Background: Anti-CD19 targeting autologous chimeric antigen receptor (CAR) T cells (CD19 CAR T) continue to demonstrate transformative potential in achieving clinical disease remission across a spectrum of autoimmune diseases (AID). However, despite an increasing number of encouraging preliminary clinical datasets, their broader application and patient reach across AID indications is constrained by their reliance on conditioning chemotherapy (CCT), which is associated with significant toxicities such as cytopenia, severe infections and secondary malignancies. Furthermore, the requirement for prolonged hospitalization, the logistical challenges, inconsistencies and cost surrounding manufacture, and continuing safety concerns (CRS and ICANS) present formidable barriers to widespread adoption. Lastly, the underlying immunological pathology of many AIDs is driven by diverse and spatially distinct populations of B and T cell subsets that cannot be entirely targeted and depleted by existing CD19 CAR T cell therapies.

Objectives: To overcome these significant limitations, we aim to develop a n e x t-generation m ulti-antigen targeting CAR T cell (NxM CAR T) that supports scalable manufacture and off-the-shelf administration without the need for CCT. We believe this approach will enable broad outpatient adoption, reduce toxicities, and promote elimination of aberrant B and T cell subsets to elicit an immunological reset, allowing for application across a wide range of AID pathologies.

Methods: Using a clonal induced pluripotent stem cell (iPSC) line, we performed a series of precise multiplexed engineering events to incorporate novel synthetic functional elements designed to provide effective targeting across specific B and T cell subsets, to enhance bio-distribution to primary, secondary and tertiary tissues, and to overcome the need for administration of CCT. Following the differentiation from iPSC clone through to T cells, we evaluated canonical CAR T cell attributes using a collection of tumor cells lines, as well as AID pateint derived peripheral blood mononuclear cells (PBMCs), across a suite of in vitro and in vivo functional assays designed to model the diverse immune cell subset populations associated with AID, including in the presence or absence of allogeneic host immune cell pressure.

Results: NxM CAR T cells consist of a clonal population of genetically edited cells that express two independent CAR motifs targeting separate antigens. These cells are deficient in endogenous TCRα chain and CD38 expression, exhibit a CD8αβ T cell phenotype comparable to that of a potent cytotoxic T cell poised for activation, and lack expression of canonical exhaustion markers following CAR-mediated activation. NxM T cells express an anti-CD19 CAR habouring a novel 1XX signaling endodomain, and an anti-BCMA CAR exhibiting potent and specific elimination of CD19+ B cells across multiple in vitro cytotoxicity assays, including those containing PBMCs from AID patients, at levels comparable to primary CD19 CAR T cells. Moreover, NxM CAR T cells demonstrate the added ability to specifically target CD19- BCMA+ plasma cells, in addition to CD19+ B cells, and achieve durable depletion of B cells and plasma cells in primary, secondary and tertiary tissues, including the bone marrow, in surrogate murine lymphoma and/or multiple myeloma xenograft tumor models. To overcome the need for administration of CCT, NxM CAR T cells also contain a novel allo-immune defense receptor (ADR), which we have previously shown to promote T cell expansion, function, and persistence in an allogeneic setting, as well as a genetic disruption of the immune synapse adhesion molecule CD58 (CD58 ^KO ). ADR/CD58 ^KO NxM CAR T cells displayed enhanced persistence and cytotoxicity in an in vitro allogeneic re-stimulation assay, relative to control cells that lack ADR/CD58 ^KO , eliminating target cells through multiple rounds of re-challenge in the presence of alloreactive PBMCs. Similarly, ADR/CD58 ^KO NxM CAR T cells maintained tumor growth inhibition and persistence in vivo in the presence of unmatched alloreactive T cells.To evaluate whether targeting CD38, a surface glycoprotein expressed on lymphocytes detected in multiple AIDs, could simultaneously target plasma cells, plasmablasts, and aberrant T cell subsets, we incorporated an anti-CD38 CAR modality into a CD19 CAR-targeted NxM CAR T cell. Using the collection of in vitro and in vivo assays described above, we highlight that this multi-antigen, multi-lymphocyte subset targeting strategy provided potent elimination of aberrant CD19+ B cells, CD38+ plasma cells and plasmablasts, and CD38+ activated lymphocytes.

Conclusion: This NxM CAR T cell platform represents a significant advancement in cell therapy approaches for the treatment of B and/or T cell driven pathologies. By targeting B cell, plasma cells, plasmablasts, and T cell subsets, in isolation or desired combinations therof, and circumventing the need for CCT, NxM CAR T cells promise to enhance therapeutic outcomes, reduce treatment-associated toxicities, and maximize patient access via outpatient treatment. Their scalability and versatility position them as a compelling therapeutic option for SLE with broader application across the AID spectrum.

REFERENCES: NIL.

Acknowledgements: NIL.

Disclosure of Interests: John Goulding Fate Therapeutics, John Reiser Fate Therapeutics, Alison O’Connor Fate Therapeutics, Daniel Morales-Mantilla Fate Therapeutics, Rina Mbofung Fate Therapeutics, Alan Williams Fate Therapeutics, Alma Gutierrez Fate Therapeutics, Mark Jelcic Fate Therapeutics, Yijia Pan Fate Therapeutics, Brian Groff Fate Therapeutics, Angela Macia Fate Therapeutics, Nicholas Brookhouser Fate Therapeutics, Trever Greene Fate Therapeutics, Sreedevi Raman Fate Therapeutics, Ramesh Janani Fate Therapeutics, Bryan Hancock Fate Therapeutics, Matthew Denholtz Fate Therapeutics, Betsy Rezner Fate Therapeutics, Ramzey Abujarour Fate Therapeutics, Lilly Wong Fate Therapeutics, Vaneet Sandhu Fate Therapeutics, Tom Lee Fate Therapeutics, Bahram Valamehr Fate Therapeutics, Jode Goodridge Fate Therapeutics.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.