Development of Plant-Derived Bispecific Monoclonal Antibody Targeting PD-L1 and CTLA-4 against Mouse Colorectal Cancer

Abstract

Checkpoint blockade immunotherapy has revolutionized cancer treatment, with monoclonal antibodies targeting immune checkpoints, yielding promising clinical benefits. However, with the advent of resistance to immune checkpoint inhibitor treatment in clinical trials, developing next-generation antibodies with potentially increased efficacy is critical. Here, we aimed to generate a recombinant bispecific monoclonal antibody for dual inhibition of programmed cell death protein 1/programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 axes. The plant system was used as an alternative platform for bispecific monoclonal antibody production. Dual variable domain immunoglobulin atezolizumab × 2C8 is a plant-derived bispecific monoclonal antibody that combines both programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 blockade into a single molecule. Dual variable domain immunoglobulin atezolizumab × 2C8 was transiently expressed in Nicotiana benthamiana and the expression level was determined to be the highest after 4 days of infiltration. The size and assembly of the purified bispecific monoclonal antibody were determined, and its function was investigated in vitro and in vivo. The molecular structures of plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 are as expected, and it was mostly present as a monomer. The plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 showed in vitro binding to programmed cell death ligand 1 and cytotoxic T-lymphocyte-associated protein 4 proteins. The antitumor activity of plant-produced bispecific monoclonal antibody was tested in vivo by treating humanized Balb/c mice bearing a CT26 colorectal tumor. Plant-produced dual variable domain immunoglobulin atezolizumab × 2C8 significantly inhibited tumor growth by reducing tumor volume and weight. Body weight changes indicated that the plant-produced bispecific monoclonal antibody was safe and tolerable. Overall, this proof of concept study demonstrated the viability of plants to produce functional plant-based bispecific immunotherapy.

Key words

Nicotiana benthamiana - Solanaceae - DVD-AT2C - bispecific antibody - checkpoint blockade - immunotherapy

