CN101550416A - Humanized single anti-Hu-ScFv18 light and heavy chain variable region gene and coding polypeptide thereof and application thereof - Google Patents
Humanized single anti-Hu-ScFv18 light and heavy chain variable region gene and coding polypeptide thereof and application thereof Download PDFInfo
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Abstract
The present invention relates to a humanized single anti-Hu-ScFv18 light and heavy chain variable region gene for resisting HAb18G/CD147 and A coding polypeptide thereof and application thereof. The invention uses a set of designed primers to humanizedly reconstruct the anti-HAb18G/CD147 single light and heavy chain variable region gene excreted from HAb18 hybridoma, and the humanized single anti-Hu-ScFv18 heavy chain and light chain variable region gene may encode a correct antibody variable region. Based on the cloned anti-HAb18G/CD 147 humanized single anti-Hu-ScFv18 light and heavy chain variable region gene, it is capable of constructing and expressing various small molecule genetic engineering antibodies such as single-chain antibody, jogged antibody, Fab antibody and the like against the HAb18G/CD 147 molecule by a genetic engineering method, so as to be used for diagnosis and treatment of hepatocarcinoma.
Description
Technical field
The present invention relates to anti-HAb18G/CD147 humanization monoclonal antibody Hu-ScFv18 light, heavy chain variable region gene and by the polypeptide of described genes encoding, and described gene and polypeptide are used for diagnosing application with the medicine for treating tumor thing in preparation.
Background technology
Malignant tumour is that current serious influences human health, threatens one of main killer of human life.According to The World Health Organization's year, the whole world had 7,600,000 people to die from malignant tumour (cancer) in 2005.In recent years, the cause of disease of cancer and clinical diagnosis and treatment research have bigger progress, but the mortality ratio of cancer is not still effectively contained.Cancer is a kind of genopathy, and it takes place to relate to a plurality of genes with evolution and through a plurality of different stages, the adjusting disorder of the number of ways relevant with cytodifferentiation, cell cycle regulating, apoptosis, vasculogenesis and transfer is relevant.The traditional cancerous tissue of clinical application is learned diagnostic method on the one hand, with indexs such as cancerous tissue classification and type, cancer nodus lymphoideus transferring rate (TNM) staging hierarchies, can't carry out early diagnosis exactly, be difficult to assess clinical prognosis, therefore, to instructing clinical formulation processing scheme to have significant limitation.On the other hand, traditional cancer operation, radiotherapy, chemotherapy can not satisfy clinical needs.Therefore accelerating to strengthen the diagnosis of research cancer and treating is the urgent requirements of people always.Since Kohler in 1975 and Milstein create the B lymphocyte hybridoma technology, various monoclonal antibodies (McAb) occur in succession, they are bringing into play positive effect aspect the fundamental research of diagnosis of disease and the clinical application, and wherein the development with monoclonal antibody is that various intractable tumours have been brought new hope.Just because of antibody drug specificity height, can be at specific target spot preparation, be applied to treat autoimmune disorder, cardiovascular diseases, transmissible disease, inflammation, particularly oncotherapy, become the focus of current global bio-pharmaceutical field research and development, annual sales amount surpasses 10,000,000,000 dollars " cookle medicine ".Successively ratify 26 kinds of antibody drug listings to U.S. FDA in 2007, have 14 to be used for the treatment of noumenal tumours such as colorectal carcinoma, mammary cancer, lymphatic cancer.As treat lymphadenomatous antibody drug Rituximab (rituximab), and single therapy total reaction rate is 50%, combined chemotherapy is efficient up to more than 80%.Antibody drug also can be used as the curative effect that the instrument that transmits medicine strengthens chemicotherapy, becomes the strongest tumour-specific bio-pharmaceutical of target and curative effect probably.Meanwhile, China National Drug Administration has also strengthened antibody drug and has examined dynamics, existing 11 kinds of antibody drugs approval listing.Especially it should be noted that the more existing antibody drugs with independent intellectual property right of China are carrying out clinical or finish clinical study, listing waits for ratification.
At present, mouse monoclonal antibody will be substituted by humanized antibody gradually, and its major cause is: mouse monoclonal antibody and human complement component binding ability are low, the CDC effect corresponding a little less than, to the kill capability of tumour cell a little less than; A little less than the Fc receptor affinity of immunocytes such as it and NK surface, a little less than the ADCC effect of mediation; The transformation period of mouse source antibody in people's circulation of blood is short, and it is shorter that it brings into play the time of ADCC and CDC effect; Mouse monoclonal antibody has immunogenicity, and the host easily produces anti-antibody reaction (AAR) and causes allergic reaction.So just seriously hindered the further application of mouse monoclonal antibody.In order to address these problems, humanized antibody has demonstrated huge advantage.At present, be that the humanization modified main method of murine antibody comprises that chimeric antibody, surface reinvent antibody, reshaped antibody to the humanized construction process of murine antibody.
Chimeric antibody: utilize the DNA recombinant technology that light, the heavy chain variable region gene insertion of mouse monoclonal antibody are contained in the expression vector of people's antibody constant region, transformed mammalian cell gives expression to human mouse chimeric antibody, its humanization degree reaches about 70%, the variable region that has intactly kept the allos monoclonal antibody, keep its affine activity to greatest extent, reduced immunogenicity.But, can bring out the anti-mouse reaction of people HAMA because human mouse chimeric antibody has still kept 30% mouse source property.
Antibody is reinvented on the surface: carry out humanization modified to murine antibody surface amino groups acid residue.The principle of this method is only to replace and the tangible zone of people's antibody SAR difference, is keeping antibody activity and is taking into account to reduce and select for use the amino acid similar to people's antibody surface residue to replace on the heterology basis; In addition, the section of being replaced should be not too much, for influencing side chain size, electric charge, hydrophobicity, do not replace thereby maybe may form the residue that hydrogen bond has influence on complementary antibody determining area (CDR) conformation as far as possible.
