WO2019137309A1 - Humanized anti-human cd146 monoclonal antibody having high neutralizing activity - Google Patents

Abstract

The present invention discloses a humanized anti-human CD146 monoclonal antibody having high neutralizing activity. The humanized anti-human CD146 monoclonal antibody having high neutralizing activity comprises a light chain variable region and a heavy chain variable region. The present invention enables preparation of a monoclonal antibody having a high degree of humanization while maintaining or enhancing the binding activity of the antibody molecule to a human CD146 receptor molecule, so as to avoid the human anti-mouse immune reaction caused by the antibody when it enters the human body as a therapeutic antibody, achieving good technical effects.

Description

Humanized anti-human CD146 monoclonal antibody with high efficiency of neutralizing activity Technical field:

The invention relates to the field of biomedicine, in particular to a humanized anti-human CD146 monoclonal antibody with high neutralizing activity.

Background technique:

CD146 is an adhesion molecule with a transmembrane structure. It is a type I membrane protein composed of 646 amino acid residues and belongs to the immunoglobulin Ig superfamily. Studies have shown that CD146 is a co-receptor of vascular endothelial growth factor receptor VEGFR-2, which regulates VEGF-induced VEGFR-2 activation and downstream signaling, thereby promoting tumor angiogenesis (MicroRNA 329 Suppresses Angiogenesis by Targeting CD146.Molecular And Cellular Biology, 2013; 33(18): 3689-99.). In addition, CD146 molecule is an important factor in promoting the conversion of inflammatory bowel disease into colorectal tumor. The pro-inflammatory cytokines TNFα and IL1β can activate the transcription of CD146, making it highly expressed on the vascular endothelial cell membrane, and then promoting inflammatory cell invasion. Run and angiogenesis lead to the continuous progression of chronic inflammation, which evolves into colorectal cancer. Through a series of tumor models related to ulcerative colitis, the researchers found that CD146, which specifically knocks out vascular endothelial cells, significantly reduced the symptoms of inflammatory bowel disease mice, and significantly reduced the infiltration of inflammatory cells and new blood vessels. In tumor models in which vascular endothelial cells specifically knock out CD146, the number and volume of tumors also decreased significantly. Targeting endothelial CD146 attenuates neuroinflammation by limiting lymphocyte extravasation to the CNS. Scientific Reports, 2013; 3:1687. Overexpression of CD146 molecules in epithelial breast cancer cells results in a very high degree of cell invasion and migration of tumor cells with a lower degree of malignancy, as well as the appearance of breast cancer stem cells. Studies have shown that CD146 promotes breast cancer progression through the mechanism of EMT, so CD146 may be a new target for the treatment of breast cancer (CD146, an epithelial-mesenchyaml 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.).

Antibodies targeting CD146 differ in their affinity to CD146 molecules due to their different structural characteristics. P1H12 is capable of binding to most 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 antibodies bind only to tumors. A blood vessel in an active state in a tissue that does not bind to a blood vessel in a normal tissue. Studies have shown that these antibodies recognize different epitopes, P1H12 recognizes a linear epitope located in the N-terminal domain, and some anti-CD146 antibodies recognize a spatial conformational epitope based on disulfide bonds, thus resulting in binding activity to different endothelial tissues. difference. CN102936283A also discloses a murine CD146 antibody capable of specifically recognizing the above human CD146 epitope.

The above-mentioned several antibodies are all murine monoclonal antibodies, have low binding ability to human complement components, and have weak killing ability to tumor cells; and have weak affinity with Fc receptors on NK and other immune cells, and the effect of ADCC is weak; Moreover, the murine antibody has a short half-life in the human blood circulation and is immunogenic, and thus the host is prone to cause many defects such as anti-antibody-induced rejection.

Currently, there is a lack of a humanized anti-human CD146 monoclonal antibody with potent neutralizing activity.

Summary of the invention:

In view of this, the present invention has been made.

In a first aspect of the invention, there is provided a light chain variable region of a humanized anti-human CD146 monoclonal antibody, the amino acid sequence of the light chain variable region being any one of SEQ ID NOs: 12-17.

In another aspect of the invention, there is provided a heavy chain variable region of a humanized anti-human CD146 monoclonal antibody, the heavy chain variable region amino acid sequence being any one of SEQ ID NOs: 1-11.

In another aspect of the present invention, there is provided a humanized anti-human CD146 monoclonal antibody having high potency neutralizing activity, the monoclonal antibody comprising at least one of the above-described light chain variable region and heavy chain variable region.

Further, the above humanized anti-human CD146 monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, the amino acid sequence of the heavy chain constant region being the amino acid sequence shown by SEQ ID NO: 18; the light chain constant region The amino acid sequence is the amino acid sequence shown by SEQ ID NO: 19.

In another aspect of the present invention, there is provided a use of the above humanized anti-human CD146 monoclonal antibody for the preparation of a medicament for the treatment of a malignant tumor or systemic autoimmune disease or neovascular disease.

In another aspect of the invention, a pharmaceutical composition for treating a malignant tumor comprising the above-described humanized anti-human CD146 monoclonal antibody and a pharmaceutically acceptable carrier is provided.

In another aspect of the invention, there is provided a use of the above humanized anti-human CD146 monoclonal antibody for the preparation of a kit or diagnostic for the detection or diagnosis of a malignant tumor or systemic autoimmune disease or neovascular disease.

The above malignant tumor is selected from cystic brain tumor, glioma, nasopharyngeal carcinoma, pancreatic cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colon cancer, liver cancer, prostate cancer, ovarian malignant tumor, cervical cancer, endometrium. Cancer, malignant melanoma, skin cancer, lymphoma, leukemia or thyroid cancer.

The above systemic autoimmune disease is selected from the group consisting of systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid gland Autoimmune disease or ulcerative colitis.

The neovascular disease is selected from the group consisting of age-related macular, multiple sclerosis, macular edema, choroidal neovascular disease, neovascular glaucoma, retinal vein occlusion, or retinal neovascular disease.

In another aspect of the invention, a nucleic acid sequence encoding the light chain variable region of a humanized anti-human CD146 monoclonal antibody having high potency neutralizing activity, wherein the nucleic acid sequence is SEQ ID NO: 31-36 One.

In another aspect of the invention, a nucleic acid sequence encoding the heavy chain variable region of the humanized anti-human CD146 monoclonal antibody having high potency neutralizing activity, wherein the nucleic acid sequence is SEQ ID NO: 20-30 One.

In another aspect of the invention, a nucleic acid sequence encoding the light chain constant region of a humanized anti-human CD146 monoclonal antibody having high potency neutralizing activity is provided, the nucleic acid sequence being SEQ ID NO:38.

In another aspect of the invention, a nucleic acid sequence encoding the heavy chain constant region of a humanized anti-human CD146 monoclonal antibody having high potency neutralizing activity is provided, the nucleic acid sequence being SEQ ID NO:37.

In another aspect of the invention, a nucleic acid sequence encoding the humanized anti-human CD146 monoclonal antibody having high neutralizing activity, wherein the nucleic acid sequence is any of the above-described light chain variable regions, heavy chains A combination of a variable region, a light chain constant region, and a heavy chain constant region nucleic acid sequence.

Further, the present invention provides an expression vector comprising a nucleic acid sequence encoding at least one of SEQ ID NOs: 20-36. Preferably, the expression vector comprises at least one of SEQ ID NOs: 20-30, and at least one of SEQ ID NOs: 31-36.

Further, the present invention provides a prokaryotic or eukaryotic host cell comprising the above expression vector.

The beneficial effects of the present invention are as follows: the humanized anti-human CD146 monoclonal antibody of the present invention has high-efficiency neutralizing activity, and the degree of humanization is prepared as long as possible while maintaining or enhancing the binding activity of the antibody molecule to the human CD146 receptor molecule. High monoclonal antibody to avoid the human anti-mouse immune response caused by the antibody as a therapeutic antibody into the human body; the present invention adopts a combination of experiment and calculation to perform site-directed mutagenesis on the surface of a single antibody, and selects a human IgG framework. Based on the CDR structure of the original antibody, site-directed mutagenesis of the framework region of IgG; the antibody structure prepared by the present invention comprises the heavy chain variable region of human IGHV3-33 and the framework FR region of the light chain variable region, without affecting the antibody Under the premise of targeting and function, the framework region is subjected to point mutation to enhance the binding activity of the antibody to the human CD146 receptor.

BRIEF DESCRIPTION OF THE DRAWINGS:

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below. Obviously, the drawings in the following description are only It is a certain embodiment of the present invention, and other drawings can be obtained from those skilled in the art without any creative work.

