CN108129567B - A kind of Humanized anti-human CD146 monoclonal antibody having efficient neutralization activity - Google Patents

A kind of Humanized anti-human CD146 monoclonal antibody having efficient neutralization activity Download PDF

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CN108129567B
CN108129567B CN201810018944.XA CN201810018944A CN108129567B CN 108129567 B CN108129567 B CN 108129567B CN 201810018944 A CN201810018944 A CN 201810018944A CN 108129567 B CN108129567 B CN 108129567B
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CN108129567A (en
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梅岩
李涛
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Nanjing Sino New Drug Research And Development Co ltd
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Nanjing Ren Nuo Pharmaceutcal Corp Ltd
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    • 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
    • C07K16/3076—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 against structure-related tumour-associated moieties
    • C07K16/3092—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 against structure-related tumour-associated moieties against tumour-associated mucins
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    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

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Abstract

The invention discloses a kind of Humanized anti-human CD146 monoclonal antibodies for having efficient neutralization activity, and the Humanized anti-human CD146 monoclonal antibody for having efficient neutralization activity includes light chain variable region and heavy chain variable region.The present invention is under the premise of keeping or enhancing antibody molecule is active in conjunction with people's CD146 acceptor molecule, prepare the high monoclonal antibody of humanization degree, the caused anti-mouse of people is immunoreacted after entering human body as therapeutic antibodies to avoid the antibody, has reached good technical effect.

Description

A kind of Humanized anti-human CD146 monoclonal antibody having efficient neutralization activity
Technical field:
The present invention relates to biomedicine fields, and in particular to a kind of Humanized anti-human CD146 for having efficient neutralization activity Monoclonal antibody.
Background technique:
CD146 is a kind of adhesion molecule with transmembrane structure, the I type memebrane protein being made of 646 amino acid residues, Belong to Mitochondrion IgG superfamily member.Research shows that CD146 is being total to for vascular endothelial growth factor receptor VEGFR-2 Receptor is adjustable the activation of the VEGFR-2 of VEGF induction and the transmitting of downstream signal, and then promotes Tumor Angiongesis
(MicroRNA 329 Suppresses Angiogenesis by Targeting CD146.Molecular and Cellular Biology,2013；33(18):3689-99.).In addition, CD146 molecule is that inflammatory bowel disease is promoted to be converted into An important factor for colorectal carcinoma, pro-inflammatory cytokine TNF α and IL1 β can activate the transcription of CD146, make it in the blood vessels High expression on chrotoplast film, and then lead to the continuing advances of chronic inflammation by promoting inflammatory cell to invade profit and angiogenesis, it drills Become colorectal cancer.By a series of relevant tumor model of ulcerative colitis, researcher has found specific knockdown blood vessel The CD146 of endothelial cell, makes the frank mitigation of inflammatory bowel disease mouse, inflammatory cell invade profit and new vessels are obvious It reduces；In the blood vessels in the tumor model of chrotoplast specific knockdown CD146, the quantity and volume of tumour can be also decreased obviously Targeting endothelial CD146 attenuates neuroinflammation by limiting lymphocyte extravasation to the CNS.ScientificReports,2013；3:1687..CD146 molecule Being overexpressed in the breast cancer cell of epithelial character makes the lower tumour cell of script grade malignancy obtain high cell invasion Transfer ability, and show the feature of breast carcinoma stem cell.Research shows that CD146 by the mechanism of EMT promote breast cancer into Exhibition, therefore CD146 may be as new target drone (CD146, an an epithelial-mesenchyaml for the treatment of breast cancer transition inducer,isassociated with triple-negative breastcancer.Proceedings of the National Academy of Sciences of the United States of America,2012；109 (4)1127-32.)。
It is the antibody of target spot because of its structure feature difference using CD146, causes it from the affinity of CD146 molecule and different. P1H12 can in conjunction with most of different types of vascular endothelial tissues (Identification of the S-Endo1 endothelial-associated antigen.Biochem Biophys Res Commun.1996；218(1):210- 6.)；Some anti-CD146 antibody only combine the blood vessel that active growth state is in tumor tissues, do not combine in normal tissue Blood vessel.Research shows that these antibody identify that different epitopes, P1H12 identify the linear list for being located at N-terminal structural domain Position, and some anti-CD146 antibody identify the space conformation epitope based on disulfide bond, therefore cause to different endothelial tissue knots Close active difference.CN102936283A also discloses a kind of source of mouse CD146 antibody, being capable of the above-mentioned people CD146 of specific recognition Epitope.
Above-mentioned several antibody are mouse monoclonal antibodies, low with human complement component binding ability, to tumour cell Killing ability is weaker；Weak with the immunocytes surface Fc receptors affinity such as NK, the ADCC effect of mediation is weaker；And source of mouse antibody Half-life short in people's blood circulation has immunogenicity, thus host is also easy to produce antiantibody and causes rejection etc. many not Foot.
Currently, lacking a kind of Humanized anti-human CD146 monoclonal antibody for having efficient neutralization activity.
Summary of the invention:
In view of this, proposing the present invention.
The first aspect of the present invention provides a kind of light chain variable region of Humanized anti-human CD146 monoclonal antibody, described Chain variable region amino acid sequence is any of SEQIDNO:12-17.