Reshaped antibody:, comprise the transplanting of CDR district, portion C DR transplanting and specific determining area (SDR) transfer by splicing structure with antigen again in conjunction with relevant residue and people's antibody in the heterologous antibody.Antibody after CDR transplants also claims reshaping antibody.Jone has equaled to report in 1986 their achievement in research about CDR grafted antibody on Nature, with complementary determining region (CDR) sequence in the mouse monoclonal antibody B1-8 monoclonal antibody V district replace to the most similar human antibody of himself framework region (FR) in corresponding CDR sequence, started this novel method.Zenapax (Daclizumab) is that this method of usefulness of drugs approved by FDA produces the humanization monoclonal antibody that is used in acute rejection or obstinate asthma.On this basis, the interaction of discovery antigen-antibodies such as Padlan is mainly finished by the amino-acid residue of CDR district 20%-33%, these zones are widely different between each antibody, be called as specificity determinant residue (specifity determining residues, SDRs), transplant with CDR and to compare, this method can reduce the mouse source sequence of antibody to greatest extent, and sequence is transplanted the active influence of antagonist drops to minimum.
Be full humanized antibody in addition: the formation of fully human antibodies starts from nineteen nineties, and obtaining full humanization antibody method at present has antibody library triage techniques, genetically engineered mouse to prepare human antibody, transfection chromosome ox etc.As be used for the adalimumab of rheumatism and the panitumumab of treatment of colon cancer is human antibody.
Mention the method that except that above-mentioned we are also shown in framework region and reinvent (frameworkpatching) in addition, super humanization methods such as (Superhumanization).These methods are mostly similar with the above-mentioned method of mentioning.Similar as super humanization method and SDR transfer methods, promptly keep the canonical structure (canonical structures) of mouse source antibody, and this structure there is important effect for the space structure of keeping parental antibody.Tan etc. have compared people source and mouse source antibody at first, the antibody close or identical to the variable region canonical structure is transplanted to corresponding people source framework region fully, has further reduced the immunogenicity for the treatment of engineered antibody, functional experiment shows that this method also can finely make antigen-binding activity keep.In the monoclonal antibody medicine of the common approval listing of U.S. food Food and Drug Admistraton (FDA) and European medicine assessment office (EMEA) in 2004-2006, more than 90% humanized antibody.In today that the idea of disease target treatment highlights day by day, the range of application of antibody drug exceeds tumour and immunological disease just gradually; In addition, along with new disease-related target antigen is found, therapeutic antibodies will play an increasingly important role; And the humanization of antibody also can further be applied clinically for it and removes the obstacles, and promptly antagonist carries out humanization modifiedly, has kept the antigenic ability of antibody recognition on the one hand.On the other hand, reduced the immunogenicity of mouse endogenous antibody, reduced " human antimouse antibody reaction (HAMA) reaction " that human body produces at the mouse endogenous antibody, thereby improved the security of antibody accordingly in clinical application.
At present aspect the diagnosis of malignant tumour, the biological diagnosis reagent of FDA approval mainly is divided into 4 classes: 1. serum carcinoma marker detection kit.2. radio-immuno-image test kit.3. immunohistochemistry test kit.4. gene diagnosis kit.Serology carcinoma marker detection kit mainly comprises the detection of AFP (alpha-fetoprotein), be used for diagnosing liver cancer and carcinoma of testis, the detection of CEA (carcinomebryonic antigen), be used for diagnosis such as diagnosing, colorectal cancer, liver cancer, mammary cancer, carcinoma of the pancreas, the detection of PSA (prostate specific antigen) is used for prostate cancer diagnosis etc.The radio-immuno-image test kit mainly comprises CEA-Scan (the CEA antibody of Tc-99m mark), is used for the in-vivo diagnostic of colorectal carcinoma, mammary cancer, lung cancer; Nofetumobab (antibody of the cancer associated antigen of the 40KD of Tc-99m mark) is used for the in-vivo diagnostic of small cell lung cancer; Oncoscint (the antibody B72.3 of the anti-Tag-72 of Indium-111 mark) is used for the in-vivo diagnostic and the Prostascint (the anti-PSMA of Indium-111 mark) of colorectal carcinoma, is used for the in-vivo diagnostic of prostate cancer.Gene diagnosis kit comprises with fluorescent probe the her-2 in the tissue and the detection of the transitional cell bladder carcinoma cell line in the urine.The immunohistochemistry test kit mainly is the her-2 detection kit, is used for the judge index before the her-2 antibody drug is used.This shows that immunohistochemistry test kit like product is less at present both at home and abroad, this carcinoma marker of HAb18G/CD147 also of no use temporarily carries out the diagnostic reagent development product.The result of study of mouse source monoclonal antibody HAb18 shows according to early stage, compare with other carcinoma markers, HAb18G/CD147 more wide spectrum, positive rate is higher, and for example Her-2 albumen only has 20%～40% at the positive expression of mammary cancer, and the positive rate of HAb18G/CD147 is 64.36%.Therefore the application of the immune diagnostic reagent of HAb18G/CD147 is wider, susceptibility is stronger.
