CN112661845B - Affinity maturation binding protein bound with CXCR4 and application thereof - Google Patents

Abstract

The invention discloses an affinity maturation binding protein combined with CXCR4 and application thereof. The invention screens a human CXCR4 affinity maturation binding protein library to obtain the affinity maturation binding protein which is combined with CXCR 4. The binding protein is an aCX13C6 affinity maturation binding protein; or a binding protein formed by combining at least one of aCX16C1 affinity matured binding protein, aCX17D5 affinity matured binding protein, aCX20B2 affinity matured binding protein and aCX20E5 affinity matured binding protein with aCX13C6 affinity matured binding protein. The invention also discloses application of the affinity maturation binding protein combined with CXCR4 in preparing autoimmune diseases and antitumor drugs. The binding protein provided by the invention is humanized, has no immunogenicity in human body, and can be better used for development and clinical application of autoimmune diseases and anticancer drugs.

Description

Affinity matured binding protein that binds to CXCR4 and its application

technical field

The invention belongs to the field of binding proteins, and particularly relates to an affinity matured binding protein that binds to CXCR4 and its application.

Background technique

Malignant tumors have always been a problem that is difficult to overcome in medicine, and it is also the "number one killer" of human health. Therefore, the treatment of tumors has always been a hot and difficult research topic. The traditional treatment of tumors is mainly through the combination of radiotherapy, chemotherapy and surgery. Although these treatments can inhibit or eliminate tumor cells, they also have damage and toxic side effects on normal cells of the body. In recent years, targeted therapy of tumors has become a research hotspot. This therapy method binds specific proteins to tumor cells, which can kill tumor cells and minimize the damage to normal cells in the body.

Chemokine receptor 4 (CXCR4) is a 7-transmembrane glycoprotein composed of 352 amino acid residues, belonging to a G protein-coupled receptor with a size of about 40 kDa. CXCR4 has been found to be expressed in 23 different types of tumors, and it is the most common chemokine receptor expressed by tumor cells. The biological axis composed of CXCR4 and its ligand SDF-1 can mediate the migration, invasion, apoptosis, angiogenesis and proliferation of tumor cells by activating various signaling pathways. Based on the close relationship between the chemokine SDF-1/CXCR4 and tumors, the development of drugs to block the SDF-1/CXCR4 axis has attracted great attention. AMD3100, a CXCR4 antagonist, has entered clinical phase I and II in the treatment of sarcomas, gliomas and brain tumors. At the same time, anti-CXCR4 antibodies have been used for tumor treatment. MDX-1338 monoclonal antibody can block the binding of SDF-1 to CXCR4 and effectively inhibit tumor metastasis. It has been used for acute leukemia, non-Hodgkin lymphoma, multiple myeloid Clinical phase I treatment of tumor. Therefore, the use of CXCR4 as a target for cancer therapy has high medical value.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide an affinity-matured binding protein that binds to CXCR4.

Another object of the present invention is to provide the above-mentioned application of the affinity matured binding protein that binds to CXCR4.

The object of the present invention is achieved by the following technical solutions: an affinity matured binding protein that binds to CXCR4, which is aCX13C6 affinity matured binding protein; or aCX16C1 affinity matured binding protein, aCX17D5 affinity matured binding protein, aCX20B2 affinity matured binding protein and aCX20E5 A binding protein formed by combining at least one of the affinity-matured binding proteins with aCX13C6 affinity-matured binding proteins;

The aCX13C6 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:1 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:2;

The aCX16C1 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:3 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:4;

The aCX17D5 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:5 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:6;

The aCX20B2 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:7 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:8;

The aCX20E5 affinity maturation binding protein includes a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:9 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:10.

The nucleotide sequence encoding the heavy chain variable region of the aCX13C6 affinity maturation binding protein is shown in SEQ ID NO: 11.

The nucleotide sequence encoding the light chain variable region of the aCX13C6 affinity maturation binding protein is shown in SEQ ID NO: 12.

The nucleotide sequence encoding the heavy chain variable region of the aCX16C1 affinity maturation binding protein is shown in SEQ ID NO: 13.

The nucleotide sequence encoding the light chain variable region of the aCX16C1 affinity maturation binding protein is shown in SEQ ID NO: 14.

The nucleotide sequence encoding the heavy chain variable region of the aCX17D5 affinity maturation binding protein is shown in SEQ ID NO: 15.

The nucleotide sequence encoding the light chain variable region of the aCX17D5 affinity maturation binding protein is shown in SEQ ID NO: 16.

The nucleotide sequence encoding the heavy chain variable region of the aCX20B2 affinity maturation binding protein is shown in SEQ ID NO: 17.

The nucleotide sequence encoding the light chain variable region of the aCX20B2 affinity maturation binding protein is shown in SEQ ID NO: 18.

The nucleotide sequence encoding the heavy chain variable region of the aCX20E5 affinity maturation binding protein is shown in SEQ ID NO: 19.

The nucleotide sequence encoding the light chain variable region of the aCX20E5 affinity maturation binding protein is shown in SEQ ID NO: 20.

Since the biological activity of the affinity-matured binding protein is determined by the gene sequences specific for the hypervariable regions in the variable regions of the light and heavy chains of the antibody, the variable regions encoding the heavy chain and the variable regions provided above can be genetically engineered. The nucleotide sequences encoding the light chain variable regions are recombined to obtain different types of affinity matured binding proteins.

The affinity-matured binding protein that binds to CXCR4 is composed of a heavy chain variable region, a linking chain and a light chain variable region.

The amino acid sequence of the aCX13C6 affinity maturation binding protein is shown in SEQ ID NO:21.

The amino acid sequence of the aCX16C1 affinity matured binding protein is shown in SEQ ID NO:22.

The amino acid sequence of the aCX17D5 affinity matured binding protein is shown in SEQ ID NO:23.

The amino acid sequence of the aCX20B2 affinity maturation binding protein is shown in SEQ ID NO:24.

The amino acid sequence of the aCX20E5 affinity maturation binding protein is shown in SEQ ID NO:25.

The nucleotide sequence encoding the above-mentioned affinity maturation binding protein that binds to CXCR4 is the nucleotide sequence encoding the aCX13C6 affinity maturation binding protein; or the nucleotide sequence encoding the aCX16C1 affinity maturation binding protein, encoding At least one of the nucleotide sequence of the aCX17D5 affinity maturation binding protein, the nucleotide sequence encoding the aCX20B2 affinity maturation binding protein, and the nucleotide sequence encoding the aCX20E5 affinity maturation binding protein and the The nucleotide sequence formed by the combination of the nucleotide sequences of the aCX13C6 affinity maturation binding protein.

The nucleotide sequence encoding the aCX13C6 affinity matured binding protein is preferably as shown in SEQ ID NO:29.

The nucleotide sequence encoding the aCX16C1 affinity matured binding protein is preferably as shown in SEQ ID NO:30.

The nucleotide sequence encoding the aCX17D5 affinity matured binding protein is preferably as shown in SEQ ID NO:31.

The nucleotide sequence encoding the aCX20B2 affinity matured binding protein is preferably as shown in SEQ ID NO:32.

The nucleotide sequence encoding the aCX20E5 affinity matured binding protein is preferably as shown in SEQ ID NO:33.

The nucleotide sequences encoding the aCX13C6 affinity maturation binding protein, aCX16C1 affinity maturation binding protein, aCX17D5 affinity maturation binding protein, aCX20B2 affinity maturation binding protein and aCX20E5 affinity maturation binding protein consist of 774, 774, 774, 774 and 774, respectively. Base composition, the corresponding encoded amino acids are 258, 258, 258, 258 and 258, respectively.

The gene encoding the heavy chain variable region of the aCX13C6 affinity maturation binding protein is composed of 381 bases, encoding 127 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is GGSFSGYE; CDR2 is INHSGST ; CDR3 is AR.

The gene encoding the light chain variable region of the aCX13C6 affinity maturation binding protein is composed of 339 bases, encoding 113 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is QSLLHSNGYNY; CDR2 is LCS ; CDR3 is MQALQPP.

The gene encoding the heavy chain variable region of the aCX16C1 affinity maturation binding protein is composed of 381 bases, encoding 127 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is GGSFSGYY; CDR2 is INHSGST ; CDR3 is AR.

The gene encoding the light chain variable region of the aCX16C1 affinity maturation binding protein is composed of 339 bases, encoding 113 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS ; CDR3 is MQALQPP.

The gene encoding the heavy chain variable region of the aCX17D5 affinity maturation binding protein is composed of 381 bases, encoding 127 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is GGSFSGYY; CDR2 is INHSGST ; CDR3 is VR.

The gene encoding the light chain variable region of the aCX17D5 affinity maturation binding protein is composed of 339 bases, encoding 113 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS ; CDR3 is MQALQPP.

The gene encoding the heavy chain variable region of the aCX20B2 affinity maturation binding protein is composed of 381 bases, encoding 127 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is GGSFSGYE; CDR2 is INHSGST ; CDR3 is AR.

The gene encoding the light chain variable region of the aCX20B2 affinity maturation binding protein is composed of 339 bases, encoding 113 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS ; CDR3 is MQALQPP.

The gene encoding the heavy chain variable region of the aCX20E5 affinity maturation binding protein is composed of 381 bases, encoding 127 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is GGSFSGYY; CDR2 is INHSGST ; CDR3 is AR.

The gene encoding the light chain variable region of the aCX20E5 affinity maturation binding protein is composed of 339 bases, encoding 113 amino acids, and the variable region contains 3 CDR (complementary determinant) regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS ; CDR3 is MQALQPP.

The preparation method of the affinity-matured binding protein that binds to CXCR4 comprises the steps of: synthesizing a nucleotide sequence encoding the affinity-matured binding protein that binds to CXCR4, cloning it into an expression vector, and recombining the nucleotide sequence. The expression vector is then transferred into host cells for inclusion body expression and purification to obtain the affinity-matured binding protein that binds to CXCR4; or the affinity-matured binding protein that binds to CXCR4 is obtained by a protein synthesis method.

The application of the CXCR4 affinity maturation binding protein in the preparation of a medicament for treating diseases characterized by high CXCR4 expression.

The high expression of CXCR4 is characterized by autoimmune diseases and cancer.

Described tumor is CXCR4 high expression tumor, including pancreatic cancer, breast cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, colorectal cancer, prostate cancer, lung cancer, ovarian tumor, cervical cancer, uterine cancer, Liver, spleen, kidney and brain tumors.

