T cell-mediated immune surveillance is critical for cancer control, yet its role in bone marrow malignancies remains poorly understood. Here, we integrate TCR profiling, HLA immunopeptidomics, and functional screening to characterize tumor-reactive T cells in the bone marrow of patients with multiple myeloma (MM) and acute myeloid leukemia (AML). These cells are transcriptionally defined by a conserved effector program distinct from the exhausted phenotype of tumor-reactive T cells in solid cancers. Immunopeptidomic profiling reveals a partially shared antigen landscape enriched for noncanonical peptides driving convergent TCR responses. We develop TFiT (tumor-reactive features in T cells), a transcriptional classifier that identifies these cells and stratifies immunotherapy, but not chemotherapy, response across independent MM and AML cohorts, supporting its specificity for T cell-mediated tumor control. These findings reveal a latent but activatable anti-tumor T cell compartment in bone marrow malignancies and provide a framework for engaging endogenous immunity in MM and AML.

Abstract Background: T cell receptor (TCR)-engineered T cell therapies targeting tumor-associated antigens such as PRAME have shown promising clinical activity in melanoma (Wermke, Nature Medicine, 2025). We identified high PRAME expression in multiple relapsed, refractory pediatric tumors in our pediatric precision oncology program INFORM. Here we report on a named-patient-use of transgenic PRAME-specific TCR T cells in a pediatric patient with exceeded curative treatment option, not eligible for any ongoing clinical trial. Case Description: A 17-year-old male with rapidly progressive, relapsed nephroblastoma after 2 prior systemic and multiple local lines of therapy presented in good general condition. Baseline imaging revealed a 16 cm (longest diameter) abdominal target lesion, multiple hepatic metastases (≤6 cm), bilateral pulmonary metastases and a solitary intracranial metastasis. Methods: High PRAME expression and HLA-A*02:01 status were assessed by bulk RNA sequencing and confirmed by immunohistochemistry. After written informed consent, the patient was treated under named-patient use. Autologous CD4⁺ and CD8⁺ T cells were enriched from leukapheresis, activated, and transduced with an investigational clinical-grade lentiviral vector using the CliniMACS Prodigy platform. The vector encoding a PRAME-specific TCR and a CD8αβ co-receptor was supplied by Immatics. After lymphodepletion with fludarabine and cyclophosphamide, freshly harvested T cells were infused at 1 × 10⁹ transduced cells/m². Results: T cell infusion led to rapid in vivo expansion, accompanied by severe cytokine release syndrome that resolved promptly with multimodal anti-cytokine therapy and corticosteroids. Early imaging demonstrated global pseudo-progression and perifocal edema of the brain lesion. An abdominal tumor biopsy on day +9 after infusion showed extensive T cell infiltration and tumor necrosis. Subsequent imaging revealed marked regression across all disease sites. Two residual pulmonary lesions resected on day +100 showed no viable tumor cells. FDG-PET demonstrated no metabolic activity in residual abdominal or hepatic lesions. Furthermore, circulating tumor-specific cell-free DNA in serum fell below the detection limit. The patient is in excellent physical condition. On day +120 PRAME-specific T cells comprised 14% of viable T cells in peripheral blood. Conclusions: Fresh point-of-care-manufactured PRAME-specific TCR T cells induced a deep antitumor response with histological, metabolic, and molecular remission evident 3 months post infusion and ongoing at 6 months follow up in a heavily pretreated pediatric patient with extensive multifocal nephroblastoma. These findings support further evaluation of PRAME-specific TCR T cell therapy in PRAME-positive pediatric cancers in a phase I/II clinical trial. Citation Format: Katharina Mair, Corinne Rossi, Jens H. Westhoff, Esther Wahlbrink, Kendra Maass, Sophia Scheuermann, Fabienne Engelmann, Simon Krost, Alexandra Tuch, Barbara Jones, Laura Fankhauser, Joachim B. Kunz, Johann Greil, Stella Okouoyo, Florian Selt, Elke Pfaff, Jutta Mattern, Maria E. Kögler, Roland Imle, Abdulsattar Alrajab, Jens-Peter Schenk, Inga Harting, Erik Winter, Patrick Günther, Markus Keßler, Astrid Burger, Juri Fuchs, Anita Schmitt, Carsten Müller-Tidow, Kathrin Schramm, Stefanie Volz, Robert J. Autry, David T. Jones, Kristian W. Pajtler, Dirk Jäger, Stefan Pfister, Thomas Grünewald, Andreas E. Kulozik, Olaf Witt, Patrick Schmidt, Christian M. Seitz. Point-of-care-manufactured PRAME-specific TCR T cell therapy induces deep remission in a heavily pre-treated pediatric patient with extensive, multifocal nephroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB326.