Another aspect of the present invention provides a kind of heavy chain variable region of Humanized anti-human CD146 monoclonal antibody, described Heavy chain variable amino acid sequence is any of SEQIDNO:1-11.
It is anti-to provide a kind of Humanized anti-human CD146 monoclonal for having efficient neutralization activity for another aspect of the present invention Body, the monoclonal antibody include at least above-mentioned light chain variable region, one kind of heavy chain variable region.
Further, above-mentioned Humanized anti-human CD146 monoclonal antibody further includes heavy chain constant region and constant region of light chain, institute The amino acid sequence for stating heavy chain constant region is amino acid sequence shown in SEQIDNO:18；The amino acid sequence of the constant region of light chain Column are amino acid sequences shown in SEQIDNO:19.
Another aspect of the present invention provides a kind of above-mentioned Humanized anti-human CD146 monoclonal antibody in preparation for treating Application in malignant tumour or Autoimmune diseases or new vessels class disease medicament.
Another aspect of the present invention, provides a kind of for treating the pharmaceutical composition of malignant tumour, and it includes above-mentioned source of people Change anti-human CD146 monoclonal antibody and pharmaceutical carrier.
Another aspect of the present invention provides a kind of above-mentioned Humanized anti-human CD146 monoclonal antibody in preparation for detecting Or the kit or the application in diagnosticum of diagnosing malignant tumor or Autoimmune diseases or new vessels class disease.
Above-mentioned malignant tumour is selected from irradiation, glioma, nasopharyngeal carcinoma, cancer of pancreas, lung cancer, the cancer of the esophagus, mammary gland Cancer, gastric cancer, colorectal cancer, liver cancer, prostate cancer, malignant tumor of ovary, cervical carcinoma, carcinoma of endometrium, malignant mela noma, skin Cancer, lymthoma, leukaemia or thyroid cancer.
Above system autoimmunity disease is selected from systemic loupus erythematosus, rheumatoid arthritis, systemic vasculitis, hard Skin disease, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, Autoimmune Thyroid Diseases or ulcer Property colitis.
Above-mentioned new vessels class disease is selected from age-related macular, multiple sclerosis, macular edema, choroidal neovascularization Disease, neovascular glaucoma, retinal vein obstruction or retina neovascular diseases.
Another aspect of the present invention provides the Humanized anti-human CD146 for having efficient neutralization activity described in a kind of coding The nucleic acid sequence of monoclonal antibody light chain variable region, the nucleic acid sequence are any of SEQIDNO:31-36.
Another aspect of the present invention provides the Humanized anti-human CD146 for having efficient neutralization activity described in a kind of coding The nucleic acid sequence of monoclonal antibody heavy variable region, the nucleic acid sequence are any of SEQIDNO:20-30.
Another aspect of the present invention provides the Humanized anti-human CD146 for having efficient neutralization activity described in a kind of coding The nucleic acid sequence of monoclonal antibody constant region of light chain, the nucleic acid sequence are SEQIDNO:38.
Another aspect of the present invention provides the Humanized anti-human CD146 for having efficient neutralization activity described in a kind of coding The nucleic acid sequence of monoclonal antibody heavy constant region, the nucleic acid sequence are SEQIDNO:37.
Another aspect of the present invention provides the Humanized anti-human CD146 for having efficient neutralization activity described in a kind of coding The nucleic acid sequence of monoclonal antibody, the nucleic acid sequence are any of the above-described kind of light chain variable region, heavy chain variable region, chain constant The combination in area, heavy chain constant region nucleic acid sequence.
Further, the present invention provides a kind of expression vector, and the expression vector includes encoding SEQIDNO:20-36 at least A kind of nucleic acid sequence.Preferably, the expression vector includes at least one of SEQIDNO:20-30 and SEQIDNO:31-36 At least one of.
Further, the present invention provides a kind of protokaryon or eukaryotic host cell including above-mentioned expression vector.
The invention has the benefit that Humanized anti-human CD146 monoclonal antibody of the invention has efficient neutralization activity, Under the premise of keeping or enhancing antibody molecule is active in conjunction with people's CD146 acceptor molecule, it is high as far as possible to prepare humanization degree Monoclonal antibody, enter the caused anti-mouse immune response of people after human body as therapeutic antibodies to avoid the antibody；The present invention Using experiment with calculating the method combined in monoclonal antibody surface progress rite-directed mutagenesis, the framework of human IgG is selected, not Change in original antibody CDR structure basis, rite-directed mutagenesis is carried out to the area framework of IgG；Antibody structure packet prepared by the present invention The heavy chain variable region of the IGHV3-33 containing people and the area frame FR of light chain variable region, do not influence antibody target tropism and it is functional before It puts, point mutation is carried out to framework region, to improve the combination activity of antibody and people's CD146 receptor.
Detailed description of the invention:
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with It obtains other drawings based on these drawings.
Fig. 1 is ELISA result of the anti-CD146 source of mouse antibody in conjunction with CD146.
Specific embodiment
Example embodiments are described in detail here, and the example is illustrated in the accompanying drawings.Following description is related to When attached drawing, unless otherwise indicated, the same numbers in different drawings indicate the same or similar elements.Following exemplary embodiment Described in embodiment do not represent all embodiments consistented with the present invention.On the contrary, they be only with it is such as appended The example of device and method being described in detail in claims, some aspects of the invention are consistent.
