AU2020101779A4

AU2020101779A4 - Bispecific antibody binding to agr2 protein and chi3l1 protein, construction, expression, purification method therefor, and use thereof

Info

Publication number: AU2020101779A4
Application number: AU2020101779A
Authority: AU
Inventors: Dawei Li; Zeling Wang
Original assignee: Shenzhen Hubio Biotechnology Co Ltd; Shanghai Jiaotong University
Current assignee: Shenzhen Hubio Biotechnology Co Ltd; Shanghai Jiaotong University
Priority date: 2020-08-12
Filing date: 2020-08-12
Publication date: 2020-09-17
Anticipated expiration: 2028-08-12

Abstract

of the Disclosure The present invention provides a bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein, the bispecific antibody includes an anti-AGR2 antibody 18A4Hu as a parent structure, and an anti-CHI3L1 antibody FRGscFv fragment connected to a C-terminus of a light chain of the anti-AGR2 antibody 18A4Hu. The present invention provides also provides construction, expression, purification method for a bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein, and use of the bispecific antibody in the field of medicine and especially in the treatment of cancer.

Description

Title

Bispecific Antibody Binding to AGR2 Protein and CHI3L1 Protein, Construction, Expression, Purification Method Therefor, and Use Thereof

Background of the Present Invention

Field of Invention

The present invention relates to a new bispecific antibody having the function of binding to AGR2 and CHI3L1, a method for construction, expression, purification, and use of the bispecific antibody in the field of medicine and especially in the treatment of cancer.

Description of Related Arts

The treatment of solid tumors is evolving from traditional therapies such as surgery, radiotherapy, and chemotherapy to biological treatments. The main disadvantages of radiotherapy and chemotherapy are the development of drug resistance and serious side effects. Currently, personal customization of biological therapy embodies the advantages. Its main advantages are strong targeting, low toxicity, good curative effect, delaying the progression of the disease, killing tumor cells that are resistant to chemotherapeutics, and it has achieved good curative effect when combined with chemotherapeutics. Under circumstances, it can improve the survival rate of patients with advanced malignant tumors. As a result, it has developed rapidly and has gradually become a first-line treatment option in some tumor fields (R. Lu et al., J. Biomed. Sci., 27: 1-30, 2020).

The development of tumor antibodies requires the identification of a specific tumor antigen. This specific antigen can be a tumor epitope antigen or a highly expressed antigen in the tumor microenvironment (Tumor Microenvironment, TME). Among the monoclonal antibodies that have been marketed, HER2 and EGFR targeted by Herceptin, Erbitux and other monoclonal antibody drugs are tumor surface antigens, like Avastin is targeted to the growth factor VEGF in TME. (M.F. Press et al., Drugs, 67: 2045-2075, 2007).

The Anterior Gradient-2 (AGR2) in this research is a kind of tumor-specific antigens that are highly expressed in tumor tissues such as lung cancer, breast cancer, prostate cancer, ovarian cancer, stomach cancer, and pancreatic cancer and the circulatory system of tumor patients. AGR2 is also called AG2, GOB-4, HAG-2, HEL-S-116, PDIA17, XAG-2. AGR2 originally found in

Xenopus laevis (G.C. Fletcher et al., Brit. J. Cancer, 88: 579-585, 2003), furthermore, the newt's tail regeneration also has the effect of AGR2. AGR2 can promote tumor metastasis and angiogenesis in TME. Highly expressed AGR2 leads to down-regulation of the tumor suppressor gene p53 response ([4] E. Pohler et al., Mol. Cell. Proteomics 3: 534-547, 2004), and also has a direct impact on cell migration, cell transformation and the invasion ability of cancer cells, and can be used as a marker protein for primary and secondary tumors (Z. Wang et al., Cancer Res., 68: 492-497, 2008; D. Liu et al., Cancer Res., 65: 3796-3805, 2005; V. Ramachandran et al., Cancer Res., 68: 7811-7818, 2008; Y. Zhang et al., Cancer Res., 70: 240-248, 2010).

Epidermal growth factor receptor (EGFR) needs to be combined with AGR2 before it can be delivered from the endoplasmic reticulum to the cell surface (A. Dong et al., J. Biol. Chem., 290: 8016-8027, 2015). Li Zheqi et al. showed that Insulin-like Growth Factor (IGF1) significantly induces AGR2 expression in MCF7 cell line through estrogen response element and leucine zipper transcription factor, promoting the growth of breast cancer (Z. Li et al., Med. Oncol., 32: 178, 2015). It was also found for the first time that AGR2 is an important regulator of the mechanism of hypoxic breast cancer cells resistant to doxorubicin(Z. Li et al., Cancer Science, 106: 1041-1049, 2015; F.R. Fritzsche et al., Clin. Cancer Res., 12: 1728-1734, 2006).

Zhu Qi et al. (Q. Zhu et al., The FEBS journal, 284: 2856-2869, 2017) showed that AGR2 has a significant role in wound healing. After skin injury, migratory epidermis and proliferative epidermis have a high expression of AGR2. AGR2 increases the migration of keratinocytes and the recruitment of fibroblasts, which significantly accelerates the wound healing process.

