CN107417792B

CN107417792B - anti-CD 40-HER2 bispecific single chain antibody and application thereof in preparation of antitumor drugs

Info

Publication number: CN107417792B
Application number: CN201710753948.8A
Authority: CN
Inventors: 付蔚华; 李卫东; 赵智成; 李川; 闫永嘉; 陆礼; 张国敬; 张骜; 黄飞
Original assignee: Tianjin Medical University General Hospital
Current assignee: Tianjin Medical University General Hospital
Priority date: 2017-08-29
Filing date: 2017-08-29
Publication date: 2020-07-03
Anticipated expiration: 2037-08-29
Also published as: CN107417792A

Abstract

The invention discloses a CD40-HER2 bispecific single chain antibody, the nucleic acid sequence of which is formed by sequentially splicing a VH fragment of a CD40 ScFv, a VL fragment of a HER2ScFv, a VH fragment of a HER2ScFv and a VL fragment of a CD40 ScFv, and the nucleic acid sequence is shown as SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively. The bispecific single-chain antibody combines tumor immunotherapy and molecular targeted therapy, and simultaneously ensures that the immune activation related to CD40 has certain tumor tissue aggregation, thereby reducing the toxicity of the whole body immune activation, exerting better anti-tumor effect and having wide application prospect.

Description

anti-CD 40-HER2 bispecific single chain antibody and application thereof in preparation of antitumor drugs

Technical Field

The invention relates to a DNA recombination technology, in particular to an anti-CD 40-HER2 bispecific single chain antibody and application thereof in preparing antitumor drugs.

Background

The malignant tumor is one of important diseases threatening the life and health of human beings, and the latest statistics of the morbidity of the malignant tumor show that the number of new malignant tumor cases is 1410 ten thousands and the number of death cases is 810 ten thousands in the whole world in 2012. With the development of science and technology, the treatment mode for malignant tumors is greatly improved, the traditional treatment mode is gradually improved and optimized, and the emerging treatment mode is continuously popularized. The direct excision of the focus by the operation method is an effective treatment mode for the initial malignant tumor and a main treatment mode for the current solid tumor, and the excision range and the excision mode of the operation are more accurate along with more scientific and reasonable typing stage of the disease after years of development. However, in some patients with advanced stage, the time for surgery is lost due to local infiltration and distant metastasis. Chemical drugs, one of the fastest fields of current tumor treatment, play a certain role in improving the quality of life and consolidating the treatment effect of surgery, and the occurrence of a conversion treatment scheme brings new hopes for patients who have lost the opportunity of surgery, but the chemotherapy drugs lack the specificity of tumor cells and tissues, and can also interfere with the biological activities of normal cells while killing the tumor cells, thereby causing various toxic and side effects. Therefore, improving the specificity of treatment and reducing the toxicity of the drug are the research targets of tumor treatment.

Molecular targeted therapy, as an emerging tumor treatment mode, has been rapidly developed in recent years and has exerted remarkable effects in the treatment of a large number of tumors. The tumor cell surface specificity expression marker is mostly used as a target spot, and the formation of the functional form of the tumor cell surface specificity expression marker is blocked or the interaction of a ligand and a receptor is inhibited through the specific combination of a small molecular compound or a large molecular antibody, so that the activation of a downstream signal path network is inhibited, and the effect of inhibiting the tumor proliferation is finally achieved. Imatinib mesylate, as a small molecule tyrosine kinase receptor inhibitor, has shown significant efficacy in the treatment of gastrointestinal stromal tumors. Human Epidermal growth factor 2(Human Epidermal growth factor 2, HER2), one of Epidermal growth factor family members, is expressed in epithelial tumors such as gastric cancer, breast cancer, colorectal cancer and prostate cancer, and it has been shown that the expression of HER2 in gastric cancer is often indicative of poor prognosis and higher malignancy. Intensive research on the action mechanism of the HER2 shows that the HER2 has no natural ligand, and can activate downstream channels including PI3K/Akt and MAPK/ERK1/2 signal channels through forming homodimers or heterodimers with other members of an epidermal growth factor family, thereby influencing the proliferation, metastasis and the like of tumors. Therefore, if HER2 dimerization can be blocked by a certain means, activation of its downstream pathway can be inhibited, and tumor proliferation and metastasis can be inhibited. Trastuzumab (Herceptin) is a humanized murine monoclonal antibody that can target HER2 by specifically binding to HER2 extracellular domain (ECD), inhibiting its homodimer formation, and thereby blocking the activation of its downstream signaling pathway. Trastuzumab has remarkable curative effect on breast cancer positively expressed by HER2 and advanced gastric cancer, and is written in NCCN guidelines as a targeted therapeutic for advanced gastric cancer standards. However, monoclonal antibody drugs have large molecular weights and poor tissue penetration, and in order to achieve satisfactory treatment concentrations, high blood concentrations are often required to be maintained, the burden of liver and kidney metabolism caused by treatment is increased, which cannot be ignored, and long-term drug resistance caused by long-term use often limits the exertion of long-term functions.

With the deep research on the occurrence and development mechanism of malignant tumor, people find the characteristics of an immunosuppressive state in a tumor microenvironment, and tumor cells can inhibit the functions of immune cells by secreting certain inhibitory cytokines, so that effect lymphocytes are incapable of achieving the effect of tumor immune escape. Therefore, activating tumor-specific immune responses in the tumor microenvironment, or being able to become a new and broader approach to tumor therapy, nivolumab, as a blocking antibody for programmed death receptor-1 (PD-1), was first successful in advanced melanoma therapy, confirming the position of immunotherapy in tumor therapy and its gradual spread in different types of tumor therapy. Tumor-associated immune escape occurs as a multi-step process of multiple cycles, which, in addition to inducing apoptosis of T lymphocytes through expression of PD-L1, may interfere in certain pathways with the relevant immune signaling pathways activated by T lymphocytes, resulting in T lymphocyte anergy. CD40/CD40L is an important secondary signal pathway, and CD40 is mainly expressed on the surface of antigen-presenting cells such as dendritic cells. CD40L is mainly expressed in T lymphocytes, and after the MHC-I antigen peptide complex presented by the antigen presenting cells binds to TCR, its expression is significantly up-regulated, thereby activating the second signal pathway, promoting maturation of dendritic cells, and further promoting presentation of tumor antigens and activation of T lymphocytes in a positive feedback manner. With the intensive research on the tumor immune escape mechanism, it is found that malignant tumors can inhibit the activation of a second signal pathway by inhibiting the expression of CD40L by T lymphocytes, so that the maturation of antigen presenting cells is hindered, and the occurrence of tumor specific immune response is inhibited. Therefore, if an agonist CD40 antibody is synthesized, it binds to CD40 on the surface of dendritic cells to promote maturation of dendritic cells, thereby activating tumor-specific T lymphocytes or achieving a tumor killing effect. Currently, several studies have demonstrated that CD 40-agonistic antibodies can suppress tumor proliferation by activating tumor immunity. CP-870, 893 has entered phase I clinical trials to inhibit the progression of pancreatic cancer. SGN-40 is an agonistic anti-CD 40 monoclonal antibody, which has a significant therapeutic effect in the treatment of various hematological and lymphatic tumors. However, some problems occur in the using process, and firstly, the problems of large molecular weight and poor tissue penetration which are commonly existed in the monoclonal antibody medicaments are faced. Meanwhile, the maintenance of high blood concentration of the immune activator can cause the disturbance of the systemic immune function and the generation of Cytokine Release Syndrome (CRS).

