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This paper indicates that murine nucleated erythroid cells (EC) are able to reduce, in a dose-dependent manner, the proliferation of both L1210 lymphoma and P815 mastocytoma cells and that the leukemia cell growth inhibitory activity of unseparated bone marrow (BM) cells may be markedly augmented by their short-term culturing with erythropoietin (Epo). These results raise the intriguing possibility to utilize erythropoesis-stimulating, therapeutic strategies with the purpose of inhibiting leukemia cell growth in the body. [ABSTRACT FROM AUTHOR]

Background: T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR., Methods: T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19)., Results: SB-mediated genetic transposition and stimulation resulted in 2,200- to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients., Conclusions: CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach., Trial Registration: Autologous, NCT00968760; allogeneic, NCT01497184; long-term follow-up, NCT01492036., Funding: National Cancer Institute, private foundations, and institutional funds. Please see Acknowledgments for details.

The efficacy of most therapeutic monoclonal antibodies (mAbs) targeting tumor antigens results primarily from their ability to elicit potent cytotoxicity through effector-mediated functions. We have engineered the fragment crystallizable (Fc) region of the immunoglobulin G (IgG) mAb, HuM195, targeting the leukemic antigen CD33, by introducing the triple mutation Ser293Asp/Ala330Leu/Ile332Glu (DLE), and developed Time-lapse Imaging Microscopy in Nanowell Grids to analyze antibody-dependent cell-mediated cytotoxicity kinetics of thousands of individual natural killer (NK) cells and mAb-coated target cells. We demonstrate that the DLE-HuM195 antibody increases both the quality and the quantity of NK cell-mediated antibody-dependent cytotoxicity by endowing more NK cells to participate in cytotoxicity via accrued CD16-mediated signaling and by increasing serial killing of target cells. NK cells encountering targets coated with DLE-HuM195 induce rapid target cell apoptosis by promoting simultaneous conjugates to multiple target cells and induce apoptosis in twice the number of target cells within the same period as the wild-type mAb. Enhanced target killing was also associated with increased frequency of NK cells undergoing apoptosis, but this effect was donor-dependent. Antibody-based therapies targeting tumor antigens will benefit from a better understanding of cell-mediated tumor elimination, and our work opens further opportunities for the therapeutic targeting of CD33 in the treatment of acute myeloid leukemia., (© 2014 by The American Society of Hematology.)

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼10(10) T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

Pancreatic carcinoma is a very aggressive disease with dismal prognosis. Although evidences for tumor-specific T cell immunity exist, factors related to tumor microenvironment and the presence of immunosuppressive cytokines in patients' sera have been related to its aggressive behavior. Carcinoembryonic Ag (CEA) is overexpressed in 80-90% of pancreatic carcinomas and contains epitopes recognized by CD4(+) T cells. The aim of this study was to evaluate the extent of cancer-immune surveillance and immune suppression in pancreatic carcinoma patients by comparing the anti-CEA and antiviral CD4(+) T cell immunity. CD4(+) T cells from 23 normal donors and 44 patients undergoing surgical resection were tested for recognition of peptides corresponding to CEA and viral naturally processed promiscuous epitopes by proliferation and cytokine release assays. Anti-CEA CD4(+) T cell immunity was present in a significantly higher number of normal donors than pancreatic cancer patients. Importantly, whereas CD4(+) T cells from normal donors produced mainly GM-CSF and IFN-gamma, CD4(+) T cells from the patients produced mainly IL-5, demonstrating a skew toward a Th2 type. On the contrary, the extent of antiviral CD4(+) T cell immunity was comparable between the two groups and showed a Th1 type. The immunohistochemical analysis of tumor-infiltrating lymphocytes showed a significantly higher number of GATA-3(+) compared with T-bet(+) lymphoid cells, supporting a Th2 skew also at the tumor site. Collectively, these results demonstrate that Th2-immune deviation in pancreatic cancer is not generalized but tumor related and suggests that the skew might be possibly due to factor(s) present at the tumor site.

The murine bone marrow (BM) cells having a certain phenotypic similarity to null natural suppressor (NS) cells have been previously established to be able to inhibit in vitro leukemic cell growth in a genetically unrestricted manner. In this study we found that the treatment of normal (C57BL/6 x DBA)F1 BM cells with a lysosomotropic agent, L-leucine methyl ester (LME), largely abrogated their ability to reduce both P815 mastocytoma and L1210 lymphoma cell proliferation, as well as their NS activity tested for suppression of mitogen (Con A or LPS)-driven spleen cell proliferation. However, after being depleted of the cells binding wheat germ agglutinin (WGA), the BM cells maintained tumor growth-inhibitory activity, while demonstrating no significant NS activity. Moreover, in contrast to T-cell blastogenesis-inhibitory NS activity of BM cells, that was greatly reduced by the addition into the culture of either neutralizing anti-interferon (IFN)-gamma antibody (Ab) or NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, natural antitumor cytostatic activity of BM cells was not found to be dependent on the presence in medium of IFN-gamma and to be associated with NO production. When incubated at suboptimal numbers with tumor cells on conic, round, and flat well bottoms for 7 h, BM cells provided the most, middle, and least (or no) tumor growth inhibition, respectively, suggesting, thereby, a significance of cell density in cytostatic process. It was also found that the BM cells cultured for 20 h with the medium conditioned by mitogen-preactivated T or B lymphocytes were significantly more suppressive to tumor cell proliferation than the BM cells cultured in medium alone. The potentiation of BM-cell cytostatic activity by T-cell-conditioned medium (CM), but not that by B-cell-CM, was found to be partially reversed by anti-IFN-gamma Ab. Finally, a noticeable tumor growth-inhibitory activity, which could be significantly enhanced upon T-cell-CM, was shown to be also attributable to BM cells from athymic BALB/c mice. Taken together, the results suggest that (1) the tumor growth inhibitory BM cells and the NS BM cells are not identical in their cell compositions, but also differ in their mechanisms of antiproliferative action; (2) a contact cell-to-cell interaction may play a significant role in BM-cell-mediated tumor growth inhibition; (3) the activated lymphocytes, through both IFN-gamma-mediated and IFN-gamma-independent pathways, are able to operatively up-regulate the cytostatic activity of BM cells; and (4) the tumor growth-inhibitory activity exhibited by the normal unmanipulated BM cells, at least in its significant part, may not be a consequence of thymus-dependent immune processes occurring normally in the body.