Guo Hao et al. (H. Guo et al., Oncogene, 36: 5098-5109, 2017) proved that secreted AGR2 promotes the angiogenesis and invasion of vascular endothelial cells and fibroblasts in TME by enhancing the activity of VEGF and fibroblast growth factor 2 (FGF-2). AGR2 directly binds to these extracellular signal molecules and enhances their homodimerization or heterodimerization effects. When AGR2 monoclonal antibody was used to block the activity of secreted AGR2 in vitro and in vivo, the growth of tumor cells was inhibited, and the combination therapy with Avastin produced a significant inhibitory effect.

Based on the above research, our laboratory independently developed an anti-AGR2 monoclonal antibody mAb18A4, which has been publicly authorized, and the international patent number is W02013/004076 Al (D. Li et al., AGR2 blocking antibody and use thereof. In, Google Patents, 2017). mAb18A4 inhibited lung cancer progression and metastasis without exerting any adverse side effects on the major organs and blood in mice. Moreover, we found that mAbl8A4 activated the p53 pathway and attenuated ERK1/2-MAPK pathway. Furthermore, mAbl8A4treated cancer cell lines showed attenuated proliferation and colony formation, enhanced apoptosis, increased p53 expression, and reduced phosphorylated ERK1/2 expression. Treatment with mAb18A4 significantly reduced tumor size and suppressed tumor metastasis in and increased the survival of different xenograft tumor models. In addition, mAbl8A4 potently suppressed AGR2-induced angiogenesis. (H. Negi et al., Cancer Lett., 449: 125-134, 2019).

The humanized monoclonal antibody 18A4Hu targeting AGR2 (generic name Agtuzumab) was efficiently expressed by the CHO cell line. This cell line has been granted a Chinese patent (Patent No. CN 104593333 B). 18A4Hu can up-regulate the p53 pathway and down-regulate the ERK1/2-MAPK pathway. It has anti-tumor effects on xenograft tumor models produced by SKOV3, H460, B16F10, and MCF-7 cell lines. (H. Guo et al., Biochem. Bioph. Res. Co., 475: 57-63, 2016).

The chitinase-like protein called chitinase 3-like-i (CHI3L1; also called Chill in mice and YKL-40 in human) has been implicated in asthma and cancer, e.g., lung cancer. It has been demonstrated that the levels of circulating CHI3L1 are increased in human asthma where they correlate with disease severity. The levels of circulating YKL-40 are increased in many malignancies. The expression of CHI3L1 is particularly high in patients with serum histological types, advanced stages and drug resistance. CHI3L1 is also an important mediator of tissue remodeling, so the expression is very high among cancer patients (B.E. Hakala et al., J. Biol. Chem., 268: 25803-25810, 1993).In these diseases, the levels of YKL-40 frequently correlate directly with disease progression and inversely with disease-free interval and survival.

CHI3L1 is higher in cancer tissues than normal tissues and is directly related to the poor prognosis of cancer patients. CHI3L1 levels are often directly related to disease progression. Studies have shown that many malignancies including prostate cancer, colon cancer, rectal cancer, ovarian cancer, kidney cancer, breast cancer, glioblastoma, lymphoma and malignant melanoma overexpress CHI3L1 (M.K. Tanwar et al., Cancer Res., 62: 4364-4368, 2002; J.S. Johansen et al., Future Oncol., : 1065-1082, 2009). Overexpressed CHI3L1 can promote the proliferation, migration and invasion of cancer cells by regulating the cell cycle (C. Chen et al., The American journal of pathology, 179: 1494-1503, 2011; I.K. Choi et al., Acta Oncol., 49: 861-864, 2010; F.D. Coffman, Crit. Rev. Cl. Lab. Sci., 45: 531-562, 2008; A.F. Hottinger et al., Ann. Neurol., 70: 163-169, 2011; Y. Chiang et al., Oncotarget, 6: 39740, 2015).

CHI3L1 is not only highly expressed in tumor tissues, but also has a related role in tumor-related immune tissues. For example, it plays a role in T cell-mediated inflammation and

IL-13-mediated inflammation, sensitization, differentiation of M2 macrophages, and immunosuppression of TAMs (C.G. Lee et al., Annu. Rev. Physiol., 73: 479-501, 2011; Q. Zhang et al., Cancer Res., 75: 4312-4321, 2015; M. Bruchard et al., Nat. Immunol., 16: 859-870, 2015; A.G. Bais et al., J. Clin. Pathol., 58: 1096-1100, 2005).

CHI3L1 is a glycoprotein associated with extracellular matrix (ECM) which causes pulmonary fibrosis. CHI3L1 regulate the TGF-j signaling pathway and has kinase activity or activation kinase to phosphorylate SMAD2 and SMAD390 (D. Kim et al., Nat. Commun., 9: 1-14, 2018).