T cells that recognize tumor-specific mutations are crucial for cancer immunosurveillance and in adoptive transfer of TILs or transgenic-TCR T cell products. However, their challenging identification and isolation limits their use in clinical practice. Therefore, novel approaches to isolate tumor-specific T cells are needed. Here, we report the isolation of neoantigen-specific CD8+ T cells from a vaccination site of a metastatic breast cancer patient who received a personalized vaccine. Based on the somatic mutations, potential MHC binding epitopes were predicted, of which 17 were selected to generate a peptide vaccine. Cutaneous biopsies were processed after the fifth vaccination cycle to obtain infiltrating lymphocytes from the vaccination site (VILs). IFNγ ELISpot revealed reactivity to four peptides used in the vaccine. Reactive T cells from VILs were non-overlapping with those detected in the blood and the tumor-microenvironment. ScTCR Seq analysis revealed the presence of a clonotype in VILs that further expanded after a round of in vitro stimulation and validated to be specific against a private mutation, namely NCOR1L1475R, presented in the context of HLA-B * 07:02, with no reactivity to the wild-type peptide. Our study shows, for the first time, that tumor mutation – specific T cells are generated at high frequencies in the vaccination site and can be isolated with standard methods for TCR screening. The easy and safe accessibility of skin biopsies overcomes the major hurdles of current TCR screening approaches and present exciting opportunities for the development of innovative immunotherapeutic strategies.

Personalized treatment has become a realistic option for tumor patients, accelerated by significantly reduced sequencing costs of tumor genomes and advances in vaccine formulations. The druggability of cancer neo-antigens caused by individual mutations is centered in this effort. We here use an adeno-associated virus (AAV)-based virus-like particle (VLP) platform to compose a neo-antigen-specific protein vaccine that is effective in a murine prevention and treatment setting. Furthermore, we show that CD4+ T cell responses that are provided by the AAV capsid are crucial for effective murine melanoma treatment. To uncover the optimal composition of a peptide vaccine we de-linked major histocompatibility complex (MHC) class II helper peptides from the capsid and formulated an efficient neo-antigen-specific vaccine, which showed the independence of CD4+ T cell response from tumor sequences. The findings are supported by clinical data of neo-antigen-vaccinated tumor patients. Our results punctuate on the significance of MHC class II epitopes for CD8+ T cell responses and suggest a future use of AAVLPs as neo-epitope vaccines in personalized cancer treatments.

T cell-mediated immune surveillance is critical for cancer control, yet its endogenous effectiveness in hematological malignancies remains limited and poorly understood. Here, we integrate single-cell T cell receptor (TCR) profiling, HLA immunopeptidomics and functional antigen mapping to dissect the specificity landscape of bone marrow lymphocytes (BMLs) in multiple myeloma (MM) and acute myeloid leukemia (AML). We identify a rare subset of tumor-reactive T cells that exhibit a stereotyped transcriptional state distinct from bystander and virus-specific populations. Across both malignancies, immunopeptidomic profiling uncovers a partially conserved antigen repertoire enriched for noncanonical peptides, including products of novel or unannotated open reading frames (nuORFs), pseudogenes, and clonotypic immunoglobulin sequences. Several of these epitopes are recurrently presented and associated with convergent TCR responses across individuals. Based on this immune architecture, we develop a TCR-intrinsic fitness model that infers BML tumor specificity from transcriptional cues and stratifies immunotherapy response across three independent patient cohorts. Together, these findings map the latent potential of endogenous anti-tumor immunity in two biologically distinct diseases and provide a framework for decoding and restoring productive immune surveillance of hematological malignancies. Highlights ### Competing Interest Statement M.J.F reports speaker honoraria from Pfizer, Roche and Kerna Ventures and is a consultant for Moonwalk Biosciences. M.P. and E.W.G. are founders of Tcelltech. S.A.C. is a member of the scientific advisory boards of Kymera, PTM BioLabs, Seer and PrognomIQ. S.F. reports consultancy fees from Illumina. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Deutsche José Carreras Leukämie-Stiftung, https://ror.org/00826gz80, 01ZI/2022 Dr. Rolf M. Schwiete Stiftung, https://ror.org/03v7rwj71, 2025-018 Else Kröner-Fresenius-Stiftung, 2025_EKMS.52 Heidelberg University, https://ror.org/038t36y30, ExU 6.1.12

