Construction of Dual- and Enhanced Dual-Antibodies Targeting CD19 in the Treatment of B-ALL and Investigation of the Relevant Mechanisms

Objective: Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by the development of immature lymphocytes. Although many patients can achieve complete remission (CR) after chemotherapy, only minority can sustain long term remission without relapse. In the past decade, chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses for B cells malignancies, however, it still encounters with substantial challenges, such as expensive cost and treatment failure. T cell-redirecting bispecific antibodies is able to build the connection between endogenous T cells and tumor cells in order to activate T cells function to eliminate cancer cells without the need for ex vivo genetic alteration or manipulation of the T cells. It can provide an off-the-shelf immune-oncology therapy, unlike the long-term preparation before CAR-T infusion. Here, we developed the novel dual-specific antibody (DuAb) and enhanced DuAb (EDuAb) with different stimulation signals to activate T cells and explored their impact on the treatment of hematologic tumors by altering the function of T cells.

Methods: The expression plasmids of dual-specific antibody targeting CD19 and CD3 (DuAb) and the enhanced DuAb (EDuAb) containing CD80 molecule were constructed by cloning heavy chain and light chain variable fragments from previously established anti-human CD19 (HI19a) and CD3 (HIT3a) monoclonal antibodies, respectively. Both DuAb and EDuAb were produced through a eukaryotic expression system. The activation of human primary T cells was evaluated after stimulated by DuAb and EDuAb in vitro, by detecting T cells proliferation, cytokine secretion, apoptosis and inhibitor marker expression. The antitumor efficacy of human primary T cells mediated by DuAb and EDuAb was evaluated through co-culture killing assay. B-ALL xenograft NSG mouse model was established to investigate the in vivo therapeutic effect.

Results: EDuAb promoted the optimal expansion of primary human T cells with low expression of inhibitor marker in vitro than DuAb did (fold change of proliferation on day 9: 61.39 vs 19.23, p<0.0001; proportion of annexin V⁺: 11.83% vs 45.13%, p<0.0001, Tim3⁺: 20.08% vs 35.37%, p=0.007, PD-1⁺: 11.6% vs 34.3%, p=0.001, Lag3⁺: 29.12% vs 51.42%, p=0.006). In addition, BCL-X_L was upregulated and IL2 secretion increased in EDuAb group. DuAb and EDuAb showed a similar capability in inducing healthy donor T cells to specifically eliminate B-ALL cell lines in vitro. At the concentration of 0.01nM, E/T ratio (2/1), the lysis of Nalm6 cells exceeded 96% after co-cultured for 48 hours. The similar ability was also observed in the patient-derived T cells. Both DuAb and EDuAb markedly suppressed tumor burden to baseline level and extended survival of B-ALL xenograft NSG mice. The median survival of vehicle, DuAb and EDuAb treatment group were 27, 38 and 45 days, respectively, which illustrated that EDuAb had a better therapeutic effect in vivo than that of DuAb (p=0.0066). The phenotype of T cells and cytokine release in peripheral blood of B-ALL xenograft NSG mouse model on day 24 were analyzed as well. The results showed that the proportion of CD8⁺ T cells and the cytokine levels, including IL2, IFN-γ and TNF-α, were higher in the EDuAb group than that of DuAb (CD8⁺: 0.746% vs 0.064%, p=0.0079; IL2: 1.862pg/ml vs 0.654pg/ml，p=0.0014; IFN-γ: 432.27pg/ml vs 1.48pg/ml, p= 0.047; TNF-α: 4.57pg/ml vs 2.08pg/ml, p=0.018). Both DuAb and EDuAb could significantly decrease the residual Nalm6 cells in PB of B-ALL xenograft NSG mice, and the residual tumor cells on day 24 were 1.61%, 0.45% and 0.29% in Vehicle, DuAb and EDuAb groups, respectively (Vehicle vs DuAb, p=0.0134; Vehicle vs EDuAb, p=0.0034; DuAb vs EDuAb, p=0.0169).

Conclusion: Both DuAb and EDuAb showed great potential for clinical application as a novel treatment method for B-ALL. Compared with DuAb, there is a significant advantage for EDuAb to promote the proliferation and survival of T cells. In addition, EDuAb showed better promising effect to eliminate tumor cells and extend survival in vivo, which provides a new insight for constructing new multi-specific antibodies.

Wang:AstraZeneca: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees.