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M. Gurney et al.
The multifunctional cell surface glycoprotein CD38, a breakthrough immunotherapeutic target in multiple myeloma, is also considered a potential target antigen in AML. In contrast to the uniformly high CD38 expression on malignant plasma cells, blast cell CD38 expression is heterogeneous although frequently exceeds that of nor- mal cell populations.10 The CD38 monoclonal antibody daratumumab has been investigated in AML and has shown promising pre-clinical activity.10 CD38 CAR-T cells have been evaluated mainly for their activity in mul- tiple myeloma and cytotoxicity against primary AML samples has also been confirmed.11 However, there remains concern about a potent myelosuppressive effect with a constituently expressed high-affinity anti-CD38 CAR due to CD38 expression on both mature myeloid cells and their precursors.11,12 To circumvent this problem, an affinity-optimized CD38 CAR has been developed to minimize the targeting of positive, but low-expressing normal cell populations.13
There is a strong biological rationale for natural killer (NK) cell-based approaches to adoptive cell transfer immunotherapy for AML. NK cells confer a component of the graft-versus-leukemia effect of allogeneic stem cell transplant and infusions of purified alloreactive NK cells have proven therapeutic potential.14-16 CAR-NK cell thera- pies are emerging as a complementary approach to CAR- T cells, with potential advantages including allogeneic cell sources and innate antigen independent anti-leukemic activity. An early clinical report of a cord-blood derived CD19 CAR-NK cell therapy has shown promising safety and efficacy in B-cell malignancies.17 We set out to devel- op and evaluate an affinity optimized CD38 CAR-NK cell therapy for AML. We first used the NK cell line KHYG-1, which has naturally low levels of CD38 expression. While allogeneic expanded NK (eNK) cell approaches are more suited to clinical translation, ex vivo NK cell expan- sion has been shown to lead to upregulation of CD38, which we also encountered using a feeder-free, inter- leukin-2-based expansion protocol.18 To reduce the antic- ipated NK cell fratricide that would occur using eNK cells, we applied CRISPR/Cas9 to disrupt the CD38 gene dur- ing NK cell expansion, creating fratricide-resistant NK cells prior to CD38 CAR expression. Both KHYG-1 and CD38 knockdown (KD) eNK approaches lead to efficient targeting of AML blasts upon CD38 CAR expression, with the degree of cytotoxicity correlating with CD38 expression. Finally, we confirm a rational combination approach utilizing all-trans retinoic acid (ATRA) to enhance CD38 expression on the AML cells. Collectively, our data support the potential of CD38 as a therapeutic target in AML and help to define a CD38 CAR-NK cell approach suited to clinical development.
Methods
Ethical statement
Healthy donor blood and AML patients’ bone marrow sam- ples were collected with written informed consent and approval from the institutional review boards at each institution (ref: CA2219). Cryopreserved samples were obtained from the biobank of Blood Cancer Network Ireland.
Cells and reagents
The cell lines THP-1, KG1a, U937 and KHYG-1 were
obtained from the American Type Culture Collection and their identities confirmed by short tandem repeat profiling (Eurofins GenomicsTM). CD38 CAR and mock KHYG-1 cells were gener- ated by retroviral transduction with genomic integration con- firmed by the inclusion of DsRed fluorescent protein. The development of the second-generation CD28-CD3ζ, opti- mized-affinity CD38 CAR was reported previously.13 Primary NK cells were isolated from healthy donor peripheral blood mononuclear cells after Ficoll-Paque density gradient centrifu- gation and negative immunomagnetic selection (NK Isolation Kit, Miltenyi BiotecTM). NK cells were expanded in NK MACS medium (Miltenyi BiotecTM) containing NK MACS supplement, 5% heat-inactivated human AB serum and 100 U/mL. inter- leukin-2 (PeproTechTM). Cultures were pre-treated for 48 h with ATRA (Sigma-AldrichTM) or dimethyl sulfoxide, in the relevant experiments.
CRISPR/Cas9 gene editing
Five days after isolation, 5x105 NK cells were electroporated with sgRNA-Cas9 complexes targeting multiple sites within the CD38 gene (Gene Knockout Kit V2, SynthegoTM) or control electroporated (MaxCyteTM GT flow transfection system). CD38-edited and control electroporated cells were expanded at a target density of 1x106 cells/mL. On day 13-15 of expansion, CD38 expression was assessed by flow cytometry. Knockdown efficiency was calculated as (% CD38-positive cells [mock elec- troporated] - % CD38-positive cells [CRISPR/Cas9 edited]).
CD38 chimeric antigen receptor mRNA electroporation
CD38 CAR mRNA was synthesized (Trilink BiotechnologiesTM) and CD38 CAR expression in primary CD38 KD and control eNK cells was achieved by electropora- tion (100 mg/mL mRNA, MaxcyteTM GT Flow Transfection System). CAR expression was confirmed by flow cytometry using anti-IgG H+L specific goat anti-human antibody (Jackson- Immuno researchTM) and biotinylated protein L stain (ACRO BiosystemsTM).
Cytotoxicity assays
Co-culture experiments involved 10,000 target cells (cell lines), or 20,000-50,000 bone marrow mononuclear cells from AML patients’ samples. NK cell numbers were determined by the desired effector to target (E:T) cell ratio. After co-culture for 18-24 h, target cell lines or bone marrow mononuclear cells were identified by flow cytometry, using a cell-tracking dye: Tag-IT BVTM proliferation and cell tracking dye (BiolegendTM) or VioletTraceTM (Thermo Fisher). Primary blast cell populations were identified as CD45int/SSClow (CD45 APC), supported by additional markers chosen based on clinical immunophenotyp- ing data. Cell death was determined using propidium iodide (PI) or LIVE/DEAD Fixable Near-IR (Life Technologies L10119) staining and reported as ‘% specific (blast) cytotoxicity’ ([sam- ple cytotoxicity – background cytotoxicity]/[100 – background cytotoxicity] x 100%) or ‘% blast cell cytotoxicity’ as indicated.
Statistical analysis
GraphPad Prism 8 software (San Diego, CA, USA) was used for statistical analysis. Comparisons were conducted using mul- tiple two-sided t-tests for cytotoxicity assays at each E:T ratio or one-way analysis of variance for cell expression data with statistical significance indicated by asterisks (*P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001). Flow cytometry data were acquired on a BD FACS Canto II and analyzed using Flow Jo V10 software and Infinicyt (CytognosTM).
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haematologica | 2022; 107(2)