The development of recombinant antibody technology brings hope for the improvement of antibody structure, people can reasonably modify the structure of an antibody according to self requirements, a single-chain antibody is one of the currently studied small molecular antibodies in the recombinant antibody, and is formed by reasonably connecting VH and VL in the antibody by using a section of flexible polypeptide. The bispecific single-chain antibody is a novel antibody form formed by reasonably splicing the heavy chain variable region and the light chain variable region of two single-chain antibodies according to a certain special conformation by using flexible short peptides on the basis of the single-chain antibody, and the double-antibody MT111 resisting CD3-CEA constructed by Oberst exerts remarkable curative effect in the treatment of colon cancer, and can play the targeted treatment effect on CEA while recruiting lymphocytes.

With the development of science, the antibody screening technology is mature day by day, and the traditional method for obtaining the antibody mainly depends on the hybridoma technology, but the technology has large workload and low screening efficiency, and the affinity of the obtained antibody is often unsatisfactory. The antibody library screening technology is one of the important technologies for screening the current antibodies, combines the phage display technology, performs fusion expression on the clone for constructing the antibody library and the capsid protein of the phage and the surface of the phage, and performs multiple rounds of screening on the clone so as to gradually improve the affinity of the clone and the antigen. However, the molecular weight of the fusion expressed protein cannot be too large due to the packaging of the capsid protein. Therefore, the phage display technology is more suitable for screening and obtaining the small-molecule single-chain antibody, and the affinity of the single-chain antibody obtained by screening is more mature. There have been several studies using phage display technology to screen for high affinity single chain antibodies. Therefore, the phage display technology can lay a foundation for obtaining the high-affinity bispecific single-chain antibody.

Disclosure of Invention

The invention aims to solve the problems that the tissue penetrability of a monoclonal antibody drug which is singly targeted and combined with HER2 is poor, the maintenance of higher blood concentration aggravates the metabolic burden of liver and kidney, and the drug resistance is caused by long-term use; the problems that the tissue penetrability of an agonistic antibody singly targeting and combined with CD40 is poor, and the maintenance of the high blood concentration of an immune activator can cause the occurrence of systemic immune dysfunction and cytokine release syndrome are solved, and the provided anti-CD 40-HER2 bispecific single chain antibody and the application thereof in preparing antitumor drugs.

The invention is realized according to the following technical scheme.

An anti-CD 40-HER2 bispecific single chain antibody having the nucleic acid sequence of SEQ id no: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

The nucleic acid sequence of the bispecific single-chain antibody is formed by splicing a VH and VL fragment of a CD40 ScFv and a VH and VL fragment of a HER2ScFv through a Linker.

The nucleic acid sequence of the bispecific single chain antibody sequentially splices a VH fragment of a CD40 ScFv, a VL fragment of a HER2ScFv, a VH fragment of a HER2ScFv and a VL fragment of a CD40 ScFv.

An application of the anti-CD 40-HER2 bispecific single chain antibody in preparing antitumor drugs.

The invention has the following beneficial effects:

the bispecific single-chain antibody has two resistances of resisting CD40 and HER2, and has high single-chain antibody affinity, combines tumor immunotherapy and molecular targeted therapy, and simultaneously ensures that the immune activation related to CD40 has certain tumor tissue aggregation, thereby reducing the toxicity of the whole body immune activation and playing a better anti-tumor effect. The bispecific single-chain antibody lays a foundation for further exploring a tumor immune escape mechanism, and opens up a new idea for antibody treatment of tumors.

Drawings

FIG. 1 is a schematic representation of the CD40 × HER2ScDb expression analysis of the invention;

FIG. 2 is a schematic diagram showing the prediction of the tertiary structure of the CD40 × HER2ScDb protein of the present invention;

FIG. 3 is a diagram of protein elution and purification by protein electrophoresis for detecting imidazole at different concentrations according to the present invention;

FIG. 4 is a graph of the flow cytometry detection of mouse dendritic cell surface marker expression under different stimuli according to the present invention;

FIG. 5 is a graph showing the difference in the level of secretion of IL-12 by dendritic cells under different stimuli according to the present invention;

FIG. 6 is a graph showing differences in IFN-. gamma.secretion levels of T lymphocytes of different groups according to the present invention;

FIG. 7 is a graph comparing the killing rates of different groups of T lymphocytes of the invention against 4T1 cells;

FIG. 8 is a graph showing the effect of different interventions of the present invention on tumor volume changes in the 4T1 mouse model;

FIG. 9 is a graph of the effect of different interventions of the invention on IFN- γ expression levels in 4T1 tumor tissue;

FIG. 10 is a graph of the effect of various interventions of the invention on IL-12 expression in 4T1 tumor tissue;

FIG. 11 is a graph showing the difference between lymphocyte infiltration and clear-Caspase-3 expression in different intervention groups, 4T1 tumor tissues, according to the present invention (IHC 200 ×);

FIG. 12 is a HER2 targeting map of the cellular immunofluorescence assay CD40 × HER2ScDb of the invention (200 ×);

FIG. 13 is a graph of the effect of different interventions of the invention on T6-17 cell proliferation and cell morphology (200 ×);

FIG. 14 is a graph comparing the inhibition rate of T6-17 proliferation by different interventions of the invention;

FIG. 15 is a graph showing the effect of Western Blot to detect different interventions on the phosphorylation levels of Akt and ERK in T6-17 cells;

FIG. 16 is a graph depicting the effect of various interventions of the invention on Akt, ERK expression and phosphorylation in T6-17 cells;

FIG. 17 is a graph of tumor growth for the T6-17 tumor model of the present invention;

FIG. 18 is a graph showing the morphological differences of different groups of T6-17 tumor cells observed by HE staining according to the present invention (400 ×).

Detailed Description

In order to further explain the technical solutions of the present invention and the technical effects achieved by the technical solutions, the present invention will be further described with reference to the accompanying drawings and embodiments.

1. Screening of anti-CD 40 high affinity single chain antibody clones

1.1 immunization of rats

According to the immunization program designed in the experiment, after the skin is disinfected along the two sides of the spine of the back of the rat, emulsified mouse recombinant CD40 protein (purchased from Beijing Yiwangzhou biotechnology 50324-M03H-50) is injected subcutaneously at multiple points, so that a small skin dome is formed under the skin, the liquid does not overflow after subcutaneous injection, the back skin of the rat is flat and does not see the skin dome before the next immunization program is performed, the back skin is slightly damaged and scabs, and the antigen absorption is proved to be good.

1.2 extraction of Total RNA from spleen of immunized rat and Synthesis of cDNA

Total RNA of rat spleen is extracted by using a Tiangen extraction kit, and the extracted product is subjected to electrophoresis, and the electrophoresis result shows two clear and bright 28S and 18S. The brightness of the 28S strip is greater than that of the 18S strip, and the 5S strip is fuzzy and invisible. The ultraviolet micro-spectrophotometer detects the RNA concentration of 990.7ng/ul, A260/A280 is 2.06, and the experimental result meets the experimental requirements.