It is only to be not intended to limit the invention merely for for the purpose of describing particular embodiments in terminology used in the present invention. It is also intended in the present invention and the "an" of singular used in the attached claims, " described " and "the" including majority Form, unless the context clearly indicates other meaning.It is also understood that term "and/or" used herein refers to and wraps It may be combined containing one or more associated any or all of project listed.
It will be appreciated that though various information, but this may be described using term first, second, third, etc. in the present invention A little information should not necessarily be limited by these terms.These terms are only used to for same type of information being distinguished from each other out.For example, not departing from In the case where the scope of the invention, the first information can also be referred to as the second information, and similarly, the second information can also be referred to as One information.Depending on context, word as used in this " if " can be construed to " ... when " or " when ... When " or " in response to determination ".
The present invention will be described in detail by way of examples below.
The Humanized strategies of the anti-human CD146 human mouse chimeric antibody of embodiment 1
Anti-human CD146 human mouse chimeric antibody is carried out using best-fit CDR constructing technology humanization modified.Source of mouse CDR, which is transplanted to, to be suitble in frame human-acceptable with chimeric antibody framework region with high sequence homology.In people The FR frame of most matched portable CDR is searched in Germline sequences database.It is tied using the primary structural sequence and 3D of antibody Structure data maintain the correct conformation of source of mouse CDR so that critical framework needed for keeping binding affinity and binding specificity is residual to determine Base.Back mutation is carried out in different humanized molecule's frames to keep the affinity of antibody, finally obtains height source of people The monoclonal antibody of change.
The database of the area the V gene of people, people IGHV3-33 and anti-human CD146 human mouse chimeric antibody weight are analyzed by BLAST Chain variable region sequence has 41% similarity (including CDRs), and framework region is preferably anti-human CD146 human mouse chimeric antibody people The framework region of source design improvement.Based on the sequence similarity of JH3minigene (Kabat Vol.II), the region germline V passes through Computer simulation is in conjunction with FR4.The WGQG of JH3minigene residue falls in the region CDR3 of anti-human CD146 human mouse chimeric antibody In.Based on sequence alignment and the influence to antibody function is considered, using 14 humanized heavy chains of IGHV3-33 Frame Design Mutant.H0 is the framework region that the CDRs of anti-human CD146 human mouse chimeric antibody is grafted directly to human antibody.H1-H11 is in H0 On the basis of transformation, be mutated on 71,69,66,30,27,28,73,78,24,48,38 position respectively.H12 is in H0 base Back mutation is carried out in 66,69 and 71 sites on plinth.H13 is on the basis of H0 above 7 27,28,30,66,69,71,73 Site carries out back mutation.H14 is that site on the basis of H0 71,69,66,30,27,28,73,78,24,48,38 carries out Back mutation.Back mutation refers to the sequence by source of people sequence amino acid inverse transition at anti-human CD146 human mouse chimeric antibody.
The area people V gene database is analyzed by BLAST, selection can with anti-human CD146 human mouse chimeric antibody light chain There is substitution of the people IGKV1-39 of 64% similitude (including CDRs) as the area FR in change area.Based on sequence similarity analysis, J- The FR4 frame of 2 minigene of region kappa (Kabat Vol.II) is most suitable.The first two of JK-2 minigene is residual Base is fallen in the anti-human area CD146 human mouse chimeric antibody CDR3.By sequence alignment and in view of issuable to antibody function It influences, obtains 6 light chain mutants containing IGKV1-39 framework region.Structure L0 is by anti-human CD146 human mouse chimeric antibody CDR is grafted directly to people's IGKV1-39 framework region.L1-44 structure and the difference of L0 are that they are respectively in 2,1,3 and 60 sites It is mutated.L5 is in 4 site simultaneous mutations.
Embodiment 2 determines specificity of the antibody of humanization design in conjunction with people's CD146 receptor by ELISA
Synthesize VH structural domain and VL structural domain according to computer simulation humanization design's result, by the VH structural domain of synthesis and VL structural domain is cloned into mammalian expression vector, which enables corresponding structural domain in complete human IgG bone It is expressed in frame.By heavy chain construct and light chain construct cotransfection into mammalian cell, the small quantities of of humanized antibody is carried out Amount preparation.The supernatant of transient transfection is subjected to ELISA detection.By albumin A or G resin, IgG purification to uniform, for It is detected in measurement based on cell.
To determine specificity of the humanized antibody in conjunction with CD146, ELISA has been carried out for CD146 and reference protein.It will The supernatant (protein concentration is about 5 μ g/ml) of above-mentioned transient transfection dilutes 20 times, 200 times, 2000 times, 20000 times and is used to connect The ELISA detection got off.Known CD146 is diluted to 1~10 μ g/ml with coating buffer, every hole adds 0.1ml, 4 DEG C overnight into Row coating.Next day washs 3 times.Add certain diluted humanized antibody expression product sample (above-mentioned supernatant) 0.1ml in above-mentioned In the reacting hole being coated with, sets 37 DEG C and be incubated for 1 hour, washing.(while doing blank, feminine gender and positive hole control) is in reacting hole In, be added enzyme mark secondary antibody (antiantibody) 0.1ml of diluted fresh, 37 DEG C incubation 30-60 minutes, washing, last is all over using DDW washing.Add substrate solution to develop the color: being added the tmb substrate solution 0.1ml of Extemporaneous in each reacting hole, 37 DEG C 10~30 points Clock.It terminates reaction: 2M sulfuric acid 0.05ml being added in each reacting hole.Result judgement: it directly can with the naked eye be seen in white background Examine result: color is deeper in reacting hole, and positive degree is stronger, and negative reaction is colourless or extremely shallow, the depth of the be in color of foundation, It is indicated with "+", "-" number.OD450 value can also be surveyed: on ELISA detector, at 450nm, being surveyed after being returned to zero with blank control wells Each hole OD450 value, it is as positive if more than 2.1 times of defined negative control OD450 value.As a result as shown in table 1- table 4.Root Preferred several groups of antibody are selected according to experimental result, as shown in table 5.
