David E. Gilham

The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens.

Human peripheral blood lymphocytes can be transduced to express antigen-dependent CD3ζ chimeric immune receptors (CIRs), which function independently of the T-cell receptor (TCR). Although the exact function of these domains is unclear, previous studies imply that an extracellular spacer region is required for optimal CIR activity. In this study, four scFvs (in the context of CIRs with or without extracellular spacer regions) were used to target the human tumor-associated antigens carcinoembryonic antigen (CEA), neural cell adhesion molecule (NCAM), the oncofetal antigen 5T4, and the B-cell antigen CD19. In all cases human T-cell populations expressing the CIRs were functionally active against their respective targets, but the anti-5T4 and anti-NCAM CIRs showed enhanced specific cytokine release and cytotoxicity only when possessing an extracellular spacer region. In contrast, the anti-CEA and anti-CD19 CIRs displayed optimal cytokine release activity only in the absence of an extracellular spacer. Interestingly, mapping of the scFv epitopes has revealed that the anti-CEA scFv binds close to the amino-terminal of CEA, which is easily accessible to the CIR. In contrast, CIRs enhanced by a spacer domain appear to bind to epitopes residing closer to the cell membrane, suggesting that a more flexible extracellular domain may be required to permit the efficient binding of such epitopes. These results show that a spacer is not necessary for optimal activity of CIRs but that the optimal design varies.

CAR-T cells and solid tumors: tuning T cells to challenge an inveterate foe

Recent reports on the impressive efficacy of adoptively transferred T cells to challenge cancer in early phase clinical trials have significantly raised the profile of T cell therapy. Concomitantly, general expectations are also raised by these reports, with the natural aspiration to deliver this therapy over a wide range of tumor indications. Chimeric antigen receptors (CARs) endow T cell populations with defined antigen specificities that function independently of the natural T cell receptor and permit targeting of T cells towards virtually any tumor. Here, we review the current clinical application of CAR-T cells and relate clinical efficacy and safety of CAR-T cell trials to parameters considered critical for CAR engineering, classified as the three Ts of CAR-T cell manipulation.

T regulatory cells in cancer: Recent advances and therapeutic potential

Importance of the field: The active suppression of immune responses against tumor is a major barrier to the likely success of cancer immunotherapy. There is now compelling evidence implicating T regulatory cells (Tregs) as being key players driving immune suppression. Elevated frequencies of Tregs within the peripheral circulation and tumor microenvironment of cancer patients correlate with poor prognosis and reduced survival. Understanding the mechanism of Treg elevation is critical for the development of new approaches aiming to modulate the frequency and function of Tregs to enhance the efficacy of cancer immune-based therapies. Areas covered in this review: This review focuses on current knowledge concerning Tregs in cancer and discusses putative mechanisms which underlie the expansion of Tregs in cancer patients. Additionally, we review current strategies to deplete/suppress Treg activity, the limitations of these strategies and future perspective for improving their efficacy. What the reader will gain: An insight of the current aspects concerning Treg subsets in cancer and an overview of recent advances in the identification of Treg-specific markers, in addition to the potential strategies to target Tregs for enhancing antitumor immunity. Take home message: Mechanisms by which Treg functions modulate the immune response to tumors are becoming further understood. However, specific markers to tumor-specific/induced Tregs are yet to be clearly identified, which is a major limitation in optimizing strategies to specifically target Tregs in cancer. Despite this, strategies aimed at modulating Tregs in patients are providing some early encouraging results supporting the overall concept and indicating that further studies are clearly warranted.

