Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment

Abstract

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue.

A Bump in the Road: How the Hostile AML Microenvironment Affects CAR T Cell Therapy

“…CXCR4 expression dictates AML blast migration, is implicated in prognosis, and is being explored as a therapeutic target (125). The CXCR4 pathway can also be involved in migration of CAR T cells to the bone marrow niche, demonstrated by improvement in bone marrow localization when CAR T cells are co-transduced with CXCR4 in preclinical studies (126).…”

CRISPR/Cas9 for the treatment of haematological diseases: a journey from bacteria to the bedside

“…One example of this strategy is the enhancement of CXCR4 expression on edited HSPCs, which has been shown to improve homing to and engraftment in the bone marrow niche. [110][111][112] Due to the requirement of CRISPR/Cas9 systems for a short sequence motif adjacent to target sites known as protospacer adjacent motif (PAM), a significant portion of the genome has not been amenable to editing. Recent efforts focussed on the generation of 'PAMless' Cas9 variants, which widely expanded the therapeutic applicability of this nuclease platform to chromosomal regions that were previously inaccessible to editing.…”

Targeting the niche: depleting haemopoietic stem cells with targeted therapy

“…Only leads to significant donor engraftment in immunocompromised mice and not immunocompetent mice [4,40,41] Anti-human mAb (clone SR-1) Conditioning allows for long term engraftment in 1 of 5 macaques with non-myeloablative irradiation or busulfan in an autologous gene modified model. Depletes normal and MDS human HSPC in an mouse xenograft model [42,43] Anti-human mAb (clone AMG 191) Depletes normal and MDS human HSPC in an mouse xenograft model, clinical trial (NCT02963064) for patients with primary immunodeficiencies to receive AMG191 conditioning prior to allo-HSCT is currently recruiting [43] Anti-mouse ADC (CD117-saporin) Combined with T cell depleting agents allowed for significant and durable engraftment in an immunocompetent mouse allo-HSCT model [50] Anti-human CD117-ADC (conjugate unspecified) Depletes human HSPC in vitro and in mouse xenografts [51] Anti-mouse CD117 CAR T cells Depletes mouse HSPC in vitro and in vivo, allow durable limited chimerism in congenic and CGD models [57] CD47 Anti-mouse CD47 FAB Depletes immunocompetent mouse HSPC when combined with anti-CD117 mAb and allows for durable chimerism in a congenic HSCT and allo-HSCT (with added T cell depletion) [47] CD123 Anti-human CD123 CAR T cells Causes ablation of healthy human haematopoiesis in a mouse xenograft, after depleation of the CAR T cells a second sex mismatch healthy human graft can be performed. Depletes co-engrafted healthy human HSPC and AML [58,59] HSPC haemopoietic stem and progenitor cells,…”

“…Success in the treatment of B cell acute lymphoblastic leukaemia, non-Hodgkin lymphoma and chronic lymphocytic leukaemia with CAR T cells demonstrate their powerful cytotoxic ability [53][54][55][56]. Murine CAR T cells directed against CD117 deplete mouse HSPC in vitro and in vivo and facilitate long term, but limited engraftment when used as conditioning in a CGD mouse model [57]. The murine derived CAR T cells initially had limited expansion and migration into the bone marrow in vivo but this was overcome by co-transducing mouse CXCR4 DNA (with the anti-CD117 CAR) and pre-treating with cyclophosphamide.…”