Compared with the prior art, the present invention has the following advantages and effects:

1. The present invention uses phage display technology to screen the affinity matured binding proteins that interact with CXCR4 in the human phage affinity matured binding protein library, and the binding proteins of human proteins can be obtained without using antigens to immunize the human body.

2. The affinity-matured binding protein provided by the present invention is of human origin and has no immunogenicity in the human body, and can be better applied to the development of anti-tumor drugs.

3. The affinity matured binding protein provided by the present invention utilizes a highly expressed prokaryotic host for protein expression, which can significantly reduce the production cost of the affinity matured binding protein and promote the application of the affinity matured binding protein.

Description of drawings

Figure 1 is a graph showing the results of 5 rounds of library screening by polyclonal phage ELISA. PBS wells are blank controls, EGFR and EpCAM are irrelevant antigen controls, and CXCR4 wells are coated with synthetic CXCR4 polypeptide; the primary antibody is the polyclonal phage-binding protein obtained by amplification and purification after each round of library screening, and the secondary antibody is HRP-labeled antibody. Antibody to phage M13. The results showed that from the first round to the fifth round of screening the antibody library, the OD450 nm value obtained by screening the library with CXCR4 peptide increased round by round, indicating that the enrichment of CXCR4 antibody clones in the library screened with CXCR4 peptide in each round gradually increased. rise.

Fig. 2 is a graph showing the results of screening positive clones bound to CXCR4 polypeptide by monoclonal phage ELISA; wherein, A is a graph of representative clones of monoclonal phage ELISA, and is the ELISA result of 32 monoclonal phages, and the primary antibody is a monoclonal phage , the secondary antibody is anti-M13-HRP; B is the specificity result of detecting the binding of positive clones to CXCR4 polypeptide by monoclonal phage ELISA using multiple irrelevant antigens as controls. Figure 2B shows that the CXCR4 affinity matured binding protein phage clone is able to specifically bind to CXCR4-Fc and CXCR4 polypeptides.

Figure 3 is the SDS-PAGE electrophoresis image after expression and purification of the affinity matured binding protein; in which, A is aCX13C6, B is aCX16C1, C is aCX17D5, D is aCX20B2, and E is aCX20E5; in the figure, lane 1 is protein Marker, lane 2 is the uninduced cell protein, lane 3 is the induced cell protein, lane 4 is the supernatant after the induced cell fragmentation, swimming lane 5 is the precipitate after the induced cell fragmentation, and swimming lane 6 is the precipitate denaturation after the induced cell fragmentation Swimming lane 7 is the column liquid, lane 8 is the washing liquid, and lanes 9-13 are the target protein eluates 1-5.

Figure 4 is a graph showing the binding results of purified CXCR4 affinity matured binding protein and antigenic CXCR4 polypeptide by ELISA; wherein *p<0.05 relative to PBS; **p<0.01 relative to PBS (n=3). The results showed that compared with the wild-type binding protein aCX82, aCX13C6 and aCX20E5 had higher binding ability to CXCR4-Fc, and aCX20E5 had higher binding ability to CXCR4 polypeptide. In addition, the binding of the five affinity-matured binding proteins to unrelated antigens was not obvious, indicating that the five affinity-matured binding proteins could specifically bind to CXCR4.

Figure 5 is a graph showing the effect of MTT assay on the effect of affinity matured binding proteins on tumor cell proliferation; among them, A is DU145, B is PC-3, and C is MDA-MB-231; *p<0.05 relative to 0 μg/mL ; **p<0.01 vs. 0 μg/mL; ***p<0.001 vs. 0 μg/mL; ****p<0.0001 vs. 0 μg/mL (n=3). The results showed that for DU145 cells, compared with wild-type binding protein aCX82, aCX20E5 could more effectively inhibit the proliferation of tumor cells at a concentration of 50 μg/mL. For PC-3 cells, the five affinity-matured binding proteins had similar inhibitory effects on tumor cell proliferation compared to aCX82. For MDA-MB-231 cells, compared with aCX82, aCX20E5 inhibited tumor cell proliferation more effectively at 50 and 100 μg/mL concentrations.

Figure 6 shows the effect of affinity maturation binding protein on the apoptosis of tumor cells DU145, PC-3 and MDA-MB-231 and the ratio of promoting tumor cell apoptosis by flow cytometry and Annexin V/PI double staining kit. Histogram; among them, A is the flow cytometry chart of DU145 cells, B is the statistics chart of the apoptotic ratio of A, C is the flow cytometry chart of PC-3 cells, D is the statistics chart of the apoptosis ratio of C, E is the flow cytometry chart of MDA-MB-231 cells, F is the statistical chart of the apoptosis ratio of E; *p<0.05 relative to the control group without binding protein; **p<0.01 relative to the control group without binding protein; * **p<0.001 vs. no binding protein control; ****p<0.0001 vs. no binding protein control (n=3). The results showed that for DU145 and MDA-MB-231 cells, aCX13C6 and aCX20E5 had a higher ability to induce apoptosis compared with the wild-type binding protein aCX82. For PC-3 cells, CX20E5 has a high ability to induce apoptosis.

Figure 7 is a graph showing the effect of the affinity matured binding protein on tumor cell migration detected by Transwell migration assay and its absorbance value change trend chart; wherein, A is a photo of the Transwell migration assay to detect the migration of affinity matured binding protein to DU145, and B is A Analysis result picture, C is the photo of Transwell migration assay to detect the migration of affinity matured binding protein to PC-3, D is the analysis result of C, E is the photo of Transwell migration assay to detect the migration of affinity matured binding protein to MDA-MB-231 Figure, F is the analysis result of E; *p<0.05 versus 0 μg/mL; **p<0.01 versus 0 μg/mL; ***p<0.001 versus 0 μg/mL; ****p<0.0001 Relative to 0 μg/mL (n=3). The results showed that for DU145 cells, the five affinity-matured binding proteins had similar inhibitory effects on tumor cell migration compared to the wild-type binding protein aCX82. For PC-3 cells, compared with aCX82, aCX13C6, aCX20B2 and aCX20E5 were more effective in inhibiting tumor cell migration at a concentration of 50 μg/mL. For MDA-MB-231 cells, compared with aCX82, aCX20E5 could more effectively inhibit the migration of tumor cells at a concentration of 100 μg/mL.

Figure 8 is a graph showing the effect of the affinity matured binding protein on the invasion of tumor cells and the change of its absorbance value using the Transwell invasion assay; wherein, A is a photo of the experiment using the Transwell invasion assay to detect the invasion of the affinity matured binding protein on DU145 cells, and B is The analysis result of A, C is the experimental photo of the invasion of MDA-MB-231 cells by the affinity matured binding protein using the Transwell invasion assay, and D is the analysis result of C. The results showed that the five affinity-matured binding proteins could inhibit the invasion of DU145 and MDA-MB-231 cells, and with the increase of the concentration of the five affinity-matured binding proteins, the invasive ability of cancer cells could gradually decrease.

Figure 9 is a graph showing the effect of two affinity-matured binding proteins on tumor growth in a mouse model of DU145 prostate cancer cells using animal experiments; wherein, A is a graph of changes in tumor volume in mice after administration, and B is administration Picture of tumor size of mice in each group at the end of the cycle, C is the result of tumor weight detection of mice in each group after the end of the dosing cycle, D is the result of HE staining and immunohistochemistry of mouse tumor tissue, E is Graph of analysis results of D data using Image Pro-Plus software; *p<0.05 vs. PBS; **p<0.01 vs. PBS (n=4-6). The results showed that at the end of the dosing cycle (day 30), aCX20E5 had a better ability to inhibit tumor cell growth in vivo compared to the wild-type binding protein aCX82, according to the tumor volume map in Figure 9A. According to the tumor weight graph in Figure 9C, aCX13C6 and aCX20E5 had lower tumor weights compared to the wild-type binding protein aCX82. According to the integrated optical density results of immunohistochemical staining in Figure 9E, compared with the wild-type binding protein aCX82, aCX20E5 can more effectively inhibit tumor cell proliferation (Ki67) and tumor angiogenesis (CD31) in vivo. The two affinity-matured binding proteins had no significant effect on the induction of tumor cell apoptosis (C-caspase-3).

Detailed ways

The present invention will be described in further detail below with reference to specific embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1 Construction of phage affinity maturation binding protein library

(1) Using wild-type binding protein aCX82 as a template (the amino acid sequence encoding aCX82 binding protein is shown in SEQ ID NO: 26, and the nucleotide sequence encoding aCX82 binding protein is shown in SEQ ID NO: 34), according to GeneMorph II Random Mutagenesis Perform error-prone PCR according to the instructions of the Kit, and amplify the target gene by the method of error-prone PCR. The primers are Error-F2 (nucleotide sequence shown in SEQ ID NO: 37) and Error-R2 (nucleotide sequence shown in SEQ ID NO: 37) NO: 38).

(2) After the PCR reaction, use agarose gel electrophoresis to identify the error-prone PCR products.

(3) Perform gel recovery of error-prone PCR products according to the instructions of EZNA Gel Extraction Kit.

(4) The pComb3XSS phage display plasmid and the error-prone PCR product were subjected to double digestion reaction, and the digestion was performed at 50°C overnight.

(5) Use agarose gel electrophoresis to identify the digestion products.

(6) According to the instructions of EZNA Gel Extraction Kit, the gel recovery of the digested product was carried out.

(7) Use T4 DNA ligase to carry out the enzymatic ligation reaction at 4°C overnight to obtain the enzymatic ligation product.

(8) Transform the enzyme-linked product into electrotransformed competent cells. Immediately after electroporation, 1 mL of pre-cooled SOC medium was added, cultured at 37°C at 220 rpm for 1 h, and then spread on LB plates containing 100 μg/mL ampicillin, and cultured at 37°C overnight.

(9) Randomly pick single clones and carry out bacterial liquid PCR identification (the primers used are Error-F2 and Error-R2). After the PCR reaction, the PCR products of bacterial liquid were identified by agarose gel electrophoresis. The bacterial liquid with the correct position of the band (about 800bp) was sent for sequencing to determine whether the library was constructed successfully.

Example 2 Preparation of helper phage

(1) Three-district line TG1 E. coli clone strain was placed on a petri dish of TYE solid medium, and the streaked petri dish was placed upside down in a 37°C incubator for about 14 hours.