Figure 1 shows the results of ELISA of binding of anti-CD146 murine antibody to CD146.

Detailed ways

Exemplary embodiments will be described in detail herein, examples of which are illustrated in the accompanying drawings. The following description refers to the same or similar elements in the different figures unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Instead, they are merely examples of devices and methods consistent with aspects of the invention as detailed in the appended claims.

The terminology used in the present invention is for the purpose of describing particular embodiments, and is not intended to limit the invention. The singular forms "a", "the" and "the" It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

It should be understood that although the terms first, second, third, etc. may be used to describe various information in the present invention, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, the first information may also be referred to as the second information without departing from the scope of the invention. Similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "when" or "when" or "in response to a determination."

The invention will be described in detail below by way of examples.

Example 1 Humanization strategy of anti-human CD146 human mouse chimeric antibody

Humanized transformation of human CD146 human and mouse chimeric antibodies was performed using the best-fit CDR construction technique. The murine CDRs are grafted into a suitable human acceptable framework with high sequence homology to the chimeric antibody framework regions. The FR framework of the most matching portable CDRs was searched in the human germline sequence database. The primary structural sequence of the antibody and the 3D structural data are utilized to determine the key framework residues required to maintain the correct conformation of the murine CDRs to maintain binding affinity and binding specificity. Back mutations are made in different humanized molecular frameworks to maintain antibody affinity, resulting in highly humanized monoclonal antibodies.

The human IGHV3-33 and anti-human CD146 human murine chimeric antibody heavy chain variable region sequences have 41% similarity (including CDRs) by BLAST analysis of the human V region gene database, and the framework region is preferably anti-human CD146 human The framework region of the humanized design of the mouse chimeric antibody. Based on the sequence similarity of JH3 minigene (Kabat Vol. II), the germline V region was combined with FR4 by computer simulation. The WGQG of the JH3 minigene residue falls within the CDR3 region of the anti-human CD146 human murine chimeric antibody. Based on sequence alignment and considering the effect on antibody function, 14 humanized heavy chain mutants were designed using the IGHV3-33 framework. H0 is a framework region in which CDRs of an anti-human CD146 human mouse chimeric antibody are directly transplanted into a human antibody. H1-H11 is a modification based on H0, and mutations are made at positions 71, 69, 66, 30, 27, 28, 73, 78, 24, 48, 38, respectively. H12 was back-mutated at positions 66, 69 and 71 on the basis of H0. H13 is a back mutation at the above seven 27, 28, 30, 66, 69, 71, and 73 loci based on H0. H14 is a back mutation at the positions of 71, 69, 66, 30, 27, 28, 73, 78, 24, 48, 38 on the basis of H0. A back mutation refers to a sequence that reverse mutates a human sequence amino acid into an anti-human CD146 human murine chimeric antibody.

Human V region gene databases were analyzed by BLAST and human IGKV1-39 with 64% similarity (including CDRs) to the human CD146 human murine chimeric antibody light chain variable region was selected as a replacement for the FR region. Based on sequence similarity analysis, the FR4 framework of J-region kappa 2 minigene (Kabat Vol. II) is most suitable. The first two residues of JK-2 minigene fall within the CDR3 region of the anti-human CD146 human murine chimeric antibody. Six light chain mutants containing the IGKV1-39 framework region were obtained by sequence alignment and taking into account the possible effects on antibody function. The structure L0 is the direct transplantation of the CDRs of the anti-human CD146 human mouse chimeric antibody into the human IGKV1-39 framework region. The L1-44 structure differs from L0 in that they mutate at positions 2, 1, 3 and 60, respectively. L5 is mutated at four sites simultaneously.

Example 2 Determination of the specificity of binding of humanized designed antibodies to human CD146 receptor by ELISA

The VH domain and the VL domain were synthesized according to the results of computer simulation of humanized design, and the synthetic VH domain and VL domain were cloned into a mammalian expression vector, which enables the corresponding domain to be in the complete human IgG backbone. Expressed in. The heavy chain construct and the light chain construct were co-transfected into mammalian cells for small batch production of humanized antibodies. The transiently transfected supernatant was subjected to ELISA. IgG was purified to homogeneity by protein A or G resin for detection in cell-based assays.