The newcomer of the HAb18G/CD147 molecule CD147 family that to be The Fourth Military Medical University cell engineering center obtain with tumour monoclonal antibody HAb18 screening people liver cancer tissue cDNA library.Analysis HAb18G/CD147 molecule has clinical meaning as the tumor markers of mammary cancer and liver cancer in the following aspects:
1) the HAb18G/CD147 molecule can be used as the diagnosis marker of cancer: Showed by immune group result, HAb18G/CD147 is at most of cancerous tissues, particularly the The positive expression rate in the cancerous tissue of epithelial origin is very high, its positive expression is only limited to cancer cells, background clear (matter is all not painted), do not have non-specific painted, and, very low in normal tissue expression, organize all negative in innocent tumour and benign lesion; Each carcinoid 1553 example after testing, wherein the positive detection rate reaches 1035 examples, and positive rate is 66.64%; And in the 239 routine healthy tissuess, positive rate only accounts for 5.18%, reflects that the HAb18G/CD147 molecule has very high susceptibility and specificity to cancer cells.This will have the important clinical meaning to the diagnosis of cancer.
2) the HAb18G/CD147 molecule can be used as the cancer early diagnosis marker: the immunohistochemical methods detected result shows that the positive rate of HAb18G/CD147 is 27.23% (12/44) in atypical hyperplasia of mammary (precancerous lesion); Liver cell atypical hyperplasia 100% (6/6); The cancer of the stomach intestinal epithelial metaplasia is 72.92% (35/48), these express the gene level change that male atypical hyperplasia pathology also may have canceration, find and diagnose these pathologies will improve the early diagnostic rate of cancer greatly, make this part patient obtain early treatment.
3) the HAb18G/CD147 molecule can be used as the histological grade and the prognosis prediction mark of cancer: early-stage Study result shows: HAb18G/CD147 is in the medullary carcinoma of mammary gland, and mucinous carcinoma is not expressed, at infitrating ductal carcinoma The positive expression rate height.Expression intensity with organize classification relevant (p=0.002).Analyzed the relation of HAb18G/CD147 molecule and other several (ER, PR, CerbB-2, p53, Ki67) indexs simultaneously, HAb18G/CD147 developed by molecule and CerbB-2 are remarkable positive correlation (P=0.000), are remarkable negative correlation (P=0.000) with PR; 106 routine clinical patients are followed up a case by regular visits to, and the expression of discovery HAb18G/CD147 and patient's transfer and recurrence, patient's existence closely related (p=0.000) illustrates that HAb18G/CD147 can be used as the bad index of predicting prognosis of breast cancer.And our early-stage Study also shows, HAb18G/CD147 significantly is lower than the expression of breaking up liver cancer low in the expression of height differentiation liver cancer, increases the recurrence that is indicating liver cancer in its expression of liver Transplantation for Hepatocellular Carcinoma postoperative.The expression intensity of above results suggest HAb18G/CD147 is to the prognosis of clinical assessment tumour, and the treatment plan of formulating individuation is significant.
Summary of the invention
One object of the present invention is to provide the humanization monoclonal antibody Hu-ScFv18 heavy chain variable region gene and the chain variable region gene of the monoclonal antibody of an anti-broad-spectrum tumor mark HAb18G/CD147; So that obtain by giving expression to specific recognition after two segments reorganization and in conjunction with the antibody activity fragment of tumor markers HAb18G/CD147.
Another object of the present invention is to described humanization monoclonal antibody Hu-ScFv18 heavy chain variable region gene and chain variable region gene and corresponding polypeptide and be used for diagnosing application with the medicine for treating tumor thing in preparation.
According to an aspect of the present invention, the invention provides humanization monoclonal antibody Hu-ScFv18 heavy chain variable region gene and chain variable region gene and its coded polypeptide of an antitumor mark HAb18G/CD147 monoclonal antibody.Humanization monoclonal antibody Hu-ScFv18 heavy chain of the present invention and chain variable region gene are that the cloned genes transformation obtains from the hybridoma cell strain HAb18 of the anti-HAb18G monoclonal antibody of existing mouse source property, wherein said heavy chain variable region gene total length is 357bp, its nucleotide sequence as in the sequence table＜400〉1 shown in, its encoded polypeptides sequence as in the sequence table＜400〉3 shown in.Described chain variable region gene total length is 333bp, its nucleotide sequence as in the sequence table＜400〉2 shown in, its encoded polypeptides sequence as in the sequence table＜400〉4 shown in, can be used for expression specificity identification after above-mentioned two gene recombination and in conjunction with the antibody activity fragment of liver cancer related antigen HAb18G.
We are existing to studies show that, HAb18G/CD147 is a universal tumour wide spectrum mark, therefore at the monoclonal antibody of the anti-HAb18G/CD147 of hybridoma HAb18 excretory carry out humanization modified, resulting humanization monoclonal antibody Hu-ScFv18 is light, heavy chain variable region gene promptly can be used for using in diagnosis and the medicine for treating tumor thing.
Description of drawings
The structural topology figure of Fig. 1 mouse source property HAb18 monoclonal antibody VH and VL.
The tomograph in mutational site, property HAb18 monoclonal antibody variable region, Fig. 2 mouse source.
The gel electrophoresis figure of Fig. 3 PCR product.
Fig. 4 recombinant plasmid enzyme is cut the gel electrophoresis figure of evaluation.
The cell ELISA result schematic diagram of Fig. 5 humanization monoclonal antibody Hu-ScFv18.
The cellular immunofluorescence result schematic diagram of Fig. 6 humanization monoclonal antibody Hu-ScFv18.
The immunohistochemical methods result schematic diagram of Fig. 7 humanization monoclonal antibody Hu-ScFv18.
