided.
- the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.
- a genetically engineered host cell comprising the polynucleotide of the seventh aspect of the invention, or a polynucleotide of the seventh aspect of the invention, is provided.
- an immunoconjugate comprising:
- a coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
- the conjugate is selected from the group consisting of: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing a detectable agent
- Product enzymes radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, pre- A drug activating enzyme (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (eg, cisplatin) or any form of nanoparticles, and the like.
- DTD DT-diaphorase
- BPHL biphenyl hydrolase-like protein
- a pharmaceutical composition comprising:
- the pharmaceutical composition is in the form of an injection.
- the pharmaceutical composition is used for preparing a medicament for treating a tumor, the tumor being selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, Colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, adrenal tumor, or bladder tumor.
- a twelfth aspect of the invention provides a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable according to the third aspect of the invention
- a light chain according to the fourth aspect of the invention an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or an immunoconjugate according to the tenth aspect of the invention
- the reagent, the detection plate or the kit is used for: detecting the CD19 protein in the sample;
- the agent is for treating or preventing a tumor expressing a CD19 protein.
- the tumor comprises: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, or adrenal gland Tumor.
- the tumor is selected from the group consisting of gastric cancer and follicular lymphoma.
- the reagent comprises a chip, an immunoparticle coated with an antibody.
- a method of detecting a CD19 protein in a sample comprising the steps of:
- a fourteenth aspect of the invention a method for preparing a recombinant polypeptide, comprising:
- a fifteenth aspect of the invention provides a method of treating a disease, comprising: administering to a subject in need thereof a therapeutically effective amount of an antibody according to the fifth aspect of the invention, as described in the sixth aspect of the invention A recombinant protein, an immunoconjugate according to the tenth aspect of the invention, or a pharmaceutical composition according to the eleventh aspect of the invention.
- FIG. 1 is a flow chart of the anti-CD19 monoclonal antibody (PGC3D6H10).
- the target cell line selected is Ramos, and the anti-CD19 monoclonal antibody has a working concentration of 1 ug/ml.
- the left panel is a negative control without anti-CD19 antibody, and the right panel is a fluorescence detection map of anti-CD19, and the positive rate is 83.83%.
- FIG. 2 is a flow chart of the anti-CD19 monoclonal antibody (PGC3D6H10).
- the target cell selected is human PBMC (peripheral blood mononuclear cells), and the anti-CD19 monoclonal antibody has a working concentration of 1 ug/ml.
- the left panel is a negative control without anti-CD19 antibody, and the right panel is a fluorescence detection map of anti-CD19.
- the positive rate was 15.44%.
- FIG. 3 is a flow chart of the anti-CD19 monoclonal antibody (PGC3D6H10).
- the target cell selected is Jurkat, and the anti-CD19 monoclonal antibody has a working concentration of 1 ug/ml.
- the left panel is a negative control without anti-CD19 antibody, and the right panel is a fluorescent assay for anti-CD19.
- the anti-CD19 antibody is hardly associated with Jurkat. Cells were combined and the positive rate was 0.54%.
- Figure 4 is a Western blot (WB) map of the anti-CD19 monoclonal antibody (PGC3D6H10).
- the working concentration of the anti-CD19 monoclonal antibody was 1 ug/ml, lanes 1 and 2 were two parallel experimental groups, the loading protein was the total protein of the lysed Ramos cells, and the lane 3 was the total protein of the lysed Jurkat cells as a negative control.
- FIG. 5 is a flow chart of the anti-CD19 monoclonal antibody (PGA6E2D5).
- the target cell line selected is Ramos, and the anti-CD19 monoclonal antibody has a working concentration of 1 ug/ml.
- the left panel is a negative control without anti-CD19 antibody, and the right panel is a fluorescence detection map of anti-CD19, and the positive rate is 82.65%.
- FIG. 6 is a flow chart of the anti-CD19 monoclonal antibody (PGA6E2D5).
- the target cell selected is Jurkat, and the anti-CD19 monoclonal antibody has a working concentration of 1 ug/ml.
- the left panel is a negative control without anti-CD19 antibody, and the right panel is a fluorescent assay for anti-CD19.
- the anti-CD19 antibody is hardly associated with Jurkat. Cells were combined and the positive rate was 0.00%.
- FIG. 7 is a flow chart of the anti-CD19 monoclonal antibody (PGB8D5C6).
- the target cell line selected was Ramos, and the anti-CD19 monoclonal antibody has a working concentration of 1 ug/ml.
- the left panel is a negative control without anti-CD19 antibody, and the right panel is a fluorescence detection map of anti-CD19, and the positive rate is 72.33%.
- FIG. 8 is a flow chart of the anti-CD19 monoclonal antibody (PGB8D5C6).
- the target cell selected is Jurkat, and the anti-CD19 monoclonal antibody has a working concentration of 1 ug/ml.
- the left panel is a negative control without anti-CD19 antibody, and the right panel is a fluorescent assay for anti-CD19.
- the anti-CD19 antibody is hardly associated with Jurkat. Cells were combined and the positive rate was 0.08%.
- Figure 9 is a graph showing the antigen affinity detection of an anti-CD19 monoclonal antibody (PGC3D6H10).
- Figure 10 is a graph showing the results of agarose gel electrophoresis analysis of the RT-PCR products of the variable regions VH and VL of the cloned anti-CD19 monoclonal antibody (clone number PGC3D6H10).
- Lane 1 is the DL5000 DNA Marker; Lane 2 is the VH gene; Lane 3 is the RT-PCR amplification VH gene negative control; Lane 4 is the VL gene; Lane 5 is the RT-PCR amplification VL gene negative control.
- Figure 11 shows random selection of 9 clones for colony PCR and agarose gel electrophoresis (clone number PGC3D6H10).
- the left side of Marker is the colony PCR identification of the corresponding randomly picked VH clone; the right side of Marker is the colony PCR identification of the corresponding randomly picked VL clone.
- the inventors have obtained a monoclonal antibody against CD19 through extensive screening through extensive and intensive research.
- the results of the experiment show that the anti-CD19 monoclonal antibody can not only effectively recognize cells with high expression of CD19 protein, but also succeed.
- the present invention has been completed on this basis.
- the anti-CD19 monoclonal antibody PGC3D6H10 of the invention can specifically target the CD19 molecular epitope on the surface of tumor cells, and can be used for preparation of antibody drugs, and the diagnosis, treatment, progression, metastasis potential and prognosis of B cell line-derived tumors The evaluation and other aspects have great potential.
- the antibodies which can be produced based on the PGC3D6H10 monoclonal antibody of the present invention include recombinant antibodies, ScFv antibodies, humanized antibodies, bispecific antibodies (BiTE), and chimeric antigen receptor antibodies (CAR).
- the CD19 molecule is mainly expressed in early B cells and is a transmembrane glycoprotein unique to the B cell line of about 95KDa. CD19 is expressed in both normal and malignant B lymphocytes and is considered to be the most reliable surface marker for a long period of coverage during B cell development.
- the amino acid sequence of the CD19 protein is:
- the term "antibody” or "immunoglobulin” is about 150,000 having the same structural features. Dalton's heterotetrameric glycoprotein consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked 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 a plurality of 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 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 .
- Particular amino acid residues form an interface between the variable regions of the light and heavy chains.
- variable means that certain portions of the variable regions of an antibody differ in sequence, which form the binding and specificity of various specific antibodies for their particular antigen. However, the variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the variable region of the light and heavy chains called the complementarity determining region (CDR) or hypervariable region. The more conserved portion of the variable region is referred to as the framework region (FR).
- the variable regions of the native heavy and light chains each comprise four FR regions which are substantially in a beta-sheet configuration and are joined by three CDRs forming a linker, in some cases forming a partial beta sheet structure.
- the CDRs in each chain are closely joined together by the FR region and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH Publ. No. 91-3242, Vol. I, pp. 647-669). (1991)).
- the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as antibody-dependent cytotoxicity of the participating antibodies.
- the "light chain" of a vertebrate antibody can be classified into one of two distinct classes (called kappa and lambda) depending on the amino acid sequence of its constant region.
- Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which may 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.