1.3PCR amplification of antibody VH, VL and Linker genes

Using the obtained cDNA as a template, carrying out PCR amplification to obtain VH and VL gene fragments, carrying out agarose gel electrophoresis on PCR products to obtain bands respectively at about 340bp and 320bp, wherein the base size is consistent with an expected result. Linker sequences were biosynthesized in Olympic and PCR amplified using them as templates. Because 24bp overlapping sequences are added at two ends of the (G1y4Ser)3, the length of a Linker is about 90bp, and a band can be seen by about 100bp when PCR amplification products are subjected to electrophoresis.

1.4 overlapping PCR splicing ScFv sequences

Splicing an ScFv sequence by adopting two-step overlapped PCR, and splicing the VH segment and a Linker in the first step; in the second step, VH + Linker was ligated to VL fragment. The length of VH is about 340bp, and the length of a splicing fragment is about 420bp by subtracting an overlapping sequence with Linker of 24 bp. And recovering the electrophoresis product of the first step, continuing splicing with the VL fragment to obtain the ScFv sequence with the base size of about 740bp, wherein the electrophoresis result shows that the construction of the ScFv is successful.

1.5 construction of recombinant plasmid pCANTAB5E-ScFv and double restriction enzyme digestion identification

The amplified pCANTAB5E plasmid and ScFv are subjected to double enzyme digestion by SfiI and NotI simultaneously, T4 ligase is used for connecting a recovered product after double enzyme digestion to construct a recombinant plasmid pCANTAB5E-ScFv, the recombinant plasmid is transformed into TG1 competent cells, a plate is coated for overnight culture, a positive strain is selected for amplification, the plasmid is extracted and subjected to double enzyme digestion identification by SfiI and NotI, and a band is respectively seen at about 4500bp and 740bp after double enzyme digestion of the recombinant plasmid. The results of double enzyme digestion identification and sequencing fully indicate that the recombinant plasmid is successfully constructed.

1.6 phage library capacity and diversity evaluation

The ScFv sequencing sequence constructed by the experiment is contrastively analyzed with the structure and characteristics of the rat antibody variable region in an IMGT coding system, and the result shows that the ScFv sequence synthesized by PCR has good structural comparison with the VH and VL genes of the rat antibody. The success of construction of ScFv synthesized by PCR splicing is shown. Infecting the recombinant phageTG1 competent cells, the capacity of the rat anti-mouse CD40-ScFv phage library was detected to be about 1.2 × 10⁷pfu/mL. Phage titer 10¹²。

18 positive single clones were randomly picked from LB-A plates and sequenced. Sequencing results show that 13 positive clones have gene insertion, SfiI and NotI enzyme cutting site sequences can be seen at the upstream and downstream of the inserted ScFv, the inserting position and the DNA sequence size of the ScFv are correct, and the recombination rate is 72.2%.

In order to evaluate the diversity of the library, 13 groups of the library are randomly extracted for construction, and the results show that the 13 positive clone sequencing results are also subjected to alignment analysis in an IMGT (international montmorillonoid transistor) coding system, so that the corresponding VH and VL have good structural contrast with the VH and VL genes of a rat antibody. BLAST comparison is carried out on the 13 groups of sequences, and the comparison result shows that no repeated sequence is found, which indicates that the diversity of the constructed library is good.

1.7 phage display Each round of screening pressure determination

And calculating the quantity of the phage input and output in each round by adopting a method of gradient dilution, top layer gel culture and plaque counting for the titer of the phage before and after each round of screening. By this process, the overall affinity of the clones in the library was gradually increased, and the enrichment effect of phage display on the clones was confirmed by the continuously increasing elution amount (see table 1).

TABLE 1 phage display screening Each round of input and output phage titer

1.8 phage display Each round of screening clone random sequencing and mutual comparison

Sequencing identification is carried out on the phages produced by the original library, the second round of screening and the third round of screening in the experiment respectively, the sequencing result shows that all clones have positive insertions, and the sequence similarity of the clones in the library is gradually improved along with the screening after the similarity grouping of multi-gene comparison is summarized. The peak of the distribution of the degree of match of the original library sequences was in the range of 81-85%, accounting for 37.2% of the total number of sequencing results. The peak value of the sequence matching degree distribution after the second round of screening is in the interval of 86-90 percent and accounts for 58.2 percent of the total number of sequencing results. The peak value of the sequence matching degree distribution after the third round of screening is in the interval of 91% -95%, and clones with completely matched sequences appear, wherein the sequence matching degree of 8#, 10#, 13# is 100%, the sequence matching degree of 6# and 9#, 12# and 14#, 15# and 20# is also 100%, and the 100% matching degree accounts for 3.2% of the sequencing result, which reflects the enrichment effect of the screening displayed by phage on the library clone sequences (see table 2).

TABLE 2 phage display clone sequences compared to each other similarity distribution after each round of screening

1.9 selection of potentially Positive clones

The analysis of the results of the mutual alignment of the clones after the third round of screening revealed that the results in which the degree of match of the clones was more than 90% were particularly concentrated among some clones, including the clones with the degree of match of 100% in the mutual alignment. This result reflects the enrichment trend of phage display on the library, while the aggregation of high similarity sequences reflects the presence of potentially positive clones. Therefore, 4#, 6#, 8#, 9#, 10#, 12#, 13#, 14#, 15#, 16#, 17#, 19# and 20# clones selected after the third round of screening are used as potential positive clones, clones with completely identical sequences are removed, and 4#, 6#, 8#, 12#, 15#, 16#, 17# and 19# clones are further screened.

1.10 phage ELISA comparison of differences in affinity between clones

Phage ELISA was performed on potential positive clones to compare the difference in affinity between different clones and the CD40 antigen, and on the premise that the protein background coating concentration is 5 mug/mL, the library is diluted by 5% BSA 1:1, and the plate washing strength is the same, the 450nmOD of the 8# clone experimental well is the largest, which reflects the best affinity between the 8# clone experimental well and the CD40 antigen. (Table 3).

TABLE 3 ANOVA to detect differences in the affinity of different clones to the CD40 antigen

1.11 gradient phage ELISA 1 st time comparing affinity differences between clones

The experiment was first run with a protein coating concentration gradient of 5. mu.g/mL, 2.5. mu.g/mL, 1.25. mu.g/mL, 0.625. mu.g/mL, 0.3125. mu.g/mL and showed: clone # 8 had the best affinity for CD40 antigen under different protein coating concentration gradients, but the decrease in protein coating concentration gradient was not significant, presumably due to too high loading phage titer and too small a coating protein gradient setting. Meanwhile, in order to prove the specificity of the positive clones for affinity with CD40, each clone is additionally provided with a BSA (bovine serum albumin) coated hole, and the OD value ratio of each clone experimental hole to the BSA hole is compared, so that the result shows that the affinity of the 8# clone and CD40 is obviously stronger than that of BSA, and when the protein coating concentration is higher than 0.625 mu g/mL, the difference of the affinities is more than 3 times (see Table 4).

