Development and Anti-tumor Study of Bispecific Antibody Drug Conjugate Targeting TPBG and EGFR

doi:10.13523/j.cb.2212010

China Biotechnology

2023, Vol. 43

Issue (5): 11-23 DOI: 10.13523/j.cb.2212010

Development and Anti-tumor Study of Bispecific Antibody Drug Conjugate Targeting TPBG and EGFR

JIN Mei-qin^1,^2,³,SHANG Cheng-zhang³,SHEN Yue-lei^2,^3,^**(

)

1 College of Pharmacy, Anhui University of Chinese Medicine, Hefei 230012, China
2 Nantong Haimen Yangtze Delta Drug Advanced Research Institute, Nantong 226133, China
3 Biocytogen Pharmaceuticals (Beijing), Beijing 102600, China

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Abstract

Objective: To develop a bispecific antibody drug conjugate (BsADC) targeting TPBG×EGFR and to investigate its anti-tumor activity in vitro and in vivo. Methods: Fully human antibodies targeting the oncofetal trophoblast glycoprotein (TPBG, also known as 5T4) and the epidermal growth factor receptor (EGFR) were acquired from the RenLite platform, which is a novel mouse model expressing the entire human antibody variable region of the heavy chain and a specific common light chain. Anti-TPBG and EGFR antibodies were assembled into anti-TPBG× EGFR bispecific antibody (BsAb) by the knobs-into-holes (KIH) technique. Structural and functional characterization of anti-TPBG×EGFR BsAb was screened by Flow Cytometry (FCM), Surface Plasmon Resonance (SPR) and High Performance Liquid Chromatography (HPLC). Anti-TPBG×EGFR BsAb and the microtubule protein inhibitor MonoMethyl auristatin E (MMAE) were assembled using the cysteine coupling strategy to generate anti-TPBG×EGFR BsADC with a drug to antibody ration (DAR) of 4. Furthermore, the in vitro cell killing and in vivo anti-tumor activity of TPBG×EGFR BsADC were explored in human-derived tumor cell lines and cell derived xenograft (CDX) models. Results: According to the GEPIA2 database, TPBG is co-expressed with EGFR in a variety of tumours. Immunofluorescence analysis of five Patient-Derived Xenografts (PDX) showed that TPBG and EGFR are co-expressed in tumour cells in varying proportions. The bispecific antibodies targeting TPBG and EGFR with high-purity were successfully developed by the RenLite co-light chain fully human antibody discovery platform. In vitro results showed that by simultaneously targeting both TPBG and EGFR on the surface of tumor cells, the bispecific antibody significantly enhanced the binding affinity (avidity), endocytosis and killing of tumor cells compared to the TPBG parental monoclonal antibody. Moreover, in the A431 (EGFR^high/TPBG^low) model, anti-TPBG×EGFR BsADC showed stronger anti-tumor activity than the parental ADCs of TPBG and EGFR in vivo, demonstrating synergistic effects. In addition, in the NCI-H292 (EGFR^moderate/TPBG^low) and DU145 (EGFR^low/TPBG^low) models, anti-TPBG×EGFR BsADC also showed strong anti-tumor activity. Conclusion: These results suggest that combining TPBG with EGFR, a rapid internalizing ADC target, is a powerful strategy for enhancing anti-tumor activity of ADCs targeting TPBG.

Key words： Epidermal growth factor receptor(EGFR) Trophoblast glycoprotein(TPBG) Bispecific antibody drug conjugate Antitumor activity

Received: 04 December 2022 Published: 01 June 2023

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	JIN Mei-qin
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Cite this article:

JIN Mei-qin, SHANG Cheng-zhang, SHEN Yue-lei. Development and Anti-tumor Study of Bispecific Antibody Drug Conjugate Targeting TPBG and EGFR. China Biotechnology, 2023, 43(5): 11-23.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2212010 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I5/11

Table 1 Co-expression of TPBG and EGFR in tumors (GEPIA2,Datas in table are Median log values)

