Engineering Novel CD19/CD22 Dual-Target CAR-T Cells for Improved Anti-Tumor Activity.

Despite high remission rates following chimeric antigen receptor T cell (CAR-T) cell therapy in B-cell acute lymphoblastic leukemia (B-ALL), relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may improve the CAR-T effect. The in vitro and in vivo leukemia model was established, and the anti-tumor effects of BiCAR-T, CD19 CAR-T, CD22 CAR-T, and LoopCAR6 cells were observed. We found that the BiCAR-T cells showed significant cytotoxicity in vitro and in vivo. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce the risk of antigen loss.

Trident cold atmospheric plasma blocks three cancer survival pathways to overcome therapy resistance.

Therapy resistance is responsible for most cancer-related death and is mediated by the unique ability of cancer cells to leverage metabolic conditions, signaling molecules, redox status, and other pathways for their survival. Interestingly, many cancer survival pathways are susceptible to disturbances in cellular reactive oxygen species (ROS) and may therefore be disrupted by exogenous ROS. Here, we explore whether trident cold atmospheric plasma (Tri-CAP), a gas discharge with exceptionally low-level ROS, could inhibit multiple cancer survival pathways together in a murine cell line model of therapy-resistant chronic myeloid leukemia (CML). We show that Tri-CAP simultaneously disrupts three cancer survival pathways of redox deregulation, glycolysis, and proliferative AKT/mTOR/HIF-1α signaling in this cancer model. Significantly, Tri-CAP blockade induces a very high rate of apoptotic death in CML cell lines and in primary CD34+ hematopoietic stem and progenitor cells from CML patients, both harboring the therapy-resistant T315I mutation. In contrast, nonmalignant controls are minimally affected by Tri-CAP, suggesting it selectively targets resistant cancer cells. We further demonstrate that Tri-CAP elicits similar lethality in human melanoma, breast cancer, and CML cells with disparate, resistant mechanisms and that it both reduces tumor formation in two mouse models and improves survival of tumor-bearing mice. For use in patients, administration of Tri-CAP may be extracorporeal for hematopoietic stem cell transplantation therapy, transdermal, or through its activated solution for infusion therapy. Collectively, our results suggest that Tri-CAP represents a potent strategy for disrupting cancer survival pathways and overcoming therapy resistance in a variety of malignancies.

Mouse Models of CMML.

Chronic myelomonocytic leukemia (CMML) is a rare and challenging type of myeloproliferative neoplasm. Poor prognosis and high mortality, associated predominantly with progression to secondary acute myeloid leukemia (sAML), is still an unsolved problem. Despite a growing body of knowledge about the molecular repertoire of this disease, at present, the prognostic significance of CMML-associated mutations is controversial. The absence of available CMML cell lines and the small number of patients with CMML make pre-clinical testing and clinical trials complicated. Currently, specific therapy for CMML has not been approved; most of the currently available therapeutic approaches are based on myelodysplastic syndrome (MDS) and other myeloproliferative neoplasm (MNP) studies. In this regard, the development of the robust CMML animal models is currently the focus of interest. This review describes important studies concerning animal models of CMML, examples of methodological approaches, and the obtained hematologic phenotypes.

Pharmacokinetic and pharmacodynamic studies of CD19 CAR T cell in human leukaemic xenograft models with dual-modality imaging.

In recent years, chimeric antigen receptor T (CAR T)-cell therapy has shown great potential in treating haematologic disease, but no breakthrough has been achieved in solid tumours. In order to clarify the antitumour mechanism of CAR T cell in solid tumours, the pharmacokinetic (PK) and pharmacodynamic (PD) investigations of CD19 CAR T cell were performed in human leukaemic xenograft mouse models. For PK investigation, we radiolabelled CD19 CAR T cell with 89 Zr and used PET imaging in the CD19-positive and the CD19-negative K562-luc animal models. For PD evaluation, optical imaging, tumour volume measurement and DNA copy-number detection were performed. Unfortunately, the qPCR results of the DNA copy number in the blood were below the detection limit. The tumour-specific uptake was higher in the CD19-positive model than in the CD19-negative model, and this was consistent with the PD results. The preliminary PK and PD studies of CD19 CAR T cell in solid tumours are instructive. Considering the less efficiency of CAR T-cell therapy of solid tumours with the limited number of CAR T cells entering the interior of solid tumours, this study is suggestive for the subsequent CAR T-cell design and evaluation of solid tumour therapy.