(2) Pick a single colony of TG1 on the petri dish and inoculate it into 5 mL of 2×TY liquid medium, and cultivate overnight at 37° C. and 250 rpm.

(3) Transfer the bacterial liquid obtained in step (2) into another 5 mL 2×TY liquid medium at a volume ratio of 1:100, and cultivate at 37° C. and 250 rpm until the bacterial liquid OD _600nm is about 0.5.

(4) The KM13 helper phage was serially diluted with PBS (10 ¹² pfu/mL to 10 ⁴ pfu/mL).

(5) Take 200 μL of the bacterial solution cultured in step (3), add 10 μL of the diluted KM13 helper phage, and place the mixture in a 37° C. water bath for 30 min to obtain mixture A.

(6) Mix low melting point agarose and 2×TY liquid medium, heat until completely melted, then incubate in water and cool to 42°C to obtain H-top medium, wherein the concentration of low melting point agarose is 1.5 %. Take 3 mL of the top medium and mix gently with the mixture A obtained in step (5), and then spread it onto a TYE solid culture plate preheated at 37°C. After the top medium was completely solidified, the culture dish was placed upside down in a 37°C incubator for overnight incubation.

(7) Pick a small plaque, inoculate it in 5 mL of TG1 bacterial solution (OD _600nm = 0.5), and cultivate for 2 hours at 37° C. and 250 rpm.

(8) Transfer the bacterial solution to another 500mL 2×TY liquid medium at a volume ratio of 1:100, and shake the bacteria at 37°C and 250rpm for 2h.

(9) kanamycin was added to a concentration of 50 μg/mL in the medium, and cultured at 30° C. and 250 rpm overnight.

(10) Centrifuge and filter the bacterial liquid to obtain a supernatant, then add a 20% w/vPEG-NaCl solution equivalent to 1/5 volume of the supernatant, mix well and distribute it in a 50 mL centrifuge tube to incubate on ice Centrifuge at 3200g for 1 h at 4°C for 30 min, discard the supernatant, resuspend the pellet with 1 mL of PBS buffer, centrifuge at 4°C and 3200g for 5 min, filter and sterilize the supernatant with a 0.45 μM filter to obtain helper phage.

Example 3 Amplification of phage affinity maturation binding protein library

(1) Thaw the bacterial library containing affinity-matured binding protein particles on ice, add 100 μL to 50 ml 2×TY medium (containing 100 μg/mL ampicillin+ and 1% w/v glucose), and incubate at 37°C, 220 rpm with shaking for 2 h , until OD _600nm =0.5.

(2) Then add 5×10 ¹¹ pfu KM13 helper phage, put it in a constant temperature water bath at 37° C. for 30 min. Centrifuge in a small high-speed centrifuge at 3200g for 10min. The pellet was gently resuspended in 2×TY liquid medium (containing 100 μg/mL Amp+ and 50 μg/mL Kana+), and cultured with shaking at 220 rpm for 20 h at 25°C.

(3) Centrifuge in a small high-speed centrifuge, centrifuge at 3200g for 20min, retain the supernatant solution, absorb 40ml of the supernatant solution, add 10ml of 20%w/v PEG-NaCl solution, mix gently, and ice bath for 2h.

(4) Centrifuge in a small high-speed centrifuge, 3200g for 30min, and retain the precipitate. The pellet was gently resuspended with a small amount of PBS buffer and centrifuged in a small high-speed centrifuge at 3200g for 10min. The obtained supernatant contains phage and can be directly used as a phage library for screening.

Example 4 Screening of CXCR4 affinity-matured binding proteins in a phage affinity-matured binding protein library

(1) The first round of screening: Coated antigen (CXCR4 polypeptide, purchased from Shanghai Botai Biotechnology Co., Ltd.), 4ml PBS buffer was added to the immune tube of the blank control group, and the same volume of antigen diluent was added to the immune tube of the experimental group (diluted with PBS buffer), the antigen concentration was 100 μg/ml, and it was placed in a refrigerator at 4°C overnight.

(2) The next day, rinse the immunotube with PBS buffer for a total of 3 times. Then, blocking solution (2% w/v BSA-PBS) was added along the tube wall and blocked at room temperature for 2 h.

(3) Rinse the immunotube with PBS buffer three times in total. (5×10 ¹² ) of the phage library (the phage-containing supernatant obtained in Example 3) was added, diluted with 2% w/v BSA-PBS, the volume was 4 ml, and it was allowed to stand at room temperature for 2 h.

(4) The first round of screening: Rinse the immunotube 10 times with PBST buffer. Screening Round 2 - Screening Round 5 Rinse the immunotube 20 times with PBST buffer. Each rinse needs to be done quickly to achieve the rinse effect.

(5) Use trypsin solution to elute the phage bound to the antigen with a concentration of 1 mg/ml and a volume of 500 μL, and continue to rotate and elute for 10 min.

(6) The phage eluate was added to the TG1 bacterial solution, placed in a constant temperature water bath at 37°C, and water bathed for 30 minutes.

(7) Gradient dilution of the bacterial solution, take 100 μL of the diluted bacterial solution and spread it on a TYE plate (containing 100 μg/mL Amp+ and 1% w/v glucose), and then cultivate it in an incubator at 37°C for ≥12 hours;

(8) Centrifuge the remaining bacterial liquid in a small high-speed centrifuge, centrifuge at 3200g for 10 minutes, gently resuspend the pellet with a small amount of 2×TY liquid medium, spread it on a TYE plate, and then culture it in an incubator at 37°C ≥ 12h.

(9) On the second day, collect the bacteria in the TYE plate of step (8): add 2 ml of 2×TY liquid medium (containing 15% glycerol), and scrape off all the colonies on the plate. 50 μL of bacterial liquid was added to 50 ml of 2×TY liquid medium (containing 100 μg/mLmp+ and 1% w/v glucose), and cultured with shaking at 37° C. and 220 rpm for 2 h until OD _600nm =0.5.

(10) Take 10 ml of bacterial solution, then add 5×10 ¹¹ pfu KM13 helper phage, put it in a constant temperature water bath at 37° C. for 30 min.

(11) Centrifuge in a small high-speed centrifuge at 4°C and 3200g for 10min. The pellet was gently resuspended in 2×TY liquid medium (containing 100 μg/mL Amp+ and 50 μg/mL Kana+), and cultured with shaking at 220 rpm for 20 h at 25°C.

(12) Centrifuge in a small high-speed centrifuge, centrifuge at 3200g for 20min, retain the supernatant solution, absorb 40ml of the supernatant solution, add 10ml of 20%w/v PEG-NaCl solution, mix gently, and ice bath for 2h.

(13) Centrifuge in a small high-speed centrifuge, 3200g for 30min, and retain the precipitate. The pellet was gently resuspended with a small amount of PBS buffer and centrifuged in a small high-speed centrifuge at 3200g for 10min. The obtained supernatant contains phage, which can be directly screened for the next round of phage library, or added with glycerol and stored at -80°C.

(14) Perform 5 rounds of screening in sequence, and perform according to the above steps (4) to (13). The antigen concentrations of the subsequent 4 rounds of screening were different. The antigen concentrations of the second and third rounds were both 50 μg/mL, and the antigen concentrations of the fourth and fifth rounds were both 25 μg/mL. The phages obtained in the previous round were screened in turn. .

Example 5 Polyclonal phage ELISA detects the enrichment results of 5 rounds of screening

(1) Coating antigen: NUNC 96-well microtiter plate was used in the experiment, and the blank control group was PBS, which was coated with PBS buffer, and the volume was 100 μL. The irrelevant antigen group was EGFR polypeptide (purchased by Shanghai Botai Biotechnology Co., Ltd.) and EpCAM polypeptide (purchased by Shanghai Botai Biotechnology Co., Ltd.), the concentration was 2 μg/mL, diluted with PBS buffer, 100 μL/well. The experimental group was CXCR4 polypeptide with a concentration of 2 μg/mL, diluted with PBS buffer, and the volume was 100 μL. Place the 96-well microtiter plate at 4°C overnight.

(2) Rinse the 96-well ELISA plate three times with PBS buffer, and shake dry the liquid on the ELISA plate each time. Then, 200 μL of 2% w/v BSA-PBS blocking solution was added, and the cells were blocked at 37° C. for 2 h.

(3) Rinse the 96-well ELISA plate three times with PBS buffer, and shake off the liquid on the ELISA plate each time. 100 μL of phage dilution solution (the phage obtained in 5 rounds was diluted into 2% BSA-PBS) was added to each well, and it was allowed to stand at room temperature for 1 h.

(4) Rinse the 96-well ELISA plate three times with PBST buffer, and shake off the liquid on the ELISA plate each time. 100 μL of anti-M13 antibody-HRP diluent (diluted anti-M13 antibody-HRP into 2% w/v BSA-PBS solution, the dilution ratio is 1:10000) was added to each well, and allowed to stand at room temperature for 1 h.

(5) Rinse the 96-well ELISA plate 3 times with PBST buffer, let stand for 2 min each time, and shake dry the liquid on the ELISA plate. Add 100 μL of TMB substrate chromogenic solution to each well, incubate in the dark for 5 min, add 50 μL of H ₂ SO ₄ at a concentration of 1 mol/L to stop the reaction, and measure the absorbance at OD _450nm .

(6) Figure 1 shows the results of 5 rounds of library screening by polyclonal phage ELISA. The phage affinity maturation binding protein library obtained by the 5th round of screening and amplification was selected for monoclonal phage ELISA. The results showed that from the first round to the fifth round of screening the antibody library, the OD _450nm value obtained by screening the library with CXCR4 peptide increased round by round, indicating that the enrichment of CXCR4 antibody clones in the library screened with CXCR4 peptide in each round gradually increased. raised.

Example 6 Picking monoclonal phage from the 5th round enriched library for ELISA validation

(1) Dip a small amount of Escherichia coli TG1 glycerol bacteria with an inoculating loop for streaking on the plate, and culture in an incubator at 37°C for ≥12 hours.

(2) Select a colony on a plate and inoculate it into 5 mL of 2×TY liquid medium (without antibiotics), and incubate with shaking overnight on a shaker at a temperature of 37° C. and a rotation speed of 220 rpm.

(3) The TG1 bacterial liquid cultured overnight was added to 2×TY liquid medium at a volume ratio of 10%, and cultured with shaking at 37° C. and 220 rpm for 2 hours.

(4) Dilute the phage gradient to 10 ^-8 , add it to the TG1 bacterial solution, and place it in a constant temperature water bath at 37°C for 30 minutes.