Table 1: supernatant dilutes 20 times of ELISA testing results
Table 2: supernatant dilutes 200 times of ELISA testing results
Antibody OD450 Antibody OD450 Antibody OD450 Antibody OD450 Antibody OD450 Antibody OD450
H0L0 2.456 H2L4 2.354 H5L2 2.357 H8L0 2.238 H10L4 2.378 H13L2 2.397
H0L1 2.378 H2L5 2.317 H5L3 2.279 H8L1 2.020 H10L5 2.315 H13L3 2.442
H0L2 2.482 H3L0 2.281 H5L4 2.291 H8L2 2.098 H11L0 2.190 H13L4 2.321
H0L3 2.469 H3L1 2.315 H5L5 2.241 H8L3 2.070 H11L1 2.326 H13L5 2.315
H0L4 2.437 H3L2 2.273 H6L0 2.230 H8L4 2.162 H11L2 2.317 H14L0 2.317
H0L5 2.368 H3L3 2.120 H6L1 2.220 H8L5 1.991 H11L3 2.236 H14L1 2.220
H1L0 2.152 H3L4 2.319 H6L2 2.306 H9L0 2.391 H11L4 2.354 H14L2 2.295
H1L1 1.844 H3L5 2.418 H6L3 2.408 H9L1 2.456 H11L5 2.485 H14L3 2.467
H1L2 2.188 H4L0 2.510 H6L4 2.311 H9L2 2.420 H12L0 1.434 H14L14 2.312
H1L3 2.168 H4L1 2.303 H6L5 2.225 H9L3 2.271 H12L1 1.056 H14L15 2.275
H1L4 2.226 H4L2 2.281 H7L0 1.364 H9L4 2.244 H12L2 1.412 Blank 0.067
H1L5 1.919 H4L3 2.390 H7L1 2.284 H9L5 2.270 H12L3 1.244 Blank 0.063
H2L0 2.329 H4L4 2.341 H7L2 2.261 H10L0 2.267 H12L4 1.345 blank89 0.061
H2L1 2.225 H4L5 2.214 H7L3 2.159 H10L1 2.195 H12L5 1.101 blank90 0.054
H2L2 2.378 H5L0 2.407 H7L4 2.222 H10L2 2.344 H13L0 2.277 blank0 0.048
H2L3 2.481 H5L1 2.440 H7L5 2.400 H10L3 2.494 H13L1 2.387 blank0 0.051
Table 3: supernatant dilutes 2000 times of ELISA testing results
Table 4: supernatant dilutes 20000 times of ELISA testing results
Antibody OD450 Antibody OD450 Antibody OD450 Antibody OD450 Antibody OD450 Antibody OD450
H0L0 0.277 H2L4 0.333 H5L2 0.278 H8L0 0.180 H10L4 0.280 H13L2 0.263
H0L1 0.333 H2L5 0.340 H5L3 0.291 H8L1 0.145 H10L5 0.262 H13L3 0.348
H0L2 0.380 H3L0 0.325 H5L4 0.300 H8L2 0.151 H11L0 0.202 H13L4 0.211
H0L3 0.292 H3L1 0.324 H5L5 0.260 H8L3 0.171 H11L1 0.288 H13L5 0.330
H0L4 0.333 H3L2 0.298 H6L0 0.182 H8L4 0.195 H11L2 0.396 H14L0 0.478
H0L5 0.361 H3L3 0.247 H6L1 0.252 H8L5 0.154 H11L3 0.351 H14L1 0.325
H1L0 0.158 H3L4 0.274 H6L2 0.240 H9L0 0.284 H11L4 0.400 H14L2 0.268
H1L1 0.127 H3L5 0.304 H6L3 0.255 H9L1 0.278 H11L5 0.358 H14L3 0.325
H1L2 0.167 H4L0 0.435 H6L4 0.225 H9L2 0.332 H12L0 0.091 H14L14 0.317
H1L3 0.190 H4L1 0.292 H6L5 0.262 H9L3 0.247 H12L1 0.084 H14L15 0.310
H1L4 0.205 H4L2 0.318 H7L0 0.093 H9L4 0.276 H12L2 0.098 Blank 0.065
H1L5 0.132 H4L3 0.326 H7L1 0.282 H9L5 0.314 H12L3 0.091 Blank 0.07
H2L0 0.295 H4L4 0.333 H7L2 0.296 H10L0 0.246 H12L4 0.091 blank89 0.052
H2L1 0.251 H4L5 0.243 H7L3 0.287 H10L1 0.277 H12L5 0.093 blank90 0.059
H2L2 0.336 H5L0 0.312 H7L4 0.212 H10L2 0.324 H13L0 0.383 blank0 0.051
H2L3 0.397 H5L1 0.279 H7L5 0.275 H10L3 0.285 H13L1 0.273 blank0 0.055
Table 5: the combination that ELISA result is best in the supernatant of different dilutions is selected
It is in consolidated statement 5 as a result, expression quantity preferable light and weight chain combinational expression higher to some affinity in above-mentioned experiment Antibody carries out further ELISA verifying.Thin thereon it will release 5000 times and 20000 times progress ELISA detections respectively.As a result such as Shown in table 6- table 7.H5L0 antibody, H10L2 antibody, H14L0 antibody, H11L3 antibody, H11L4 antibody, H13L0 antibody, H0L2 are anti- Body, H11L2 antibody, H9L2 antibody, H13L4 antibody, H2L3 antibody, H0L4 antibody, H9L0 antibody, H2L4 antibody, H2L2 antibody, H7L3 antibody, H0L5 antibody, H4L0 antibody, H12L0 antibody, H14L1 are currently preferred antibody.