- the term "monoclonal antibody (mAb)” refers to an antibody obtained from a substantially homogeneous population, ie, the individual antibodies contained in the population are identical except for a few naturally occurring mutations that may be present. Monoclonal antibodies are highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal” indicates the identity of the antibody and is obtained from a substantially homogeneous population of antibodies, which should not be construed as requiring any particular method for producing the antibody.
- the present invention also includes a monoclonal antibody having the corresponding amino acid sequence of the anti-CD19 protein monoclonal antibody, a monoclonal antibody having the variable region chain of the anti-CD19 protein monoclonal antibody, and other proteins having these chains or Protein conjugates and fusion expression products.
- the invention encompasses any protein or protein conjugate having a light chain and a heavy chain comprising a hypervariable region (complementarity determining region, CDR) and a fusion expression product (ie, an immunoconjugate and a fusion expression product), as long as The hypervariable regions are identical or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.
- immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules with the anti-CD19 protein monoclonal antibody or A conjugate formed by the combination of its fragments.
- the invention also includes the same A cell surface marker or antigen to which a CD19 protein monoclonal antibody or fragment thereof binds.
- the present invention encompasses not only intact monoclonal antibodies, but also immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
- immunologically active antibody fragments such as Fab or (Fab') 2 fragments; antibody heavy chains; antibody light chains.
- variable region are used interchangeably with “complementarity determining region (CDR).
- the heavy chain variable region of the antibody comprises the following three complementarity determining region CDRs:
- CDR1 the amino acid sequence of which is Its coding nucleotide sequence is,
- amino acid sequence of the heavy chain variable region is:
- the heavy chain of the antibody comprises the above-described heavy chain variable region and heavy chain constant region, and the heavy chain constant region may be of murine or human origin.
- VL light chain variable region
- the light chain variable region of an antibody according to the invention has a complementarity determining region CDR selected from the group consisting of:
- amino acid sequence of the light chain variable region is:
- the light chain of the antibody comprises the above-described light chain variable region and light chain constant region, and the light chain constant region may be of murine or human origin.
- the terms “antibody of the invention”, “protein of the invention”, or “polypeptide of the invention” are used interchangeably and refer to an antibody that specifically binds to a CD19 protein, for example, having a heavy chain variable region (eg, SEQ ID A protein or polypeptide of NO.: amino acid sequence of 2) and/or a light chain variable region (such as the amino acid sequence of SEQ ID NO.: 4). They may or may not contain an initial methionine.
- a heavy chain variable region eg, SEQ ID A protein or polypeptide of NO.: amino acid sequence of 2
- a light chain variable region such as the amino acid sequence of SEQ ID NO.: 4
- the antibody is a murine or human murine chimeric monoclonal antibody against the CD19 protein
- the heavy chain constant region and/or the light chain constant region thereof may be a humanized heavy chain constant region or Light chain constant region.
- the humanized heavy chain constant region or light chain constant region is a heavy chain constant region or a light chain constant region of human IgGl, IgG2 or the like.
- the invention also provides other proteins or fusion expression products having the antibodies of the invention.
- the invention encompasses any protein or protein conjugate having a heavy chain and a light chain comprising a variable region and a fusion expression product (ie, an immunoconjugate and a fusion expression product), as long as the variable region is conjugated to an antibody of the invention
- the variable regions of the heavy and light chains are identical or at least 90% homologous, preferably at least 95% homologous.
- variable regions which are divided into four framework regions (FR), four
- FR framework regions
- the amino acid sequence of FR is relatively conservative and is not directly involved in the binding reaction.
- CDRs form a cyclic structure in which the ⁇ -sheets formed by the FRs are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
- the amino acid sequence of the same type of antibody can be compared to determine which amino acids constitute the FR or CDR regions.
- variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest since at least some of them are involved in binding antigen. Accordingly, the invention includes those molecules having a light chain and heavy chain variable region of a monoclonal antibody having a CDR, as long as the CDR thereof is 90% or more (preferably 95% or more, optimally 98%) with the CDR identified herein. The homology of the above).
- the present invention encompasses not only intact monoclonal antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies with other sequences. Therefore, the present invention also includes fragments and derivatives of the antibodies. Things and analogues.
- fragment refers to a polypeptide that substantially retains the same biological function or activity of an antibody of the invention.
- the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) a polypeptide formed by fusion, or (iv) a polypeptide formed by fused an additional amino acid sequence to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed by the 6
- the antibody of the present invention refers to a polypeptide comprising the above CDR regions having CD19 protein binding activity.
- the term also encompasses variant forms of a polypeptide comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include, but are not limited to, one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acid deletions , Insertion and/or Substitution, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
- the function of the protein is generally not altered.
- the addition of one or several amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
- the term also encompasses active fragments and active derivatives of the antibodies of the invention.
- Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing to the DNA encoding the antibody of the present invention under high or low stringency conditions.
- the encoded protein, and the polypeptide or protein obtained using an antiserum against the antibody of the present invention.
- the invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof.
- the invention also includes fragments of the antibodies of the invention.
- the fragment will have at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of the antibody of the invention.
- “conservative variant of the antibody of the present invention” means having up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3, compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
- the present invention also provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof.
- the polynucleotide of the present invention may be in the form of DNA or RNA.
- DNA forms include cDNA, genomic DNA or synthetic DNA.
- DNA can be single-stranded or double-stranded.
- the DNA can be a coding strand or a non-coding strand.
- the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO.: 1, 3, 8, 9, 10, 14, 15, 16 or a degenerate variant.
- a "degenerate variant” in the present invention refers to an amino acid sequence encoding the same as the polypeptide of the present invention, but with SEQ ID NO.: 1, 3, 8, 9, 10, 14, 15, A nucleic acid sequence differing in the coding region sequence shown by 16.
- Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences) and non-coding sequences .
- polynucleotide encoding a polypeptide can be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising additional coding and/or non-coding sequences.
- the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
- the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
- stringent conditions means: (1) hybridization and elution at a lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60 ° C; or (2) hybridization a denaturing agent such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 90% identity between the two sequences, more It is good that hybridization occurs more than 95%.
- the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide represented by SEQ ID NO.: 2 and/or SEQ ID NO.: 4.
- the full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
- One possible method is to synthesize related sequences by artificial synthesis, especially when the fragment length is short. Usually, by synthesizing multiple small segments first, then connecting again A very long sequence of fragments can be obtained.
- the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
- the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
- the biomolecule (nucleic acid, protein, etc.) to which the present invention relates includes biomolecules existing in an isolated form.
- DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
- the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
- mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
- the invention also relates to vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences. These vectors can be used to transform appropriate host cells to enable them to 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.
- a prokaryotic cell such as a bacterial cell
- a lower eukaryotic cell such as a yeast cell
- a higher eukaryotic cell such as a mammalian cell.
- Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, and the like.
- Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
- the host is a prokaryote such as E. coli
- competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
- Another method is to use MgCl 2 .
- Conversion can also be carried out by electroporation if desired.
- the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
- the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
- the medium used in the culture may be selected from various conventional media depending on the host cell used.
- the cultivation is carried out under conditions suitable for the growth of the host cell.
- the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
- the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
- the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super 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.
- the antibodies of the invention may be used alone or in combination or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
- Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (electron computed tomography) contrast agents, or capable of producing detectable products Enzyme.
- Therapeutic agents that can bind or conjugate to the antibodies of the invention include, but are not limited to: 1. Radionuclides (Koppe et al, 2005, Cancer metastasis reviews 24, 539); Drug toxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. Cytokines such as IL-2, etc. (Gillies et al.
- the invention also provides a composition.
- the composition is a pharmaceutical composition comprising the above antibody or active fragment thereof or a fusion protein thereof, and a pharmaceutically acceptable carrier.
- these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
- the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
- the pharmaceutical composition of the present invention can be directly used for binding to a CD19 protein molecule, and thus can be used for the prevention and treatment of tumors.
- other therapeutic agents can be used simultaneously.
- the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and pharmacy An acceptable carrier or excipient.
- Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
- the pharmaceutical preparation should be matched to the mode of administration.
- the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
- compositions such as injections and solutions are preferably prepared under sterile conditions.
- the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
- the polypeptides of the invention may also be used with other therapeutic agents.
- 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 per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
- specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
- the present invention also provides a hybridoma cell strain which can produce the monoclonal antibody against CD19 protein of the present invention; preferably, the present invention provides a high titer hybridoma cell strain against a monoclonal antibody against CD19 protein.