Based on the above mechanism of CHI3L1, an anti-CHI3L1 monoclonal antibody FRG has been produced. It has been verified by the C57BL/6J mouse melanoma lung metastasis model that the FRG monoclonal antibody significantly reduces the lung metastasis of melanoma and the development of primary lung cancer in terms of prevention and treatment (J.A. Elias et al., Methods and compositions relating to anti-CHI3L1 antibody reagents. In, Google Patents, 2019.)

Summary of the Present Invention

The present invention provides a bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein, a method for construction, expression, purification, and use of the bispecific antibody in the field of medicine and especially in the treatment of cancer.

The present invention adopts the following technical solutions:

The first aspect of the present invention provides a bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein.

Preferably, the bispecific antibody includes an anti-AGR2 antibody 18A4Hu as a parent structure, and an anti-CHI3L1 antibody FRGscFv fragment connected to a C-terminus of a light chain of the anti-AGR2 antibody 18A4Hu.

Preferably, the amino acid sequence of 18A4Hu heavy chain variable region is set forth in SEQ ID NO.16.

Preferably, the amino acid sequence of 18A4Hu light chain variable region is set forth in SEQ ID NO.17.

Preferably, the amino acid sequence of FRGscFv heavy chain variable region is set forth in SEQ ID NO.18.

Preferably, the amino acid sequence of FRGscFv light chain variable region is set forth in SEQ ID NO.19.

A second aspect of the present invention provides a method for constructing a bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein, including connecting a sequence of an anti-CHI3L1scFv to a C-terminus of a light chain fragment of an anti-AGR2 antibody through a Linker1, and internally connecting the anti-CHI3L1scFv via a Linker2.

Preferably, the nucleotide sequence of the anti-AGR2 antibody variable heavy chain is set forth in SEQ ID NO.1, the nucleotide sequence of anti-AGR2 antibody variable light chain is set forth in SEQ ID NO.2, the nucleotide sequence of anti-AGR2 antibody is set forth in SEQ ID NO.3, the nucleotide sequence of anti-CHI3L1scFv variable heavy chain is set forth in SEQ ID NO.4, the nucleotide sequence of anti-CHI3L1scFv variable light chain is set forth in SEQ ID NO.5, The nucleotide sequence of anti-CHI3L1scFv is set forth in SEQ ID NO.6, the nucleotide sequence of the Linker1 is set forth in SEQ ID NO.7, and the nucleotide sequence of Linker2 is set forth in SEQ ID NO.8.

Preferably, the anti-AGR2 antibody is 18A4Hu, and the anti-CHI3L1scFv is FRGscFv.

Preferably, the primers for constructing the bispecific antibody include a primer L-FRG-F having a nucleotide sequence as set forth in SEQ ID NO. 11, a primer L-VECTOR-R having a nucleotide sequence as set forth in SEQ ID NO. 12, a primer L-VECTOR-F having a nucleotide sequence as set forth in SEQ ID NO. 13, and a primer L-FRG-R having a nucleotide sequence as set forth in SEQ ID NO. 14.

A third aspect of the present invention provides a method for expressing a bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein, including expressing the bispecific antibody in Expi293F TM cell and secreting the bispecific antibody into a culture medium.

Preferably, the method for expressing the bispecific antibody according to further including selecting a secretory expression signal peptide.

Preferably, a nucleotide sequence of the secretory expression signal peptide for the heavy chain is set forth in SEQ ID NO.9, and a nucleotide sequence of the secretory expression signal peptide for the light chain is set forth in SEQ ID NO.10.

A fourth aspect of the present invention provides a method for purifying the bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein, including purifying the bispecific antibody by Protein G.

A fifth aspect of the present invention provides the use of a bispecific antibody having a function of binding to AGR2 protein and CHI3L1 protein in the treatment of cancer.

Brief Description of the Drawings

Fig. 1 shows the schematic diagram of AGR2-CHI3LBsAb-BFLA.

Fig. 2 shows the AGR2-CHI3L1BsAb-BFLA plasmid construction. A. Insert fragment FRGscFv PCR amplification. B. 18A4Hu vector amplification.

Fig. 3AB show the expression of EGFP after 24 hours.

Fig. 3CDE show a comparison of different puromycin concentration to cell screening.

Fig.3F shows the preliminary confirmation of antibody size.

Fig. 3GHI show cell lines with high fluorescence intensity and high antibody expression.

Fig. 4 shows the characterization of AGR2-CHI3LBsAb-BFLA antibody.

Fig. 5 shows BFLA affinity to AGR2 and CHI3L1.

Fig. 6 shows MTT assay of AGR2, 18A4Hu and BFLA on the proliferation of B16-F1O cells.

Fig. 7 shows that AGR2-CHI3L1BsAb-BFLA inhibits H460 growth in the H460 and THP-1 co-cultured experiment.

Detailed Description of the Preferred Embodiments

The followings are embodiments of the characterization and methods of the present invention. Given the general description provided above, various other embodiments may be practiced.

