Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1–dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.
Despite the recent success of CAR T cells targeting CD19 and CD22 in hematological malignancies, the production of CAR T cells still requires an extensive manufacturing process. The well-established NK-92 cell line provides a promising alternative to produce CAR-modified effector cells in a GMP-compliant, costeffective way. NK-92 can be redirected against a variety of surface antigens by our adapter CAR (AdCAR) system utilizing biotinylated antibodies (bAb) as adapter molecules. Selected bAb were capable of inducing significant AdCAR NK-92-mediated lysis of non-Hodgkin lymphoma (NHL) and mantle-cell lymphoma (MCL) cell lines as well as primary MCL and chronic lymphocytic leukemia (CLL) cells. AdCAR specificity was proven using a JeKo-1 CD19/CD20 knockout antigen-loss model. Moreover, through combinations of bAb, AdCAR NK-92 cells are capable of combatting tumor antigen evasion mechanisms. In conclusion, we successfully generated the AdCAR NK-92 cell line which can be manufactured as an "offthe-shelf, on-demand" product allowing universal and tunable tumor targeting.
Chimeric antigen receptor (CAR)-T therapy holds great promise to sustainably improve cancer treatment. However, currently, a broad applicability of CAR-T cell therapies is hampered by limited CAR-T cell versatility and tractability and the lack of exclusive target antigens to discriminate cancerous from healthy tissues. To achieve temporal and qualitative control on CAR-T function, we engineered the Adapter CAR (AdCAR) system. AdCAR-T are redirected to surface antigens via biotin-labeled adapter molecules in the context of a specific linker structure, referred to as Linker-Label-Epitope. AdCAR-T execute highly specific and controllable effector function against a multiplicity of target antigens. In mice, AdCAR-T durably eliminate aggressive lymphoma. Importantly, AdCAR-T might prevent antigen evasion by combinatorial simultaneous or sequential targeting of multiple antigens and are capable to identify and differentially lyse cancer cells by integration of adapter molecule-mediated signals based on multiplex antigen expression profiles. In consequence the AdCAR technology enables controllable, flexible, combinatorial, and selective targeting.
Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-β. TGF-β plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-β within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-β, both in vitro and in vivo . Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-β and tumor cell killing by targeting CD66c as TAA in vivo . In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-β.
Despite tremendous clinical success of chimeric antigen receptor (CAR) expressing T cell (CAR-T) therapies, targeting B-phenotypic antigens in ALL, CLL, NHL or multiple myeloma, there are still major limitations for broader clinical application. CAR-Ts are capable to generate a specific immune response against defined surface-expressed antigens leading to sustained depletion of target antigen expressing tissues e.g. B cells. While B cell function can be substituted by repetitive IgG infusions, prolonged depletion of vitally essential tissues is not compatible with life. In AML for instance, most promising target antigens are expressed along myeloid lineage differentiation, limiting the therapeutic applicability. Therefore, CAR-Ts targeting essential shared antigens must allow tight regulation of CAR-T function and/or be able to differentiate between cancerous and healthy tissue. To address these issues, we have developed the adapter CAR-T cell (aCAR-T) system. By splitting antigen recognition and CAR-T activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combine target antigens) regulation of CAR-T function. aCAR-Ts are based on the unique properties of a novel scFv targeting a "neo"-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated to novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. We were able to demonstrate that aCAR-Ts allow simultaneous targeting of various antigens ("OR"-gate) , preventing antigen evasion by selection of antigen or epitope-loss variants. In the present study we intended to investigate whether aCAR-Ts are capable to identify and differentiate target cells due to versatile antigen expression profiles ("AND"-gate). In theory, AMs against different target antigens can be assembled on the surface of a target cell , leading to aCAR-T activation independent of the targeted antigens (surface painting) , by binding to the presented LLE-tags. Therefore, combinatorial AMs treatment might allow to translate complex and multiple antigen-dependent target cell identification into an aCAR-T activation. To test this hypothesis, we have generated LLE-AMs against ALL/NHL - (CD10, CD19, CD20, CD22, CD37, CD138, ROR1) and AML - (CD32, CD33, CD38, CD123, CD135, CD305, CLL1) associated antigens. Individual threshold concentrations for aCAR-T activation by different AMs, targeti ng the model cell lines Nalm 6 (ALL), JeKo1 (NHL), HL-60 , U973 and Molm13 ( all AML), have been analyzed. Cut offs were found to be between 10 and 100 pg/ml, dependent on target expression and target cell line. Importantly, combinations of 2, 3 or 5 AMs, targeting different antigens expressed on the same target cell, cause target- cell lysis at concentrations below the activation threshold for single AMs (exemplified for HL-60 in Figure A) . Our results clearly demonstrate an additive effect in combining different AMs to hurdle the activation threshold. Moreover, in a JeKo 1 CD19 and/or CD20 knock out (KO) antigen-loss model, combinations of AMs targeting CD19, CD20 and ROR1 can differentiate between wild type and antigen -1 (CD19 or CD20 KO) or antigen -2 (CD19 and CD20 KO) variants in medi ating target- cell lysis, even though at least one target antigen is expressed. Finally, we found that combinations of CD10, CD19, CD22 and CD138 sufficiently eliminate Nalm-6 BCP-ALL cells, while sparing healthy B cells in co - culture experiments. Similar results were obtained in co - culture experiments of HL-60 AML cells with monocytes, neutrophils as well as CD34- enriched hematopoietic progenitor cells, applying combinations of CD32, CD33, CD38, CD123, CD305 and CLL1. Co - culturing experiments using autologous blasts, monocytes, neutrophils and aCAR-Ts are ongoing. Together, our results indicate that aCAR-Ts in combination with selected AM combinations might have the ability to identify and specifically eliminate cancer cells based on complex antigen expression profiles. This would have major implications for clinical translation, enabling combinatorial therapy, essential to avoid antigen evasion, and the possibility to spare vitally essential tissue from elimination. Figure. Figure. Disclosures Seitz: Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Lock:Miltenyi Biotec: Employment. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Lang:Miltenyi Biotec: Patents & Royalties, Research Funding. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.