Embodiment
Mouse source property HAb18 monoclonal antibody is light, the heavy chain variable region gene clone
Used cell strain is the plant height avidity that The Fourth Military Medical University cell engineering research centre adopts traditional fusion method to obtain, (the biomaterial preservation proves the anti-human hepatocellular carcinoma monoclonal antibody of the mouse source property HAb18 hybridoma cell strain of high specific: the classification name of biomaterial: human liver cancer monoclonal antibody HAb18 hybridoma cell strain, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, address: Zhong Guan-cun, BeiJing, China, preservation date: on December 6th, 1999, deposit number: CGMCCNO.0426), this antibody has passed through the requirement of Nat'l Pharmaceutical ﹠ Biological Products Control Institute's " preparation of human mouse monoclonal antibody and key Quality Control ", its antigen is the HAb18G molecule, and its excretory antibody molecule hypotype is IgG1.Utilize this cell strain to extract total RNA, can increasing by reverse transcription and PCR reaction, mouse source property monoclonal antibody HAb18 is light, heavy chain variable region gene.Concrete grammar and result are open in following article and patent: the foundation of this cell strain can be referring to " monoclonal antibody communication ", 1989; 2:33-36, Chen Zhinan, Liu Yanfang, Yang Jizhen etc.; This gene inventor's patent: CN021144710 has obtained the authorization." structure of HAb18G molecule single-chain antibody gene and the secreting, expressing in intestinal bacteria ", Chinese Journal of Immunology, 2003 7 phases.
Mouse source property HAb18 monoclonal antibody variable region sequences and architecture computer analysis
The inventor utilizes the nucleotide sequence (patent: CN021144710) translate of gene order translation software ORFfinder with the monoclonal antibody HAb18 variable region of heavy chain VH and the variable region of light chain VL in mouse source, simultaneously the rule according to antibody gene database Kabat, Abm, Chothia and Contact marks the complementary determining region CDR district of antibody and the SDR district that represents with rectangle frame, and " starlet " (Stars) represents CDR district different in disparate databases (seeing Table 1).
Table 1 mouse source property HAb18 monoclonal antibody variable region sequences and analysis thereof
Utilize the gene order DNA PLOT of antibody germline gene database VBase2 that its nucleotide sequence is analyzed, determined that HAb18VH has used the V of Igh-VJ606VH6 family gene fragment, VL has used the V of IGKV19/28 family gene fragment.The Non-redundant data storehouse nr of the homogenic database Genbank BlastP that compares, tissue-derived people Homo sapiens and mouse Mus musculus two classes selected respectively.The definite the highest people source of similarity score, each 100 of mouse source antibody variable region sequences selected added up, search the difference residue in the mouse source sequence, utilize SWISS-MODEL that HAb18 variable region homology mould is built then, the three-dimensional structure mould of HAb18 is built with structural analysis be.Finally obtain from PDB, to select and HAb18VH, 10 antibody structures that VL similarity score aggregate value is the highest.Utilizing the match function of structural database Swiss-PdbViewer 3.7 is that template is carried out the space splicing with HAb18VH, VL with this antibody structure, obtain the calculating energy value in the field of force with software Gromos96, use software Discover to carry out molecular mechanics optimization on the butt joint model of minimum energy value, obtain the accurate three-dimensional model in mouse HAb18 variable region.
Further analyze the variable region three-dimensional structure of antibody, the same From Template of HAb18VH is 1v7mH, 1v7nK, 1d1fH, 1wz1H as can be known, the same From Template of VL is 1ncdL, 1ncaL, 1iaiL, 1ztxL simultaneously, selection has the 1wz1 of 73.3% similarity as the splicing template, and result's total energy after the GROMOS96 field of force is optimized is-8820.85KJ/Mol.After the Discover molecular mechanics is optimized, check that with Procheck obtaining the G-factor value is 0.13
Every index of representation model is in the statistics scope of correspondence.The three-dimensional model of building according to mould calculates mouse monoclonal antibody variable region apparent surface's accessibility, hydrogen bond action, and the rendering architecture topological diagram is seen Fig. 1, and wherein the square frame table shows the residue that side chain exposes; Circular frame table presentation surface accessibility is less than 30% residue; The triangle frame table shows the interactional residue of participation VH-VL; Ash colour specification difference residue; Abnormal residue in the different residue of black table differential; Arrow is represented intramolecular hydrogen bond (NH → OC).Preliminary decision sudden change candidate locus, the CDR-FR junction participates in forming the residue of hydrogen bond, and forms the residue that hydrogen bond keeps sphaeroprotein β laminated structure in the antibody framework district, and above residue is all kept.Check non-SDR residue among the CDR on this basis, find to have only the ThrL056 exposed area of CDR2 among the VL to be 62.6% and not participate in hydrogen bond action, can be used as the sudden change candidate locus.By above analysis, the candidate mutational site that finally obtains is divided three classes: devoid of risk site, β lamella site, the outside, CDR position point specifically see Table 2.By observing the HAb18 model, find that candidate's mutational site is distributed in CDR in lateral areas and the FR district, away from CDR, see Fig. 2.
Table 2 selects to carry out the residue site of rite-directed mutagenesis
Reuse apparent surface's accessibility that Naccess is calculation of parameter heavy chain HAb18VH, light chain HAb18VL and mouse monoclonal antibody HAb18 with the van der Waals radius.Utilize the bond energy analysis tool FindHBond among the software Chimera to analyze HAb18VH, VL intramolecularly and intermolecular hydrogen bonding interaction.Final determine the sudden change candidate locus, build and analyze by the mould of the humanized antibody that similarly sudden change produced at last, the humanized VH and the VL gene (seeing sequence table) of the correct antibody variable region that obtains to encode.Make up the humanized antibody model with same method, use Swiss-PdbViewer 3.7 that the two molecule is superimposed, and to calculate RMSDall atom be 0.21
Illustrate and introduce the CDR conformation that does not change former mouse source antibody behind the sudden change residue.
