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The tumor microenvironment hinders CAR T-cell access, activation, and persistence at the tumor site, thereby impacting on the therapeutic efficacy. To tackle these obstacles, ongoing efforts are focusing on further engineering CAR T-cells to enhance their homing, fitness, long-term persistence, and antitumor activity. Advances in genetic modification have prompted the development of armored CAR T-cells equipped with a combination of synergistic elements strengthening their function. These include cytokine release, chemokine receptor expression, immune checkpoint inhibition, gene-editing of inhibitory molecules, or metabolic reprogramming, among others. Multiarmored CAR T-cells may allow addressing the unmet clinical needs of patients with solid tumors or hard-to-treat hematological malignancies who do not benefit from conventional CAR T-cell therapy. Accordingly, several clinical trials are currently assessing the safety and efficacy of these novel CAR constructs.

In spite of the remarkable progress in basic and preclinical studies of acute myeloid leukemia (AML), the five-year survival rate of AML patients remains poor, highlighting the urgent need for novel and synergistic therapies. Over the past decade, increased attention has been focused on identifying suitable immunotherapeutic strategies for AML, and in particular on targeting leukemic cells and their progenitors. However, recent studies have also underlined the important contribution of the leukemic microenvironment in facilitating tumor escape mechanisms leading to disease recurrence. Here, we describe the immunological features of the AML niche, with particular attention to the crosstalk between the AML blasts and the cellular components of the altered tumor microenvironment (TME) and the mechanisms of immune escape that hamper the therapeutic effects of the most advanced treatments. Considering the AML complexity, immunotherapy approaches may benefit from a rational combination of complementary strategies aimed at preventing escape mechanisms without increasing toxicity.

Acute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML remains unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T therapy in AML has been hampered by several factors including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche, where chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within the niche, could improve T cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells facilitating disease eradication. Specifically, we engineered conventional CD33.CAR-cytokine induced killer cells (CIKs) with the wild-type CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of WHIM syndrome patients. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell conditioned medium with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs co-expressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a non-canonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.

Chimeric Antigen Receptor (CAR) cytokine-induced killer (CIK) cell therapy is a promising treatment for acute myeloid leukemia (AML). Specifically, it is crucial to improve CAR CIK-cells infiltration ability into the bone marrow (BM) niche to eradicate leukemia stem cells (LSCs) at their location. Actually, BM mesenchymal stromal cells (MSCs) interact with LSCs, residing in the niche, releasing different chemokines and soluble factors. The chemokine ligand 12 (CXCL12), produced by MSCs, and its chemokine receptor 4 (CXCR4) regulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 overexpressed on blasts, promoting their homing in the niche. On the contrary, CXCR4 expression is drastically downregulated on CIKs during culture. Combining the expression of CD33.CAR and CXCR4 might facilitate CAR-CIKs homing to the BM and subsequent leukemia eradication. We designed two bicistronic Sleeping Beauty transposon vectors: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. We observed both CD33.CAR(2A)CXCR4-CIKs (n=22, P<0.0001) and CXCR4(IRES)CD33.CAR-CIKs (n=9, P<0.0001) maintained CXCR4 overexpression during culture, whereas in CD33.CAR-CIKs was drastically downregulated (n=22). However, CD33.CAR expression was lower in CXCR4(IRES)CD33.CAR-CIKs (n=8, P<0.0001) compared with CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=11) exhibit a significant co-expression of both proteins against control (P=0.001). Chemotaxis assays toward recombinant CXCL12 confirmed both CXCR4(IRES)CD33.CAR-CIKs (n=7, P=0.01) and CD33.CAR(2A)CXCR4-CIKs (n=8, P=0.0006) displayed a migration advantage over CD33.CAR-CIKs (n=12) with a mean percentage of migration of 58.5% and 67.2% respectively, compared to 40.1%. Interestingly, CD33.CAR(2A)CXCR4-CIKs (n=2) showed an increased specific chemotactic response toward HD- (n=3) and AML-MSCs (n=2) supernatants, as demonstrated by the use of CXCR4 antagonist Plerixafor. Moreover, CXCR4(IRES)CD33.CAR

