Biology subjects

Abken, H.

Publications and source records attributed to Abken, H..

6 recordsLinked to original sources

bioRxiv2026

Multicenter preclinical validation of next-generation CAR T cells: a strategy for harmonization, reproducibility, and its feasibility in clinical translation

PurposeClinical translation of CAR T cell therapies has accelerated, yet preclinical evidence still often originates from single-center studies lacking sufficient robustness. Preclinical confirmatory multicenter studies have been proposed to improve the translational success, but their feasibility in cellular therapies remains unexplored. MethodsWe performed a confirmatory multicenter study validating C-C-motive-receptor-8 (CCR8) overexpression in CAR T cells--a strategy previously shown to enhance solid tumor infiltration. In vitro experiments covering activation, cytotoxicity, and migration using three CAR constructs were conducted across two centers with harmonized materials, preregistered protocols, randomization, and blinding. ResultsThe data from the two centers confirmed key findings of the exploratory study: CCR8 overexpression in anti-EpCAM and anti-mesothelin CAR T cells leads to enhanced selective migration towards a CCL1-gradient, while not compromising antigen-specific T cell activatory capacity and cytotoxicity in vitro. The study furthermore broadened the applicability of CCR8 overexpression to anti-CEA CAR T cells. ConclusionsThis first-of-its-kind preclinical confirmatory CAR T study demonstrates the feasibility of a multicenter confirmation in cellular therapy, with technical and logistical challenges resolved through transparent communication between all parties involved. Both exploratory and confirmatory studies aim to downselect CAR candidates with the highest clinical success potential, as they compete for limited resources in preclinical research. It is therefore mandatory to clarify the extent of replications required to validate the experimental methodology and identify CAR candidates with most likelihood of success. TRANSLATIONAL RELEVANCEPreclinical evidence for novel CAR T cell therapeutic strategies relies mostly on exploratory single-center studies lacking robustness, with recent findings substantiating their limited predictive value for cellular therapies tested outside hematology. Here, the function of CCR8-armored CARs in vitro was confirmed in a preclinical confirmatory multicenter study, demonstrating the feasibility of such studies in adding value to the transition of preclinical concepts to clinical development. Our first-of-its-kind study may contribute to define new routes for preclinical testing and further raises the general question of what level of preclinical evidence is reasonably achievable in an academic context. It indicates the need for strong collaborative efforts to realize dedicated preclinical infrastructure for clinical translation of reprogrammed immune cellular therapeutics.

synthetic biology↗

bioRxiv2025

Dextran-based T-cell expansion nanoparticles for manufacturing CAR T cells with augmented efficacy

Adoptive T cell therapy (ACT) using chimeric antigen receptor (CAR) engineered T cells is currently being explored in multiple cancer types beyond leukemia/lymphoma. A key step in CAR-T cell manufacturing is the activation and expansion of T cells, which facilitates viral transduction, however, may hamper T cell fitness and reduce in vivo persistence. We developed "T-Expand" for T cell activation and expansion, comprising dextran-based nanoparticles (NPs) conjugated with anti-CD3 and anti-CD28 antibodies. The NPs triggered robust polyclonal expansion of human T cells with efficiency in the range of commercial microbeads (Dynabeads). Engineered in presence of T-Expand, CD19 CAR T cells exhibited enhanced proliferative capacity, cytotoxicity and persistence in vitro, and furthermore, showed superior anti-lymphoma activity in mouse models resulting in complete tumor clearance at one fourth of the CAR T cell dose. Importantly, T-Expand is biocompatible with no observed toxicity, circumventing removal steps after T cell expansion compared to DynabeadsTM. As a biocompatible T cell expansion platform, T-Expand simplifies the manufacturing process while enhancing T cell persistence and functionality, thereby holding promise for increasing clinical efficacy of CAR T cell therapy. O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/648181v1_ufig1.gif" ALT="Figure 1"> View larger version (74K): org.highwire.dtl.DTLVardef@35337corg.highwire.dtl.DTLVardef@c75199org.highwire.dtl.DTLVardef@1be184forg.highwire.dtl.DTLVardef@12e1683_HPS_FORMAT_FIGEXP M_FIG Graphical abstract/Cover figureIllustration of CAR T cell manufacturing using T-Expands ex vivo. C_FIG