(5) Take a small amount of bacterial liquid and spread it on a TYE plate (containing 100 μg/mL Amp+ and 1% w/v glucose), and then cultivate it in an incubator at 37°C for ≥12 hours;

(6) Select monoclonal colonies from the plate and inoculate it into a 96-well culture plate, add 200 μL of 2×TY liquid medium (containing 100 μg/mL Amp+ and 1% w/v glucose) to each well, and cultivate overnight at 37° C. and 220 rpm with shaking.

(7) Draw 5 μL of bacterial liquid from each well of the 96-well plate, inoculate it into a new culture plate, and add 200 μL of 2×TY liquid medium (containing 100 μg/mL Amp+ and 1% w/v glucose) to each well, at 37°C, Shaking at 220rpm for 2h. The old culture plate was used to preserve glycerol bacteria, 30% glycerol was added to each well, and it was placed in a -80°C refrigerator.

(8) Add the bacterial solution from the 96-well plate to 1.5mL EP tubes one by one, add 50μL of KM13 auxiliary phage dilution solution (diluted with 2×TY liquid medium) to each 1.5mL EP tube, and place them in a constant temperature water bath at 37°C in the water bath for 30min.

(9) Centrifuge in a small high-speed centrifuge at 3200g for 10min. The pellet was gently resuspended in 2×TY liquid medium (containing 100 μg/mL Amp+ and 50 μg/mL Kana+), and cultured with shaking at 220 rpm for 20 h at 25°C.

(10) Centrifuge in a small high-speed centrifuge, centrifuge at 3200g for 10min, retain the supernatant, and store at 4°C.

(11) Coating antigen: NUNC 96-well microtiter plate was used in the experiment, and the blank control group was PBS, which was coated with PBS buffer, and the volume was 100 μL. The irrelevant antigen group was EpCAM polypeptide at a concentration of 2 μg/ml, diluted with PBS buffer, and the volume was 100 μL. The experimental group was CXCR4 polypeptide with a concentration of 2 μg/ml, diluted with PBS buffer, and the volume was 100 μL. Place the 96-well microtiter plate at 4°C overnight.

(12) Rinse the 96-well ELISA plate 3 times with PBS buffer, and shake dry the liquid on the ELISA plate each time. 200 μL of 2% BSA-PBS blocking solution was added, and the cells were blocked at 37° C. for 2 h.

(13) Rinse the 96-well ELISA plate three times with PBS buffer, and shake dry the liquid on the ELISA plate each time. Add 100 μL of monoclonal phage antibody to each well and let stand for 1 h at room temperature.

(14) Rinse the 96-well ELISA plate three times with PBST buffer, and shake dry the liquid on the ELISA plate each time. Add 100 μL of anti-M13-HRP conjugate (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., dilute anti-M13-HRP antibody into 2% BSA-PBS solution, the dilution ratio is 1:10000) to each well, and let stand for 1 h at room temperature .

(15) Rinse the 96-well ELISA plate 3 times with PBST buffer again, let stand for 2 min each time, and shake dry the liquid on the ELISA plate. Add 100 μL of TMB substrate chromogenic solution to each well, let stand in the dark for 5 min, stop the reaction with 50 μL of H ₂ SO ₄ at a concentration of 1 mol/L, and measure the absorbance at OD _450nm .

(16) 992 single clones were randomly selected in the experiment, and 75 positive clones were obtained. Figure 2A is a graph of representative clones for the monoclonal phage ELISA, ELISA results for 32 of the monoclonal phages. Figure 2B is a graph showing the binding of positive clones to CXCR4 detected by monoclonal phage ELISA using multiple irrelevant antigens as controls. Figure 2B shows that the CXCR4 affinity matured binding protein phage clone is able to specifically bind to CXCR4-Fc and CXCR4 polypeptides.

(17) Send positive clones to BGI for sequencing. The sequencing results were analyzed in DNAMAN, the same nucleotide sequence was excluded, and 5 different sequences were obtained, which were named aCX13C6, aCX16C1, aCX17D5, aCX20B2, aCX20E5, and the amino acid sequences were as SEQ ID NO: 21, SEQ ID NO: 22. Shown in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.

Example 7 Expression and purification of affinity matured binding proteins

(1) Expression of affinity matured binding protein inclusion bodies

1) BL21 (DE3) competent cells and the plasmid pET28a-sumo containing the target gene (the target gene is the protein obtained by screening in Example 6, the gene sequence encoding the protein is cloned into pET28a-sumo through HindIII and XhoI, and the recombinant Plasmid preparation was synthesized by Suzhou Jinweizhi Company) and thawed on ice. Pipette 1 μL pET28a-sumo plasmid into a 1.5 mL EP tube containing 100 μL BL21(DE3) competent cells, mix gently, and place on ice for 30 min. Then placed in a constant temperature water bath at 42°C, heat-shocked for 2 min, and finally placed on ice for 2 min. 900 μL of LB liquid medium was added, and the cells were cultured with shaking at 37° C. and 220 rpm for 1 h. Centrifuge at 12000g for 2min, remove 900μL of supernatant, resuspend the pellet gently with the remaining 100μL, spread on LB plate (containing 50μg/mL Kana+), and then incubate at 37°C for 12h.

2) Select a colony on a plate and inoculate it into 5 mL of LB liquid medium (containing 50 μg/mL Kana+), and cultivate overnight at 37°C and 220 rpm with shaking.

3) Inoculate 4ml of bacterial liquid into 400ml of LB liquid medium (containing 50μg/mL Kana+), shake and culture at 37°C and 220rpm for 2.5h, until the OD _600nm is 0.6-1.0, take 1mL of bacterial liquid for subsequent target protein SDS- Identification by PAGE gel electrophoresis (uninduced bacterial protein sample). The bacterial solution was added with IPTG (working concentration of 0.5 mM) to induce expression, and the expression was induced at 30°C and 220 rpm for 6 h. 1 mL of bacterial solution was taken for subsequent identification of target protein by SDS-PAGE gel electrophoresis (induced bacterial protein sample).

4) Centrifuge the bacterial liquid in a high-speed refrigerated centrifuge at 4°C and 5000 g for 5 min to retain the bacterial precipitate.

5) Resuspend the bacterial cell pellet in a 50 mL beaker with 25 mL of pre-cooled sterilization buffer, and then perform sonication. Ultrasonic crusher parameter setting: power 40%, work 4s, stop 8s, working time is 40min. After ultrasonic fragmentation, centrifuge in a high-speed refrigerated centrifuge, centrifuge at 15000g for 30 min at 4°C, and take 20 μL of supernatant for subsequent identification of target protein by SDS-PAGE gel electrophoresis (the supernatant sample of bacterial cell fragmentation after induction), and retain the precipitate. This precipitate is the inclusion body.

6) The inclusion body precipitate was resuspended with 30 mL of inclusion body washing solution A, added to a rotor, and stirred at 4° C. overnight to obtain a suspension. Centrifuge in a high-speed refrigerated centrifuge, centrifuge at 15000g for 30 min at 4°C, retain the precipitate, and take 20 μL for subsequent identification of the target protein by SDS-PAGE gel electrophoresis (bacterial fragmentation and sedimentation samples after induction).

7) Resuspend the inclusion body pellet with 30 mL of inclusion body washing solution B, and stir at 4°C for 1.5 h. Centrifuge in a high-speed refrigerated centrifuge, 4°C, 15000g for 30min, and retain the precipitate.

8) Resuspend the pellet with denaturing buffer and stir at room temperature for 2.5h. Centrifuge in a high-speed refrigerated centrifuge, centrifuge at 20000g for 30 min at 4°C, and collect the supernatant, which is the inclusion body dissolution and denaturation solution. Take 20 μL for the subsequent identification of the target protein by SDS-PAGE gel electrophoresis (the sample of the precipitated denaturation solution after the induced bacterial cell disruption).

(2) Purification of affinity matured binding proteins

1) Equilibrate the column: Take a piece of Ni-NTA His·Bind Resin purification resin, add 10 mL of denaturing buffer to pass through the column.

2) Sample loading: The inclusion body was dissolved and denatured solution was passed through the column, the target protein was adsorbed on the column, and 20 μL was taken for subsequent identification of the target protein by SDS-PAGE gel electrophoresis (the sample passed through the column).

3) Add another 10 mL of denaturing buffer to pass through the column, and wash the residual sample on the side wall of the column.

4) Add 20 mL of washing buffer to pass through the column, and take 20 μL for subsequent identification of target protein by SDS-PAGE gel electrophoresis (sample of washing liquid).

5) Add 10 mL of elution buffer to elute the target protein, collect it in 1.5 mL EP tubes, collect 1 mL of the column fluid in each tube, collect 5 tubes, and take 20 μL of each EP tube for subsequent SDS-PAGE coagulation of the target protein. Identification by gel electrophoresis (eluate sample).

6) Measure the protein concentration and purity of the collected eluate with Nanodrop 2000.

(3) Refolding of affinity matured binding proteins

1) Add the collected eluate to a 5kDa dialysis bag, and fasten both ends of the dialysis bag with rubber bands to prevent liquid leakage. A certain amount of air should be left in the dialysis bag to prevent the dialysis bag from bursting during dialysis.

2) Transfer the dialysis bag to the beaker, immerse the whole in the protein renaturation solution, and stir and dialyze at 4°C for 12 hours. Replace the renaturing solution and continue dialysis for 12h. The urea concentration in the protein renaturation solution was successively decreased from 8M to 0M, and dialyzed overnight in each urea concentration gradient renaturation solution.

3) After the dialysis, the samples were taken out, the protein concentration was detected and stored in a -80°C refrigerator.

(4) SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining

1) Prepare the separating glue with a concentration of 12% (w/v), add the prepared separating glue to the position about 2-3cm from the top of the glue filling mold, then add anhydrous ethanol to press the line, leave it for 20min, etc. The separating gel solidifies.

2) After pouring out the absolute ethanol, add 5% (w/v) stacking gel to the top, insert a comb, stand still for 30 minutes, wait for the stacking gel to solidify, remove the comb, and prepare for sample loading.

3) Put the prepared gel plate in the electrophoresis tank and add electrophoresis solution.

4) Add the protein sample collected in the above-mentioned affinity-matured binding protein expression and purification steps to 5× SDS-PAGE protein loading buffer to make the final concentration of the buffer 1×, and heat at 100° C. for 10 min to complete the sample preparation.

