CN110818797B

CN110818797B - Recombinant antibody of anti-human CA153 protein

Info

Publication number: CN110818797B
Application number: CN201810904816.5A
Authority: CN
Inventors: 崔鹏; 何志强; 孟媛; 游辉
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2018-08-09
Filing date: 2018-08-09
Publication date: 2022-11-04
Anticipated expiration: 2038-08-09
Also published as: CN110818797A

Abstract

The invention relates to a novel isolated binding protein containing an antigen binding domain of a CA153 protein, and researches on the preparation, application and the like of the binding protein. The binding protein has strong activity and high affinity with the human CA153 protein, and can be widely applied to the field of detection of the CA153 protein.

Description

Recombinant antibody of anti-human CA153 protein

Technical Field

The invention relates to the technical field of immunization, in particular to a recombinant antibody of anti-human CA153 protein.

Background

Breast Cancer (Breast Cancer) is one of the most common malignancies in women, with about 120 million women suffering from Breast Cancer each year worldwide, and about 50 million deaths from Breast Cancer. In recent years, the incidence of breast cancer in China is on the trend of increasing year by year, and the pathogenic characteristics of the breast cancer are that the onset age is younger, the early stage is more asymptomatic, the disease period is relatively late when the patient is in treatment, and the survival period is shortened due to metastasis, so that early diagnosis and early treatment are the key points for improving the survival rate of patients.

How to improve the early diagnosis rate of breast cancer is a problem which is commonly concerned by scholars at home and abroad at present. The clinical use of serum markers associated with breast cancer is limited by their low sensitivity and specificity when used alone for early diagnosis. In recent years, the detection of tumor markers is increasingly applied to clinical diagnosis and treatment of malignant tumors, and various tumor markers related to breast cancer exist. CA153 is a tumor marker recognized at present and specific to breast cancer, is over-expressed in breast cancer, is not related to pathological types, and is related to clinical staging, tumor size, axillary lymph nodes condition and estrogen-progestogen receptors. CA153 is a variant of the epithelial surface glycoprotein of breast cells and is an important marker of breast cancer. The assay for CA153 can assist in the monitoring of treatment in breast cancer patients, and its dynamic assay can help in early detection of recurrence after treatment in stage II and III breast cancer patients, and monitoring the responsiveness of breast cancer metastasis patients to treatment. It is the most useful index for detecting postoperative recurrence of breast cancer, and CA153 is obviously increased in 96% of patients with local or systemic recurrence before various detections in radiology and clinic. The increased concentration in serum is also closely related to the severity of the tumor; otherwise; its value is reduced, and its therapeutic effect is obvious. The clinical methods for detecting the level of CA153 include enzyme-linked immunosorbent assay (ELISA), chemiluminescence, colloidal gold and the like, and different methods have respective advantages and disadvantages, but all require specific monoclonal antibodies aiming at CA 153. The traditional clinical diagnosis uses monoclonal antibodies of murine origin.

The existing CA153 antibody has low activity and poor affinity, and cannot be well applied to the detection of CA153 protein, so there is a strong need in the art for an antibody that can effectively and specifically bind to and detect CA 153.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention relates to a novel isolated binding protein containing a CA153 antigen binding domain, and researches on the preparation, application and the like of the binding protein.

The antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences of seq id nos: or; has at least 80% sequence identity to the complementarity determining regions of the amino acid sequence described below and has K with the CA153 protein _D ≤1.89×10 ^-8 Affinity of mol/L;

the CDR-VH1 is K-X1-S-X2-Y-T-F-T-X3-Y-D-I-N, wherein,

x1 is A, D or E, X2 is G or D, X3 is T or S;

CDR-VH2 is W-I-Y-P-X1-D-G-S-X2-K-F-N-X3-K-F,

x1 is K, G or R, X2 is I or L, X3 is D or E;

CDR-VH3 is X1-R-E-G-P-X2-Y-A-M-D-Y, wherein,

x1 is V or A, X2 is Q, N or G;

the complementarity determining region CDR-VL1 is H-A-X1-Q-N-X2-N-V-W-L-S, wherein,

x1 is S or T, X2 is L, V or I;

the CDR-VL2 of the complementarity determining region is Y-X1-A-S-X2-L-H-T-G, wherein,

x1 is R or K, X2 is K or N;

CDR-VL3 is Q-Q-X1-Q-S-X2-P-R-T, wherein,

x1 is D, Y or G, and X2 is S or Y.

