CN112661845A - Affinity maturation binding proteins that bind to CXCR4 and uses thereof - Google Patents

Affinity maturation binding proteins that bind to CXCR4 and uses thereof Download PDF

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CN112661845A
CN112661845A CN202011563448.6A CN202011563448A CN112661845A CN 112661845 A CN112661845 A CN 112661845A CN 202011563448 A CN202011563448 A CN 202011563448A CN 112661845 A CN112661845 A CN 112661845A
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CN112661845B (en
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魏星
周晓欣
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Jinan University
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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 maturation binding proteins that bind to CXCR4 and uses thereof
Technical Field
The invention belongs to the field of binding protein, and particularly relates to affinity maturation binding protein combined with CXCR4 and application thereof.
Background
Malignant tumor is a problem which is difficult to overcome in medicine, and is a first killer for human health, so that the treatment of the tumor is always a hotspot and a difficulty of research. The traditional tumor treatment scheme mainly combines radiotherapy, chemotherapy, operative treatment and the like. These treatments, while capable of inhibiting or eliminating tumor cells, also have deleterious and toxic effects on normal cells of the body. In recent years, the targeted therapy of tumor has become a hot research point, and the therapeutic method is to combine specific protein to tumor cells, which can kill the tumor cells and minimize the damage of normal cells of the body.
Chemokine receptor 4(CXCR4) is a 7-transmembrane glycoprotein consisting of 352 amino acid residues, belonging to the G protein-coupled receptor, of approximately 40kDa in size. CXCR4 has now been found to be expressed in 23 different types of tumors, the most common chemokine receptor expressed by tumor cells. The biological axis formed by CXCR4 and its ligand SDF-1 can mediate tumor cell migration, invasion, apoptosis, angiogenesis and proliferation by activating various signal pathways. Based on the close relationship between the chemokine SDF-1/CXCR4 and tumors, the development of drugs for blocking the SDF-1/CXCR4 axis has attracted great attention. The antagonist AMD3100 of CXCR4 has entered clinical stages I and II in the treatment of sarcomas, gliomas and brain tumors. Meanwhile, an antibody against CXCR4 is used for treating tumors, and the MDX-1338 monoclonal antibody can block SDF-1 from binding to CXCR4 and effectively inhibit tumor metastasis, so that the antibody is currently used for clinical phase I treatment of acute leukemia, non-Hodgkin lymphoma and multiple myeloma. Therefore, the CXCR4 is utilized as a target for cancer treatment, and has high medical value.
Disclosure of Invention
The primary object of the present invention is to overcome the disadvantages and drawbacks of the prior art and to provide an affinity matured binding protein which binds to CXCR 4.
It is another object of the present invention to provide the above use of affinity maturation binding proteins that bind to CXCR 4.
The purpose of the invention is realized by the following technical scheme: an affinity maturation binding protein that binds to CXCR4 is the 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 aCX13C6 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 1 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 2;
the aCX16C1 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 3 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 4;
the aCX17D5 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 5 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 6;
the aCX20B2 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 8;
the aCX20E5 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 9 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 10.
The nucleotide sequence of the heavy chain variable region of the aCX13C6 affinity maturation binding protein is shown as SEQ ID NO. 11.
The nucleotide sequence of the variable region of the light chain of the aCX13C6 affinity maturation binding protein is shown as SEQ ID NO. 12.
The nucleotide sequence of the heavy chain variable region of the aCX16C1 affinity maturation binding protein is shown as SEQ ID NO. 13.
The nucleotide sequence of the variable region of the light chain of the aCX16C1 affinity maturation binding protein is shown as SEQ ID NO. 14.
The nucleotide sequence of the heavy chain variable region of the aCX17D5 affinity maturation binding protein is shown as SEQ ID NO. 15.
The nucleotide sequence of the variable region of the light chain of the aCX17D5 affinity maturation binding protein is shown as SEQ ID NO 16.
The nucleotide sequence of the heavy chain variable region of the aCX20B2 affinity maturation binding protein is shown as SEQ ID NO. 17.
The nucleotide sequence of the variable region of the light chain of the aCX20B2 affinity maturation binding protein is shown as SEQ ID NO. 18.
The nucleotide sequence of the heavy chain variable region of the aCX20E5 affinity maturation binding protein is shown as SEQ ID NO. 19.
The nucleotide sequence of the variable region of the light chain of the aCX20E5 affinity maturation binding protein is shown as SEQ ID NO: 20.
Since the biological activity of the affinity maturation binding protein is determined by the gene sequences specific for the hypervariable regions in the light chain and heavy chain variable regions of the antibody, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region as provided above can be recombined by genetic engineering methods to obtain different types of affinity maturation binding proteins.
The affinity maturation binding protein combined with CXCR4 consists of heavy chain variable region, connecting chain and light chain variable region.
The amino acid sequence of the aCX13C6 affinity maturation binding protein is shown as 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 as SEQ ID NO. 24.
The amino acid sequence of the aCX20E5 affinity maturation binding protein is shown as SEQ ID NO. 25.
The nucleotide sequence for coding the affinity maturation binding protein which is combined with CXCR4 is the nucleotide sequence for coding the aCX13C6 affinity maturation binding protein; or a nucleotide sequence formed by combining at least one of a nucleotide sequence for coding the aCX16C1 affinity maturation binding protein, a nucleotide sequence for coding the aCX17D5 affinity maturation binding protein, a nucleotide sequence for coding the aCX20B2 affinity maturation binding protein and a nucleotide sequence for coding the aCX20E5 affinity maturation binding protein with a nucleotide sequence for coding the aCX13C6 affinity maturation binding protein.
The nucleotide sequence of the aCX13C6 affinity maturation binding protein is preferably shown as SEQ ID NO. 29.
The nucleotide sequence of the aCX16C1 affinity maturation binding protein is preferably shown as SEQ ID NO. 30.
The nucleotide sequence of the aCX17D5 affinity maturation binding protein is preferably shown as SEQ ID NO. 31.
The nucleotide sequence of the aCX20B2 affinity maturation binding protein is preferably shown as SEQ ID NO. 32.
The nucleotide sequence of the aCX20E5 affinity maturation binding protein is preferably shown as SEQ ID NO. 33.
The nucleotide sequences for coding the aCX13C6 affinity maturation binding protein, the aCX16C1 affinity maturation binding protein, the aCX17D5 affinity maturation binding protein, the aCX20B2 affinity maturation binding protein and the aCX20E5 affinity maturation binding protein respectively consist of 774, 774 and 774 bases, and correspondingly coded amino acids are 258, 258 and 258 respectively.
The gene for encoding the heavy chain variable region of the aCX13C6 affinity maturation binding protein consists of 381 bases, encodes 127 amino acids, and the variable region contains 3 CDR (complementary determining cluster) regions: CDR1 is GGSFSGYE; CDR2 is INHSGST; CDR3 is AR.
The gene encoding the light chain variable region of aCX13C6 affinity matured binding protein consists of 339 bases and encodes 113 amino acids, and the variable region contains 3 CDR regions: CDR1 is QSLLHSNGYNY; CDR2 is LCS; CDR3 is MQALQPP.
The gene for encoding the heavy chain variable region of the aCX16C1 affinity maturation binding protein consists of 381 bases, encodes 127 amino acids, and the variable region contains 3 CDR (complementary determining cluster) regions: CDR1 is GGSFSGYY; CDR2 is INHSGST; CDR3 is AR.
The gene encoding the light chain variable region of the aCX16C1 affinity matured binding protein consists of 339 bases and encodes 113 amino acids, and the variable region contains 3 CDR (complementary determining cluster) regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS; CDR3 is MQALQPP.
The gene for encoding the heavy chain variable region of the aCX17D5 affinity maturation binding protein consists of 381 bases and encodes 127 amino acids, and the variable region contains 3 CDR (complementary determining cluster) regions: CDR1 is GGSFSGYY; CDR2 is INHSGST; CDR3 is VR.
The gene encoding the light chain variable region of aCX17D5 affinity matured binding protein consists of 339 bases and encodes 113 amino acids, and the variable region contains 3 CDR regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS; CDR3 is MQALQPP.
The gene for encoding the heavy chain variable region of the aCX20B2 affinity maturation binding protein consists of 381 bases and encodes 127 amino acids, and the variable region contains 3 CDR (complementary determining cluster) regions: CDR1 is GGSFSGYE; CDR2 is INHSGST; CDR3 is AR.
The gene encoding the light chain variable region of the aCX20B2 affinity matured binding protein consists of 339 bases and encodes 113 amino acids, and the variable region contains 3 CDR (complementary determining cluster) regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS; CDR3 is MQALQPP.
The gene for encoding the heavy chain variable region of the aCX20E5 affinity maturation binding protein consists of 381 bases and encodes 127 amino acids, and the variable region contains 3 CDR (complementary determining cluster) regions: CDR1 is GGSFSGYY; CDR2 is INHSGST; CDR3 is AR.
The gene encoding the light chain variable region of the aCX20E5 affinity matured binding protein consists of 339 bases and encodes 113 amino acids, and the variable region contains 3 CDR (complementarity determining cluster) regions: CDR1 is QSLLHSNGYNY; CDR2 is LGS; CDR3 is MQALQPP.