Abstract Innovative immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor infiltrating lymphocytes (TILs) have shown great success in the treatment of solid tumors and hematological malignancies. Although treatment of multiple myeloma with CAR T cells can induce deep responses, relapses frequently occur due to antigen escape and limited CAR T cell persistence. TCR-engineered T cells may show prolonged persistence in vivo and could mediate sustained antitumor effects. A further benefit of TCR transgenic T cells is the ability to target intracellular antigens that are inaccessible to CAR T cells, expanding the range of potential targets for immunotherapy. In our project, we propose to identify T cell receptors (TCRs) specifically targeting autologous myeloma cells. Tumor-reactive T cells were identified using the Bruker Cellular Analysis Lightning® platform, allowing simultaneous functional analysis of up to 1500 individual T cell/target cell interactions on a chip. Reactive T cells were identified upon detection of secreted cytokines (IFNγ, TNFα, IL2) and measurement of 4-1BB (CD137) surface expression. Tumor-reactive T cells showing various cytokine secretion patterns and 4-1BB expression profiles were detected in each myeloma patient (on average 11.9 T cells out of 1243 cells tested per assay run). Individual tumor-reactive T cells have been isolated and their TCRs were sequenced. TCR sequences of tumor-reactive T cells were mapped to single-cell RNA sequencing data of T cells from the same patiens to reveal a gene expression signature of myeloma-reactive T cells. TCR genes of reactive T cells were cloned and overexpressed in autologous T cells for functional validation and analysis of tumor derived neoepitope specificity. In summary, we present a pipeline allowing identification of myeloma-recognizing T cells and recovery of bona fide tumor-reactive TCRs eligible for patient-individualized T cell therapy. Citation Format: Tim Robin Wagner, Niklas Kehl, Simon Steiger, Michael Kilian, Bruno Schönfelder, Tamara Boschert, Katharina Lindner, Patrick Schmidt, Karsten Rippe, Hartmut Goldschmidt, Marc-Steffen Raab, Michael Platten, Mirco Friedrich, Stefan B. Eichmüller. Identification of tumor-reactive T cell receptors through functional single cell interaction analyses for personalized T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 14.

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β2-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients’ HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β2m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8+ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.

Within the last decade CAR-T cells have changed the landscape of treatment regimen for leukemia and myeloma, which is reflected by the first FDA approval of this class of living drugs in 2017. As of today, the clinical trial situation aims on CAR-T applicability in solid tumors, which is more arduous due to antigen heterogeneity and limited CAR-T persistence. More personalized and multi-target oriented cellular products may offer a solution to overcome these problems but then CAR identification and selection display the major bottleneck in the drug development process. Usually, matching scFvs are selected from targeting screens of a phage-display library and hits are subsequently cloned in CAR backbones and tested for functionality and possible limiting factors as the occurrence of tonic signaling. This makes the whole process very time consuming and laborious. Within this project, we propose a novel CAR-T selection method that rapidly shortens the discovery procedure. We have developed a full length CAR library in nS/MARt DNA vectors that is electroporated in a Jurkat reporter cell line reflecting entirely its full diversity. By this, we can quickly identify the amount of tonic signaling CARs and exclude them from further selection. For on-target selection we first perform a bulk pre-selection followed by a single cell functionality screening using the Berkeley Lights Lightning™ device. This allows us to export hits as clonal viable cells that undergo long length Nanopore CAR-RNA sequencing. Our findings show the feasibility of our approach and that it can shorten the timeframe needed for the full selection process from weeks to days. Citation Format: Eren Boga, Luisa Berger, Alice De Roia, Inka Zörnig, Robert Embacher, Stefan B. Eichmüller, Dirk Jäger, Richard Harbottle, Patrick Schmidt. A combination approach of a cellular library and single cell microfluidics analysis for the rapid selection of CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1769.