Humanization monoclonal antibody Hu-ScFv18 is light, the amplification and the checking of weight chain variabl area sequence
Method with overlapping PCR (Over-lapping PCR), in conjunction with top mouse source property monoclonal antibody HAb18 variable region sequences and architecture computer analysis, stronger to the potential immunogenicity, but the candidate locus that does not influence antibody structure is carried out rite-directed mutagenesis, thereby obtains humanization monoclonal antibody Hu-ScFv18 weight chain variabl area sequence.10 primer sequences of the humanization monoclonal antibody Hu-ScFv18 variable region gene pcr amplification of design are as follows respectively:
Part1
Primer P1-5
5ATGACCATGATTACGCCAAG?3
Primer P1-3
5AACACAAGACAGGCGCATAGATCCT?3
Primer P2-5
5AGGATC?tATGCGCCTGTCTTGTGTT3
Primer P2-3
5AGTGTCTTCAGTTCTTAAGTTG?3
Primer P3-5
5CAACTTAAGAACTGAAGACACT?3
Primer P3-3
5AGTAAGGATCCACCGCCACCCGAGCC?3
Part2
Primer P4-5.3
5CGGTGGATCCGGTGGCGGTGGCTCGGGCGGTGGCGGTTCG?3
Primer P4-5.2
5CGGTGGCGGTTCGAGTATTGTGATGACCCAGACTCCCAC?3
Primer P4-5.1
5ATGACCCAGACTCCCACAAGCCTGGTTGT3
Primer P4-3
5GATGTGCGGCCGCGCGTTTGATTTCCA?3
The concrete steps of pcr amplification reaction are carried out according to a conventional method, use above-mentioned each gene required to primer amplification respectively.Reaction system is: template 2.5ul, dNTP (each 0.4mM), 10 * Buffer 5ul, ExTaq archaeal dna polymerase 1.25u, 5 ' end and 3 ' end each 5ul of primer (about 30pmol) add water to 50ul, mixing, instantaneous centrifugal after, add 1-2 drop of liquid paraffin, put on the PCR instrument and react.Reaction conditions: 94 ℃ of 1min, 54 ℃ of 1min, 72 ℃ of 1min, 35 circulations, last 72 ℃ are extended 10min.At first, be that masterplate (is seen inventor's article and patent: " structure of HAb18G molecule single-chain antibody gene and the secreting, expressing in intestinal bacteria ", Chinese Journal of Immunology, 2003 7 phases with 18-ScFv; Chinese patent: CN021144710), amplification utilizes three couples of pairing primer P1-5/P1-3 respectively, primer P2-5/P2-3, and primer P3-5/P3-3 obtains product P 1 (Fig. 3, swimming lane 2), product P 2 (Fig. 3, swimming lane 3), product P 3 (Fig. 3, swimming lane 4); After three product P 1, P2, P3 are mixed, with increase the once more product P art1 (Fig. 3, swimming lane 5) of the about 510pb of acquisition of pairing primer P1-5/P3-3.Be masterplate with 18-ScFv once more subsequently, obtain intermediate product P4.1 with primer P4-3/P4-5.1; Intermediate product P4.1 with purifying is a masterplate again, obtains product P 4.2 with primer P4-3/P4-5.2; P4.2 with purifying is a masterplate at last, obtains product P art2 (Fig. 3, swimming lane 6) with P4-3/P4-5.3.Two fragment gene Patt1+Part2 that obtain are utilized BamH1 digestion, connect, end product directly with P4-3 and P1-5 amplification, obtains target gene Hu-ScFv18, its theoretical big or small 870bp, (Fig. 3 is shown in swimming lane 7 arrows).
Purpose fragment T carrier cloning order-checking scheme is as follows: with reclaiming after the PCR product gel electrophoretic separation, be connected into the pMD18-T carrier.The ligation system is: pMD18-T carrier 1ul, PCR product gel purifying heavy chain (or light chain) 3ul, deionized water 1ul, connect damping fluid 5ul, 4 ℃ are spent the night behind the mixing, transformed into escherichia coli JM109, the screening recombinant clone adopts the order-checking of universal sequencing primer thing then.Sudden change back gene Hu-ScFv18 be put in the T carrier sequence verification correct after, be put into expression vector pCANT-AB5e by Sfi I/Not I double digestion, Sfi I/Not I double digestion qualification result as shown in Figure 4: wherein clone 1-6 and all inserted the Hu-ScFv18 sequence; Swimming lane 7 is the ScFv18 contrasts (ScFv18/pCANT) in mouse source, the 8th, and negative control.Can find to clone 1-6 from this result and all insert correct Hu-ScFv18.To clone 1 recombinant plasmid transfection Escherichia coli coli expressing protein at last; Expressing protein can be by Ni-NTA chromatography column enriching and purifying (by supplier: the standard step of Pharmacia Corporation be carried out).The albumen that obtains can detect the activity of expressing protein with Western-blot and immunofluorescence, and then the reliability of humanized's sequence of being obtained of checking.