immunology↗

bioRxiv2024

Development of antigen-dextramers for detection and evaluation of CAR T cells

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed the treatment landscape of hematologic cancers by engineering T cells to specifically target and destroy cancer cells. Monitoring CAR T cell activity and function is essential for optimizing therapeutic outcomes, but existing tools for CAR detection are often limited in specificity and functional assessment capability. MethodsWe developed antigen-dextramers by conjugating multiple CAR-specific antigens to a dextran backbone. The dextramers were compared to previously reported antigen-tetramers for their ability to stain and detect CAR T cells. Because these multimers incorporate the CAR target antigen, they uniquely enable assessment of CAR T cell functionality by facilitating binding and activation analyses. We tested the staining and functional properties of the multimers across a range of CAR constructs with different affinities, using flow cytometry, microscopy, and NFAT-luciferase reporter assays. ResultsThe antigen-dextramers demonstrated high specificity and sensitivity in staining CAR T cells, with adjustable antigen density to optimize binding. Antigen-dextramers also enabled effective clustering and subsequent activation of CARs, showing their utility as both a staining and functional assessment tool. The dextramers revealed that CARs with different affinities and clustering tendencies displayed varied binding and activation in response to different antigen densities. ConclusionAntigen-dextramers offer a dual advantage as versatile reagents for both staining and functional analysis of CAR T cells. Their capacity to engage CARs with the specific antigen provides a valuable platform for evaluating CAR functionality, informing CAR design improvements, and enhancing therapeutic precision.

bioengineering↗

bioRxiv2024

Drug-controlled CAR-T cells through the regulation of cell-cell interactions

CAR T-cell therapy is constrained by on-target, off-tumor toxicities as well as cellular exhaustion due to chronic antigen exposure. CARs comprising small-molecule controlled switches can enhance both safety and therapeutic efficacy but are limited by the scarcity of non-immunogenic protein elements responsive to non-immunosuppressive, clinically approved drugs with favorable pharmacodynamics. Here, we combine rational design and library-based optimization of a protein-protein interaction (PPI) of human origin to develop venetoclax-controlled Drug-Regulated Off-switch PPI (DROP)-CARs. DROP-CARs enable dose-dependent release of the tumor-targeting scFv and consequent T-cell dissociation from the target tumor cell. Additionally, we present proof-of-concept for a dual DROP-CAR controlled by different small molecules, as well as for logic-gated synthetic receptors enabling STAT3 signaling. We demonstrate in vitro and in vivo function of DROP-CAR T cells and conclude that the approach holds promise for clinical application.

synthetic biology↗

bioRxiv2023

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells

I.Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3{zeta} (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3{zeta} gene as the CARs activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCR{gamma}/{delta} T cells, regulatory T cells, and NK cells. In T cells, CD3{zeta} in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3{zeta}-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3{zeta}-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC-edited CAR-T cells, integration of a Her2-CAR into CD3{zeta} conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3{zeta} gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3{zeta} gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key pointsO_LIIntegration of {zeta}-deficient CARs into CD3{zeta} gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use C_LIO_LICD3{zeta}-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions C_LI

immunology↗

bioRxiv2020

CD19-targeting CAR T cells potently redirected to kill solid tumor cells

Successful CAR T cell therapy for the treatment of solid tumors requires exemplary CAR T cell expansion, persistence and fitness, and the ability to target tumor antigens safely. Here we address this constellation of critical attributes for successful cellular therapy by using integrated technologies that simplify development and derisk clinical translation. We have developed a CAR-CD19 T cell that secretes a CD19-anti-Her2 bridging protein. This cell therapy strategy exploits the ability of CD19-targeting CAR T cells to interact with CD19 on normal B cells to drive expansion, persistence and fitness. The secreted bridging protein potently binds to Her2-positive tumor cells, mediating CAR-CD19 T cell cytotoxicity in vitro and in vivo. Because of its short half-life, the secreted bridging protein will selectively accumulate at the site of highest antigen expression, ie. at the tumor. Bridging proteins that bind to multiple different tumor antigens have been created. Therefore, antigen-bridging CAR-CD19 T cells incorporate critical attributes for successful solid tumor cell therapy. This platform can be exploited to attack tumor antigens on any cancer.

cancer biology↗