CD371-Targeted CAR T-Cells Secreting Interleukin-18 Exhibit Robust Expansion and Disease Clearance in Patients with Refractory Acute Myeloid Leukemia
Authors: Geyer Mark Blaine , DeWolf Susan , Mi Xiaoli , Shaffer Brian C. , Cadzin Briana , McAvoy Devin , Hosszu Kinga K. , Weis Kenyon , Lorenc Rachel , Lewis Alexander M. , Girotra Narina N. , Burns Erin R. , Um Jasmine S. , Yoo Sarah , Katsamakis Zoe , Wang Xiuyan , Riviere Isabelle , Curran Kevin J. , Park Jae H. , van den Brink Marcel R.M. , Abdel-Wahab Omar , Brentjens Renier , Daniyan Anthony F. 
Published: December 2024

Background: Patients (pts) with (w/) relapsed and refractory (R/R) acute myeloid leukemia (AML) have a dismal prognosis. Chimeric antigen receptor T cell (CAR T) therapy for AML has been limited by lack of optimal surface antigen targets, myelosuppression, and hostile tumor microenvironment (TME). CD371 is expressed on myeloid blasts and leukemia-initiating cells in most pts w/ AML. We designed next generation CAR T cells w/ fully human CD371-specific scFv (CD371), modified CD28 costimulatory domain to limit T cell exhaustion (SAVVY), wildtype CD3z, and constitutive secretion of interleukin 18 (IL-18) to enhance CAR T cytotoxicity and induce host immune response (CD371-SAVVYz-IL18).

Methods: We initiated a phase I clinical trial of CD371-SAVVYz-IL18 in pts w/ R/R AML (NCT06017258). Pts underwent leukapheresis for T cell collection and received fludarabine/cyclophosphamide conditioning + CD371-SAVVYz-IL18 (3x105 CAR Ts/kg, pts 1-2; 3x104/kg, pts 3-5). Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were defined by ASTCT consensus grading (G) and other toxicities by CTCAE v5.0. Responses were evaluated by ELN criteria. We performed spectral flow cytometry (FACS), 5'-based CITE-seq to detect RNA, TCRs, and surface proteins in single cells (n=26 samples, 251,642 cells) and measured 96 cytokines by Luminex FLEXMAP in serial samples to study CAR T products and their effect on AML and TME.

Results: 5 pts (4 F/1 M) were enrolled/treated in the pilot study w/ median (med) age 32 yrs (range [r] 16-45). All received prior high-dose cytarabine-containing therapy and venetoclax. Three relapsed post-allogeneic hematopoietic cell transplant (alloHCT). Med production time was 7 days (d). All had measurable AML pre-treatment w/ uniform (n=4) or partial (n=1) CD371.

CAR T expansion was seen in all pts w/ highest vector copy number (med: 23 k/mL, r: 15-1164 k/mL) and peak IL-18 levels (med: 28.4 k pg/mL, r: 1.3 k-58.2 k pg/mL) d7-14 post-CAR T, and not clearly correlated w/ AML burden. CRS was manageable (G1-2, n=3; G3-4, n=2) w/ med onset d2; only 1 experienced ICANS (G3). 3 dose-limiting toxicities observed overall, 2 at higher (3x105 CAR T/kg) dose (prolonged hypoplasia w/ cytopenias, #1; G4 CRS, #2) and 1 at lower dose (G3 reduced LV ejection fraction lasting 16d, #5).

Three pts (all w/ prior alloHCT) achieved best response of morphologic leukemia free state (MLFS), minimal residual disease (MRD) negative by FACS, w/ first response at med d15, hypocellular in pt #1/#2 (&lt;5%), normocellular in pt #5 (40-50%), w/ MLFS sustained at 4 weeks post-CAR T. Pt #1 received CD34-selected stem cell boost from her HLA-identical sibling on d36 due to ongoing cytopenias and disseminated Fusarium infection; she subsequently relapsed and died d73. Pt #2 proceeded to second alloHCT d45, but subsequently relapsed and died d219. Pt #5 experienced adenovirus reactivation (had prior CD34-selected alloHCT) and died d57 of multi-organ failure w/out evidence of AML. All pts w/ detectable AML post-CAR T (n=4) had CD371 expression.

CITE-seq and FACS showed CAR T products consisted predominantly of CD4 proliferating and central memory T cells. CD8 effector memory T cells became dominant CAR T population by d14 in the 3 responders. CD4 and CD8 CAR Ts were polyclonal based on TCRs and acquired cytotoxic profile post-infusion (↑ of GZMB, PRF1, IFN-γ, and GNLY) w/ gene set enrichment analyses revealing active IFN-γ and -α responses; CD8 CAR Ts also acquired NK-like features (CD56, CD16, and KIRs). Notably, all 3 responders were post-alloHCT; CAR Ts were of donor origin in the 2 pts w/ opposite sex donors based on X/Y mismatch. NK cells expanded in responders post-CAR T w/ differential expression of activation and cytotoxic markers, and enrichment of genes associated w/ IFN responses. Non-responders had limited CD8 CAR T and NK cell expansion.

Conclusions: CD371-SAVVYz-IL18 CAR T exhibits robust expansion and activity at low doses in pts w/ highly refractory AML, but w/ risk of CRS and prolonged cytopenias. We plan to treat next pts at the 3x104 cells/kg dose and intensify preventive CRS interventions. IL-18 secretion may provide autocrine stimulation for CAR T expansion, including expansion of low frequency cytotoxic CD8+ T cells, as well as activation of host NK cells to promote anti-leukemic activity. Prompt alloHCT post-CAR T may prevent complications of prolonged cytopenias and consolidate response.