Antithymidylate resistance enables transgene selection and cell survival for T cells in the presence of 5-fluorouracil and antifolates.

Antithymidylates (AThy) constitute a class of drugs used in the treatment of cancers such as lung, colon, breast and pancreas. These drugs inhibit DNA synthesis by targeting the enzymes dihydrofolate reductase (DHFR) and/or thymidylate synthase (TYMS). AThys effectively inhibit cancer cells, and also inhibit T cells, preventing anticancer immunity, which might otherwise develop from AThy-induced cancer destruction. We establish that T cells expressing mutant DHFR--DHFR L22F, F31S (DHFR(FS))--and/or mutant TYMS--TYMS T51S, G52S (TYMS(SS))-effectively survive in toxic concentrations of AThys methotrexate, pemetrexed and 5-fluorouracil. Furthermore, we show that DHFR(FS) permitted rapid selection of an inducible suicide transgene in T cells. These findings demonstrate that AThy resistances prevent AThy cytotoxicity to T cells while permitting selection of important transgenes. This technological development could enhance in vitro and in vivo survival and selection of T-cell therapeutics being designed for a broad range of cancers.

Manufacture of T cells using the Sleeping Beauty system to enforce expression of a CD19-specific chimeric antigen receptor.

T cells can be reprogrammed to redirect specificity to tumor-associated antigens (TAAs) through the enforced expression of chimeric antigen receptors (CARs). The prototypical CAR is a single-chain molecule that docks with TAA expressed on the cell surface and, in contrast to the T-cell receptor complex, recognizes target cells independent of human leukocyte antigen. The bioprocessing to generate CAR(+) T cells has been reduced to clinical practice based on two common steps that are accomplished in compliance with current good manufacturing practice. These are (1) gene transfer to stably integrate the CAR using viral and nonviral approaches and (2) activating the T cells for proliferation by crosslinking CD3 or antigen-driven numeric expansion using activating and propagating cells (AaPCs). Here, we outline our approach to nonviral gene transfer using the Sleeping Beauty system and the selective propagation of CD19-specific CAR(+) T cells on AaPCs.

Immunotherapy for chronic lymphocytic leukemia in the era of BTK inhibitors.

Understanding the pathogenesis of CLL has uncovered a plethora of novel targets for human application of monoclonal antibodies, engineered T cells, or inhibitors of signal transduction pathways. The B-cell receptor signaling pathway is being actively explored as a therapeutic target in CLL. Ibrutinib, an inhibitor of Bruton's tyrosine kinase is showing impressive responses in heavily pre-treated high-risk CLL, whether alone or in combination with MoAbs or chemotherapy. Other key components of the BCR pathway, namely PI3K-δ, are also being targeted with novel therapies with promising results as well. Future trials would likely evaluate ibrutinib in the front-line setting. Moreover, improvements in allogeneic HCT mostly by continuing to reduce associated toxicity as well as incorporating cellular therapies such as autologous CLL tumor vaccines, among others, will continue to expand. This is also the case for the next generation of chimeric antigen receptor therapy for CLL once genetically modified T cells are available at broad scale and with improved efficacy. As our ability to further refine and integrate these therapies continues to improve, and we gain further knowledge from gene sequencing, we anticipate that treatment algorithms will continue to be revised to a more personalized approach to treat this disease with improved efficacy and devoid of unnecessary toxicity.

The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor.

Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on the measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen-presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with an SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells.

Adoptive cellular immunotherapy for childhood malignancies.

Clinical trials have established that T cells have the ability to prevent and treat pathogens and tumors. This is perhaps best exemplified by engraftment of allogeneic T cells in the context of hematopoietic stem-cell transplantation (HSCT), which for over the last 50 years remains one of the best and most robust examples of cell-based therapies for the treatment of hematologic malignancies. Yet, the approach to infuse T cells for treatment of cancer, in general, and pediatric tumors, in particular, generally remains on the sidelines of cancer therapy. This review outlines the current state-of-the-art and provides a rationale for undertaking adoptive immunotherapy trials with emphasis on childhood malignancies.

Preparing clinical grade Ag-specific T cells for adoptive immunotherapy trials.