Embodiment 1 Construction of AGR2-CHI3L1BsAb-BFLA eukaryotic expression vector

In the present invention, the Appended IgG bispecific antibody targeting the AGR2 protein and the CHI3L1 protein in the TME is named AGR2-CHI3LBsAb-BFLA.

1. AGR2-CHI3L1BsAb-BFLA insert fragment and vector fragment construction

The specific construction scheme of AGR2-CHI3L1BsAb-BFLA is: the sequence of anti-CHI3LscFv is connected to the C-terminus of the light chain fragment of anti-AGR2 through a linker (Linkerl), Anti-CHI3L1scFv is internally connected via linker2 (Linker2). The schematic diagram is shown in Fig. 1.

The nucleotide sequence of anti-AGR2 antibody variable heavy chain is shown as SEQ ID NO.1:

ATGGACTGGACCTGGAGCATCCTTTTCTTGGTGGCAGCAGCAACAGGTGCCCAC TCCCAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCTTCAGTGA AGGTTTCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACATGGACTGGGTTCGA CAGGCCCCTGGACAGGGCCTTGAGTGGATTGGAGATATTAATCCTAACTATGACACTAC TAGCTACAACCAGAAGTTCCAGGGCAGAGTGACAATGACTGTGGACAAGTCCACGAGC ACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACTGCAGTCTATTACTGTGC AAGATCGATGATGGGATATGGTTCCCCTATGGACTACTGGGGTCAAGGCACACTGGTCA CCGTCTCCTCA

The nucleotide sequence of anti-AGR2 antibody variable light chain is shown as SEQ ID NO.2:

ATGGACATGAGGGTTCCTGCTCAGCTCCTGGGACTCCTGCTGCTCTGGCTCCCA GGTGCCAGATGTGAGATTGTGCTGACACAGTCTCCTGCCACCTTATCTCTCTCTCCAGG GGAGAGGGCCACCCTGAGCTGCAGGGCCAGCAAGAGTGTCAGTACATCTGGCTATAGT TATATGCACTGGTACCAACAGAAACCAGGCCAGGCCCCCAGACTCCTCATCTATCTTGC ATCTAACCTAGAATCTGGGATCCCTGCTAGATTCAGTGGCAGTGGGTCTGGGACAGACT TCACACTCACCATCAGCAGGCTGGAACCTGAGGACTTCGCTGTGTATTACTGTCAGCAC ATTAGAGAGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA

The nucleotide sequence of anti-AGR2 antibody is shown as SEQ ID NO.3:

ATGGACATGAGGGTTCCTGCTCAGCTCCTGGGACTCCTGCTGCTCTGGCTCCCA GGTGCCAGATGTGAGATTGTGCTGACACAGTCTCCTGCCACCTTATCTCTCTCTCCAGG GGAGAGGGCCACCCTGAGCTGCAGGGCCAGCAAGAGTGTCAGTACATCTGGCTATAGT TATATGCACTGGTACCAACAGAAACCAGGCCAGGCCCCCAGACTCCTCATCTATCTTGC ATCTAACCTAGAATCTGGGATCCCTGCTAGATTCAGTGGCAGTGGGTCTGGGACAGACT TCACACTCACCATCAGCAGGCTGGAACCTGAGGACTTCGCTGTGTATTACTGTCAGCAC ATTAGAGAGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGAACTG TGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTG CCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAG GTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA ACACAAACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAG AGCTTCAACAGGGGAGAGTGTATGGACTGGACCTGGAGCATCCTTTTCTTGGTGGCAGC AGCAACAGGTGCCCACTCCCAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAG CCTGGAGCTTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACATTCACTGACTACAA CATGGACTGGGTTCGACAGGCCCCTGGACAGGGCCTTGAGTGGATTGGAGATATTAATC CTAACTATGACACTACTAGCTACAACCAGAAGTTCCAGGGCAGAGTGACAATGACTGT GGACAAGTCCACGAGCACAGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACT GCAGTCTATTACTGTGCAAGATCGATGATGGGATATGGTTCCCCTATGGACTACTGGGG TCAAGGCACACTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCC TGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCAGCCCTGGGCTGCCTGGTCAA GGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGC GTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT GACCGTGCCCTCCAGCAGCTTGGACACCCAGACCTACATCTGCAACGTGAATCACAAG CCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACA CATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGT GGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGG CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA ACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT CCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAA GAGCCTCTCCCTGTCTCCGGGTAAATAG

The nucleotide sequence of Anti-CHI3LscFv variable heavy chain is shown as SEQ ID NO.4: CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCCGTGAAGG TGAGCTGCAAGGCTTCCGGCTATACCTTTACCAATTATGGCATGGATTGGGTGAGGCAG

GCTCCCGGCCAGGGCTTGGAGTGGATCGGCGATATCAATACCTACACCGGCGAGCCTA GCTACAACCAGAAGTTCCAGGGCAGGGTGACCATGACCGTGGACAAGAGCACCAGCAC CGCCTATATGGAGCTGAGCTCCCTGAGGTCCGAGGACACCGCCGTGTACTATTGCGCTA GGCTGGGCTATGGCAAGTTTTACGTGATGGACTATTGGGGCCAGGGCACCCTGGTGACC GTGAGCTCCGCCAGCACCAAGGGC

