Abstract B031: Development of antimyeloma immunotherapy by exploiting modified antibodies specific for A2/NY-ESO-1
Authors: Ochi Toshiki , Maruta Masaki , Tanimoto Kazushi , Asai Hiroaki , Saitou Takashi , Yakushijin Yoshihiro , Fujiwara Hiroshi , Imamura Takeshi , Takenaka Katsuto , Yasukawa Masaki 
Published: February 2019

Abstract
Background: T-cell therapy can be a promising treatment option even in patients with refractory malignancies including myeloma. NY-ESO-1 is a well-known cancer-testis antigen which is expressed by refractory myeloma cells, and a NY-ESO-1_157-165 peptide presented by an HLA-A*02:01 molecule (A2/NY-ESO-1_157) has been demonstrated. Adoptive transfer therapy using T-cells modified with T-cell receptor (TCR) specific for A2/NY-ESO-1_157 successfully induced clinical responses in patients with advanced myeloma. However, TCR-transduced T-cells are laborious to generate and possess the cross-reactivity induced by mispaired and/or introduced TCRs, resulting in increase of unwanted toxicities. T-cell therapy utilizing modified antibodies containing single chain fragment variables (scFvs), such as chimeric antigen receptor (CAR) and bispecific antibody (BiTE) would overcome the issues concerning TCR-T therapy and expand clinical versatility of T-cell therapy targeting NY-ESO-1 in the treatment of refractory myeloma. In this study, we have generated both CAR and BiTE which recognize A2/NY-ESO-1_157, and assessed their anti-myeloma reactivity and cross-reactivity in vitro and in vivo. Methods: Expression of NY-ESO-1 in a panel of myeloma cell lines was examined by real-time PCR and Western blotting. Based on the structure of previously reported monoclonal antibody specific for A2/NY-ESO-1_157 (clone: 3M4E5), we newly synthesized an A2/NY-ESO-1_157-specific scFv. Second generation CAR possessing an scFv linked with CD28 and CD3z was generated. A BiTE composed of an A2/NY-ESO-1_157-specific scFv and a CD3e-binding scFv was also generated. A2/NY-ESO-1_157-specific reactivity mediated by CAR and BiTE-redirected T-cells were assessed by A2/NY-ESO-1_157 tetramer and multiple cytokine assays. Specific lysis of targeT-cells by those T-cells was measured by standard Cr-release assay. Alanine scanning of NY-ESO-1_157 peptide was performed, and nine peptides homologous to NY-ESO-1_157 were synthesized. Cross-reactivity of CAR and BiTE-redirected T-cells for these peptides and NY-ESO-1_157 peptide presented by HLA-A2 alleles was evaluated. NOG mice engrafted with a luciferase-transduced A2+NY-ESO-1+ myeloma cell line (U266/SLR) were treated with CAR-T-cells or T-cells with BiTE, and tumor sizes were measured by bioluminescence imaging assays. Results: Three out of six myeloma cell lines we tested abundantly expressed NY-ESO-1 mRNA and protein. CAR-T-cells established from five out of five donors showed A2/NY-ESO-1_157-specific reactivity. These gene-modified T-cells recognized and killed targeT-cells which naturally process and present A2/NY-ESO-1_157, resulting in anti-myeloma reactivity to A2+NY-ESO-1+ U266 myeloma cells. Newly generated BiTE successfully engaged A2/NY-ESO-1_157 expressing targeT-cells with CD3+ T-cells, thereby peripheral T-cells produced multiple cytokines against A2+NY-ESO-1+ targeT-cells, and lysed them. CAR and BiTE-redirected T-cells can possess cross-reactivity for some of homologous peptides and NY-ESO-1_157 peptide presented by HLA-A2 alleles. Functional avidity of BiTE-redirected T-cells for A2/NY-ESO-1_157 was comparable with that of CAR-T-cells. Importantly, tumor growth was suppressed by intravenous injection of CAR-T-cells and T-cells in combination with BiTE, and their antitumor effects were similarly observed. Conclusions: T-cells redirected with CAR and BiTE both successfully showed anti-myeloma reactivity in an A2/NY-ESO-1_157-specific manner. An A2/NY-ESO-1_157-specific BiTE displayed a potential to induce sufficient antitumor T-cell responses against myeloma cells in vivo. These two scFv-based modalities also require to pay attention to unwanted cross-reactivity; however, they can provide efficacious and flexible options for the treatment of HLA-A2-positive patients with refractory myeloma.
Citation Format: Toshiki Ochi, Masaki Maruta, Kazushi Tanimoto, Hiroaki Asai, Takashi Saitou, Yoshihiro Yakushijin, Hiroshi Fujiwara, Takeshi Imamura, Katsuto Takenaka, Masaki Yasukawa. Development of antimyeloma immunotherapy by exploiting modified antibodies specific for A2/NY-ESO-1 [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B031.