Side-population (SP) analysis identifies precursor cells in normal and malignant tissues. Cells with this phenotype have increased resistance to many cytotoxic agents, and may represent a primary drug-resistant population in malignant diseases. To discover whether drug-resistant malignant SP cells are nonetheless sensitive to immune-mediated killing, we first established the presence of a malignant CD5(+)CD19(+) SP subset in the blood of 18/21 subjects with B-cell chronic lymphocytic leukemia (B-CLL). We examined the fate of these cells in six of these individuals who received autologous human CD40 ligand and interleukin-2 (hCD40L/IL-2) gene-modified tumor cells as part of a tumor vaccine study. Vaccinated patients showed an increase in B-CLL-reactive T cells followed by a corresponding decline in circulating CD5(+)CD19(+) SP cells. T-cell lines and clones generated from vaccinated patients specifically recognized B-CLL SP tumor cells. Elimination of SP cells is likely triggered by their increased expression of target antigens, such as receptor for hyaluronan-mediated motility (RHAMM), after stimulation of the malignant cells by hCD40L, as CD8(+) RHAMM-specific T cells could be detected in the peripheral blood of immunized patients and were associated with the decline in B-CLL SP cells. Hence, malignant B cells with a primary drug-resistant phenotype can be targeted by T- cell-mediated effector activity after immunization of human subjects.

There has been interest in generating T cells expressing chimeric artificial receptors (CARs) targeting CD19/CD20 antigens to treat B-cell lymphomas. If successful, however, this approach would likely impair humoral immunity because T cells may persist long-term. Most low-grade lymphoma and chronic lymphocytic leukemia (B-CLL) cells express monoclonal immunoglobulins carrying either kappa or lambda light chains. We, therefore, explored whether T lymphocytes could be genetically modified to target the tumor-associated light chain, sparing B lymphocytes expressing the reciprocal light chain, and consequently reduce impairment of humoral immunity. We found that T lymphocytes expressing the anti-kappa light chain CAR showed cytotoxic activity against Igkappa(+) tumor cell lines and B-CLL cells both in vitro and in vivo. We also found that the incorporation of the CD28 endodomain within the CAR enhanced the in vitro and in vivo expansion of transgenic T cells after tumor-associated antigen stimulation. Free Igkappa(+) did not compromise the ability of redirected T lymphocytes to eliminate Igkappa(+) tumors because these free immunoglobulins served to sustain proliferation of CAR-CD28 transgenic T cells. Thus, adoptive transfer of T lymphocytes targeting the appropriate light chain could be a useful immunotherapy approach to treat B-lymphocyte malignancies that clonally express immunoglobulin without entirely compromising humoral immunity.

Background aims. Several studies have demonstrated that the immunogenicity of chronic lymphocytic leukemia (CLL) cells can be increased by manipulation of the CD40/CD40-ligand (CD40L) pathway. Although immunologic, and perhaps clinical, benefits have been obtained with an autologous CLL tumor vaccine obtained by transgenic expression of CD40L and interleukin (IL)-2, there is little information about the optimal gene transfer strategies. Methods. We compared two different CLL vaccines prepared by adenoviral gene transfer and plasmid electroporation, analyzing their phenotype and immunostimulatory activity. Results. We found that higher expression of transgenic CD40L was mediated by adenoviral gene transfer than by plasmid transduction, and that adenoviral transfer of CD40L was associated with up-regulation of the co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54. In contrast, transgenic IL-2 secretion was greater following plasmid transduction. These phenotypic differences in the vaccines were associated with different functionality, both ex vivo and following administration to patients. Thus adenoviral vaccines induced greater activation of leukemia-reactive T cells ex vivo than plasmid vaccines. In treated patients, specific T-cell (T helper 1 (Th1) and T helper 2 (Th2)) and humoral anti-leukemia responses were detected following administration of the adenoviral vaccine (n = 15), while recipients of the plasmid vaccine (n = 9) manifested only a low-level Th2 response. Progression-free survival at 2 years was 46.7% in the adenoviral vaccine recipients, versus 11.1 % in those receiving plasmid vaccine. Conclusions. CLL vaccines expressing the same transgenes but produced by distinct methods of gene transfer may differ in the polarity of the immune response they induce in patients

Cancer precursor/progenitor cells may initiate and sustain the growth of tumors, but evidence for their existence in human disease is indirect, relying on their in vitro properties and animal models. More directly, specific elimination of these rare cells from cancer patients should produce a delayed but progressive disappearance of differentiated malignant progeny. Here, we describe selective eradication of a putative precursor population in a patient with B-cell chronic lymphocytic leukemia, followed 6 months later by a progressive loss of mature tumor cells without further treatment. This outcome supports the presence of a rare population of precursor/progenitor cells in human malignancies, and suggests benefit from their removal.