The nucleotide sequence of Anti-CHI3LscFv variable light chain is shown as SEQ ID NO.5:

GAGATCGTGCTGACCCAGTCCCCCGCTACCCTGTCCCTGAGCCCCGGAGAGCG GGCTACCCTGAGCTGTCGGGCCAGCCAGTCCCTGGTGCACTCCAATGGCAATACCTATA TGCACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTCGGCTGCTGATCTATAAGGTGAGC AATCTGGAGAGCGGCATCCCTGCCCGGTTTTCCGGCAGCGGCAGCGGAACCGACTTCAC CCTGACCATCTCCAGGCTGGAGCCTGAGGATTTCGCTGTGTACTACTGTAGCCAGAGCA CCCACGTGACCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTGGC TTGA

The nucleotide sequence of Anti-CHI3L1scFv is shown as SEQ ID NO.6:

CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCTTCCGT GAAGGTGAGCTGCAAGGCTTCCGGCTATACCTTTACCAATTATGGCATGGATTGGGTGA GGCAGGCTCCCGGCCAGGGCTTGGAGTGGATCGGCGATATCAATACCTACACCGGCGA GCCTAGCTACAACCAGAAGTTCCAGGGCAGGGTGACCATGACCGTGGACAAGAGCACC AGCACCGCCTATATGGAGCTGAGCTCCCTGAGGTCCGAGGACACCGCCGTGTACTATTG CGCTAGGCTGGGCTATGGCAAGTTTTACGTGATGGACTATTGGGGCCAGGGCACCCTGG TGACCGTGAGCTCCGCCAGCACCAAGGGCGAGATCGTGCTGACCCAGTCCCCCGCTAC CCTGTCCCTGAGCCCCGGAGAGCGGGCTACCCTGAGCTGTCGGGCCAGCCAGTCCCTGG TGCACTCCAATGGCAATACCTATATGCACTGGTATCAGCAGAAGCCCGGCCAGGCTCCT CGGCTGCTGATCTATAAGGTGAGCAATCTGGAGAGCGGCATCCCTGCCCGGTTTTCCGG CAGCGGCAGCGGAACCGACTTCACCCTGACCATCTCCAGGCTGGAGCCTGAGGATTTCG CTGTGTACTACTGTAGCCAGAGCACCCACGTGACCTGGACCTTCGGCGGCGGCACCAAG CTGGAGATCAAGAGGACCGTGGCTTGA

Anti-CHI3LscFv is linked with anti-AGR2 light chain fragment C terminal by Linker1. The nucleotide sequence of Linker1 is shown as SEQ ID NO.7:

GTGGAGGGAGGCTCCGGAGGATCTGGCGGCTCCGGAGGTAGCGGCGGAGTGGA T

Anti-CHI3LIscFv is internally connected via linker2 (Linker2). The nucleotide sequence of Linker2 is shown as SEQ ID NO.8:

GGCGGAGGAGGAAGCGGCGGAGGAGGCTCTGGCGGAGGAGGTAGCGGAGGCG GAGGCTCT

In order to make a bispecific antibody expressed in Expi293FTM cell and successfully secreted into the culture medium, the signal peptide expressed by antibody secretion was selected for this example.

The nucleotide sequence of the secretory expression signal peptide for the heavy chain is shown as SEQ ID NO.9:

ATGGACATGAGGGTTCCTGCTCAGCTCCTGGGACTCCTGCTGCTCTGGCTCCCA GGTGCCAGATGT

The nucleotide sequence of the secretory expression signal peptide for the light chain is shown as SEQ ID NO.10:

ATGGACTGGACCTGGAGCATCCTTTTCTTGGTGGCAGCAGCAACAGGTGCCCAC TCC

2. Construction of AGR2-CHI3L1BsAb-BFLA eukaryotic expression vector

Construction and expression of the bispecific antibody, the anti-AGR2 antibody 18A4Hu with patent authorization W02013/004076 was selected as the parent structure, and the anti-CHI3L1 antibody FRGscFv fragment was connected to the C-terminus of the 18A4Hulight chain. In order to construct the bispecific antibody, the primers shown in Table 1 were designed respectively. All the primers and the gene template required for amplification were synthesized by Shanghai Sunny Biotechnology Co., Ltd.

