Monica Casucci

Loss of Mismatched HLA in Leukemia after Stem-Cell Transplantation

BACKGROUND Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease. METHODS We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures. RESULTS In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donors haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed. CONCLUSIONS After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donors antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse.

CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma

Genetically targeted T cells promise to solve the feasibility and efficacy hurdles of adoptive T-cell therapy for cancer. Selecting a target expressed in multiple-tumor types and that is required for tumor growth would widen disease indications and prevent immune escape caused by the emergence of antigen-loss variants. The adhesive receptor CD44 is broadly expressed in hematologic and epithelial tumors, where it contributes to the cancer stem/initiating phenotype. In this study, silencing of its isoform variant 6 (CD44v6) prevented engraftment of human acute myeloid leukemia (AML) and multiple myeloma (MM) cells in immunocompromised mice. Accordingly, T cells targeted to CD44v6 by means of a chimeric antigen receptor containing a CD28 signaling domain mediated potent antitumor effects against primary AML and MM while sparing normal hematopoietic stem cells and CD44v6-expressing keratinocytes. Importantly, in vitro activation with CD3/CD28 beads and interleukin (IL)-7/IL-15 was required for antitumor efficacy in vivo. Finally, coexpressing a suicide gene enabled fast and efficient pharmacologic ablation of CD44v6-targeted T cells and complete rescue from hyperacute xenogeneic graft-versus-host disease modeling early and generalized toxicity. These results warrant the clinical investigation of suicidal CD44v6-targeted T cells in AML and MM.

Adoptive immunotherapy with genetically modified lymphocytes in allogeneic stem cell transplantation

Hematopoietic stem cell transplantation from a healthy donor (allo‐HSCT) represents the most potent form of cellular adoptive immunotherapy to treat malignancies. In allo‐HSCT, donor T cells are double edge‐swords: highly potent against residual tumor cells, but potentially highly toxic, and responsible for graft versus host disease (GVHD), a major clinical complication of transplantation. Gene transfer technologies coupled with current knowledge on cancer immunology have generated a wide range of approaches aimed at fostering the immunological response to cancer cells, while avoiding or controlling GVHD. In this review, we discuss cell and gene therapy approaches currently tested in preclinical models and in clinical trials.

Overcoming the toxicity hurdles of genetically targeted T cells

Abstract The recent successes of clinical trials with T cells genetically modified with either clonal T cell receptors or chimeric antigen receptors have also highlighted their potential toxicities. The aim of this focused review was to describe the adverse events observed in these clinical trials and to link them to the complex biology of genetically targeted T cells. Finally, strategies to overcome these toxicities will be proposed and discussed, including the use of suicide genes and other innovative gene therapy strategies.

Graft-versus-leukemia effect of HLA-haploidentical central-memory t-cells expanded with leukemic APCs and modified with a suicide gene

Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T(CM)) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC-expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC-expanded T cells modified with a suicide gene in the setting of haplo-HSCT.

Clinical pharmacology of CAR-T cells: Linking cellular pharmacodynamics to pharmacokinetics and antitumor effects.

Adoptive cell transfer of T cells genetically modified with tumor-reactive chimeric antigen receptors (CARs) is a rapidly emerging field in oncology, which in preliminary clinical trials has already shown striking antitumor efficacy. Despite these premises, there are still a number of open issues related to CAR-T cells, spanning from their exact mechanism of action (pharmacodynamics), to the factors associated with their in vivo persistence (pharmacokinetics), and, finally, to the relative contribution of each of the two in determining the antitumor effects and accompanying toxicities. In light of the unprecedented curative potential of CAR-T cells and of their predicted wide availability in the next few years, in this review we will summarize the current knowledge on the clinical pharmacology aspects of what is anticipated to be a brand new class of biopharmaceuticals to join the therapeutic armamentarium of cancer doctors.

Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells

In the clinic, chimeric antigen receptor–modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity. In humanized mice with high leukemia burden, CAR T cell–mediated clearance of cancer triggered high fever and elevated IL-6 levels, which are hallmarks of CRS. Human monocytes were the major source of IL-1 and IL-6 during CRS. Accordingly, the syndrome was prevented by monocyte depletion or by blocking IL-6 receptor with tocilizumab. Nonetheless, tocilizumab failed to protect mice from delayed lethal neurotoxicity, characterized by meningeal inflammation. Instead, the IL-1 receptor antagonist anakinra abolished both CRS and neurotoxicity, resulting in substantially extended leukemia-free survival. These findings offer a therapeutic strategy to tackle neurotoxicity and open new avenues to safer CAR T cell therapies.IL-1 blockade prevents cytokine-release syndrome and neurotoxicity by CAR T cells.

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting.

Exploiting Secreted Luciferases to Monitor Tumor Progression In Vivo.

Secreted luciferases provide a simple, accurate, and cost-effective tool to monitor tumor response after treatment in small animal models. This protocol describes all the steps required to generate human tumor cell lines expressing this reporter gene and to monitor in vivo tumor progression after injection in immunocompromised mice by means of serial peripheral blood sampling and analysis.

512. The Cytokine Release Syndrome Crucially Contributes to the Anti-Leukemic Effects of CD44v6 CAR-T Cells

Background: Despite the remarkable clinical results of CD19 CAR-T cells in B-cell leukemias, their long-term efficacy is limited by the emergence of CD19-loss escape variants. Moreover, whether the cytokine release syndrome (CRS) is necessary for durable remissions is a matter of debate. Currently available xenograft models in NSG mice are not suited for studying the antitumor effects of CAR-T cells beyond 3-4 weeks, because of xenograft-versus-host disease (X-GVHD). Moreover, since NSG mice lack functional myeloid cells, the CRS does not develop. Aim: To verify whether the CRS contributes to the antileukemic effects of CAR in an innovative xenotolerant mouse model.Results: NSG mice triple transgenic for human IL-3, GM-CSF and SCF (NSG-3GS) were sub-lethally irradiated and injected intra-liver with human HSCs soon after birth, enabling an accelerated and better balanced lympho-hematopoietic reconstitution compared with NSG mice. Reconstituting human T cells were single CD4+/CD8+ T cells, representing all memory sub-populations. After ex vivo isolation and activation with CD3/CD28-beads and IL-7/IL-15, NSG-3GS T cells were transduced with a CD44v6 CAR, retaining an early-differentiated (stem-cell/central-memory) phenotype and full antitumor functionality against acute myeloid leukemia (AML). NSG-3GS-derived CD44v6 CAR T cells were subsequently infused in tumor-bearing secondary recipients previously humanized with autologous HSCs. CAR-T cells persisted in vivo for at least 6 months and mediated durable leukemia remissions (P<0.001 vs controls) in the absence of X-GVHD. Tumor clearance associated with an acute malaise syndrome, characterized by high fevers and a surge in human IL-6 levels, which was lethal in 30% of the mice. Differently from CD19 CAR-T cells, the CRS by CD44v6 CAR-T cells was significantly anticipated (3 vs 8 days), coinciding with human CD44v6+ monocyte depletion. In humanized mice, previous myeloid-cell depletion by clodronate administration completely prevented this syndrome, but associated with late leukemia relapses. Conversely, mice developing the CRS entered a state of durable and profound remission, as demonstrated by prolonged observation times and secondary transplantation. Conclusions: By using an innovative xenotolerant mouse model, we have demonstrated that the CRS is needed for sustained antileukemic effects by CD44v6 CAR-T cells.