Generation of TIM3 inhibitory single‑domain antibodies to boost the antitumor activity of chimeric antigen receptor T cells

Yang,Liu; Chen,Xin; Wang,Qian; Zhu,Yuankui; Wu,Changfa; Ma,Xuqian; Zuo,Dianbao; He,Huixia; Huang,Le; Li,Jingwen; Xia,Chunjiao; Hu,Sheng; Yang,Xiaoqing; Feng,Mingqian

doi:10.3892/ol.2021.12803

Generation of TIM3 inhibitory single‑domain antibodies to boost the antitumor activity of chimeric antigen receptor T cells

Authors:
- Liu Yang
- Xin Chen
- Qian Wang
- Yuankui Zhu
- Changfa Wu
- Xuqian Ma
- Dianbao Zuo
- Huixia He
- Le Huang
- Jingwen Li
- Chunjiao Xia
- Sheng Hu
- Xiaoqing Yang
- Mingqian Feng
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Affiliations: College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China, Clinical Testing Branch, Hongshan District Chinese Medicine Hospital, Wuhan, Hubei 430000, P.R. China, Department of Internal Medicine‑Oncology, Hubei Cancer Hospital, Wuhan, Hubei 430079, P.R. China, Clinical Laboratory, Hospital of Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China
Published online on: May 20, 2021 https://doi.org/10.3892/ol.2021.12803
Article Number: 542
Copyright: © Yang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Targeting inhibitory immune checkpoint molecules has significantly altered cancer treatment regimens. T cell immunoglobulin and mucin domain 3 (TIM3) is one of the major inhibitory immune checkpoints expressed on T cells. Blocking the engagement of TIM3 and its inhibitory ligand galectin‑9 may potentiate the effects of immunotherapy or overcome the adaptive resistance to the therapeutic blockade of programmed cell death protein 1, cytotoxic T‑lymphocyte‑associated protein 4, B‑ and T‑lymphocyte attenuator and lymphocyte‑activation gene 3, amongst others, as each of these immune checkpoints harbors unique properties that set it apart from the rest. Heavy chain variable fragment (VH)‑derived single‑domain antibodies (sdAbs) represent a class of expanding drug candidates. These sdAbs have unique advantages, including their minimal size in the antibody class, ease of expression, broad scope for modular structure design and re‑engineering, and excellent tumor penetration. In the present study, two sdAbs, TIM3‑R23 and TIM3‑R53, were generated by immunizing rabbits with the recombinant extracellular domain of TIM3 and applying phage display technology. These sdAbs were easily expressed in mammalian cells. The purified sdAbs were able to bind to both recombinant and cell surface TIM3, and blocked it from binding to the ligand galectin‑9. In vivo studies demonstrated that TIM3‑R53 was able to potentiate the antitumor activity of chimeric antigen receptor T cells that targeted mesothelin. In conclusion, the results of the present study suggested that TIM3‑R53 may be a novel and attractive immune checkpoint inhibitor against TIM3, which is worthy of further investigation.