The preparation method of the affinity maturation binding protein combined with CXCR4 comprises the following steps: encoding the nucleotide sequence of the affinity maturation binding protein combined with CXCR4 by gene synthesis, cloning the nucleotide sequence to an expression vector, transferring the recombined expression vector to a host cell for carrying out inclusion body expression and purification to obtain the affinity maturation binding protein combined with CXCR 4; or the affinity maturation binding protein combined with CXCR4 is obtained by a protein synthesis method.
The CXCR4 affinity maturation binding protein can be used for preparing medicines for treating the diseases characterized by the high expression of CXCR 4.
The CXCR4 is highly expressed and characterized by autoimmune diseases and cancers.
The tumor is CXCR4 high expression tumor, including pancreatic cancer, breast cancer, bladder cancer, esophageal cancer, nasopharyngeal carcinoma, head and neck cancer, gastric cancer, colorectal cancer, prostatic cancer, lung cancer, ovarian tumor, cervical cancer, uterine cancer, liver cancer, spleen cancer, kidney cancer and brain tumor.
Compared with the prior art, the invention has the following advantages and effects:
1. the invention screens affinity mature binding protein interacting with CXCR4 in a humanized phage affinity mature binding protein library by a phage display technology, and can obtain the binding protein of human protein without using antigen to immunize human body.
2. The affinity maturation binding protein provided by the invention is humanized, has no immunogenicity in human body, and can be better applied to the development of antitumor drugs.
3. The affinity maturation binding protein provided by the invention utilizes a high-expression prokaryotic host to carry out protein expression, can obviously reduce the production cost of the affinity maturation binding protein, and promotes the application of the affinity maturation binding protein.
Drawings
FIG. 1 is a graph showing the results of 5 rounds of library screening in the polyclonal phage ELISA assay. PBS wells were blank control, EGFR and EpCAM were irrelevant antigen control, and CXCR4 wells were coated with synthetic CXCR4 polypeptide; the primary antibody is polyclonal phage binding protein obtained by amplification and purification after each round of library screening, and the secondary antibody is an antibody of HRP-labeled anti-phage M13. The results show that the OD450 nm value obtained by screening the library by using the CXCR4 polypeptide increases from round 1 to round 5, which indicates that the enrichment of CXCR4 antibody clones in the library obtained by screening by using the CXCR4 polypeptide increases gradually.
FIG. 2 is a graph of the results of monoclonal phage ELISA screening for positive clones that bind to CXCR4 polypeptide; wherein, A is a diagram of representative clones of monoclonal phage ELISA, and is ELISA results of 32 monoclonal phage, the primary antibody is monoclonal phage, and the secondary antibody is anti-M13-HRP; b is a graph of the specificity results of detecting binding of positive clones to CXCR4 polypeptide by monoclonal phage ELISA using multiple unrelated antigens as controls. The results in figure 2B indicate that CXCR4 affinity maturation binding protein phage clones were able to specifically bind to CXCR4-Fc and CXCR4 polypeptides.
FIG. 3 is a SDS-PAGE electrophoresis of affinity matured binding protein expression after purification; wherein, A is aCX13C6, B is aCX16C1, C is aCX17D5, D is aCX20B2, E is aCX20E 5; in the figure, lane 1 is a protein Marker, lane 2 is an uninduced bacterial protein, lane 3 is an induced bacterial protein, lane 4 is a broken supernatant of the induced bacterial, lane 5 is a broken precipitate of the induced bacterial, lane 6 is a denatured solution of the broken precipitate of the induced bacterial, lane 7 is a column-passing solution, lane 8 is a washing solution, and lanes 9 to 13 are target protein eluents 1 to 5.
FIG. 4 is a graph showing the results of detecting the binding of purified CXCR4 affinity matured binding protein to the antigen CXCR4 polypeptide by ELISA; wherein p <0.05 vs PBS; p <0.01 vs PBS (n-3). The results show that aCX13C6 and aCX20E5 have higher binding capacity with CXCR4-Fc and aCX20E5 has higher binding capacity with CXCR4 polypeptide compared with wild-type binding protein aCX 82. Furthermore, the binding of 5 affinity matured binding proteins to unrelated antigens was not apparent, suggesting that 5 affinity matured binding proteins were capable of specifically binding to CXCR 4.
FIG. 5 is a graph showing the results of testing the effect of affinity maturation binding proteins on the proliferative capacity of tumor cells using the MTT assay; wherein, A is DU145, B is PC-3, C is MDA-MB-231; p <0.05 versus 0 μ g/mL; p <0.01 versus 0 μ g/mL; p <0.001 to 0 μ g/mL; p <0.0001 vs. 0 μ g/mL (n-3). The results show that, for DU145 cells, aCX20E5 was more effective at inhibiting tumor cell proliferation than wild-type binding protein aCX82 at a concentration of 50 μ g/mL. For PC-3 cells, 5 affinity mature binding proteins had similar effects on inhibiting tumor cell proliferation compared to aCX 82. For MDA-MB-231 cells, aCX20E5 was more effective at inhibiting tumor cell proliferation than aCX82 at concentrations of 50 and 100 μ g/mL.
FIG. 6 is a bar graph of the effect of affinity matured binding protein on the apoptosis of tumor cells DU145, PC-3 and MDA-MB-231 and the ratio of promotion of tumor cell apoptosis using a flow cytometric analyzer and Annexin V/PI double staining kit; wherein, A is a flow cytometry detection graph of DU145 cells, B is an apoptosis proportion statistical graph of A, C is a flow cytometry detection graph of PC-3 cells, D is an apoptosis proportion statistical graph of C, E is a flow cytometry detection graph of MDA-MB-231 cells, and F is an apoptosis proportion statistical graph of E; p <0.05 vs no binding protein control group; p <0.01 vs control no binding protein; p <0.001 vs control group without binding protein; p <0.0001 relative to control no binding protein (n-3). The results indicate that aCX13C6 and aCX20E5 have a higher ability to induce apoptosis in DU145 and MDA-MB-231 cells compared to wild-type binding protein aCX 82. For PC-3 cells, CX20E5 has a higher ability to induce apoptosis.
FIG. 7 is a graph showing the effect of affinity maturation binding protein on tumor cell migration and the trend of absorbance change thereof, which was measured by a Transwell migration assay; wherein A is a photo picture of affinity matured binding protein detected by a Transwell migration experiment on migration of DU145, B is an analysis result picture of A, C is a photo picture of affinity matured binding protein detected by a Transwell migration experiment on migration of PC-3, D is an analysis result picture of C, E is a photo picture of affinity matured binding protein detected by a Transwell migration experiment on migration of MDA-MB-231, and F is an analysis result picture of E; p <0.05 versus 0 μ g/mL; p <0.01 versus 0 μ g/mL; p <0.001 to 0 μ g/mL; p <0.0001 vs. 0 μ g/mL (n-3). The results show that 5 affinity mature binding proteins have a similar effect of inhibiting tumor cell migration compared to wild-type binding protein aCX82 for DU145 cells. For PC-3 cells, aCX13C6, aCX20B2 and aCX20E5 were more effective in inhibiting tumor cell migration when used at a concentration of 50 μ g/mL than when used at aCX 82. For MDA-MB-231 cells, aCX20E5 was more effective at inhibiting tumor cell migration when used at a concentration of 100 μ g/mL than aCX 82.
FIG. 8 is a graph showing the results of detecting the effect of affinity maturation binding protein on tumor cell invasion and its absorbance change using a Transwell invasion assay; wherein, A is a picture of an experiment photo for detecting the invasion of the affinity maturation binding protein to DU145 cells by adopting a Transwell invasion experiment, B is a picture of an analysis result of A, C is a picture of an experiment photo for detecting the invasion of the affinity maturation binding protein to MDA-MB-231 cells by adopting a Transwell invasion experiment, and D is a picture of an analysis result of C. The results show that 5 affinity maturation binding proteins can inhibit invasion of DU145 and MDA-MB-231 cells, and the invasion capacity of cancer cells can be gradually reduced along with the increase of the concentration of the 5 affinity maturation binding proteins.
FIG. 9 is a graph of the results of animal experiments to examine the effect of 2 affinity maturation binding proteins on tumor growth in a mouse model of DU145 prostate cancer cells; wherein, A is a curve chart of the change of the tumor volume of the mice after administration, B is a picture of the tumor size of each group of mice at the end of the administration period, C is a picture of the tumor weight detection result of each group of mice after the end of the administration period, D is a picture of the HE staining and immunohistochemistry result of the tumor tissues of the mice, and E is a picture of the analysis result of D data by using Image Pro-Plus software; p <0.05 vs PBS; p <0.01 vs PBS (n-4-6). The results show that, according to the tumor volume plot of fig. 9A, aCX20E5 has better ability to inhibit tumor cell growth in vivo at the end of the dosing cycle (day 30) compared to wild-type binding protein aCX 82. According to the tumor weight plot of fig. 9C, aCX13C6 and aCX20E5 had lower tumor weights compared to wild-type binding protein aCX 82. From the integrated optical density results of the immunohistochemical staining of fig. 9E, aCX20E5 was able to inhibit tumor cell proliferation (Ki67) and tumor tissue angiogenesis (CD31) more effectively in vivo than wild-type binding protein aCX 82. The 2 affinity mature binding proteins had no significant effect on the induction of tumor cell apoptosis (C-caspase-3).
