Tessa Gargett

The inducible caspase-9 suicide gene system as a "safety switch" to limit on-target, off-tumor toxicities of chimeric antigen receptor T cells

Immune modulation has become a central element in many cancer treatments, and T cells genetically engineered to express chimeric antigen receptors (CAR) may provide a new approach to cancer immunotherapy. Autologous CAR T cells that have been re-directed toward tumor-associated antigens (TAA) have shown promising results in phase 1 clinical trials, with some patients undergoing complete tumor regression. However, this T-cell therapy must carefully balance effective T-cell activation, to ensure antitumor activity, with the potential for uncontrolled activation that may produce immunopathology. An inducible Caspase 9 (iCasp9) “safety switch” offers a solution that allows for the removal of inappropriately activated CAR T cells. The induction of iCasp9 depends on the administration of the small molecule dimerizer drug AP1903 and dimerization results in rapid induction of apoptosis in transduced cells, preferentially killing activated cells expressing high levels of transgene. The iCasp9 gene has been incorporated into vectors for use in preclinical studies and demonstrates effective and reliable suicide gene activity in phase 1 clinical trials. A third-generation CAR incorporating iCasp9 re-directs T cells toward the GD2 TAA. GD2 is over-expressed in melanoma and other malignancies of neural crest origin and the safety and activity of these GD2-iCAR T cells will be investigated in CARPETS and other actively recruiting phase 1 trials.

Different cytokine and stimulation conditions influence the expansion and immune phenotype of third-generation chimeric antigen receptor T cells specific for tumor antigen GD2

BACKGROUND AIMS Chimeric antigen receptor (CAR) T cells are a novel immunotherapy for cancer. To achieve anti-tumor efficacy, these cells must survive, expand, and persist after infusion into patients, functions that are reportedly best achieved by cells with a stem or central-memory rather than effector-memory phenotype. We have developed third-generation CAR T cells specific for the tumor-associated antigen GD2 for use in a phase I clinical trial. We investigated the optimal cell culture conditions for CAR T-cell production, and here we describe the relative effects of 3 activation and cytokine conditions on CAR T-cell expansion, effector function and phenotype. METHODS Peripheral blood mononuclear cells were activated by anti-CD3 and anti-CD28 or anti-CD3 and Retronectin. Activated cells were transduced with the CAR-encoding retroviral vector and expanded in either interleukin (IL)-2 or IL-7 and IL-15. Immune phenotype and expansion were tracked throughout the culture, and transduction efficiency, and subsequent GD2-specific effector functions were evaluated by flow cytometry and cytotoxic T lymphocytes assay. RESULTS CD3/Retronectin stimulation with IL-2 resulted in poorer activation, expansion and Th1 cytokine secretion of CAR T cells than CD3/CD28 stimulation with either IL-2 or IL-7 and IL-15. However, CAR T cells cultured in CD3/CD28/IL7/IL-15 and CD3/Retronectin/IL-2 had superior cytotoxic T lymphocyte activity and a more stem-like phenotype. DISCUSSION The combination of CD3 and CD28 with IL-7 and IL-15 gave the best balance of CAR T-cell expansion and potent GD2-specific effector functions while retaining a stem/memory phenotype, and these growth conditions will therefore be used to manufacture CAR T cells for our phase I clinical trial.

BRAF and MEK inhibition variably affect GD2-specific chimeric antigen receptor (CAR) T-cell function in vitro

