Advanced Materials : Construction of a Novel Bispecific Antibody to Enhance Antitumor Activity against Lung Cancer

Construction of a Novel Bispecific Antibody to Enhance

Antitumor Activity against Lung Cancer

Wei Yin, Junjie Zhu, Diego Gonzalez-Rivas, Meinoshin Okumura, Gaetano Rocco,Harvey Pass, Gening Jiang, and Yang Yang*

HER2 and VEGF are closely related to the progression of several tumors. The inhibitor simultaneously targeting these two proteins will effectively inhibit the progression of tumors. Here, a bispecific antibody, termed as YY0411, targeting both HER2 and VEGF as a potent anticancer therapeutic antibody is reported. YY0411 is the first bispecific antibody constructed in IgG-Decoy receptor format. It efficiently identifies and combines both HER2 and VEGF protein. YY0411 is believed to be a candidate tumor suppressor as it significantly inhibits the colony formation ability of human cancer cells (Calu-3, MDA-MB-453, and NCI-N87 cells). The phosphorylation of HER2 and VEGF downstream components are also decreased in these cells with the treatment of YY0411. Similar to other antibodies, YY0411 has the ability to promote the secretion of IFN-γ by T lymphocytes. In addition, YY0411 significantly inhibits the growth of Calu-3 cells-induced xenograft in nude mice. This work demonstrates that YY0411 may be a potential anti-lung cancer drug.

The cancer burden induced by lung cancer is rapidly growing. Although advanced progression on diagnosis and treatment has been achieved, it is still the leading cause of cancer death around the world.[1a,b,2a] Resection is the best choice for early staged patients, while some of them have a risk of recurrence.[2b,c,3a] Chemotherapy, which is applied for patients with lesions in advanced stages, is restricted with the drug- resistance and side effect.[2d,e] Therefore, there is still an urgent need to develop effective therapeutic drugs.
   With the implementation of tobacco control measures, smoking-driven lung cancer has been decreasing. Biological studies indicate that dysregulation of cell signaling pathway has become the main cause of lung cancer.[4,5] The focus of research and development of anti- lung cancer drugs has also shifted to target the critical components of aberrant signaling pathways. Tumor-associated antigens (TAAs) in critical signaling pathway which can be recognized by immunocyte provide potential targets for treatment.[2f,g] A number of monoclonal antibodies have been approved for marketing. Although monoclonal antibody develops a high specificity inhibitory effect on cancer,[2h,i] defects such as off- target cannot be ignored. Bispecific anti- body (BsAb) is a recombinant antibody which has two kinds of specific antigen binding sites. It has enhanced binding specificity with cancer cells as it can simultaneously bind to different antigens. Com- pared to monoclonal antibody, bispecific antibody has a higher therapeutic effect. Currently, there are already two bispecific antibodies, Catumaxomab (Trion Pharma) and Blinatumomab (Amgen lnc.), which are approved by European Medicines Agency (EMA) and Food and Drug Administration (FDA). Catumaxomab targets both CD3 and Epithelial cell adhesion molecule (EPCAM), while Amgen targets CD19 and CD13.


Dr. W. Yin
Key laboratory of Oral Biomedical engineering of Ministry of education Hospital of Stomatology
School of Stomatology Wuhan University Wuhan 430079, China
J. Zhu, Dr. D. Gonzalez-Rivas, Prof. G. Jiang, Dr. Y. Yang Department of Thoracic Surgery
Shanghai Pulmonary Hospital
507 Zhengmin Road, Shanghai 200433, China E-mail: timyangsh@tongji.edu.cn
Dr. D. Gonzalez-Rivas
Department of thoracic surgery and Minimally Invasive Thoracic Surgery Unit (UCTMI).
Coruña University Hospital, Coruña 15706, Spain

Prof. M. Okumura Hospital Director
Toneyama National Hospital Osaka 560-8552, Japan
Prof. G. Rocco Thoracic Service Department of Surgery
Memorial Sloan-Kettering Cancer Center NY 10065, USA
Prof. H. Pass
Department of Cardiothoracic Surgery NYU Langone Medical Center
NY 10016, USA
Dr. Y. Yang
Institute for Advanced Study Tongji University
1239 Siping Road, Shanghai 200430, China

この論文著者のORCID識別番号は、https://doi.org/10.1002/adma.201805437にある。

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adma.201805437.