TABLE 4 differences in the affinity to BSA affinity ratios of different clones CD40

1.12 comparison of affinity differences between clones by gradient phage ELISA 2 nd time

This experiment extended the concentration gradient of CD40 protein coating to 2.5. mu.g/mL, 0.5. mu.g/mL, 0.1. mu.g/mL, 0.02. mu.g/mL, respectively. And the dilution ratio of the loaded phage particles is improved, and the phage particles are loaded after being diluted by 5% BSA 1: 3. The results show that the affinity of all clones to the CD40 antigen is linearly changed along with the decrease of the concentration gradient of the coating protein. Similarly, the ratio of the OD values at different concentrations to the OD value of BSA affinity showed that the affinity of clone 8# to CD40 was significantly stronger than that of BSA, and the ratio was higher for different background protein concentrations than for other clones, confirming that the affinity of clone 8# to background protein was stronger than that of the remaining clones at each concentration gradient, and attempting to perform the next experiment using clone 8# as the final positive clone (see table 5).

TABLE 5 differences in the affinity to BSA affinity ratios of different clones CD40

1.138# Positive clone sequencing results and analysis

And analyzing the sequencing result of the 8# clone obtained by final screening, wherein the sequence has the full length of 714bp, and the whole sequence comprises a heavy chain variable region (345bp), a light chain variable region (324bp) and (Gly4Ser)345 bp. Enzyme cutting sites Sfi I and Not I inserted by visible genes at the upstream and downstream of the sequences and Linker connected with heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region and light chain variable region sequences are aligned on line by using IMGT antibody structure

(http：//www.imgt.org/IMGT_vquest/vquest？livret＝0&Option＝ratIg)

Shows that: the heavy chain variable region sequence was matched with "Ratnor IGHV5-29 × 01F" to 95.2%, and the light chain variable region sequence was matched with "Ratnor IGKV10S12 × 01F" to 98.57%, confirming that both the heavy chain variable region and the light chain variable region sequences of clone # 8 have high homology with rat-derived IgG VH and VL.

The sequencing results for clone # 8 were as follows:

note: underlined + italic: sfi I and Not I restriction sites; gray background color: 8^#Cloned heavy chain variable region and light chain variable region; underlining: (Gly)₄Ser)₃Linker sequence

2 anti-CD 40 × HER2 bispecific single chain antibody construction and protein expression

2.1PCR amplification of VH and VL fragments of clone # 8CD40 ScFv

And performing PCR amplification by using the screened 8# clone phagemid as a template to respectively obtain CD40VH and VL fragments, wherein the size of an amplification product of the VH is based on the VH fragment, enzyme cutting site Nde I and downstream splicing sticky end are added, and the theoretical length is 366 bp. The agarose electrophoresis result shows that a clear single band is seen between 250-500bp, and the size of the band is consistent with the expected size, thereby confirming that the VH segment is successfully amplified. The amplification of the CD40VL fragment is carried out by the same method, the size of the amplification product is based on the fragment, the restriction enzyme cutting site SalI and the upstream splicing sticky end are added, the theoretical length is 345bp, a clear single band can be seen between 250 bp and 500bp in the agarose electrophoresis result, the size of the band is consistent with the theory, and the successful amplification of the VL fragment is verified.

2.2PCR amplification of the full Length HER2ScFv sequence

The full length of HER2ScFv sequence is inserted into pUC57 plasmid as template, PCR amplification is carried out to obtain the full length of HER2ScFv sequence, agarose electrophoresis can show a bright single band about 750bp, the size of the band is consistent with the sequence length 765bp of sequencing result, and the successful amplification of HER2ScFv sequence is verified.

HER2ScFv gene sequence

GCAGATATTCAGATGACTCAGAGCCCTTCTTCACTGTCAGCCAGCGTTGGAGACCGTGTTACAATCACTTGCCGCGCCAGCCAGGACGTGAACACCGCCGTTGCGTGGTATCAGCAGAAACCAGGCAAGGCCCCTAAACTGCTGATCTATTCAGCGAGCTTTCTGTACAGTGGCGTGCCATCACGCTTCTCCGGATCTCGTAGTGGCACAGATTTTACACTGACTATCAGCTCCCTGCAGCCTGAAGACTTCGCAACCTATTACTGCCAGCAGCACTACACCACACCGCCCACATTTGGTCAGGGGACTAAGGTTGAGCTGAAACGGGCTACTCCGTCCCACAACTCTCATCAGGTCCCAAGCGCCGGCGGTCCAACCGCAAATTCCGGTGAAGTTAAGCTGGTCGAGTCTGGGGGAGGCCTGGTGCAGCCCGGTGGGAGCCTGCGTCTGTCCTGTGCCACCTCTGGCTTCAACATTAAGGATACATATATCCATTGGGTCCGCCAGGCACCAGGAAAAGGCCTGGAATGGGTGGCTCGTATCTATCCTACCAATGGTTATACACGGTACGCCGATAGCGTGAAGGGGCGCTTCACCATCAGTGCAGACACTTCAAAAAACACCGCTTATCTGCAGATGAATAGTCTGCGTGCAGAGGACACCGCTGTTTATTACTGCAGCCGGTGGGGAGGCGATGGCTTTTATGCGATGGATTACTGGGGTCAGGGGACTACCGTGACCGTTTCTAGTGGTGGGGGAGGCTCC

2.3 overlapping PCR splicing of CD40 × HER2ScDb

According to the experimental design of obtaining CD40 × HER2ScDb by OE-PCR splicing, firstly, HER2ScFv + CD40VL is spliced by two-step overlapped PCR, the theoretical length of the HER2ScFv (765bp) + Gly4Ser Linker (15bp) + CD40VL (324bp) ═ 1104bp), a single clear band can be seen in about 1000bp of agarose electrophoresis results, the size of the band is consistent with the theoretical size, and the successful sequence splicing is verified.

On the basis, the splicing of CD40VH and HER2ScFv + CD40VL is continuously carried out, the theoretical length is that CD40VH (345bp) + Gly4Ser Linker (15bp) + HER-2ScFv + CD40VL (1104bp) ═ 1464bp, agarose electrophoresis can see a single clear band between 1000-2000bp, and the size of the single clear band is consistent with the theoretical size of CD40 × HER2ScDb, so that the successful PCR amplification is confirmed.

The conformation selection is based on that there are multiple conformations of specific Single-chain antibodies, and the connection sequence and connection mode of the two antibody V regions are different, which can be divided into five types, namely Diabody, Single chain in Diabody, Tandem ScFv, Tandem Diabody and Dual-affinity targeting molecules (DARTs), Tandem ScFv is a Single-chain protein, the vector construction and protein induction expression process is simple and easy to obtain, and the Linker length of the V region is sufficient, the correct folding of the two antibodies will not interfere with each other, but it is also shown that Tandem ScFv is too long due to its Linker connection, which often results in uncontrollable heavy chain and light chain cross-assembly between the two antibodies, which affects the function thereof, the design of Diabody and DART connects heavy chain and light chain variable regions of the two antibodies, and shortens the Linker length therebetween, which forces the two antibodies to pair due to too close to match, sequencing and light chain splicing, which makes the Linker and light chain variable region combination of the two antibodies pass through PCR splicing experiments, which the design of heavy chain splicing and light chain splicing, which the heavy chain splicing sequences are not easily obtained by the same PCR, and the heavy chain splicing, the heavy chain splicing reaction sequence of the two heavy chain splicing sequences are more mature polypeptide sequence, which is more than the heavy chain splicing, the heavy chain splicing sequence of the heavy chain splicing of the same, which is not easily occurs, the same.