O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy

INTRODUCTION Alkylating agents are frequently used in the chemotherapy of many types of cancer. This group of drugs mediates cell death by damaging DNA and therefore, understandably, cellular DNA repair mechanisms can influence both their antitumour efficacy and their dose-limiting toxicities. SOURCES OF DATA This review focuses on the mechanism of action of the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT) and its exploitation in cancer therapy and reviews the current literature. AREAS OF AGREEMENT MGMT can provide resistance to alkylating agents by DNA damage reversal. Inhibition of tumour MGMT by pseudosubstrates to overcome tumour resistance is under clinical evaluation. In addition, MGMT overexpression in haematopoietic stem cells has been shown in animal models to protect normal cells against the myelosuppressive effects of chemotherapy: this strategy has also entered clinical trials. AREAS OF CONTROVERSY MGMT inhibitors enhance the myelotoxic effect of O(6)-alkylating drugs and therefore reduce the maximum-tolerated dose of these agents. Retroviral vectors used for chemoprotective gene therapy are associated with insertional mutagenesis and leukaemia development. GROWING POINTS The results of ongoing preclinical and clinical research involving various aspects of MGMT modulation should provide new prospects for the treatment of glioma, melanoma and other cancer types. AREAS TIMELY FOR DEVELOPING RESEARCH Tissue- and tumour-specific approaches to the modulation of MGMT together with other DNA repair functions and in combination with immuno- or radiotherapy are promising strategies to improve alkylating agent therapy.

Building Better Chimeric Antigen Receptors for Adoptive T Cell Therapy

The last few years have seen the transfer of two decades of research into Chimeric Antigen Receptors (CARs) into clinical trials. Despite this extensive research, there is still a great deal of debate into the optimal design strategy for these, primarily, anti-cancer entities. The archetypal CAR consists of a single-chain antibody fragment, specific to a tumour-associated antigen, fused to a component of the T-cell receptor complex (typically CD3zeta) which on antigen binding primes the engrafted T-cell for anti-tumour activity. The modular nature of these artificial receptors has enabled researchers to modify aspects of their structure, including the extracellular spacer, transmembrane and cytoplasmic domain, to achieve laboratory defined optimal activity. Despite this there is no consensus on the optimal structure, a problem exacerbated by conflicting results using identical receptors. In this review, we provide a structural overview of CAR development and highlight areas that require further refinement. We also attempt to identify possible reasons for conflicting results in the hope that this information will inspire future rational design strategies for optimal tumour targeting using CARs.

The optimal antigen response of chimeric antigen receptors harboring the CD3zeta transmembrane domain is dependent upon incorporation of the receptor into the endogenous TCR/CD3 complex.

Chimeric Ag receptors (CARs) expressed in T cells permit the redirected lysis of tumor cells in an MHC-unrestricted manner. In the Jurkat T cell model system, expression of a carcinoembryonic Ag-specific CD3ζ CAR (MFEζ) resulted in an increased sensitivity of the transduced Jurkat cell to generate cytokines when stimulated through the endogenous TCR complex. This effect was driven through two key characteristics of the MFEζ CAR: 1) receptor dimerization and 2) the interaction of the CAR with the endogenous TCR complex. Mutations of the CAR transmembrane domain that abrogated these interactions resulted in a reduced functional capacity of the MFEζ CAR to respond to carcinoembryonic Ag protein Ag. Taken together, these results indicate that CARs containing the CD3ζ transmembrane domain can form a complex with the endogenous TCR that may be beneficial for optimal T cell activation. This observation has potential implications for the future design of CARs for cancer therapy.

Primary polyclonal human T lymphocytes targeted to carcino-embryonic antigens and neural cell adhesion molecule tumor antigens by CD3zeta-based chimeric immune receptors.

Antigen-specific T lymphocytes are attractive as potential anticancer agents. The generation of large numbers of antigen-specific T cells is possible through the use of gene therapy to express targeting receptors on the T lymphocyte. Activated T lymphocytes were transduced to express carcino-embryonic antigen or neural cell adhesion molecule targeted CD3ζ chimeric immune receptors. The chimeric receptors were expressed as homodimers and also as heterodimers with the native CD3ζ. T lymphocyte populations were expanded in the absence of selection for the modified cells and were shown to produce cytokines when cultured in the presence of immobilized purified protein antigen. These lymphocytes also responded by cytokine production and cytolytic activity when challenged with tumor-cell lines expressing the antigen recognized by the chimeric immune receptor. The cytolytic activity appears to be largely perforin mediated. Furthermore, soluble carcino-embryonic antigen did not interfere with the functional activity of the carcino-embryonic antigen-targeted lymphocytes. Long-term (5-day) stimulation of the modified lymphocytes by protein antigen resulted in reduced viability similar to that induced by anti-CD3 antibodies alone. Viability was improved by a costimulatory signal indicating that such signals may be vital in the maintenance of long-term functional activity of receptor modified T lymphocytes.