An important advantage is that the binding protein is highly active and has a high affinity for the human CA153 protein.

Detailed Description

The present invention may be understood more readily by reference to the following description of some embodiments of the invention and the detailed description of the examples included therein.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such embodiments are necessarily varied. It is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Noun definitions

"isolated binding protein comprising an antigen binding domain" broadly refers to all proteins/protein fragments that comprise a CDR region. The term "antibody" includes polyclonal and monoclonal antibodies and antigenic compound-binding fragments of these antibodies, including Fab, F (ab') 2, fd, fv, scFv, diabodies and minimal recognition units of antibodies, as well as single chain derivatives of these antibodies and fragments. The type of antibody can be selected from IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional) and humanized (humanized) antibodies, as well as related synthetic isoforms (antibodies). The term "antibody" is used interchangeably with "immunoglobulin".

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site. The light or heavy chain variable region (VL or VH) is composed of framework regions interrupted by three hypervariable regions, termed "complementarity determining regions" or "CDRs". The extent of framework regions and CDRs has been precisely defined, for example, in Kabat (see Sequences of Proteins of Immunological Interest), E.Kabat et al, U.S. department of Health and Human Services (U.S. department of Health and Human Services), (1983), and Chothia. The framework regions of the antibody, which constitute the combination of the essential light and heavy chains, serve to locate and align the CDRs, which are primarily responsible for binding to the antigen.

As used herein, the "framework" or "FR" regions mean the regions of the antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into adjacent regions (FR 1, FR2, FR3 and FR 4) separated by CDRs.

Typically, the variable domains VL/VH of the heavy and light chains are obtained by linking the CDRs and FRs numbered as follows in a combinatorial arrangement: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

As used herein, the term "purified" or "isolated" in relation to a polypeptide or nucleic acid means that the polypeptide or nucleic acid is not in its native medium or native form. Thus, the term "isolated" includes a polypeptide or nucleic acid that is removed from its original environment, e.g., from its natural environment if it is naturally occurring. For example, an isolated polypeptide is generally free of at least some proteins or other cellular components that are normally bound to or normally mixed with it or in solution. Isolated polypeptides include naturally-produced such polypeptides contained in cell lysates, in purified or partially purified form, recombinant polypeptides, such polypeptides expressed or secreted by cells, and such polypeptides in heterologous host cells or cultures. In connection with a nucleic acid, the term isolated or purified indicates, for example, that the nucleic acid is not in its natural genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a heterologous host cell).

Exemplary embodiments of the invention

The present invention relates to an isolated binding protein comprising an antigen binding domain, wherein the antigen binding domain comprises at least one complementarity determining region selected from the group consisting of amino acid sequences recited in seq id nos: or; has at least 80% sequence identity with the complementarity determining region of the following amino acid sequence and has K with CA153 protein _D ≤1.89×10 ^-8 Affinity of mol/L;

CDR-VH1 is K-X1-S-X2-Y-T-F-T-X3-Y-D-I-N, wherein,

x1 is A, D or E, X2 is G or D, X3 is T or S;

CDR-VH2 is W-I-Y-P-X1-D-G-S-X2-K-F-N-X3-K-F,

x1 is K, G or R, X2 is I or L, X3 is D or E;

CDR-VH3 is X1-R-E-G-P-X2-Y-A-M-D-Y, wherein,

x1 is V or A, X2 is Q, N or G;

CDR-VL1 is H-A-X1-Q-N-X2-N-V-W-L-S, wherein,

x1 is S or T, X2 is L, V or I;

x1 is R or K, X2 is K or N;

CDR-VL3 is Q-Q-X1-Q-S-X2-P-R-T, wherein,

x1 is D, Y or G, and X2 is S or Y.

In some embodiments, the antigen binding domain has at least 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to a complementarity determining region of an amino acid sequence having a K with CA153 protein _D ≤1.89×10 ^-8 mol/L，K _D The value can also be selected to be 1.0 × 10 ^-9 mol/L、2.0×10 ^-9 mol/L、3.0×10 ^-9 mol/L、4.0×10 ^-9 mol/L、5.0×10 ^-9 mol/L、6.0×10 ^-9 mol/L、7.0×10 ^-9 mol/L、8.0×10 ^-9 mol/L、9.0×10 ^-9 mol/L、1.0×10 ^-10 mol/L、3.0×10 ^-10 mol/L、5.0×10 ^- ¹⁰ mol/L、7.0×10 ^-10 mol/L、8.7×10 ^-10 mol/L、9.0×10 ^-10 Affinity of mol/L; or 8.7X 10 ^-10 mol/L≤K _D ≤1.89×10 ^-8 mol/L。

Wherein the affinity is determined according to the method of the present specification.