2.4 construction and identification of prokaryotic expression vector of CD40 × HER2ScDb

pET-28a is selected as a prokaryotic expression vector for protein expression, after double enzyme digestion treatment is carried out on the vector and a CD40 × HER2ScDb fragment, T4 DNA ligase carries out plasmid recombination, agarose electrophoresis is carried out on the recombined plasmid to show that a band with the size slightly larger than that of an empty pET-28a plasmid can be seen in 3500 bp-5500 bp, the success of recombination is verified, and the double enzyme digestion identification is further carried out on the recombined plasmid, the result shows that after Nde I and Sal I double enzyme digestion is carried out on the recombined plasmid, a single band can be seen between 1000 bp-2000 bp and about 5500bp respectively, the single band respectively conforms to the size (1464bp) of a bispecific single-chain antibody fragment and the size (5369bp) of a linear pET-28a plasmid, the insertion position of CD40 × HER2ScDb gene is verified to be correct, and the construction of the recombined plasmid pET-28a-CD40 × HER2ScDb is successful.

2.5 sequencing and sequence analysis of the CD40 × HER2ScDb Gene

Sequencing and identifying the successfully constructed pET-28a-CD40 × HER2ScDb recombinant plasmid, wherein the total length of an effective sequence is 1464bp, enzyme digestion sites NdeI and Sal I for constructing and inserting a prokaryotic expression vector and a Linker positioned between CD40VH and HER2ScFv and between CD40VL and HER2ScFv can be respectively found at the upstream and the downstream of the sequence, and the insertion sequence of the gene sequence is confirmed to be consistent with the expectation.

2.6 prediction of translation results of CD40 × HER2ScDb Gene sequences

The prediction of the translation result of the CD40 × HER2ScDb sequence is carried out by using DNASTAR software, wherein the prediction comprises enzyme cutting sites at two sides, the full length of a gene sequence is 1476bp, the number of amino acids after protein translation is 492, and the inserted connecting LinkerGly4Ser is translated correctly, so that the complete reading frame, no codon frameshift, deletion mutation and no stop codon of the CD40 × HER2ScDb sequence are shown.

The CD40 × HER2ScDb nucleic acid sequence is as follows:

CATATGGTCCAGCTGCAGCAGTCTGGTGGAGGCTTAGTGCAGCCTGGAAGGTCCCTAAAACTCCCCTGTGCAGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGGCTGGAGTGGGTCGCAAGCATTAGTTATGATGGTAGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGAGATAATGCAAAAAGCACCCTATACCTGCAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGCGGAAGACACAGTAGCTACTTTGATTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGCGGAGGCAGCGCAGATATTCAGATGACTCAGAGCCCTTCTTCACTGTCAGCCAGCGTTGGAGACCGTGTTACAATCACTTGCCGCGCCAGCCAGGACGTGAACACCGCCGTTGCGTGGTATCAGCAGAAACCAGGCAAGGCCCCTAAACTGCTGATCTATTCAGCGAGCTTTCTGTACAGTGGCGTGCCATCACGCTTCTCCGGATCTCGTAGTGGCACAGATTTTACACTGACTATCAGCTCCCTGCAGCCTGAAGACTTCGCAACCTATTACTGCCAGCAGCACTACACCACACCGCCCACATTTGGTCAGGGGACTAAGGTTGAGCTGAAACGGGCTACTCCGTCCCACAACTCTCATCAGGTCCCAAGCGCCGGCGGTCCAACCGCAAATTCCGGTGAAGTTAAGCTGGTCGAGTCTGGGGGAGGCCTGGTGCAGCCCGGTGGGAGCCTGCGTCTGTCCTGTGCCACCTCTGGCTTCAACATTAAGGATACATATATCCATTGGGTCCGCCAGGCACCAGGAAAAGGCCTGGAATGGGTGGCTCGTATCTATCCTACCAATGGTTATACACGGTACGCCGATAGCGTGAAGGGGCGCTTCACCATCAGTGCAGACACTTCAAAAAACACCGCTTATCTGCAGATGAATAGTCTGCGTGCAGAGGACACCGCTGTTTATTACTGCAGCCGGTGGGGAGGCGATGGCTTTTATGCGATGGATTACTGGGGTCAGGGGACTACCGTGACCGTTTCTAGTGGTGGGGGAGGCTCCGGCGGTGGTGGGTCTGACATTGAGCTCACCCAGTCTCCATCCACATTGCCTGCATCTCTGGGAGAGAGAGTCACCATCAGTTGCAGAGCAAGTCAGAGTATTAGCAATAGTTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACGCCTGATCTACTCTACATCCACTTTAGAATCTGGTGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCTCCATCAGCAGTCTTGAGTCTGAAGATTTTGCAATGTATTACTGTCTACAGTATGCTACTTATCCTCACACGTTCGGTTCTGGGACCAAGCTGGAAATAAAACGGGTCGAC

the amino acid sequence of CD40 × HER2ScDb protein expression is as follows:

HMVQLQQSGGGLVQPGRSLKLPCAASGFTFSDYYMAWVRQAPTKGLEWVASISYDGSSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCGRHSSYFDYWGQGTTVTVSSGGGGSADIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVELKRATPSHNSHQVPSAGGPTANSGEVKLVESGGGLVQPGGSLRLSCATSGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTTVTVSSGGGGSGGGGSDIELTQSPSTLPASLGERVTISCRASQSISNSLNWYQQKPDGTVKRLIYSTSTLESGVPSRFSGSGSGTDYSLSISSLESEDFAMYYCLQYATYPHTFGSGTKLEIKRVD

2.7 molecular Structure prediction of CD40 × HER2ScDb protein physicochemical Properties

The result of CD40 × HER2ScDb induced expression was analyzed according to the expression region of pET28a carrier protein, after IPTG induction, ribosome was bound to the ribosome binding site (ribosome-binding site RBS) at position 307, translation was started at the first initiation codon ATG (position 294) after the ribosome binding site, and when the translation length was 19 amino acids, the translation reached the enzyme cutting site NdeI (position 238), the inserted bispecific single chain antibody was translated to 492 amino acids at the enzyme cutting site Sal I (position 179), and the translation was ended by continuing to extend 13 amino acids to the termination codon TGA (position 137). The analysis showed that the actual protein expression length of CD40 × HER2ScDb after IPTG induction was 524 amino acids, including upstream and downstream His-tag for protein labeling, for the subsequent purification of the protein (see FIG. 1).

Using ExPASy (A), (B), (C), (Dhttp://web.expasy.org/protparam/) The physicochemical properties of the protein were evaluated by on-line detection, the molecular weight of the protein was 56.548Kda, and the average hydrophobic index (GRAVY) was-0.458, indicating that the CD40 × HER2ScDb protein is a hydrophilic protein, which is consistent with the prediction result of DNASTAR-protein on the hydrophilic region of the protein, and the isoelectric Point (PI) was 8.58.

The result shows that the CD40 × HER2ScDb protein contains 11 helices α, 24 β folds which are regionally spaced and distributed in the whole peptide segment, and β corners with different sizes and irregular curls exist between α helices and β folding units, and the analysis of the hydrophily and the hydrophobicity of the protein shows that the hydrophilic region is distributed in a larger area in the protein.