Detailed Description
The present invention will be described in further detail below with reference to specific examples and drawings, but the embodiments of the present invention are not limited thereto.
Example 1 construction of phage affinity maturation binding protein library
(1) Wild-type binding protein aCX82 is used as a template (the amino acid sequence of the coded aCX82 binding protein is shown as SEQ ID NO:26, and the nucleotide sequence of the coded aCX82 binding protein is shown as SEQ ID NO: 34), Error-prone PCR is carried out according to the instruction of the GeneMorph II Random Mutagenesis Kit, and a target gene is amplified by an Error-prone PCR method, wherein primers are Error-F2 (the nucleotide sequence is shown as SEQ ID NO: 37) and Error-R2 (the nucleotide sequence is shown as SEQ ID NO: 38).
(2) After the PCR reaction is finished, the error-prone PCR product is identified by agarose gel electrophoresis.
(3) Gel recovery of error-prone PCR products was performed according to the EZNA Gel Extraction Kit instructions.
(4) The pComb3XSS phage display plasmid and the error-prone PCR product are subjected to double enzyme digestion reaction and are digested at 50 ℃ overnight.
(5) The cleavage products were identified by agarose gel electrophoresis.
(6) Gel recovery of the cleavage products was performed according to the EZNA Gel Extraction Kit instructions.
(7) The enzyme-linked product was obtained by performing the enzyme-linked reaction with T4 DNA ligase overnight at 4 ℃.
(8) The enzyme linked product is transformed into electroporation competent cells. Immediately after the electroporation, 1mL of precooled SOC medium was added, incubated at 37 ℃ and 220rpm for 1 hour, and then plated on LB plates containing 100. mu.g/mL of ampicillin, and incubated overnight at 37 ℃.
(9) Single clones were randomly picked and identified by PCR in the presence of a bacterial suspension (primers used were Error-F2 and Error-R2). After the PCR reaction is finished, identifying the PCR product of the bacterial liquid by agarose gel electrophoresis. The correct band (about 800bp) was sequenced to determine if the library was successfully constructed.
Example 2 preparation of helper phage
(1) TG1 E.coli clones were streaked in a three-region on a Petri dish of TYE solid medium, and the streaked Petri dish was placed upside down in an incubator at 37 ℃ for about 14 hours.
(2) TG1 single colonies on the plates were picked and inoculated into 5mL of 2 XTY liquid medium and incubated overnight at 37 ℃ and 250 rpm.
(3) Transferring the bacterial liquid obtained in the step (2) into another 5mL of 2 XTY liquid culture medium according to the volume ratio of 1:100, culturing at 37 ℃ and 250rpm until the bacterial liquid OD600nmAbout 0.5.
(4) KM13 helper phage was diluted in PBS in a gradient (10)12pfu/mL～104pfu/mL)。
(5) And (3) adding 10 mu L of diluted KM13 helper phage into 200 mu L of the bacterial liquid obtained by culturing in the step (3), and placing the mixture in a water bath kettle at 37 ℃ for 30min to obtain a mixture A.
(6) The low melting agarose and 2 × TY broth were mixed, heated to complete melting, and then incubated in water and cooled to 42 ℃ to give the top layer (H-top) medium, wherein the concentration of low melting agarose was 1.5%. Taking 3mL of the top layer culture medium, mixing the mixture A obtained in the step (5) well, and then spreading the mixture on a TYE solid culture plate preheated at 37 ℃. After the top layer of culture medium was completely solidified, the dish was placed upside down in an incubator at 37 ℃ for overnight culture.
(7) A small plaque was picked and inoculated into 5mL TG1 bacterial suspension (OD)600nm0.5), cultured at 37 ℃ and 250rpm for 2 h.
(8) The bacterial liquid is transferred to another 500mL of 2 XTY liquid culture medium according to the volume ratio of 1:100, and the bacterial liquid is shaken at the temperature of 37 ℃ and the rpm of 250 for 2 h.
(9) Kanamycin was added to a concentration of 50. mu.g/mL in the medium, and cultured overnight at 30 ℃ and 250 rpm.
(10) Centrifuging and filtering the bacterial liquid to obtain a supernatant, adding 20% w/v PEG-NaCl solution with the volume equivalent to 1/5 of the supernatant, mixing uniformly, subpackaging in a 50mL centrifuge tube for incubation for 1h on ice, centrifuging for 30min at 4 ℃ and 3200g, discarding the supernatant, then re-suspending and precipitating with 1mL PBS buffer solution, centrifuging for 5min at 4 ℃ and 3200g, and filtering and sterilizing the supernatant by using a 0.45 mu M filter to obtain the auxiliary phage.
Example 3 amplification of phage affinity maturation binding protein libraries
(1) The bacterial library containing the affinity maturation binding protein plasmid was thawed on ice, 100. mu.L was added to 50mL of 2 XTY medium (containing 100. mu.g/mL ampicillin + and 1% w/v glucose) and incubated at 37 ℃ for 2h with shaking at 220rpm until OD600nm＝0.5。
(2) Then 5X 10 of11pfu KM13 helper phage was placed in a 37 ℃ constant temperature water bath for 30 min. The centrifugation was carried out in a small high-speed centrifuge at 3200g for 10 min. The pellet was gently resuspended in 2 XTY liquid medium (containing 100. mu.g/mL Amp + and 50. mu.g/mL Kana +) and incubated at 25 ℃ for 20h with shaking at 220 rpm.
(3) Centrifuging in a small high-speed centrifuge at 3200g for 20min, keeping the supernatant, sucking 40ml of the supernatant, adding 10ml of 20% w/v PEG-NaCl solution, mixing gently, and ice-cooling for 2 h.
(4) Centrifuging in a small high-speed centrifuge at 3200g for 30min, and retaining precipitate. The pellet was gently resuspended in a small amount of PBS buffer, centrifuged in a small high speed centrifuge, 3200g for 10 min. The obtained supernatant contains phage and can be directly used as phage library for screening.
Example 4 screening of phage affinity maturation binding protein libraries for CXCR4 affinity maturation binding proteins
(1) Screening in round 1: the antigen (CXCR4 polypeptide, purchased from Shanghai Boragi Biotech Co., Ltd.) was coated in 4ml of PBS buffer to the immunization tube of the blank control group, and the same volume of antigen dilution (diluted with PBS buffer) was added to the immunization tube of the experimental group at an antigen concentration of 100. mu.g/ml, and the mixture was placed in a refrigerator at 4 ℃ overnight.
(2) The next day, the immune tubes were washed 3 times with PBS buffer. Blocking solution (2% w/v BSA-PBS) was then added along the tube wall and blocked for 2h at room temperature.
(3) The immune tubes were washed 3 times with PBS buffer. Adding (5X 10)12) The phage library (phage-containing supernatant obtained in example 3) was diluted with 2% w/v BSA-PBS in a volume of 4ml and allowed to stand at room temperature for 2 hours.
(4) Screening in round 1: the immune tubes were washed 10 times with PBST buffer. Round 2 selection-round 5 selection the immune tubes were washed 20 times with PBST buffer. The flushing is carried out rapidly every time, so that the flushing effect can be achieved.
(5) Eluting phage combined with antigen with trypsin solution at concentration of 1mg/ml and volume of 500 μ L, continuously performing rotary elution for 10min, retaining supernatant, and storing in refrigerator at 4 deg.C.
(6) Adding the phage eluate into TG1 bacterial solution, placing in 37 deg.C water bath, and water-bathing for 30 min.
(7) Diluting the bacterial liquid in a gradient manner, coating 100 mu L of diluted bacterial liquid on a TYE (containing 100 mu g/mL Amp + and 1% w/v glucose), and culturing in an incubator at 37 ℃ for more than or equal to 12 h;
(8) the remaining bacterial liquid was centrifuged in a small high-speed centrifuge at 3200g for 10min, resuspended in a small amount of 2 × TY liquid medium, spread on a TYE plate, and cultured in an incubator at 37 ℃ for 12h or more.
(9) On day 2, the bacteria in the TYE plates of step (8) were collected: 2ml of 2 XTY liquid medium (containing 15% glycerol) was added and the colonies on the plate were scraped off. 50 μ L of the inoculum solution was added to 50mL of 2 XTY liquid medium (containing 100 μ g/mL Amp + and 1% w/v glucose) and cultured at 37 ℃ for 2 hours at 220rpm with shaking until OD600nm＝0.5。
(10) Taking 10ml of bacterial liquid, adding 5X 1011pfu KM13 helper phage was placed in a 37 ℃ constant temperature water bath for 30 min.
(11) Centrifuging in a small high-speed centrifuge at 4 deg.C and 3200g for 10 min. The pellet was gently resuspended in 2 XTY liquid medium (containing 100. mu.g/mL Amp + and 50. mu.g/mL Kana +) and incubated at 25 ℃ for 20h with shaking at 220 rpm.
(12) Centrifuging in a small high-speed centrifuge at 3200g for 20min, keeping the supernatant, sucking 40ml of the supernatant, adding 10ml of 20% w/v PEG-NaCl solution, mixing gently, and ice-cooling for 2 h.