In some embodiments:

in the complementarity determining region CDR-VH1, X3 is S;

in the CDR-VH2, X3 is E;

in the complementarity determining region CDR-VH3, X1 is V;

in the complementarity determining region CDR-VL1, X1 is S;

in the complementarity determining region CDR-VL2, X1 is K;

in the CDR-VL3, X2 is Y.

In some embodiments, in the complementarity determining region CDR-VH1, X1 is a and X2 is G.

In some embodiments, in the complementarity determining region CDR-VH1, X1 is E and X2 is G.

In some embodiments, in the complementarity determining region CDR-VH1, X1 is D and X2 is G.

In some embodiments, in the complementarity determining region CDR-VH1, X1 is a and X2 is D.

In some embodiments, in the complementarity determining region CDR-VH1, X1 is E and X2 is D.

In some embodiments, in the complementarity determining region CDR-VH1, X1 is D and X2 is D.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is K and X2 is I.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is G and X2 is I.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is R and X2 is I.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is K and X2 is L.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is G and X2 is L.

In some embodiments, in the complementarity determining region CDR-VH2, X1 is R and X2 is L.

In some embodiments, in the complementarity determining region CDR-VH3, X2 is Q.

In some embodiments, in the complementarity determining region CDR-VH3, X2 is G.

In some embodiments, in the complementarity determining region CDR-VH3, X2 is L.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is L.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is I.

In some embodiments, in the complementarity determining region CDR-VL1, X2 is K.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is K.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is N.

In some embodiments, in the complementarity determining region CDR-VL2, X2 is I.

In some embodiments, in the complementarity determining region CDR-VL3, X1 is D.

In some embodiments, in the complementarity determining region CDR-VL3, X1 is Y.

In some embodiments, in the complementarity determining region CDR-VL3, X1 is G.

In some embodiments, the binding protein includes at least 3 CDRs (e.g., 3 CDRs of a heavy chain, or 3 CDRs of a light chain); alternatively, the binding protein comprises at least 6 CDRs.

In some embodiments, the binding protein is a whole antibody comprising a variable region and a constant region.

In some embodiments, the binding protein is a "functional sheet" of an antibodySegments ", e.g. Nanobodies, F (ab') ₂ Fab', fab, fv, scFv, diabody and antibody minimal recognition unit.

scFv (sc = single chain), bispecific antibodies (diabodies).

The term "functional fragment" as used herein refers in particular to an antibody fragment having the same specificity for CA153 as the parent antibody. In addition to the above functional fragments, any fragment having an increased half-life is also included.

These functional fragments typically have the same binding specificity as the antibody from which they are derived. Those skilled in the art will conclude from the description of the invention that the antibody fragment of the invention can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by chemical reductive cleavage of disulfide bonds.

Antibody fragments can also be obtained by peptide synthesis by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers, such as those sold by Applied BioSystems and the like.

In addition to the amino acid sequences disclosed herein above, the framework regions may be derived from human species to constitute humanized antibodies.

In some embodiments, the binding protein further comprises an antibody constant region sequence.

In some embodiments, the constant region sequence is selected from the group consisting of sequences of any one of the constant regions IgG1, igG2, igG3, igG4, igA, igM, igE, igD.

In some embodiments, the species of the constant region is from a cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken scoop, or human.

In some embodiments, the constant region is murine;

the light chain constant region sequence is shown as SEQ ID NO 1;

the heavy chain constant region sequence is shown in SEQ ID NO. 2.

According to one aspect of the invention, the invention also relates to an isolated nucleic acid molecule, which is DNA or RNA, encoding a binding protein as described above.

According to one aspect of the invention, the invention also relates to a vector comprising a nucleic acid molecule as described above.

The invention further comprises at least one nuclear construct, e.g. a plasmid, further an expression plasmid, encoding a nucleic acid molecule as described above, the construction of which vector will be described in one embodiment of the present application.

According to one aspect of the invention, the invention also relates to a host cell transformed with a vector as described above.

The host cell may be a eukaryotic cell, such as a mammalian cell.

In some embodiments, the host cell is a CHO cell.