To determine the specificity of binding of humanized antibodies to CD146, ELISA was performed against CD146 and control proteins. The above transiently transfected supernatant (protein concentration of about 5 μg/ml) was diluted 20-fold, 200-fold, 2000-fold, and 20,000-fold for the next ELISA assay. The known CD146 was diluted to 1 to 10 μg/ml with a coating buffer, 0.1 ml per well, and coated at 4 ° C overnight. Wash 3 times the next day. 0.1 ml of a sample of the humanized antibody expression product (the above supernatant) diluted with a certain amount was added to the above-mentioned coated reaction well, and the mixture was incubated at 37 ° C for 1 hour, and washed. (Also blank, negative and positive well control) In the well, 0.1 ml of freshly diluted enzyme-labeled secondary antibody (anti-antibody) was added, incubated at 37 ° C for 30-60 minutes, washed, and finally washed with DDW. Adding substrate liquid color: 0.1 ml of the temporarily prepared TMB substrate solution was added to each reaction well at 37 ° C for 10 to 30 minutes. The reaction was terminated: 0.05 ml of 2 M sulfuric acid was added to each reaction well. The result was judged: the result can be directly observed with the naked eye on a white background: the darker the color in the reaction well, the stronger the positive degree, and the negative reaction is colorless or extremely light, according to the depth of the color, with "+", "- The number indicates. The OD450 value can also be measured: on the ELISA detector, the OD450 value of each well is measured at 450 nm with the blank control well, and if it is greater than 2.1 times the OD450 value of the specified negative control, it is positive. The results are shown in Tables 1 to 4. Preferred groups of antibodies were selected based on the experimental results, as shown in Table 5.

Table 1: Supernatant dilution 20 times ELISA test results

antibody	OD450	antibody	OD450	antibody	OD450	antibody	OD450	antibody	OD450	antibody	OD450
H0L0	2.508	H2L4	2.510	H5L2	2.403	H8L0	2.533	H10L4	2.612	H13L2	2.599

H0L1	2.463	H2L5	2.484	H5L3	2.410	H8L1	2.479	H10L5	2.449	H13L3	2.451
H0L2	2.415	H3L0	2.361	H5L4	2.337	H8L2	2.452	H11L0	2.369	H13L4	2.411
H0L3	2.537	H3L1	2.427	H5L5	2.368	H8L3	2.473	H11L1	2.473	H13L5	2.476
H0L4	2.480	H3L2	2.369	H6L0	2.319	H8L4	2.449	H11L2	2.358	H14L0	2.416
H0L5	2.372	H3L3	2.244	H6L1	2.176	H8L5	2.378	H11L3	2.317	H14L1	2.341
H1L0	2.473	H3L4	2.404	H6L2	2.335	H9L0	2.501	H11L4	2.379	H14L2	2.468
H1L1	2.537	H3L5	2.501	H6L3	2.473	H9L1	2.535	H11L5	2.557	H14L3	2.582
H1L2	2.445	H4L0	2.509	H6L4	2.513	H9L2	2.550	H12L0	2.427	H14L14	2.454
H1L3	2.381	H4L1	2.367	H6L5	2.387	H9L3	2.460	H12L1	2.325	H14L15	2.447
H1L4	2.390	H4L2	2.393	H7L0	2.286	H9L4	2.374	H12L2	2.226	Blank	0.069
H1L5	2.337	H4L3	2.449	H7L1	2.434	H9L5	2.460	H12L3	2.256	Blank	0.062
H2L0	2.389	H4L4	2.371	H7L2	2.377	H10L0	2.367	H12L4	2.290	Blank89	0.059
H2L1	2.221	H4L5	2.345	H7L3	2.276	H10L1	2.338	H12L5	2.168	Blank90	0.066
H2L2	2.422	H5L0	2.413	H7L4	2.429	H10L2	2.412	H13L0	2.452	Blank0	0.055
H2L3	2.521	H5L1	2.523	H7L5	2.502	H10L3	2.566	H13L1	2.545	Blank0	0.067

Table 2: Supernatant dilution 200 times ELISA test 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 dilution 2000 times ELISA test results

antibody	OD450	antibody	OD450	antibody	OD450	antibody	OD450	antibody	OD450	antibody	OD450
H0L0	1.562	H2L4	1.795	H5L2	1.756	H8L0	1.081	H10L4	1.625	H13L2	1.576