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1	Hamid O, Robert C, Ribas A, Hodi FS, Walpole E, Daud A, Arance AS, Brown E, Hoeller C, Mortier L, et al: Antitumour activity of pembrolizumab in advanced mucosal melanoma: A post-hoc analysis of KEYNOTE-001, 002, 006. Br J Cancer. 119:670–674. 2018. View Article : Google Scholar : PubMed/NCBI
2	Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, Patnaik A, Aggarwal C, Gubens M, Horn L, et al: Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 372:2018–2028. 2015. View Article : Google Scholar : PubMed/NCBI
3	Motzer RJ, Escudier B, McDermott DF, George S, Hammers HJ, Srinivas S, Tykodi SS, Sosman JA, Procopio G, Plimack ER, et al: Nivolumab versus everolimus in advanced renal-cell carcinoma. N Engl J Med. 373:1803–1813. 2015. View Article : Google Scholar : PubMed/NCBI
4	Langer CJ, Gadgeel SM, Borghaei H, Papadimitrakopoulou VA, Patnaik A, Powell SF, Gentzler RD, Martins RG, Stevenson JP, Jalal SI, et al: Carboplatin and pemetrexed with or without pembrolizumab for advanced, non-squamous non-small-cell lung cancer: A randomised, phase 2 cohort of the open-label KEYNOTE-021 study. Lancet Oncol. 17:1497–1508. 2016. View Article : Google Scholar : PubMed/NCBI
5	Mcintire JJ, Umetsu SE, Akbari O, Potter M, Kuchroo VK, Barsh GS, Freeman GJ, Umetsu DT and Dekruyff RH: Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat Immunol. 2:1109–1116. 2001. View Article : Google Scholar : PubMed/NCBI
6	Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB and Kuchroo VK: The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol. 6:1245–1252. 2005. View Article : Google Scholar : PubMed/NCBI
7	Anderson AC, Anderson DE, Bregoli L, Hastings WD, Kassam N, Lei C, Chandwaskar R, Karman J, Su EW, Hirashima M, et al: Promotion of tissue inflammation by the immune receptor Tim-3 expressed on innate immune cells. Science. 318:1141–1143. 2007. View Article : Google Scholar : PubMed/NCBI
8	Ndhlovu LC, Lopez-Vergès S, Barbour JD, Jones RB, Jha AR, Long BR, Schoeffler EC, Fujita T, Nixon DF and Lanier LL: Tim-3 marks human natural killer cell maturation and suppresses cell-mediated cytotoxicity. Blood. 119:3734–3743. 2012. View Article : Google Scholar : PubMed/NCBI
9	Phong BL, Avery L, Sumpter TL, Gorman JV, Watkins SC, Colgan JD and Kane LP: Tim-3 enhances FcεRI-proximal signaling to modulate mast cell activation. J Exp Med. 212:2289–2304. 2015. View Article : Google Scholar : PubMed/NCBI
10	Han G, Chen G, Shen B and Li Y: Tim-3: An activation marker and activation limiter of innate immune cells. Front Immunol. 4:4492013. View Article : Google Scholar : PubMed/NCBI
11	Sabatos-Peyton CA, Nevin J, Brock A, Venable JD, Tan DJ, Kassam N, Xu F, Taraszka J, Wesemann L, Pertel T, et al: Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy. Oncoimmunology. 7:e13856902017. View Article : Google Scholar : PubMed/NCBI
12	Dolina JS, Braciale TJ and Hahn YS: Liver-primed CD8+ T cells suppress antiviral adaptive immunity through galectin-9-independent T-cell immunoglobulin and mucin 3 engagement of high-mobility group box 1 in mice. Hepatology. 59:1351–1365. 2014. View Article : Google Scholar : PubMed/NCBI
13	Markwick LJ, Riva A, Ryan JM, Cooksley H, Palma E, Tranah TH, Manakkat Vijay GK, Vergis N, Thursz M, Evans A, et al: Blockade of PD1 and TIM3 restores innate and adaptive immunity in patients with acute alcoholic hepatitis. Gastroenterology. 148:590–602.e10. 2015. View Article : Google Scholar : PubMed/NCBI
14	Khodabakhsh F, Behdani M, Rami A and Kazemi-Lomedasht F: Single-domain antibodies or nanobodies: A class of next-generation antibodies. Int Rev Immunol. 37:316–322. 2018. View Article : Google Scholar : PubMed/NCBI
15	Wesolowski J, Alzogaray V, Reyelt J, Unger M, Juarez K, Urrutia M, Cauerhff A, Danquah W, Rissiek B, Scheuplein F, et al: Single domain antibodies: Promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 198:157–174. 2009. View Article : Google Scholar : PubMed/NCBI
16	Elverdi T and Eskazan AE: Caplacizumab as an emerging treatment option for acquired thrombotic thrombocytopenic purpura. Drug Des Devel Ther. 13:1251–1258. 2019. View Article : Google Scholar : PubMed/NCBI
17	Scully M, Cataland SR, Peyvandi F, Coppo P, Knöbl P, Kremer Hovinga JA, Metjian A, de la Rubia J, Pavenski K, Callewaert F, et al: Caplacizumab treatment for acquired thrombotic thrombocytopenic purpura. N Engl J Med. 380:335–346. 2019. View Article : Google Scholar : PubMed/NCBI