According to one aspect of the invention, the invention also relates to a method for producing a binding protein as described above, said method comprising the steps of:

the host cells as described above are cultured in a medium and under suitable culture conditions, and the binding protein so produced is recovered from the medium or from the cultured host cells.

According to one aspect of the invention, the invention also relates to the use of a binding protein as described above for the preparation of a diagnostic agent or kit for the diagnosis of breast cancer, lung cancer, ovarian cancer, lung adenocarcinoma or colorectal cancer.

According to one aspect of the invention, the invention also relates to a method of detecting CA153 protein in a test sample, comprising:

a) Contacting a CA153 protein in the test sample with a binding protein of claim above under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) Detecting the presence of said immune complex, the presence of said complex indicating the presence of said CA153 protein in said test sample;

in this embodiment, the binding protein may be labeled with an indicator that indicates the strength of the signal, so that the complex is readily detected.

In some embodiments, in step a), a second antibody is further included in the immune complex, the second antibody binding to the binding protein;

in this embodiment, the binding protein is in the form of a first antibody that forms a partner antibody with the second antibody for binding to a different epitope of CA 153;

the second antibody may be labeled with an indicator that indicates the intensity of the signal, so that the complex is easily detected.

In some embodiments, in step a), a second antibody is further included in the immune complex, which second antibody binds to the CA153 protein;

in this embodiment, the binding protein serves as an antigen for the second antibody, which may be labeled with an indicator of signal intensity to allow the complex to be readily detected.

In some embodiments, the indicator that indicates signal intensity comprises any one of a fluorescent substance, a quantum dot, a digoxigenin-labeled probe, biotin, a radioisotope, a radiocontrast agent, a paramagnetic ion fluorescent microsphere, an electron dense substance, a chemiluminescent label, an ultrasound contrast agent, a photosensitizer, colloidal gold, or an enzyme.

In some embodiments, the fluorescent species comprises Alexa 350, alexa 405, alexa 430, alexa 488, alexa 555, alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, 5-carboxy-2 ',4',5',7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, cascade Blue, cy2, cy3, cy5, cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), oregon Green 488, oregon Green 500, oregon Green514, pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, rhodamine, and mixtures thereof azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate or chelate, diamine, bispyanine glycoside, la Jolla Blue dye, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas red.

In some embodiments, the radioisotope comprises ¹¹⁰ In、 ¹¹¹ In、 ¹⁷⁷ Lu、 ¹⁸ F、 ⁵² Fe、 ⁶² Cu、 ⁶⁴ Cu、 ⁶⁷ Cu、 ⁶⁷ Ga、 ⁶⁸ Ga、 ⁸⁶ Y、 ⁹⁰ Y、 ⁸⁹ Zr、 ⁹⁴ mTc、 ⁹⁴ Tc、 ⁹⁹ mTc、 ¹²⁰ I、 ¹²³ I、 ¹²⁴ I、 ¹²⁵ I、 ¹³¹ I、 ^154-158 Gd、 ³² P、 ¹¹ C、 ¹³ N、 ¹⁵ O、 ¹⁸⁶ Re、 ¹⁸⁸ Re、 ⁵¹ Mn、 ⁵² mMn、 ⁵⁵ Co、 ⁷² As、 ⁷⁵ Br、 ⁷⁶ Br、 ⁸² mRb and ⁸³ sr.

In some embodiments, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase, and glucose oxidase.

In some embodiments, the fluorescent microspheres are: the polystyrene fluorescent microsphere is internally wrapped with rare earth fluorescent ion europium.

According to one aspect of the invention, the invention also relates to a kit comprising a binding protein as described above.

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are commercially available, and are not indicated by manufacturers.

Example 1

Restriction enzyme, prime Star DNA polymerase, was purchased from Takara in this example. MagExtractor-RNA extraction kit was purchased from TOYOBO. The SMARTERTM RACE cDNA Amplification Kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen corporation. Primer synthesis and gene sequencing were done by Invitrogen.