• References

• 1 Shiravand Y, Khodadadi F, Kashani SMA, Hosseini-Fard SR, Hosseini S, Sadeghirad H, Ladwa R, OʼByrne K, Kulasinghe A. Immune checkpoint inhibitors in cancer therapy. Curr Oncol 2022; 29: 3044-3060
• 2 Twomey JD, Zhang B. Cancer immunotherapy update: FDA-approved checkpoint inhibitors and companion diagnostics. AAPS J 2021; 23: 39
• 3 Torka P, Barth M, Ferdman R, Hernandez-Ilizaliturri FJ. Mechanisms of resistance to monoclonal antibodies (mAbs) in lymphoid malignancies. Curr Hematol Malig Rep 2019; 14: 426-438
• 4 Chen S, Li J, Li Q, Wang Z. Bispecific antibodies in cancer immunotherapy. Hum Vaccin Immunother 2016; 12: 2491-2500
• 5 Rudd CE, Taylor A, Schneider H. CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol Rev 2009; 229: 12-26
• 6 Kotanides H, Li Y, Malabunga M, Carpenito C, Eastman SW, Shen Y, Wang G, Inigo I, Surguladze D, Pennello AL, Persaud K, Hindi S, Topper M, Chen X, Zhang Y, Bulaon DK, Bailey T, Lao Y, Han B, Torgerson S, Chin D, Sonyi A, Haidar JN, Novosiadly RD, Moxham CM, Plowman GD, Ludwig DL, Kalos M. Bispecific targeting of PD-1 and PD-L1 enhances T-cell activation and antitumor immunity. Cancer Immunol Res 2020; 8: 1300-1310
• 7 Dovedi SJ, Mazor Y, Elder M, Hasani S, Wang B, Mosely S, Jones D, Hansen A, Yang C, Wu Y, Achour I, Durham N, Browne G, Murray T, Hair J, Morrow M, Rainey G, Kunkel MJ, Gooya J, Freeman D, Herbst R, Wilkinson R. MEDI5752: A novel bispecific antibody that preferentially targets CTLA-4 on PD-1 expressing T-cells. Cancer Res 2018; 78: 2776
• 8 Shum E, Reilley M, Najjar Y, Daud A, Thompson J, Baranda J, Harvey RD, Shields A, Cohen E, Pant S, Leidner R, Mita A, Cohen R, Chmielowski B, Stein M, Hu-Lieskovan S, Fleener C, Ding Y, Bao L, Chollate S, Shorr J, Clynes R, Hickingbottom B. 523 Preliminary clinical experience with XmAb20717, a PD-1 x CTLA-4 bispecific antibody, in patients with advanced solid tumors. J Immunother Cancer 2021; 9: A553-A553
• 9 Millward M, Frentzas S, Gan HK, Prawira A, Tran B, Coward J, Jin X, Li B, Wang M, Kwek KY, Xia Y, Desai J. 1021O Safety and antitumor activity of AK104, a bispecific antibody targeting PD-1 and CTLA-4, in patients with mesothelioma which is relapsed or refractory to standard therapies. Ann Oncol 2020; 31: S705-S706
• 10 Berezhnoy A, Sumrow BJ, Stahl K, Shah K, Liu D, Li J, Hao SS, De Costa A, Kaul S, Bendell J, Cote GM, Luke JJ, Sanborn RE, Sharma MR, Chen F, Li H, Diedrich G, Bonvini E, Moore PA. Development and preliminary clinical activity of PD-1-guided CTLA-4 blocking bispecific DART molecule. Cell Rep Med 2020; 1: 100163
• 11 Richardson G, Kichenadasse G, Ganju V, Xu J, Van H, Kong P, Yang F, Wei Y, Lu Y, Guo K, Donato L, Xu T, Coward J. MA06.09 preliminary safety, efficacy results of KN046 (bispecific Anti-PD-L1/CTLA4) in subjects with rare thoracic tumors. J Thorac Oncol 2021; 16: S154-S155
• 12 Ma J, Mo Y, Tang M, Shen J, Qi Y, Zhao W, Huang Y, Xu Y, Qian C. Bispecific antibodies: From research to clinical application. Front Immunol 2021; 12: 626616
• 13 Komarova TV, Sheshukova EV, Kosobokova EN, Kosorukov VS, Shindyapina AV, Lipskerov FA, Shpudeiko PS, Byalik TE, Dorokhov YL. The biological activity of bispecific trastuzumab/pertuzumab plant biosimilars may be drastically boosted by disulfiram increasing formaldehyde accumulation in cancer cells. Sci Rep 2019; 9: 16168
• 14 Shanmugaraj B, I Bulaon CJ. Phoolcharoen W. Plant molecular farming: A viable platform for recombinant biopharmaceutical production. Plants (Basel) 2020; 9: 842
• 15 Komarova TV, Sheshukova EV, Dorokhov YL. Plant-made antibodies: Properties and therapeutic applications. Curr Med Chem 2019; 26: 381-395
• 16 Nandi S, Kwong AT, Holtz BR, Erwin RL, Marcel S, McDonald KA. Techno-economic analysis of a transient plant-based platform for monoclonal antibody production. MAbs 2016; 8: 1456-1466
• 17 Mir-Artigues P, Twyman RM, Alvarez D, Cerda Bennasser P, Balcells M, Christou P, Capell T. A simplified techno-economic model for the molecular pharming of antibodies. Biotechnol Bioeng 2019; 116: 2526-2539
• 18 Gleba YY, Tusé D, Giritch A. Plant viral vectors for delivery by Agrobacterium . Curr Top Microbiol Immunol 2014; 375: 155-192
• 19 Komarova TV, Baschieri S, Donini M, Marusic C, Benvenuto E, Dorokhov YL. Transient expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccines 2010; 9: 859-876
• 20 Rattanapisit K, Bulaon CJI, Strasser R, Sun H, Phoolcharoen W. In vitro and in vivo studies of plant-produced Atezolizumab as a potential immunotherapeutic antibody. Sci Rep 2023; 13: 14146
• 21 Bulaon CJI, Khorattanakulchai N, Rattanapisit K, Sun H, Pisuttinusart N, Strasser R, Tanaka S, Soon-Shiong P, Phoolcharoen W. Antitumor effect of plant-produced anti-CTLA-4 monoclonal antibody in a murine model of colon cancer. Front Plant Sci 2023; 14: 1149455