Fig.1 Expression and co-expression of TPBG and EGFR in PDX Different proportions of tumour cells co-stained with TPBG and EGFR are observed in two non-small cell lung cancers, two pancreatic cancers and one breast cancer PDX

Fig.2 anti-TPBG×EGFR BsAb assembly expression process and characterization (a)Process diagram for the construction of anti-TPBG×EGFR-BsADC. anti-TPBG × EGFR BsAb are constructed by the RenLite platform in combination with KIH technology and then coupled to vc-MMAE by partial reduction of interchain cysteines to obtain BsADC with a DAR of 4 (b) SDS-PAGE result (c) Molecular weight of anti-TPBG×EGFR-BsAb (d) Purity of anti-TPBG×EGFR-BsAb. Peak time of 4.215 min for anti-TPBG×EGFR-BsAb

Fig.3 Affinity of anti-TPBG×EGFR bispecific antibodies for TPBG and EGFR human antigens

Fig.4 HIC-HPLC and SEC-HPLC detection for anti-TPBG×EGFR BsADC BsADC (a) HIC-HPLC analysis of DAR value distribution of anti-TPBG × EGFR BsADC (b) SEC-HPLC analysis of SEC-HPLC analysis of anti-TPBG x EGFR BsADC purity

Fig.5 Binding activity of anti-TPBG×EGFR BsAb to multiple tumor cell lines Red:Isotype control;Blue: anti-TPBG-mAb;Orange:anti-EGFR-mAb;Green:anti-TPBG×EGFR-BsAb

Fig.6 Endocytotic activity of anti-TPBG×EGFR BsAb in A431, NCI-H226, NCI-H292 In all three cell lines, a different degree of improvement in BsAb endocytosis is observed compared to anti-TPBG mAb

Fig.7 Klling activity of anti-TPBG×EGFR BsADC in NCI-H292 and A431 The cells were treated with ADC for 3 days

Table 2 In vitro IC₅₀

Fig.8 In vivo efficacy data of anti-TPBG×EGFR BsADC in a CDX mouse model ^* P<0.05, ^** P<0.01, ^*** P<0.001, ^**** P<0.000 1;NS: P>0.05


[1]	Chau C H, Steeg P S, Figg W D. Antibody-drug conjugates for cancer. The Lancet, 2019, 394(10200): 793-804. doi: 10.1016/S0140-6736(19)31774-X

[2]	Hammood M, Craig A W, Leyton J V. Impact of endocytosis mechanisms for the receptors targeted by the currently approved antibody-drug conjugates (ADCs): a necessity for future ADC research and development. Pharmaceuticals (Basel, Switzerland), 2021, 14(7): 674.

[3]	Tong J T W, Harris P W R, Brimble M A, et al. An insight into FDA approved antibody-drug conjugates for cancer therapy. Molecules (Basel, Switzerland), 2021, 26(19): 5847. doi: 10.3390/molecules26195847

[4]	Zhao Y, Wang Y X. 5T 4 oncotrophoblast glycoprotein: Janus molecule in life and a novel potential target against tumors. Cellular & Molecular Immunology, 2007, 4(2): 99-104.

[5]	Alam S M K, Jasti S, Kshirsagar S K, et al. Trophoblast glycoprotein (TPBG/5T4) in human placenta: expression, regulation, and presence in extracellular microvesicles and exosomes. Reproductive Sciences (Thousand Oaks, Calif.), 2018, 25(2): 185-197.

[6]	Wan Y, Sapra P, Bolton J, et al. Combination treatment with an antibody-drug conjugate (A1mcMMAF) targeting the oncofetal glycoprotein 5T4 and carboplatin improves survival in a xenograft model of ovarian cancer. Targeted Oncology, 2019, 14: 465-477. doi: 10.1007/s11523-019-00650-8 pmid: 31332693

[7]	Shor B, Kahler J, Dougher M, et al. Enhanced antitumor activity of an anti-5T 4 antibody-drug conjugate in combination with PI3K/mTOR inhibitors or taxanes. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 2016, 22(2): 383-394. doi: 10.1158/1078-0432.CCR-15-1166

[8]	Shapiro G I, Vaishampayan U N, LoRusso P, et al. First-in-human trial of an anti-5T 4 antibody-monomethylauristatin conjugate, PF-06263507, in patients with advanced solid tumors. Investigational New Drugs, 2017, 35(3): 315-323. doi: 10.1007/s10637-016-0419-7

[9]	Smith R A, Zammit D J, Damle N K, et al. ASN004, A 5T4-targeting scFv-fc antibody-drug conjugate with high drug-to-antibody ratio, induces complete and durable tumor regressions in preclinical models. Molecular Cancer Therapeutics, 2021, 20(8): 1327-1337. doi: 10.1158/1535-7163.MCT-20-0565 pmid: 34045226

[10]	Andreev J, Thambi N, Perez Bay A E, et al. Bispecific antibodies and antibody-drug conjugates (ADCs) bridging HER2 and prolactin receptor improve efficacy of HER2 ADCs. Molecular Cancer Therapeutics, 2017, 16(4): 681-693. doi: 10.1158/1535-7163.MCT-16-0658 pmid: 28108597

[11]	Burgess A W. EGFR family: structure physiology signalling and therapeutic targets. Growth Factors (Chur, Switzerland), 2008, 26(5): 263-274. doi: 10.1080/08977190802312844

[12]	Wang Z X. ErbB receptors and cancer. Methods in Molecular Biology (Clifton, N J), 2017, 1652: 3-35.

[13]	Kazan J M, Pollato-Blanco A, Lukacs G L, et al. Measuring EGFR plasma membrane density, stability, internalization, and recycling in alive adherent cells by cell surface ELISA. STAR Protocols, 2022, 3(3): 101475. doi: 10.1016/j.xpro.2022.101475

[14]	Xu Y R, Lee J, Tran C, et al. Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system. mAbs, 2015, 7(1): 231-242. doi: 10.4161/19420862.2015.989013

[15]	Kozuki T. Skin problems and EGFR-tyrosine kinase inhibitor. Japanese Journal of Clinical Oncology, 2016, 46(4): 291-298. doi: 10.1093/jjco/hyv207 pmid: 26826719

[16]	Southall P J, Boxer G M, Bagshawe K D, et al. Immunohistological distribution of 5T 4 antigen in normal and malignant tissues. British Journal of Cancer, 1990, 61(1): 89-95. doi: 10.1038/bjc.1990.20 pmid: 2404511

[17]	李谦, 梁晓莹, 李国柱, 等. 双特异性抗体副产物去除策略. 中国生物工程杂志, 2022, 42(10): 60-69.

[17]	Li Q, Liang X Y, Li G Z, et al. Insight into the purification strategies for removing the byproducts of bispecific antibodies. China Biotechnology, 2022, 42(10): 60-69.

[18]	Schunselaar L M, Monkhorst K, van der Noort V, et al. Trophoblast glycoprotein is associated with a favorable outcome for mesothelioma and a target for antibody drug conjugates. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer, 2018, 13(10): 1577-1587. doi: 10.1016/j.jtho.2018.06.008

[19]	Stern P L, Harrop R. 5T 4 oncofoetal antigen: an attractive target for immune intervention in cancer. Cancer Immunology, Immunotherapy, 2017, 66(4): 415-426. doi: 10.1007/s00262-016-1917-3

[20]	Shim H. Bispecific antibodies and antibody-drug conjugates for cancer therapy: technological considerations. Biomolecules, 2020, 10(3): 360. doi: 10.3390/biom10030360

[21]	Yu J F, Fang T, Yun C Y, et al. Antibody-drug conjugates targeting the human epidermal growth factor receptor family in cancers. Frontiers in Molecular Biosciences, 2022, 9: 847835. doi: 10.3389/fmolb.2022.847835

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