Using I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER)The three-level structure of the protein is predicted, five simulated prediction results are given according to the primary structure of CD40 × HER2ScDb, a model1 with the best simulation degree is selected as the final prediction result by comparing C-score of different models, the structure is shown in figure 2, the modeled C-score value is-1.58 and is positioned in a confidence interval, the estimated value of TM-score is 0.52 +/-0.15, meanwhile, a large number of β folds, β corners and irregular curls can be seen in the three-level structure diagram, and the three folds are mutually folded to form a structure with a plurality of grooves, which is basically consistent with the prediction of the secondary structure.

2.8 inducible expression and purification of CD40 × HER2ScDb protein

The induction expression pre-experiment of protein is carried out by using IPTG with different concentrations, thalli cracking sediment and supernatant after each group of induction expression are simultaneously loaded for SDS-PAGE denaturing electrophoresis, and the result shows that 0.5mmol/L IPTG protein induction can achieve the best induction effect, the expression of protein is mostly expressed in the form of inclusion bodies, the inclusion bodies are firstly denatured and dissolved by using urea, and then the concentration of the urea is reduced by gradient for slow renaturation.

2.9Western Blot to detect the expression of the target protein His-tag

The expression of a protein carrying His-tag is detected by Western Blot on the thallus protein after induction expression, and the specificity of the expression of the target protein can be determined by the expression of the His-tag only in the bispecific single-chain antibody protein.

2.10CD40 × HER2ScDb Ni-NTA Sepharose purification

The target protein with His-tag label is purified by using Ni-NTA agarose, imidazole elution concentrations of 50, 100 and 200mmol/L are set, and SDS-PAGE denaturing electrophoresis is carried out, so that the result shows that (see figure 3, wherein,

lanes

1, 2 and 3 are protein electrophoresis results after elution of 50, 100 and 200mmol/L imidazole respectively), protein impurity bands after purification and elution of the Ni-NTA agarose are obviously reduced, an obvious band exists at a position of 55Kd, and the size of the purified protein is consistent with the molecular weight of the target protein. Meanwhile, the best elution effect can be obtained by 100mmol/L imidazole.

3 anti-CD 40 × HER2 bispecific single chain antibody immune activation

3.1 flow cytometry detection of expression levels of mouse DC cell surface markers in different intervention groups

The flow cytometry detection results show that the expression levels of the Ag + CD40 × HER2ScDb groups, including CD 8093.5% + -3.1%, CD 8686.0% + -3.2% and MHC-II 92.3% + -2.8%, of the surface markers are significantly higher than those of the NS group (CD 8031.54% + -2.6%, CD 8630.04% + -4.1%, MHC-II 40.10% + -3.6%) and the Ag group (CD 8062.79% + -3.0%, CD 8649.30% + -5.6%, MHC-II 88.4% + -4.1%) which have statistical significance (P < 0.05), but the expression levels of the indexes are not statistically different (P > 0.05) compared with the Ag + TNF- α group (CD 8094.76% + -2.6%, CD 8687.0% + -3.3%, MHC-II 93.5% + -4.2%) (see FIGS. 4 and 6).

TABLE 6 comparison of dendritic cell surface marker expression rates under different stimuli

Note:^#the group CDS0 expression was statistically different from the Ag-CD40 × HER2ScDb group

^▲The group of CD86 expression was statistically different from the Ag-CD40 × HER2ScDb group

^◆The group of MHC-II expression was statistically significant compared to the Ag-CD40 × HER2ScDb group

3.2ELISA detection of IL-12 cytokine levels in the supernatant of DC cells from different groups of mice

In order to detect the accelerating effect of different interventions on DC maturation, ELISA detects the expression level of IL-12 in cell supernatant, in the Ag + CD40 × HER2ScDb group, the expression level of IL-12 in DC is obviously up-regulated, the difference is statistically significant (P is less than 0.05) compared with the Ag group and the NS group, and the expression level of IL-12 is not significantly different (P is more than 0.05) compared with the Ag + TNF- α group in a positive control group, which reflects that the bispecific single-chain antibody can accelerate dendritic cell maturation and stimulate IL-12 secretion (see figure 5 and Table 7).

TABLE 7 comparison of differences in IL-12 expression by mouse dendritic cells

Note:^★the difference between the Ag + CD40 × HER2ScDb group and the two pairs of them is statistically significant

3.3ELISA detection of differences in IFN- γ expression levels after Co-culture of different groups of T cells

The DC of the Ag + CD40 × HER2ScDb group can significantly activate CD3+ T lymphocytes of mice and up-regulate the expression of IFN-gamma cytokines, the concentration of IFN-gamma in the cell culture supernatant is (549.96 +/-27.10) pg/mL, and the difference has statistical significance (P < 0.05) compared with the NS group and the Ag group, and the expression level of IFN-gamma is slightly lower but not statistically different (P > 0.05) compared with the Ag + TNF- α group (575.53 +/-41.98) pg/mL (see FIG. 6 and Table 8).

TABLE 8 differential comparison of IFN- γ expression in mouse T-neighbor cells

Note:^◆the difference between the Ag + CD40 × HER2ScDb group and the two pairs of them is statistically significant

3.4 killing of 4T1 tumor cells by tumor-specific T lymphocytes

T lymphocytes activated by different stimuli are respectively recovered and are co-cultured with 4T1 tumor cells, and CCK8 detects the killing effect of the T lymphocytes of different groups on the tumor cells, the killing rate of the Ag + CD40 × HER2ScDb group is (75.65 +/-2.53)%, the killing rate is obviously higher than that of the Ag group, the difference has statistical significance (P is less than 0.05), and compared with the Ag + TNF- α, the difference has no statistical significance (see figure 7 and table 9) (P is more than 0.05).

TABLE 9 comparison of the killing rate of different groups of T lymphocytes against 4T1 tumor cells

Note:^■the Ag + CD40 × HER2ScDb group has statistical significance with the two-two comparison difference

3.5 tumor growth curves and comparisons of tumor model in different groups of 4T1 mice

All models were stable to tumor on Day 5 post subcutaneous tumor implantation in mice, treated for 1 st time, and tumor volume size was measured for each group, with results showing that the initial tumor size was similar and the difference was not statistically significant (P > 0.05). treatment was followed every 3 days with tumor volume changes recorded, 3 days after completion of the 7 th treatment on Day 23 of tumors, the last tumor size measurement was performed and model animals were sacrificed. the results show that CD40 × HER2ScDb 1 group (750 μ g/kg), CD40 × HER2ScDb2 group (1.5mg/kg), and CD40mAb (5mg/kg) group all slowed tumor growth compared to NS group, and no statistical difference in tumor size among groups before the 4 th treatment (Day 14). starting from the 4 th treatment, CD 9696 2ScDb 1 group and CD 396348 HER2 group could inhibit tumor growth on CD 6326 sbb 3926 group with a statistically significant difference in tumor inhibition effect of CD 2, but no difference in CD 2 (CD 5 mg/CD 638) and CD 2 b 2. the CD 5mg/kg group showed a statistically similar effect as seen in CD 2 mAb (CD 2).