Table 1. Primers used in bispecific antibody gene cloning primer sequence SEQ ID L-FRG-F CACAAAGAGCTTCAACAGGGGAGA 11 GTGTGTGGAGGGAGGCTCCGGAGG L-VECTOR-R CCTCCGGAGCCTCCCTCCACAC 12 ACTCTCCCCTGTTGAAGCTCTTTGTG L-VECTOR-F CTGGAGATCAAGAGGACCGTG 13 GCTTGAGGATCCGCCCCTCTCC L-FRG-R GGAGAGGGGCGGATCCTCAAG CCACGGTCCTCTTGATCTCCAG

For the cloning of AGR2-CHI3L1BsAb-BFLA, the second antibody scFv was directionally connected with the C-terminus of 18A4Hu light chain by homologous recombination. First, primers L-FRG-F and L-FRG-R were used to amplify FRGscFv fragments, and then primers L-VECTOR-F and L-VECTOR-R were used to amplify anti-AGR2 antibody 18A4Hu vector fragments. After amplification, NovoRec@ plus One-step PCR Cloning Kit (Novoprotein Scientific Inc, NR05) was used to recombine FRGscFv fragments and 18A4Hu vector fragments. Splicing FRGscFv and 18A4Hu vectors to construct new bispecific antibody plasmids, transforming E. coli DH5a, extracting a mini-preparation of recombinant plasmids to identify the size, and using PCR to identify positive clones, and choosing the positive recombinant plasmids for sequencing and identification. Through sequencing, it was found that the full-length gene sequence of anti-AGR2-CHI3L1 was correct and all were in line with expectations.

Complementary nucleotide sequence of AGR2-CHI3L1BsAb-BFLA light chain is shown as SEQ ID NO.15:

ATGGACATGAGGGTTCCTGCTCAGCTCCTGGGACTCCTGCTGCTCTGGCTCCCA GGTGCCAGATGTGAGATTGTGCTGACACAGTCTCCTGCCACCTTATCTCTCTCTCCAGGG GAGAGGGCCACCCTGAGCTGCAGGGCCAGCAAGAGTGTCAGTACATCTGGCTATAGTTA TATGCACTGGTACCAACAGAAACCAGGCCAGGCCCCCAGACTCCTCATCTATCTTGCATC TAACCTAGAATCTGGGATCCCTGCTAGATTCAGTGGCAGTGGGTCTGGGACAGACTTCAC ACTCACCATCAGCAGGCTGGAACCTGAGGACTTCGCTGTGTATTACTGTCAGCACATTAG AGAGCTTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGAACTGTGGCT GCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCT GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGA TAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA AACTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTC AACAGGGGAGAGTGTGTGGAGGGAGGCTCCGGAGGATCTGGCGGCTCCGGAGGTAGCG GCGGAGTGGATCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGC TTCCGTGAAGGTGAGCTGCAAGGCTTCCGGCTATACCTTTACCAATTATGGCATGGATTG GGTGAGGCAGGCTCCCGGCCAGGGCTTGGAGTGGATCGGCGATATCAATACCTACACCG GCGAGCCTAGCTACAACCAGAAGTTCCAGGGCAGGGTGACCATGACCGTGGACAAGAG CACCAGCACCGCCTATATGGAGCTGAGCTCCCTGAGGTCCGAGGACACCGCCGTGTACT ATTGCGCTAGGCTGGGCTATGGCAAGTTTTACGTGATGGACTATTGGGGCCAGGGCACCC TGGTGACCGTGAGCTCCGCCAGCACCAAGGGCGGCGGAGGAGGAAGCGGCGGAGGAG GCTCTGGCGGAGGAGGTAGCGGAGGCGGAGGCTCTGAGATCGTGCTGACCCAGTCCCC CGCTACCCTGTCCCTGAGCCCCGGAGAGCGGGCTACCCTGAGCTGTCGGGCCAGCCAGT CCCTGGTGCACTCCAATGGCAATACCTATATGCACTGGTATCAGCAGAAGCCCGGCCAGG CTCCTCGGCTGCTGATCTATAAGGTGAGCAATCTGGAGAGCGGCATCCCTGCCCGGTTTT CCGGCAGCGGCAGCGGAACCGACTTCACCCTGACCATCTCCAGGCTGGAGCCTGAGGAT TTCGCTGTGTACTACTGTAGCCAGAGCACCCACGTGACCTGGACCTTCGGCGGCGGCAC CAAGCTGGAGATCAAGAGGACCGTGGCTTGA

Complementary nucleotide sequence of AGR2-CHI3L1BsAb-BFLA heavy chain is shown as SEQ ID NO.16:

ATGGACTGGACCTGGAGCATCCTTTTCTTGGTGGCAGCAGCAACAGGTGCCCAC TCCCAGGTCCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCTTCAGTGA AGGTTTCCTGCAAGGCTTCTGGATACACATTCACTGACTACAACATGGACTGGGTTCGAC AGGCCCCTGGACAGGGCCTTGAGTGGATTGGAGATATTAATCCTAACTATGACACTACTA GCTACAACCAGAAGTTCCAGGGCAGAGTGACAATGACTGTGGACAAGTCCACGAGCAC AGCCTACATGGAGCTCAGCAGCCTGAGATCTGAGGACACTGCAGTCTATTACTGTGCAA GATCGATGATGGGATATGGTTCCCCTATGGACTACTGGGGTCAAGGCACACTGGTCACCG TCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCAGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT GACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCC TACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTG GACACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACA AGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT GAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCAT GATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACC AGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGC CCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTAC ACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAG CAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGA TGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAA TAG

Embodiment 2 Expression and purification of anti-AGR2-CHI3L1BsAb-BFLA

The recombinant plasmids sequenced correctly were arranged for large-scale extraction of plasmids and used for transfection of Expi293F TM cells. Bispecific antibody plasmid was stably transfected into Expi293F TM (Gibco, A14527). Transfection result can be evaluated by EGFP fluorescence shows in the Fig.3.

Stable transfection steps:

Plating before transfection: It is recommended to seed the cells 24 hours before transfection, which will provide normal cell metabolism and increase the possibility of foreign DNA intake.

The weight ratio of the plasmid to PEI is DNA: PEI = 1:3.

The PEI was added to the diluted DNA and mixed.

Incubate at room temperature for 15-20 minutes.

Pre-warmed DMEM without serum and antibiotics was added to the PEI-DNA mixture to the required volume.

The mixture was directly added to the cells along the wall.

PEI-DNA mixture was replaced with a complete cell culture medium containing 10% fetal bovine serum and antibiotics.

The expression of EGFP after 24 hours was detected by microscope to quantify the transfection efficiency (Fig.3AB).

After 48h, the transfected cells were treated with 2[g/ml puromycin medium to screen stable cell. Fig.3CDE is showing a comparison of different puromycin concentration to cell screening.

After 3 days of puromycin treatment, single bright fluorescent cells were picked from a large plate into a 96-well plate.

After culturing the cells in a 96-well plate for 3-5 days. Cells lysate from the 96-well plate showed the preliminary confirmation of antibody size (Fig.3F)

Single bright fluorescent cells were picked 3 round until cell lines with high fluorescence intensity and high antibody expression are selected (Fig.3GHI).

The amino acid sequence of 18A4Hu heavy chain variable region is shown as SEQ ID NO.17:

MDWTWSILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMD WVRQAPGQGLEWIGDINPNYDTTSYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYY CARSMMGYGSPMDYWGQGTLVTVSS

The amino acid sequence of 18A4Hu light chain variable region is shown as SEQ ID NO.18:

MDMRVPAQLLGLLLLWLPGARCEIVLTQSPATLSLSPGERATLSCRASKSVSTSGYS YMHWYQQKPGQAPRLLIYLASNLESGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQHIREL PRTFGGGTKLEIK

The amino acid sequence of FRGscFv heavy chain variable region is shown as SEQ ID NO.19:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMDWVRQAPGQGLEWIGDINTY TGEPSYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARLGYGKFYVMDYWGQG TLVTVSSASTKG

The amino acid sequence of FRGscFv light chain variable region is shown as SEQ ID NO.20:

EIVLTQSPATLSLSPGERATLSCRASQSLVHSNGNTYMHWYQQKPGQAPRLLIYKVS NLESGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCSQSTHVTWTFGGGTKLEIKRTVA*

FRGscFv is linked with 18A4Hu light chain fragment C terminal by Linker1. The amino acid sequence of Linker1 is shown as SEQ ID NO.21: VEGGSGGSGGSGGSGGVD

FRGscFv is internally connected via linker2 (Linker2). The amino acid sequence of Linker2 is shown as SEQ ID NO.22: GGGGSGGGGSGGGGSGGGGS

The amino acid sequence of AGR2-CHI3L1BsAb-BFLA heavy chain is shown as SEQ ID NO.23:

MDWTWSILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMD WVRQAPGQGLEWIGDINPNYDTTSYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYY CARSMMGYGSPMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLDTQTYICNVNHKPSNTKVDKKV EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

The amino acid sequence of AGR2-CHI3L1BsAb-BFLA light chain is shown as SEQ ID NO.24:

MDMRVPAQLLGLLLLWLPGARCEIVLTQSPATLSLSPGERATLSCRASKSVSTSGYS YMHWYQQKPGQAPRLLIYLASNLESGIPARFSGSGSGTDFTLTISRLEPEDFAVYYCQHIREL PRTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKLYACEVTHQGLSSPVTKSFNRGECVEGG SGGSGGSGGSGGVDQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMDWVRQAPGQG LEWIGDINTYTGEPSYNQKFQGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCARLGYGKFY VMDYWGQGTLVTVSSASTKGGGGGSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATL SCRASQSLVHSNGNTYMHWYQQKPGQAPRLLIYKVSNLESGIPARFSGSGSGTDFTLTISRLE PEDFAVYYCSQSTHVTWTFGGGTKLEIKRTVA*

Embodiment 3 Characterization of AGR2-CHI3L1BsAb-BFLA antibody.

Fig.4 depicts the characterization of AGR2-CHI3L1BsAb-BFLA antibody.

Fig.4A demonstrates purified antibody analysis in western blot. Fig.4BC depicts AGR2-CHI3L1BsAb-BFLA detection in denaturing and non-denaturing conditions.