(13) Centrifuging in a small high-speed centrifuge at 3200g for 30min, and retaining precipitate. The pellet was gently resuspended in a small amount of PBS buffer, centrifuged in a small high speed centrifuge, 3200g for 10 min. The resulting supernatant contains phage and can be directly subjected to the next round of phage library screening, or glycerol can be added and stored at-80 ℃.
(14) The 5 rounds of screening were performed sequentially according to the above steps (4) to (13). The antigen concentration of the subsequent 4 rounds of screening is different, the antigen concentration of the 2 nd round and the 3 rd round are both 50 mu g/mL, the antigen concentration of the 4 th round and the 5 th round are both 25 mu g/mL, and the screening is sequentially carried out from the phage obtained in the previous round.
Example 5 polyclonal phage ELISA assay enrichment results from 5 rounds of screening
(1) Coating antigen: the experiment used NUNC 96-well enzyme plates, the blank control group was PBS, coated with PBS buffer, and the volume was 100. mu.L. The irrelevant antigen groups were EGFR polypeptide (purchased from Shanghai Borate Biotech) and EpCAM polypeptide (purchased from Shanghai Borate Biotech), the concentration was 2. mu.g/mL, diluted with PBS buffer, and 100. mu.L/well. The experimental group was CXCR4 polypeptide at a concentration of 2. mu.g/mL, diluted in PBS buffer and a volume of 100. mu.L. The 96-well microplate was left at 4 ℃ overnight.
(2) And washing the 96-hole ELISA plate with PBS buffer solution for 3 times, and drying the liquid on the ELISA plate each time. Then 200. mu.L of 2% w/v BSA-PBS blocking solution was added and blocked at 37 ℃ for 2 h.
(3) And washing the 96-hole ELISA plate with PBS buffer solution for 3 times, and drying the liquid on the ELISA plate each time. mu.L of phage dilution (2% BSA-PBS from 5 rounds) was added to each well and allowed to stand at room temperature for 1 h.
(4) The 96-well elisa plate was washed 3 times with PBST buffer solution, and the liquid on the elisa plate was spun off each time. mu.L of anti-M13 antibody-HRP dilution (anti-M13 antibody-HRP diluted in 2% w/v BSA-PBS at a dilution ratio of 1: 10000) was added to each well and allowed to stand at room temperature for 1 h.
(5) And (3) washing the 96-hole ELISA plate by using PBST buffer solution for 3 times, standing for 2min each time, and drying liquid on the ELISA plate. Adding 100 μ L of TMB substrate developing solution into each well, incubating for 5min in dark, adding 50 μ L of 1mol/L H2SO4The reaction was terminated and OD was measured450nmAnd (4) light absorption value.
(6) FIG. 1 is a diagram showing the results of 5 rounds of library screening by polyclonal phage ELISA, and the phage affinity maturation binding protein library obtained by 5 th round of screening and amplification is selected for monoclonal phage ELISA. The results show that OD obtained from screening the library with CXCR4 polypeptide in the screening of antibody libraries from round 1 to round 5450nmThe values increased from run to run, indicating that there was a gradual increase in enrichment of CXCR4 antibody clones in the libraries obtained from each run of screening with CXCR4 polypeptide.
Example 6 monoclonal phage picked from round 5 enriched library for ELISA validation
(1) A small amount of Escherichia coli TG1 glycerol strain is dipped by an inoculating loop and streaked, and the strain is cultured in an incubator at 37 ℃ for more than or equal to 12 hours.
(2) Colonies on one plate were selected and inoculated into 5mL of 2 XTY liquid medium (containing no antibiotics) and cultured overnight on a shaker at 37 ℃ and 220 rpm.
(3) The overnight-cultured TG1 strain was added to 2 XTY liquid medium at a volume ratio of 10% and cultured with shaking at 220rpm at 37 ℃ for 2 hours.
(4) Gradient dilution of phage to 10-8Adding into TG1 bacterial solution, placing in 37 deg.C water bath, and water-bathing for 30 min.
(5) Coating a small amount of the bacterial liquid on a TYE plate (containing 100 mu g/mL Amp + and 1% w/v glucose), and then culturing in an incubator at 37 ℃ for more than or equal to 12 h;
(6) monoclonal colonies selected from the plates were inoculated into 96-well plates, 200. mu.L of 2 XTY liquid medium (containing 100. mu.g/mL Amp + and 1% w/v glucose) was added to each well, and cultured overnight at 37 ℃ with shaking at 220 rpm.
(7) mu.L of the inoculum was aspirated from each well of a 96-well plate, inoculated into a new plate, and 200. mu.L of 2 XTY liquid medium (containing 100. mu.g/mL Amp + and 1% w/v glucose) was added to each well, and cultured at 37 ℃ for 2 hours with shaking at 220 rpm. The old culture plate was used for preserving glycerol bacteria, 30% glycerol was added to each well, and the plate was placed in a refrigerator at-80 ℃.
(8) The 96-well plate was individually added to 1.5mL of EP tubes, and 50. mu.L of KM13 helper phage dilution (diluted with 2 XTY liquid medium) was added to each 1.5mL of EP tubes, and the mixture was placed in a 37 ℃ thermostat water bath and water-bathed for 30 min.
(9) The centrifugation was carried out in a small high-speed centrifuge at 3200g for 10 min. The pellet was gently resuspended in 2 XTY liquid medium (containing 100. mu.g/mL Amp + and 50. mu.g/mL Kana +) and incubated at 25 ℃ for 20h with shaking at 220 rpm.
(10) Centrifuging in a small high-speed centrifuge at 3200g for 10min, retaining supernatant, and storing at 4 deg.C.
(11) Coating antigen: the experiment used NUNC 96-well enzyme plates, the blank control group was PBS, coated with PBS buffer, and the volume was 100. mu.L. The irrelevant antigen group was EpCAM polypeptide at a concentration of 2. mu.g/ml, diluted with PBS buffer, and the volume was 100. mu.L. The experimental group was CXCR4 polypeptide at a concentration of 2. mu.g/ml diluted in PBS buffer in a volume of 100. mu.L. The 96-well microplate was left at 4 ℃ overnight.
(12) And washing the 96-hole ELISA plate with PBS buffer solution for 3 times, and drying the liquid on the ELISA plate each time. Add 200. mu.L of 2% BSA-PBS blocking solution and block for 2h at 37 ℃.
(13) And washing the 96-hole ELISA plate with PBS buffer solution for 3 times, and drying the liquid on the ELISA plate each time. Add 100. mu.L of monoclonal phage antibody to each well and let stand at room temperature for 1 h.
(14) The 96-well elisa plate was washed 3 times with PBST buffer solution, and the liquid on the elisa plate was spun off each time. mu.L of anti-M13-HRP conjugate (obtained from Beijing Yiqiao Shenzhou Biotech, diluted anti-M13-HRP antibody to 2% BSA-PBS at a dilution ratio of 1: 10000) was added to each well and allowed to stand at room temperature for 1 hour.
(15) And (3) washing the 96-hole ELISA plate with PBST buffer solution for 3 times, standing for 2min each time, and drying liquid on the ELISA plate. Adding 100 μ L of TMB substrate developing solution into each well, standing in dark for 5min, and adding 50 μ L of 1mol/L H2SO4The reaction was terminated and OD was measured450nmAnd (4) light absorption value.
(16) 992 single clones were randomly picked in the experiment, and 75 positive clones were obtained. FIG. 2A is a graph of representative clones of a monoclonal phage ELISA, showing ELISA results for 32 of the monoclonal phages. Figure 2B is a graph of the binding of positive clones to CXCR4 using a monoclonal phage ELISA with multiple unrelated antigens as controls. The results in figure 2B indicate that CXCR4 affinity maturation binding protein phage clones were able to specifically bind to CXCR4-Fc and CXCR4 polypeptides.
(17) The positive clones were sent to Huada Gene Co for sequencing. The sequencing results were analyzed in DNAMAN, excluding the same nucleotide sequence, to obtain 5 different sequences, named aCX13C6, aCX16C1, aCX17D5, aCX20B2, aCX20E5, respectively, with the amino acid sequences shown in SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24 and SEQ ID NO 25.
Example 7 expression purification of affinity maturation binding proteins
(1) Expression of affinity matured binding protein inclusion bodies
1) BL21(DE3) competent cells and plasmid pET28a-sumo containing the gene of interest (the gene of interest is the protein selected in example 6, the gene sequence encoding the protein was cloned into pET28a-sumo by HindIII and XhoI, and the recombinant plasmid was prepared by Kinzhi, Suzhou, and synthesized) were thawed on ice. Pipette 1. mu.L of pET28a-sumo plasmid into a 1.5mL EP tube containing 100. mu.L of BL21(DE3) competent cells, mix gently, and stand on ice for 30 min. Then placing in a water bath kettle with constant temperature of 42 deg.C, thermally shocking for 2min, and finally standing on ice for 2 min. 900. mu.L of LB liquid medium was added thereto, and shaking culture was carried out at 37 ℃ and 220rpm for 1 hour. Centrifugation at 12000g for 2min, aspiration of 900. mu.L of supernatant, gently resuspending the pellet with the remaining 100. mu.L, plating on LB plates (containing 50. mu.g/mL Kana +), and then culturing in an incubator at 37 ℃ for 12 h.
