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 2 of 14 | ACKUN-FARMMER Et Al.

BackgroundSuccessful 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.MethodsIn 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.ResultsWe 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.ConclusionsCD33 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 numberclinicaltrials.gov; NCT03971799.

Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

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 Kds being in the µM range.

Though childhood acute lymphoblastic leukemia (ALL) is highly treatable, there remain subsets of pediatric ALL with very poor prognoses. Infant ALL, found in children under the age of 1, is difficult to treat due to the scarcity of cases impeding the ability of even the largest pediatric oncology centers from gaining experience in treating the disease, the more aggressive initial clinical presentation, as well as the inability for these young patients to tolerate toxicities associated with chemotherapeutic regimens and procedures. Despite being only 5% of total ALL cases, 80% of infant ALL cases are marked by mixed lineage leukemia (MLL/KMT2A) rearrangements. In KMT2A rearranged (KMT2Ar) ALL, FLT3 is the most differentially expressed gene that distinguishes KMT2Ar ALL from non-KMT2Ar ALL making FLT3 an attractive target for infant ALL. CD19 and CD22 targeting chimeric antigen receptor (CAR) T cell therapy has demonstrated outstanding responses in phase 1 clinical trials for relapsed/refractory B ALL, leading to tremendous interest in testing other CAR targets. Here we explore FLT3 CAR as a potential treatment for B ALL and the unexpected finding that FLT3 CAR T cells induce lineage switch of an infant ALL from a B to T cell phenotype. We generated a FLT3-targeting CAR consisting of a FLT3 binding domain derived from a well-characterized anti-human FLT3 antibody coupled to 4-1BB costimulatory and CD3-zeta activation domains. In vitro studies confirmed that human T cells expressing the FLT3 CAR produced interferon-gamma and interleukin-2 after co-culture with KMT2Ar B ALL SEM and infant B ALL KOPN8. FLT3 CAR T cells eliminated ALL in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with high FLT3 expressing SEM. KOPN8, which expresses lower levels of FLT3 when treated with FLT3 CAR T cells showed an initial clearance of leukemia in NSG mice, however relapsed with ALL that no longer expressed FLT3 or B cell marker CD19. Interestingly, this loss of FLT3 and CD19 happened concurrently with gain of T cell markers (CD3+ and either CD4+ or CD8+). The durability of this T cell phenotype was transient because when T lineage switched KOPN8 was cultured ex vivo without immune pressure, the KOPN8 cells reverted to the parental B ALL phenotype (FLT3+, CD19+, CD3 neg, CD4 neg, CD8 neg) suggesting that the ability to lineage switch is not a selection of a clone that genetically does not express B cell markers while expressing T cell markers, but rather a potential epigenetic mechanism driving the cell lineage change. Contrary to reports from CD19 CAR treated KMT2Ar B ALL that switched to a myeloid phenotype, these cells did not upregulate myeloid markers (CD33, CD11b). Taken together these data imply that lineage switch driven by CAR T cell immune pressure may cause different types of lineage switch based on the target of the CAR. Furthermore, using CAR T cell immunotherapy we may be able to interrogate the biology of leukemia. Citation Format: Christopher D. Chien, Lila Yang, Sang M. Nguyen, Christopher T. Sauter, Kazusa Ishii, Feng Shen, Sarah K. Tasian, Terry J. Fry. FLT3 chimeric antigen receptor T cell therapy induces B to T cell lineage switch in infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1630.