- the structure of the antibody of the present invention (such as the heavy chain variable region and the light chain variable region of the antibody) can be readily known to those skilled in the art, and then The monoclonal antibodies of the invention are prepared by recombinant methods.
- Antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art.
- an antigen of the invention can be administered to an animal to induce production of a monoclonal antibody.
- monoclonal antibodies hybridoma technology can be used (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol. 6: 511, 1976; Kohler et al, Eur. J. Immunol .6: 292, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981) or may be prepared by recombinant DNA method (U.S. Patent No. 4,816,567).
- myeloma cells are those which are efficiently fused, produce stable, high levels of production of cell-supporting antibodies by selected antibodies, and are sensitive to the culture medium (HAT medium matrix), including myeloma cell lines, such as rodents.
- Myeloma cell line including myeloma cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, San Diego, California, USA) and SP-2, NZ0 or X63-Ag8 -653 cells (available from American Type Culture Collection, Rockville, Maryland, USA).
- Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J.
- the medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies having the desired specificity, for example, by in vitro binding assays such as enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- the location of the antibody-expressing cells can be detected by FACS.
- Hybridoma clones can then be subcloned by limiting dilution steps and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986) 59- 103 pages).
- Suitable media for use in this purpose include, for example, DMEM or RPMI-1640 medium.
- hybridoma cells can be grown as ascites tumors in animals.
- the monoclonal antibodies secreted by the subclones are appropriately isolated from the culture medium, ascites or serum by a conventional immunoglobulin purification process, for example, protein A-Sepharose, hydroxyphosphorus. Gray stone chromatography, gel electrophoresis, dialysis or affinity chromatography.
- the present invention provides a monoclonal antibody against the CD19 protein.
- the monoclonal antibody is prepared by culturing a hybridoma cell method. The supernatant of the hybridoma cell culture was taken, crude IgG was extracted by saturated ammonium sulfate precipitation method, and the crude antibody was purified by affinity chromatography column (Protein G-Sephrose).
- the monoclonal antibody is prepared by the method of producing monoclonal antibodies using Balb/C mouse ascites.
- the hybridoma cells were inoculated into the peritoneal cavity of the sensitized mice, and the abdomen was significantly enlarged in about 10 days.
- Ascites was taken and crudely extracted by saturated ammonium sulfate precipitation, and then the crude antibody was purified by affinity chromatography (Protein G-Sephrose).
- High affinity, low immunogenic, highly efficient recombinant or humanized antibodies can be prepared by murine monoclonal antibodies. Humanization of murine antibodies can preserve antibody variable region fusion with human antibody constant region to increase antibody affinity; or engineer antibody structure to construct ScFv or antibody-containing variable regions and portions that retain only antibody variable regions The Fab of the constant region increases the percentage of absorption and half-life of the antibody in the body.
- the immunoglobulin carries a detectable label. More preferably, the label is selected from the group consisting of a colloidal gold label, a colored label or a fluorescent label.
- the colloidal gold label can be carried out by methods known to those skilled in the art.
- the monoclonal antibody to the CD19 protein is labeled with colloidal gold to provide a colloidal gold-labeled monoclonal antibody.
- the CD19 protein monoclonal antibody of the present invention has good specificity, high potency and affinity.
- test board of the present invention can be made by using a conventional test plate preparation method by using a test plate material commonly used in the art.
- the invention relates to an immunoassay plate for detecting CD19 protein, comprising a test strip and a support plate supporting the test strip, such as a PVC polyester plate;
- the test strip is made of filter paper, chromatography material, nitrocellulose membrane and
- the absorbent paper is sequentially laminated, and the overlapping portion can be fixedly connected by a conventional method such as tape;
- the chromatographic material is pre-coated with a colloidal gold-labeled or colored-labeled CD19 protein monoclonal antibody or polyclonal antibody, preferably by colloid.
- the colloidal gold-labeled CD19 protein monoclonal antibody is pre-coated with a CD19 protein monoclonal antibody solution labeled with a concentration of 0.5-1.5 mg/ml colloidal gold.
- the amount is 50 ⁇ l/cm 2 ; the preferred concentration is 0.5 or 1.5 mg/ml, 50 ⁇ l/cm 2 ;
- test plate was placed flat, and the sample was dropped on the filter paper.
- the sample was about 120 ⁇ l, and the chromatographic results were observed within 3 to 5 minutes. The result is judged based on the position of the streaks that appear.
- Invalid There is no ribbon in the quality control area and detection area or no ribbon appears in the quality control area and a ribbon appears in the detection area, indicating that the detection method is wrong or the detection board is deteriorated or invalid, and the detection board should be replaced.
- the present invention relates to a method for detecting a tumor in a sample dissolved in cells and/or tissues.
- the method steps are substantially as follows: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of CD19 protein in the dissolved sample.
- the sample used in the method of the invention may be any sample comprising cells present in the cell preservation solution, as used in liquid-based cell assays. Detection is preferably carried out using the Western Blot method.
- the invention also provides a kit comprising an antibody (or a fragment thereof) of the invention or a detection plate of the invention, in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffer Agent Wait.
- the invention further designs a detection kit for detecting the level of CD19 protein, the kit comprising an antibody for recognizing a CD19 protein, a lysis medium for dissolving the sample, detecting a common reagent and a buffer required, such as various buffers, detection Mark, detect substrates, etc.
- the test kit can be an in vitro diagnostic device.
- the anti-CD19 monoclonal antibody PGC3D6H10 obtained by extensive screening in the present invention can efficiently recognize cells highly expressing CD19 protein;
- the PGC3D6H10 monoclonal antibody of the present invention is capable of recognizing a natural CD19 molecule on the cell surface.
- the PGC3D6H10 monoclonal antibody of the present invention can be successfully used for Western Blot detection.
- the PGC3D6H10 monoclonal antibody of the present invention has an extremely high affinity and has an affinity for a target protein of more than 20 times that of a conventional antibody.
- mice Female BLB/c mice were immunized with B cell line tumor cells or CD19 eukaryotic recombinant protein antigen, and serum ELISA, WB, and FC positive mice were selected, and spleen cells were extracted and fused with myeloma cells. Hybridoma cells were formed; hybridoma cells were cultured in HAT medium, and monoclonal cells positive for high-throughput flow screening were subjected to ELISA rescreening to obtain 36 positive clones, followed by WB rescreening to obtain positive clones. 9 strains were subcloned into WB positive cells.
- the positive monoclonal cells identified in step 1 were injected into the mice for ascites production, and the produced ascites was purified by Protein A chromatography to obtain an anti-CD19 monoclonal antibody.
- the specificity of the antibodies was identified by flow cytometry, immunofluorescence and Western Blot. The specific operations are as follows:
- Lymphoma cell line Ramos purchased from ATCC
- human PBMC peripheral blood mononuclear cells isolated by Ficol method and negative control Jurkat cell line (purchased from ATCC, USA)
- 4% paraformaldehyde room temperature were collected.
- 3% BSA blocking solution prepared in PBS was placed at room temperature for 30 min, incubated with anti-CD19 monoclonal antibody (1 ug/ml) for 1 h at room temperature, and washed twice with PBST, FITC or Alexa Fluor 647-goat anti-mouse IgG (H+ L)
- the secondary antibody was incubated for 1 h in the dark, washed 3 times with PBST, the unbound fluorescent secondary antibody was removed, and the CD19 positive rate was detected by flow cytometry.
- Lymphoma cell line Ramos with high expression of CD19, and human PBMC (peripheral blood mononuclear cells) isolated by Ficol method and negative control Jurkat cell line were collected, 4% paraformaldehyde was fixed at room temperature for 20 min, and 3% BSA blocking solution was prepared in PBS.
- the cells were incubated at room temperature for 30 min, incubated with anti-CD19 monoclonal antibody (1 ug/ml) for 1 h at room temperature, washed twice with PBST, and incubated with FITC or Alexa Fluor 488-goat anti-mouse IgG (H+L) for 1 h in the dark, PBST centrifuged. The cells were washed twice, DAPI was stained for 10 min at room temperature, washed three times with PBST, the unbound dye was removed, and Confocal was used to detect CD19 staining.