H0L1	1.538	H2L5	1.671	H5L3	1.569	H8L1	0.905	H10L5	1.503	H13L3	1.782
H0L2	1.950	H3L0	1.583	H5L4	1.542	H8L2	0.935	H11L0	1.255	H13L4	1.572
H0L3	1.598	H3L1	1.614	H5L5	1.367	H8L3	1.019	H11L1	1.663	H13L5	1.568
H0L4	1.825	H3L2	1.522	H6L0	1.320	H8L4	1.075	H11L2	1.615	H14L0	1.433
H0L5	1.747	H3L3	1.425	H6L1	1.332	H8L5	0.953	H11L3	1.644	H14L1	1.500
H1L0	0.889	H3L4	1.576	H6L2	1.437	H9L0	1.672	H11L4	1.699	H14L2	1.291
H1L1	0.555	H3L5	1.646	H6L3	1.459	H9L1	1.630	H11L5	1.825	H14L3	1.672
H1L2	0.911	H4L0	1.987	H6L4	1.320	H9L2	1.771	H12L0	0.439	H14L14	1.527
H1L3	1.10	H4L1	1.506	H6L5	1.401	H9L3	1.550	H12L1	0.295	H14L15	1.570
H1L4	1.165	H4L2	1.421	H7L0	0.372	H9L4	1.472	H12L2	0.437	Blank	0.066
H1L5	0.726	H4L3	1.579	H7L1	1.440	H9L5	1.419	H12L3	0.378	Blank	0.076
H2L0	1.531	H4L4	1.649	H7L2	1.622	H10L0	1.455	H12L4	0.415	Blank89	0.048
H2L1	1.515	H4L5	1.318	H7L3	1.636	H10L1	1.513	H12L5	0.303	Blank90	0.053
H2L2	1.612	H5L0	1.563	H7L4	1.257	H10L2	1.591	H13L0	1.533	Blank0	0.047
H2L3	1.768	H5L1	1.493	H7L5	1.613	H10L3	1.646	H13L1	1.446	Blank0	0.05

Table 4: 20,000 ELISA results of supernatant dilution

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 best combination of ELISA results in supernatants with different dilutions

20x

200x

2000x

20000x

Antibody combination	OD450	Antibody combination	OD450	Antibody combination	OD450	Antibody combination	OD450
H10L4	2.612	H4L0	2.51	H4L0	1.987	H14L0	0.478
H13L2	2.599	H10L3	2.494	H0L2	1.95	H4L0	0.435
H14L3	2.582	H11L5	2.485	H0L4	1.825	H11L4	0.4
H10L3	2.566	H0L2	2.482	H11L5	1.825	H2L3	0.397
H11L5	2.557	H2L3	2.481	H2L4	1.795	H11L2	0.396
H9L2	2.55	H0L3	2.469	H13L3	1.782	H13L0	0.383
H13L1	2.545	H14L3	2.467	H9L2	1.771	H0L2	0.38
H0L3	2.537	H0L0	2.456	H2L3	1.768	H0L5	0.361
H1L1	2.537	H9L1	2.456	H5L2	1.756	H11L5	0.358
H9L1	2.535	H13L3	2.442	H0L5	1.747	H11L3	0.351

Based on the results in Table 5, some of the antibodies with higher affinity and better expression of light and heavy chain in the above experiments were further tested by ELISA. The supernatant was diluted 5000 times and 20,000 times, respectively, for ELISA detection. The results are shown in Table 6 - Table 7. H5L0 antibody, H10L2 antibody, H14L0 antibody, H11L3 antibody, H11L4 antibody, H13L0 antibody, H0L2 antibody, H11L2 antibody, H9L2 antibody, H13L4 antibody, H2L3 antibody, H0L4 antibody, H9L0 antibody, H2L4 antibody, H2L2 antibody, H7L3 antibody, H0L5 antibody The H4L0 antibody, the H12L0 antibody, and H14L1 are preferred antibodies of the present invention.

The light and heavy chain combined with the above-mentioned affinity and high expression amount expresses the antibody, and the amino acid sequence comprises the following:

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. 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.

The nucleic acid sequence corresponding to the above amino acid sequence is as follows:

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. 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.