APDL1-CART cells exhibit strong PD-L1-specific activity against leukemia cells.

Chimeric antigen receptor (CAR) T cells target specific tumor antigens and lyse tumor cells in an MHC-independent manner. However, the efficacy of CAR-T cell and other cancer immunotherapies is limited by the expression of immune-checkpoint molecules such as programmed death-ligand 1 (PD-L1) on tumor cells, which binds to PD-1 receptors on T cells leading to T cell inactivation and immune escape. Here, we incorporated a PD-L1-targeted single-chain variable fragment (scFv) fusion protein sequence into a CAR vector to generate human anti-PD-L1-CAR-T cells (aPDL1-CART cells) targeting the PD-L1 antigen. Unlike control T cells, aPDL1-CART cells significantly halted the expansion and reduced the viability of co-cultured leukemia cells (Raji, CD46, and K562) overexpressing PD-L1, and this effect was paralleled by increased secretion of IL-2 and IFN-γ. The antitumor efficacy of aPDL1-CART cells was also evaluated in vivo by co-injecting control T cells or aPDL1-CART cells along with PDL1-CA46 cells to generate subcutaneous xenografts in NCG mice. Whereas large tumors developed in mice inoculated with PDL1-CA46 cells alone or together with control T cells, no tumor formation was detected in xenografts containing aPDL1-CART cells. Our data suggest that immune checkpoint-targeted CAR-T cells may be useful for controlling and eradicating immune-refractory hematological malignancies.

A modular and controllable T cell therapy platform for acute myeloid leukemia.

Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

Loss of T-cell quiescence by targeting Slfn2 prevents the development and progression of T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes. Despite significant improvement in the treatment of T-ALL, approximately 20% of children and most adults undergo relapse. Previous findings demonstrated that loss of T-cell quiescence due to a mutation in the Slfn2 gene (elektra) leads to acquisition of an aberrant developmental program by which T-cells lose their renewal capabilities and undergo apoptosis. Here we show that the elektra mutation in Slfn2 completely prevents a severe lymphoproliferative disease caused by overexpression of BCL2 in combination with Fas deficiency in mice. Moreover, Slfn2 impaired-function protects mice from experimental disease similar to human T-ALL by severely impairing the proliferation potential and survival of leukemic T-cells, partially by activation of the p53 tumor suppressor protein. Our study suggest that in certain malignancies, such as T-ALL, a novel therapeutic strategy may be applied by imposing aberrant development of leukemic cells. Furthermore, as the elektra mutation in Slfn2 seems to impair only T-cells and monocytes, targeting Slfn2 is expected to be harmless to other cell types, and thereby could be a promising target for treating malignancies. Together our results demonstrate the potential of targeting Slfn2 and its human paralog for T-ALL treatment.

Combination of nanoparticle-based therapeutic vaccination and transient ablation of regulatory T cells enhances anti-viral immunity during chronic retroviral infection.

BACKGROUND: Regulatory T cells (Tregs) have been shown to limit anti-viral immunity during chronic retroviral infection and to restrict vaccine-induced T cell responses. The objective of the study was to assess whether a combinational therapy of nanoparticle-based therapeutic vaccination and concomitant transient ablation of Tregs augments anti-viral immunity and improves virus control in chronically retrovirus-infected mice. Therefore, chronically Friend retrovirus (FV)-infected mice were immunized with calcium phosphate (CaP) nanoparticles functionalized with TLR9 ligand CpG and CD8(+) or CD4(+) T cell epitope peptides (GagL85-93 or Env gp70123-141) of FV. In addition, Tregs were ablated during the immunization process. Reactivation of CD4(+) and CD8(+) effector T cells was analysed and the viral loads were determined. RESULTS: Therapeutic vaccination of chronically FV-infected mice with functionalized CaP nanoparticles transiently reactivated cytotoxic CD8(+) T cells and significantly reduced the viral loads. Transient ablation of Tregs during nanoparticle-based therapeutic vaccination strongly enhanced anti-viral immunity and further decreased viral burden. CONCLUSION: Our data illustrate a crucial role for CD4(+) Foxp3(+) Tregs in the suppression of anti-viral T cell responses during therapeutic vaccination against chronic retroviral infection. Thus, the combination of transient Treg ablation and therapeutic nanoparticle-based vaccination confers robust and sustained anti-viral immunity.