Abstract BACKGROUND Novel concepts in immunotherapy have evolved with the identification of potential (neo)epitopes and/or combinations with immune checkpoint inhibitors (ICI). In contrast to many other solid tumors, ICI have not improved outcome for patients with glioblastoma (GB) in phase 3 studies. However, antigen-specific T cells induced by vaccines or adoptively transferred as chimeric antigen receptor (CAR) T cells produced encouraging responses in early clinical trials and case series. From one of these trials, the Actively Personalized Vaccine Consortium (GAPVAC) European Clinical Trial 101, we have identified a human leukocyte antigen (HLA)-A2-restricted T cell receptor (TCR) targeting protein tyrosine phosphatase receptor type zeta 1 (PTPRZ1). PTPRZ1 is strongly overexpressed in malignant gliomas, especially GB, and analyses of intratumoral heterogeneity revealed that the level of PTPRZ1 overexpression is strongly associated with cancer stemness. The PTPRZ1 epitope is naturally presented on HLA-A2. MATERIAL AND METHODS INVENT4GB is a German, multicenter, phase 1, first-in-human, first-of-kind dose-escalation window-of-opportunity investigator-initiated trial in recurrent GB, eligible for re-resection, assessing safety and feasibility of therapy with intravenously (iv) and intraventricularly (icv) infused TCR-engineered T cells (TCR-T). FPI is scheduled for 2024. HLA-A*02:01 positive patients will receive one neoadjuvant iv followed by up to three adjuvant icv infusions of PTPRZ1-specific TCR-transgenic autologous T cell therapy (TCR-T-001) with cyclophosphamide- and fludarabine-based lymphodepletion prior to iv infusion. Patient autologous T cells will be engineered by non-integrating nano scaffold matrix attachment region (SMAR) TCR DNA vectors. Icv infusions will be performed via intraventricular catheter implanted during re-resection. Biomarker discovery includes magnetic resonance imaging (MRI) and longitudinal flow cytometry-based immune monitoring of blood and cerebrospinal fluid. Primary endpoints are feasibility and safety (determination of maximum tolerated dose/ MTD), key secondary endpoints are progression-free survival according to the Immunotherapy Response Assessment in Neurooncology (iRANO) criteria. RESULTS TCR-T-001 robustly recognizes target-expressing cells in vitro and is efficacious in preclinical tumor models. Preclinical data and the trial concept will be presented. CONCLUSION INVENT4GB is a first-of-kind TCR-engineered T cell therapy for GB patients.

Supplementary Figures 1-3 from The Proteasome Inhibitor Bortezomib Sensitizes Melanoma Cells toward Adoptive CTL Attack

Abstract Innovative immunotherapy approaches such as adoptive transfer of chimeric antigen receptor (CAR) T cells or tumor infiltrating lymphocytes (TILs) have shown great success in the treatment of solid tumors and hematological malignancies. Although treatment of multiple myeloma with CAR T cells can induce deep responses, relapses frequently occur due to antigen escape and limited CAR T cell persistence. TCR-engineered T cells may mediate sustained antitumor effects upon recognition of intracellular targets, thereby significantly increasing the range of relevant target antigens. In our project, we propose to identify T cell receptors (TCRs) specifically targeting autologous myeloma cells. Tumor-reactive T cells were identified using the Berkeley Lights Lightning platform, allowing simultaneous functional analysis of up to 1500 individual T cell/target cell interactions per run. Reactive T cells have been identified upon detection of secreted chemokines (IFNγ, TNFα, IL-2) and by measurement of CD137 surface expression. Tumor-reactive T cells showing various cytokine secretion patterns and CD137 expression profiles could be detected in each myeloma patient (7 to 26 of approx.1200 cells tested per patient). Individual tumor reactive T cells have been isolated for TCR sequencing. Recovered TCR genes will be cloned and overexpressed in autologous T cells for functional validation and analysis of tumor derived neoepitope specificity. In summary, we present a pipeline allowing identification of myeloma-recognizing T cells and recovery of bona fide tumor-reactive TCRs eligible for patient-individualized T cell therapy. Citation Format: Tim R. Wagner, Eren Boğa, Patrick Schmidt, Wolfram Osen, Michael Platten, Hartmut Goldschmidt, Marc S. Raab, Mirco J. Friedrich, Stefan B. Eichmüller. Identification of myeloma-specific T cell receptors by functional single cell interaction analyses. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4071.

