Systematic preclinical evaluation of CD33-directed chimeric antigen receptor T cell immunotherapy for acute myeloid leukemia defines optimized construct design.

BACKGROUND: Successful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent inability to achieve cure with conventional chemotherapy. Initial experiences with CD19 CAR T cell immunotherapy for patients with B-cell malignancies highlighted the critical impact of intracellular costimulatory domain selection (CD28 vs 4-1BB (CD137)) on CAR T cell expansion and in vivo persistence that may impact clinical outcomes. However, the impact of costimulatory domains on the efficacy of myeloid antigen-directed CAR T cell immunotherapy remains unknown. METHODS: In this preclinical study, we developed six CAR constructs targeting CD33, a highly expressed and validated AML target, comprised of one of three single-chain variable fragments with CD3ζ and either CD28 or 4-1BB costimulatory domains. We systematically compared the preclinical in vitro and in vivo efficacy of T cells lentivirally transduced with CD33 CAR constructs (CD33CARTs) against human AML. RESULTS: We observed potent in vitro cytokine production and cytotoxicity of CD33CARTs incubated with human CD33+ AML cell lines, as well as robust in vivo antileukemia activity in cell line and childhood AML patient-derived xenograft (PDX) models. Gemtuzumab-based CD33CARTs were unexpectedly toxic in vivo in animal models despite observed in vitro anti-leukemia activity. CD28-based CD33CARTs consistently induced more robust inhibition of leukemia proliferation in AML cell line and PDX models than did 4-1BB-based CD33CARTs. A 'best-in-class' lintuzumab-CD28/CD3ζ CAR construct was thus selected for clinical translation. CONCLUSIONS: CD33 is a critical antigen for potential immunotherapeutic targeting in patients with AML. Based on this rigorous preclinical evaluation, our validated clinical grade lintuzumab-CD28/CD3ζ CD33CART immunotherapy is now under evaluation in a first-in-child/first-in-human phase 1 clinical trial for children and adolescents/young adults with relapsed/refractory AML. TRIAL REGISTRATION NUMBER: clinicaltrials.gov; NCT03971799.

Reduction of leukemic burden via bone-targeted nanoparticle delivery of an inhibitor of C-chemokine (C-C motif) ligand 3 (CCL3) signaling.

Leukemias are challenging diseases to treat due, in part, to interactions between leukemia cells and the bone marrow microenvironment (BMME) that contribute significantly to disease progression. Studies have shown that leukemic cells secrete C-chemokine (C-C motif) ligand 3 (CCL3), to disrupt the BMME resulting in loss of hematopoiesis and support of leukemic cell survival and proliferation. In this study, a murine model of blast crisis chronic myelogenous leukemia (bcCML) that expresses the translocation products BCR/ABL and Nup98/HoxA9 was used to determine the role of CCL3 in BMME regulation. Leukemic cells derived from CCL3-/- mice were shown to minimally engraft in a normal BMME, thereby demonstrating that CCL3 signaling was necessary to recapitulate bcCML disease. Further analysis showed disruption in hematopoiesis within the BMME in the bcCML model. To rescue the altered BMME, therapeutic inhibition of CCL3 signaling was investigated using bone-targeted nanoparticles (NP) to deliver Maraviroc, an inhibitor of C-C chemokine receptor type 5 (CCR5), a CCL3 receptor. NP-mediated Maraviroc delivery partially restored the BMME, significantly reduced leukemic burden, and improved survival. Overall, our results demonstrate that inhibiting CCL3 via CCR5 antagonism is a potential therapeutic approach to restore normal hematopoiesis as well as reduce leukemic burden within the BMME.

Expression, refolding and spectroscopic characterization of fibronectin type III (FnIII)-homology domains derived from human fibronectin leucine rich transmembrane protein (FLRT)-1, -2, and -3.

The fibronectin leucine rich transmembrane (FLRT) protein family consists in humans of 3 proteins, FLRT1, -2, and -3. The FLRT proteins contain two extracellular domains separated by an unstructured linker. The most membrane distal part is a leucine rich repeat (LRR) domain responsible for both cis- and trans-interactions, whereas the membrane proximal part is a fibronectin type III (FnIII) domain responsible for a cis-interaction with members of the fibroblast growth factor receptor 1 (FGFR1) family, which results in FGFR tyrosine kinase activation. Whereas the structures of FLRT LRR domains from various species have been determined, the expression and purification of recombinant FLRT FnIII domains, important steps for further structural and functional characterizations of the proteins, have not yet been described. Here we present a protocol for expressing recombinant FLRT-FnIII domains in inclusion bodies in Escherichia coli. His-tags permitted affinity purification of the domains, which subsequently were refolded on a Ni-NTA agarose column by reducing the concentration of urea. The refolding was confirmed by circular dichroism (CD) and 1H-NMR. By thermal unfolding experiments we show that a strand-strand cystine bridge has significant effect on the stability of the FLRT FnIII fold. We further show by Surface Plasmon Resonance that all three FnIII domains bind to FGFR1, and roughly estimate a Kd for each domain, all Kd s being in the µM range.