3.6ELISA detection of IFN-. gamma.cytokine expression levels in tumor tissues of different intervening mice

The ELISA method detects the level of IFN-gamma in the supernatant of single cell suspension of tumor tissues, and the result shows that the CD40 result shows that the concentration of IFN-gamma in the tumor tissues of the cell suspension supernatant group 2 is 273.44 +/-44.60 pg/mL, and compared with the normal saline group, the HER2ScFv group and the CD40 × HER2ScDb 1 group, the difference is statistically significant (P is less than 0.05). compared with the CD40mAb group, the difference is not statistically different (P is more than 0.05) (see figure 9 and table 11).

TABLE 11 Effect of different interventions on IFN-. gamma.expression levels in 4T1 tumor tissue

Note:^▲the CD40 × HER2ScDb2 group has statistical significance with the two-two comparison difference

3.7ELISA detection of the expression level of IL-12(p70) cytokine in tumor tissues of different intervening mice

Another part of the tumor tissue single cell suspension supernatant was subjected to ELISA to detect the expression level of IL-12(P70) therein, and the results of comparing the differences among groups showed that the IL-12(P70) concentration in the tumor tissue of CD40 × HER2ScDb2 group was 448.85 + -51.10 pg/mL, the difference was statistically significant (P < 0.05) compared to the NS group and the HER-2ScFv group, the difference was not statistically significant (P > 0.05) compared to the CD40mAb group and the CD40 × HER2ScDb 1 group, but the IL-12 level was relatively low in the CD40 × HER2ScDb 1 group compared to the CD40mAb and the difference was statistically significant (P ═ 0.046). the above results showed that the immune activation effect similar to that of CD40 (5mg/kg) was obtained at the CD40 × HER2ScDb dose of 1.5mg/kg (see FIG. 10 and Table 12).

TABLE 12 Effect of different interventions on IL-12 expression in 4T1 tumor tissue

Note:^#the Ag + CD40 × HER2ScDb group has statistical significance with the two-two comparison difference

3.8HE staining to detect lymphocyte infiltration in different groups of tumor tissues

The tumor tissues of different stem groups are subjected to HE staining, and observed under a mirror, the shapes of 4T1 tumor cells are irregular, the cell volumes are relatively large, the cell nuclei are large, part of the cells are obviously nucleolus, the cells are closely arranged and complete, the shapes of the NS group and the HER2ScFv group are good, and no obvious lymphocyte infiltration is seen, the shapes of the cells of the CD40 ScFv group, the CD40 × HER2ScDb group and the CD40mAb group are obviously lymphocyte infiltration, the lymphocytes are regularly circular under the mirror, more in number and aggregated, the cell nuclei are regularly circular, small and deep-stained, and mononuclear cells or lymphocytes stained with the nuclei can be seen in part of capillary vessels around the cells, the tumor cells in the corresponding areas are loosely arranged, the shapes of the tumor cells in the partial areas are incomplete, and cell necrosis areas without the nuclei can be even seen, so that the bispecific single-chain antibody can activate the lymphocytes and collect the lymphocytes in the tumor tissues by nests to kill the tumor cells (see figure 11).

3.9IHC detection of the expression level of Cleaved-caspase-3 in tumor cells

The results of immunohistochemical staining on the expression of Cleaved-Caspase-3 in tumor cells show that the expression of Cleaved-Caspase-3 in the CD40 × HER2ScDb group, CD40mAb and CD40 ScFv group is obviously up-regulated, the expression is mainly localized in cytoplasm, but some Caspase-3 can be translocated after being activated, so that the nucleus of some cells is stained, and the expression of Cleaved-Caspase-3 in HER2ScFv group and NS group 4T1 cells is not significant, so that the bispecific single-chain antibody can activate tumor-specific effector T lymphocytes in tumor tissues, promote the secretion of granzyme, up-regulate the expression of Cleaved-Caspase-3 in the tumor cells and induce the apoptosis of the tumor cells (see figure 11).

4 anti-CD 40 × HER2 bispecific single chain antibody HER2 targeting function verification

4.1 cellular immunofluorescence detection of targeting of CD40 × HER2ScDb and HER2

Immunofluorescence experiments of T6-17 cells are respectively carried out by using quantum dot labeled trastuzumab, HER2ScFv, CD40 ScFv and CD40 × HER2ScDb, and the results show that the labeled trastuzumab can be combined with HER2 on the surface of T6-17 cells and becomes red under the excitation of blue light channels, meanwhile, the bispecific single-chain antibody and HER2ScFv can also be distributed along T6-17 cell membranes, and the CD40 ScFv has no affinity for surface molecules of T6-17 cells.

4.2 Effect of different interventions on T6-17 cell proliferation and morphology

Different interventions on log-phase-grown T6-17 cells, including NS group, CD40 ScFv group (2.5. mu.g/mL), HER2ScFv group (2.5. mu.g/mL), CD40 × HER2ScDb 1 group (2.5. mu.g/mL), CD40 × HER2ScDb2 group (5. mu.g/mL), and trastuzumab group (15. mu.g/mL), were performed under the microscope, and it was found that the NS group, T6-17 cells, were in normal form, cells were in spindle or polygon form, and were mostly in adherent growth, and were closely arranged, the CD 84 HER2ScDb group, with increasing therapeutic dose, gradually decreased cell density and lost normal form, and partially increased cell volume, dysregulation of nuclear plasma ratio (see FIG. 13, A.NS group; B.40 ScFv group (2.5. mu.5. mu.g/mL), C.HER2 ScFv group (2.5. mu.25. mu.8540. mu.8940. g/mL), and No. 19. mu.38 g/mL).

4.3CCK8 testing the inhibition rate of different interventions on the proliferation of T6-17 cells

The inhibition of T6-17 cell proliferation by different interventions was examined by staining live cells with CCK8 reagent, and the results showed that the tumor inhibition rate of CD40 × HER2ScDb2 group (5. mu.g/mL) was (86.89 + -6.35)%, compared to HER2ScFv (2.5. mu.g/mL) (80.18 + -1.97)% and trastuzumab (15. mu.g/mL) (85.56 + -1.73)%, the difference was not statistically significant (P > 0.05) (see FIG. 14 and Table 13).

TABLE 13 comparison of inhibition rates of proliferation of T6-17 by different interventions

Note:^■the difference between CD40 × HER2ScDb 2(5 mu g/mL) and the two-two comparison thereof has statistical significance

4.4WB detection of the phosphorylation level of PI3K/Akt signaling pathway-related molecules in different groups of tumor cells

The results of Western Blot detection of the expression and phosphorylation levels of HER-2 related tyrosine kinase signaling pathway molecules ERK and Akt show that CD40 × HER2ScDb, HER-2ScFv and trastuzumab can inhibit the expression of Akt and ERK and inhibit the corresponding phosphorylation process, and the difference is statistically significant compared with NS group (see FIG. 15 and 16, A: NS group in FIG. 15; B: CD40 ScFv group (2.5. mu.g/mL); C: CD40 × HER2ScDb 1 group (2.5. mu.g/mL); D: CD40 × HER2ScDb2 group (5. mu.g/mL); E: HER-2ScFv group (2.5. mu.g/mL); F: trastuzumab group (15. mu.g/mL); F: 15. mu.5. g/mL) in FIG. 16: the difference in expression of different indices in each group is statistically significant; the difference is not increased compared with that of the CD 8672. No. 5. sub.72. the expression of the CD-ScFv group; and No. 5. sub. the CD 8672. the CD 2. sub. the difference is not found in each group; and the difference is not found in the CD 2. sub. 2. sub. the expression of the CD 2. sub. 10. sub. the expression of the CD 2. sub.