The purified AGR2-CHI3L1BsAb-BFLA bispecific antibody was analyzed by western blot. The antibody showed double bands under reducing conditions. The theoretical molecular weight of the heavy chain recombinant protein is 51.9KD, and the theoretical molecular weight of the light chain is 55.4KD. The purified AGR2-CHI3L1BsAb-BFLA bispecific antibody was analyzed by SDS-PAGE. The antibody showed a single electrophoretic band under non-reducing conditions and concentration is around 2mg/mL<purified antibody<1mg/mL.

Purification of AGR2-CHI3LIBsAb-BFLA by Protein G

Stable cells were washed twice with 4ml xPBS/serum-free DMEM/OPTI-MEM to achieve the purpose of washing away the remaining serum.

Cells were added 6ml serum-free medium with antibiotic and puromycin, 37°C incubated 48h, purified the supernatant with Protein G.

Purification process:

The cell supernatant was collected and filtered with a 0.45 m filter to remove cell debris. ) The supernatant was mixed with binding buffer as 1:1 so that the salt concentration and pH of the supernatant are like the column material, which helps protein binding to the column material better.

The supernatant was added to the column and flow through supernatant was collected.

The column was washed with 15-3OmL binding buffer.

Antibody binding to the column was eluted by adding lml of elution buffer to the column with a pipette each time. The antibody was collected in lml tube E1-E7 containing 100L of pre-cooled neutralizing solution.

Reconstitute the column with 20ml of binding buffer.

Embodiment 4 Sensitivity and specificity of AGR2-CHI3L1BsAb-BFLA against AGR2 and CHI3L1 detected by Western blot

AGR2-CHI3L1BsAb-BFLA as a primary antibody successfully detected AGR2 and CHI3L1.

Embodiment 5 In-vitro study shows that AGR2-CHI3L1BsAb-BFLA inhibits cancer cell growth

1. Fig. 6 depicts the MTT assay of AGR2, 18A4Hu and BFLA on the proliferation of B16-F10 cells.

2. Cell co-culture revealed that antibodies inhibit tumor cells growth effectively. H460(DiO green) and THP-1(DiL red) cocultured experiment shows that immune cells could inhibit the proliferation of cancer cells with the help of antibodies.

Where any or all of the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components. A reference herein to a patent document or any other matter identified as prior art, is not to be taken as an admission that the document or other matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Claims

The claims defining the invention are as follows:

1. Abispecific antibody, wherein the bispecific antibody has a function of binding toAGR2 protein and CHI3L1 protein.

2. The bispecific antibody according to claim 1, wherein the bispecific antibody comprises an anti-AGR2 antibody 18A4Hu as a parent structure, and an anti-CHI3L1 antibody FRGscFv fragment connected to a C-terminus of a light chain of the anti-AGR2 antibody 18A4Hu.

3. A method for constructing the bispecific antibody according to claim 1, comprising connecting a sequence of an anti- CHI3L1scFv to a C-terminus of a light chain fragment of an anti-AGR2 antibody through a Linker1, and internally connecting the anti-CHI3L1scFv via a Linker2.

4. The method according to claim 3, wherein a nucleotide sequence of the anti-AGR2 antibody variable heavy chain is set forth in SEQ ID NO.1, a nucleotide sequence of the anti-AGR2 antibody variable light chain is set forth in SEQ ID NO.2, a nucleotide sequence of the anti-AGR2 antibody is set forth in SEQ ID NO.3, a nucleotide sequence of the anti-CHI3L1scFv variable heavy chain is set forth in SEQ ID NO.4, a nucleotide sequence of the anti-CHI3L1scFv variable light chain is set forth in SEQ ID NO.5, and a nucleotide sequence of the anti-CHI3L1scFv is set forth in SEQ ID NO.6.

5. The method according to claim 3, wherein the nucleotide sequence of the Linker1 is set forth in SEQ ID NO.7, and the nucleotide sequence of the Linker2 is set forth in SEQ ID NO.8.

6. The method according to claim 3, wherein the anti-AGR2 antibody is 18A4Hu, and the anti-CHI3L1scFv is FRGscFv.

7. The method according to claim 6, wherein the primers for constructing the bispecific antibody comprises a primer L-FRG-F having a nucleotide sequence as set forth in SEQ ID NO. 11, a primer L-VECTOR-R having a nucleotide sequence as set forth in SEQ ID NO. 12, a primer L-VECTOR-F having a nucleotide sequence as set forth in SEQ ID NO. 13, and a primer L-FRG-R having a nucleotide sequence as set forth in SEQ ID NO. 14.

8. A method for expressing the bispecific antibody according to claim 1, comprising expressing the bispecific antibody in Expi293F TM cell and secreting the bispecific antibody into a culture medium.

9. The method according to claim 8, further comprising selecting a secretory expression signal peptide, wherein a nucleotide sequence of the secretory expression signal peptide for the heavy chain is set forth in SEQ ID NO.9, and a nucleotide sequence of the secretory expression signal peptide for the light chain is set forth in SEQ ID NO.10.

10. A method for purifying the bispecific antibody according to claim 1, comprising purifying the bispecific antibody by Protein G.

11. Use of the bispecific antibody according to claim 1 in the treatment of cancer.

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