   In order to develop a more powerful anti-lung cancer drug, we analyzed the expression profiles of lung cancer patients from Department of Thoracic Surgery, Shanghai Pulmonary Hospital affiliated with Tongji University. Our data suggested that overexpression of HER2 was detected in 29.8% lung cancer patients, while VEGF was expressed in 60.1% lung cancer patients (unpublished data). It is well known that overexpression of HER2 is closely related to the progression of breast cancer, ovarian cancer, and lung cancer.[2j–l] Cancer with high expression of HER2 has strong metastatic and invasive ability, while the sensitivity to chemotherapy of HER2 overexpressed cancer is poor. VEGF is an effective angiogenesis stimulating factor which is of great significance in the progression and metastasis of cancer.[3b] It positively expresses in lung cancer and other cancers.[2m] VEGF-targeted cancer angiogenesis inhibitors can efficiently inhibit angiogenesis, block the nutrition supply for cancer, and limit the progression and metastasis of cancer.[2n] Therefore, we aimed to construct a bispecific anti- body targeting both HER2 and VEGF.
  Considering that HER2 and VEGF monoclonal antibodies have been used in clinical practice for many years, we first synthesized the available monoclonal antibody sequence of HER2 and VEGF, respectively. We prepared them as intermediates in immunoglobulin(IgG)-like form or single-chain antibody form (scFv). However, these were all failed in passing transient expression and antibody binding test. In detail, after transiently transfected into mammalian cells, the recognition ability of scFv form of HER2 antibody decreased by twofold to fivefold. Moreover, protein precipitation occurred during the purification dialysis.
  The bispecific antibody containing scFV form of VEGF antibody was not expressed. The problem in expression was not improved even if the order of the heavy and light chains interchanged. Thus, bispecific antibody in the form of scFV was defective. We continued to try several other methods but results were not satisfactory to us.

Figure 1. Structure of simultaneous targeting of HER2 and VEGF bispecific antibody (YY0411). YY0411 is an IgG-DR form antibody. The light chain and heavy chain were linked by a flexible ligation short peptide.

Figure 2. a) YY0411 efficiently recognizes HER2. b) YY0411 efficiently recognizes VEGF. c,d) YY0411 shows high affinity with human HER2 or VEGF. e,f) YY0411 shows high affinity with human HER2 and VEGF.

   Vascular endothelial growth factor receptor (VEGFR) is a high-affinity receptor that specifically binds to VEGF and plays an important role in promoting VEGF-induced angiogenesis. Therefore, we  wondered  if  we could block VEGF signal by targeting VEGFR. Decoy receptor refers to a special receptor which shares the  similar  structure with a functional receptor. It binds to a ligand without signals transmission  ability  as its cytoplasmic region lacks necessary domain. It negatively regulates functional receptor through competitively binding to ligand. We synthesized VEGFR and linked  it to the IgG form of HER2 (Figure 1). Its transient  expression  level  was  increased to around 200 mg L-1, which was significantly better than the scFv form of antibody. In addition,  more  than  90%  purification  was achieved by Protein A affinity chromatography. Thus, it was advantageous for expression and purification when the C-terminus of bispecific antibody was VEGFR. Western blotting assay also confirmed that the bispecific antibody, which we named  it YY0411, efficiently identified both HER2 and VEGF protein (Figure 2a,b). Based on the abovementioned results, we concluded that YY0411 was the first bispecific antibody targeting both HER2 and VEGF. It was an IgG-Decoy receptor (IgG- DR) structure antibody. VEGF receptor sequence which had the ability of binding VEGF was linked with an IgG form of HER2 antibody by a flexible ligation short peptide, (GGGGS)4. Glycine (G) is the smallest amino acid in molecular weight  and shortest in side chain. It can increase the flexibility. While serine (S) is the most hydrophilic amino acid. The 3D structure analysis indicated that the fusion protein of HER2 and VEGFR remained independent and maintained its biological activity (Figure 1).
   After constructing the candidate bispecific molecule, we determined the binding ability of YY0411 with HER2 and VEGF. We first analyzed the ability of YY0411 to bind to a single target. As shown in Figure 2c,d, YY0411 had a target binding capacity comparable to commercially available HER2 or VEGF monoclonal antibodies. Next, we tested their ability to bind to both HER2 and VEGF. Only YY0411 recognized both targets in a dose-dependent manner (Figure 2e,f). Therefore, we concluded that YY0411 performed better in binding with HER2, VEGF, HER2-VEGF as well as VEGF-HER2 than commercially available HER2 and/or VEGF monoclonal antibody.
   Having determined the binding ability of YY0411, we further analyzed the effect of it on colony formation ability of HER2 positive cancer cells. As shown in Figure 3a, compared with the joint application of commercially available HER2 and VEGF monoclonal antibody, YY0411 significantly inhibited the colony formation of human cancer cells (Calu-3, MDA-MB-453, and NCI-N87 cells).
To precisely elaborate the role of YY0411 on tumor progression, we observed the effect of YY0411 on the growth of Calu-3 cells-induced xenograft in vivo. When tumor volume of xenograft reached 200 mm3, antibody (YY0411, commercial HER2 antibody, commercial VEGF antibody, and commercial HER2+VEGF antibody) was administered every three days through intraperitoneal injection. As shown in Figure 3b,although all of YY0411, HER2 antibody, and VEGF antibody exhibited inhibition on tumor growth, YY0411 was the most significant tumor inhibitor. Xenograft with the administration of YY0411 exhibited the smallest volumes.