18	Shinozaki N, Hashimoto R, Fukui K and Uchiyama S: Efficient generation of single domain antibodies with high affinities and enhanced thermal stabilities. Sci Rep. 7:57942017. View Article : Google Scholar : PubMed/NCBI
19	AVMA Panel on Euthanasia. American Veterinary Medical Association, . 2000 Report of the AVMA panel on euthanasia. J Am Vet Med Assoc. 218:669–696. 2001. View Article : Google Scholar : PubMed/NCBI
20	Feng M, Gao W, Wang R, Chen W, Man YG, Figg WD, Wang XW, Dimitrov DS and Ho M: Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma. Proc Natl Acad Sci USA. 110:E1083–E1091. 2013. View Article : Google Scholar : PubMed/NCBI
21	Zhang YF and Ho M: Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples. MAbs. 9:419–429. 2017. View Article : Google Scholar : PubMed/NCBI
22	Kaneko O, Gong L, Zhang J, Hansen JK, Hassan R, Lee B and Ho M: A binding domain on mesothelin for CA125/MUC16. J Biol Chem. 284:3739–3749. 2009. View Article : Google Scholar : PubMed/NCBI
23	Baghdadi M, Takeuchi S, Wada H and Seino K: Blocking monoclonal antibodies of TIM proteins as orchestrators of anti-tumor immune response. MAbs. 6:1124–1132. 2014. View Article : Google Scholar : PubMed/NCBI
24	Herrera-Camacho I, Anaya-Ruiz M, Perez-Santos M, Millán- Pérez Peña L, Bandala C and Landeta G: Cancer immunotherapy using anti-TIM3/PD-1 bispecific antibody: A patent evaluation of EP3356411A1. Expert Opin Ther Pat. 29:587–593. 2019. View Article : Google Scholar : PubMed/NCBI
25	Liu JF, Wu L, Yang LL, Deng WW, Mao L, Wu H, Zhang WF and Sun ZJ: Blockade of TIM3 relieves immunosuppression through reducing regulatory T cells in head and neck cancer. J Exp Clin Cancer Res. 37:442018. View Article : Google Scholar : PubMed/NCBI
26	Zhou G, Sprengers D, Boor PPC, Doukas M, Schutz H, Mancham S, Pedroza-Gonzalez A, Polak WG, de Jonge J, Gaspersz M, et al: Antibodies against immune checkpoint molecules restore functions of tumor-infiltrating T cells in hepatocellular carcinomas. Gastroenterology. 153:1107–1119.e10. 2017. View Article : Google Scholar : PubMed/NCBI
27	Harding JJ, Moreno V, Bang YJ, Hong MH, Patnaik A, Trigo J, Szpurka AM, Yamamoto N, Doi T, Fu S, et al: Blocking TIM-3 in treatment-refractory advanced solid tumors: A phase Ia/b study of LY3321367 with or without an anti-PD-L1 antibody. Clin Cancer Res. 27:2168–2178. 2021. View Article : Google Scholar : PubMed/NCBI
28	Kim JE, Patel MA, Mangraviti A, Kim ES, Theodros D, Velarde E, Liu A, Sankey EW, Tam A, Xu H, et al: Combination therapy with Anti-PD-1, anti-TIM-3, and focal radiation results in regression of murine gliomas. Clin Cancer Res. 23:124–136. 2017. View Article : Google Scholar : PubMed/NCBI
29	Ngiow SF, von Scheidt B, Akiba H, Yagita H, Teng MW and Smyth MJ: Anti-TIM3 antibody promotes T cell IFN-γ-mediated antitumor immunity and suppresses established tumors. Cancer Res. 71:3540–3551. 2011. View Article : Google Scholar : PubMed/NCBI
30	Kurtulus S, Sakuishi K, Ngiow SF, Joller N, Tan DJ, Teng MW, Smyth MJ, Kuchroo VK and Anderson AC: TIGIT predominantly regulates the immune response via regulatory T cells. J Clin Invest. 125:4053–4062. 2015. View Article : Google Scholar : PubMed/NCBI
31	Zhang D, Jiang F, Zaynagetdinov R, Huang H, Sood VD, Wang H, Zhao X, Jenkins MH, Ji Q, Wang Y, et al: Identification and characterization of M6903, an antagonistic anti-TIM-3 monoclonal antibody. Oncoimmunology. 9:17449212020. View Article : Google Scholar : PubMed/NCBI
32	Homayouni V, Ganjalikhani-Hakemi M, Rezaei A, Khanahmad H, Behdani M and Lomedasht FK: Preparation and characterization of a novel nanobody against T-cell immunoglobulin and mucin-3 (TIM-3). Iran J Basic Med Sci. 19:1201–1208. 2016.PubMed/NCBI
33	Jiang Z, Jiang X, Chen S, Lai Y, Wei X, Li B, Lin S, Wang S, Wu Q, Liang Q, et al: Anti-GPC3-CAR T cells suppress the growth of tumor cells in patient-derived xenografts of hepatocellular carcinoma. Front Immunol. 7:6902017. View Article : Google Scholar : PubMed/NCBI
34	Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK and Anderson AC: Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and restore anti-tumor immunity. J Exp Med. 207:2187–2194. 2010. View Article : Google Scholar : PubMed/NCBI
35	Koyama S, Akbay EA, Li YY, Herter-Sprie GS, Buczkowski KA, Richards WG, Gandhi L, Redig AJ, Rodig SJ, Asahina H, et al: Adaptive resistance to therapeutic PD-1 blockade is associated with upregulation of alternative immune checkpoints. Nat Commun. 7:105012016. View Article : Google Scholar : PubMed/NCBI