T cell receptors (TCRs) play a crucial role in orchestrating cellular immunity in both health and disease, particularly in the context of cancer. Although immunotherapy and stem cell transplantation have shown promising clinical responses in hematological malignancies, these have not been linked to the presence of endogenous tumor reactive T cells. Moreover, the specificities and phenotypes of such tumor recognizing TCRs in these largely low immunogenic entities are unknown. In this study, we addressed this gap by developing an approach to map TCR specificities and transcriptional phenotypes of human bone marrow-resident T cells directly ex vivo, focusing on patients who were treatment-naïve at the time of biopsy. This allowed us to generalize our findings beyond immune checkpoint inhibitor-treated patients, a limitation of previous studies in solid cancers. To discover T cells that specifically recognize cancer cells or viral epitopes, we developed a multiplexed profiling approach using microfluidic reaction chambers, optical cytokine detection and TCR sequencing of patient-derived primary T cells. We were able to confidently map TCR recognition of 25,957 bone marrow-resident T cells from n=20 hematological cancer patients. Based on the acquired functional data and reverse phenotyping of screened TCRs to their original cell state in situ, we built a bone marrow T cell atlas charting >300,000 expression profiles in human bone marrow-resident T cells, their TCR specificities as well as their longitudinal clonal dynamics upon therapeutic perturbations in n=40 patients with newly diagnosed multiple myeloma (MM) and acute myeloid leukemia (AML). Our approach detected tumor-reactive T cell clones in the bone marrow of all screened individuals with varying frequency (1-5% of T cells), which were largely found to display a CD8+ effector-memory or progenitor-exhausted phenotype. In contrast, non-tumor reactive clones were either CD4+ or enriched for viral specificities. We identified a conserved signature of anti-tumor reactivity across patients. This signature included the ITGB1 gene, which encodes CD29 and was uniformly expressed in tumor reactive T cells. Importantly, ITGB1 expression was specifically restricted to tumor reactive, compared to virus-specific or bystander T cells. In two independent mouse tumor models, we found that CD29 protein was significantly upregulated on homed T cells carrying tumor reactive TCRs compared to non-reactive TCRs. A causal link between tumor reactive T cells and clinical response to hematological cancers would be strengthened by finding that these cells specifically recognize autologous cancer epitopes and clonally expand as part of a clinical anti-tumor response. We therefore cloned patient-individualized TCR libraries and performed immunoprecipitation of MHC class I : peptide complexes from cancer cells. We validated TCR binding against ~1-20 epitopes per patient, which were mostly derived from cancer/testis antigens (CTAs) and novel open reading frames (nORFs). Finally, we classified tumor reactive TCRs in three clinical cohorts: Notably, we observed selective expansion of tumor reactive TCRs upon autologous stem cell transplantation, which was associated with deep clinical response in newly diagnosed MM patients. Conversely, the failure of tumor reactive TCRs to clonally expand on-treatment underlined clinical non-response to bispecific BCMAxCD3 antibody therapy (MM) or combination therapy with immune checkpoint inhibitors and Azacytidine (AML). We show here that, unexpectedly, clinically relevant endogenous anti-tumor reactivity lies in a rare subset of bone marrow-resident T cells. The recurrent detection of these immunotherapy-responsive TCRs in patients with MM or AML revealed the selective clonal expansion of tumor-reactive TCRs following autologous stem cell transplantation. We describe a conserved gene signature of tumor reactivity that diverges from lymphocytes infiltrating solid tumors and includes CD29 as an indicator of antigen-specific T cell homing. Our findings facilitate the antigen-agnostic identification of tumor-reactive TCRs with potential for future patient-individualized cell therapies and suggest the relevance of immune responses against alternative epitopes, such as cancer/testis antigens, in hematological malignancies with low mutational loads.