Cancer immunotherapy has long been used in the treatment of metastatic melanoma, and an anti-CTLA-4 monoclonal antibody treatment has recently been approved by the US Food and Drug Administration. Targeted therapies such as small molecule kinase inhibitors targeting deregulated mitogen-activated protein kinase (MAPK) signaling have markedly improved melanoma control in up to 50% of metastatic disease patients and have likewise been recently approved. Combination therapies for melanoma have been proposed as a way to exploit the high-level but short-term responses associated with kinase inhibitor therapies and the low-level but longer-term responses associated with immunotherapy. Cancer immunotherapy now includes adoptive transfer of autologous tumor-specific chimeric antigen receptor (CAR) T cells and this mode of therapy is a candidate for combination with small molecule drugs. This paper describes CART cells that target GD2-expressing melanoma cells and investigates the effects of approved MAPK pathway-targeted therapies for melanoma [vemurafenib (Vem), dabrafenib (Dab), and trametinib (Tram)] on the viability, activation, proliferation, and cytotoxic T lymphocyte activity of these CAR T cells, as well as on normal peripheral blood mononuclear cells. We report that, although all these drugs lead to inhibition of stimulated T cells at high concentrations in vitro, only Vem inhibited T cells at concentrations equivalent to reported plasma concentrations in treated patients. Although the combination of Dab and Tram also resulted in inhibition of T-cell effector functions at some therapeutic concentrations, Dab itself had little adverse effect on CAR T-cell function. These findings may have implications for novel therapeutic combinations of adoptive CAR T-cell immunotherapy and MAPK pathway inhibitors.

Induction of antigen-positive cell death by the expression of Perforin, but not DTa, from a DNA vaccine enhances the immune response

The failure of traditional protein‐based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti‐viral T cell‐mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross‐presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine‐targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T‐cell responses to the HIV‐1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis‐inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

DNA vaccines encoding membrane‐bound or secreted forms of heat shock protein 70 exhibit improved potency

Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage‐associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane‐bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag‐specific T‐cell responses. Our results demonstrated the ability of membrane‐bound and secreted HSP70 to significantly enhance gag‐specific T‐cell responses and increase the breadth of T‐cell responses to include subdominant epitopes. Membrane‐bound or secreted HSP70 also significantly improved the multifunctionality of HIV‐specific T cells and T‐cell proliferation, which is important for maintaining T‐cell integrity. Most importantly, the inclusion of membrane‐bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.

Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein

The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell‐mediated immune responses. In contrast, the co‐expression of LUC with the rotavirus non‐structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC‐specific humoral and cell‐mediated immunity. On the other hand, co‐expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine‐targeted cells after ID delivery.

GM-CSF signalling blockade and chemotherapeutic agents act in concert to inhibit the function of myeloid-derived suppressor cells in vitro

Immune evasion is a recently defined hallmark of cancer, and immunotherapeutic approaches that stimulate an immune response to tumours are gaining recognition. However tumours may evade the immune response and resist immune‐targeted treatment by promoting an immune‐suppressive environment and stimulating the differentiation or recruitment of immunosuppressive cells. Myeloid‐derived suppressor cells (MDSC) have been identified in a range of cancers and are often associated with tumour progression and poor patient outcomes. Pancreatic cancer in particular supports MDSC differentiation via the secretion of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and MDSC are believed to contribute to the profoundly immune‐suppressive microenvironment present in pancreatic tumours. MDSC‐targeted therapies that deplete or inhibit this cell population have been proposed as a way to shift the balance in favour of a tumour‐clearing immune response. In this study, we have modelled MDSC differentiation and function in vitro and this has provided us with the opportunity to test a range of potential MDSC‐targeted therapies to identify candidates for further investigation. Using in vitro modelling we show here that the combination of GM‐CSF‐signalling blockade and gemcitabine suppresses both the MDSC phenotype and the inhibition of T‐cell function by MDSC.

Loss of long term protection with the inclusion of HIV pol to a DNA vaccine encoding gag.

Traditional vaccine strategies that induce antibody responses have failed to protect against HIV infection in clinical trials, and thus cell-mediated immunity is now an additional criterion. Recent clinical trials that aimed to induce strong T cell responses failed to do so. Therefore, to enhance induction of protective T cell responses, it is crucial that the optimum antigen combination is chosen. Limited research has been performed into the number of antigens selected for an HIV vaccine. This study aimed to compare DNA vaccines encoding either a single HIV antigen or a combination of two antigens, using intradermal vaccination of C57BL/6 mice. Immune assays were performed on splenocytes, and in vivo protection was examined by challenge with a chimeric virus, EcoHIV, able to infect mouse but not human leukocytes, at 10 days (short term) and 60 days (long term) post final vaccination. At 60 days there was significantly lower frequency of induced antigen-specific CD8(+) T cells in the spleens of pCMVgag-pol-vaccinated mice compared with mice which received pCMVgag only. Most importantly, short term viral control of EcoHIV was similar for pCMVgag and pCMVgag-pol-vaccinated mice at day 10, but only the pCMVgag-vaccinated significantly controlled EcoHIV at day 60 compared with pCMV-vaccinated mice, showing that control was reduced with the inclusion of the HIV pol gene.

