Franziska Füchsl

Chimeric antigen receptor (CAR) T cells exhibit high response rates in B-cell malignancies, but most patients eventually relapse. A key mechanism of treatment failure is the loss or downregulation of tumor antigen expression, yet strategies to modulate cell surface levels of CAR T-cell targets remain largely unexplored. Here, we identify B-cell maturation antigen (BCMA), a central CAR T-cell target in multiple myeloma (MM), as a highly short-lived protein that undergoes K48-linked polyubiquitylation at the plasma membrane, leading to its p97-dependent degradation via the ubiquitin-proteasome system (UPS). This previously unprecedented mechanism of plasma membrane protein regulation enables significant enhancement of BCMA expression via proteasome inhibitors (PIs). The clinically approved PI carfilzomib (CFZ) significantly enhances the efficacy of BCMA-directed CAR T cells against both PI-sensitive and -refractory MM cells in vitro and in vivo. Notably, CFZ treatment of 10 patients with BMCA CAR T-cell therapy relapse, under the CFZ after BCMA CAR T-cell (CarCAR) protocol, resulted in increased BCMA expression in all patients. However, clinical responses were observed only in those with residual and/or expanding CAR T cells, suggesting restored CAR T-cell function. These findings provide a rationale for the use of CFZ treatment in relapsed or refractory MM after BCMA CAR T-cell therapy, advocate for future trials combining CFZ with BCMA CAR T cells, and provide a framework for exploring UPS-dependent degradation of other immunotherapy antigens.

Background: Optimizing T cell activation strength is emerging as a critical factor in improving adoptive cellular therapy (ACT). We previously reported that neoantigen-specific T cell receptor (TCR) clonotypes from a patient with metastatic melanoma exhibited enhanced resilience to repeated stimulation when initially activated at moderate levels. Methods: Building on these observations, we applied transient, low-dose MEK inhibition (MEKi) to fine-tune T cell signal strength during early activation. We evaluated this combinatorial strategy in vitro using co-cultures of CD8+ T cells engineered with patient-derived neoantigen-specific TCRs, alongside chimeric antigen receptor-T cells, bispecific T cell engagers, and non-engineered tumor-infiltrating lymphocytes (TILs). In vivo efficacy was evaluated in a xenograft model with intravenous TCR-T cell transfer and systemic low-dose MEKi. Results: MEKi co-treatment induced a more tempered activation profile that enhanced T cell proliferation, fitness, and persistence under strong stimulation. These effects were consistent across various in vitro and in vivo models for engineered T cells as well as primary melanoma-derived TILs. MEKi dampened the pro-inflammatory T cell activation profile, most notably diminishing tumor necrosis factor (TNF) secretion, mechanistically driven by coordinated and selective disruption of the key transcriptional regulators nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and nuclear factor of activated T cells (NFAT) while partly preserving activator protein 1 (AP-1) activity. Conclusion: These findings highlight moderate activation as a critical determinant of engineered T cell long-term performance. Low-dose MEKi offers a therapeutic tool for fine-tuning T cell activation and enhancing ACT efficacy.

Neoantigen-specific T cell receptors (neoTCRs) promise safe, personalized anti-tumor immunotherapy. However, detailed assessment of neoTCR-characteristics affecting therapeutic efficacy is mostly missing. Previously, we identified diverse neoTCRs restricted to different neoantigens in a melanoma patient. In this work, we now combine single-cell TCR-sequencing and RNA-sequencing after neoantigen-specific restimulation of peripheral blood-derived CD8⁺ T cells of this patient. We detect neoTCRs with specificity for the previously detected neoantigens and perform fine-characterization of neoTCR-transgenic (tg) T cells in vitro and in vivo. We describe a heterogeneous spectrum of TCR-intrinsic activation patterns in response to a shared neoepitope ranging from previously detected more highly frequent neoTCRs with moderate activation to rare ones with initially stronger activation. Experimental restimulation of adoptively transferred neoTCR-tg T cells in a xenogeneic rechallenge tumor model demonstrates superior anti-tumor responses of moderate neoTCR-tg T cells upon repeated tumor contact. These insights have significant implications for the selection of TCRs for therapeutic engineering of TCR-tg T cells.

