Development of a gene edited next-generation hematopoietic cell transplant to enable acute myeloid leukemia treatment by solving off-tumor toxicity

Abstract

Immunotherapy of acute myeloid leukemia (AML) has been challenging because the lack of tumor-specific antigens results in "on-target, off-tumor" toxicity. To unlock the full potential of AML therapies, we used CRISPR-Cas9 to genetically ablate the myeloid protein CD33 from healthy donor hematopoietic stem and progenitor cells (HSPCs), creating tremtelectogene empogeditemcel (trem-cel). Trem-cel is a HSPC transplant product designed to provide a reconstituted hematopoietic compartment that is resistant to anti-CD33 drug cytotoxicity. Here, we describe preclinical studies and process development of clinical-scale manufacturing of trem-cel. Preclinical data showed proof-of-concept with loss of CD33 surface protein and no impact on myeloid cell differentiation or function. At clinical scale, trem-cel could be manufactured reproducibly, routinely achieving >70% CD33 editing with no effect on cell viability, differentiation, and function. Trem-cel pharmacology studies using mouse xenograft models showed long-term engraftment, multilineage differentiation, and persistence of gene editing. Toxicology assessment revealed no adverse findings, and no significant or reproducible off-target editing events. Importantly, CD33-knockout myeloid cells were resistant to the CD33-targeted agent gemtuzumab ozogamicin in vitro and in vivo. These studies supported the initiation of the first-in-human, multicenter clinical trial evaluating the safety and efficacy of trem-cel in patients with AML (NCT04849910).

Keywords: CD33; CRISPR-Cas9; IND-enabled; acute myeloid leukemia; genome engineering; hematopoietic stem cell; immunotherapy; pharmacology/toxicology.

Graphical abstract

Figure 1

Molecular consequence of CD33-KO (A) HL-60 cells were electroporated with the Cas9-RNP (CD33-KO) or without the Cas9-RNP (control); n = 3 per condition. Editing frequency was assessed by ICE analysis, transcript expression by ddPCR (CD33 normalized to glucuronidase beta and percentage of averaged control), and CD33 cell surface protein expression by flow cytometry (percentage of control). Data are represented as mean ± SD. Comparisons were performed using Student’s t test; ∗p < 0.05. (B) Frequency of the most common indels in HL-60 CD33-KO cells 7 days post-electroporation, as a percentage of total editing events. Data are represented as mean ± SD, n = 3. (C) DNA sequence of the CD33 target region, gRNA+PAM ±5 nucleotides. Dash represents deletion, “C” represents the identified nucleotide insertion. Each indel results in an amino acid substitution at position 143 of the coding sequence, leading to a downstream frameshift (fs) and subsequent termination (Ter). The number following “Ter” represents the number of amino acids following the insertion.

Figure 2

CD33 KO does not impact HSPC populations or myeloid function (A) HSPC subpopulation frequency (percentage of total live cells) at 48 h post-electroporation in CD33-KO and control human CD34+ HSPCs. Mean ± SD from three donors shown. Statistical analysis was performed using ANOVA with Šidák multiple comparisons test (left panel). Editing frequencies in the overall HSPC population (bulk) and each of the subpopulations were plotted as mean ± SD from three donors (middle panel). Indel spectra from bulk and each subpopulation were plotted as mean ± SD from three donors (right panel). MLP, multi-lymphoid progenitors; CMP, common myeloid progenitors; MPP, multipotent progenitors; LT-HSCs, long-term hematopoietic stem cells. (B) CD33-KO and control mobilized peripheral blood human CD34+ HSPCs from multiple donors were induced to differentiate down granulocytic or monocytic lineages. Editing frequency was assessed by ICE analysis. Flow cytometry was used to identify CD33⁺ cells (indicated by percentages). Data are represented as mean ± SD. (C) Myeloid function assessment in CD34+ HSPCs in vitro after myeloid differentiation. Phagocytic function of differentiated cells is shown as uptake of fluorescent E. coli bioparticles. Each dot represents one replicate; mean ± SD shown by box and error bars. Statistical analysis was performed using two-way ANOVA comparing conditions with or without addition of cytochalasin: ∗∗∗∗p < 0.0001. Inflammatory cytokine secretion (IL-6, TNFα) was evaluated. Each dot represents one replicate; mean ± SD shown by box and error bars. Statistical analysis was performed using two-way ANOVA comparing unstimulated/basal condition versus lipopolysaccharide (LPS) stimulation, or basal condition versus R848 stimulation: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Two-way ANOVA with the Šidák multiple comparisons test was performed to compare CD33-KO versus control for each stimulation condition. IL-6, interleukin-6; LPS, lipopolysaccharide; TNFα, tumor necrosis factor alpha.