Encoded novel forms of HSP70 or a cytolytic protein increase DNA vaccine potency

In humans, DNA vaccines have failed to demonstrate the equivalent levels of immunogenicity that were shown in smaller animals. Previous studies have encoded adjuvants, predominantly cytokines, within these vaccines in an attempt to increase antigen-specific immune responses. However, these strategies have lacked breadth of innate immune activation and have led to disappointing results in clinical trials. Damage associated molecular patterns (DAMPs) have been identified as pattern recognition receptor (PRR) agonists. DAMPs can bind to a wide range of PRRs on dendritic cells (DCs) and thus our studies have aimed to utilize this characteristic to act as an adjuvant in a DNA vaccine approach. Specifically, HSP70 has been identified as a DAMP, but has been limited by its lack of accessibility to PRRs in and on DCs. Here, we discuss the promising results achieved with the inclusion of membrane-bound or secreted HSP70 into a DNA vaccine encoding HIV gag as the model immunogen.

Comment on “KB004, a first in class monoclonal antibody targeting the receptor tyrosine kinase EphA3, in patients with advanced hematologic malignancies: Results from a phase 1 study”

The article by Swords et al. [1] is informative in confirming he safety and tolerability of the anti-EphA3 monoclonal antibody mAb) KB004 in refractory hematologic malignancies, including cute Myeloid Leukemia (AML) and myelodysplastic syndromes. pecific target antigens enriched on leukemic cells, including the utative CD34+CD38− leukemic stem cell (LSC) population, but isplaying low to no endogenous expression on normal healthy issues are difficult to identify. We investigated the oncofetal antien EphA3 as a potential leukemia-specific target antigen in highly efractory, poor prognosis haematologic malignancies AML and last crisis Chronic Myeloid Leukemia (BC CML). The authors define EphA3 positivity as >10% nucleated bone arrow cells by immunohistochemistry using an EphA3 antiody (5E11F2). In order to investigate the utility of EphA3 as a eukemia-specific target antigen, we performed a comprehensive ssessment of EphA3 expression in various leukemic cell subpoplations, including the rare, resistant LSC presumed responsible or maintenance of the leukemia. Mononuclear cells from bone arrow (AML; ≥50% myeloblasts, n = 32) or peripheral blood (BC ML; ≥20% blasts, n = 10) were stained with anti-EphA3 mAb IIIA4, hich is the parent molecule for KB004 (1:5000 of 2 mg/mL; a ind gift from Professor Andrew Boyd, QIMR, Australia), and mAb gainst myeloid leukemia antigens (Fig. 1) purchased from BD iosciences, San Jose, CA, Beckman Coulter, Fullerton, CA, or BioLeend, San Diego, CA, USA. Cells (2 × 105) were acquired with the D LSRFortessaTM X-20 (BD Biosciences) and data analysed with lowJo v10.1 (FlowJo, LLC, Ashland, OR, USA). Data is given as edian ± standard deviation. We observed low EphA3 expression in both AML and BC CML eukemic cell subpopulations (Fig. 2). In the AML patient cohort, he median percentage of CD13+CD33+ pan-leukemic EphA3+ AML ells was 0.40 ± 0.65%. The primitive stem cell marker CD34, is a eterminant of poor prognosis in AML and also following allogeneic aematopoietic stem cell transplantation [2]. In our analysis, 44% of he investigated AML samples were CD34+ (presenting with >20% ositive CD34+ cells) and 56% had CD34− blasts.