The activity checking of humanization monoclonal antibody Hu-ScFv18 variable region sequences expressing protein
Utilize SfiI/NotI to carry out double digestion phasmid expression vector pCANT-AB5e, light, the heavy chain variable region gene of the humanization monoclonal antibody Hu-ScFv18 variable region sequences of being cloned with the present invention reassemble into solubility Hu-ScFv18 expression vector and carry out conventional prokaryotic expression and purifying. with the negative contrast of normal people's CCL 13 QSG-7701, liver cancer cell FHCC-98 is a sample, detects the activity of expression product-solubility Hu-ScFv18 with indirect ELISA.ELISA result shows, expression product energy and liver cancer cell FHCC-98 combination, but can not combine (see figure 5) with normal liver cell.This explanation purified product has obtained the Hu-ScFv18 small molecular antibody of expressing.In addition, with the immunofluorescence experiment (see figure 6) of this solubility Hu-ScFv18 small molecular antibody of expressing in conjunction with above-mentioned two kinds of cells, show that but expressed solubility Hu-ScFv18 small molecular antibody specificity is combined on the cytolemma of cancer cells, and be not joined to normal surface of hepatocytes, the explanation that this is also indirect expressed products one Hu-ScFv18 have certain biologic activity.The immunohistochemical methods test-results is shown (see figure 7): the Hu-ScFv18 small molecular antibody can combine with liver cancer tissue, and with the normal liver tissue debond, show this humanization monoclonal antibody Hu-ScFv18 have identification, in conjunction with the ability of hepatocellular carcinoma, have the potential that develops into anti-liver cancer preparation.By information biology, as follows: as to form amino acid number (Number of amino acids): 256 to the property prediction of this solubility Hu-ScFv18 small molecular antibody; Theoretic molecular weight (Molecular weight): 27329.1; Theoretic iso-electric point (Theoretical pI):: 6.79; The negative electrification residue (Asp+Glu) that wherein contains: 22; Positive charged residue (Arg+Lys): 21; Final molecular formula is: C1195H1829N3390383S8, and promptly total atom is several 3754; The N terminal sequence originates in E (Glu). and the transformation period is estimated about 1.0 hours at reticulocyte in the body, at yeast about 30 minutes; And intestinal bacteria at least greater than 10.0 hours.Fat index (Aliphatic Index) 60.55.
At present, the research of tumour monoclonal antibody application facet mainly concentrates on the application of (1) tumour monoclonal antibody in tumor associated antigen research: the development of monoclonal antibody provides an effective means of finding new tumor associated antigen or tumour antigen novel site.These antigens can be used for clinical Serological testing, have the meaning of certain diagnosis and evaluation result of treatment.(2) tumour monoclonal antibody targeted drug research: with the monoclonal antibody is that radio immuno imaging diagnosis, the guiding chemotherapy of carrier and the radiotherapy of leading have obtained substantial progress in recent years.Mainly contain: 1. chemicals-monoclonal antibody cross-linking agent; 2. cytotoxin-monoclonal antibody cross-linking agent; 3. radionuclide-monoclonal antibody cross-linking agent etc.
Protein drug so that humanization monoclonal antibody Hu-ScFv18 of the present invention is light, heavy chain variable region gene reconstitutes a definite form can be directly used in particularly kinds of tumor of diseases related, as liver cancer, and mammary cancer, the diagnosis of lung cancer etc. and targeted therapy.In addition,, heavy chain variable region gene encoded polypeptides light with humanization monoclonal antibody Hu-ScFv18 of the present invention and derivative thereof are as carrier, crosslinked cytotoxic drug, toxin, radionuclide, enzyme and the biological response modifier etc. of going up can be used for the diseases related particularly diagnosis and the treatment of tumour as targeted drug.
, heavy chain variable region gene encoded polypeptides light from humanization monoclonal antibody Hu-ScFv18 of the present invention, the novel antibody that can reassemble into mainly contains following several form: (1) humanized antibody.Humanization modified at this gene structure, the mouse source property that has not only reduced the variable region has kept specificity and the avidity of mouse MAb simultaneously again.(2) small molecular antibody.Mainly contain by VH-CH1 and VL-C1 form Fab antibody, with a peptide linker (GLy4Ser)
3Or other flexible joints can connect the VH gene and the VL gene forms single-chain antibody; The single domain antibody of forming with non covalent bond be combined into Fv fragment antibody, by VH or functional domain of VL by VH and VL; Atom that constitutes by single CDR etc.(3) multivalence miniantibody.Mainly contain double-stranded antibody, (ScFv)
2, Flex miniantibody, LD miniantibody, F (ab ')
2, F (ab ')
3, (ScFv)
4Deng.Because the polyvalent antigen binding site is arranged, the avidity height, molecular size is moderate, can penetrate tumor tissues, also slower characteristics of the clearance rate in kidney and have high clinical value.(4) bi-specific antibody.Be the antibody that a class has dual specificity and dual-use function, claim bifunctional antibody again, said gene can provide one of them target spot.(5) recombinant antibody fusion proteins.Gene fragments such as Fab or Fv, with having that other protein genes such as the toxin of non-antibody or enzyme are connected to form a kind of recombinant protein of specific biological activity guiding target site.(6) recombinant immunotoxin.The gene recombination of encoding antibody and toxin is produced, and characteristics are efficient, and non-special toxicity is low, and the body internal stability is good, easily penetrate use in tumour and the body safer.(7) phage antibody.The V district gene of Ig is connected after transfecting host bacterium with last gene III of filobactivirus DNA or gene VIII, makes its fusion protein product at film surface outer casing protein expression Fab or ScFv.By this product is taken turns the affine absorption of related antigen more, therefrom wash in a pan and sift out more optimal specific antibody.