2) Colonies on one plate were selected and inoculated into 5mL of LB liquid medium (containing 50. mu.g/mL Kana +), and cultured overnight at 37 ℃ with shaking at 220 rpm.
3) Inoculating 4mL of the bacterial liquid into 400mL of LB liquid medium (containing 50. mu.g/mL Kana +), culturing at 37 ℃ for 2.5h at 220rpm with shaking until OD600nmThe concentration is 0.6-1.0, and 1mL of the bacterial liquid is used for subsequent SDS-PAGE gel electrophoresis identification of target proteins (uninduced mycoprotein samples). Adding IPTG (working concentration of 0.5mM) into the bacterial liquid for induced expression, carrying out induced expression for 6h at 30 ℃ and 220rpm, and taking 1mL of bacterial liquid for subsequent target protein SDS-PAGE gel electrophoresis identification(induced mycoprotein sample).
4) Centrifuging the bacterial liquid in a high-speed refrigerated centrifuge at 4 deg.C and 5000g for 5min, and retaining thallus precipitate.
5) The pellet was resuspended in a 50mL beaker with 25mL of pre-cooled lysis buffer and then sonicated. Setting parameters of the ultrasonic crusher: the power is 40%, the work is 4s, the stop is 8s, and the work time is 40 min. After the ultrasonic crushing is finished, centrifuging in a high-speed refrigerated centrifuge at 4 ℃ for 30min at 15000g, taking 20 mu L of supernatant for subsequent target protein SDS-PAGE gel electrophoresis identification (after induction, thalli are crushed into supernatant samples), and keeping precipitates, namely inclusion bodies.
6) Resuspend the inclusion body pellet with 30mL of inclusion body wash A, add rotor, stir overnight at 4 ℃ to obtain suspension. Centrifuging at 4 deg.C and 15000g for 30min, collecting 20 μ L for subsequent SDS-PAGE gel electrophoresis identification of target protein (induced thallus fragmentation precipitation sample).
7) Resuspend the inclusion pellet with 30mL of inclusion wash B and stir at 4 ℃ for 1.5 h. Centrifuging in a high-speed refrigerated centrifuge at 4 deg.C and 15000g for 30min, and retaining precipitate.
8) The pellet was resuspended in denaturing buffer and stirred at room temperature for 2.5 h. Centrifuging in a high-speed refrigerated centrifuge at 4 deg.C for 30min at 20000g, and collecting supernatant as inclusion body dissolving denaturation solution. 20 μ L of the suspension was used for subsequent SDS-PAGE identification of the target protein (sample of the denatured solution of the pellet after disruption of the induced cells).
(2) Purification of affinity maturation binding proteins
1) Column equilibration: a piece of Ni-NTA His Bind Resin was purified and 10mL of denaturing buffer was added to the column.
2) Loading: the inclusion body dissolved denaturation solution passes through a column, the target protein is adsorbed on the column, and 20 mu L of the inclusion body dissolved denaturation solution is taken for subsequent SDS-PAGE gel electrophoresis identification of the target protein (a column passing solution sample).
3) An additional 10mL of denaturing buffer was added to the column and the remaining sample on the side wall of the column was washed.
4) 20mL of the washing buffer is added to pass through the column, and 20 mu L of the washing buffer is taken for subsequent SDS-PAGE gel electrophoresis identification of the target protein (washing buffer sample).
5) The target protein was eluted by adding 10mL of elution buffer, and collected in 1.5mL EP tubes, 1mL of column eluate was collected in each tube, 5 tubes were collected, and 20. mu.L of each EP tube was used for subsequent SDS-PAGE gel electrophoresis identification of the target protein (eluate sample).
6) The protein concentration and purity of the collected eluate were measured with Nanodrop 2000.
(3) Renaturation of affinity maturation binding proteins
1) And adding the collected eluent into a 5kDa dialysis bag, tightly binding two ends of the dialysis bag by using rubber bands, strictly preventing liquid leakage, and reserving a certain amount of air in the dialysis bag so as to prevent the dialysis bag from bursting in dialysis.
2) The dialysis bag was transferred to a beaker, and the whole was immersed in a protein renaturation solution and dialyzed at 4 ℃ with stirring for 12 hours. The renaturation solution is replaced, and the dialysis is continued for 12 h. The urea concentration in the protein renaturation solution is gradually reduced from 8M to 0M, and dialysis is carried out in each urea concentration gradient renaturation solution overnight.
3) After dialysis, the sample was removed, the protein concentration was measured and stored in a freezer at-80 ℃.
(4) SDS-PAGE gel electrophoresis and Coomassie blue staining
1) Preparing 12% (w/v) separating gel, adding the prepared separating gel to the position of the glue-pouring mould which is about 2-3cm away from the top, adding absolute ethyl alcohol, pressing the line, standing for 20min, and waiting for the separating gel to solidify.
2) Pouring off anhydrous ethanol, adding 5% (w/v) concentrated gel to the top, inserting a comb, standing for 30min, and removing the comb to prepare for sample application after the concentrated gel is solidified.
3) And (3) placing the prepared rubber plate in an electrophoresis tank, and adding electrophoresis liquid.
4) And (3) adding a 5 x SDS-PAGE protein loading buffer solution into the protein sample collected in the step of expressing and purifying the affinity maturation binding protein, enabling the final concentration of the buffer solution to be 1 x, and heating at 100 ℃ for 10min to finish the preparation of the sample.
5) And (3) loading 5 mu L of prepared sample on a rubber plate of an electrophoresis tank, adding a protein marker, and replacing 120V running separation gel after 80V running concentration gel is finished.
6) Taking out the gel, adding Coomassie brilliant blue staining solution, dyeing for 10min, removing the staining solution with clear water, and cooking with medium fire in a microwave oven for 5 min.
7) Adding a decolorizing solution, placing on a decolorizing shaking table for decolorizing overnight, occasionally replacing the decolorizing solution until a clear target strip is seen, and taking a picture for analysis. The results are shown in FIG. 3: FIG. 3A is an electrophoretogram of aCX13C6 affinity maturation binding protein, which has a molecular weight of approximately 45 kDa; FIG. 3B is an electrophoretogram of aCX16C1 affinity maturation binding protein, which has a molecular weight of approximately 45 kDa; FIG. 3C is an electrophoretogram of aCX17D5 affinity maturation binding protein, which has a molecular weight of approximately 45 kDa; FIG. 3D is an electrophoretogram of aCX20B2 affinity maturation binding protein, which has a molecular weight of approximately 45 kDa; FIG. 3E is an electrophoretogram of aCX20E5 affinity matured binding protein, which has a molecular weight of approximately 45 kDa.
Example 8 detection of binding of purified CXCR4 affinity matured binding protein to antigen CXCR4 polypeptide using ELISA
(1) Coating antigen: the experiment used NUNC 96-well enzyme plates, the blank control group was PBS, coated with PBS buffer, and the volume was 100. mu.L. A plurality of unrelated antigens (IFN, NGF, CD28, CD31, CSF1R, ICAM-1, EGFR-Fc, EpCAM-Fc, and CXCR4-Fc were set up, where IFN, NGF, CD28, CD31, CSF1R, ICAM-1, EGFR-Fc, and EpCAM-Fc were purchased from Beijing Queen Biotech, Inc., and CXCR4-Fc was purchased from Beijing Bethes Biotech, Inc.) as specific controls at a concentration of 2 μ g/ml, diluted in PBS buffer, at a volume of 100 μ L. The experimental group was CXCR4 polypeptide diluted at a concentration of 2. mu.g/ml in PBS buffer in a volume of 100. mu.L.
(2) And washing the 96-hole ELISA plate with PBS buffer solution for 3 times, and drying the liquid on the ELISA plate each time. Then 200. mu.L of 2% w/v BSA-PBS blocking solution was added and blocked at 37 ℃ for 2 h.
(3) And washing the 96-hole ELISA plate with PBS buffer solution for 3 times, and drying the liquid on the ELISA plate each time. mu.L of the antibody (20. mu.g/mL) was added to a well of a 96-well plate and allowed to stand at room temperature for 1 hour.
(4) The 96-well elisa plate was washed 3 times with PBST buffer solution, and the liquid on the elisa plate was spun off each time. mu.L of anti-His-HRP (diluted with 2% w/v BSA-PBS, ratio 1: 5000) was added to a well of a 96-well microplate and incubated for 1h at room temperature.
(5) And (3) washing the 96-hole ELISA plate by using PBST buffer solution for 3 times, standing for 2min each time, and drying liquid on the ELISA plate. Adding 100 mu L of TMB substrate developing solution into a 96-hole enzyme label plate, incubating for 10min in a dark place, and adding 1mol/L of H into the hole of the 96-hole enzyme label plate2SO4The reaction was stopped in solution and OD was measured450nmThe absorbance of (a). The results in fig. 4 show that aCX13C6 and aCX20E5 have higher binding ability to CXCR4-Fc and aCX20E5 has higher binding ability to CXCR4 polypeptide compared to wild-type binding protein aCX 82. Furthermore, the binding of 5 affinity matured binding proteins to unrelated antigens was not apparent, suggesting that 5 affinity matured binding proteins were capable of specifically binding to CXCR 4.
