Background rhIL-7-hyFc is a hybrid Fc-fused recombinant human interleukin-7 (NT-I7; efineptakin-alfa) with enhanced bioactivity. In a previous study, we found that a systemic administration of rhIL-7-hyFc induced antitumor effect by increasing CD8+ T cells in the tumor microenvironment. rhIL-7-hyFc monotherapy increased not only PD-1+ tumor-reactive but also intratumoral PD-1- bystander CD8+ T cells. Therefore, we hypothesized that the activation of PD-1- bystander T cells in tumors would enhance the antitumor activity of rhIL-7-hyFc. Here we evaluated the antitumor effect of combination therapy with rhIL-7-hyFc and a bispecific antibody (bsAb), anti-PD-L1xCD3ε, targeting both a tumor-associated antigen (PD-L1) and a T-cell stimulatory antigen (CD3ε).
Methods In vitro cell culture. For analysis of T cell activation and cytotoxicity, splenocytes were isolated from PD-L1 knock-out (KO) mice and co-cultured with either wild type (MC-38WT) and PD-L1-depleted (MC-38ΔPD-L1) tumor cells in the presence of bsAb for 48 hours. In vivo treatment. Tumor-bearing mice were treated subcutaneously (s.c.) with 1.25 mg/kg of rhIL-7-hyFc. An indicated dose of bsAb was daily treated intravenous (i.v.) or intratumoral (i.t.) route starting from 3 days after the rhIL-7-hyFc treatment for a total 5 times.Preparation of tumor-infiltrating cells. Tumor tissues were harvested after 7 days of rhIL-7-hyFc treatment. Single-cell suspensions were prepared through mechanical separation followed by collagenase D and DNAse I treatment.
Results Anti-PD-L1xCD3ε bsAb induced the PD-L1-specific activation and cytotoxicity of CD8+ T cells in vitro (figure 1).rhIL-7-hyFc combined with a systemic administration of bsAb enhanced antitumor responses, although loss of body-weight was shown with high-dose bsAb combination (figure 2)The combination of rhIL-7-hyFc with a systemic administration of bsAb increased not only the frequency of CD8+ T cells in tumors but also the PD-1- bystander CD8+ T cells with enhanced expression of a Granzyme B (figure 3).Intratumoral administration of high-dose bsAb enhanced antitumor response of rhIL-7-hyFc without body-weight loss (figure 4).
Conclusions The combination treatment of anti-PD-L1xCD3ε bsAb with rhIL-7-hyFc enhances antitumor efficacy.Both systemic and intratumoral administration of bsAb with rhIL-7-hyFc augments antitumor effects, and intratumoral administration induced less weight loss than systemic administration.The activation of PD-1- bystander CD8+ T cells in tumors by the combination of bsAb and rhIL-7-hyFc suggests that antitumor response may be partially mediated by the targeted activation of bystander CD8+ T cells. Our results serve as a proof-of-concept that the combination of rhIL-7-hyFc, a strong T cell amplifier, with bsAb, a tumor-targeted T-cell stimulator, would be a promising strategy for cancer immunotherapy.
Acknowledgements This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT)(NRF-2020M3H1A1075314) and the grants from Research Institute of NeoImmuneTech, Inc.
Ethics Approval This study was approved by POSTECH institutional animal care and use committee; approval number POSTECH-2020-0057.
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