- Lymphoma cell line high-expression CD19 Ramos T lymphoblastic leukemia cell line Jurkat
- protein lysate was lysed on ice for 30 min, the supernatant was centrifuged, the supernatant protein concentration was determined by BSA method, and loading buffer was added, 10% SDS-PAGE, 120V. Gel electrophoresis for 90 min, transfer film 400 mA, 90 min.
- the KD affinity constant of the murine anti-human CD19 monoclonal antibody with the clone PGC3D6H10 and the eukaryotic recombinant rCD19 protein was 434.6 pM (Fig. 9), compared to the conventional nanomolar antibody with affinity constant (for example, the other monoclonal antibodies of the present invention have an affinity constant that is more than 20 times higher.
- the present invention screened a monoclonal antibody hybridoma cell line (cloning number PGC3D6H10) capable of secreting an effective binding to the CD19 surface protein, and prepared an antibiotic.
- a monoclonal antibody to CD19 that has strong antigen affinity and antigen specificity.
- the cell strain used is a hybridoma cell strain obtained by the above method capable of secreting a high affinity and high specificity anti-CD19 monoclonal antibody, and the corresponding number is PGC3D6H10, and the corresponding antibody molecular subtype is: heavy chain IgG1 type, light Chain ⁇ type.
- the hybridoma cells in the logarithmic growth phase were 2 ⁇ 10 6 , and the total RNA was extracted by Trizol method.
- a small amount was quantified by Nanodrop and 1% non-denaturing agarose gel electrophoresis, followed by reverse transcription of cDNA using SuperScript.III First-Strand Synthesis System for RT-PCR kit (K1622, Thermo), and amplification of anti-CD19 with specific primers.
- a heavy or light chain variable region of an antibody gene was subjected to 1% agarose gel electrophoresis and recovered by gel. The recovered heavy chain variable region and light chain variable region were cloned into the sequencing vector pCR2.1, and sequenced, and the sequencing results were analyzed for homology and structure.
- Primer design The inventors selected a mouse-IgG (Mouse Ig-Primer Set, Novagen) primer for heavy chain variable region cloning, and a mouse-IgG ⁇ (Mouse Ig-Primer Set, Novagen) universal primer for light chain variable region cloning.
- mouse-IgG Mouse Ig-Primer Set, Novagen
- mouse-IgG ⁇ Mouse Ig-Primer Set, Novagen
- Reverse transcription PCR Reverse transcription of cDNA using total RNA of hybridoma cells as a template, as follows:
- the cDNA obtained by reverse transcription was used as a template, and the antibody heavy chain and light chain variable regions were synthesized using specific primers.
- Step 4 PCR product cloning and sequencing
- the heavy chain variable region and the light chain variable region nucleotide fragment product gel were recovered (09114KE1, AxyGEN), and then TA cloned according to the system in Table 4, and ligated overnight at 16 ° C, and respectively constructed on the sequencing vector. According to the system in Table 5, the colonies were picked and identified by colony PCR. The clones with positive bands were selected by 1% agarose gel electrophoresis, and the plasmid (07714KA1, AxyGEN) was sequenced.
- an antibody gene sequence (SEQ ID NO.) of a monoclonal antibody hybridoma cell line capable of secreting a CD19 surface protein is efficiently obtained by antibody gene extraction of monoclonal hybridoma cells (Fig. 10-11, clone No. PGC3D6H10). :1-4, clone number PGC3D6H10), PGC3D6H10 single
- the sequencing results of the cloned antibodies are as follows.
- VH nucleotide sequence of PGC3D6H10 monoclonal antibody VH nucleotide sequence of PGC3D6H10 monoclonal antibody:
- VH-encoding amino acid sequence of PGC3D6H10 monoclonal antibody VH-encoding amino acid sequence of PGC3D6H10 monoclonal antibody:
- VL nucleotide sequence of PGC3D6H10 monoclonal antibody VL nucleotide sequence of PGC3D6H10 monoclonal antibody:
- VL-encoding amino acid sequence of PGC3D6H10 monoclonal antibody VL-encoding amino acid sequence of PGC3D6H10 monoclonal antibody:
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Abstract
本发明提供了一种抗CD19单克隆抗体、其制备方法和应用。本发明的抗CD19的单克隆抗体不仅能够有效识别高表达CD19蛋白的细胞,而且能够成功用于Western Blot检测,亲和力检测表明,该单克隆抗体具有极高的与靶蛋白的亲和力。
Description
本发明属于生物医药领域,具体地说,本发明涉及抗CD19单克隆抗体及其制备方法。
CD19在正常及恶性B淋巴细胞中均有表达,被视为B细胞发育过程中一个涵盖阶段较长的最为可靠的表面标记物。在正常淋巴组织中,CD19表达于生发中心的B细胞和滤泡树突状细胞、套细胞、滤泡间T细胞区的树突状大细胞。事实上CD19分子主要表达于早期的B细胞,是一个95KDa左右的B细胞系特有的跨膜糖蛋白。
作为B细胞在其分化和增殖期间表达的细胞表面分子。CD19通常被认为是治疗B细胞失调或疾病如B细胞恶性肿瘤,自身免疫病和移植排斥的治疗靶点,其在B细胞系恶性肿瘤上表达,B细胞系恶性肿瘤包括但不限于非霍奇金淋巴瘤、慢性淋巴细胞白血病和急性成淋巴细胞性白血病。因此本领域技术人员致力于开发了与CD19特异性结合的抗体。
然而,目前的针对CD19蛋白的抗体性能尚难以令人满意。因此本领域需要开发新的、性能优异针对上述靶点的CD19蛋白抗体试剂或抗体药物,以满足临床上的需求。
发明内容
本发明的目的在于提供一种抗CD19单克隆抗体、其制备方法及其应用。
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:
SEQ ID NO:5所示的CDR1,
SEQ ID NO:6所示的CDR2,和
SEQ ID NO:7所示的CDR3。
在另一优选例中,所述重链可变区具有SEQ ID NO:2所示的氨基酸序列。
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和重链恒定区。
在另一优选例中,所述的重链恒定区为人源或鼠源的。
本发明的第三方面,提供了一种抗体的轻链可变区,所述轻链可变区具有选自下组的互补决定区CDR:
SEQ ID NO:11所示的CDR1’,
SEQ ID NO:12所示的CDR2’,和
SEQ ID NO:13所示的CDR3’。
在另一优选例中,所述的轻链可变区具有SEQ ID NO:4所示的氨基酸序列。
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四方面所述的轻链可变区和轻链恒定区。
在另一优选例中,所述轻链的恒定区为人源或鼠源的。
本发明的第五方面,提供了一种抗体,所述抗体具有:
(1)如本发明第一方面所述的重链可变区;和/或
(2)如本发明第三方面所述的轻链可变区。
在另一优选例中,所述抗体具有:如本发明第二方面所述的重链;和/或如本发明第四方面所述的轻链。
在另一优选例中,所述的抗体为特异性抗CD19蛋白的抗体。
在另一优选例中,所述的抗体包括:单链抗体、双链抗体、单克隆抗体、嵌合抗体(如人鼠嵌合抗体)、鼠源抗体、人源化抗体、双特异性抗体(BiTE)以及嵌合抗原受体抗体(CAR)。
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:
(i)如本发明第一方面所述的重链可变区的序列、如本发明第二方面所述的重链的序列、如本发明第三方面所述的轻链可变区的序列、如本发明第四方面所述的轻链的序列、或如本发明第五方面所述的抗体的序列;以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中,所述的标签序列包括6His标签。
在另一优选例中,所述的重组蛋白特异性地与CD19蛋白结合。
本发明的第七方面,提供了一种多核苷酸,它编码选自下组的多肽:
(1)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的轻链、或如本发明第五方面所述的抗体;或
(2)如本发明第六方面所述的重组蛋白。
在另一优选例中,所述的多核苷酸具有SEQ ID NO.:1、3、8、9、10、14、15、或16所示的序列。
本发明的第八方面,提供了一种载体,它含有本发明第七方面所述的多核苷酸。
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。
本发明的第九方面,提供了一种遗传工程化的宿主细胞,它含有本发明第八方面所述的载体或基因组中整合有本发明第七方面所述的多核苷酸。
本发明的第十方面,提供了一种免疫偶联物,该免疫偶联物含有:
(a)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、如本发明第五方面所述的抗体、或如本发明第六方面所述的重组蛋白;和
(b)选自下组的偶联部分:可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中,所述偶联物选自:荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如,DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如,顺铂)或任何形式的纳米颗粒等。
本发明的第十一方面,提供了一种药物组合物,它含有:
(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、如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或如本发明第十方面所述的免疫偶联物;以及
(ii)药学上可接受的载体。
在另一优选例中,所述的药物组合物为注射剂型。
在另一优选例中,所述的药物组合物用于制备治疗肿瘤的药物,所述的肿瘤选自下组:胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、肾上腺肿瘤、或膀胱肿瘤。
本发明的第十二方面,提供了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、如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或如本发明第十方面所述的免疫偶联物的用途,用于制备药剂、试剂、检测板或试剂盒;
所述试剂、检测板或试剂盒用于:检测样品中CD19蛋白;
所述药剂用于治疗或预防表达CD19蛋白的肿瘤。
在另一优选例中,所述肿瘤包括:胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、或肾上腺肿瘤。
在另一优选例中,所述肿瘤选自:胃癌和滤泡性淋巴瘤。
在另一优选例中,所述的试剂包括芯片、包被抗体的免疫微粒。
本发明的第十三方面,提供了一种检测样品中CD19蛋白的方法,所述方法包括步骤:
(1)将样品与本发明第五方面所述的抗体接触;
(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蛋白。
本发明的第十四方面,提供了一种重组多肽的制备方法,该方法包含:
(a)在适合表达的条件下,培养本发明第九方面所述的宿主细胞;
(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。
本发明的第十五方面,提供了一种治疗疾病的方法,所述方法包括:给需要的对象施用治疗有效量的如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的免疫偶联物、或本发明第十一方面所述的药物组合物。
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。
图1为抗CD19单克隆抗体(PGC3D6H10)的流式检测图。
其中选用的目的细胞系是Ramos,抗CD19单克隆抗体工作浓度为1ug/ml,左图是不加抗CD19抗体的阴性对照,右图为抗CD19的荧光检测图,阳性率83.83%。
图2为抗CD19单克隆抗体(PGC3D6H10)的流式检测图。
其中选用的目的细胞是人PBMC(外周血单核细胞),抗CD19单克隆抗体工作浓度为1ug/ml,左图是不加抗CD19抗体的阴性对照,右图为抗CD19的荧光检测图,阳性率15.44%。
图3为抗CD19单克隆抗体(PGC3D6H10)的流式检测图。
其中选用的目的细胞是Jurkat,抗CD19单克隆抗体工作浓度为1ug/ml,左图是不加抗CD19抗体的阴性对照,右图为抗CD19的荧光检测图,抗CD19抗体几乎不会与Jurkat细胞发生结合,阳性率为0.54%。
图4为抗CD19单克隆抗体(PGC3D6H10)的免疫印迹(WB)图。
其中抗CD19单克隆抗体工作浓度为1ug/ml,泳道1和2是两个平行实验组,上样蛋白为裂解Ramos细胞的总蛋白,泳道3是作为阴性对照的裂解Jurkat细胞的总蛋白。
图5为抗CD19单克隆抗体(PGA6E2D5)的流式检测图。
其中选用的目的细胞系是Ramos,抗CD19单克隆抗体工作浓度为1ug/ml,左图是不加抗CD19抗体的阴性对照,右图为抗CD19的荧光检测图,阳性率82.65%。
图6为抗CD19单克隆抗体(PGA6E2D5)的流式检测图。
其中选用的目的细胞是Jurkat,抗CD19单克隆抗体工作浓度为1ug/ml,左图是不加抗CD19抗体的阴性对照,右图为抗CD19的荧光检测图,抗CD19抗体几乎不会与Jurkat细胞发生结合,阳性率为0.00%。
图7为抗CD19单克隆抗体(PGB8D5C6)的流式检测图。
其中选用的目的细胞系是Ramos,抗CD19单克隆抗体工作浓度为1ug/ml,左图是不加抗CD19抗体的阴性对照,右图为抗CD19的荧光检测图,阳性率72.33%。
图8为抗CD19单克隆抗体(PGB8D5C6)的流式检测图。
其中选用的目的细胞是Jurkat,抗CD19单克隆抗体工作浓度为1ug/ml,左图是不加抗CD19抗体的阴性对照,右图为抗CD19的荧光检测图,抗CD19抗体几乎不会与Jurkat细胞发生结合,阳性率为0.08%。
图9为抗CD19单克隆抗体(PGC3D6H10)的抗原亲和力检测图。
图10为克隆抗CD19单克隆抗体(克隆号为PGC3D6H10)可变区VH和VL的RT-PCR产物琼脂糖凝胶电泳图分析结果图。
泳道1为DL5000DNA Marker;泳道2为VH基因;泳道3为RT-PCR扩增VH基因阴性对照;泳道4为VL基因;泳道5为RT-PCR扩增VL基因阴性对照。
图11为随机的选取9个克隆做菌落PCR,琼脂糖凝胶电泳图(克隆号为PGC3D6H10)。
其中Marker左边是对应的随机挑取的VH克隆进行的菌落PCR鉴定;Marker右边是对应的随机挑取的VL克隆进行的菌落PCR鉴定。
本发明人通过广泛而深入的研究,经过大量的筛选,获得一种抗CD19的单克隆抗体,实验结果表明,该抗CD19的单克隆抗体不仅能够有效识别高表达CD19蛋白的细胞,而且能够成功用于Western Blot检测。亲和力检测表明,该单克隆抗体与靶蛋白的亲和力是常规抗体的20倍以上。在此基础上完成了本发明。
本发明的抗CD19单克隆抗体PGC3D6H10可特异性靶向肿瘤细胞表面的CD19分子表位,可用于抗体药物制备,这些抗体药物对B细胞系来源的肿瘤的诊断治疗,病情进展,转移潜能以及预后的评价等方面具有巨大的潜能。基于本发明的PGC3D6H10单克隆抗体所能制备的抗体有重组抗体,ScFv抗体,人源化抗体,双特异性抗体(BiTE)以及嵌合抗原受体抗体(CAR)等。
CD19蛋白
CD19分子主要表达于早期的B细胞,是一个95KDa左右的B细胞系特有的跨膜糖蛋白。CD19在正常及恶性B淋巴细胞中均有表达,被视为B细胞发育过程中一个涵盖阶段较长的最为可靠的表面标记物。
在本发明的一个优选的实施方式中,所述CD19蛋白的氨基酸序列为:
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。