Light, heavy chain variable region gene encoded polypeptides reorganization or deutero-antibody molecule with above-mentioned humanization monoclonal antibody Hu-ScFv18 are carrier, and crosslinked immune conjugate or the targeted drug of the effector substance of various anticancer or anti-inflammatories and forming mainly contains following several form: (1) antibody-nucleic conjugate.This conjugate tumor tissues part of nucleic can being led effectively, thus the normal tissue injury that causes because of the radiotherapy external radiation exposure and relevant untoward reaction reduced, and can position diagnosis and targeted therapy to tumour.This method is radio-immuno-image and radioimmunotherapy.Have with monoclonal antibody link coupled nucleic commonly used
125I,
131I,
111In,
90Y,
99Tc
m,
188Re and
186Re etc.(2) antibody-chemotherapeutics conjugate.This conjugate tumour that can lead specifically can lower the damage to healthy tissues, reduces the toxic side effect of chemotherapeutics.Normal link coupled chemotherapeutics is as the phosphoamide in the alkylating agent; Methotrexate in the antimetabolic, 5 FU 5 fluorouracil; Antibiotics Zorubicin, pidorubicin, daunorubicin; Plant class vincristine(VCR), mitomycin etc.(3) antibody-toxin conjugated thing.This conjugate claims immunotoxin again.Its killer cell effect is strong and do not rely on biological auxiliary mechanism.Its killing tumor cells mechanism and radiotherapy, the chemotherapy difference so the tumour of general radiotherapy, chemotherapy effect difference can be used, has a extensive future.Toxin commonly used has ricin, diphtheria toxin, abrin, Saponaria officinalis toxin, pseudomonas extracellular toxin, streptolysin, pore-forming protein etc.(4) antibody-biological response modifier (BRM) conjugate.BRM regulates the immunological competence and the killing tumor cell of body separately and has obtained curative effect preferably.But owing to only have part to arrive the tumour target site after in its input body, its lethal effect is not in full use and toxic side effect.With BRM and monoclonal antibody coupling, carry it by monoclonal antibody and arrive the effect that target site is brought into play killing tumor cells, make the effect of these factors stronger, and more single-minded.BRM commonly used has INF and IL-2 etc.(5) antibody targeted enzymolysis prodrug.The conjugate of antibody and drug specificity activating enzymes is injected in the body, inject prodrug behind the certain hour at interval, make it change into high density antitumour activity medicine with killing tumor cell at tumor locus.At present, can be used as the glutamine derivative that phenylformic acid hydrogen mustard is arranged, phosphoric acid Podophyllum emodi var chinense ethylidene ring, phosphoric acid mitomycin, glucuronide daunorubicin, Zorubicin, 5-flurocytosine and the cynnematin mustargen etc. of prodrug.Activating enzymes have carboxypeptidase G
2, alkaline phosphatase, penicillin amidase, β-Nei Xiananmei, Isocytosine deaminase, beta-glucosidase enzyme and aminopeptidase etc.(6) immunoliposome.The surface energy that MAb is coupled to liposome fully combines the characteristic of MAb and antigen-specific bonded guidance quality and lipid physical efficiency parcel high amount of drug together, and relevant medicine in the embedding then can improve drug targeting and curative effect again.
Sequence table
＜110〉Chen Zhinan
＜120〉light, the heavy chain variable region gene of humanization monoclonal antibody Hu-ScFv18 and its coded polypeptide and application thereof
<160>4
<210>1
<211>357
<212>DNA
<220>
<221>V_region
<222>(1)...(357)
<400>1
gaa?gtg?aag?ctt?gag?gag?tct?gga?gga?ggc?ttg?gtg?caa?cct?gga?gga?48
tct?atg?cgc?ctg?tct?tgt?gtt?gcc?tct?gga?ttc?act?ttt?agt?gac?gcc?96
tgg?atg?gac?tgg?gtc?cgc?cag?tct?cca?gag?aag?gga?ctt?gag?tgg?gtt?144
gct?gaa?att?aga?agc?aaa?gct?aat?aat?cat?gca?cca?tac?tat?act?gag?192
tct?gtg?aaa?ggg?agg?ttc?acc?atc?tca?cga?gat?gat?tcc?aag?agt?att?240
atc?tac?ctg?caa?atg?aac?aac?tta?aga?act?gaa?gac?act?ggc?att?tat?288
tac?tgt?acc?agg?gat?agc?acg?gct?acc?cac?tgg?ggc?caa?ggg?act?ctg?336
gtc?act?gtc?tct?tca?gct?agc?357
<210>2
<211>333
<212>DNA
<220>
<221>V_region
<222>(1)...(333)
<400>2
agt?att?gtg?atg?acc?cag?act?ccc?aca?agc?ctg?gtt?gta?tca?gca?gga?48
gac?agg?gtt?acc?ata?acc?tgc?aag?gcc?agt?cag?agt?gtg?att?aat?gat?96
gta?gct?tgg?tac?caa?cag?aag?cca?ggg?cag?tct?cct?aaa?ctg?ctg?ata?144
ttc?tat?gca?tcc?aat?cgc?aac?act?gga?gtt?cct?gat?cgc?ttc?act?ggc?192
agt?gga?tat?ggg?acg?gat?ttc?act?ttc?acc?atc?agc?act?gtg?cag?gct?240
gaa?gac?ctg?gca?gtt?tat?ttc?tgt?cag?cag?gat?tat?agt?cct?cca?ttc?288
acg?ttc?ggc?tcg?ggg?aca?aag?ctg?gaa?atc?aaa?cgc?gcg?gcc?gca?333
<210>3
<211>119
<212>PRT
<220>
<221>V_segment
<400>3
Glu?Val?Lys?Leu?Glu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly?16
Ser?Met?Arg?Leu?Ser?Cys?Val?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Asp?Ala?32
Trp?Met?Asp?Trp?Val?Arg?Gln?Ser?Pro?Glu?Lys?Gly?Leu?Glu?Trp?Val?48
Ala?Glu?Ile?Arg?Ser?Lys?Ala?Asn?Asn?His?Ala?Pro?Tyr?Tyr?Thr?Glu?64
Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asp?Ser?Lys?Ser?Ile?80
Ile?Tyr?Leu?Gln?Met?Asn?Asn?Leu?Arg?Thr?Glu?Asp?Thr?Gly?Ile?Tyr?96
Tyr?Cys?Thr?Arg?Asp?Ser?Thr?Ala?Thr?His?Trp?Gly?Gln?Gly?Thr?Leu?112
Val?Thr?Val?Ser?Ser?Ala?Ser?119
<210>4
<211>111
<212>PRT
<213>mouse
<220>
<221>V_segment
<400>4
Ser?Ile?Val?Met?Thr?Gln?Thr?Pro?Thr?Ser?Leu?Val?Val?Ser?Ala?Gly?16
Asp?Arg?Val?Thr?Ile?Thr?Cys?Lys?Ala?Ser?Gln?Ser?Val?Ile?Asn?Asp?32
Val?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ser?Pro?Lys?Leu?Leu?Ile?48
Phe?Tyr?Ala?Ser?Asn?Arg?Asn?Thr?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly?64
Ser?Gly?Tyr?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Thr?Val?Gln?Ala?80
Glu?Asp?Leu?Ala?Val?Tyr?Phe?Cys?Gln?Gln?Asp?Tyr?Ser?Pro?Pro?Phe?96
Thr?Phe?Gly?Ser?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Ala?Ala?Ala?111
Claims (5)
1, the heavy chain variable region gene of the humanization monoclonal antibody Hu-ScFv18 of anti-HAb18G/CD147, it has sequence table＜400〉1 sequence.