5) Load 5 μL of the prepared sample onto the gel plate of the electrophoresis tank, add protein marker, run the stacking gel at 80V, and then change to 120V to run the separation gel.

6) Take out the glue, add Coomassie brilliant blue staining solution to dye for 10 minutes, replace the staining solution with water, and cook in a microwave oven for 5 minutes with medium heat.

7) Add the decolorizing solution, place it on a decolorizing shaker to decolorize overnight, and change the decolorizing solution from time to time until a clear target band is seen, and take pictures for analysis. The results are shown in Figure 3: Figure 3A is the electropherogram of aCX13C6 affinity-matured binding protein, the protein molecular weight is about 45kDa; Figure 3B is the electrophoresis image of aCX16C1 affinity-matured binding protein, the protein molecular weight is about 45kDa; Figure 3C is aCX17D5 affinity-matured binding protein The electrophoresis image of the protein, the molecular weight of the protein is about 45kDa; Figure 3D is the electrophoresis image of the aCX20B2 affinity matured binding protein, the protein molecular weight is about 45kDa; Figure 3E is the electrophoresis image of the aCX20E5 affinity matured binding protein, the protein molecular weight is about 45kDa.

Example 8 Detecting the binding of purified CXCR4 affinity-matured binding protein to antigenic CXCR4 polypeptide by ELISA

(1) Coating antigen: NUNC 96-well microtiter plate was used in the experiment, and the blank control group was PBS, which was coated with PBS buffer, and the volume was 100 μL. Set multiple unrelated antigens (IFN, NGF, CD28, CD31, CSF1R, ICAM-1, EGFR-Fc, EpCAM-Fc and CXCR4-Fc, where IFN, NGF, CD28, CD31, CSF1R, ICAM-1, EGFR-Fc and EpCAM-Fc were purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., and CXCR4-Fc was purchased from Beijing Baipsis Biotechnology Co., Ltd.) as a specificity control at a concentration of 2 μg/ml, diluted with PBS buffer, and the volume was 100 μL . The experimental group was CXCR4 polypeptide at a concentration of 2 μg/ml, diluted with PBS buffer, and the volume was 100 μL.

(2) Rinse the 96-well ELISA plate three times with PBS buffer, and shake dry the liquid on the ELISA plate each time. Then, 200 μL of 2% w/v BSA-PBS blocking solution was added, and the cells were blocked at 37° C. for 2 h.

(3) Rinse the 96-well ELISA plate three times with PBS buffer, and shake off the liquid on the ELISA plate each time. 100 μL of antibody (concentration of 20 μg/mL) was added to the wells of a 96-well microtiter plate, and allowed to stand at room temperature for 1 h.

(4) Rinse the 96-well ELISA plate three times with PBST buffer, and shake off the liquid on the ELISA plate each time. 100 μL of anti-His-HRP (diluted with 2% w/v BSA-PBS solution, with a ratio of 1:5000) was added to the wells of a 96-well microtiter plate, and incubated at room temperature for 1 h.

(5) Rinse the 96-well ELISA plate 3 times with PBST buffer, let stand for 2 min each time, and shake dry the liquid on the ELISA plate. Add 100 μL of TMB substrate chromogenic solution to the 96-well ELISA plate, incubate in the dark for 10 min, add 1 mol/L H ₂ SO ₄ solution to the wells of the 96-well ELISA plate to stop the reaction, and measure the absorbance at OD _450nm . The results in Figure 4 show that, compared with the wild-type binding protein aCX82, aCX13C6 and aCX20E5 have higher binding ability to CXCR4-Fc, and aCX20E5 has higher binding ability to CXCR4 polypeptide. In addition, the binding of the five affinity-matured binding proteins to unrelated antigens was not obvious, indicating that the five affinity-matured binding proteins could specifically bind to CXCR4.

Example 9 Obtaining negative control binding protein

In the preliminary experiments of our laboratory, the phage display technology was used to use human epidermal growth factor receptor 2 (Her2) and vascular endothelial growth factor (VEGF) synthetic polypeptides as antigens. For clones that do not bind to the corresponding antigens, negative control binding proteins aHer2-13C1 and aVE201 were obtained respectively, which were used as negative controls. The negative control binding proteins aHer2-13C1 and aVE201 are respectively shown as SEQ ID NO:27 and SEQ ID NO:28 amino acid sequences; the nucleotide sequences encoding aHer2-13C1 and aVE201 are respectively as SEQ ID NO:35 and SEQ ID NO:36 shown.

Example 10 MTT assay was used to detect the effect of purified CXCR4 affinity maturation binding protein on tumor cell proliferation

(1) Collect tumor cells in logarithmic growth phase (human prostate cancer DU145 cells, human prostate cancer PC-3 cells and human breast cancer MDA-MB-231 cells), adjust the cell concentration to 2.5×10 ⁴ cells/ml, Seed into 96-well cell culture plates at 5000 cells per well. Cells were allowed to adhere by incubating overnight at 37°C, 5% _CO2 in a cell incubator.

(2) The old medium was then sucked away, DMEM medium containing 1% v/v FBS was added, and the culture medium was starved for 6 hours.

(3) Experimental grouping: 5 CXCR4 affinity maturation binding proteins (aCX13C6, aCX16C1, aCX17D5, aCX20B2 and aCX20E5), 1 positive control binding protein (wild-type binding protein aCX82) and 2 negative control binding proteins (aVE201 and aHer2- 13C1). Three kinds of cancer cells were co-treated with SDF-1 (working concentration of 100ng/ml, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) at different concentrations (0, 25, 50, 100 μg/mL). Incubate for 72h in a cell incubator with 5% CO ₂ .

(4) Add 30 μL of MTT solution to each well, and continue to culture for 4 h in a cell incubator at 37° C. and 5% CO ₂ .

(5) Take out the 96-well plate, aspirate the cell culture medium, add 200 μL of DMSO to each well, and place the 96-well cell culture plate on a shaker for 10 minutes, in order to dissolve the crystals, and finally measure the absorbance at OD570nm. Figure 5 shows that, for DU145 cells, compared with wild-type binding protein aCX82, aCX20E5 can more effectively inhibit the proliferation of tumor cells at a concentration of 50 μg/mL. For PC-3 cells, the five affinity-matured binding proteins had similar inhibitory effects on tumor cell proliferation compared to aCX82. For MDA-MB-231 cells, compared with aCX82, aCX20E5 inhibited tumor cell proliferation more effectively at 50 and 100 μg/mL concentrations.

Example 11 Detection of the effect of purified CXCR4 affinity maturation binding protein on tumor cell apoptosis by flow cytometry and Annexin V/PI double staining kit

(1) Collect tumor cells DU145, PC-3 and MDA-MB-231 in logarithmic growth phase, adjust the cell concentration to 2.5×10 ⁵ cells/ml, and inoculate them into 6-well cell culture plates, 2.5×10 ⁵ per well cells. Cells were allowed to adhere by incubating overnight at 37°C, 5% _CO2 in a cell incubator.

(2) The old medium was then sucked away, DMEM medium containing 1% v/v FBS was added, and the culture medium was starved for 6 hours.

(3) Experimental grouping: no binding protein control group, 5 CXCR4 affinity matured binding proteins (aCX13C6, aCX16C1, aCX17D5, aCX20B2 and aCX20E5), 1 positive control binding protein (wild-type binding protein aCX82) and 2 negative control binding proteins proteins (aVE201 and aHer2-13C1). Three cancer cells were co-treated with SDF-1 (working concentration of 100 ng/ml, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) at a concentration of 50 μg/mL, and the cells were cultured at 37 °C, 5% CO ₂ . Incubate for 48h.

(4) Aspirate the old medium, wash the cells with PBS buffer, aspirate the PBS buffer, and add trypsin to digest the cells. Digestion was terminated by the addition of 10% v/v FBS in DMEM medium. The cells were gently pipetted, the cell suspension was collected, centrifuged at 1024 g for 5 min, and the pellet was retained.

(5) Wash the cells again with PBS buffer, centrifuge at 1024g for 5 min, and retain the pellet. Cells were washed 2 times consecutively.

(6) Add 195 μL of 1× binding buffer to gently resuspend the cells, add 5 μL of Annexin V-FITC, and incubate at room temperature for 15 minutes in the dark.

(7) Add 200 μL of 1× binding buffer to gently resuspend the cells, centrifuge in a small high-speed centrifuge, and centrifuge at 4°C and 1000 g for 5 min to retain the pellet.

(8) Add 190 μL of 1× binding buffer to gently resuspend the cells, add 10 μL of PI, and mix gently.

(9) Use flow cytometry to detect cell apoptosis. The results in Figure 6 show that, for DU145 and MDA-MB-231 cells, aCX13C6 and aCX20E5 have a higher ability to induce apoptosis compared with the wild-type binding protein aCX82. For PC-3 cells, CX20E5 has a high ability to induce apoptosis.

Example 12 Transwell migration assay was used to detect the effect of purified CXCR4 affinity maturation binding protein on tumor cell migration

(1) Collect tumor cells DU145, PC-3 and MDA-MB-231 in logarithmic growth phase, add DMEM medium containing 1% v/vFBS, and culture for 6 h under starvation. The cells were digested, centrifuged and resuspended in DMEM medium containing 1% v/v FBS.

(2) Experimental grouping: 5 CXCR4 affinity maturation binding proteins (aCX13C6, aCX16C1, aCX17D5, aCX20B2 and aCX20E5), 1 positive control binding protein (wild-type binding protein aCX82) and 2 negative control binding proteins (aVE201 and aHer2- 13C1). 200 μL (5×10 ⁴ ) of cell suspensions were co-treated with different concentrations (0, 25, 50, 100 μg/mL) and SDF-1 (the working concentration was 100 ng/ml, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.). cells, DMEM medium containing 1% v/v FBS), were added to the upper chamber of the Transwell chamber. The DMEM medium with 20% 1% v/v FBS was added to the lower chamber, and cultured in an incubator at 37°C and 5% CO ₂ for 24 h.