Table 6: ELISA results for binding of CD146 after dilution of the preferred supernatant by 5000 fold

antibody	OD450 value	antibody	OD450 value	antibody	OD450 value
H0L2	0.724	H7L3	0.711	H13L0	0.714
H0L4	0.757	H9L0	0.785	H13L4	0.524
H0L5	0.772	H9L2	0.592	H14L0	0.701

H2L2	0.747	H10L2	0.736	H14L1	0.777
H2L3	0.903	H11L2	1.003	Blank	0.063
H2L4	0.767	H11L3	0.735	Blank	0.058
H4L0	0.77	H11L4	0.881	Blank0	0.047
H5L0	0.706	H12L0	0.147	Blank0	0.052

Table 7: ELISA results for the binding of the preferred supernatant to 20,000 fold after dilution 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

Example 3: Binding of humanized antibody series to CD146 for BIACORE analysis

The purpose of this example was to analyze the binding affinity of humanized antibodies to human CD146 using BIACORE. The humanized antibodies are H5L0 antibodies (including amino acid sequences SEQ ID NO 1 and SEQ ID NO 12), H10L2 antibodies (including amino acid sequences SEQ ID NO 2 and SEQ ID NO 13 ), H 14 L0 antibodies (including amino acid sequences SEQ ID NO 3 and SEQ ID NO 12 ), H11L3 antibodies (including amino acid sequence SEQ ID NO 4 and SEQ ID NO 14) H11L4 antibody (including amino acid sequences SEQ ID NO 4 and SEQ ID NO 15) H13L0 antibody (including amino acid sequences SEQ ID NO 5 and SEQ ID NO 12), H0L2 antibody (including amino acid sequences SEQ ID NO 6 and SEQ ID NO 13), H11L2 antibody (including amino acid sequences SEQ ID NO 4 and SEQ ID NO 13 ), H9L2 antibody (including amino acid Sequences SEQ ID NO 7 and SEQ ID NO 13), H13L4 antibodies (including amino acid sequences SEQ ID NO 5 and SEQ ID NO 15 ), H 2 L 3 antibodies (including amino acid sequences SEQ ID NO 8 and SEQ ID NO 14 ), H0 L4 antibodies (including amino acid sequences SEQ ID NO 6 and SEQ ID NO 15 ), H9L0 antibodies (including amino acid sequences SEQ ID NO 7 and SEQ ID NO 12), H2L4 antibody (comprising amino acid sequences SEQ ID NO 8 and SEQ ID NO 15), H2L2 antibody (comprising amino acid sequences SEQ ID NO 8 and SEQ ID NO 13), H7L3 antibody (including amino acid sequences SEQ ID NO 9 and SEQ ID NO 14).

The analysis was performed on a BIACORE 3000 instrument that was performed before each new chip was prepared and passed the system test. All operations were carried out at 25 ° C with HBS-EP (GE Healthcare BR-1006-69/5 m MHEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005%surfactant P20, pH 7.4) as working buffer. The target protein A covalently coupled to the CM5 chip by a primary amine chemical property (resonance unit of about 4000 RU's) was analyzed against a CD146 human mouse chimeric antibody and a humanized antibody in a similar resonance unit (100-200 RU's). A control with a reference surface, i.e., unconjugated capture antibody, was also prepared for each chip. Sensinggrams of the cycles performed using different analyte concentrations were obtained for kinetic analysis. One cycle consists of a monoclonal antibody that is captured on the surface, a transient stationary phase with a flow-through buffer, and a subsequent defined concentration of analyte (ECD or subdomain protein). Injection of the surface bound analyte (3-4 minutes) gave the binding moiety in the curve. The buffer was then eluted (3-4 minutes) and the dissociation data was recorded. At the end of the cycle, the regeneration solution (10 mM glycine pH 1.5) provided by the capture kit was injected to remove the captured antibody/analyte, but did not significantly affect the capture of the capture antibody by subsequent capture of the monoclonal antibody.

The general method of affinity analysis is as follows: First, the chip is prepared and tested for resonance units (RU) captured at different monoclonal antibody concentrations. A kinetic cycle is then performed, in which the monoclonal antibody is captured to a level of approximately 100 RU, allowing the assay protein to bind and then dissociate, and the surface is regenerated to remove all proteins except covalently coupled. A series of such cycles were performed at six different analytical protein concentrations (typically 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM). Multiple rounds of buffer circulation were performed prior to analysis of the protein to ensure cycle consistency, and in some experiments, buffer circulation was used as a "double reference." The analyte used was human CD146. During the kinetic test cycle, the mimetic coupled surface provides a reference that is subtracted from the running specific antibody-analyte RU data to remove artifacts from the buffer. For each concentration in the kinetic curve set, the data for the buffer-only cycle is subtracted from the data from the cycle containing the analyte to give a double reference to some runs. The curve data thus obtained was globally fitted to the Langmuir model using the BIAEVALUATION software (v.3.2). The interaction sensory curve was generated using BIAEVALUATIONTM software, and the dynamics evaluation was performed using the graph. The experimental results are shown in Table 8.

