Off the shelf T cell therapies for hematologic malignancies.

Adoptive transfer of autologous CAR-T cells can induce durable remissions in patients with relapsed/refractory hematologic malignancies. However, multiple challenges exist for manufacturing CAR-T cells from patients with advanced disease including inability to manufacture a product, disease progression or death while waiting for the CAR-T product to be available, and heterogeneity among autologous CAR-T products that contributes to unpredictable and variable clinical activity. Healthy donor T cells can provide a source for production of universal CAR-T cells when combined with gene editing to prevent expression of endogenous TCRs and avoid generation of GvHD in HLA mismatched recipients.

Interferon Gamma Induces Changes in Natural Killer (NK) Cell Ligand Expression and Alters NK Cell-Mediated Lysis of Pediatric Cancer Cell Lines.

Natural killer (NK) cells have therapeutic potential for cancer due to their capacity for targeting tumor cells without prior sensitization. Our laboratory has developed an NK cell expansion protocol that generates large quantities of NK cells for therapeutic infusion that secret 20 times the amount of interferon gamma (IFNγ) than resting NK cells. IFNγ can upregulate major histocompatibility complex (MHC)-class I, an inhibitory ligand for NK cells, but can also upregulate intercellular adhesion molecule 1 (ICAM-1) which promotes NK:target cell interaction for an efficient lysis.

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells.

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.

Antibody Fc engineering improves frequency and promotes kinetic boosting of serial killing mediated by NK cells.

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.

Transcription of the activating receptor NKG2D in natural killer cells is regulated by STAT3 tyrosine phosphorylation.

Signal transducer and activator of transcription 3 (STAT3) is considered a negative regulator of inflammation, as inhibition of STAT3 signaling enhances antitumor immunity. However, STAT3 activation is a key oncogenic pathway in natural killer (NK)-lineage large granular lymphomas, and we recently reported enhanced proliferation and function of human NK cells activated with IL-21, which signals primarily through STAT3. These IL-21-expanded NK cells also have increased NKG2D expression, which led us to focus our investigation on whether STAT3 regulates NKG2D.

Manufacture of clinical-grade CD19-specific T cells stably expressing chimeric antigen receptor using Sleeping Beauty system and artificial antigen presenting cells.

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.

Decitabine has a biphasic effect on natural killer cell viability, phenotype, and function under proliferative conditions.

DNA hypermethylation resulting in aberrant epigenetic silencing plays an important role in the oncogenesis of many cancer types, including acute myelogenous leukemia (AML).(4) The modulation of NK cell receptors and their cognate ligands is a known mechanism of immune escape in AML, and some membrane proteins, such as killer immunoglobulin-like receptors (KIR), are known to be transcriptionally regulated by DNA methylation of their promoter regions. Thus, restoring proper expression of immunoreceptors or their ligands with immunosensitizing drugs is an attractive approach to improving cancer immunotherapy.

Membrane-bound IL-21 promotes sustained ex vivo proliferation of human natural killer cells.

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening.

Expansion, purification, and functional assessment of human peripheral blood NK cells.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).

Carcinoembryonic antigen-specific but not antiviral CD4+ T cell immunity is impaired in pancreatic carcinoma patients.

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.

Cell therapy of comatose states.

We demonstrated that liquor from adult humans can maintain proliferative activity of cells of immature nervous tissue in vitro. The paper presents the results of a retrospective clinical study of the efficiency of cell therapy in the treatment of II-III degree comatose patients with severe brain injury. Cell suspension consisting of cells derived from immature nervous and hemopoietic tissues was injected into the recipient subarachnoidal space through a cerebrospinal puncture.

Cell therapy of brain stroke.

Cell suspension consisting of cells from immature nervous and hemopoietic tissues was subarachnoidally transplanted to 10 patients with brain stroke consequences. Clinical effect of different degree was attained in all patients. Six months after cell therapy functional activity significantly increased in contrast to clinically comparable control group.

Erythroid cells in suppressing leukemia cell growth.

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.

Erythroid cells in immunoregulation: characterization of a novel suppressor factor.

Nucleated erythroid cells (EC) have been previously reported to possess a potent natural suppressor (NS) activity for B-cell responses. In this study, we demonstrate that murine EC are able to reduce not only lipopolysaccharide (LPS)-driven B-cell proliferation, but also proliferative and cytotoxic T-cell responses generated in a primary allogeneic mixed lymphocyte culture (MLC); and that a soluble low molecular weight factor may be involved in such EC-derived immunoregulation. In addition, the erythroid cell-derived suppressor factor (ESF) was found to be capable of effectively reducing the allergen-driven proliferation of peripheral blood mononuclear cells (PBMC) isolated from allergic patients.

Soluble factor and cell-cell interaction in cytostasis induced by bone marrow cells.

Cell-cell interaction and soluble low-molecular-weight products are probably involved in in vitro inhibition of leukemic cell growth by bone marrow cells.

Tumor growth inhibitory and natural suppressor activities of murine bone marrow cells: a comparative study.

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.