Alloimmune Defense Receptor Combined with Genetic Ablation of Adhesion Ligand CD58 Is a Comprehensive Approach to Promote Functional Persistence of Allogeneic Cell Therapies without Conditioning Chemotherapy
Authors: Williams Alan , Mbofung Rina , Morales-Mantilla Daniel , Groff Brian , O'Connor Alison , Li Binzhong , Janani Ramesh , Rezner Betsy , Jelcic Mark , Pan Yijia , Lee Tom , Malmberg Karl-Johan , Mamonkin Maksim , Goulding John , Goodridge Jode , Valamehr Bahram 
Published: December 2024

Clinical administration of chimeric antigen receptor (CAR) T cell and CAR NK cell therapies rely on conditioning chemotherapy (CCT) to deplete the host immune system, maximize access to homeostatic cytokines, and promote cell expansion and functional persistence. However, CCT also elicits pan-immune cell cytopenia, increases susceptibility to infection and malignancy, and requires costly patient hospitalization and administration of therapy in tertiary healthcare settings. Indeed, the administration of off-the-shelf CAR T cell and CAR NK cell therapies to patients without requiring CCT has the potential to significantly reduce treatment-related toxicities and cost of care, and, importantly, extend patient reach by enabling treatment in primary or outpatient community healthcare settings.
To eliminate the need for CCT, we established a unique approach through the cooperative action of a novel alloimmune defense receptor (ADR), a synthetic receptor that targets cell surface expression of 4-1BB (CD137) to eliminate alloreactive immune cells, and the knock-out of CD58 (CD58KO), a unidirectional synapse-stabilizing ligand where disruption attenuates the ability of alloreactive immune cells to properly function. To enable the distinct ability for consistent manufacture and uniform production of an off-the-shelf available, multiplexed-edited CAR T cell, we generated iPSC-derived T cells (iT cells), produced from a clonal master iPSC line engineered to homogeneously express ADR, to be completely deficient for the expression of CD58, and to express a CAR targeting B cell maturation antigen (BCMA) (ADR+/CD58KO CAR iT cells; ADR and CAR surface expression &gt;95%, CD58 not detected).
In an MLR assay containing saturated levels of allogeneic peripheral blood mononuclear cells (PBMCs) from an unmatched donor, ADR+/CD58KO CAR iT cells exhibited resistance to host-mediated rejection and uniquely demonstrated expansion in cell number over the duration of the assay, while the co-culture with control CAR iT cells (lacking ADR and with intact CD58) were eliminated. To further increase the stringency of our assessment and generate an environment to promote supraphysiological allogeneic rejection by immune cells, we substituted allogeneic PBMCs with T cells from an unmatched donor that were primed and enriched for alloreactivity (primed-T cells) against iT cells. As observed with allogeneic PBMCs, control CAR iT cells co-cultured with primed-T cells were depleted within a few days and expression of 41BB was detected on a large subset of the primed-T cell population in culture. In contrast, ADR+/CD58KO CAR iT cells durably persisted over the entire culture period, with the primed-T cells in the co-culture significantly reduced in number (p&lt;0.05) and without detectable 41BB expression (not detected vs 47% 41BB+ in control).
To confirm ADR+/CD58KO CAR iT cells retain potent anti-tumor efficacy under alloreactive pressure, we performed a rigorous tri-culture MLR study that combined anti-BCMA CAR iT cells with daily-dosing of tumor cells expressing BCMA in the presence of primed-T cells. In the absence of primed-T cells, co-cultures with repeat restimulation of BCMA+ tumor cells were equally eliminated by either CAR iT cells or ADR+/CD58KO CAR iT cells over the course of the assay. In the presence of primed-T cells, the tri-culture assay with control CAR iT cells showed rapid loss of tumor control with associated expansion of 41BB+ primed-T cells. In contrast, ADR+/CD58KO CAR iT cells demonstrated tumor growth inhibition equivalent to the control culture lacking primed-T cells, durably persisted through the end of the assay, and completely suppressed the 41BB+ primed-T cell population. Similar results were observed in allogeneic xenograft tumor models assessing the control of lymphoma and leukemia burden in vivo in the presence of alloreactive PBMCs and T cells.
Collectively, we demonstrate the potential of CAR iT cells incorporating the novel combination of ADR-arming and CD58KO to elicit a robust cytotoxic response against diseased cells in the presence of an intact endogenous immune compartment. The effective use of cell-based immunotherapies without requiring administration of CCT to patients facilitates broad clinical application across multiple lines of therapy, including in combination with standard-of-care agents that are used for treatment of patients with newly-diagnosed disease.