Example 9 obtaining of negative control binding protein
In the early-stage experiment of the laboratory, a phage display technology is adopted, polypeptide synthesized by human epidermal growth factor receptor 2(Her2) and Vascular Endothelial Growth Factor (VEGF) is used as an antigen, a humanized binding protein phage library is screened by adopting the method, 2 clones which are not combined with the corresponding antigen are selected by ELISA, negative control binding proteins aHer2-13C1 and aVE201 are respectively obtained and are used as negative controls. Negative control binding proteins aHer2-13C1 and aVE201 have amino acid sequences shown as SEQ ID NO 27 and SEQ ID NO 28, respectively; the nucleotide sequences encoding aHer2-13C1 and aVE201 are shown in SEQ ID No. 35 and SEQ ID No. 36, respectively.
Example 10 MTT assay was used to examine the effect of purified CXCR4 affinity maturation binding protein on tumor cell proliferation
(1) Collecting tumor cells (human prostatic cancer DU145 cells, human prostatic cancer PC-3 cells and human breast cancer MDA-MB-231 cells) in logarithmic growth phase, and adjusting cell concentration to 2.5 × 104One per ml, seeded into 96-well cell culture plates, 5000 cells per well. At 37 deg.C, 5% CO2The cells were allowed to adhere to the wall overnight in the cell culture chamber of (1).
(2) Then, the old medium was aspirated, and DMEM medium containing 1% v/v FBS was added thereto, followed by starvation for 6 hours.
(3) Grouping experiments: 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 aHer2-13C 1). 3 cancer cells were treated separately at different concentrations (0, 25, 50, 100. mu.g/mL) with SDF-1 (working concentration 100ng/mL, available from Beijing Yiqiao Shenzhou technologies, Inc.) at 37 ℃ with 5% CO2The cells were cultured in the cell culture chamber for 72 hours.
(4) mu.L of MTT solution was added to each well at 37 ℃ with 5% CO2The cell culture box of (2) is continuously cultured for 4 hours.
(5) The 96-well plate was removed, the cell culture broth was aspirated, 200. mu.L DMSO was added to each well, the 96-well cell culture plate was placed on a shaker for 10min with rapid shaking in order to dissolve the crystals, and finally the absorbance of OD570nm was measured. The results in FIG. 5 show that aCX20E5 was more effective in inhibiting tumor cell proliferation compared to wild-type binding protein aCX82 when used at a concentration of 50. mu.g/mL for DU145 cells. For PC-3 cells, 5 affinity mature binding proteins had similar effects on inhibiting tumor cell proliferation compared to aCX 82. For MDA-MB-231 cells, aCX20E5 was more effective at inhibiting tumor cell proliferation than aCX82 at concentrations of 50 and 100 μ g/mL.
Example 11 detection of the Effect of purified CXCR4 affinity maturation binding protein on apoptosis of tumor cells Using flow cytometric Analyzer and Annexin V/PI double staining kit
(1) Collecting DU145, PC-3 and MDA-MB-231 in logarithmic growth phase, adjusting cell concentration to 2.5 × 105One/ml, inoculated into 6-well cell culture plates, 2.5X 10 per well5And (4) cells. At 37 deg.C, 5% CO2The cells were allowed to adhere to the wall overnight in the cell culture chamber of (1).
(2) Then, the old medium was aspirated, and DMEM medium containing 1% v/v FBS was added thereto, followed by starvation for 6 hours.
(3) Grouping experiments: control group without binding protein, 5 CXCR4 affinity maturation binding proteins (aCX13C6, aCX16C1, aCX 17D)5. aCX20B2 and aCX20E5), 1 positive control binding protein (wild-type binding protein aCX82) and 2 negative control binding proteins (aVE201 and ahr 2-13C 1). 3 cancer cells were treated with SDF-1 (working concentration 100ng/mL, available from Beijing Yiqiao Shenzhou technologies Co., Ltd.) at a concentration of 50. mu.g/mL, respectively, and 5% CO at 37 ℃2The cells were cultured in a cell culture chamber for 48 hours.
(4) Old medium was aspirated, cells were washed with PBS buffer, PBS buffer was aspirated, and cells were digested with pancreatin. Digestion was stopped in DMEM medium with 10% v/v FBS. Gently blow the cells, collect the cell suspension, centrifuge at 1024g for 5min, and retain the pellet.
(5) The cells were washed again with PBS buffer, centrifuged at 1024g for 5min and the pellet was retained. Cells were washed 2 times in succession.
(6) Add 195. mu.L of 1 × binding buffer to gently resuspend the cells, add 5. mu.L of Annexin V-FITC, and incubate for 15min at room temperature in the dark.
(7) Add 200 u L1 x binding buffer gently heavy suspension cells, in a small high speed centrifuge centrifugation, 4 degrees, 1000g centrifugation for 5min, retention of precipitation.
(8) Add 190. mu.L of 1 × binding buffer to gently resuspend the cells, add 10. mu.L of PI, and gently mix.
(9) Apoptosis was detected using a flow cytometer. The results in FIG. 6 show that aCX13C6 and aCX20E5 have a higher ability to induce apoptosis in DU145 and MDA-MB-231 cells compared to wild-type binding protein aCX 82. For PC-3 cells, CX20E5 has a higher ability to induce apoptosis.
Example 12 Using the Transwell migration assay to examine the effect of purified CXCR4 affinity maturation binding protein on tumor cell migration
(1) Tumor cells DU145, PC-3 and MDA-MB-231 were harvested in logarithmic growth phase, added to DMEM medium containing 1% v/v FBS and starved for 6 h. Cells were digested, centrifuged and resuspended in DMEM medium containing 1% v/v FBS.
(2) Grouping experiments: 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 aHer2-13C 1). 200 μ L (5X 10) of cell suspensions co-treated with different concentrations (0, 25, 50, 100 μ g/mL) and SDF-1 (working concentration 100ng/mL, available from Beijing Yiqiao Shenzhou technologies, Inc.) respectively4Cells, DMEM medium with 1% v/v FBS), were added to the upper chamber of the Transwell chamber. The lower chamber was filled with 20% 1% v/v FBS DMEM medium at 37 deg.C with 5% CO2The culture box is used for culturing for 24 hours.
(3) The Transwell chamber was removed, the cell culture medium in the upper and lower chambers was aspirated, and the cells that did not migrate in the upper chamber were gently wiped off with a cotton swab. The lower chamber was used to fix the cells using 4% w/v paraformaldehyde in a volume of 500. mu.L for a fixing period of 10 min. Then 4% paraformaldehyde was aspirated and the cells were stained with 0.1% w/v crystal violet stain in a volume of 500. mu.L for 30 min. The Transwell cells were washed with ultrapure water 3 times each, and the cells were placed under a microscope for observation and photographing. Adding 33% acetic acid solution into each well, eluting with a volume of 100 μ L, rapidly shaking on a shaker, and measuring OD570nmThe absorbance of (a). The results in fig. 7 show that 5 affinity mature binding proteins have a similar effect on inhibiting tumor cell migration compared to wild-type binding protein aCX82 for DU145 cells. For PC-3 cells, aCX13C6, aCX20B2 and aCX20E5 were more effective in inhibiting tumor cell migration when used at a concentration of 50 μ g/mL than when used at aCX 82. For MDA-MB-231 cells, aCX20E5 was more effective at inhibiting tumor cell migration when used at a concentration of 100 μ g/mL than aCX 82.
Example 13 Using the Transwell invasion assay to examine the effect of purified CXCR4 affinity maturation binding protein on tumor cell invasion
The procedure was as in the Transwell migration test in example 12, except that an additional Matrigel matrix was applied. The inner surface of a Transwell chamber (8 μm pore size, 24-well plate) was coated with Matrigel 50 μ g/well and placed in a cell incubator to gel for 12 h. The results in FIG. 8 show that 5 affinity maturation binding proteins can inhibit the invasion of DU145 and MDA-MB-231 cells, and the invasion capacity of cancer cells can be gradually reduced with the concentration of the 5 affinity maturation binding proteins.
Example 14 Effect of CXCR4 affinity maturation binding proteins on mouse models of prostate cancer
(1) According to the in vitro function experiment result of CXCR4 affinity maturation binding protein, a mouse DU145 cell prostate cancer model is selected and constructed, and aCX13C6 affinity maturation binding protein and aCX20E5 affinity maturation binding protein are selected for animal experiments.
(2) Subjecting DU145 cells to subculture, when the cells grow to logarithmic phase, trypsinizing and collecting the cells, washing the cells with PBS, and adding appropriate amount of PBS to adjust the cell density to 5 × 107/mL。
(3) 100 μ L of the cell suspension was aspirated and injected subcutaneously into the axilla of a mouse (4-week-old male Balb/C nude mouse, purchased from Beijing Huafukang Biotech GmbH) for tumor formation. Mice were housed in SPF-grade animal laboratories.