1.1 primers

Amplifying Heavy Chain and Light Chain 5' RACE primers:

SMARTER II A Oligonucleotide：

5’>AAGCAGTGGTATCAACGCAGAGTACXXXXX<3’；

5'-RACE CDS Primer(5'-CDS)：5’>(T) ₂₅ VN<3’(N＝A,C,G,orT；V＝A,G,orC)；

Universal Primer A Mix(UPM):

5’>CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT<3’

Nested Universal Primer A(NUP):

5’>AAGCAGTGGTATCAACGCAGAGT<3’

mIg-kR：5’>CTAACACTCATTCCTGTTGAAGCTCTTGACAAT<3’。

mIg-HR：5’>TCATTTACCAGGAGAGTGGGAGAGGC<3’。

1.2 cloning and sequencing of antibody variable region genes

Extracting RNA from hybridoma cell strain secreting Anti-CA 153D 2 monoclonal antibody by using MagExtractor-RNA extraction kit, and extracting RNA by using SMARTER ^TM The RACE cDNA Amplification Kit and SMARTER II A Oligonucleotide and 5' -CDS primer in the Kit are used for first strand cDNA synthesis, and the obtained first strand cDNA product is used as PCR Amplification template. The Light Chain gene was amplified with Universal Primer A Mix (UPM), nested Universal Primer A (NUP) and mIg-kR primers, and the Heavy Chain gene was amplified with Universal PrimerAmplification was performed with r A Mix (UPM), nested Universal Primer A (NUP) and mIg-HR primers. The primer pair of Light Chain amplifies about 0.7KB of target band, and the primer pair of Heavy Chain amplifies about 1.4KB of target band. The product was purified and recovered by agarose gel electrophoresis, and the product was subjected to A addition reaction with rTaq DNA polymerase, inserted into pMD-18T vector, transformed into DH 5. Alpha. Competent cells, and after colonies were grown, 4 clones of the Heavy Chain and Light Chain gene clones, respectively, were transferred to Invitrogen corporation for sequencing.

1.3 sequence analysis of Anti-CA 153D 2 antibody variable region genes

Putting the gene sequence obtained by sequencing in an IMGT antibody database for analysis, and analyzing by using VNTI11.5 software to determine that the genes amplified by the heavy Chain primer pair and the Light Chain primer pair are correct, wherein in the gene fragment amplified by the Light Chain, the VL gene sequence is 378bp, belongs to VkII gene family, and a leader peptide sequence of 57bp is arranged in front of the VL gene sequence; in the gene fragment amplified by the Heavy Chain primer pair, the VH gene sequence is 411bp, belongs to a VH1 gene family, and has a leader peptide sequence of 57bp in front.

1.4 construction of recombinant antibody expression plasmid

pcDNA ^TM 3.4

vector is a constructed recombinant antibody eukaryotic expression vector, and multiple cloning enzyme cutting sites such as HindIII, bamHI, ecoRI and the like are introduced into the expression vector and named as pcDNA3.4A expression vector, and the vector is called as 3.4A expression vector for short in the following; according to the sequencing result of the antibody gene in the pMD-18T, the Heavy Chain and Light Chain gene specific primers of the Anti-Dengue NS 1D 9 antibody are designed, wherein the two ends of the primers are respectively provided with HindIII and EcoRI enzyme cutting sites and protective bases, and the primers are as follows:

CA153-9D2-HF：5’>CCCAAGCTTATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATG<3’；

CA153-9D2-HR:5’>CACGAATTCTTACTAACACTCATTCCTGTTGAAGCTCTTGACAATG<3’；

CA153-9D2-LF:5’>CATGAAGCTTATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATG<3’；

CA153-9D2-LR:5’>CATGGAATTCTTACTAACACTCATTCCTGTTGAAGCTCTTGACA<3’；

a0.72 KB Light Chain gene fragment and a 1.4KB Heavy Chain gene fragment were amplified by PCR amplification. The gene fragments of the Heavy Chain and the Light Chain are subjected to double enzyme digestion by HindIII/EcoRI respectively, the 3.4A vector is subjected to double enzyme digestion by HindIII/EcoRI, the gene of the Heavy Chain and the gene of the Light Chain are respectively connected into the 3.4A expression vector after the fragments and the vector are purified and recovered, and recombinant expression plasmids of the Heavy Chain and the Light Chain are respectively obtained.

Example two

Recombinant antibody expression plasmid transient transfection CHO cell and expression supernatant antibody activity identification

Plasmid was diluted to 400ng/ml with ultrapure water and CHO cells were conditioned at 1.43X 10 ⁷ cells/ml in centrifuge tube, 100ul plasmid mixed with 700ul cells, transferred to electric rotor, transferred to 10ml of CD CHO AGT containing medium, cultured in 37 degree shaker (8% CO) ₂ Seismic amplitude 150); the cell viability was measured by sampling every day, and when the cell viability was less than 50%, the cell culture supernatant was centrifuged to obtain the antibody (having the light and heavy chains having the sequences shown in SEQ ID NOS: 3 and 4).