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(参见Kabat等,NIH Publ.No.91-3242,卷I,647-669页(1991))。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。
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。
如本文所用,术语“单克隆抗体(单抗)”指从一类基本均一的群体获得的抗体,即该群体中包含的单个抗体是相同的,除少数可能存在的天然发生的突变外。单克隆抗体高特异性地针对单个抗原位点。而且,与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同,各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外,单克隆抗体的好处还在于它们是通过杂交瘤培养来合成的,不会被其它免疫球蛋白污染。修饰语“单克隆”表示了抗体的特性,是从基本均一的抗体群中获得的,这不应被解释成需要用任何特殊方法来生产抗体。
本发明还包括具有所述的抗CD19蛋白单克隆抗体的相应氨基酸序列的单克隆抗体、具有所述的抗CD19蛋白单克隆抗体可变区链的单克隆抗体,以及具有这些链的其他蛋白质或蛋白质偶联物及融合表达产物。具体地,本发明包括具有含超变区(互补决定区,CDR)的轻链和重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物),只要该超变区与本发明的轻链和重链的超变区相同或至少90%同源性,较佳地至少95%同源性。
如本领域技术人员所知,免疫偶联物及融合表达产物包括:药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与所述的抗CD19蛋白单克隆抗体或其片段结合的而形成的偶联物。本发明还包括与所述的抗
CD19蛋白单克隆抗体或其片段结合的细胞表面标记物或抗原。
本发明不仅包括完整的单克隆抗体,还包括具有免疫活性的抗体片段,如Fab或(Fab’)2片段;抗体重链;抗体轻链。
如本文所用,术语“重链可变区”与“VH”可互换使用。
如本文所用,术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中,所述抗体的重链可变区包括以下三个互补决定区CDR:
在另一优选例中,所述重链可变区的氨基酸序列为:
其编码核苷酸序列为:
在本发明的一个优选的实施方式中,所述抗体的重链包括上述重链可变区和重链恒定区,所述重链恒定区可以为鼠源或人源。
如本文所用,术语“轻链可变区”与“VL”可互换使用。
在本发明的一个优选的实施方式中,根据本发明的抗体的轻链可变区,具有选自下组的互补决定区CDR:
在另一优选例中,所述的轻链可变区的氨基酸序列为:
其编码核苷酸序列为:
在本发明的一个优选的实施方式中,所述抗体的轻链包括上述轻链可变区和轻链恒定区,所述轻链恒定区可以为鼠源或人源。
在本发明中,术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用,都指特异性结合CD19蛋白的抗体,例如具有重链可变区(如SEQ ID NO.:2的氨基酸序列)和/或轻链可变区(如SEQ ID NO.:4的氨基酸序列)的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
在另一优选例中,所述的抗体为抗CD19蛋白的鼠或人鼠嵌合单克隆抗体,它的重链恒定区和/或轻链恒定区可以是人源化的重链恒定区或轻链恒定区。更优选地,所述的人源化的重链恒定区或轻链恒定区为人IgG1、IgG2等的重链恒定区或轻链恒定区。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地,本发明包括具有含可变区的重链和轻链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物),只要该可变区与本发明抗体的重链和轻链的可变区相同或至少90%同源性,较佳地至少95%同源性。
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。
本发明抗体的重链和/或轻链的可变区特别令人感兴趣,因为它们中至少部分涉及结合抗原。因此,本发明包括那些具有带CDR的单克隆抗体轻链和重链可变区的分子,只要其CDR与此处鉴定的CDR具有90%以上(较佳地95%以上,最佳地98%以上)的同源性。
本发明不仅包括完整的单克隆抗体,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。
如本文所用,术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的多肽,或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物,例如聚乙二醇)融合所形成的多肽,或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列,或与6His标签形成的融合蛋白)。根据本文的教导,这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有CD19蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于):一个或多个(通常为1-50个,较佳地1-30个,更佳地1-20个,最佳地1-10个)氨基酸的缺失、插入和/或取代,以及在C末端和/或N末端添加一个或数个(通常为20个以内,较佳地为10个以内,更佳地为5个以内)氨基酸。例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变蛋白质的功能。又比如,在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括:同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽,如包含人抗体或其片段的融合蛋白。除了几乎全长的多肽外,本发明还包括了本发明抗体的片段。通常,该片段具有本发明抗体的至少约50个连续氨基酸,较佳地至少约50个连续氨基酸,更佳地至少约80个连续氨基酸,最佳地至少约100个连续氨基酸。
在本发明中,“本发明抗体的保守性变异体”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1进行氨基酸替换而产生。
表1
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。编码成熟多肽的编码区序列可以与SEQ ID NO.:1、3、8、9、10、14、15、16所示的编码区序列相同或者是简并的变异体。如本文所用,“简并的变异体”在本发明中是指编码具有与本发明的多肽相同的氨基酸序列,但与SEQ ID NO.:1、3、8、9、10、14、15、16所示的编码区序列有差别的核酸序列。
编码本发明的成熟多肽的多核苷酸包括:只编码成熟多肽的编码序列;成熟多肽的编码序列和各种附加编码序列;成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸,也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50%,较佳地至少70%,更佳地至少80%相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中,“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2×SSC,0.1%SDS,60℃;或(2)杂交时加有变性剂,如50%(v/v)甲酰胺,0.1%小牛血清/0.1%Ficoll,42℃等;或(3)仅在两条序列之间的相同性至少在90%以上,更好是95%以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与SEQ ID NO.:2和/或SEQ ID NO.:4所示的成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是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。此外,还可将重链的编码序列和表达标签(如6His)融合在一起,形成融合蛋白。
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。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前,已经可以完全通过化学合成来得到编码本发明蛋白(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。
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。
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。代表性例子有:大肠杆菌,链霉菌属;鼠伤寒沙门氏菌的细菌细胞;真菌细胞如酵母;果蝇S2或Sf9的昆虫细胞;CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时,能吸收DNA的感受态细胞可在指数生长期后收获,用CaCl2法处理,所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要,转化也可用电穿孔的方法进行。当宿主是真核生物,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养,表达本发明的基因所编码的多肽。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、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。
本发明的抗体可以单独使用,也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的的可检测标记物包括但不限于:荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于:1.放射性核素(Koppe等,2005,癌转移评论(Cancer metastasis reviews)24,539);2.生
