Combinatorial Targeting of Multiple Shared Antigens By Adapter-CAR-T Cells (aCAR-Ts) Allows Target Cell Discrimination and Specific Lysis Based on Differential Expression Profiles

Abstract

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.

Adapter chimeric antigen receptor (AdCAR)-engineered NK-92 cells: an off-the-shelf cellular therapeutic for universal tumor targeting

“…Other universal CAR approaches with T cells, including the presented adapter CAR, as well as NK cells have already shown promising results. 32 , 35 , 36 , 53 , 54 However, CAR systems using adapter molecules that are foreign to the human body can cause immunogenic reactions and make clinical translation difficult. Even CAR systems utilizing endogenous molecules as tag such as biotin may face challenges due to the potential immunogenicity of avidin and streptavidin as well as their high affinity for physiological biotin in the clinical setting.…”

“…The AdCAR is 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 monoclonal antibodies as linkers, referred to as linker-label-epitope (LLE), whose context is introduced in the patent application EP3315511A1. [35][36][37] NK-92 cell-mediated target cell lysis is the result of a twostep process: antibody-specific binding to the target cell surface and binding of the AdCAR-modified NK-92 cells to the bAb (Figure 1a). AdCAR specificity relies solely on the bAb used, leading to almost unlimited possibilities in tumor antigen targeting.…”

Emerging Immunotherapy for Acute Myeloid Leukemia

“…The adapter CAR-T (aCAR-T) is a new concept in which antigen recognition of CAR-T cells is split into multiple agents (e.g., CD32, CD33, CD38, CD123, CD135, CD305, and CLL1 for AML) by linker-label-epitopes (LLEs) that allows qualitative regulation of CAR-T function (for example, recognition of four or more antigens to activate and three or less not to do). Preclinical studies of aCAR-T are ongoing, and the results are awaited [ 114 ]. Potential IO targets of AML are schematically displayed in Figure 2 .…”

Immunotherapy with NK cells: recent developments in gene modification open up new avenues

“…Separating antigen recognition and CAR activation facilitates universal, as well as combinatorial, immunotargeting. 71 , 72 …”