Figure 3

Trem-cel manufacturing at clinical scale and characterization (A) Critical quality attributes of 30 batches of independently manufactured trem-cel products (dose, viability, purity, editing efficiency, and residual T cells) from two different sites. Each point represents a single batch and upper and lower horizontal lines indicated the standard error. (B) Quantification of BFU-E, CFU-GM/G/M, and CFU-GEMM colonies plated at 200 cells/plate for control and trem-cel-derived samples (seven batches each). The average number of colonies from two technical replicates across all batches is plotted. The horizontal lines represent mean of all batches. All p-values (Wilcoxon matched-pair signed rank test) for trem-cel versus control for all colony types were >0.05. (C) Loss of CD33 surface protein was assessed by flow cytometry in control and trem-cel-derived cells differentiated to monocytes (five batches each). Each symbol represents a single batch, the horizontal lines represent the mean ± SD. (D) In silico and laboratory-based approaches used to nominate off-target editing events. Combined Fisher exact test (p value <0.05) and >0.2% indel frequency difference between edited and control samples were used to determine that no significant off-target sites were observed.

Figure 4

In vivo pharmacology study (A) Experimental schema of xenotransplant study. (B) Chimerism and multilineage differentiation by flow cytometry. Levels of human CD45+ cell engraftment, and lymphoid (B cells: CD19+; T cells: CD3+) and (C) myeloid lineages in BM harvested from mice 16 weeks after engraftment of trem-cel (two independent batches). Each symbol represents one mouse; mean ± SD are shown by box and error bars. ∗∗∗∗p < 0.0001; ns, not significant (p > 0.05) by unpaired t test (n = 12 mice per condition). (D) (left panels) Total editing rates for all samples from two independent batches of trem-cel in BM from trem-cel engrafted and control mice. Cells used for engraftment (input) were included as an additional control. (right panels) Distribution of indel species in trem-cel-treated BM samples and trem-cel input cells. (E) Enumeration of BFU-E, CFU-GM/G/M, and CFU-GEMM colonies from engrafted samples plated at 5 × 10⁴ cells/well (two independent batches). Mean ± SD shown by box and error bars; each dot represents one mouse. Top row represents trem-cel batch 1, bottom row represents trem-cel batch 2. (F) Single cell-derived CFU colonies were genotyped and categorized as biallelically edited, monoallelically edited, or unedited. The numbers indicated on the pie chart represent the percentage and number of colonies of each type.

Figure 5

Trem-cel in vivo toxicology study (A) Experimental schema of in vivo toxicology study. (B) Levels of human CD45+ cell engraftment, and lymphoid (B cells: CD19+) and myeloid lineages in BM harvested from mice 20 weeks after engraftment of trem-cel. Data are shown separately for male and female mice. Each dot represents one mouse. Boxes and error bars show mean ± SD. (C). (left panel) Total editing rates in input samples and BM from trem-cel-treated mice for all samples (right panel) Distribution of indel species in input samples and BM samples from trem-cel-treated mice.

Figure 6

CD33-KO cells are resistant to GO cytotoxicity (A) HL-60 CD33-KO and control cells were treated with increasing concentrations of GO for 72 h and the number of viable cells was determined by flow cytometry. (B) Following 7 days of myeloid differentiation to either granulocytes or monocytes, CD34⁺ HSPC CD33-KO and control cells were exposed to increasing concentrations of GO for 72 h. Live cell percentages were determined by flow cytometry and dose-response curves were determined. IC₅₀ values were compared between CD33-KO and control cells using the unpaired Student’s t test (p = 0.0475 for granulocytic cultures and p = 0.0003 for monocytic cultures). (C). Three separate batches of CD33-KO CD34+ HSPCs were assessed (one research-scale, two clinical-scale trem-cel). CD33-KO cells were diluted with unedited cells to generate an editing titration curve from 10% to 90% editing frequency, with 90% being CD33-KO cells alone. Following 9 days of in vitro myeloid differentiation to monocytes, CD33 surface protein expression (flow cytometry) was correlated with CD33 editing frequency (ICE) (left panel). Cells were also exposed to increasing concentrations of GO for 72 h and live cell percentages were determined by flow cytometry, a representative sample (batch 1) is shown in the middle panel. IC₅₀ values were plotted for CD33-KO and control cells (right panel). (D) Experimental schema of the in vivo GO-challenge studies from two independent batches of CD33-KO cells. (E) Batch 1 and (F) Batch 2. Levels of human CD45+ cell engraftment and lymphoid (CD19+) and myeloid lineages among total human leukocytes in the BM of CD33-KO engrafted and control animals were analyzed by flow cytometry. Each dot represents one mouse. Boxes and error bars show mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test was used to compare between mice that received the same cells but different treatment (GO or vehicle), or to compare between mice that received the same treatment but different cells (CD33KO or control cells). (G). Input cell editing frequency determined by ICE.