Above-mentioned affinity is higher, the preferable light and weight chain combinational expression antibody of expression quantity, the following institute of the amino acid sequence for including It states:
H5:SEQ ID NO.1；H10:SEQ ID NO.2；H14:SEQ ID NO.3；H11:SEQ ID NO.4；H13: SEQ ID NO.5；H0:SEQ ID NO.6；H9:SEQ ID NO.7；H2:SEQ ID NO.8；H7:SEQ ID NO.9；H4: SEQ ID NO.10；H12:SEQ ID NO.11；L0:SEQ ID NO.12；L2:SEQ ID NO.13；L3:SEQ ID NO.14；L4:SEQ ID NO.15；L1:SEQ ID NO.16；L5:SEQ ID NO.17.
Nucleic acid sequence corresponding to above-mentioned amino acid sequence is as described below:
H5:SEQ ID NO.20；H10:SEQ ID NO.21；H14:SEQ ID NO.22；H11:SEQ ID NO.23； H13:SEQ ID NO.24；H0:SEQ ID NO.25；H9:SEQ ID NO.26；H2:SEQ ID NO.27；H7:SEQ ID NO.28；H4:SEQ ID NO.29；H12:SEQ ID NO.30；L0:SEQ ID NO.31；L2:SEQ ID NO.32；L3:SEQ ID NO.33；L4:SEQ ID NO.34；L1:SEQ ID NO.35；L5:SEQ ID NO.36.
Table 6: the ELISA result after preferred 5000 times of supernatant dilution in conjunction with CD146
Table 7: the ELISA result after preferred 20000 times of supernatant dilution in conjunction with CD146
Antibody OD450 value Antibody OD450 value Antibody OD450 value
H0L2 0.259 H7L3 0.261 H13L0 0.246
H0L4 0.266 H9L0 0.284 H13L4 0.196
H0L5 0.284 H9L2 0.219 H14L0 0.242
H2L2 0.28 H10L2 0.268 H14L1 0.273
H2L3 0.346 H11L2 0.36 Blank 0.056
H2L4 0.278 H11L3 0.265 Blank 0.059
H4L0 0.281 H11L4 0.316 blank0 0.051
H5L0 0.254 H12L0 0.081 blank0 0.055
Embodiment 3: the combination of humanized antibody series and CD146 carry out BIACORE analysis
The purpose of the present embodiment is that the binding affinity of humanization modified antibody Yu people CD146 is analyzed with BIACORE.It is described Humanization modified antibody be that H5L0 antibody (comprising amino acid sequence SEQIDNO1 and SEQIDNO12), H10L2 antibody (include Amino acid sequence SEQIDNO2 and SEQIDNO13), H14L0 antibody (include amino acid sequence SEQIDNO3 and SEQIDNO12), H11L3 antibody (including amino acid sequence SEQIDNO4 and SEQIDNO14) H11L4 antibody (includes amino acid sequence SEQIDNO4 And SEQIDNO15) H13L0 antibody (include amino acid sequence SEQIDNO5 and SEQIDNO12), H0L2 antibody (includes amino acid Sequence SEQIDNO6 and SEQIDNO13), H11L2 antibody (include amino acid sequence SEQIDNO4 and SEQIDNO13), H9L2 it is anti- Body (include amino acid sequence SEQIDNO7 and SEQIDNO13), H13L4 antibody (comprising amino acid sequence SEQIDNO5 and SEQIDNO15), H2L3 antibody (including amino acid sequence SEQIDNO8 and SEQIDNO14), H0L4 antibody (include amino acid sequence Arrange SEQIDNO6 and SEQIDNO15), H9L0 antibody (include amino acid sequence SEQIDNO7 and SEQIDNO12), H2L4 antibody (include amino acid sequence SEQIDNO8 and SEQIDNO15), H2L2 antibody (comprising amino acid sequence SEQIDNO8 and SEQIDNO13), H7L3 antibody (including amino acid sequence SEQIDNO9 and SEQIDNO14).