Functional co-expression of CYP2D6 and human NADPH-cytochrome P450 reductase in escherichia coli

The polymorphic human CYP2D6 has been co-expressed with human NADPH-cytochrome P450 oxidoreductase in Escherichia coli in order to generate a functional recombinant monooxygenase system for the study of xenobiotic metabolism. The two cDNAs were co-expressed from separate, compatible plasmids with different antibiotic selection markers. The CYP2D6 could be detected in bacterial cells at levels up to 700 nmol I-1 culture by Fe(2+)-CO versus Fe2+ difference spectroscopy, exhibiting the characteristic absorbance peak at 450 nm. Immunoblotting demonstrated the presence of both proteins in bacterial membranes, where they were expressed at levels significantly higher than those found in human liver microsomes. Membrane content was 150-200 pmol CYP2D6 (determined spectrally) and 100-230 pmol CYP-reductase (determined enzymatically) per mg protein. Critically, the two co-expressed proteins were able to couple to form a NADPH-dependent monooxygenase which metabolized the CYP2D6 substrate bufuralol (Vmax 3.30 nmol min-1 mg-1 protein; K(m) 11.1 microM) in isolated membrane fractions. This K(m) value was similar to the K(m) determined in human liver microsomes. Activity could be inhibited by the specific inhibitor quinidine. Of greater significance however, was the finding that intact E. coli cells, even in the absence of exogenous NADPH, were able to metabolize bufuralol at rates almost as high as those measured in membranes (4.6 +/- 0.4 min-1 versus 5.7 +/- 0.2 min-1 at 50 microM substrate). Such recombinant strains will greatly facilitate the molecular characterization of allelic variants of cytochrome P450 isoenzymes.

Targeted immunotherapy of cancer with CAR T cells: achievements and challenges

The adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a relatively new but promising approach in the field of cancer immunotherapy. This therapeutic strategy is based on the genetic reprogramming of T cells with an artificial immune receptor that redirects them against targets on malignant cells and enables their destruction by exerting T cell effector functions. There has been an explosion of interest in the use of CAR T cells as an immunotherapy for cancer. In the pre-clinical setting, there has been a considerable focus upon optimizing the structural and signaling potency of the CAR while advances in bio-processing technology now mean that the clinical testing of these gene-modified T cells has become a reality. This review will summarize the concept of CAR-based immunotherapy and recent clinical trial activity and will further discuss some of the likely future challenges facing CAR-modified T cell therapies.

Retroviral transduction of human peripheral blood lymphocytes with bcl-x(L) promotes in vitro lymphocyte survival in pro-apoptotic conditions.

The prolonged in vivo survival of genetically modified effector cells is crucial to the success of any (gene-modified) adoptive cellular immunotherapy approach. In cancer clinical trials to date, however, the detection of surviving circulating gene-modified T cells has required highly sensitive techniques. In vitro studies of T cell co-stimulation have shown that up-regulation of the anti-apoptosis gene Bcl-XL by ligation of CD28 promotes T cell survival, but not proliferation. Here we have investigated the ability to modulate resistance to apoptosis and improve cell survival by transducing human peripheral blood lymphocytes using a retroviral vector that expresses Bcl-XL. We show that Jurkat cells transduced with Bcl-XL retrovirus were partially resistant to Fas (CD95) antibody-induced apoptosis. Subsequent in vitro assays with transduced primary human lymphocytes demonstrates that over-expression of Bcl-XL promotes the survival of lymphocytes cultured in the absence of interleukin-2. Activation-induced apoptosis with anti-CD3ɛ antibody, OKT3 is also modulated. Furthermore, Bcl-XL over-expression in human lymphocytes delays the onset of apoptosis induced by long-term co-culture with tumour cell lines. Despite this improved in vitro survival, in a preliminary experiment to assess safety, no signs of malignancy or autoimmunity were observed in NOD/SCID mice injected with Bcl-XL transduced lymphocytes. These results indicate that expression of Bcl-XL in lymphocyte therapy either alone or in conjunction with an additional therapeutic gene could enhance persistence of cells in vivo thereby potentially improving the clinical outcome of adoptive cellular therapy.