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Abstract
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).
MC-38WT and MC-38ΔPD-L1tumor cells were cultured in vitro. (a) PD-L1 expression levels on each cell line. (b) Splenocytes isolated from PD-L1 KO mice were co-cultured with indicated tumor cells (E:T = 20:1) in the presence of bsAb. Expression levels of activation markers, such as CD69 and CD25, on the CD8+ T cells were analyzed by flow cytometry. (c) Cytotoxicity against tumors was analyzed in the presence of bsAb. Cytotoxicity was calculated using the formula: [1 - live target cells(sample)/live target cells(control)] × 100
(a-b) Mice bearing MC-38 tumors were treated with different doses of bsAb (i.v.) as indicated in (a) (n = 5 per group). (b) Shown are mean tumor growth curves (left) and body-weight changes (right). (c-d) Mice bearing MC-38 tumors were treated either 1.25 mg/kg of rhIL-7-hyFc (s.c.), indicated doses of bsAb (i.v.), or combination of each therapy as indicated in (c). In the case of combination therapy with 1 ug bsAb, mice were treated only for the first 3 doses of bsAb because of body-weight loss (n = 5–7 per group). (d) Shown are mean tumor growth curves (left) and body-weight changes (right). Arrows indicate the dosing of bsAb. Data are represented as mean ± SEM. Statistical significance was analyzed by two-way ANOVA with bonferroni’s multiple comparisons for (b and d). *P<0.05;**P<0.01;***P<0.001
(a) Experimental scheme for the analysis of tumor-infiltrating T cells (n = 4 per group). (b) Frequencies of CD8+, CD4+Foxp3- T helper (Th), and CD4+Foxp3+ T regulatory (Treg) cells among CD45+ cells. (c) Frequencies of CD4+Foxp3+ Treg cells among CD4+ T cells. (d) The ratio of CD8+ T cells to Treg cells. (e) Frequencies of PD-1- cells among CD8+ T cells. (f) Frequencies of Granzyme B (GzmB) expressing cells among PD-1+ or PD-1- CD8+ T cells. Data are represented as mean ± SD. Statistical significance was analyzed by one-way ANOVA with bonferroni’s multiple comparisons. *P<0.05;**P<0.01;***P<0.001
(a-b) Mice bearing MC-38 tumors were treated i.t. with bsAb as indicated in (a) (n = 6–7 per group). (b) Shown are mean tumor growth curves (left) and body-weight changes (right). (c-d) Mice bearing MC-38 tumors were treated either 1.25 mg/kg of rhIL-7-hyFc (s.c.), indicated doses of BsAb (i.t.), or combination of each therapy as indicated in (c). (n = 9–10 per group). (d) Shown are mean tumor growth curves (left) and body-weight changes (right). Arrows indicate the dosing of bsAb. Data are represented as mean ± SEM. Statistical significance was analyzed by two-way ANOVA with bonferroni’s multiple comparisons for tumor growth graphs. *P<0.05;**P<0.01;***P<0.001
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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