The production of clinical-grade T cells for adoptive immunotherapy has evolved from the ex vivo numerical expansion of tumor-infiltrating lymphocytes to sophisticated bioengineering processes often requiring cell selection, genetic modification and other extensive tissue culture manipulations, to produce desired cells with improved therapeutic potential. Advancements in understanding the biology of lymphocyte signaling, activation, homing and sustained in vivo proliferative potential have redefined the strategies used to produce T cells suitable for clinical investigation. When combined with new technical methods in cell processing and culturing, the therapeutic potential of T cells manufactured in academic centers has improved dramatically. Paralleling these technical achievements in cell manufacturing is the development of broadly applied regulatory standards that define the requirements for the clinical implementation of cell products with ever-increasing complexity. In concert with academic facilities operating in compliance with current good manufacturing practice, the prescribing physician can now infuse T cells with a highly selected or endowed phenotype that has been uniformly manufactured according to standard operating procedures and that meets federal guidelines for quality of investigational cell products. In this review we address salient issues related to the technical, immunologic, practical and regulatory aspects of manufacturing these advanced T-cell products for clinical use.

Manufacturing of gene-modified cytotoxic T lymphocytes for autologous cellular therapy for lymphoma.

BACKGROUND: The production of therapeutic T-cell populations for adoptive immunotherapy of cancer requires extensive ex vivo cell processing, including the isolation or creation of Ag-specific T cells and their subsequent propagation to clinically relevant numbers. These procedures must be performed according to the principles of current good manufacturing practices (cGMP) for phase I clinical trials to ensure the identity, purity potency and safety of the cellular product. In this report we describe our approach to manufacturing and characterizing bulk populations of gene-modified autologous T cells for use in treating follicular lymphoma. METHODS: PBMC from healthy donors, obtained after informed consent, were stimulated in vitro with Ab to CD3epsilon (OKT3) and recombinant human IL-2 and then electroporated with plasmid DNA containing a human CD19-specific chimeric Ag receptor (CAR) gene and HSV-1 thymidine kinase (TK) gene. Stably transfected cells were selected in cytocidal concentrations of hygromycin B over multiple 14-day stimulation culture cycles and then cryopreserved. Vials of cryopreserved/selected T cells were used to initiate T-cell expansion cultures to produce cell products for clinical infusion. These cultures were characterized for phenotype, function and suitability for use in adoptive immunotherapy studies. RESULTS: Our results demonstrate that bulk populations of gene-modified T cells derived from peripheral blood of healthy donors express CD19+ chimeric Ag receptor at low levels and can specifically lyse CD19+ target cells in vitro. These cells display a differentiated T-effector phenotype, are sensitive to ganciclovir-mediated killing and display a non-transformed phenotype. TCR Vbeta usage indicated that all populations tested were polyclonal. Ex vivo cell expansion from cryopreserved cell banks is sufficient to produce doses of between 5 x 10(9) and 1 x 10(10) cells/run. One of three transductions resulted in a population of cells that was not suitable for infusion but was identified during release testing. No populations displayed any evidence of bacterial, fungal or mycoplasma contamination. DISCUSSION: We have established a manufacturing strategy that is being used to produce T cells for a phase I clinical trial for follicular lymphoma. Genetically modified T cells have been characterized by cell-surface marker phenotype, functional activity against CD19+ targets and requisite safety testing. These pre-clinical data confirm the feasibility of this approach to manufacturing T-cell products.

Engineered CD20-specific primary human cytotoxic T lymphocytes for targeting B-cell malignancy.

BACKGROUND: Immunotherapy for B-cell lymphomas has evolved significantly with the advent of CD20-targeted Ab-based therapeutics. Strategies to invoke or augment cellular anti-lymphoma immune responses may also have considerable therapeutic potential and serve to further augment the clinical efficacy of MAbs. METHODS: We report here the aquisition by priming human cytotoxic T lymphocyte (CTL) effectors of re-directed CD20 specificity by their genetic modification to express a chimeric immunoreceptor consisting of an anti-CD20 single chain Ab extracellular domain molecularly fused to the T-cell receptor complex CD3-zeta cytoplasmic tail (scFvFczeta). Peripheral blood-derived human T-cells were transduced with naked DNA plasmid vector by electoporation then selected for G418 resistance. RESULTS: Following cloning in limiting dilution and ex vivo expansion to large numbers scFvFczeta+ TCRalpha/beta+ CD4- CD8+ CTL display re-directed HLA-unresricted CD20-specific lymphoma cell cytolysis proportional to the cell-surface density of the chimeric immunoreceptor. Engineered CTL clones are also activated through the chimeric immunoreceptor to produce Tc1 cytokines (IFN-gamma) upon co-culture with CD20+ lymphoma stimulator cells. Additionally, CD20-specific CTL proliferate in the presence of lymphoma stimulators and IL-2 (5 U/mL). DISCUSSION: These studies provide the rationale for exploring the clinical utility of adoptive therapy with CD20-specific CTL as a component of immunotherapeutic targeting of CD20+ malignancy.