• 22 Rattanapisit K, Phakham T, Buranapraditkun S, Siriwattananon K, Boonkrai C, Pisitkun T, Hirankarn N, Strasser R, Abe Y, Phoolcharoen W. Structural and in vitro functional analyses of novel plant-produced anti-human PD1 antibody. Sci Rep 2019; 9: 15205
• 23 Phetphoung T, Malla A, Rattanapisit K, Pisuttinusart N, Damrongyot N, Joyjamras K, Chanvorachote P, Phakham T, Wongtangprasert T, Strasser R, Chaotham C, Phoolcharoen W. Expression of plant-produced anti-PD-L1 antibody with anoikis sensitizing activity in human lung cancer cells via., suppression on epithelial-mesenchymal transition. PLoS One 2022; 17: e0274737
• 24 Phakham T, Bulaon CJI, Khorattanakulchai N, Shanmugaraj B, Buranapraditkun S, Boonkrai C, Sooksai S, Hirankarn N, Abe Y, Strasser R, Rattanapisit K, Phoolcharoen W. Functional characterization of pembrolizumab produced in Nicotiana benthamiana using a rapid transient expression system. Front Plant Sci 2021; 12: 736299
• 25 Akbarzadeh-Sharbaf S, Yakhchali B, Minuchehr Z, Shokrgozar MA, Zeinali S. Expression enhancement in trastuzumab therapeutic monoclonal antibody production using genomic amplification with methotrexate. Avicenna J Med Biotechnol 2013; 5: 87-95
• 26 Brodzik R, Glogowska M, Bandurska K, Okulicz M, Deka D, Ko K, van der Linden J, Leusen JH, Pogrebnyak N, Golovkin M, Steplewski Z, Koprowski H. Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells. Proc Natl Acad Sci U S A 2006; 103: 8804-8809
• 27 Westerhof LB, Wilbers RHP, van Raaij DR, van Wijk CZ, Goverse A, Bakker J, Schots A. Transient expression of secretory IgA in planta is optimal using a multi-gene vector and may be further enhanced by improving joining chain incorporation. Front Plant Sci 2016; 6: 1200
• 28 Paul M, Reljic R, Klein K, Drake PM, van Dolleweerd C, Pabst M, Windwarder M, Arcalis E, Stoger E, Altmann F, Cosgrove C, Bartolf A, Baden S, Ma JK. Characterization of a plant-produced recombinant human secretory IgA with broad neutralizing activity against HIV. MAbs 2014; 6: 1585-1597
• 29 Esqueda A, Jugler C, Chen Q. Chapter Eleven – Design and expression of a bispecific antibody against dengue and chikungunya virus in plants. In: OʼDell WB, Kelman Z. eds. Meth enzymol. Academic Press; 2021: 223-238
• 30 Rosenberg AS. Effects of protein aggregates: An immunologic perspective. AAPS J 2006; 8: E501-E507
• 31 Fesinmeyer RM, Hogan S, Saluja A, Brych SR, Kras E, Narhi LO, Brems DN, Gokarn YR. Effect of ions on agitation- and temperature-induced aggregation reactions of antibodies. Pharm Res 2009; 26: 903-913
• 32 Amano M, Hasegawa J, Kobayashi N, Kishi N, Nakazawa T, Uchiyama S, Fukui K. Specific racemization of heavy-chain cysteine-220 in the hinge region of immunoglobulin gamma 1 as a possible cause of degradation during storage. Anal Chem 2011; 83: 3857-3864
• 33 Coward J, Ganju V, Behzadigohar R, Kwong K, Xu J, Van H, Kong P, Yang F, Chen L, Guo K, Liu M, Zhu D, Donato LK, Xu T, Richardson GE. Preliminary safety, efficacy, and pharmacokinetics (PK) results of KN046 (bispecific anti-PD-L1/CTLA4) from a first-in-human study in subjects with advanced solid tumors. J Clin Oncol 2019; 37: 2554
• 34 Liu Y, Zheng P. Preserving the CTLA-4 checkpoint for safer and more effective cancer immunotherapy. Trends Pharmacol Sci 2020; 41: 4-12
• 35 Pang X, Huang Z, Zhong T, Zhang P, Wang ZM, Xia M, Li B. Cadonilimab, a tetravalent PD-1/CTLA-4 bispecific antibody with trans-binding and enhanced target binding avidity. MAbs 2023; 15: 2180794
• 36 Jakob CG, Edalji R, Judge RA, DiGiammarino E, Li Y, Gu J, Ghayur T. Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig^™) molecule. MAbs 2013; 5: 358-363
• 37 Digiammarino EL, Harlan JE, Walter KA, Ladror US, Edalji RP, Hutchins CW, Lake MR, Greischar AJ, Liu J, Ghayur T, Jakob CG. Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design. MAbs 2011; 3: 487-494
• 38 Wu C, Ying H, Bose S, Miller R, Medina L, Santora L, Ghayur T. Molecular construction and optimization of anti-human IL-1alpha/beta dual variable domain immunoglobulin (DVD-Ig) molecules. MAbs 2009; 1: 339-347
• 39 Bulaon CJI, Sun H, Malla A, Phoolcharoen W. Therapeutic efficacy of plant-produced Nivolumab in transgenic C57BL/6-hPD-1 mouse implanted with MC38 colon cancer. Biotechnol Rep (Amst) 2023; 38: e00794
• 40 Sato Y, Fu Y, Liu H, Lee MY, Shaw MH. Tumor-immune profiling of CT-26 and Colon 26 syngeneic mouse models reveals mechanism of anti-PD-1 response. BMC Cancer 2021; 21: 1222
• 41 Wei SC, Levine JH, Cogdill AP, Zhao Y, Anang NAS, Andrews MC, Sharma P, Wang J, Wargo JA, Peʼer D, Allison JP. Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpoint blockade. Cell 2017; 170: 1120-1133.e1117
• 42 Zhang J, Yi J, Zhou P. Development of bispecific antibodies in China: Overview and prospects. Antib Ther 2020; 3: 126-145
• 43 Chen Q, He J, Phoolcharoen W, Mason HS. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum Vaccin 2011; 7: 331-338