Figure 3. a) YY0411 significantly inhibits colony formation ability of human NCI-N87, Calu-3, and MDA-MB-453 cancer cells. The significant difference between YY0411 group and joint application of HER2 and VEGF monoclonal antibody is observed (*P < 0.05). b) YY0411 decreases the tumorigenicity of Calu-3 cells in vivo. The significant difference between YY0411 group and other groups is observed (*P < 0.05).

Figure 4. a) YY0411 inhibits the activation of HER2- and VEGF-mediated signaling pathways. b) YY0411 promotes human T lymphocytes to secret IFN-γ (*P < 0.05).

   After observing its antitumor effect, we further analyzed the molecular mechanism underlying this effect. We detected the activation of downstream molecules of HER2 and VEGF. In HUVEC cells, the phosphorylation of VEGFR2 was strongly inhibited by YY0411, while in Calu-3 cells, the activation of AKT decreased with the application of YY0411 (Figure 4a). The inhibitory ability of YY0411 on HER2 and VEGF downstream molecules was significantly stronger than the two monoclonal antibodies alone or in combination. Considering that antibodies tended to stimulate and activate host immune cells, we also investigated the level of IFN-γ secretion by T lymphocytes with or without the application of YY0411. Our findings confirmed that YY0411 promoted T lymphocytes to secrete IFN-γ (Figure 4b). IFN-γ plays a critical role in inhibiting the development and progression of several tumors. Therefore, we believed that our findings demonstrated that the ability of YY0411 in inhibiting tumors was comprehensive.
   In summary, we successfully constructed the first bispecific antibody targeting both HER2 and VEGF. YY0411 had potential application for both early stage and advanced stages patients. Considering the recent advancement of nanomaterials in diagnosis,[6a,b] for patients with early stage lesion, after determining genotype and expression level of HER2 and VEGF through real-time quantitative polymerase chain reaction (QPCR) and protein quantification with the fresh resected cancer tissues, patients with HER2 overexpression and VEGF expression would benefit from the application of YY0411. Furthermore, for patients with advanced stage lesion, YY0411 has the potential to improve the survival through blocking both HER2 and VEGF and inhibiting the progression of lesions.