4.5 Effect of CD40 × HER2ScDb on T6-17 tumor proliferation in nude mice

All models were stable to tumor on day 4 after subcutaneous implantation in nude mice, the first treatment was given while measuring the tumor volume size of each group, and the results showed that the initial tumor size of each experimental group was similar and the difference was not statistically significant (P > 0.05). the subsequent treatment was performed every 4 days while recording the change in tumor volume, the last treatment was given on day 16 after the implantation, and the measurement of the last tumor size was performed 4 days later and the model mice were sacrificed.

4.6HE staining to detect the Effect of different interventions on T6-17 tumor cell morphology

Pathological sections are carried out on each group of tumor tissues, HE staining is carried out, and the difference of the forms of the tumor cells of each group is observed, the result shows that T6-17 cells of NS group are in normal cell state, the cell nuclei are complete, the cell connection is tight, part of the cells can see nucleolus, and the proliferation of the cells is active, the cell forms of most tumor tissue areas of CD40 ScFv group are complete, part of the cells lose normal forms, a small amount of mononuclear cells and inflammatory cell infiltration can be seen in the process, the cell visual field of a plurality of CD40 × HER2ScDb groups can be observed, diffuse necrosis can be seen in the tumor tissues, the cells of most areas can lose normal forms, the phenomenon of nuclear compaction is obvious, scattered nuclear fragments can be seen, even some areas of the cells can only see nuclear contour, the cell lines can not reach HER2ScFv group and trastuzumab group, the cell forms of HER2ScFv group and the trastuzumab group are similar (see, A, NS group, B, CD40 ScFv group, C, HER-2ScFv group, D, CD40 × ScFv group, CD 1 group, CD 6323 group, bispecific antibody group, chromatin group, Tsf7 group, Tsa-9, single chain trastuzumab group, polyclonal antibody in vivo proliferation inhibition result can be confirmed, single chain T-9 in the results.

Sequence listing

<110> general Hospital of Tianjin medical university

<120> anti-CD 40-HER2 bispecific single chain antibody and application thereof in preparation of antitumor drugs

<130>12

<160>2

<170>SIPOSequenceListing 1.0

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<211>1476

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<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<220>

<221>N_region

<222>(1)..(1476)

<400>1

catatggtcc agctgcagca gtctggtgga ggcttagtgc agcctggaag gtccctaaaa 60

ctcccctgtg cagcctcagg attcactttc agtgactatt acatggcctg ggtccgccag 120

gctccaacga aggggctgga gtgggtcgca agcattagtt atgatggtag tagcacttac 180

tatcgagact ccgtgaaggg ccgattcact atctccagag ataatgcaaa aagcacccta 240

tacctgcaaa tggacagtct gaggtctgag gacacggcca cttattactg cggaagacac 300

agtagctact ttgattactg gggccaaggg accacggtca ccgtctcctc aggtggcgga 360

ggcagcgcag atattcagat gactcagagc ccttcttcac tgtcagccag cgttggagac 420

cgtgttacaa tcacttgccg cgccagccag gacgtgaaca ccgccgttgc gtggtatcag 480

cagaaaccag gcaaggcccc taaactgctg atctattcag cgagctttct gtacagtggc 540

gtgccatcac gcttctccgg atctcgtagt ggcacagatt ttacactgac tatcagctcc 600

ctgcagcctg aagacttcgc aacctattac tgccagcagc actacaccac accgcccaca 660

tttggtcagg ggactaaggt tgagctgaaa cgggctactc cgtcccacaa ctctcatcag 720

gtcccaagcg ccggcggtcc aaccgcaaat tccggtgaag ttaagctggt cgagtctggg 780

ggaggcctgg tgcagcccgg tgggagcctg cgtctgtcct gtgccacctc tggcttcaac 840

attaaggata catatatcca ttgggtccgc caggcaccag gaaaaggcct ggaatgggtg 900

gctcgtatct atcctaccaa tggttataca cggtacgccg atagcgtgaa ggggcgcttc 960

accatcagtg cagacacttc aaaaaacacc gcttatctgc agatgaatag tctgcgtgca 1020

gaggacaccg ctgtttatta ctgcagccgg tggggaggcg atggctttta tgcgatggat 1080

tactggggtc aggggactac cgtgaccgtt tctagtggtg ggggaggctc cggcggtggt 1140

gggtctgaca ttgagctcac ccagtctcca tccacattgc ctgcatctct gggagagaga 1200

gtcaccatca gttgcagagc aagtcagagt attagcaata gtttaaactg gtatcagcag 1260

aaaccagatg gaactgttaa acgcctgatc tactctacat ccactttaga atctggtgtc 1320

ccatcaaggt tcagtggcag tgggtctggg acagattatt ctctctccat cagcagtctt 1380

gagtctgaag attttgcaat gtattactgt ctacagtatg ctacttatcc tcacacgttc 1440

ggttctggga ccaagctgga aataaaacgg gtcgac 1476

<210>1

<211>492

<212>PRT

<213>2 Ambystoma laterale x Ambystoma jeffersonianum

<220>

<221>VARSPLIC

<222>(1)..(492)

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His Met Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly

1 5 10 15

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

20 25 30

Tyr Tyr Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp

35 40 45

Val Ala Ser Ile Ser Tyr Asp Gly Ser Ser Thr Tyr Tyr Arg Asp Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Gly Arg His Ser Ser Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ala Asp Ile Gln Met Thr

115 120 125

Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile

130 135 140

Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln

145 150 155 160

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe

165 170 175

Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr

180 185 190

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

195 200 205

Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly

210 215 220

Thr Lys Val Glu Leu Lys Arg Ala Thr Pro Ser His Asn Ser His Gln

225 230 235 240

Val Pro Ser Ala Gly Gly Pro Thr Ala Asn Ser Gly Glu Val Lys Leu

245 250 255

Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

260 265 270

Ser Cys Ala Thr Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp

275 280 285

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr

290 295 300

Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe

305 310 315 320

Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn

325 330 335

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly

340 345 350

Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val

355 360 365

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

370 375 380

Glu Leu Thr Gln Ser Pro Ser Thr Leu Pro Ala Ser Leu Gly Glu Arg

385 390 395 400

Val Thr Ile Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Ser Leu Asn

405 410 415

Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Arg Leu Ile Tyr Ser

420 425 430

Thr Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

435 440 445

Ser Gly Thr Asp Tyr Ser Leu Ser Ile Ser Ser Leu Glu Ser Glu Asp

450 455 460

Phe Ala Met Tyr Tyr Cys Leu Gln Tyr Ala Thr Tyr Pro His Thr Phe

465 470 475 480

Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg Val Asp

485 490

Claims

1. An anti-CD 40-HER2 bispecific single chain antibody characterized in that: the nucleic acid sequence of the bispecific single chain antibody is shown in SEQ ID NO: 1, and the amino acid sequence is shown as SEQ ID NO: 2, respectively.

2. An application of the anti-CD 40-HER2 bispecific single chain antibody of claim 1 in preparing an antitumor drug.