(3) Take out the Transwell chamber, suck out the cell culture medium in the upper chamber and the lower chamber respectively, and gently wipe off the cells that have not migrated in the upper chamber with a cotton swab. The lower chamber was fixed with 4% w/v paraformaldehyde, the volume was 500 μL, and the fixation time was 10 min. Then, 4% paraformaldehyde was aspirated, and the cells were stained with 0.1% w/v crystal violet staining solution in a volume of 500 μL for 30 min. The Transwell chambers were washed with ultrapure water, each chamber was washed three times, and the chambers were placed under a microscope for observation and photography. Add 33% acetic acid solution to each well for elution, the volume is 100 μL, put it on a shaker for rapid shaking, and finally measure the absorbance at OD _570nm . The results in Figure 7 show that, for DU145 cells, the five affinity-matured binding proteins have similar inhibitory effects on tumor cell migration compared to the wild-type binding protein aCX82. For PC-3 cells, compared with aCX82, aCX13C6, aCX20B2 and aCX20E5 were more effective in inhibiting tumor cell migration at a concentration of 50 μg/mL. For MDA-MB-231 cells, compared with aCX82, aCX20E5 could more effectively inhibit the migration of tumor cells at a concentration of 100 μg/mL.

Example 13 Transwell invasion assay was used to detect the effect of purified CXCR4 affinity maturation binding protein on tumor cell invasion

The method steps are the same as the Transwell migration experiment in Example 12, except that the step of laying Matrigel matrigel is added. The inner surface of a Transwell chamber (8 μm pore size, 24-well plate) was coated with Matrigel 50 μg/well, and put into a cell culture incubator to form a gel for 12 h. The results in Figure 8 show that the five affinity-matured binding proteins can inhibit the invasion of DU145 and MDA-MB-231 cells. With the increase of the concentration of the five affinity-matured binding proteins, the invasion ability of cancer cells can be gradually reduced.

Example 14 Effect of CXCR4 affinity-matured binding protein on mouse prostate cancer model

(1) According to the in vitro functional experiment results of the CXCR4 affinity matured binding protein, a mouse DU145 cell prostate cancer model was selected, and aCX13C6 affinity matured binding protein and aCX20E5 affinity matured binding protein were selected for animal experiments.

(2) Subculture DU145 cells, when the cells grow to log phase, trypsinize and collect the cells, after washing the cells with PBS, add an appropriate amount of PBS to adjust the cell density to 5×10 ⁷ /mL.

(3) Aspirate 100 μL of the cell suspension, and inject the suspension into the armpit of mice (4-week-old male Balb/C nude mice, purchased from Beijing Huafukang Biotechnology Co., Ltd.) by subcutaneous injection to create tumors. Mice were kept in SPF animal laboratory.

(4) The mouse tumor volume was measured every three days, and the longest side (length) and the shortest side (width) of the mouse tumor were measured. According to the formula tumor volume = length × width ² × 0.5.

(5) When the tumor volume of 36 mice increased to an average of about 100 mm ³ , they were randomly divided into 6 groups with 6 mice in each group. Group 1 was blank control group (PBS), group 1 was negative control binding protein group (aVE201), group 2 was positive control group (DDP and aCX82), and group 2 was experimental affinity mature binding protein group (aCX13C6 and aCX20E5). Administer via tail vein injection using an insulin syringe. The dose of each mouse in the experimental affinity matured binding protein group and the negative control binding protein group was 10 mg/kg, the dose of each mouse in the DDP group was 2 mg/kg, and the blank control group was injected with 100 μL PBS buffer. liquid. The dosing cycle was 30 days, and the mice were administered once every 3 days and the tumor volume and body weight of the mice were recorded.

(6) After the experiment, the mice were sacrificed, the tumors of each group of mice were excised, the tumor weight was measured and photographed, and then the tumor tissue was subjected to H&E staining and immunohistochemical analysis. According to the tumor volume graph in Figure 9A, aCX20E5 had a better ability to inhibit tumor cell growth in vivo at the end of the dosing cycle (day 30) compared to the wild-type binding protein aCX82. According to the tumor size photo map in Figure 9B, the tumor volume of the aCX13C6 and aCX20E5 groups was slightly smaller than that of the wild-type binding protein aCX82 group. According to the tumor weight graph in Figure 9C, aCX13C6 and aCX20E5 had lower tumor weights compared to the wild-type binding protein aCX82. According to the results of HE staining and immunohistochemistry in Figure 9D, no obvious histopathological abnormalities were found in the aCX13C6 and aCX20E5 groups. According to the integrated optical density results of immunohistochemical staining in Figure 9E, compared with the wild-type binding protein aCX82, aCX20E5 can more effectively inhibit tumor cell proliferation (Ki67) and tumor angiogenesis (CD31) in vivo. The two affinity-matured binding proteins had no significant effect on the induction of tumor cell apoptosis (C-caspase-3).

The configuration method of the reagents used in the embodiment is as follows:

(1) LB solid medium: 2 g of peptone, 1 g of yeast powder, 2 g of NaCl, and 4 g of agar powder.

Measure the above reagents on an electronic balance, dissolve them in 200 mL of ultrapure water, mix well, and then pack them, and sterilize at 121°C for 20 minutes under high temperature and high pressure. When the temperature of the medium drops to about 50 °C, add the corresponding antibiotics, the working concentration of ampicillin is 100 μg/ml, and the working concentration of kanamycin is 50 μg/ml, mix gently, and pour the plate. Let stand for a period of time, and after the plate is solidified, wrap the plate and store it in a 4°C refrigerator temporarily.

(2) LB liquid medium: 1 g of NaCl, 1 g of peptone, and 0.5 g of yeast powder.

Measure the above reagents on an electronic balance, dissolve them in 100 mL of ultrapure water, mix gently, and then dispense them into test tubes, and sterilize at 121°C for 20 minutes under high temperature and high pressure.

(3) TYE solid medium: peptone 5g, yeast powder 2.5g, NaCl 4g, agar powder 6g.

Measure the above reagents on an electronic balance, dissolve them in 400 mL of ultrapure water, mix gently, and then dispense them, and sterilize at 121°C for 20 minutes under high temperature and high pressure. When the temperature of the medium drops to about 50°C, add ampicillin and a final concentration of 1% glucose solution, mix gently, and pour the plate. Let stand for a period of time, and after the plate is solidified, wrap the plate and store it in a 4°C refrigerator temporarily.

(4) 2×TY medium: 8 g of peptone, 5 g of yeast powder, and 2.5 g of NaCl.

Measure the above reagents on an electronic balance, dissolve them into 500 mL of ultrapure water, mix gently, and then dispense them, and sterilize at 121°C for 20 minutes. Store in a 4°C refrigerator temporarily.

(5) PBS buffer: Na ₂ HPO ₄ ·12H ₂ O 4.5 g, NaCl 4 g, KH ₂ PO ₄ 0.12 g, KCl 0.1 g.

Measure the above reagents on an electronic balance, dissolve them into 300mL of ultrapure water, adjust the pH to 7.4, and continue to add ultrapure water to make up to 500mL. Sterilize at high temperature and autoclave at 121°C for 20min. Store in a 4°C refrigerator temporarily.

(6) PBST: 0.5mL of Tween-20 was added to 500mL of PBS buffer and mixed gently. Sterilize at 121 °C for 20 min at high temperature and autoclave, and store in a 4 °C refrigerator temporarily.

(7) Kanamycin (storage concentration is 50 mg/mL): Measure 0.5 g of kanamycin powder on an electronic balance, dissolve it in 10 mL of ultrapure water, mix and dissolve, filter and sterilize, and put it in - Temporary storage in a 20°C refrigerator.

(8) Ampicillin (storage concentration is 100mg/mL): Measure 0.5g of ampicillin powder on an electronic balance, dissolve it in 5mL of ultrapure water, mix and dissolve, filter and sterilize, and put it in a -20℃ refrigerator for a while live.

(9) Bacteria breaking buffer: NaCl 8.77g, Tris base 6.06g, 0.5M EDTA 4ml, PMSF (100×) 10ml.

Measure the above reagents on an electronic balance, dissolve them into 800 mL of ultrapure water, adjust the pH to 7.5, and continue to add ultrapure water to make the volume to 1L. Store in a 4°C refrigerator temporarily.

(10) Inclusion body washing solution A: NaCl 1.76g, Tris base 1.22g, Triton X-100 4mL, 0.5M EDTA 0.8mL.

Measure the above reagents on an electronic balance, dissolve them in 180 mL of ultrapure water, mix gently and then dispense, add ultrapure water to dilute the solution to 200 mL, and store it in a 4°C refrigerator temporarily.

(11) Inclusion body washing solution B: NaCl 1.76 g, Tris base 1.22 g, Triton X-100 4 mL, PMSF (100×) 2 mL.

Measure the above reagents on an electronic balance, dissolve them in 180 mL of ultrapure water, mix gently and then dispense, add ultrapure water to dilute the solution to 200 mL, and store it in a 4°C refrigerator temporarily.

(12) Inclusion body denaturation buffer: 240.24 g of urea, 7.3 g of NaCl, 1.21 g of Tris base, 5 mL of PMSF (100×), 0.345 mL of 2-mercaptoethanol (2-Hydroxy-1-ethanethiol).

Measure the above reagents on the electronic balance, dissolve them into 300mL of ultrapure water, adjust the pH to 7.5, and continue to add ultrapure water to make up the volume to 500mL. Store in a 4°C refrigerator temporarily.

(13) Washing buffer: Measure 0.68g of imidazole powder on an electronic balance, dissolve it into 5ml of inclusion body denaturation buffer, and after mixing, pipette 200 μL into 19.8ml of denaturation buffer, that is, wash buffer.

(14) Elution buffer: Measure 0.68g of imidazole powder on an electronic balance, dissolve it into 5ml of inclusion body denaturation buffer, and after mixing, add 1ml to 9ml of denaturation buffer, that is, elution buffer.

(15) 1mol/L Tris-HCl: Measure 0.1g of Tris-base powder on an electronic balance, dissolve it into 900mL of ultrapure water, mix gently, adjust the pH to 8.8 or 6.8, add ultrapure water to make the solution constant to 1L. Store in a 4°C refrigerator temporarily.

(16) 10% APS solution: Measure 2g of APS powder on an electronic balance, dissolve it into 20mL of ultrapure water, mix gently and then dispense. Store in -20°C refrigerator temporarily.

(17) 5×SDS-PAGE running buffer: 94 g of glycine, 30.2 g of Tris base, and 5 g of SDS.

Measure the above reagents on an electronic balance, dissolve them into 900 mL of ultrapure water, mix gently, add ultrapure water to make the solution 1L, and store it in a 4°C refrigerator temporarily.

(18) Coomassie brilliant blue staining solution: 0.5 g of Coomassie brilliant blue, 125 mL of isopropanol, and 50 mL of acetic acid.

