Background Cancer immunotherapy exploiting T cell-activation approaches, such as CAR-T cells, T cell engaging CD3-bispecific antibodies (CD3 BsAbs), have demonstrated significant clinical benefit to patients with hematopoietic malignancies. However, use of T-cell activating therapies in patients with late-stage disease, refractory/relapsed disease, and patients with solid tumors remains challenging. Traditionally, T cell-activating approaches activate mature T cells, which are either CD4⁺ or CD8⁺. The currently approved CD3 BsAb, the CD3 binding domain derived from SP34 or OKT3 preferentially binds and activates CD4⁺ T cells. We hypothesize that a T cell engager that selectively binds and activates CD8⁺ T cells may lead to the reduced cytokine release and enhanced antitumor efficacy.

Methods CD19 was used as a target to test our hypothesis. A series of TCE recombinant proteins were generated including CD3 x CD19, CD8 x CD19, and CD3 x CD8 x CD19. In these TCEs, the CD19 binding domain was kept constant, while a variety of CD3 and CD8 Abs with different affinity and potency were tested. The biological properties of the above antibodies were evaluated in vitro and in vivo.

Results In vitro: The CD3 x CD19 engager showed preferential CD4⁺ T cell binding, CD4⁺ T cell activation and target killing. The CD8 x CD19 engager showed high affinity binding to CD8⁺ T cells, but no significant target killing. The CD3 x CD8 x CD19 tri-specific T cell engager (CD8 TCE) showed selective CD8⁺ T cell binding (80% to >99%). When comparing the bi-specific CD3 x CD19 TCE with the CD3 x CD8 x CD19 tri-specific TCE, a 2–4 log increase of CD8 binding affinity, a 1–3 log increase of CD8 T cell activation, a 6- to 100-fold enhanced target killing, and an increased IFNg and TNFa release in CD8⁺ T cells, was observed with the CD8 TCE. In vivo: In the mouse xenograft tumor model, the CD8 TCE showed dose-dependent antitumor activities. Furthermore, monkeys treated with CD8 TCE by single IV (2 hrs, 40 mg/kg, n=3) or by repeat SC (400 mg/kg, n=6) dosing, had significant peripheral B cell depletion, rapid CD8⁺ T cell redistribution, and low levels of cytokine release. We observed safe and tolerable SC dosing that also showed consistently low levels of cytokine release.

Conclusions These results demonstrate a novel tri-specific CD8 TCE that selectively activates CD8⁺ T cells. This novel biologic may have improved safety and enhanced efficacy for T-cell activating cancer treatment.

Ethics Approval The in vivo studies were approved by Institutional Animal Care and Use Committee of KYLIN lab Co., Ltd.; approval number KL02–009-2022.