The utilization of adeno-associated viruses (AAVs) as vaccines has primarily been focused on the induction of antibody responses. Displaying antigens derived from HER2 or HPV on the AAV capsid surface lead to strong humoral immune responses. However, the potential of AAVs as T cell inducing vaccines has been poorly investigated. Model antigens have been inserted into the capsid of AAVs to analyze T cell responses, but these studies rather focused on preventing vector immunity for an improved tolerance of gene therapies. By using this as a starting point, we set out to test AAVs as T cell inducing vaccines with the prospect of targeting cancer neoantigens. In first experiments, the general properties of the vaccination strategy were estimated by displaying the ovalbumin-derived model antigen SIINFEKL on the surface of adeno-associated virus-like particles (AAVLPs). Upon injection of the AAVLP-SIINFEKL vaccine, mice developed strong CD8+ T cell responses against the displayed antigen. Highest immune responses were achieved by subcutaneous hock injection of AAVLPs, adjuvanted by Montanide ISA 51 VG. SIINFEKL-specific T cell responses peaked around three weeks after vaccination, whereas a memory subset remained present for long term. The anti-tumor efficacy of the vaccine was shown by injecting SIINFEKL-expressing B16F10 melanoma cells subcutaneously into mice, in which the tumor was completely rejected after vaccination. Interestingly, the induction of CD8+ T cell responses and the tumor protection depended on the presence of CD4+ T cells. Accordingly, T helper epitopes were identified in the AAVLP capsid sequence. In addition to the initial tests, a set of murine neoantigens, derived from B16F10 cells, were displayed on AAVLPs. In direct comparison to a peptide vaccine, which did not have an effect on tumor growth, the AAVLP vaccine had a significant impact on the tumor growth rate. In conclusion, AAVLPs show promising effects as T cell vaccines. The vaccination strategy can be used to induce cytotoxic T cell responses in general and anti-tumor effects in particular. AAV capsid-specific helper epitopes is one inherent advantage, since the AAVLP vaccine delivers immune stimulation within the viral particle itself. Thus, antigen-displaying AAVLPs could be an alternative to current gold standards in the field of neoantigen vaccines and have a prospect for future clinical applications. Citation Format: Lasse Neukirch, Patrick Schmidt, Inka Zörnig, Dirk Jäger, Silke Uhrig-Schmidt. CD8+ T cell-mediated tumor rejection by an Adeno-associated virus-like particle (AAVLP) vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3563.

The compelling need to provide adoptive cell therapy (ACT) to an increasing number of oncology patients within a meaningful therapeutic window makes the development of an efficient, fast, versatile, and safe genetic tool for creating recombinant T cells indispensable. In this study, we used nonintegrating minimally sized DNA vectors with an enhanced capability of generating genetically modified cells, and we demonstrate that they can be efficiently used to engineer human T lymphocytes. This vector platform contains no viral components and is capable of replicating extrachromosomally in the nucleus of dividing cells, providing persistent transgene expression in human T cells without affecting their behavior and molecular integrity. We use this technology to provide a manufacturing protocol to quickly generate chimeric antigen receptor (CAR)-T cells at clinical scale in a closed system and demonstrate their enhanced anti- tumor activity in vitro and in vivo in comparison to previously described integrating vectors.

Dear Editors, We have read with interest the comments by Schmitt et al1 to our study of the “cost of decentralized CAR T-cell production in an academic setting.”2 While the letter confirms the sensitivity of the topic, in our view it is, at least in part, a result of misunderstandings by its authors. Importantly, our study is not based on “scientific modeling” (as suggested in the letter) but rather on the application of standard cost accounting techniques using empirical data.3 Our objective has been to increase the transparency of one cost component often cited as a reason for certain pricing policies,4, 5 and to illuminate the potential to reduce manufacturing cost and improve efficiency offered by an alternative to commercial products and their high acquisition costs. Unfortunately, in their letter, the authors do not contribute to the much-needed transparency,6 as they claim that in their experience the (undefined) “real-life financial effort … differs dramatically” from our analysis, but do not offer any quantitative information to support their claim. For clarification, let us first reiterate that our report deals with just one cost component, that is, the fixed and variable cost arising from decentralized chimeric antigen receptor (CAR) T-cell production in our setting. This should not be confused with pricing and reimbursement policies. Especially in commercial settings, the latter will also be influenced by the cost of research and development, by administrative overheads including marketing and sales expenditures, as well as by the expected return on investment. We note that “clinical development” (the last issue raised in the letter by Schmitt et al1) represents a cost category clearly different from the cost of production. In sum, the letter of Schmitt et al1 does not contribute to an increased transparency of CAR T-cell manufacturing costs. However, although perhaps inadvertently, it illustrates the potential for future cost reductions due to technological improvements (“learning curve effects,” such as the use of semiautomated closed systems, as well as nonviral vector systems) and standardization, in addition to economies of scale and scope to be expected in the future. In addition to his primary employment with the DKFZ, Michael Schlander is Chairman & Scientific Director of the nonprofit Institute for Innovation & Valuation in Health Care (InnoVal-HC) in Wiesbaden/Germany, which accepts funding under an unrestricted educational grants policy only. In the context of his involvement with non–cancer-related projects at InnoVal-HC, he has received travel expenses and honoraria for lectures and presentations. The other authors declared no potential conflict of interest.

