Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors

Our plain-English summary of Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors, by S. Stock, M.R. Benmebarek, A.K. Kluever, et al (Journal for Immunotherapy of Cancer, 2022).

The study

• Authors: S. Stock, M.R. Benmebarek, A.K. Kluever, D. Darowski, C. Jost, K.G. Stubenrauch, J. Benz, A. Freimoser-Grundschober, E. Moessner, P. Umana, M. Subklewe, S. Endres, C. Klein, S. Kobold
• Journal: Journal for Immunotherapy of Cancer
• Published: 2022;10(7)
• Cited by: 29 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

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(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

In plain English

This was a preclinical laboratory study. The authors built a second-generation chimeric antigen receptor (CAR) with CD28 and CD3zeta signalling domains that recognises the P329G mutation in the Fc part of human IgG1 antibodies, and transduced it into primary human T cells. These CAR T cells were tested together with P329G LALA-mutated antibodies against EGFR, mesothelin (MSLN) or HER2, using pancreatic (SUIT-2-MSLN), mesothelioma (MSTO-MSLN-LUC) and breast cancer (HCC1569, MCF-7 HER2 OE) cell lines, and in female NSG mice (83 in total). In the EGFR comparison, P329G-CAR T cells produced IFN-gamma and killed tumour cells only with the P329G LALA-mutated antibody, whereas CD16-CAR T cells, described by the authors as binding human IgG non-selectively, responded only to the glycoengineered antibody. Soluble mesothelin did not reduce killing, although at the lower binder concentration of 0.1 micrograms per mL cytokine production dropped (p=0.04). In mice, P329G-CAR T cells plus the MSLN antibody reduced mesothelioma tumour growth (p less than 0.0001) but without complete eradication; with the HER2 antibody, treated mice had complete tumour eradication. The authors concluded that the platform showed activity and modularity in solid tumour models.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: preclinical laboratory study: in vitro cell culture experiments with engineered human T cells plus xenograft mouse experiments
• Who was studied: Primary human T cells from independent donors (n=3 per experiment), human tumour cell lines (pancreatic SUIT-2-MSLN, mesothelioma MSTO-MSLN-LUC, breast HCC1569 and MCF-7 HER2 OE), and 83 female immunodeficient NSG mice aged 2-5 months
• Exposure: P329G-targeting CAR T cells combined with human IgG1 antibodies carrying P329G LALA Fc mutations directed against EGFR, mesothelin or HER2 (trastuzumab-derived), compared with CD16-ECD-CAR, MSLN-CAR, HER2-CAR and CD33-CAR T cells; binders at 1 microgram per mL in vitro and 5 or 20 micrograms per mouse intraperitoneally twice weekly
• Main results:
  • P329G-CAR T cells released IFN-gamma only with the P329G LALA-mutated EGFR antibody, not with the glycoengineered EGFR antibody
  • In the same EGFR comparison, CD16-CAR T cells showed the opposite pattern, responding only to the glycoengineered EGFR antibody; the authors state CD16-CAR T cells bind human IgG non-selectively
  • Added polyclonal human IgG (1 mg/mL) decreased IFN-gamma production by P329G-CAR T cells but increased it for CD16-CAR T cells
  • Soluble mesothelin did not cause unspecific activation or reduce cytotoxicity of P329G-CAR T cells, even at supraphysiological concentrations
  • At 0.1 micrograms per mL binder, soluble mesothelin reduced cytokine production by P329G-CAR T cells
  • In the mesothelioma (MSTO-MSLN-LUC) xenograft, P329G-CAR T cells plus 20 micrograms MSLN binder reduced tumour growth compared with no binder or CD33-CAR T cells; no complete eradication occurred in this model
  • In the HER2-expressing HCC1569 breast cancer xenograft, mice given P329G-CAR T cells with the HER2 binder, or HER2-CAR T cells, had complete tumour eradication and 100% survival
  • Surviving mice re-challenged with MSLN-expressing tumour and then given the MSLN binder had reduced tumour growth versus comparison groups
• The authors' conclusion: The authors concluded that they had demonstrated the activity, potency, modularity and reversibility of this P329G-targeting CAR T cell platform in different solid tumour models, and that further preclinical evaluation is needed
• Limitations the authors state:
  • The mesothelioma model was highly ulcerative, so no complete tumour eradication was seen there
  • Because trastuzumab does not cross-react with mouse tissue, on-target off-tumour toxicity on normal tissues could not be assessed
  • Whether this platform shares the limitations of conventional mesothelin-directed or other targeted CAR T cells remains unanswered
  • The authors state further preclinical work is needed before translation

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: EGFR, Mesothelin, HER2, Mesothelioma, Cytokine.

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