Analysis carries out on BIACORE3000 instrument, which carries out before prepared by each new chip and passed through system Test.All operationss 25 DEG C carry out, with HBS-EP (GEHealthcareBR-1006-69/5mMHEPES, 150mMNaCl, 3.4mMEDTA, 0.005%surfactantP20, pH7.4) it is used as working buffer solution.It (resonates using by primary amine chemical property Unit be about 4000RU ' s) target protein A of the covalent coupling on CM5 chip at similar resonating member (100-200RU ' s) Confrontation CD146 human mouse chimeric antibody and humanized antibody are analyzed.Each chip is also prepared for i.e. not even with reference surface The control of connection capture antibody.Obtain the induction curve figure (Sensorgrams) of the circulation carried out using different analyte concentrations with Just dynamic analysis is carried out.One circulation by monoclonal antibody be captured on the surface, with flowing working buffer solution carry out it is short Temporary stationary phase and subsequent limiting concentration analyte (ECD or subdomain albumen) are bonded.Injection is tied with surface The analyte (3-4 minutes) of conjunction obtains the bound fraction in curve.Then buffer elution (3-4 minutes) are carried out, record dissociation Data.Circulation ends here, and the actified solution (10mM glycine pH1.5) that injection capture kit provides captures to remove Antibody/analyte, but not significantly affect capture antibody and carry out the capture of monoclonal antibody again in subsequent circulation.
The conventional method of affinity analysis is as follows: firstly, preparing chip and testing it to different MAb concentrations The resonance units (RU) of lower capture.Then kinetic loop is executed, monoclonal antibody is captured to the water of general 100RU in circulation It is flat, allow to analyze albumen and be combined then dissociation, surface is regenerated except the whole albumen for eliminating covalent coupling.With 6 kinds of different analysis protein concentrations (usually 200nM, 100nM, 50nM, 25nM, 12.5nM and 6.25nM) carry out it is a series of this Kind circulation.More wheel buffer circulations are carried out before analyzing albumen to guarantee the consistency of circulation, in some tests, buffer Circulation is by as " dual reference ".The analyte used is people CD146.In dynamic test cyclic process, analogies coupling Surface provide reference, which is subtracted from the specific antibody being currently running-analyte RU data to remove buffer band The illusion come.To each concentration in kinetic curve group, subtract from the data of the circulation containing analyte containing only buffer Circulation data, thus to it is certain operation do dual reference.It will be obtained in this way using BIAEVALUATION software (v.3.2) The curve data overall situation be fitted 11 Lang Gemiaoer (Langmuir) model.Utilize BIAEVALUATIONTM Software Create phase interaction With induction curve figure, dynamic assessment is carried out with curve graph.Experimental result is as shown in table 8.
Table 8: anti-CD146 human mouse chimeric antibody and humanized antibody and people CD146 receptor affinity analyze result
Antibody Analyte Ka(1/Ms) Kd(1/s) KD(M)
H5L0 People CD146 7.98x104 6.48x10-3 8.11x10-8
H10L2 People CD146 2.76x105 1.42x10-2 5.15x10-8
H14L0 People CD146 1.62x105 3.30x10-3 2.03x10-8
H11L3 People CD146 3.31x105 8.88x10-3 2.68x10-8
H11L4 People CD146 9.65x104 7.83x10-3 8.11x10-8
H13L0 People CD146 7.22x104 5.57x10-3 7.71x10-8
H0L2 People CD146 1.13x105 1.06x10-2 9.39x10-8
H11L2 People CD146 1.20x105 8.65x10-3 7.21x10-8
H9L2 People CD146 1.60x105 9.05x10-3 5.65x10-8
H13L4 People CD146 1.59x105 5.52x10-3 3.48x10-8
H2L3 People CD146 1.51x105 9.70x10-3 6.41x10-8
H0L4 People CD146 2.95x105 1.10x10-2 3.72x10-8
H9L0 People CD146 1.50x105 8.92x10-3 5.95x10-8
H2L4 People CD146 1.55x105 1.03x10-2 6.61x10-8
H2L2 People CD146 1.58x105 1.00x10-2 6.37x10-8
H7L3 People CD146 1.10x105 9.51x10-3 8.67x10-8
Anti- CD146 human mouse chimeric antibody People CD146 8.34x104 3.28x10-3 3.94x10-8
Table 8 shows the data that BIACORE is obtained, and can be seen that the humanization designed by computer simulation from KD value Antibody (H14L0) and humanized antibody (H11L3) are best to the affinity of people CD146, are higher than source of mouse antibody in conjunction with activity, are not Come the good candidate antibody for developing therapeutic anti-CD146 monoclonal antibody.In above data, H13L4, H0L4, H10L2, H9L2, The affinity and source of mouse of the monoclonal antibody and CD146 that obtain after several humanization designs of H9L0, H2L2, H2L3, H2L4 are anti- Body is very nearly the same.It proves antibody provided by the present invention, antibody can be improved in the case where affinity is constant or even increases Humanization degree.
