Article Text

Download PDFPDF

450 Combination of rhIL-7-hyFc and anti-PD-L1xCD3ε bispecific antibody enhances antitumor response in mice
  1. Sujeong Park1,
  2. Ji-Hae Kim1,
  3. Yeon-Woo Kang1,
  4. Kun-Joo Lee1,
  5. Dain Moon1,
  6. Nara Tae2,
  7. Yunji Park3,
  8. Dae Hee Kim2,
  9. Byung Ha Lee4,
  10. Donghoon Choi5,
  11. Se Hwan Yang4 and
  12. Seung-Woo Lee6
  1. 1Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea
  2. 2Scripps Korea Antibody Institute, KNU CHUNCHEON CAMPUS, Seoul, Republuc of Korea
  3. 3POSTECH Biotech Center, POSTECH, Pohang, Republic of Korea
  4. 4NeoImmuneTech, Inc., MD, USA
  5. 5Research Institute, NeoImmuneTech co. ltd., Pohang, Republic of Korea
  6. 6Department of Life Sciences, POSTECH, Pohang, Republic of Korea

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).

Abstract 450 Figure 1

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

Abstract 450 Figure 2

(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

Abstract 450 Figure 3

(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

Abstract 450 Figure 4

(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.

http://creativecommons.org/licenses/by-nc/4.0/

This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.

Statistics from Altmetric.com

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.