(4) Tumor volumes were measured in mice every three days, and the longest side (length) and shortest side (width) of the tumor in mice were measured. Tumor volume is length x width according to formula2×0.5。
(5) When 36 mice tumor volume increased to average 100mm3On the left and right, they were randomly divided into 6 groups of 6 individuals. Group 1 was a blank control group (PBS), group 1 was a negative control binding protein group (aVE201), group 2 was a positive control group (DDP and aCX82), and group 2 was an experimental affinity maturation binding protein group (aCX13C6 and aCX20E 5). Administration was by tail vein injection using an insulin syringe. Wherein the administration amount of each mouse of the experimental affinity maturation binding protein group and the negative control binding protein group is 10mg/kg, the administration amount of each mouse of the DDP group is 2mg/kg, and each mouse of the blank control group is injected with 100 mu L of PBS buffer. The dosing period was 30 days, once every 3 days and the tumor volume and body weight of the mice were recorded.
(6) After completion of the experiment, mice were sacrificed, tumors from each group of mice were stripped, tumor weights were measured and photographed, and then tumor tissues were subjected to H & E staining and immunohistochemical analysis. According to the tumor volume plot of fig. 9A, aCX20E5 has better ability to inhibit tumor cell growth in vivo at the end of the dosing cycle (day 30) compared to wild-type binding protein aCX 82. From the photographic picture of fig. 9B tumor size, the tumor volumes of aCX13C6 and aCX20E5 groups were slightly smaller than the wild-type binding protein aCX82 group. According to the tumor weight plot of fig. 9C, aCX13C6 and aCX20E5 had lower tumor weights compared to wild-type binding protein aCX 82. From the graph of the results of HE staining and immunohistochemistry of fig. 9D, no significant histopathological abnormalities were found in groups aCX13C6 and aCX20E 5. From the integrated optical density results of the immunohistochemical staining of fig. 9E, aCX20E5 was able to inhibit tumor cell proliferation (Ki67) and tumor tissue angiogenesis (CD31) more effectively in vivo than wild-type binding protein aCX 82. The 2 affinity mature binding proteins had no significant effect on the induction of tumor cell apoptosis (C-caspase-3).
The reagents used in the examples were configured as follows:
(1) LB solid medium: 2g of peptone, 1g of yeast powder, 2g of NaCl and 4g of agar powder.
Weighing the above reagents on an electronic balance, dissolving into 200mL of ultrapure water, mixing uniformly, subpackaging, and sterilizing at 121 ℃ for 20min under high temperature and high pressure. When the temperature of the culture medium is reduced to about 50 ℃, adding corresponding antibiotics, wherein the working concentration of ampicillin is 100 mu g/ml, the working concentration of kanamycin is 50 mu g/ml, gently mixing, and pouring the mixture into a flat plate. Standing for a period of time, wrapping the flat plate after the flat plate is solidified, and temporarily storing in a refrigerator at 4 ℃.
(2) LB liquid medium: NaCl 1g, peptone 1g, and yeast powder 0.5 g.
Weighing the above reagents on an electronic balance, dissolving in 100mL of ultrapure water, gently mixing, subpackaging in test tubes, and sterilizing at 121 deg.C under high temperature and pressure for 20 min.
(3) TYE solid medium: 5g of peptone, 2.5g of yeast powder, 4g of NaCl and 6g of agar powder.
Weighing the above reagents on an electronic balance, dissolving into 400mL of ultrapure water, slightly mixing uniformly, subpackaging, and sterilizing at 121 ℃ for 20min under high temperature and high pressure. When the temperature of the medium was reduced to about 50 ℃, ampicillin and a 1% glucose solution were added thereto, and the mixture was gently mixed and poured into a plate. Standing for a period of time, wrapping the flat plate after the flat plate is solidified, and temporarily storing in a refrigerator at 4 ℃.
(4)2 × TY medium: peptone 8g, yeast powder 5g, NaCl 2.5 g.
Weighing the above reagents on an electronic balance, dissolving into 500mL of ultrapure water, slightly mixing uniformly, subpackaging, and sterilizing at 121 ℃ for 20min under high temperature and high pressure. Storing in a refrigerator at 4 deg.C.
(5) PBS buffer: na (Na)2HPO4·12H2O 4.5g、NaCl 4g、KH2PO4 0.12g、KCl 0.1g。
The above reagent was weighed out on an electronic balance, dissolved in 300mL of ultrapure water, the pH was adjusted to 7.4, and ultrapure water was continuously added to a constant volume of 500 mL. Sterilizing at 121 deg.C under high temperature and high pressure for 20 min. Storing in a refrigerator at 4 deg.C.
(6) PBST: 0.5mL of Tween-20 was added to 500mL of PBS buffer and gently mixed. Sterilizing at 121 deg.C under high temperature and high pressure for 20min, and storing in refrigerator at 4 deg.C.
(7) Kanamycin (stock concentration 50 mg/mL): measuring 0.5g kanamycin powder on an electronic balance, dissolving in 10mL ultrapure water, uniformly mixing and dissolving, filtering and sterilizing, and temporarily storing in a refrigerator at the temperature of-20 ℃.
(8) Ampicillin (stock concentration 100 mg/mL): taking 0.5g of ampicillin powder on an electronic balance, dissolving in 5mL of ultrapure water, mixing uniformly, filtering, sterilizing, and storing in a refrigerator at-20 deg.C.
(9) And (3) breaking the bacteria buffer solution: 8.77g of NaCl, 6.06g of Tris base, 4ml of 0.5M EDTA and 10ml of PMSF (100X).
The above reagent was measured on an electronic balance, dissolved in 800mL of ultrapure water, adjusted to pH 7.5, and then added with ultrapure water to a constant volume of 1L. Storing in a refrigerator at 4 deg.C.
(10) Inclusion body cleaning solution A: NaCl 1.76g, Tris base 1.22g, Triton X-1004 mL, 0.5M EDTA 0.8 mL.
Measuring the reagent on an electronic balance, dissolving the reagent into 180mL of ultrapure water, slightly mixing the reagent uniformly, subpackaging the mixture, adding the ultrapure water to fix the volume of the solution to 200mL, and temporarily storing the solution in a refrigerator at 4 ℃.
(11) Cleaning fluid B of inclusion body: NaCl 1.76g, Tris base 1.22g, Triton X-1004 mL, PMSF (100X) 2 mL.
Measuring the reagent on an electronic balance, dissolving the reagent into 180mL of ultrapure water, slightly mixing the reagent uniformly, subpackaging the mixture, adding the ultrapure water to fix the volume of the solution to 200mL, and temporarily storing the solution in a refrigerator at 4 ℃.
(12) Inclusion body denaturation buffer: 240.24g of urea, 7.3g of NaCl, 1.21g of Tris base, 5mL of PMSF (100X) and 0.345mL of 2-mercaptoethanol (2-Hydroxy-1-ethanethiol).
The above reagent was measured on an electronic balance, dissolved in 300mL of ultrapure water, adjusted to pH 7.5, and then added with ultrapure water to a constant volume of 500 mL. Storing in a refrigerator at 4 deg.C.
(13) Washing with a miscellaneous buffer solution: 0.68g of imidazole powder is weighed in an electronic balance, dissolved in 5ml of inclusion body denaturation buffer solution, mixed evenly, and added into 19.8ml of denaturation buffer solution, namely impurity washing buffer solution, by sucking 200 mu L.
(14) Elution buffer: 0.68g of imidazole powder is weighed on an electronic balance, dissolved in 5ml of inclusion body denaturation buffer solution, mixed evenly, absorbed with 1ml and added into 9ml of denaturation buffer solution, namely elution buffer solution.
(15)1mol/L Tris-HCl: measuring 0.1g of Tris-base powder on an electronic balance, dissolving the Tris-base powder into 900mL of ultrapure water, gently mixing, adjusting the pH to 8.8 or 6.8, and adding the ultrapure water to fix the volume of the solution to 1L. Storing in a refrigerator at 4 deg.C.
(16) 10% APS solution: weighing 2g of APS powder on an electronic balance, dissolving the APS powder into 20mL of ultrapure water, slightly mixing the APS powder and the ultrapure water uniformly, and subpackaging. Storing in refrigerator at-20 deg.C.
(17)5 XSDS-PAGE running buffer: 94g of glycine, 30.2g of Tris alkali and 5g of SDS.
Measuring the reagent on an electronic balance, dissolving the reagent into 900mL of ultrapure water, slightly mixing the reagent and the ultrapure water, adding the ultrapure water to fix the volume of the solution to 1L, and temporarily storing the solution in a refrigerator at 4 ℃.
(18) Coomassie brilliant blue staining solution: 0.5g of Coomassie brilliant blue, 125mL of isopropanol and 50mL of acetic acid.
Measuring the reagent on an electronic balance, dissolving the reagent into 300mL of ultrapure water, slightly mixing the reagent and the ultrapure water, adding the ultrapure water to fix the volume of the solution to 500mL, and temporarily storing the solution at room temperature.
(19) SDS-PAGE destaining solution: 50mL of acetic acid and 25mL of ethanol.
Measuring the reagent on an electronic balance, dissolving the reagent into 300mL of ultrapure water, slightly mixing the reagent and the ultrapure water, adding the ultrapure water to fix the volume of the solution to 500mL, and temporarily storing the solution at room temperature.
(20)5 × protein loading buffer: SDS 2.5g, bromophenol blue 0.12g, glycerol 12.5mL, 1mol/L Tris-HCl 6.25mL, beta-mercaptoethanol 0.5 mL.
Measuring the reagent on an electronic balance, dissolving the reagent into 15mL of ultrapure water, slightly mixing the reagent and the ultrapure water, adding the ultrapure water to fix the volume of the solution to 25mL, and temporarily storing the solution in a refrigerator at 4 ℃. The beta-mercaptoethanol is added just before use.
(21) IPTG: weighing 1.2g of IPTG powder on an electronic balance, dissolving into 10mL of sterile water, mixing uniformly, filtering for sterilization, and temporarily storing in a refrigerator at the temperature of-20 ℃.
(22)1mol/L H2SO4: 5.6ml of 98% concentrated sulfuric acid was slowly added to 94.4ml of ultrapure water, and the mixture was stirred slowly with a glass rod, and after the solution was cooled to room temperature, it was kept at room temperature.
(23) PMSF (100 ×): 0.87g of PMSF powder is weighed in an electronic balance, dissolved in 50mL of isopropanol and placed in a refrigerator at the temperature of minus 20 ℃ for temporary storage.
(24) 2% BSA-PBS solution: 2g of BSA powder was weighed on an electronic balance, dissolved in 100ml of PBS buffer, filtered and sterilized, and stored temporarily in a refrigerator at 4 ℃.
(25) 30% of glycerin: measuring 30ml glycerol with a measuring cylinder, adding 70ml ultrapure water, mixing, sterilizing at 121 deg.C under high pressure for 20min, and storing in 4 deg.C refrigerator.
(26) PEG-NaCl: 100g of PEG-6000 and 73g of NaCl are weighed in an electronic balance, dissolved in 500ml of ultrapure water, sterilized at the high temperature of 121 ℃ for 20min under high pressure, and placed at room temperature for temporary storage.
(27) Protein renaturation liquid: 8.383g of NaCl, 2.42g of Tris alkali, 0.6g of oxidized glutathione and 0.8g of reduced glutathione are measured in an electronic balance, the reagents are dissolved in 400mL of ultrapure water, a certain amount of urea (8M/6M/4M/2M/1M/0.5M/0.25M/0M) is added, after the reagents are fully mixed, the pH value of the solution is adjusted to 7.5 by using dilute hydrochloric acid, the solution is added with ultrapure water to a constant volume of 1L, and the solution is placed in a refrigerator at 4 ℃ for temporary storage.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
<110> river-south university
<120> affinity maturation binding protein binding to CXCR4 and uses thereof
<160> 38
<170> SIPOSequenceListing 1.0
<223> aCX13C6 amino acid sequence of heavy chain variable region of 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 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> aCX13C6 amino acid sequence of light chain variable region of 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> aCX16C1 amino acid sequence of heavy chain variable region of 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> aCX16C1 amino acid sequence of light chain variable region of 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> aCX17D5 amino acid sequence of heavy chain variable region of affinity maturation 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> aCX17D5 amino acid sequence of light chain variable region of affinity maturation 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> aCX20B2 amino acid sequence of heavy chain variable region of 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 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> aCX20B2 amino acid sequence of light chain variable region of 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> aCX20E5 amino acid sequence of heavy chain variable region of affinity maturation 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> aCX20E5 amino acid sequence of light chain variable region of affinity maturation 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 the 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
ccagggaagg 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 the 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
ccagggaagg 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 maturation 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 the 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
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 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 the 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 the 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
ccagggaagg 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 light chain variable region of aCX20E5 affinity maturation 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> aCX13C6 amino acid sequence of affinity maturation 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 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> aCX16C1 amino acid sequence of affinity maturation 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> aCX17D5 amino acid sequence of affinity maturation 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> aCX20B2 amino acid sequence of affinity maturation 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 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> aCX20E5 amino acid sequence of affinity maturation 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> aCX82 amino acid sequence of 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> aVE201 amino acid sequence of 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 maturation binding protein
<400> 29
caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60
acctgcgctg tctatggtgg gtccttcagt ggttacgagt ggagctggat ccgccagccc 120
ccagggaagg 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 maturation binding protein
<400> 30
caggtgcagc tacagcattg gggcgcagga ctgttgaagc ctgaggagac 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 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 maturation 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 maturation 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 maturation binding protein
<400> 33
caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagag 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 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 aCX 82-binding protein
<400> 34
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 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 (9)

1. An affinity matured binding protein that binds to CXCR4, wherein: is 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 aCX13C6 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 1 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 2;
the aCX16C1 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 3 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 4;
the aCX17D5 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 5 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 6;
the aCX20B2 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 7 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 8;
the aCX20E5 affinity maturation binding protein comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 9 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 10.
2. The affinity matured binding protein that binds to CXCR4 of claim 1, wherein:
the amino acid sequence of the aCX13C6 affinity maturation binding protein is shown as SEQ ID NO: 21;
the amino acid sequence of the aCX16C1 affinity maturation binding protein is shown as SEQ ID NO. 22;
the amino acid sequence of the aCX17D5 affinity maturation binding protein is shown as SEQ ID NO. 23;
the amino acid sequence of the aCX20B2 affinity maturation binding protein is shown as SEQ ID NO. 24;
the amino acid sequence of the aCX20E5 affinity maturation binding protein is shown as SEQ ID NO. 25.
3. The affinity matured binding protein that binds to CXCR4 of claim 1, wherein:
the nucleotide sequence of the heavy chain variable region of the aCX13C6 affinity maturation binding protein is shown as SEQ ID NO. 11;
the nucleotide sequence of the variable region of the light chain of the aCX13C6 affinity maturation binding protein is shown as SEQ ID NO. 12;
the nucleotide sequence of the heavy chain variable region of the aCX16C1 affinity maturation binding protein is shown as SEQ ID NO 13;
the nucleotide sequence of the variable region of the light chain of the aCX16C1 affinity maturation binding protein is shown as SEQ ID NO. 14;
the nucleotide sequence of the heavy chain variable region of the aCX17D5 affinity maturation binding protein is shown as SEQ ID NO. 15;
the nucleotide sequence of the variable region of the light chain of the aCX17D5 affinity maturation binding protein is shown as SEQ ID NO. 16;
the nucleotide sequence of the heavy chain variable region of the aCX20B2 affinity maturation binding protein is shown as SEQ ID NO. 17;
the nucleotide sequence of the variable region of the light chain of the aCX20B2 affinity maturation binding protein is shown as SEQ ID NO. 18;
the nucleotide sequence of the heavy chain variable region of the aCX20E5 affinity maturation binding protein is shown as SEQ ID NO. 19;
the nucleotide sequence of the variable region of the light chain of the aCX20E5 affinity maturation binding protein is shown as SEQ ID NO: 20.
4. A nucleotide sequence encoding the affinity matured binding protein of claim 2 which binds to CXCR4, wherein: the nucleotide sequence is a nucleotide sequence encoding the aCX13C6 affinity maturation binding protein; or a nucleotide sequence formed by combining at least one of a nucleotide sequence encoding the aCX16C1 affinity maturation binding protein, a nucleotide sequence encoding the aCX17D5 affinity maturation binding protein, a nucleotide sequence encoding the aCX20B2 affinity maturation binding protein and a nucleotide sequence encoding the aCX20E5 affinity maturation binding protein with a nucleotide sequence encoding the aCX13C6 affinity maturation binding protein;
the nucleotide sequence of the aCX13C6 affinity maturation binding protein is shown as SEQ ID NO. 29;
the nucleotide sequence of the aCX16C1 affinity maturation binding protein is shown as SEQ ID NO. 30;
the nucleotide sequence of the aCX17D5 affinity maturation binding protein is shown as SEQ ID NO. 31;
the nucleotide sequence of the aCX20B2 affinity maturation binding protein is shown as SEQ ID NO: 32;
the nucleotide sequence of the aCX20E5 affinity maturation binding protein is shown as SEQ ID NO. 33.
5. The method of making an affinity matured binding protein that binds to CXCR4 of claim 2 comprising the steps of: encoding the nucleotide sequence of the affinity maturation binding protein combined with CXCR4 by gene synthesis, cloning the nucleotide sequence to an expression vector, transferring the recombined expression vector to a host cell for carrying out inclusion body expression and purification to obtain the affinity maturation binding protein combined with CXCR 4; or the affinity maturation binding protein combined with CXCR4 is obtained by a protein synthesis method.
6. Use of the CXCR4 affinity matured binding protein of any one of claims 1 to 3 in the preparation of a medicament for the treatment of a disease characterized by high expression of CXCR 4.
7. Use of the CXCR4 affinity maturation binding protein of claim 6 in the manufacture of a medicament for the treatment of a disease characterized by high expression of CXCR4 wherein: the CXCR4 is highly expressed and characterized by autoimmune diseases and cancers.
8. Use of the CXCR4 affinity maturation binding protein of claim 7 in the manufacture of a medicament for the treatment of a disease characterized by high expression of CXCR4 wherein: the cancer is a CXCR4 high expression tumor.
9. Use of the CXCR4 affinity maturation binding protein of claim 8 in the manufacture of a medicament for the treatment of a disease characterized by high expression of CXCR4 wherein: the tumor comprises pancreatic cancer, breast cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, head and neck cancer, gastric cancer, colorectal cancer, prostatic cancer, lung cancer, ovarian tumor, cervical cancer, uterine cancer, liver cancer, spleen cancer, kidney cancer and brain tumor.
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