Cord Derived 8F4CAR-iNKT Cells to Prevent Acute Myeloid Leukemia Relapse and Graft Vs. Host Disease after Allogeneic Stem Cell Transplantation

While allogeneic stem cell transplant (ASCT) is a curative therapy for acute myeloid leukemia (AML), only 50% of patients reach long-term survival with cancer relapse and graft vs host disease (GvHD) being the primary contributors to mortality. An innovative approach with the potential to prevent both ASCT comorbidities is needed. Invariant natural killer T (iNKT) cells are powerful immune regulators that are directly correlated with the reduction of GvHD onset in humans and are well documented to ameliorate GvHD in murine models. Previous work from our group demonstrated cord blood-derived iNKT cells can suppress GvHD more efficiently than adult-derived iNKT cells primarily through the expression of IL-10. Thus, we hypothesize the addition of 8F4 chimeric antigen receptor (8F4CAR), specific for the AML-associated antigen PR1 presented by HLA/A201, to cord-derived iNKT cells will result in an off-the-shelf cell product that retains immunoregulatory properties and lyses AML cells. Here we report an expansion protocol tailored to generate cord-derived 8F4CAR-iNKT cells as well as in vitro functional data to support our hypothesis.

We enriched iNKT cells from the mononuclear cells of buffy coats and cord units (n=4) with magnetic beads coated with anti-iNKTCR antibody. Enriched cells were stimulated with irradiated allogeneic dendritic cells (DCs) in the presence of α-Galactosylceramide (αGalCer) and IL-2 on day 0, followed by transduction with retrovirus harboring 8F4CAR on day 3. On day 14, iNKT cells were restimulated with αGalCer pulsed DCs and IL-2. After two antigenic stimulations, cord-derived untranslated (UT) and 8F4CAR-iNKT cells underwent 77,119 and 88,930 fold change to a final number of 5.3x10⁷ and 6.1x10⁷ respectively compared to adult-derived UT and 8F4CAR-iNKT cells that underwent a 1,079 and 1,083 fold change to a final number of 4.8x10⁷ and 4.5x10⁷ respectively. Both adult and cord-derived UT and 8F4CAR-iNKT cells are highly pure with averages at or above 95% for all groups and retain high expression of 8F4CAR at 79.7% and 81.9% respectively. Interestingly, CD62L expression is significantly increased on cord UT (40.8%) and 8F4CAR (37.2%) iNKT cells compared to adult UT (15.3%) and 8F4CAR (13.8%) iNKT cells suggesting potentially better in vivo persistence of cord-derived iNKT cells than adult-derived.

Next, we evaluated the cytotoxic activity of 8F4CAR-iNKT cells against leukemia mediated via native iNKTCR or 8F4CAR recognition. We demonstrate both cord and adult 8F4CAR-iNKT cells exhibit >95% antigen-specific lysis of PR1 presenting leukemia cell lines. In addition, native iNKTCR-mediated killing of CD1d⁺ targets is preserved and enhanced in 8F4CAR compared to UT iNKT cells. Lastly, we assessed the cytokine production profile of UT and 8F4CAR-iNKT cells upon stimulation via iNKTCR or CAR. We found, in response to iNKTCR stimulation, the cytokine production profile of 8F4CAR-iNKT cells was similar to their UT counterparts both in Th1/Th2 type cytokines and quantity produced. Importantly, we observe that cord-derived 8F4CAR-iNKT cells retain the ability to produce IL-10, but do so only upon iNKTCR stimulation suggesting the preservation of the potent immune-regulatory properties of cord-derived iNKT cells via the iNKTCR-CD1d signaling axis.

In summary, cord-derived 8F4CAR-iNKT cells can be generated in greater numbers than adult iNKT cells with equivalent purity and 8F4CAR expression. Both cord and adult-derived iNKT cells display potent cytotoxic activity against AML cell lines via both iNKTCR and 8F4CAR. Notably, cord-derived 8F4CAR-iNKT cells maintain an immunosuppressive cytokine production profile upon iNKTCR-CD1d signaling, but abrogate IL-10 release upon 8F4CAR-mediated cytolysis, suggesting a dual-functionality in GvHD prevention while not compromising GvL. The dual-functionality of cord-derived 8F4CAR-iNKT cells is currently being evaluated via xenogenic leukemia/GvHD murine model.

Shpall:Fibroblasts and FibroBiologics: Consultancy; Navan: Consultancy; axio: Consultancy; NY blood center: Consultancy; Affimed: Other: License agreement; Bayer: Honoraria; Takeda: Patents & Royalties; adaptimmune: Consultancy.