It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, may be used also With several improvements and modifications are made to the present invention, these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Sequence table
<110>Nanjing Ren Nuo pharmaceutcal corporation, Ltd
<120>a kind of Humanized anti-human CD146 monoclonal antibody for having efficient neutralization activity
<130> 2017
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 2
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 3
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Tyr Ile Phe Thr Asn Tyr
20 25 30
Trp Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Phe Thr Leu Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 4
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 5
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe Thr Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Phe Thr Leu Ser Val Asp Lys Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 6
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 7
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 7
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 8
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 9
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 9
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 10
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 11
<211> 118
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 11
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Gly Thr Asp Ile Thr Tyr Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Phe Thr Leu Ser Val Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Gly Gly Tyr Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr
100 105 110
Met Val Thr Val Ser Ser
115
<210> 12
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ile Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 13
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 13
Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ile Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 14
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ile Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 15
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ile Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 16
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 16
Asp Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ile Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 17
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 17
Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Ile Ser
20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg
85 90 95
Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
<210> 18
<211> 330
<212> PRT
<213>homo sapiens (homo sapiens)
<400> 18
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 19
<211> 107
<212> PRT
<213>homo sapiens (homo sapiens)
<400> 19
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
<210> 20
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggcta caccttctcc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccatc tcccgcgaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 21
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggctt caccttctcc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg gatcgcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccatc tcccgcgaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 22
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgcca cctccggcta catcttcacc aactactgga tccactgggt gaagcaggcc 120
cccggcaagg gcctggagtg gatcgcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggcaa gttcaccctg tccgtggaca agtccaagaa caccgcctac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 23
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggctt caccttctcc aactactgga tccactgggt gaagcaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccatc tcccgcgaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 24
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggcta catcttcacc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggcaa gttcaccctg tccgtggaca agtccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 25
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggctt caccttctcc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccatc tcccgcgaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 26
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgcca cctccggctt caccttctcc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccatc tcccgcgaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 27
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggctt caccttctcc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccctg tcccgcgaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 28
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggctt caccttctcc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccatc tcccgcgaca agtccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 29
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggctt caccttcacc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccatc tcccgcgaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 30
<211> 354
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
caggtgcagc tggtggagtc cggcggcggc gtggtgcagc ccggccgctc cctgcgcctg 60
tcctgcgccg cctccggctt caccttctcc aactactgga tccactgggt gcgccaggcc 120
cccggcaagg gcctggagtg ggtggcccgc atctaccccg gcaccgacat cacctactac 180
aacgagaagt tcaagggccg cttcaccctg tccgtggaca actccaagaa caccctgtac 240
ctgcagatga actccctgcg cgccgaggac accgccgtgt actactgcgc ccgctccggc 300
ggctactggt acttcgacgt gtggggcgcc ggcaccatgg tgaccgtgtc ctcc 354
<210> 31
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgcgtgacc 60
atcacctgcc gcgcctccaa gtccgtgtcc atctccggct actcctacat gcactggtac 120
cagcagaagc ccggcaaggc ccccaagctg ctgatctacc tggcctccaa cctggagtcc 180
ggcgtgccct cccgcttctc cggctccggc tccggcaccg acttcaccct gaccatctcc 240
tccctgcagc ccgaggactt cgccacctac tactgccagc actcccgcga gctgccctac 300
accttcggcc agggcaccaa gctggagatc aagcgc 336
<210> 32
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gagatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgcgtgacc 60
atcacctgcc gcgcctccaa gtccgtgtcc atctccggct actcctacat gcactggtac 120
cagcagaagc ccggcaaggc ccccaagctg ctgatctacc tggcctccaa cctggagtcc 180
ggcgtgccct cccgcttctc cggctccggc tccggcaccg acttcaccct gaccatctcc 240
tccctgcagc ccgaggactt cgccacctac tactgccagc actcccgcga gctgccctac 300
accttcggcc agggcaccaa gctggagatc aagcgc 336
<210> 33
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
gacatcgtga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgcgtgacc 60
atcacctgcc gcgcctccaa gtccgtgtcc atctccggct actcctacat gcactggtac 120
cagcagaagc ccggcaaggc ccccaagctg ctgatctacc tggcctccaa cctggagtcc 180