Upon analysis, the complementarity determining region (WT) of the heavy chain:

CDR-VH1 is K-A (X1) -S-D (X2) -Y-T-F-T-T (X3) -Y-D-I-N

CDR-VH2 is W-I-Y-P-G (X1) -D-G-S-I (X2) -K-F-N-D (X3) -K-F;

CDR-VH3 is A (X1) -R-E-G-P-N (X2) -Y-A-M-D-Y;

complementarity determining regions of the light chain:

CDR-VL1 is H-A-T (X1) -Q-N-L (X2) -N-V-W-L-S;

CDR-VL2 is Y-R (X1) -A-S-K (X2) -L-H-T-G;

CDR-VL3 is Q-Q-D (X1) -Q-S-S (X2) -P-R-T;

wherein X1, X2 and X3 are all the sites to be mutated.

TABLE 1 mutation sites related to antibody Activity

The inventors performed the above-described mutation of the CDR sites in WT to obtain a more active antibody.

Diluting goat anti-mouse IgG 1ug/ml with the coating solution to coat the microplate, wherein each well is 100uL, and the temperature is 4 ℃ overnight; the next day, washing with the washing solution for 2 times, and patting dry; add blocking solution (20% BSA +80% PBS), 120uL per well, 37 deg.C, 1h, pat dry; adding diluted CA153 monoclonal antibody, 100 uL/hole, 37 deg.C, 60min; throwing off liquid in the plate, patting dry, adding 20% of mouse negative blood, sealing each hole at 120ul,37 ℃ for 1h; throwing off liquid in the plate, patting dry, adding 100 times diluted CA153 antigen (the constant value of the CA153 in tumor ascites is 8.2 KU/ml), wherein each hole is 100uL,37 ℃ and 40min; washing with washing solution for 5 times, and drying; adding a CA153 monoclonal antibody (1; adding a developing solution A (50 uL/hole), adding a developing solution B (50 uL/hole), and carrying out 10min; adding stop solution at 50 uL/hole; OD readings were taken at 450nm (reference 630 nm) on the microplate reader.

TABLE 2 analysis of antibody Activity data

"-" indicates no activity.

As can be seen from Table 2, since the activity of mutation 1 was most effective, mutation sites with better potency were selected by using mutation 1 as the framework sequence (ensuring that the activity of the antibody obtained by the screening was similar to that of mutation 1, and the antibody activity was. + -. 10%), and some results are as follows.

TABLE 3 mutation sites related to antibody affinity

Affinity analysis of mutation sites

Using AMC sensors, purified antibodies were diluted to 10ug/ml with PBST, and CA153 ascites specimens (tumor abdomen CA153 fixed at 8.2 KU/ml) were diluted in PBST in a gradient: 400U/ml, 200U/ml, 100U/ml, 50U/ml, 25U/ml, 12.5U/ml, 6.25U/ml, 0U/ml;

the operation flow is as follows: equilibration for 60s in buffer 1 (PBST), immobilized antibody for 300s in antibody solution, incubation for 180s in buffer 2 (PBST), binding for 420s in antigen solution, dissociation for 1200s in buffer 2, sensor regeneration with 10mM GLY solution at pH 1.69 and buffer 3, and data output (KD represents the equilibrium dissociation constant, i.e. affinity; kon represents the association rate; kdis represents the dissociation rate. Calculations assume that 1U/ml of CA153 ascites specimens corresponds to 1 nmol/ml).

Table 4 affinity assay data

As can be seen from table 4, the mutation sites listed in table 3 have little effect on the affinity of the antibody.

To verify the above results, the above experiment was repeated using WT as a backbone sequence, and affinity verification of the mutation site was performed, and some results are as follows.

TABLE 5 mutations with WT as backbone

Table 6 affinity assay data

From the analyses in tables 5 and 6, the association between the mutation site and other sites was not significant on the premise that the antibody activity was ensured.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Dongguan City of Pengzhi Biotech Co., ltd

<120> recombinant antibody against human CA153 protein

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 107

<212> PRT

<213> Mus musculus

<400> 1

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

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Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

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Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 2

<211> 324

<212> PRT

<213> Mus musculus

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Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

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Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

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<213> Artificial sequence

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Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Asp Leu Gly

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Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Leu Asn Val Trp

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Leu Ser Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile

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Tyr Lys Ala Ser Asn Leu His Thr Gly Asp Pro Ser Arg Ile Ser Gly