Table 8: Affinity analysis results of anti-CD146 human mouse chimeric antibody and humanized antibody and human CD146 receptor

antibody	Analyte	Ka (1/Ms)	Kd (1/s)	KD(M)
H5L0	Human CD146	7.98x10 4	6.48x10 -3	8.11x10 -8
H10L2	Human CD146	2.76x10 5	1.42x10 -2	5.15x10 -8
H14L0	Human CD146	1.62x10 5	3.30x10 -3	2.03x10 -8
H11L3	Human CD146	3.31x10 5	8.88x10 -3	2.68x10 -8
H11L4	Human CD146	9.65x10 4	7.83x10 -3	8.11x10 -8
H13L0	Human CD146	7.22x10 4	5.57x10 -3	7.71x10 -8
H0L2	Human CD146	1.13x10 5	1.06x10 -2	9.39x10 -8
H11L2	Human CD146	1.20x10 5	8.65x10 -3	7.21x10 -8
H9L2	Human CD146	1.60x10 5	9.05x10 -3	5.65x10 -8
H13L4	Human CD146	1.59x10 5	5.52x10 -3	3.48x10 -8
H2L3	Human CD146	1.51x10 5	9.70x10 -3	6.41x10 -8
H0L4	Human CD146	2.95x10 5	1.10x10 -2	3.72x10 -8
H9L0	Human CD146	1.50x10 5	8.92x10 -3	5.95x10 -8
H2L4	Human CD146	1.55x10 5	1.03x10 -2	6.61x10 -8
H2L2	Human CD146	1.58x10 5	1.00x10 -2	6.37x10 -8
H7L3	Human CD146	1.10x10 5	9.51x10 -3	8.67x10 -8
anti-CD146 human and mouse chimeric antibody	Human CD146	8.34x10 4	3.28x10 -3	3.94x10 -8

Table 8 shows the data obtained by BIACORE. From the KD value, the humanized antibody (H14L0) and humanized antibody (H11L3) designed by computer simulation have the best affinity for human CD146, and the binding activity is higher than that of the mouse. Antibodies are good candidate antibodies for the development of therapeutic anti-CD146 mAbs in the future. In the above data, several humanized designs of H13L4, H0L4, H10L2, H9L2, H9L0, H2L2, H2L3, and H2L4 have similar affinities to CD146 and are comparable to murine antibodies. It is proved that the antibody provided by the present invention can increase the degree of humanization of the antibody without changing the affinity or even increasing the affinity.

It should be noted that those skilled in the art can make various modifications and changes to the present invention without departing from the spirit and scope of the invention.

Claims (10)

1. A humanized anti-human CD146 monoclonal antibody comprising at least one of SEQ ID NOs: 1-17.
2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody comprises a light chain variable region, and the light chain variable region amino acid sequence is any one of SEQ ID NOs: 12-17.
3. The monoclonal antibody according to claim 1 or 2, wherein the monoclonal antibody comprises a heavy chain variable region, and the heavy chain variable region amino acid sequence is any one of SEQ ID NOs: 1-11.
4. The monoclonal antibody according to claim 1, wherein the monoclonal antibody further comprises a heavy chain constant region and a light chain constant region.
5. The monoclonal antibody according to claim 4, wherein the amino acid sequence of the heavy chain constant region is the amino acid sequence of SEQ ID NO: 18.
6. The monoclonal antibody according to claim 4, wherein the amino acid sequence of the light chain constant region is the amino acid sequence of SEQ ID NO: 19.
7. Use of a humanized anti-human CD146 monoclonal antibody according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of a malignant tumor or systemic autoimmune disease or neovascular disease.
8. A pharmaceutical composition for treating a tumor comprising the humanized anti-human CD146 monoclonal antibody of any of claims 1-6 and a pharmaceutically acceptable carrier.
9. An expression vector comprising a nucleic acid sequence encoding at least one of SEQ ID NOs: 20-36.
10. The expression vector according to claim 9, wherein said expression vector comprises at least one of SEQ ID NOs: 20-30, and at least one of SEQ ID NOs: 31-36.

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