Abstract The capability to introduce Chimeric Antigen Receptors (CARs) into naïve Human T-Cells represents one of the most promising therapeutic strategies for the treatment of cancer. However, virus mediated adoptive cell therapy (ACT) remain severely limited by two factors: the long lead time and high cost of GMP virus manufacture, and the virus safety profiles. What if the entire ACT process could be sped up, made safer and more cost-effective by at least an order of magnitude? We have invented a novel DNA Vector platform based on scaffold/matrix attachment region (S/MAR) component that provides the opportunity to efficiently generate genetically engineered T-cells. This system is based on a nanovector technology. It contains no immunogenic and comprises only clinically approved sequences. It is easy, simple and cost-efficient to produce. Critically, it does not integrate and replicates autonomously and extrachromosomally in the nuclei of dividing primary human cells, thus avoiding the inherent risk of integrative mutagenesis. Through a process of iterative CpG depletion, selection marker minimalisation, empirical promoter design and elimination of cryptic eukaryotic signals our nano-S/MARt DNA Vector (nS/MARt) can be efficiently transfected into primary human T Cells. nS/MARt vectors are designed to remain stably expressed, and in addition to having the best in class safety profile, they also demonstrate enhanced performance as a biopharmaceutical. Human T-cells engineered to express the CAR receptor against the carcinoembryonic antigen (CEA) using a nS/MARt vector provide more effective killing of human cancer cells in vitro than those engineered with integrative lentivirus. These results hold in vivo, where nS/MARt transfected CAR Tcells outperform the lentivirally transduced cells, attenuating tumour growth and extending mouse survival. Moreover, in pre-clinical studies, the comparison with the FDA approved drug Kymriah®,T cells modified with nS/MARt vectors harbouring the expression of a CD19 CAR are comparable to those engineered with the viral vector. Notably, we have also taken steps to evaluate nS/MARt's scalability and have succeeded in manufacturing a clinically relevant number of CAR-T Cells (2 × 107CAR+ T-cells per kilo, we estimate the production for an individual of 80 Kg). The extension of the results from mice to patients-scale required a 1000x scale up for the processing of T-cell transfection while halving the time for production to hit a meaningful therapeutic window. We have developed a novel manufacturing protocol where nS/MARt vectors can be used "off the shelf" for CAR-T therapy to generate a clinically relevant number of modified cells in just seven days. The delivery of our DNA to CD3+ cells, reaches ~60-70% with cell viability of 60%, that increases in the days that follow the cell electroporation. Thus, the most significant benefit will be for the patients that will be able to access the nS/MARt mediated therapy in 1 week. To translate this technology into a clinical reality a fermentation process that allows the preparation of 2.6 g/L of pure, supercoiled DNA was optimised. There is a pressing need to offer ACT to more oncology patients, and we believe that this novel DNA Vector system provides a unique and innovative approach to this therapeutic strategy for cancer therapy. Citation Format: Matthias Bozza, Alice De Roia, Aileen Berger, Alexandra Tuch, Patrick Schmidt, Richard Harbottle. A non-integrating, non-viral DNA Nanovector platform for the safe, persistent, and rapid manufacture of recombinant T-cells for Adoptive cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4066.

Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy with high acquisition costs, and it has raised concerns about affordability and sustainability in many countries. Furthermore, the current centralized production paradigm for the T cells is less than satisfactory. Therefore, several countries are exploring alternative T-cell production modes. Our study is based on the T-cell production experience in a nonprofit setting in Germany. We first identified the work steps and main activities in the production process. Then we determined the fixed costs and variable costs. Main cost components included personnel and technician salaries, expenditure on equipment, a clean room, as well as production materials. All costs were calculated in 2018 euros and converted into U.S. dollars. For a clean room with one machine for closed and automated manufacturing installed, annual fixed costs summed up to approximately euro438 098 ($584 131). The variable cost per production was roughly euro34 798 ($46 397). At the maximum capacity of one machine, total cost per product would be close to euro60 000 ($78 849). As shown in the scenario analysis, if three machines were to be installed in the clean room, per production cost could be as low as euro45 000 (roughly $59905). If a cheaper alternative to lentivirus was used, per production total cost could be further reduced to approximately euro33 000 (roughly $44309). Decentralized T-cell production might be a less costly and more efficient alternative to the current centralized production mode that requires a high acquisition cost.