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Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Arg Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Asp Gln Ser Tyr Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala

100 105 110

Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly

115 120 125

Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile

130 135 140

Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu

145 150 155 160

Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser

165 170 175

Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr

180 185 190

Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser

195 200 205

Phe Asn Arg Asn Glu Cys

210

<210> 4

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<213> DIQMNQSPSSLSADLGDTITITCHASQNLNVWLSWYQQKPGNIPKLLIYKASNLHTGDPSRISGSGSGTGFTLTIRSLQPEDIATYYCQQDQSYPRTFGGGTKLEIK

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Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Leu Val Lys Ile Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Thr Tyr

20 25 30

Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Tyr Pro Gly Asp Gly Ser Ile Lys Phe Asn Asp Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Gly Pro Asn Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro

115 120 125

Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly

130 135 140

Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn

145 150 155 160

Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr

180 185 190

Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser

195 200 205

Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro

210 215 220

Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro

225 230 235 240

Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys

245 250 255

Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp

260 265 270

Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met

290 295 300

His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser

305 310 315 320

Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly

325 330 335

Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln

340 345 350

Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe

355 360 365

Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu

370 375 380

Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe

385 390 395 400

Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn

405 410 415

Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr

420 425 430

Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

Claims

1. An isolated binding protein comprising an antigen binding domain, wherein the antigen is a CA153 protein, and wherein the antigen binding domain comprises complementarity determining region CDR-VH1, complementarity determining region CDR-VH2, complementarity determining region CDR-VH3, complementarity determining region CDR-VL1, complementarity determining region CDR-VL2, and complementarity determining region CDR-VL3;

CDR-VH1 is K-X1-S-X2-Y-T-F-T-X3-Y-D-I-N, wherein X3 is S;

CDR-VH2 is W-I-Y-P-X1-D-G-S-X2-K-F-N-X3-K-F, wherein X3 is E;

CDR-VH3 is X1-R-E-G-P-X2-Y-A-M-D-Y, wherein X1 is V;

CDR-VL1 is H-A-X1-Q-N-X2-N-V-W-L-S, wherein X1 is S;

the CDR-VL2 of the complementarity determining region is Y-X1-A-S-X2-L-H-T-G, wherein X1 is K;

CDR-VL3 is Q-Q-X1-Q-S-X2-P-R-T, wherein X2 is Y;

the mutation site of each complementarity determining region is selected from any one of the following combinations of mutations:

site of the body CDR-VH1 X1/X2 CDR-VH2 X1/X2 CDR-VH3 X2 CDR-VL1 X2 CDR-VL2 X2 CDR-VL3 X1 Mutation 1 A/D G/I N L K D Mutation 1-1 E/D R/I Q I N Y Mutations 1-2 D/D K/I G V K G Mutations 1 to 3 A/G G/L N L N Y Mutations 1 to 4 E/G R/L Q I K G Mutations 1-5 D/G K/L G V N D Mutations 1 to 6 A/D G/I N I K G Mutations 1 to 7 E/D R/I G V N D Mutations 1 to 8 D/D G/I Q L K G Mutations 1-9 A/G G/L Q V N D Mutations 1-10 E/G R/L G I K Y Mutations 1 to 11 D/G K/L N V N G Mutations 1 to 12 A/D G/I N I K Y Mutations 1-13 E/D R/L N L N G Mutations 1 to 14 D/D K/L G V K D Mutations 1-15 A/G R/L Q I N G Mutations 1-16 E/G K/I N V K D Mutations 1-17 D/G R/I N I N Y Mutations 1-18 A/D G/L G L K D Mutations 1-19 E/D K/L Q V N Y Mutations 1-20 D/D R/L N I K G Mutations 1-21 A/G K/I G L N Y Mutations 1-22 E/G G/I Q V K G Mutations 1-23 D/G R/I N I N D Mutations 1-24 A/D G/L Q V K G Mutations 1-25 E/D R/L N I N D Mutations 1-26 D/D K/I G L K Y Mutations 1-27 A/G G/I Q V N D Mutations 1-28 E/G R/I N I K Y Mutations 1-29 D/G K/L G L N G Mutations 1-30 A/D R/L G I K Y Mutations 1-31 E/D K/I Q L N G Mutations 1-32 D/D G/I N V K D Mutations 1-33 A/G R/I G I N G Mutations 1-34 E/G K/L Q L K D Mutations 1-35 D/G G/L N V N Y Mutations 1 to 36 A/D G/L Q L K D Mutations 1-37 E/D K/I N V N Y Mutations 1-38 D/D R/I G I K G Mutations 1-39 A/G K/L N L N Y Mutations 1-40 E/G G/L Q V K G Mutations 1-41 D/G R/L G I N D Mutations 1-42 A/D K/I N V K G Mutations 1-43 E/D G/I G I N D Mutations 1-44 D/D K/L Q L K Y Mutations 1-45 A/G G/L G V N D Mutations 1-46 E/G R/L Q I K Y Mutations 1-47 D/G K/I N L N G Mutations 1-48 A/D G/I Q I K Y Mutations 1-49 E/D K/L N L N G Mutations 1-50 D/D G/L G V K D Mutations 1-51 A/G R/L Q I K G Mutations 1-52 E/G K/I N L K D Mutations 1-53 D/G G/I G V N Y

