Protection of CD33-modified hematopoietic stem cell progeny from CD33-directed CAR T cells in rhesus macaques

Abstract

The treatment of monogenetic disorders, such as hemoglobinopathies and lysosomal storage diseases, has markedly improved with the advent of cell and gene therapies, particularly allogeneic or gene-modified autologous stem cell transplantations. However, therapeutic efficacy is reliant on maintaining engraftment above a critical threshold. To maintain such engraftment levels, we and others have pursued approaches to shield edited cells from antibody or chimeric antigen receptor (CAR) T-cell-mediated selection. Here, we focused on CD33, which is expressed early on hematopoietic stem and progenitor cells (HSPCs) as well as on myeloid progenitors. Rhesus macaques were engrafted with HSPCs edited to ablate CD33 using either CRISPR/CRISPR-associated protein 9 or adenine base editor. Both editing strategies showed similar post-transplant recovery kinetics and yielded equivalent levels of engraftment. We then created a V-set domain-specific CAR construct (CAR33), validated its functionality in vitro, and treated both animals with autologous CAR33 T cells. CAR33 T cells expanded after infusion and caused specific depletion of CD33WT but not CD33null progeny, leading to a transient enrichment for gene-edited cells in the blood. No depletion was seen in the bone marrow stem cell compartment with CD34+CD90+ HSCs expressing lower levels of CD33 in comparison to monocytes. Thus, we show proof of concept and safety of an epitope editing-based enrichment/protection strategy in macaques.

Graphical abstract

Figure 1.

HSPCs are amenable to both CRISPR/Cas9 and ABE/Cas9-mediated CD33 editing, engrafting long-term and maintaining multilineage potential. (A) CFC assay of transplanted HSPCs. Sorted on (1) CD34⁺, (2) CD34⁺CD90⁺, (3) CD34⁺CD90⁻, and (4) CD34⁺CD45RA⁺. (B) Editing of single colonies collected from CFC assay. (C) Schematic of BM transplant and tracking of hematologic recovery. (D) Representative table of transplant information. (E) Hematologic recovery of A17039 and A18038 as measured by time to recovery of platelets and neutrophils. Vertical dashed line represents day of transplant. Horizontal lines represent threshold of recovery (solid line) and healthy blood ranges (dashed lines). (F) Recovery of total WBC compartment with CD33-edited cells (top) and the granulocyte compartment with CD33-modified cells (bottom) measured by ddPCR/NGS or FACS, respectively. BFU-E, burst forming unit erythroid; G, granulocyte; GEMM, granulocyte, erythrocyte, macrophage, megakaryocyte; GM, granulocyte, macrophage; M, macrophage; QC, quality control; WBC, white blood cell; WT, wild type.

Figure 2.

In vitro validation of a human/NHP cross-reactive CD33-directed CAR construct. (A) Representative schematic of CAR33 design with bicistronic tCD19 reporter. (B) CRA results of human CAR33 T cells cultured with CD33⁺ or CD33⁻ ML-1 cells. (C) xCELLigence killing assay of macaque CAR33 T cells cultured with CD33^+/− LLCMK2 cells. IgG4, immunoglobulin G4.

Figure 3.

CAR33 expands in vivo and demonstrated divergent toxicity profiles in 2 RMs. (A) Representative schematic of CAR33 production and treatment of 2 RMs. (B) Values of total T cells and CAR33 T cells infused per kilogram bodyweight for both macaques and days after transplant (DPT) to CAR treatment. (C) Post-CAR cytokine response in both treated animals as measured by IL-6 and CRP. Colored X’s indicate treatment with toci (green), dexa (blue), and anakinra (orange). (D) Distribution of PB immune lineages before and immediately after CAR33 treatment. Population proportions determined by flow cytometry and matched to absolute cell counts determined by complete blood counts. Dex, dexamethasone; DPT, days post-transplant; NK cell, natural killer cell; toci, tocilizumab.

Figure 4.

CAR33 treatment led to complete ablation of CD33⁺ events while sparing CD33^null cells. (A) Depletion of CD33⁺ granulocytes in the PB in both treatment animals. Scaled proportion of CD33⁺ events by flow cytometry with absolute cell counts by complete blood counts (CBC). (B) Measured CD33 editing in bulk WBCs before and immediately after CAR33 treatment. Editing measured by ddPCR (A17039) and MiSeq (A18038). (C) Proportions of CD33⁺ and CD33⁻ granulocytes after CAR33 treatment. Granulocyte identity and CD33 positivity determined by flow cytometry and scaled using absolute cell counts from CBC.

Figure 5.

CD33 is differentially expressed in RM but not human HSPCs. (A) mRNA expression from bulk sequencing of HSCs and EMPs from 3 human donors (left) and 3 macaque donors (right) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. (B) Representative flow plot of the canonical HSC marker CD90 against CD33 in human (top) and rhesus (bottom). (C) Calculated antigen per cell on HSCs, EMPs, and LMPs in human (left) and rhesus (right). mRNA, messenger RNA.