To reduce the toxicity of anti-CTLA-4, Pai and Simons et al. created an anti-CTLA-4 with dual variable domains (DVD), in which the inner variable domain (ID) targeting CTLA-4 was connected via a cleavable linker to the outer domain (OD) specific for the prostate stem cell antigen; the OD blocked binding by the ID. DVD ameliorated toxicities observed with anti-CTLA-4 by preserving tissue-resident Tregs and preventing systemic T cell activation. DVD accumulated at higher levels in TRAMP-C2 tumors than anti-CTLA-4, and linker cleavage by protease present in the tumor led to depletion of intratumoral Tregs and effective antitumor immunity.

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 FcgammaR-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.

Author Info: (1) Department of Hematology and Oncology, School of Medicine, UCSF, San Francisco, California, USA. (2) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (3) AbbVie Bioresearch Center

Author Info: (1) Department of Hematology and Oncology, School of Medicine, UCSF, San Francisco, California, USA. (2) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (3) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (4) Department of Radiology and Biomedical Imaging, School of Medicine, UCSF, San Francisco, California, USA. (5) Department of Radiology and Biomedical Imaging, School of Medicine, UCSF, San Francisco, California, USA. (6) Department of Radiology and Biomedical Imaging, School of Medicine, UCSF, San Francisco, California, USA. (7) Department of Hematopathology, School of Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA. (8) Department of Hematology and Oncology, School of Medicine, UCSF, San Francisco, California, USA. (9) Department of Hematology and Oncology, School of Medicine, UCSF, San Francisco, California, USA. (10) Department of Hematology and Oncology, School of Medicine, UCSF, San Francisco, California, USA. (11) Department of Hematology and Oncology, School of Medicine, UCSF, San Francisco, California, USA. (12) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (13) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (14) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (15) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (16) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (17) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (18) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (19) AbbVie Inc., North Chicago, Illinois, USA. (20) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (21) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (22) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (23) AbbVie Inc., North Chicago, Illinois, USA. (24) AbbVie Bioresearch Center, Worcester, Massachusetts, USA. (25) Department of Hematology and Oncology, School of Medicine, UCSF, San Francisco, California, USA. Parker Immunotherapy Institute, Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

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