Experimental Section

  Cells and Reagents: HUVEC, Calu-3, MDA-MB-453, and NCI-N87 cells were obtained from ATCC and cultured in mediums recommend by ATCC. Rabbit antibodies against VEGF, p-VEGFR, HER2, AKT, ρ-AKT(Ser308), ρ-AKT(Thr473), ρ-GSK3b, ρ-mTOR as well as mouse antibodies against β-actin (A2228 Sigma) were purchased from the indicated manufacturers.
 Generation of Bispecific Antibodies: After synthesizing DNA fragments encoding light chain and heavy chain of human HER2 and VEGFR, these DNA fragments were cloned into the pcDNA3.1 expression vector. The light chain and heavy chain DNA (mass ratio = 2:1) were transfected into HEK293T cells through calcium phosphate transfection. The candidate bispecific molecules were purified from the supernatant of the culture medium with affinity chromatography gel column (Protein A). The 3D structure of molecule was analyzed with GENO3D (https://geno3d-prabi.ibcp.fr/cgi-bin/geno3d_ automat.pl?page=/GENO3D/geno3d_home.html). The ability of the candidate bispecific molecules to recognize HER2 and VEGF were determined through western blotting assay in MDA-MB-453 and HUVEC cells.
 Binding Affinities to HER2 and VEGF: The ELISA assay was used to compare the binding affinities of candidate bispecific molecules with HER2 and VEGF. Human recombinant HER2 and/or VEGF were coated in 96-well plates for 20 h at 4 °C. Then it was blocked with PBS (including 1% bovine serum albumin, BSA) for 2 h at 37 °C and washed with PBS-T. The plates were incubated with either of YY0411, commercial HER2, commercial VEGF, or commercial HER2+VEGF antibody for 2 h at 37 °C following by 43 the peroxidase-conjugated antihuman IgG Fab antibody. After incubation with 3,3¢,5,5¢-tetramethylbenzidine (TMB) substrate reagent, the binding affinity was determined through detecting the absorbance at 450 nm.
 Cell Proliferation Assay: HUVEC, Calu-3, MDA-MB-453, and NCI-N87 cells were seeded on 96-well plates for 20 h. Then candidate bispecific molecules and commercial HER2 and/or VEGF antibody were added into culture medium. Cell proliferation was determined by CKK8 assays at day 4 following the recommended protocol.
 Activation of HER2 and VEGF Signaling Pathway: HUVEC and Calu-3 cells were seeded on 6-well plates for 20 h. Then candidate bispecific molecules and commercial HER2 and/or VEGF antibody were added into culture medium. HUVEC cells were stimulated with recombinant human VEGF. Calu-3 cells were stimulated with recombinant human HER2. Cells were lysed in lysis buffer, and phosphorylation of VEGFR2 and AKT were analyzed through western blotting.
 IFN-γ Expression Assay: T lymphocytes suspension was cultured with or without candidate bispecific molecules at 37 °C for 72 h. The concentration of IFN-γ was determined through ELISA kit.
 Xenograft Model: The athymic immunodeficient Balb/c nude mice, which were eight week old, were used to analyze the effect of YY0411 on the progression of Calu-3 cells-induced tumor. Calu-3 cells (1 x 107) cells were injected subcutaneously into the flank.
 Antibody (YY0411, 2 mg kg-1), commercial HER2 antibody (following manufacturer’s recommendation), commercial VEGF antibody (following manufacturer’s recommendation), and commercial HER2+VEGF antibody) was administered every three days through intraperitoneal injection when the tumor volume reached 200 mm3. Tumor volume was continually recorded every day. Specimens were harvested at day 21 post injection.
 Statistics: The tumor volume of mice xenograft was analyzed by two-way ANOVA. One-way ANOVA was used to analyze the results of cell viability assay, cell invasion assays, and colony formation assays. All data were analyzed by the SPSS package for Windows (Version 18.0, Chicago, IL). The P-value <0.05 was considered statistically significant.

Acknowledgements

 This work was supported by National Natural Science Foundation of China (81602412; 51872205; 81501750), Fundamental Research Funds for the Central Universities, Training plan of outstanding young medical talents, Shanghai Municipal Commission of Health and Family Planning (2017YQ050), Scientific research project of Shanghai Municipal Commission of Health and Family Planning (2016Y0121), Natura Scientific Foundation of Shanghai (134119b1002), Outstanding young scientific researcher of Shanghai pulmonary hospital, Natural Scientific Foundation of Hubei (2018002971272), and key young and middle-aged medical talents in Wuhan city. All animal experiments were approved by the Institutional Review Board (IRB) of Shanghai Pulmonary Hospital, and all protocols of animal studies conformed to the Guide for the Care and Use of Laboratory Animals.

Conflict of Interest

 Y.Y. and W.Y. are inventors on two patents (201810721654.1 and 15854645) that cover the findings discussed.

Keywords

bispecific antibodies, HER2, IgG-Decoy receptors, lung cancer, VEGF

Received: August 20, 2018
Revised: September 11, 2018
Published online:


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