2, by the polypeptide product of claim 1 genes encoding, it has sequence table＜400〉3 sequence.
3, the chain variable region gene of the humanization monoclonal antibody Hu-ScFv18 of anti-HAb18G/CD147, it has sequence table＜400〉2 sequence.
4, by the polypeptide product of claim 3 genes encoding, it has sequence table＜400〉4 sequence.
5, claim 1 or 3 described genes are used for diagnosing and the application of medicine for treating tumor thing in preparation.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229671A (en) * | 2011-06-07 | 2011-11-02 | 南京医科大学 | Anti-IMMT anthropogenic Fab antibody and application thereof in preparing anti-liver cancer drugs |
| CN103343141A (en) * | 2013-06-20 | 2013-10-09 | 陈志南 | Restructured newcastle disease virus/genome plasmid for expressing humanized chimeric antibody cHAb18 and application thereof in anti-tumor treatment |
| CN104086654A (en) * | 2014-07-04 | 2014-10-08 | 中国人民解放军第四军医大学 | Humanization modified anti-CD147 chimeric antibody HcHAb18 and application thereof |
| CN105820250A (en) * | 2016-04-29 | 2016-08-03 | 中国人民解放军第四军医大学 | Anti-BASIGIN humanized antibody and application thereof |
| CN105999294A (en) * | 2016-05-12 | 2016-10-12 | 上海柏运医疗器材有限公司 | CD147 targeting drug-delivery system and preparation and application thereof |
| CN113234173A (en) * | 2015-08-11 | 2021-08-10 | 南京传奇生物科技有限公司 | Chimeric antigen receptors targeting BCMA and methods of use thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1186445C (en) * | 2002-03-15 | 2005-01-26 | 陈志南 | Light chain or heavy chain variable region gene of human liver cancer monoclonal antibody HAb18 and its application |
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2009
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102229671A (en) * | 2011-06-07 | 2011-11-02 | 南京医科大学 | Anti-IMMT anthropogenic Fab antibody and application thereof in preparing anti-liver cancer drugs |
| CN102229671B (en) * | 2011-06-07 | 2013-04-10 | 南京医科大学 | Anti-IMMT anthropogenic Fab antibody and application thereof in preparing anti-liver cancer drugs |
| CN103343141A (en) * | 2013-06-20 | 2013-10-09 | 陈志南 | Restructured newcastle disease virus/genome plasmid for expressing humanized chimeric antibody cHAb18 and application thereof in anti-tumor treatment |
| CN104086654A (en) * | 2014-07-04 | 2014-10-08 | 中国人民解放军第四军医大学 | Humanization modified anti-CD147 chimeric antibody HcHAb18 and application thereof |
| CN113234173A (en) * | 2015-08-11 | 2021-08-10 | 南京传奇生物科技有限公司 | Chimeric antigen receptors targeting BCMA and methods of use thereof |
| CN105820250A (en) * | 2016-04-29 | 2016-08-03 | 中国人民解放军第四军医大学 | Anti-BASIGIN humanized antibody and application thereof |
| WO2017186182A1 (en) * | 2016-04-29 | 2017-11-02 | Fourth Military Medical University | Humanized anti-basigin antibodies and the use thereof |
| CN109476760A (en) * | 2016-04-29 | 2019-03-15 | 中国人民解放军第四军医大学 | Anti- BASIGIN antibody of humanization and application thereof |
| CN105820250B (en) * | 2016-04-29 | 2019-04-30 | 中国人民解放军第四军医大学 | A kind of anti-BASIGIN humanized antibody and its application |
| CN109476760B (en) * | 2016-04-29 | 2021-11-12 | 中国人民解放军第四军医大学 | Humanized anti-BASIGIN antibodies and uses thereof |
| CN105999294A (en) * | 2016-05-12 | 2016-10-12 | 上海柏运医疗器材有限公司 | CD147 targeting drug-delivery system and preparation and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101550416B (en) | 2012-01-25 |
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Effective date of registration: 20160204 Address after: 710032 Changle West Road, Shaanxi, China, No. 169, No. Patentee after: The Fourth Military Medical University of the Chinese People's Liberation Army Address before: 710032 center of cell engineering, The Fourth Military Medical University, 17 West Changle Road, Xi'an, Shaanxi Patentee before: Chen Zhinan |