。

2. An isolated binding protein comprising an antigen binding domain, wherein said antigen is a CA153 protein, and wherein said antigen binding domain comprises complementarity determining region CDR-VH1, complementarity determining region CDR-VH2, complementarity determining region CDR-VH3, complementarity determining region CDR-VL1, complementarity determining region CDR-VL2, and complementarity determining region CDR-VL3;

CDR-VH1 is K-X1-S-X2-Y-T-F-T-X3-Y-D-I-N, wherein X3 is T;

CDR-VH2 is W-I-Y-P-X1-D-G-S-X2-K-F-N-X3-K-F, wherein X3 is D;

CDR-VH3 is X1-R-E-G-P-X2-Y-A-M-D-Y, wherein X1 is A;

CDR-VL1 is H-A-X1-Q-N-X2-N-V-W-L-S, wherein X1 is T;

CDR-VL2 is Y-X1-A-S-X2-L-H-T-G, wherein X1 is R;

CDR-VL3 is Q-Q-X1-Q-S-X2-P-R-T, wherein X2 is S;

site of the body CDR-VH1 X1/X2 CDR-VH2 X1/X2 CDR-VH3 X2 CDR-VL1 X2 CDR-VL2 X2 CDR-VL3 X1 WT A/G K/I Q L K D WT 1-1 E/G G/I N V N Y WT 1-5 D/D R/L G I N D WT 1-10 E/D R/L Q I K Y WT 1-20 D/G R/L N I K G

。

3. The binding protein according to any one of claims 1 to 2, wherein the binding protein is F (ab') ₂ Fab', fab, fv, scFv and diabody.

4. The binding protein according to any one of claims 1 to 2, further comprising an antibody constant region sequence.

5. The binding protein according to claim 4, wherein said constant region sequence is selected from the group consisting of sequences of any one of the constant regions of IgG1, igG2, igG3, igG4, igA, igM, igE, and IgD.

6. The binding protein of claim 4, wherein the species of said constant region is from a bovine, equine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

7. The binding protein of claim 6, wherein the species source of said constant region is a bovine.

8. The binding protein of claim 6, wherein said species source of the constant region is turkey or turkey.

9. The binding protein according to claim 6, wherein said constant region is derived from a mouse.

10. The binding protein according to claim 9, wherein the light chain constant region sequence is set forth in SEQ ID NO 1;

the heavy chain constant region sequence is shown in SEQ ID NO 2.

11. An isolated nucleic acid molecule which is DNA or RNA and which encodes the binding protein of any one of claims 1 to 10.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A host cell transformed with the vector of claim 12.

14. A method of producing the binding protein of any one of claims 1 to 10, comprising the steps of:

culturing the host cell of claim 13 in a culture medium and under suitable culture conditions, and recovering the binding protein so produced from the culture medium or from the cultured host cell.

15. Use of the binding protein of any one of claims 1 to 10 in the preparation of a CA153 protein detection reagent.

16. Use of the binding protein of any one of claims 1 to 10 in the preparation of a kit for detecting CA153 protein in a test sample, comprising:

a) Contacting a CA153 protein in the test sample with a binding protein of any one of claims 1 to 10 under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex; and

b) Detecting the presence of said immune complex, the presence of said complex indicating the presence of said CA153 protein in said test sample.

17. The use of claim 16, wherein in step a) the immune complex further comprises a second antibody, said second antibody binding to said binding protein.

18. The use of claim 16, wherein the immune complex further comprises a second antibody that binds to the CA153 protein.

19. A kit comprising the binding protein of any one of claims 1 to 10.