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CAR T-cells for AML
Institute reported a phase I clinical trial of autologous NKG2D-CAR T-cells in seven patients with AML.21 Their first-generation construct (CAR-NKG2D-CD3ζ) uses the naturally-occurring NKG2D receptor as the antigen-bind- ing domain, with endogenous DAP10 expression provid- ing co-stimulation.21 CART-NKG2D cells were successful- ly manufactured in all seven patients, and median infused CAR+ cells varied across four dose levels (1x106, 3x106, 1x107, 3x107) without preceding lymphodepletion. No dose-limiting toxicities were observed. Biological activity in vivo was manifest by cytokine perturbations and CAR- transgene detection, though persistence of the CART population was limited and no objective clinical respons- es were observed, with all patients requiring subsequent therapies for AML progression. In a follow-up phase I trial of CART-NKG2D for AML and myelodysplastic syn- drome (MDS), higher doses of CART-NKG2D are being infused (3x108, 1x109 and 3x109)21 and of the two AML patients reported to date, one achieved an objective clini- cal response. This patient initially achieved a morpholog- ical leukemia-free state (MLFS) and was subsequently able to proceed to alloHSCT and at the time of reporting remains in CR with 100% donor chimerism.22 The same group is planning a related phase I study evaluating the safety and efficacy of combining their anti-NKG2D CART with azacytidine in treatment-naïve patients with AML/MDS who are ineligible for alloHSCT or intensive therapy (clinicaltrials.gov identifier: 03612739). Azacytidine has been shown to enhance expression of NKG2D lig- ands on AML blasts by reversing hypermethylation.23 It remains to be seen if azacytidine will also cause upregu- lation of NKG2D ligands on healthy tissues, which could lead to unforeseen toxicity. This trial was not open to enrollment at the time of writing.
Identifying new target antigens
The lack of ideal antigens for CART therapy in AML spurred a search for new ones. Investigators from the Memorial Sloan-Kettering Cancer (MSKCC) sought to identify sets of antigens suitable for targeting with a com- binatorial CART strategy,24 by which CAR T cells express a dual-specific CAR (or 2 different CAR transduced into the same cell) directed against two different target anti- gens with non-overlapping expression in normal tissues. When both antigens are encountered by the dual-specific CART, the potency of cell killing is increased relative to that seen if only one antigen is present; the design of the combinatorial CART must therefore comprise two target antigens that are specifically co-expressed in malignant cells. The MSKCC group identified four promising tar- gets, ADGRE2, CCR1, CD70 and LILRB2, which satisfied their criteria for suitable off-tumor expression that also demonstrated AML cell killing in vitro when two targets were present on the same cells, and are thus potentially amenable to a combinatorial CAR approach.24
The antigen CLL-1, which was targeted in the above- mentioned combinatorial approach in combination with CD33 by Liu et al.8 also has potential utility as a stand- alone antigen, with pre-clinical data suggesting that dif- ferential expression between malignant and healthy blasts may be sufficient to preserve hematopoeisis.25,26 No clinical CAR T-cell trials targeting CLL-1 alone are cur- rently open, though a phase I trial evaluating a CLL1-CD3 bi-specific antibody (clinicaltrials.gov identifier: 03038230) may be informative as to the feasibility of this approach.
Bone marrow transplant with a gene-edited allograft followed by chimeric antigen receptor T cells
The provision of a rescue alloHSCT after in vivo deple- tion of the CART population is a potential solution to the problem of hematopoietic toxicity, though it may create a new problem, which is placing a limit on the duration of in vivo persistence of the CART population. The accrued experience with CART-19 in B-ALL suggests that the optimal period of time for CART persistence for dis- ease response is at least 2-3 months,27 or potentially as long as 8-10 months.1 A new approach to allow long-term CART against myeloid antigens, such as CD33, is to edit out the antigen from allogeneic donor HSPC, which are then transplanted into the patient with AML. Following engraftment, the patient may then be treated with CART33 manufactured from the same allogeneic donor. Our research group has demonstrated that CD33 can be removed from HSPC using CRISPR/Cas9 without impair- ment of hematopoietic or immunological function, and that the CD33 knock-out HSPC (and their progeny) are impervious to attack by CART33 both in vitro and in vivo in murine and non-human primate models.28 This treat- ment approach, currently under development at the University of Pennsylvania (see Figure 1 for a summary) would allow for long-term CART persistence while also protecting normal hematopoiesis. It remains to be seen if this approach is feasible from both a manufacturing and a clinical perspective, and indeed what the clinical conse- quences of CD33 depletion would be in the bone marrow compartment, and by the depletion of CD33+ tissue resi- dent macrophages. Several publications have demonstrat- ed that donor-derived cells eventually repopulate the vis- ceral resident macrophage niche; specifically, Kupffer cells,29 pulmonary alveolar cells,30 and microglia.31 Data are limited as to the time taken for repopulation of these cells, with published data in the lung suggesting this may take 2-3 months after alloHSCT alone.30 It is noted that the main toxicities associated with gemtuzumab ozogamcicin (Mylotarg®, an anti-CD33 antibody-drug conjugate) have been the expected hematopoietic toxici- ty, and also hepatotoxicity and veno-occlusive disease, with both being likely attributable to the calicheamicin component of the therapy, rather than due to targeting of CD33+ hepatic cells.32 There is evidence to suggest that CD33 has a role in the modulation of inflammatory and immune response,33 and the potential for this to impact the incidence of severity of CRS response when CART33 are administered after engraftment with CD33KO-HSPC will be actively considered during the conduct of the trial.
Other immunotherapy options for acute myeloid leukemia
Given the challenges of treating AML with CAR T cells, as outlined above, many investigator groups are also exploring other types of adoptive cellular therapy that have different therapeutic mechanisms, and may be less hampered than this issue of antigen specificity that are currently limiting CAR T-cell therapy for AML. By trans- ducing T cells with the a and β chains of a known speci- ficity T-cell receptor (TCR), engineered TCR cells (TCR- T) can be endowed with specificity to known tumor- associated antigens (TAA) or conceivably, neoantigens. The TCR chains may be cloned from patients or normal donors that exhibit an immune response to the TAA, and may be further affinity-enhanced in order to confer enhanced reactivity to the target.34-36
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