ggcgtgccct cccgcttctc cggctccggc tccggcaccg acttcaccct gaccatctcc 240
tccctgcagc ccgaggactt cgccacctac tactgccagc actcccgcga gctgccctac 300
accttcggcc agggcaccaa gctggagatc aagcgc 336
<210> 34
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
gacatccaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgcgtgacc 60
atcacctgcc gcgcctccaa gtccgtgtcc atctccggct actcctacat gcactggtac 120
cagcagaagc ccggcaaggc ccccaagctg ctgatctacc tggcctccaa cctggagtcc 180
ggcgtgcccg cccgcttctc cggctccggc tccggcaccg acttcaccct gaccatctcc 240
tccctgcagc ccgaggactt cgccacctac tactgccagc actcccgcga gctgccctac 300
accttcggcc agggcaccaa gctggagatc aagcgc 336
<210> 35
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
gacctgcaga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgcgtgacc 60
atcacctgcc gcgcctccaa gtccgtgtcc atctccggct actcctacat gcactggtac 120
cagcagaagc ccggcaaggc ccccaagctg ctgatctacc tggcctccaa cctggagtcc 180
ggcgtgccct cccgcttctc cggctccggc tccggcaccg acttcaccct gaccatctcc 240
tccctgcagc ccgaggactt cgccacctac tactgccagc actcccgcga gctgccctac 300
accttcggcc agggcaccaa gctggagatc aagcgc 336
<210> 36
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
gagctggtga tgacccagtc cccctcctcc ctgtccgcct ccgtgggcga ccgcgtgacc 60
atcacctgcc gcgcctccaa gtccgtgtcc atctccggct actcctacat gcactggtac 120
cagcagaagc ccggcaaggc ccccaagctg ctgatctacc tggcctccaa cctggagtcc 180
ggcgtgcccg cccgcttctc cggctccggc tccggcaccg acttcaccct gaccatctcc 240
tccctgcagc ccgaggactt cgccacctac tactgccagc actcccgcga gctgccctac 300
accttcggcc agggcaccaa gctggagatc aagcgc 336
<210> 37
<211> 990
<212> DNA
<213>homo sapiens (homo sapiens)
<400> 37
gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc cacctccggc 60
ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt gaccgtgtcc 120
tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct gcagtcctcc 180
ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg cacccagacc 240
tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa ggtggagccc 300
aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct gctgggcggc 360
ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc ccgcaccccc 420
gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa gttcaactgg 480
tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc cccgcgagga gcagtacaac 540
tccacctacc gcgtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 600
gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa gaccatctcc 660
aaggccaagg gccagccccg cgagccccag gtgtacaccc tgcccccctc ccgcgacgag 720
ctgaccaaga accaggtgtc cctgacctgc ctggtgaagg gcttctaccc ctccgacatc 780
gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac cccccccgtg 840
ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa gtcccgctgg 900
cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960
cagaagtccc tgtccctgtc ccccggcaag 990
<210> 38
<211> 320
<212> DNA
<213>homo sapiens (homo sapiens)
<400> 38
cgcaccgtgg ccgccccctc cgtgttcatc ttccccccct ccgacgagca gctgaagtcc 60
ggcaccgcct ccgtggtgtg cctgctgaac aacttctacc cccgcgaggc caaggtgcag 120
tggaaggtgg acaacgccct gcagtccggc actcccagga gtccgtgacc gagcaggact 180
ccaaggactc cacctactcc ctgtcctcca ccctgaccct gtccaaggcc gactacgaga 240
agcacaaggt gtacgcctgc gaggtgaccc accagggcct gtcctccccc gtgaccaagt 300
ccttcaaccg cggcgagtgc 320

Claims (7)

1. a kind of Humanized anti-human CD146 monoclonal antibody, it is characterised in that: the monoclonal antibody include light chain variable region and Heavy chain variable region: where the amino acid sequence of light chain variable region is SEQ ID NO.12, and the amino acid sequence of heavy chain variable region is SEQ ID NO.3；Or, the amino acid sequence of light chain variable region is SEQ ID NO.14, the amino acid sequence of heavy chain variable region is SEQ ID NO.4；Or, the amino acid sequence of light chain variable region is SEQ ID NO.15, the amino acid sequence of heavy chain variable region is SEQ ID NO.5；Or, the amino acid sequence of light chain variable region is SEQ ID NO.15, the amino acid sequence of heavy chain variable region is SEQ ID NO.6；Or, the amino acid sequence of light chain variable region is SEQ ID NO.13, the amino acid sequence of heavy chain variable region is SEQ ID NO.2；Or, the amino acid sequence of light chain variable region is SEQ ID NO.13, the amino acid sequence of heavy chain variable region is SEQ ID NO.7；Or, the amino acid sequence of light chain variable region is SEQ ID NO.12, the amino acid sequence of heavy chain variable region is SEQ ID NO.7；Or, the amino acid sequence of light chain variable region is SEQ ID NO.13, the amino acid sequence of heavy chain variable region is SEQ ID NO.8；Or, the amino acid sequence of light chain variable region is SEQ ID NO.14, the amino acid sequence of heavy chain variable region is SEQ ID NO.8；Or, the amino acid sequence of light chain variable region is SEQ ID NO.15, the amino acid sequence of heavy chain variable region is SEQ ID NO.8。
2. monoclonal antibody according to claim 1, it is characterised in that: the monoclonal antibody further includes heavy chain constant region And constant region of light chain.
3. monoclonal antibody according to claim 2, it is characterised in that: the amino acid sequence of the heavy chain constant region is Amino acid sequence shown in SEQIDNO:18.
4. monoclonal antibody according to claim 2, it is characterised in that: the amino acid sequence of the constant region of light chain is Amino acid sequence shown in SEQIDNO:19.
5. any Humanized anti-human CD146 monoclonal antibody is in preparation for treating malignant tumour in claim 1-4 Or the application in Autoimmune diseases or new vessels class disease medicament.
6. a kind of for treating the pharmaceutical composition of tumour, it includes any Humanized anti-human CD146 of such as claim 1-5 is mono- Clonal antibody and pharmaceutical carrier.
7. a kind of expression vector, the expression vector includes coding: the nucleic acid sequence of SEQ ID NO.22, SEQ ID NO.31； Or, including the nucleic acid sequence of SEQ ID NO.23, SEQ ID NO.33；Or, including SEQ ID NO.24, SEQ ID NO.34 Nucleic acid sequence；Or, including the nucleic acid sequence of SEQ ID NO.25, SEQ ID NO.34；Or, including SEQ ID NO.21, SEQ The nucleic acid sequence of ID NO.32；Or, including the nucleic acid sequence of SEQ ID NO.26, SEQ ID NO.32；Or, including SEQ ID The nucleic acid sequence of NO.26, SEQ ID NO.31；Or, including the nucleic acid sequence of SEQ ID NO.27, SEQ ID NO.32；Or, packet Include the nucleic acid sequence of SEQ ID NO.27, SEQ ID NO.33；Or, including the nucleic acid of SEQ ID NO.27, SEQ ID NO.34 Sequence.
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