Dataset Information

High-affinity CD16-polymorphism and Fc-engineered antibodies enable activity of CD16-chimeric antigen receptor-modified T cells for cancer therapy.

ABSTRACT:

Background

CD16-chimeric antigen receptors (CAR) T cells recognise the Fc-portion of therapeutic antibodies, which can enable the selective targeting of different antigens. Limited evidence exists as to which CD16-CAR design and antibody partner might be most effective. We have hypothesised that the use of high-affinity CD16 variants, with increased Fc-terminus antibody affinity, combined with Fc-engineered antibodies, would provide superior CD16-CAR T cell efficacy.

Methods

CD16-CAR T (wild-type or variants) cells were co-cultured with Panc-1 pancreatic cancer, Raji lymphoma or A375 melanoma cells in the presence or absence of anti-CD20, anti-MCSP, wild-type or the glycoengineered antibody variants. The endpoints were proliferation, activation, and cytotoxicity in vitro.

Results

The CD16 158 V variant of CD16-CAR T cells showed increased cytotoxic activity against all the tested cancer cells in the presence of the wild-type antibody directed against MCSP or CD20. Glycoengineered antibodies enhanced CD16-CAR T cell activity irrespective of CD16 polymorphisms as compared with the wild-type antibody. The combination of the glycoengineered antibodies with the CD16-CAR 158 V variant synergised as seen by the increase in all endpoints.

Conclusion

These results indicate that CD16-CAR with the high-affinity CD16 variant 158 V, combined with Fc-engineered antibodies, have high anti-tumour efficacy.

SUBMITTER: Rataj F

PROVIDER: S-EPMC6325122 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

High-affinity CD16-polymorphism and Fc-engineered antibodies enable activity of CD16-chimeric antigen receptor-modified T cells for cancer therapy.

Rataj Felicitas F Jacobi Severin J SJ Stoiber Stefan S Asang Florian F Ogonek Justyna J Tokarew Nicholas N Cadilha Bruno L BL van Puijenbroek Erwin E Heise Constanze C Duewell Peter P Endres Stefan S Klein Christian C Kobold Sebastian S

British journal of cancer 20181115 1

<h4>Background</h4>CD16-chimeric antigen receptors (CAR) T cells recognise the Fc-portion of therapeutic antibodies, which can enable the selective targeting of different antigens. Limited evidence exists as to which CD16-CAR design and antibody partner might be most effective. We have hypothesised that the use of high-affinity CD16 variants, with increased Fc-terminus antibody affinity, combined with Fc-engineered antibodies, would provide superior CD16-CAR T cell efficacy.<h4>Methods</h4>CD16- ...[more]

PMID: 30429531

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Project description:BackgroundChimeric 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.MethodsA 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.ResultsUnlike 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.ConclusionsP329G-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.

Project description:The neonatal Fc receptor (FcRn) is a key membrane protein that plays an integral role in serum immunoglobulin (IgG) recycling, which extends the half-life of antibody. In addition, FcRn is known to traffic antigen-bound immunoglobulins (Ag-IgGs), and to interact with immune complexes to facilitate the antigen cross-presentation of peptides derived from the immune complexes in antigen-presenting cells (APCs). Studies on the IgG-FcRn molecular interactions have primarily focused on the Fc region, and only recently have shown the potential impact of the antigen-binding fragment physiochemical properties on FcRn binding. However, the effect of the antigen physiochemical properties on IgG structure as it relates to Ag-IgG-FcRn binding is not well understood. Here we used an IgG-peptide antigen complex as a model system to investigate the structural effects of the antigen's physiochemical properties on the IgG structure, and the subsequent effects of Ag-IgG-FcRn interactions. We used hydroxyl radical footprinting-mass spectrometry to investigate the structural impact on an IgG upon antigen binding, and observed that the physicochemical properties of the antigen differentially induce conformational changes in the IgG FcRn binding region. The extent of these structural changes directly correlates to the magnitude of the affinity differences between the Ag-IgG complexes and FcRn. Moreover, the antigen's physicochemical properties differentially induce structural differences within the Ag-IgG-FcRn ternary complex. We also provide electron microscopy data that shows corroborating Fab-FcRn interactions, and confirms the hypothesis of potential 2:1 FcRn:IgG binding stoichiometry. These data demonstrate antigen-induced Fc structural rearrangements affect both the affinity toward FcRn and the trimeric antigen-IgG-FcRn complex, providing novel molecular insights in the first steps toward understanding interactions of FcRn-containing large(r)-sized immune complex.

Dataset Information

High-affinity CD16-polymorphism and Fc-engineered antibodies enable activity of CD16-chimeric antigen receptor-modified T cells for cancer therapy.

Background

Methods

Results

Conclusion

Publications

High-affinity CD16-polymorphism and Fc-engineered antibodies enable activity of CD16-chimeric antigen receptor-modified T cells for cancer therapy.

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