(1) Stock S (2) Benmebarek MR (3) Kluever AK (4) Darowski D (5) Jost C (6) Stubenrauch KG (7) Benz J (8) Freimoser-Grundschober A (9) Moessner E (10) Umana P (11) Subklewe M (12) Endres S (13) Klein C (14) Kobold S

Journal for immunotherapy of cancer

Stock et al. created a scFv-based second-generation CAR targeting the validated P329G L234A/L235A IgG1Fc effector-silencing mutations present in developing and approved human therapeutic antibodies. P329G-directed CAR T cells combined with EGFR-, mesothelin-, or HER2/neu-specific “adapter” IgG1s that carried the P329G Fc mutations mediated specific responses against mesothelioma, pancreatic, and breast cancer cells expressing the respective antigens in vitro and in mouse cancer models. While endogenous IgG or soluble target had no impact, removing or swapping “adapter” IgG1s reversed or refocused the CAR T cell effects, demonstrating modularity.

Contributed by Paula Hochman

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has proven its clinical utility in hematological malignancies. Optimization is still required for its application in solid tumors. Here, the lack of cancer-specific structures along with tumor heterogeneity represent a critical barrier to safety and efficacy. Modular CAR T cells indirectly binding the tumor antigen through CAR-adaptor molecules have the potential to reduce adverse events and to overcome antigen heterogeneity. We hypothesized that a platform utilizing unique traits of clinical grade antibodies for selective CAR targeting would come with significant advantages. Thus, we developed a P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human antibodies containing P329G L234A/L235A (LALA) mutations for Fc silencing. METHODS: A single chain variable fragment-based second generation P329G-targeting CAR was retrovirally transduced into primary human T cells. These CAR T cells were combined with IgG1 antibodies carrying P329G LALA mutations in their Fc part targeting epidermal growth factor receptor (EGFR), mesothelin (MSLN) or HER2/neu. Mesothelioma, pancreatic and breast cancer cell lines expressing the respective antigens were used as target cell lines. Efficacy was evaluated in vitro and in vivo in xenograft mouse models. RESULTS: Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, P329G-targeting CAR T cells revealed specific effector functions only when combined with antibodies carrying P329G LALA mutations in their Fc part. P329G-targeting CAR T cells cannot be activated by an excess of human IgG. P329G-directed CAR T cells combined with a MSLN-targeting P329G-mutated antibody mediated pronounced in vitro and in vivo antitumor efficacy in mesothelioma and pancreatic cancer models. Combined with a HER2-targeting antibody, P329G-targeting CAR T cells showed substantial in vitro activation, proliferation, cytokine production and cytotoxicity against HER2-expressing breast cancer cell lines and induced complete tumor eradication in a breast cancer xenograft mouse model. The ability of the platform to target multiple antigens sequentially was shown in vitro and in vivo. CONCLUSIONS: P329G-targeting CAR T cells combined with antigen-binding human IgG1 antibodies containing the P329G Fc mutation mediate pronounced in vitro and in vivo effector functions in different solid tumor models, warranting further clinical translation of this concept.

Author Info: (1) Department of Medicine IV, Division of Clinical Pharmacology, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany sophia.stock@med.uni-muenchen.de sebastia

Author Info: (1) Department of Medicine IV, Division of Clinical Pharmacology, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany sophia.stock@med.uni-muenchen.de sebastian.kobold@med.uni-muenchen.de christian.klein.ck1@roche.com. Department of Medicine III, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany. German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany. (2) Department of Medicine IV, Division of Clinical Pharmacology, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany. National Cancer Institute (NCI), Bethesda, Maryland, USA. (3) Department of Medicine IV, Division of Clinical Pharmacology, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany. (4) Roche Innovation Center Zurich, Roche Pharma Research & Early Development, Schlieren, Switzerland. Innovent Biologics (Suzhou) Co., Ltd, Suzhou, Jiangsu, China. (5) Roche Innovation Center Zurich, Roche Pharma Research & Early Development, Schlieren, Switzerland. Athebio AG, Schlieren, Switzerland. (6) Roche Innovation Center Munich, Penzberg, Germany. (7) Roche Innovation Center Basel, Basel, Switzerland. (8) Roche Innovation Center Zurich, Roche Pharma Research & Early Development, Schlieren, Switzerland. (9) Roche Innovation Center Zurich, Roche Pharma Research & Early Development, Schlieren, Switzerland. (10) Roche Innovation Center Zurich, Roche Pharma Research & Early Development, Schlieren, Switzerland. (11) Department of Medicine III, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany. German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany. (12) Department of Medicine IV, Division of Clinical Pharmacology, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany. German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany. Einheit fŸr Klinische Pharmakologie (EKLiP), Helmholtz Zentrum MŸnchen, German Research Center for Environmental Health (HMGU), Neuherberg, Germany. (13) Roche Innovation Center Zurich, Roche Pharma Research & Early Development, Schlieren, Switzerland sophia.stock@med.uni-muenchen.de sebastian.kobold@med.uni-muenchen.de christian.klein.ck1@roche.com. (14) Department of Medicine IV, Division of Clinical Pharmacology, University Hospital, Ludwig Maximilian University (LMU), Munich, Germany sophia.stock@med.uni-muenchen.de sebastian.kobold@med.uni-muenchen.de christian.klein.ck1@roche.com. German Cancer Consortium (DKTK), Partner Site Munich, Munich, Germany. Einheit fŸr Klinische Pharmakologie (EKLiP), Helmholtz Zentrum MŸnchen, German Research Center for Environmental Health (HMGU), Neuherberg, Germany.

Citation: J Immunother Cancer 2022 Jul 10: Epub

Link to PUBMED: http://www.ncbi.nlm.nih.gov/pubmed/35902133

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