Measure the above reagents on an electronic balance, dissolve them in 300mL of ultrapure water, mix gently, add ultrapure water to make the solution volume to 500mL, and store at room temperature temporarily.

(19) SDS-PAGE destaining solution: 50 mL of acetic acid and 25 mL of ethanol.

Measure the above reagents on an electronic balance, dissolve them in 300mL of ultrapure water, mix gently, add ultrapure water to make the solution volume to 500mL, and store at room temperature temporarily.

(20) 5× protein loading buffer: SDS 2.5g, bromophenol blue 0.12g, glycerol 12.5mL, 1mol/L Tris-HCl 6.25mL, β-mercaptoethanol 0.5mL.

Measure the above reagents on an electronic balance, dissolve them in 15mL of ultrapure water, mix gently, add ultrapure water to make the solution volume to 25mL, and store in a 4°C refrigerator temporarily. β-Mercaptoethanol was added just before use.

(21) IPTG: Measure 1.2 g of IPTG powder on an electronic balance, dissolve it into 10 mL of sterile water, mix well, filter sterilize, and temporarily store it in a -20°C refrigerator.

(22) 1mol/LH ₂ SO ₄ : slowly add 5.6 ml of 98% concentrated sulfuric acid to 94.4 ml of ultrapure water, stir slowly with a glass rod, and store the solution temporarily at room temperature after cooling to room temperature.

(23) PMSF (100×): Measure 0.87 g PMSF powder on an electronic balance, dissolve it into 50 mL of isopropanol, and temporarily store it in a -20°C refrigerator.

(24) 2% BSA-PBS solution: weigh 2 g of BSA powder on an electronic balance, dissolve it into 100 ml of PBS buffer, filter and sterilize it, and put it in a 4°C refrigerator for temporary storage.

(25) 30% glycerin: Measure 30ml of glycerol with a graduated cylinder, add 70ml of ultrapure water and mix well, sterilize at 121°C for 20 minutes under high temperature and high pressure, and store in a 4°C refrigerator temporarily.

(26) PEG-NaCl: Measure 100g PEG-6000 and 73g NaCl on an electronic balance, dissolve in 500ml ultrapure water, sterilize at 121°C for 20min under high temperature and high pressure, and store at room temperature temporarily.

(27) Protein renaturation solution: measure 8.383g NaCl, 2.42g Tris base, 0.6g oxidized glutathione and 0.8g reduced glutathione on an electronic balance, and dissolve the above reagents into 400mL of ultrapure water , and then add a certain amount of urea (8M/6M/4M/2M/1M/0.5M/0.25M/0M), after fully mixing, use dilute hydrochloric acid to adjust the pH of the solution to 7.5, and add ultrapure water to dilute the solution to 1L, temporarily stored in a 4°C refrigerator.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

<110> Jinan University

<120> Affinity mature binding protein binding to CXCR4 and its application

<160> 38

<170> SIPOSequenceListing 1.0

<223> Amino acid sequence of heavy chain variable region of aCX13C6 affinity maturation binding protein

<400> 1

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Glu Trp Ser Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<223> Amino acid sequence of light chain variable region of aCX13C6 affinity maturation binding protein

<400> 2

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Cys Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Ala

<223> Amino acid sequence of heavy chain variable region of aCX16C1 affinity maturation binding protein

<400> 3

Gln Val Gln Leu Gln His Trp Gly Ala Gly Leu Leu Lys Pro Glu Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Asp Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<223> Amino acid sequence of light chain variable region of aCX16C1 affinity maturation binding protein

<400> 4

Asp Ile Val Met Thr Gln Ser Pro Val Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

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

35 40 45

Thr Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Ala

<223> Amino acid sequence of heavy chain variable region of aCX17D5 affinity matured binding protein

<400> 5

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<223> Amino acid sequence of light chain variable region of aCX17D5 affinity matured binding protein

<400> 6

Asp Ile Val Met Thr Leu Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Cys Gln Ser Leu Leu His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Ala

<223> Amino acid sequence of heavy chain variable region of aCX20B2 affinity maturation binding protein

<400> 7

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Glu Trp Ser Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asp Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<223> Amino acid sequence of light chain variable region of aCX20B2 affinity maturation binding protein

<400> 8

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Ala

<223> Amino acid sequence of heavy chain variable region of aCX20E5 affinity matured binding protein

<400> 9

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asn Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<223> Amino acid sequence of light chain variable region of aCX20E5 affinity matured binding protein

<400> 10

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

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

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Ala

<223> Nucleotide sequence encoding heavy chain variable region of aCX13C6 affinity maturation binding protein

<400> 11

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttacgagt ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc a 381

<223> Nucleotide sequence encoding light chain variable region of aCX13C6 affinity maturation binding protein

<400> 12

gatattgtga tgacacagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttgcattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgtgttc tcatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggatt tattactgca tgcaagctct acaacctccg 300

ctcactttcg gcggagggac caagctggag atcaaagca 339

<223> Nucleotide sequence encoding heavy chain variable region of aCX16C1 affinity maturation binding protein

<400> 13

caggtgcagc tacagcattg gggcgcagga ctgttgaagc ctgaggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg ggaccgtggc 360

accctggtca ctgtctcctc a 381

<223> Nucleotide sequence encoding light chain variable region of aCX16C1 affinity matured binding protein

<400> 14

gatattgtga tgacacagtc tccagtctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttgcattgg 120

tacctgcaga agccagggca gtctacacag ctcctgatct atttgggttc tcatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggatt tattactgca tgcaagctct acaacctccg 300

ctcactttcg gcggagggac caagctggag atcaaagca 339

<223> Nucleotide sequence encoding heavy chain variable region of aCX17D5 affinity maturation binding protein

<400> 15

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatg actgtgtgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc a 381

<223> Nucleotide sequence encoding light chain variable region of aCX17D5 affinity maturation binding protein

<400> 16

gatattgtga tgacactgtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtcttgtca gagcctcctg catagtaatg gatacaacta tttgcattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc tcatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggatt tattactgca tgcaagctct acaacctccg 300

ctcactttcg gcggagggac caagctggag atcaaagca 339

<223> Nucleotide sequence encoding heavy chain variable region of aCX20B2 affinity maturation binding protein

<400> 17

caggtgcagc tacagcattg gggcgcagga gtgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttacgagt ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac cgactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc a 381

<223> Nucleotide sequence encoding light chain variable region of aCX20B2 affinity maturation binding protein

<400> 18

gatattgtga tgacacagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttgcattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc tcatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggatt tattactgca tgcaagctct acaacctccg 300

ctcactttcg gcggagggac caagctggag atcaaagca 339

<223> Nucleotide sequence encoding heavy chain variable region of aCX20E5 affinity maturation binding protein

<400> 19

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagag cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

aactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc a 381

<223> Nucleotide sequence encoding the light chain variable region of aCX20E5 affinity-matured binding protein

<400> 20

gatattgtga tgacacagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttgcattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc tcatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggatt tattactgca tgcaagctct acaacctccg 300

ctcactttcg gcggagggac caagctggag atcaaagca 339

<223> Amino acid sequence of aCX13C6 affinity-matured binding protein

<400> 21

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Glu Trp Ser Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg

130 135 140

Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

145 150 155 160

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

165 170 175

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

180 185 190

Ser Pro Gln Leu Leu Ile Tyr Leu Cys Ser His Arg Ala Ser Gly Val

195 200 205

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

210 215 220

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

225 230 235 240

Ala Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ala

<223> Amino acid sequence of aCX16C1 affinity matured binding protein

<400> 22

Gln Val Gln Leu Gln His Trp Gly Ala Gly Leu Leu Lys Pro Glu Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Asp Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg

130 135 140

Gln Asp Ile Val Met Thr Gln Ser Pro Val Ser Leu Pro Val Thr Pro

145 150 155 160

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

165 170 175

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

180 185 190

Ser Thr Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val

195 200 205

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

210 215 220

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

225 230 235 240

Ala Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ala

<223> Amino acid sequence of aCX17D5 affinity matured binding protein

<400> 23

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg

130 135 140

Gln Asp Ile Val Met Thr Leu Ser Pro Leu Ser Leu Pro Val Thr Pro

145 150 155 160

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Cys Gln Ser Leu Leu His

165 170 175

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

180 185 190

Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val

195 200 205

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

210 215 220

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

225 230 235 240

Ala Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ala

<223> Amino acid sequence of aCX20B2 affinity matured binding protein

<400> 24

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Glu Trp Ser Trp Ile Arg Gln Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asp Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg

130 135 140

Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

145 150 155 160

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

165 170 175

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

180 185 190

Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val

195 200 205

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

210 215 220

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

225 230 235 240

Ala Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ala

<223> Amino acid sequence of aCX20E5 affinity matured binding protein

<400> 25

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asn Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg

130 135 140

Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

145 150 155 160

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

165 170 175

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

180 185 190

Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val

195 200 205

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

210 215 220

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

225 230 235 240

Ala Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ala

<223> Amino acid sequence of aCX82 binding protein

<400> 26

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

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

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg

130 135 140

Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

145 150 155 160

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

165 170 175

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

180 185 190

Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val

195 200 205

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

210 215 220

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

225 230 235 240

Ala Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ala

<223> Amino acid sequence of aHER2-13C1 negative control binding protein

<400> 27

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

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Val Ser

20 25 30

Ser Glu Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Gly Ile Leu Ala Gly Asp Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Phe Thr Ser Gly Gln Gly Ser Leu Arg Ser Asp Pro

100 105 110

Ile Arg Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala

115 120 125

Ala

<223> Amino acid sequence of aVE201 negative control binding protein

<400> 28

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

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Val Ser

20 25 30

Asn Glu Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ser Ile Thr Asp Gln Ser Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Gly Gln Arg Arg Arg Gln Met His Ser Tyr Lys Val

100 105 110

Ser Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<223> Nucleotide sequence encoding aCX13C6 affinity-matured binding protein