A Toll-like receptor 2 agonist-fused antigen enhanced antitumor immunity by increasing antigen presentation and the CD8 memory T cells population.

The induction of long-lived effector CD8+ T cells is key to the development of efficient cancer vaccines. In this study, we demonstrated that a Toll-like receptor 2 (TLR2) agonist-fused antigen increased antigen presentation via TLR2 signaling and induced effector memory-like CD8+ T cells against cancer after immunization. The N-terminus of ovalbumin (OVA) was biologically fused with a bacterial lipid moiety TLR2 agonist to produce a recombinant lipidated ovalbumin (rlipo-OVA). We demonstrated that rlipo-OVA activated bone marrow-derived dendritic cells (BM-DCs) maturation and increased antigen presentation by major histocompatibility complex (MHC) class I via TLR2. After immunization, rlipo-OVA skewed the immune response towards T helper (Th) 1 and induced OVA-specific cytotoxic T lymphocyte (CTL) responses. Moreover, immunization with rlipo-OVA induced higher numbers of effector memory (CD44+CD62L-) CD8+ T cells compared with recombinant ovalbumin (rOVA) alone or rOVA mixed with the TLR2 agonist Pam3CSK4. Accordingly, the CD27+CD43+ effector memory CD8+ T cells expressed high levels of the long-lived CD127 marker. The administration of rlipo-OVA could inhibit tumor growth, but the anti-tumor effects were lost after the depletion of CD8 or CD127 cells in vivo. These findings suggested that the TLR2 agonist-fused antigen induced long-lived memory CD8+ T cells for efficient cancer therapy.

Characterization of a bispecific FLT3 X CD3 antibody in an improved, recombinant format for the treatment of leukemia.

FLT3 is a receptor-tyrosine-kinase that is expressed on leukemic cells of the myeloid and lymphoid lineage rather specifically. We here report on the construction and selection of bispecific FLT3 X CD3 antibodies in a new recombinant format, termed Fabsc, that resembles the normal antibody structure more closely than the well-established bispecific single chain (bssc)-format. Our preferred antibody, which emerged from an initial selection procedure utilizing different FLT3- and CD3-antibodies, contains the FLT3-antibody 4G8 and the CD3-antibody UCHT1. The 4G8 X UCHT1 Fabsc-antibody was found to be superior to a bssc-antibody with identical specificities with respect to (i) affinity to the target antigen FLT3, (ii) production yield by transfected cells, and (iii) the diminished formation of aggregates. T-cell activation in the presence and absence of cultured leukemic cells and killing of these cells was comparable for both molecules. In addition, the 4G8 X UCHT1 Fabsc-antibody was found to induce T-cell activation and efficient killing of leukemic blasts in primary peripheral blood mononuclear cell (PBMC) cultures of acute myeloid leukemia (AML) patients. In these experiments, the bispecific molecule was clearly superior to an Fc-optimized monospecific FLT3-antibody described previously, indicating that within PBMC of AML patients the recruitment of T cells is more effective than that of natural killer cells.

Checkpoint blockade immunotherapy relies on T-bet but not Eomes to induce effector function in tumor-infiltrating CD8+ T cells.

