Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity
Chien-Chun Steven Pai, … , Gillian Kingsbury, Lawrence Fong
Chien-Chun Steven Pai, … , Gillian Kingsbury, Lawrence Fong
Published December 10, 2018
Citation Information: J Clin Invest. 2019;129(1):349-363. https://doi.org/10.1172/JCI123391.
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Research Article Immunology Oncology

Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity

Abstract

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte–associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4–binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1–/– mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2–bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.

Authors

Chien-Chun Steven Pai, Donald M. Simons, Xiaoqing Lu, Michael Evans, Junnian Wei, Yung-hua Wang, Mingyi Chen, John Huang, Chanhyuk Park, Anthony Chang, Jiaxi Wang, Susan Westmoreland, Christine Beam, Dave Banach, Diana Bowley, Feng Dong, Jane Seagal, Wendy Ritacco, Paul L. Richardson, Soumya Mitra, Grace Lynch, Pete Bousquet, John Mankovich, Gillian Kingsbury, Lawrence Fong

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Figure 3

Protease-cleavable linkers can act as a switch to convert the specificity of a DVD from a tumor antigen to a therapeutic target.

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Protease-cleavable linkers can act as a switch to convert the specificit...
A panel of DVDs was made to identify the optimal linker for conditional activation. All DVDs had identical ODs (anti-PSCA) and IDs (anti-CTLA4), but used different linkers. (A) Schematic showing the linker sequences used to optimize cleavage by MT-SP1. (B) Reducing SDS-PAGE of DVDs before and after incubation with MT-SP1. CTL indicates DVD made with linkers lacking the MT-SP1 cleavage site (LSGSDN/SGSDN). Asterisk indicates candidate DVD (5037) with optimal linker lengths that was advanced for further testing. (C and D) After incubation with MT-SP1, intact DVDs or cleaved DVDs were incubated with HEK293 cells overexpressing PSCA (C) or CTLA4 (D) and specific bindings were detected by secondary labeling and FACS. (E) Intact or cleaved DVDs were incubated with HEK293 cells overexpressing CTLA4 in the presence of CD86, and specific binding of CD86 to the cells was detected by flow cytometry. Each in vitro experiment was conducted 3 times independently. A representative figure from each in vitro experiment is shown. (F) Mice were implanted with s.c. TRAMP-C2 tumors and injected with either anti-CTLA4 or anti-CTLA4 DVD. The representative figure from each treatment arm demonstrates the biodistribution of anti-CTLA4 and anti-CTLA4 DVD at 48 hours by PET/CT. The upper portion is the transverse plane of the CT scan. The highlight portion is the tumor. The lower panel is the coronal plane of the CT scan. (G) Quantification of PET/CT tissue distribution of anti-CTLA4 and anti-CTLA4 DVD at 48 hours. Data were conducted with 2 independent experiments. Each treatment arm was collected from 8 mice per group for the anti-CTLA4–treated group and 4 mice per group for the anti-CTLA4 DVD–treated group. Bars represent mean ± SEM. *P < 0.05, 2 tailed Student’s t test.