物毒(Chaudhary等,1989,自然(Nature)339,394;Epel等,2002,癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)51,565);3.细胞因子如IL-2等(Gillies等,1992,美国国家科学院院刊(PNAS)89,1428;Card等,2004,癌症免疫学和免疫治疗(Cancer Immunology and Immunotherapy)53,345;Halin等,2003,癌症研究(Cancer Research)63,3202);4.金纳米颗粒/纳米棒(Lapotko等,2005,癌症通信(Cancer letters)239,36;Huang等,2006,美国化学学会杂志(Journal of the American Chemical Society)128,2115);5.病毒颗粒(Peng等,2004,基因治疗(Gene therapy)11,1234);6.脂质体(Mamot等,2005,癌症研究(Cancer research)65,11631);7.纳米磁粒;8.前药激活酶(例如,DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL));10.化疗剂(例如,顺铂)或任何形式的纳米颗粒等。
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,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。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。
本发明的药物组合物可直接用于结合CD19蛋白分子,因而可用于预防和治疗肿瘤。此外,还可同时使用其他治疗剂。
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。
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8毫克/千克体重,较佳地该剂量是约10微克/千克体重-约1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。
杂交瘤细胞株
本发明还提供了可生产本发明针对CD19蛋白单克隆抗体的杂交瘤细胞株;优选的,本发明提供了高效价的针对CD19蛋白单克隆抗体的杂交瘤细胞株。
在获得生产本发明的CD19蛋白单克隆抗体的杂交瘤之后,本领域技术人员可以方便地利用该杂交瘤细胞株制备抗体。此外,本领域技术人员还可很方便地获知本发明的抗体的结构(比如抗体的重链可变区和轻链可变区),然后可
通过重组方法来制备本发明的单克隆抗体。
单克隆抗体的制备
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。例如,本发明抗原,可被施用于动物以诱导单克隆抗体的产生。对于单克隆抗体,可利用杂交瘤技术来制备(见Kohler等人,Nature 256;495,1975;Kohler等人,Eur.J.Immunol.6:511,1976;Kohler等人,Eur.J.Immunol.6:292,1976;Hammerling等人,In Monoclonal Antibodies and T Cell Hybridomas,Elsevier,N.Y.,1981)或可用重组DNA法(美国专利号4,816,567)制备。
代表性的骨髓瘤细胞是有效融合、通过选择的抗体产生细胞支持抗体的稳定高水平产生、且对培养基(HAT培养基基质)敏感的那些骨髓瘤细胞,包括骨髓瘤细胞株,例如鼠类的骨髓瘤细胞株,包括衍生自MOPC-21和MPC-11小鼠肿瘤的骨髓瘤细胞株(可购自Salk Institute Cell Distribution Center,圣地亚哥,加利福尼亚,美国)以及SP-2、NZ0或X63-Ag8-653细胞(可购自American Type Culture Collection,洛克维尔,马里兰,美国)。人骨髓瘤和小鼠-人杂合骨髓瘤细胞株也已被描述用于产生人单克隆抗体[Kozbor,J.Immunol.,133:3001(1984);Brodeur等,单克隆抗体的生产技术和应用(Monoclonal Antibodies Production Techniques and Applications),51-63页(Marcel Dekker,Inc.,纽约,1987)]。
对杂交瘤细胞生长于其中的培养基进行分析以检测具有所需特异性的单克隆抗体的产生,如,通过体外结合分析例如,酶联免疫吸附分析(ELISA)或放射免疫分析(RIA)。表达抗体的细胞的位置可用FACS进行检测。然后,可将杂交瘤克隆通过有限稀释步骤形成亚克隆(subcloned),并通过标准方法生长(Goding,单克隆抗体(Monoclonal Antibodies):原则和实践(Principles and Practice),Academic Press(1986)59-103页)。为了达到这一目的而使用的适合的培养基包括,例如,DMEM或RPMI-1640培养基。此外,杂交瘤细胞可在动物体内作为腹水瘤生长。
由亚克隆分泌的单克隆抗体从培养基、腹水或血清中通过常规的免疫球蛋白纯化工艺适当地得到分离,这些纯化工艺为例如,蛋白A-琼脂糖法(protein A-Sepharose)、羟基磷灰石层析、凝胶电泳、透析或亲和层析。
本发明提供了一种针对CD19蛋白的单克隆抗体。在本发明的一个优选的方案中,单克隆抗体采用培养杂交瘤细胞方法制备。取杂交瘤细胞培养的上清液,经饱和硫酸铵沉淀法粗提出IgG,再将粗提的抗体经亲和层析柱(Protein G-Sephrose)纯化。
本发明的一个优选的方案中,单克隆抗体采用Balb/C小鼠腹水生产单克隆抗体的方法制备。将约杂交瘤细胞接种到致敏的小鼠腹腔内,10天左右可见腹部明显胀大。抽取腹水,经饱和硫酸铵沉淀法粗提后,再将粗提的抗体经亲和层析柱(Protein G-Sephrose)纯化。
可以通过鼠源单抗来制备高亲和力,低免疫源性,高效的重组或人源化抗体。对鼠源抗体进行人源化改造,可保留抗体可变区与人源抗体恒定区融合,提高抗体亲和力;或改造抗体结构,构建只保留抗体可变区的ScFv或含抗体可变区和部分
恒定区的Fab,可提高抗体在体内的吸收百分比和半衰期。
标记的免疫球蛋白
在本发明的一个优选例中,所述免疫球蛋白带有可检测标记物。更佳地,所述的标记物选自下组:胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中,CD19蛋白的单克隆抗体用胶体金标记,得到胶体金标记的单克隆抗体。
本发明的CD19蛋白单克隆抗体具有很好的特异性,很高的效价和亲和力。
检测板及其材料
本发明的检测板可采用本领域常用的检测板材料,采用常规的检测板制备方法制成。
本发明检测CD19蛋白的免疫检测板,包括测试条和支撑测试条的支撑板,如可采用PVC聚脂胶板等;所述的测试条由滤样纸、层析材料、硝酸纤维素膜和吸水纸依次搭接组成,搭接部位可以采用常规的方法,如胶带等固定连接;其中:层析材料预包被胶体金标记或有色标记的CD19蛋白单克隆抗体或多克隆抗体,优选被胶体金标记的CD19蛋白单克隆抗体,硝酸纤维素膜上吸附检测线和质控线;
在一个优选的方案中:层析材料上预包被胶体金标记的CD19蛋白单克隆抗体是采用浓度为0.5-1.5mg/ml胶体金标记的CD19蛋白单克隆抗体溶液进行预包被的,包被量为50μl/cm2;优选的浓度为0.5或1.5mg/ml,50μl/cm2;
检测方法与结果判定
平放检测板,将试样滴在滤样纸上,试样约120μl,3~5min内观察层析结果。根据出现的条纹位置来判断结果。
阴性:质控区、检测区均出现明显的色带,示为阴性;
阳性:只在质控区出现明显色带,而在检测区无色带,示为阳性;
无效:质控区、检测区无任何色带或在质控区未出现色带而在检测区出现色带,表明检测方法错误或检测板变质或失效,应重新换取检测板检测。
方法和样本
本发明涉及用于在以细胞和/或组织溶解的样本检测肿瘤的方法。该方法步骤大致如下:获得细胞和/或组织样本;将样本溶解在介质中;检测在所述溶解的样本中CD19蛋白的水平。本发明方法所使用的样本可以是存在于细胞保存液中的包括细胞的任何样本,正如在液基细胞检测法中所使用的。优选地使用Western Blot方法进行检测。
试剂盒
本发明还提供了一种指含有本发明的抗体(或其片段)或本发明的检测板的试剂盒,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。
本发明进一步设计用于检测CD19蛋白水平的检测试剂盒,该试剂盒包括识别CD19蛋白的抗体,用于溶解样本的裂解介质,检测所需的通用试剂和缓冲液,如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
本发明的主要优点在于:
(1)本发明中经过大量筛选获得的抗CD19单克隆抗体PGC3D6H10能够有效识别高表达CD19蛋白的细胞;
(2)本发明PGC3D6H10单克隆抗体能够识别细胞表面的天然CD19分子。
(3)本发明PGC3D6H10单克隆抗体能够成功用于Western Blot检测。
(4)本发明PGC3D6H10单克隆抗体具有极高的亲和力,与靶蛋白的亲和力是常规抗体的20倍以上。
下面结合具体实施例,进一步详陈本发明。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详细条件的实验方法,通常按照常规条件如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,否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1
步骤一:杂交瘤细胞的制备
用B细胞系肿瘤细胞或CD19真核重组蛋白抗原分别免疫雌性健康的BALB/c小鼠,挑选出血清ELISA,WB,FC检测呈阳性的小鼠,对其提取脾脏细胞,与骨髓瘤细胞融合,形成杂交瘤细胞;将杂交瘤细胞在HAT培养基中培养,对高通量流式筛选呈阳性的单克隆细胞进行ELISA复筛,获得阳性克隆36株,再进行WB复筛,获得阳性克隆9株,对WB阳性细胞进行亚克隆,经过2次亚克隆,排除亚克隆过程中返阴细胞株6株,最终筛选出能够稳定分泌CD19抗体的单克隆细胞3株(包括:PGC3D6H10单克隆抗体细胞株、PGA6E2D5单克隆抗体细胞株、PGA6D5C6单克隆抗体细胞株);运用亚型鉴定试剂盒,鉴定单克
隆抗体的亚型。
步骤二:抗CD19单克隆抗体的制备
(1)单克隆抗体的生物学鉴定
将步骤一鉴定合格的阳性的单克隆细胞注射到小鼠体内进行腹水生产,将产生的腹水通过Protein A层析纯化后得到抗CD19单克隆抗体。采用流式细胞术,免疫荧光术和Western Blot鉴定抗体的特异性。具体操作如下:
(1.1)流式细胞术检测抗体的特异性
收集高表达CD19的淋巴瘤细胞系Ramos(购自美国ATCC),以及Ficol法分离的人PBMC(外周血单核细胞)以及阴性对照Jurkat细胞系(购自美国ATCC),4%多聚甲醛室温固定20min,PBS配制的3%BSA封闭液中室温放置30min,用抗CD19单克隆抗体(1ug/ml)室温孵育1h,PBST离心洗涤2次,FITC或Alexa Fluor 647-羊抗鼠IgG(H+L)二抗避光共孵育1h,PBST离心洗涤3次,去掉未结合上的荧光二抗,流式细胞仪检测CD19阳性率。
(1.2)免疫荧光术检测抗体的特异性
收集高表达CD19的淋巴瘤细胞系Ramos,以及Ficol法分离的人PBMC(外周血单核细胞)以及阴性对照Jurkat细胞系,4%多聚甲醛室温固定20min,PBS配制的3%BSA封闭液中室温放置30min,用抗CD19单克隆抗体(1ug/ml)室温孵育1h,PBST离心洗涤2次,FITC或Alexa Fluor 488-羊抗鼠IgG(H+L)二抗避光共孵育1h,PBST离心洗涤2次,DAPI室温核染10min,PBST离心洗涤3次,去掉未结合上的染料,Confocal检测CD19染色情况。
(1.3)Western Blot检测抗体的特异性
收集高表达CD19的淋巴瘤细胞系Ramos,T淋巴白血病细胞系Jurkat,蛋白裂解液冰上裂解30min,离心取上清,BSA法测定上清蛋白浓度,加入loading Buffer,10%SDS-PAGE,120V凝胶电泳90min,转膜400mA,90min。5%脱脂牛奶室温封闭1h,anti-CD19单克隆抗体(1ug/ml)4℃孵育过夜,PBST(含0.1%Tween-20的PBS)洗涤3次,HRP-羊抗鼠IgG(H+L)二抗室温孵育1h,PBST洗涤3次,ECL法显影,曝光2min。
实验结果:
对所获得的3株阳性单克隆抗体(编号:PGC3D6H10、PGA6E2D5、PGB8D5C6)分别进行流式检测和Western Blot实验的应用,结果如图1-8示(图1-4,克
隆号PGC3D6H10;图5-6,PGA6E2D5;图7-8,PGB8D5C6)。从这些应用数据可以看出,克隆号为PGC3D6H10的单克隆抗体应用效果最佳,流式细胞术分析中,Ramos的CD19染色阳性率达到83.83%(图1),而且PGC3D6H10单克隆抗体的Western Blot应用结果为阳性,说明PGC3D6H10单克隆抗体能够成功用于Western Blot中进行相关抗原的检测,而PGA6E2D5单克隆抗体和PGB8D5C6单克隆抗体的Western Blot应用结果为阴性,无法用于Western Blot检测。
通过进一步的抗体亲和力分析,克隆号为PGC3D6H10的鼠抗人CD19单克隆抗体与真核重组rCD19蛋白的KD亲和力常数为434.6pM(图9),较于常规的亲和力常数为纳摩尔级的抗体(如,本发明中其它单克隆抗体)亲和力常数高出20倍以上。
结合上述流式检测和Western Blot实验的应用结果,以及亲和力测定分析,本发明筛选到了一株可以分泌有效结合CD19表面蛋白的单克隆抗体杂交瘤细胞株(克隆号PGC3D6H10),并制备出了抗CD19的单克隆抗体,这一抗体具有较强的抗原亲和力和抗原特异性。
实施例2抗CD19单克隆抗体重链和轻链可变区基因的克隆
所用细胞株为采用上述方法获得的能分泌高亲和力、高特异性的抗CD19单克隆抗体的杂交瘤细胞株,对应的编号为PGC3D6H10,所对应的抗体分子亚型为:重链IgG1型,轻链κ型。
取对数生长期的杂交瘤细胞2×106,Trizol法提取细胞总RNA,
取少量用Nanodrop定量及1%非变性琼脂糖凝胶电泳检测,随后用SuperScript.III First-StrandSynthesis System for RT-PCR试剂盒(K1622,Thermo)反转录cDNA,用特异性引物扩增抗CD19抗体基因的重链或轻链可变区。将含有相应重链可变区或轻链可变区片段的PCR反应产物经1%琼脂糖凝胶电泳,胶回收。将回收得到的相应重链可变区和轻链可变区克隆至测序载体pCR2.1,并进行测序,对测序结果进行同源性和结构分析。
具体操作步骤:
(1)RT-PCR扩增CD19抗体轻链和重链可变区
引物设计:本发明人选用mouse-IgG(Mouse Ig-Primer Set,Novagen)引物进行重链可变区克隆,mouse-IgGκ(Mouse Ig-Primer Set,Novagen)通用引物进行轻链可变区克隆。
1.1杂交瘤细胞总RNA提取:Trizol法;
1.2反转录PCR:以杂交瘤细胞总RNA为模板,反转录cDNA,具体操作如下:
1.2.1按表1配成20ul体系,轻轻混匀,离心,42℃孵育60min,70℃终止反应5min,分装,-20℃保存。
表1
成分 | 体积(ul) |
RNA | 1 |
Oligo(dT)18Primer | 1 |
5×Reaction Buffer | 4 |
RiblockTMRNA酶抑制剂 | 1 |
10mM dNTP MIX | 2 |
RevertAidTM逆转录酶 | 1 |
H2O | 10 |
1.3抗体可变区特异引物PCR,具体操作如下:
按表2,表3中的反应体系,以反转录得到的cDNA为模板,用特异引物合成抗体重链和轻链可变区。
表2
成分 | 体积(ul) |
dNTP(10mM) | 5 |
10×Buffer | 5 |
Taq | 0.5 |
5’-primers | 1 |
3’-primers | 1 |
cDNA | 1 |
H2O | 36.5 |
表3
步骤四:PCR产物克隆和测序
将PCR得到的重链可变区和轻链可变区核苷酸片段产物胶回收(09114KE1,AxyGEN)后,按表四中体系进行TA克隆,16℃连接过夜,分别构建到测序载体上,按表五中体系挑取单克隆菌落进行菌落PCR鉴定,1%琼脂糖凝胶电泳选取有阳性条带的克隆,提质粒(07714KA1,AxyGEN)测序。
表4
表5
成分 | 体积(ul) |
dNTP(10mM) | 5 |
10×Buffer | 5 |
Taq | 0.5 |
5’-primers | 1 |
3’-primers | 1 |
克隆 | 1 |
H2O | 36.5 |
本实施例通过单克隆杂交瘤细胞的抗体基因提取(图10-11,克隆号PGC3D6H10),来获得可以分泌有效结合CD19表面蛋白的单克隆抗体杂交瘤细胞株的抗体基因序列(SEQ ID NO.:1-4,克隆号PGC3D6H10),PGC3D6H10单
克隆抗体的测序结果如下。
PGC3D6H10单克隆抗体的VH核苷酸序列:
PGC3D6H10单克隆抗体的VH编码氨基酸序列:
PGC3D6H10单克隆抗体的VL核苷酸序列:
PGC3D6H10单克隆抗体的VL编码氨基酸序列:
表6PGC3D6H10单克隆抗体的的重链可变区
表7PGC3D6H10单克隆抗体轻链可变区
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。
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:SEQ ID NO:5所示的CDR1,SEQ ID NO:6所示的CDR2,和SEQ ID NO:7所示的CDR3。
- 一种抗体的重链,其特征在于,所述的重链具有权利要求1所述的重链可变区和重链恒定区。
- 一种抗体的轻链可变区,其特征在于,所述轻链可变区具有选自下组的互补决定区CDR:SEQ ID NO:11所示的CDR1’,SEQ ID NO:12所示的CDR2’,和SEQ ID NO:13所示的CDR3’。
- 一种抗体的轻链,其特征在于,所述的轻链具有权利要求3所述的轻链可变区和轻链恒定区。
- 一种抗体,其特征在于,所述抗体具有:(1)如权利要求1所述的重链可变区;和/或(2)如权利要求3所述的轻链可变区;优选地,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。
- 一种重组蛋白,其特征在于,所述的重组蛋白具有:(i)如权利要求1所述的重链可变区的序列、如权利要求2所述的重链的序列、如权利要求3所述的轻链可变区的序列、如权利要求4所述的轻链的序列、或如权利要求5所述的抗体的序列;以及(ii)任选的协助表达和/或纯化的标签序列。
- 一种多核苷酸,其特征在于,它编码选自下组的多肽:(1)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(2)如权利要求6所述的重组蛋白。
- 一种载体,其特征在于,它含有权利要求7所述的多核苷酸。
- 一种遗传工程化的宿主细胞,其特征在于,它含有权利要求8所述的载体或基因组中整合有权利要求7所述的多核苷酸。
- 一种免疫偶联物,其特征在于,该免疫偶联物含有:(a)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、如本发明第五方面所述的抗体、或如本发明第六方面所述的重组蛋白;和(b)选自下组的偶联部分:可检测标记物、药物、毒素、细胞因子、放射性核 素、或酶。
- 一种药物组合物,其特征在于,它含有:(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、如权利要求5所述的抗体、如权利要求6所述的重组蛋白、或如权利要求10所述的免疫偶联物;以及(ii)药学上可接受的载体。
- 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6所述的重组蛋白、或如权利要求10所述的免疫偶联物的用途,用于制备药剂、试剂、检测板或试剂盒;所述试剂、检测板或试剂盒用于:检测样品中CD19蛋白;所述药剂用于治疗或预防表达CD19蛋白的肿瘤。
- 如权利要求12所述的用途,其特征在于,所述肿瘤包括:胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、或肾上腺肿瘤。
- 一种重组多肽的制备方法,其特征在于,该方法包含:(a)在适合表达的条件下,培养权利要求9所述的宿主细胞;(b)从培养物中分离出重组多肽,所述的重组多肽是权利要求5所述的抗体或权利要求6所述的重组蛋白。
- 一种治疗疾病的方法,其特征在于,所述方法包括:给需要的对象施用治疗有效量的如权利要求5所述的抗体、如权利要求6所述的重组蛋白、或如权利要求10所述的免疫偶联物、或权利要求11所述的药物组合物。
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