<400> 29

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttacgagt ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc agcggccgca ataacatcgt ataatgtgta ctatacgaag 420

ttattggcgc gccaggatat tgtgatgaca cagtctccac tctccctgcc cgtcacccct 480

ggagagccgg cctccatctc ctgcaggtct agtcagagcc tcctgcatag taatggatac 540

aactatttgc attggtacct gcagaagcca gggcagtctc cacagctcct gatctatttg 600

tgttctcatc gggcctccgg ggtccctgac aggttcagtg gcagtggatc aggcacagat 660

tttacactga aaatcagcag agtggaggct gaggatgttg ggatttatta ctgcatgcaa 720

gctctacaac ctccgctcac tttcggcgga gggaccaagc tggagatcaa agca 774

<223> Nucleotide sequence encoding aCX16C1 affinity-matured binding protein

<400> 30

caggtgcagc tacagcattg gggcgcagga ctgttgaagc ctgaggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg ggaccgtggc 360

accctggtca ctgtctcctc agcggccgca ataacttcgt ataatgtgta ctatacgaag 420

ttattggcgc gccaggatat tgtgatgaca cagtctccag tctccctgcc cgtcacccct 480

ggagagccgg cctccatctc ctgcaggtct agtcagagcc tcctgcatag taatggatac 540

aactatttgc attggtacct gcagaagcca gggcagtcta cacagctcct gatctatttg 600

ggttctcatc gggcctccgg ggtccctgac aggttcagtg gcagtggatc aggcacagat 660

tttacactga aaatcagcag agtggaggct gaggatgttg ggatttatta ctgcatgcaa 720

gctctacaac ctccgctcac tttcggcgga gggaccaagc tggagatcaa agca 774

<223> Nucleotide sequence encoding aCX17D5 affinity matured binding protein

<400> 31

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatg actgtgtgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc agcggccgca ataacttcgt ataatgtgta ctatacgaag 420

ttattggcgc gccaggatat tgtgatgaca ctgtctccac tctccctgcc cgtcacccct 480

ggagagccgg cctccatctc ctgcaggtct tgtcagagcc tcctgcatag taatggatac 540

aactatttgc attggtacct gcagaagcca gggcagtctc cacagctcct gatctatttg 600

ggttctcatc gggcctccgg ggtccctgac aggttcagtg gcagtggatc aggcacagat 660

tttacactga aaatcagcag agtggaggct gaggatgttg ggatttatta ctgcatgcaa 720

gctctacaac ctccgctcac tttcggcgga gggaccaagc tggagatcaa agca 774

<223> Nucleotide sequence encoding aCX20B2 affinity-matured binding protein

<400> 32

caggtgcagc tacagcattg gggcgcagga gtgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttacgagt ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac cgactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc agcggccgca ataacttcgt ataatgtgta ctatacgaag 420

ttattggcgc gccaggatat tgtgatgaca cagtctccac tctccctgcc cgtcacccct 480

ggagagccgg cctccatctc ctgcaggtct agtcagagcc tcctgcatag taatggatac 540

aactatttgc attggtacct gcagaagcca gggcagtctc cacagctcct gatctatttg 600

ggttctcatc gggcctccgg ggtccctgac aggttcagtg gcagtggatc aggcacagat 660

tttacactga aaatcagcag agtggaggct gaggatgttg ggatttatta ctgcatgcaa 720

gctctacaac ctccgctcac tttcggcgga gggaccaagc tggagatcaa agca 774

<223> Nucleotide sequence encoding aCX20E5 affinity-matured binding protein

<400> 33

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagag cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

aactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc agcggccgca ataacttcgt ataatgtgta ctatacgaag 420

ttattggcgc gccaggatat tgtgatgaca cagtctccac tctccctgcc cgtcacccct 480

ggagagccgg cctccatctc ctgcaggtct agtcagagcc tcctgcatag taatggatac 540

aactatttgc attggtacct gcagaagcca gggcagtctc cacagctcct gatctatttg 600

ggttctcatc gggcctccgg ggtccctgac aggttcagtg gcagtggatc aggcacagat 660

tttacactga aaatcagcag agtggaggct gaggatgttg ggatttatta ctgcatgcaa 720

gctctacaac ctccgctcac tttcggcgga gggaccaagc tggagatcaa agca 774

<223> Nucleotide sequence encoding aCX82 binding protein

<400> 34

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccaggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc agcggccgca ataacttcgt ataatgtgta ctatacgaag 420

ttattggcgc gccaggatat tgtgatgaca cagtctccac tctccctgcc cgtcacccct 480

ggagagccgg cctccatctc ctgcaggtct agtcagagcc tcctgcatag taatggatac 540

aactatttgc attggtacct gcagaagcca gggcagtctc cacagctcct gatctatttg 600

ggttctcatc gggcctccgg ggtccctgac aggttcagtg gcagtggatc aggcacagat 660

tttacactga aaatcagcag agtggaggct gaggatgttg ggatttatta ctgcatgcaa 720

gctctacaac ctccgctcac tttcggcgga gggaccaagc tggagatcaa agca 774

<223> Nucleotide sequence encoding aHER2-13C1 negative control binding protein

<400> 35

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatatagc gttagctctg agaatatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaggcattt tggcgggaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

tttacgtcgg gtcaggggtc gttgcggtcc gaccccatcc ggtcttgggg tcagggaacc 360

ctggtcaccg tctcgagcgc ggccgca 387

<223> Nucleotide sequence encoding aVE201 negative control binding protein

<400> 36

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagttagc gttagcaatg aggctatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaagcatta ctgaccaaag cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

gggcagcgtc gtaggcagat gcattcgtac aaggtcagct cttggggtca gggaaccctg 360

gtcaccgtct cgagcgcggc cgca 384

<223> Nucleotide sequence of Error-F2 forward primer

<400> 37

actggcccag gcggcccaag cttgccaggt gcagctacag c 41

<223> Nucleotide sequence of Error-R2 reverse primer

<400> 38

actggccggc ctggccctcg agtttgatct ccagcttggt ccctcc 46

Claims (6)

1. An affinity maturation binding protein combined with CXCR4, characterized in that: it is aCX13C6 affinity maturation binding protein; or aCX16C1 affinity maturation binding protein, aCX17D5 affinity maturation binding protein, aCX20B2 affinity maturation binding protein and aCX20E5 affinity maturation binding protein at least one binding protein formed in combination with aCX13C6 affinity matured binding protein; The aCX13C6 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:1 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:2; The aCX16C1 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:3 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:4; The aCX17D5 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:5 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:6; The aCX20B2 affinity maturation binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:7 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:8; The aCX20E5 affinity maturation binding protein includes a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:9 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:10. 2. the affinity maturation binding protein combined with CXCR4 according to claim 1, is characterized in that: The amino acid sequence of the aCX13C6 affinity maturation binding protein is shown in SEQ ID NO: 21; The amino acid sequence of the aCX16C1 affinity maturation binding protein is shown in SEQ ID NO: 22; The amino acid sequence of the aCX17D5 affinity maturation binding protein is shown in SEQ ID NO: 23; The amino acid sequence of the aCX20B2 affinity maturation binding protein is shown in SEQ ID NO: 24; The amino acid sequence of the aCX20E5 affinity maturation binding protein is shown in SEQ ID NO:25. 3. the nucleic acid of the affinity maturation binding protein that encodes the described binding protein of CXCR4 of claim 1, is characterized in that: The sequence of the nucleic acid encoding the heavy chain variable region of the aCX13C6 affinity maturation binding protein is shown in SEQ ID NO: 11; The sequence of the nucleic acid encoding the light chain variable region of the aCX13C6 affinity maturation binding protein is shown in SEQ ID NO: 12; The sequence of the nucleic acid encoding the heavy chain variable region of the aCX16C1 affinity maturation binding protein is shown in SEQ ID NO: 13; The sequence of the nucleic acid encoding the light chain variable region of the aCX16C1 affinity maturation binding protein is shown in SEQ ID NO: 14; The sequence of the nucleic acid encoding the heavy chain variable region of the aCX17D5 affinity maturation binding protein is shown in SEQ ID NO: 15; The sequence of the nucleic acid encoding the light chain variable region of the aCX17D5 affinity maturation binding protein is shown in SEQ ID NO: 16; The sequence of the nucleic acid encoding the heavy chain variable region of the aCX20B2 affinity maturation binding protein is shown in SEQ ID NO: 17; The sequence of the nucleic acid encoding the light chain variable region of the aCX20B2 affinity maturation binding protein is shown in SEQ ID NO: 18; The sequence of the nucleic acid encoding the heavy chain variable region of the aCX20E5 affinity maturation binding protein is shown in SEQ ID NO: 19; The sequence of the nucleic acid encoding the light chain variable region of the aCX20E5 affinity maturation binding protein is shown in SEQ ID NO:20. 4. the nucleic acid encoding the affinity maturation binding protein of claim 2, wherein the nucleic acid is the nucleic acid encoding the aCX13C6 affinity maturation binding protein; or the aCX16C1 affinity maturation encoding At least one of the nucleic acid of the binding protein, the nucleic acid encoding the aCX17D5 affinity matured binding protein, the nucleic acid encoding the aCX20B2 affinity matured binding protein and the nucleic acid encoding the aCX20E5 affinity matured binding protein and the nucleic acid encoding the aCX20E5 affinity matured binding protein. Nucleic acids formed by combining nucleic acids of aCX13C6 affinity-matured binding proteins; The sequence of the nucleic acid encoding the aCX13C6 affinity maturation binding protein is shown in SEQ ID NO: 29; The sequence of the nucleic acid encoding the aCX16C1 affinity maturation binding protein is shown in SEQ ID NO: 30; The sequence of the nucleic acid encoding the aCX17D5 affinity maturation binding protein is shown in SEQ ID NO: 31; The sequence of the nucleic acid encoding the aCX20B2 affinity maturation binding protein is shown in SEQ ID NO: 32; The sequence of the nucleic acid encoding the aCX20E5 affinity matured binding protein is shown in SEQ ID NO:33. 5. the preparation method of the affinity maturation binding protein that is combined with CXCR4 according to claim 2, comprises the steps: by gene synthesis coding the nucleic acid of the affinity maturation binding protein that is combined with CXCR4, it is cloned into an expression vector, and then Transfer the reconstituted expression vector into host cells for inclusion body expression and purification to obtain the affinity maturation binding protein that binds to CXCR4; or obtain the affinity maturation binding protein that binds to CXCR4 by a method of protein synthesis . 6. the application of the affinity maturation binding protein that binds to CXCR4 described in claim 1 or 2 in the preparation of the medicine that treats the disease that CXCR4 high expression is characterized, it is characterized in that: described CXCR4 high expression is that characteristic disease is pancreatic cancer , breast, bladder, esophagus, nasopharyngeal, head and neck, stomach, colorectal, prostate, lung, ovarian, cervical, uterine, liver, kidney and brain tumors.

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