CCR1-Targeting CAR T Cells for Acute Myeloid Leukemia

• Published in: Blood Vol. 142; no. Supplement 1; p. 4808
• Main Authors: Chen, Sophia, Boardman, Alexander P., James, Scott E., van den Brink, Marcel R.M.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 02.11.2023
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-184464

BACKGROUND Chimeric antigen receptor (CAR) T cell therapy is highly effective in patients with B cell malignancies and multiple myeloma, but initial clinical trials with CARs targeting acute myeloid leukemia (AML) have shown less successful results. Challenges inherent to using CAR T cells for AML include lack of a leukemia-specific target antigen due to heterogeneous antigen expression and potential on-target/off-tumor toxicity due to co-expression of target antigens on normal myeloid cells. C-C chemokine receptor 1 (CCR1, also known as CD191), a G protein-coupled receptor that binds to members of the C-C chemokine family, is a promising AML-associated antigen that is reported to be expressed on over 75% of AML samples and minimally expressed on hematopoietic stem/progenitor cells and T cells. The present study aimed to explore the potential of CCR1 as a novel CAR target antigen using a syngeneic mouse model that allows investigation of both efficacy and toxicity of the CAR T cells. METHODS AND RESULTS We developed a CAR based on the single chain variable fragment (scFv) KM5907 targeting both mouse and human CCR1. We constructed a second-generation CCR1-CAR containing a CD28 hinge, a CD28 transmembrane domain, a CD3 zeta signaling domain, and a CD28 co-stimulatory domain. We mutated the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 zeta chain to retain only a single active membrane-proximal (1XX) ITAM, based on a prior study showing that the 1XX ITAM format enhanced CAR T cell activity by reducing T cell exhaustion. Primary murine T cells were retrovirally transduced and CCR1-CAR expression was assessed by flow cytometry. CCR1-CAR T cells promoted effective elimination of BM185 leukemia cells overexpressing murine CCR1 in vitro. Similarly, primary human T cells transduced with the CCR1-CAR exhibited potent cytotoxicity against C1498 leukemia cells overexpressing human CCR1 in vitro. To evaluate anti-leukemia activity of the CCR1-CAR T cells in vivo, BALB/c mice were sublethally irradiated and engrafted with 1x10 5 BM185 leukemia cells modified to overexpress murine CCR1. Two days later, mice were treated with 2x10 6 CCR1-CAR T cells and monitored for weight changes and survival. Growth of BM185 leukemia cells (expressing firefly luciferase) and expansion of CAR T cells (expressing gaussia luciferase) were assessed via two-color bioluminescence imaging (BLI). On day 6, mice were bled retro-orbitally and serum was analyzed for cytokine levels. The CCR1-CAR T cells demonstrated potent anti-leukemia activity and T cell expansion in vivo, promoting improved overall survival compared with control mice that received only tumor cells (Figure 1). CCR1-CAR T cell activity was accompanied by mild, self-limited toxicity, characterized by transient weight loss (Figure 2), but not significant elevation of serum levels of cytokines associated with cytokine release syndrome (CRS) such as IL-6 and TNF-α. BLI of the CCR1-CAR T cells revealed enhanced trafficking into the bone marrow and spleen followed by proliferation in irradiated mice even in the absence of tumor, suggesting that CCR1-CAR T cells are stimulated by CCR1 antigen expressed on non-leukemic host cells in addition to leukemia cells. CONCLUSIONS In summary, CCR1-CAR T cells represent a promising therapeutic strategy for AML that to our knowledge has not yet been evaluated in preclinical or clinical studies. We generated CCR1-CAR T cells that demonstrated potent anti-leukemia activity in vitro and in vivo in a syngeneic mouse model. Clinical signs of self-limited toxicity were not associated with elevated levels of inflammatory cytokines and might be caused by an on-target/off-tumor effect, which warrants further investigations to better characterize the safety profile of CCR1-CAR T cells. The CCR1-CAR T cells also displayed potent cytotoxicity in vitro via human CCR1, which will facilitate clinical translation of the findings. A CCR1-CAR may also offer benefit in other hematologic malignancies with reported CCR1 expression such as multiple myeloma or Hodgkin's lymphoma. James:MSKCC: Patents & Royalties: Pending patents related to leucine zipper sorting technology. van den Brink:Thymofox: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Da Volterra: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Wolters Kluwer: Patents & Royalties; Juno Therapeutics: Other: IP licensing; DKMS (a non-profit organization): Membership on an entity's Board of Directors or advisory committees; Lygenesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pluto Immunotherapeutics: Consultancy, Current holder of stock options in a privately-held company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ceramedix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Notch Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Frazier Healthcare Partners: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Rheos Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Seres Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: IP licensing , Research Funding. 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