Neoantigen-specific T cell receptors (neoTCRs) promise a safe, highly personalized therapeutic approach in anti-tumor immunotherapy. Substantial progress has been made regarding their identification whereas detailed functional assessment of single TCR characteristics impacting therapeutic efficacy is lacking. We previously identified and functionally characterized neoTCRs specific for neoepitopes derived from KIF2C and SYTL4 demonstrating differences in functional avidity in a patient with metastatic melanoma. In this work, we now combined single-cell TCR- and RNA-sequencing using stimulated peripheral blood derived CD8 ⁺ T cells of this patient and thereby identified two new neoTCRs recognizing the previously identified mutated epitope KIF2C ^P13L . Analyzing patient-derived neoTCR expressing T cells, we detected distinct activation patterns as a measure for substantial heterogeneity within oligoclonal T cell responses towards neoantigens upon specific ex vivo-restimulation. Moreover, neoTCR-transgenic T cells from healthy donors were employed for detailed in vitro and in vivo fine-characterization focusing on TCR-intrinsic functional patterns. Most importantly, in a xenogeneic mouse model experimentally simulating rechallenge of tumor infiltrating lymphocytes (TILs) after adoptive T cell transfer, we found that T cells expressing neoTCRs with a moderate activation profile provide a stable and more sustained anti-tumor response upon repeated in vivo tumor challenge as compared to neoTCRs with a stronger, burst-like reactivity. These insights have significant implications for engineering TCR-transgenic T cells for therapeutic purposes. One Sentence Summary: Combining TCR specificity linked single-cell transcriptomics with in vitro and in vivo characterization of transgenic T cells helps to decipher functional potential and persistence of neoantigen-specific T cell receptors (TCRs) for TCR-transgenic T cell-based adoptive cellular anti-tumor immunotherapy.

T cells are important players in the antitumor immune response. Over the past few years, the adoptive transfer of genetically modified, autologous T cells—specifically redirected toward the tumor by expressing either a T cell receptor (TCR) or a chimeric antigen receptor (CAR)—has been adopted for use in the clinic. At the moment, the therapeutic application of CD19- and, increasingly, BCMA-targeting-engineered CAR-T cells have been approved and have yielded partly impressive results in hematologic malignancies. However, employing transgenic T cells for the treatment of solid tumors remains more troublesome, and numerous hurdles within the highly immunosuppressive tumor microenvironment (TME) need to be overcome to achieve tumor control. In this review, we focused on the challenges that these therapies must face on three different levels: infiltrating the tumor, exerting efficient antitumor activity, and overcoming T cell exhaustion and dysfunction. We aimed to discuss different options to pave the way for potent transgenic T cell-mediated tumor rejection by engineering either the TME or the transgenic T cell itself, which responds to the environment.

Despite the substantial improvement of therapeutic approaches, multiple myeloma (MM) remains mostly incurable. However, immunotherapeutic and especially T cell-based approaches pioneered the therapeutic landscape for relapsed and refractory disease recently. Targeting B-cell maturation antigen (BCMA) on myeloma cells has been demonstrated to be highly effective not only by antibody-derived constructs but also by adoptive cellular therapies. Chimeric antigen receptor (CAR)-transgenic T cells lead to deep, albeit mostly not durable responses with manageable side-effects in intensively pretreated patients. The spectrum of adoptive T cell-transfer covers synthetic CARs with diverse specificities as well as currently less well-established T cell receptor (TCR)-based personalized strategies. In this review, we want to focus on treatment characteristics including efficacy and safety of CAR- and TCR-transgenic T cells in MM as well as the future potential these novel therapies may have. ACT with transgenic T cells has only entered clinical trials and various engineering strategies for optimization of T cell responses are necessary to overcome therapy resistance mechanisms. We want to outline the current success in engineering CAR- and TCR-T cells, but also discuss challenges including resistance mechanisms of MM for evading T cell therapy and point out possible novel strategies.

Background Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation. Methods Three neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient’s immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient’s TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing. Results Selected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation. Conclusions We performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.