Development of T-Cell Therapy By Exploiting Modified Antibodies Specific for A2/NY-ESO-1 for Refractory Myeloma

Background: Clinical trials have demonstrated that T-cell-based immunotherapy can suppress tumor growth even in patients with refractory malignancies. Multiple myeloma (MM) still remains incurable and many patients experience disease progression even though novel therapeutic agents have been developed. NY-ESO-1 is a well-known cancer-testis antigen which is expressed by myeloma cells in about half of MM patients, especially in aggressive cases, and NY-ESO-1_157-165 peptide presented by an HLA-A*02:01 molecule (A2/NY-ESO-1₁₅₇) on myeloma cell surfaces have been shown. In a recent clinical trial, adoptive therapy using T cells modified with T-cell receptor (TCR) specific for A2/NY-ESO-1₁₅₇ in combination with autologous stem cell transplantation successfully induced clinical responses in 80% patients with advanced MM. However, generation of TCR-transduced T cells are laborious, and potentially bearing a risk of lethal adverse events due to the cross-reactivity of expressing TCR. Modified antibodies; chimeric antigen receptor (CAR) and bispecific antibody (BsAb) which mediate antitumor T-cell responses have shown promise in clinical trials. We hypothesized that these two modalities enable to expand the 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 BsAb which recognize A2/NY-ESO-1₁₅₇, and assessed their anti-myeloma reactivity in vitro .

Methods: Based on the structure of previously reported monoclonal antibody specific for A2/NY-ESO-1₁₅₇ (clone: 3M4E5), we newly synthesized two different A2/NY-ESO-1₁₅₇-specific single chain fragment variable (scFv) genes encoding variable regions of a light (L) and a heavy (H) chain in order, and vice versa (HL). Second generation CAR with each scFv linked with CD28 and CD3ζ was generated. A BsAb composed of each A2/NY-ESO-1₁₅₇-specific scFv and a CD3ε-reactive scFv which can stimulate peripheral T cells was also generated. A2/NY-ESO-1₁₅₇-specific reactivity mediated by CAR-T cells were assessed by A2/NY-ESO-1₁₅₇ tetramer and multiple cytokine assays. Target-specific cytokine production induced by peripheral T cells in the presence of BsAbs was similarly examined. Specific lysis of target cells mediated by T cells redirected with modified antibodies was measured by standard ⁵¹Cr-release assay. Expression of NY-ESO-1 in a panel of myeloma cell lines was examined by real-time PCR and western blotting.

Results: Three out of six myeloma cell lines we tested abundantly expressed NY-ESO-1 mRNA and protein. CAR-transduced T cells established from five out of five donors showed A2/NY-ESO-1₁₅₇-specific reactivity. These gene-modified T cells recognized and killed target cells which naturally process and present A2/NY-ESO-1₁₅₇ on their cell surfaces, resulting in anti-myeloma reactivity to A2⁺NY-ESO-1⁺ U266 myeloma cells. LAGE-1 is a well-known homolog of NY-ESO-1, and fully shares the sequence of NY-ESO-1_157-165 peptide. As expected, CAR-T cells recognized and killed A2⁺LAGE-1⁺ target cells. Newly generated BsAbs successfully linked A2/NY-ESO-1₁₅₇ expressing targets and CD3⁺ T cells. Functional avidity of LH-BsAb-stimulated peripheral T cells for A2/NY-ESO-1₁₅₇ was higher than that of T cells stimulated with HL-BsAb, and was comparable with that mediated by CAR-T cells. BsAb-stimulated T cells produced multiple cytokines against both A2⁺NY-ESO-1⁺ and A2⁺LAGE-1⁺ target cells, and lysed both of them. Importantly, compared with CAR-T cells, BsAb-stimulated T cells showed the superior A2/NY-ESO-1₁₅₇-reactive cytokine production capacity.

Conclusions: T cells redirected with CAR and BsAb both successfully showed anti-myeloma reactivity in an A2/NY-ESO-1₁₅₇-specific manner. An A2/NY-ESO-1₁₅₇-reactive BsAb displayed a potential to induce sufficient antitumor T-cell responses against myeloma cells. In addition to TCR-gene therapy targeting NY-ESO-1, these two scFv-based modalities would be able to provide efficacious and flexible options for the treatment of patients with refractory MM.