Coinhibitory receptor blockade is a promising strategy to boost T-cell immunity against a variety of human cancers. However, many patients still do not benefit from this treatment, and responders often experience immune-related toxicities. These issues highlight the need for advanced mechanistic understanding to improve patient outcomes and uncover clinically relevant biomarkers of treatment efficacy. However, the T-cell-intrinsic signaling pathways engaged during checkpoint blockade treatment are not well defined, particularly for combination approaches. Using a murine model to study how effector CD8(+) T-cell responses to tumors may be enhanced in a tolerizing environment, we identified a critical role for the T-box transcription factor T-bet. Combination blockade of CTLA-4, PD-1, and LAG-3 induced T-bet expression in responding tumor/self-reactive CD8(+) T cells. Eradication of established leukemia using this immunotherapy regimen depended on T-bet induction, which was required for IFNγ production and cytotoxicity by tumor-infiltrating T cells, and for efficient trafficking to disseminated tumor sites. These data provide new insight into the success of checkpoint blockade for cancer immunotherapy, revealing T-bet as a key transcriptional regulator of tumor-reactive CD8(+) T-cell effector differentiation under otherwise tolerizing conditions.

Effects of iron depletion on CALM-AF10 leukemias.

Iron, an essential nutrient for cellular growth and proliferation, enters cells via clathrin-mediated endocytosis. The clathrin assembly lymphoid myeloid (CALM) protein plays an essential role in the cellular import of iron by clathrin-mediated endocytosis. CALM-AF10 leukemias harbor a single copy of the normal CALM gene and therefore may be more sensitive to the growth-inhibitory effect of iron restriction compared with normal hematopoietic cells. We found that CALM heterozygous (CALM(HET)) murine fibroblasts exhibit signs of iron deficiency, with increased surface transferrin receptor levels and reduced growth rates. CALM(HET) hematopoietic cells are more sensitive in vitro to iron chelators than their wild type counterparts. Iron chelation also displayed toxicity toward cultured CALM(HET)CALM-AF10 leukemia cells, and this effect was additive to that of chemotherapy. In mice transplanted with CALM(HET)CALM-AF10 leukemia, we found that dietary iron restriction reduced tumor burden in the spleen. However, dietary iron restriction, used alone or in conjunction with chemotherapy, did not increase survival of mice with CALM(HET)CALM-AF10 leukemia. In summary, although CALM heterozygosity results in iron deficiency and increased sensitivity to iron chelation in vitro, our data in mice do not suggest that iron depletion strategies would be beneficial for the therapy of CALM-AF10 leukemia patients.

Therapeutic efficacy of an Fc-enhanced TCR-like antibody to the intracellular WT1 oncoprotein.

PURPOSE: RMFPNAPYL (RMF), a Wilms' tumor gene 1 (WT1)-derived CD8 T-cell epitope presented by HLA-A*02:01, is a validated target for T-cell-based immunotherapy. We previously reported ESK1, a high avidity (Kd < 0.2 nmol/L), fully-human monoclonal antibody (mAb) specific for the WT1 RMF peptide/HLA-A*02:01 complex, which selectively bound and killed WT1(+) and HLA-A*02:01(+) leukemia and solid tumor cell lines. EXPERIMENTAL DESIGN: We engineered a second-generation mAb, ESKM, to have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) function due to altered Fc glycosylation. ESKM was compared with native ESK1 in binding assays, in vitro ADCC assays, and mesothelioma and leukemia therapeutic models and pharmacokinetic studies in mice. ESKM toxicity was assessed in HLA-A*02:01(+) transgenic mice. RESULTS: ESK antibodies mediated ADCC against hematopoietic and solid tumor cells at concentrations below 1 µg/mL, but ESKM was about 5- to 10-fold more potent in vitro against multiple cancer cell lines. ESKM was more potent in vivo against JMN mesothelioma, and effective against SET2 AML and fresh ALL xenografts. ESKM had a shortened half-life (4.9 days vs. 6.5 days), but an identical biodistribution pattern in C57BL/6J mice. At therapeutic doses of ESKM, there was no difference in half-life or biodistribution in HLA-A*02:01(+) transgenic mice compared with the parent strain. Importantly, therapeutic doses of ESKM in these mice caused no depletion of total WBCs or hematopoetic stem cells, or pathologic tissue damage. CONCLUSIONS: The data provide proof of concept that an Fc-enhanced mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target, and support further development of this mAb against an important intracellular oncogenic protein.

Eradicating acute myeloid leukemia in a Mll(PTD/wt):Flt3(ITD/wt) murine model: a path to novel therapeutic approaches for human disease.

The coexpression of the MLL partial tandem duplication (PTD) and the FLT3 internal tandem duplication (ITD) mutations associate with a poor outcome in cytogenetically normal acute myeloid leukemia (AML). In mice, a double knock-in (dKI) of Mll(PTD/wt) and Flt3(ITD/wt) mutations induces spontaneous AML with an increase in DNA methyltransferases (Dnmt1, 3a, and 3b) and global DNA methylation index, thereby recapitulating its human AML counterpart. We determined that a regulator of Dnmts, miR-29b, is downregulated in bone marrow of dKI AML mice. Bortezomib exerted a dose-dependent increase in miR-29b expression in AML blasts ex vivo, followed by decreased Dnmts, reduced proliferation, and increased apoptosis. In vivo, bortezomib was not active against dKI AML, yet liposomal-encapsulated bortezomib, as a single agent, reversed downregulation of miR-29b in vivo and induced a long-term (90-day) disease-free remission in 80% of dKI AML mice that exhibited high leukemic burden at the start of therapy, yet showed no signs of relapse at autopsy. Taken together, these data support that liposomal bortezomib, as a single agent, eradicates Mll(PTD/wt):Flt3(ITD/wt) AML in mouse and may represent a powerful and potentially curative approach to high-risk human disease.

Biological response modifiers influence structure function relationship of hematopoietic stem and stromal cells in a mouse model of leukemia.

Biological response modifiers (BRMs) can alter interactions between the immune system and cancer cells to boost, direct, or restore the body's ability to fight disease. Mice with ethylnitrosourea- (ENU) induced leukemia were here used to monitor the therapeutic efficacy of lipopolysaccaride (LPS), Bacillus Calmette Guerin (BCG) and sheep erythrocytes (SRBC). Flow cytometry based CD34+ positivity analysis, clonogenicity, proliferation and ultrastructure studies using scanning electron microscopy (SEM) of stem cells in ENU induced animals with and without BRMs treatment were performed. BRMs improved the stem-stromal relationship structurally and functionally and might have potential for use as an adjunct in human stem cell therapy.

The novel combination of sirolimus and bortezomib prevents graft-versus-host disease but maintains the graft-versus-leukemia effect after allogeneic transplantation.

BACKGROUND: We have previously shown that bortezomib induces a depletion of alloreactive T cells and allows the expansion of T cells with suppressive properties. In the current study, we analyzed the potential synergistic effect of bortezomib in conjunction with sirolimus in order to reduce-graft-versus-host disease without hampering graft-versus-leukemia effect in the allogeneic transplant setting. DESIGN AND METHODS: We evaluated the effect of sirolimus, bortezomib or the combination of both in the proliferation and activation of in vitro stimulated T lymphocytes. Pathways involved in this synergy were also analyzed using Western blot assays. Finally, BALB/c mice receiving C57BL/6 allogeneic donor bone marrow with splenocytes were used to measure in vivo the effect of this novel combination on the risk of graft-versus-host disease. RESULTS: The combination of both drugs synergistically inhibited both activation and proliferation of stimulated T cells. Also, the production of Th1 cytokines (IFN γ, IL-2 and TNF) was significantly inhibited. This effect was due, at least in part, to the inhibition of Erk and Akt phosphorylation. In vivo, the combination reduced the risk of graft-versus-host disease without hampering graft-versus-leukemia effect, as shown in mice receiving graft-versus-host disease prophylaxis with sirolimus plus bortezomib being infused with tumor WEHI cells plus C57BL/6 donor BM and splenocytes. CONCLUSIONS: The current study reveals a synergistic effect of the combination sirolimus and bortezomib to prevent graft-versus-host disease while maintaining the graft-versus-leukemia effect.