Minhtran Ngo

Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer

Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a randomized phase 2 dose-finding trial (n = 30). Radiologists infused low- or high-dose JX-594 into liver tumors (days 1, 15 and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P = 0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose-related survival in individuals with HCC.

Accelerated production of antigen-specific T cells for preclinical and clinical applications using gas-permeable rapid expansion cultureware (G-Rex).

The clinical manufacture of antigen-specific cytotoxic T lymphocytes (CTLs) for adoptive immunotherapy is limited by the complexity and time required to produce large numbers with the desired function and specificity. The culture conditions required are rigorous, and in some cases only achieved in 2-cm2 wells in which cell growth is limited by gas exchange, nutrients, and waste accumulation. Bioreactors developed to overcome these issues tend to be complex, expensive, and not always conducive to CTL growth. We observed that antigen-specific CTLs undergo 7 to 10 divisions poststimulation. However, the expected CTL numbers were achieved only in the first week of culture. By recreating the culture conditions present during this first week—low frequency of antigen-specific T cells and high frequency of feeder cells—we were able to increase CTL expansion to expected levels that could be sustained for several weeks without affecting phenotype or function. However, the number of 24-well plates needed was excessive and cultures required frequent media changes, increasing complexity and manufacturing costs. Therefore, we evaluated novel gas-permeable culture devices (G-Rex) with a silicone membrane at the base allowing gas exchange to occur uninhibited by the depth of the medium above. This system effectively supports the expansion of CTL and actually increases output by up to 20-fold while decreasing the required technician time. Importantly, this amplified cell expansion is not because of more cell divisions but because of reduced cell death. This bioprocess optimization increased T-cell output while decreasing the complexity and cost of CTL manufacture, making cell therapy more accessible.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications

BACKGROUND AIMS Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. METHODS We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). RESULTS Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. CONCLUSIONS We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.

Phase 1 Study of Intratumoral Pexa-Vec (JX-594), an Oncolytic and Immunotherapeutic Vaccinia Virus, in Pediatric Cancer Patients

Pexa-Vec (pexastimogene devacirepvec, JX-594) is an oncolytic and immunotherapeutic vaccinia virus designed to destroy cancer cells through viral lysis and induction of granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven tumor-specific immunity. Pexa-Vec has undergone phase 1 and 2 testing alone and in combination with other therapies in adult patients, via both intratumoral and intravenous administration routes. We sought to determine the safety of intratumoral administration in pediatric patients. In a dose-escalation study using either 10(6) or 10(7) plaque-forming units per kilogram, we performed one-time injections in up to three tumor sites in five pediatric patients and two injections in one patient. Ages at study entry ranged from 4 to 21 years, and their cancer diagnoses included neuroblastoma, hepatocellular carcinoma, and Ewing sarcoma. All toxicities were ≤ grade 3. The most common side effects were sinus fever and sinus tachycardia. All three patients at the higher dose developed asymptomatic grade 1 treatment-related skin pustules that resolved within 3-4 weeks. One patient showed imaging evidence suggestive of antitumor biological activity. The two patients tested for cellular immunoreactivity to vaccinia antigens showed strong responses. Overall, our study suggests Pexa-Vec is safe to administer to pediatric patients by intratumoral administration and could be studied further in this patient population.

Broadly-specific Cytotoxic T Cells Targeting Multiple HIV Antigens Are Expanded From HIV+ Patients: Implications for Immunotherapy

Antiretroviral therapy (ART) is unable to eradicate human immunodeficiency virus type 1 (HIV-1) infection. Therefore, there is an urgent need to develop novel therapies for this disease to augment anti-HIV immunity. T cell therapy is appealing in this regard as T cells have the ability to proliferate, migrate, and their antigen specificity reduces the possibility of off-target effects. However, past human studies in HIV-1 infection that administered T cells with limited specificity failed to provide ART-independent, long-term viral control. In this study, we sought to expand functional, broadly-specific cytotoxic T cells (HXTCs) from HIV-infected patients on suppressive ART as a first step toward developing cellular therapies for implementation in future HIV eradication protocols. Blood samples from seven HIV+ patients on suppressive ART were used to derive HXTCs. Multiantigen specificity was achieved by coculturing T cells with antigen-presenting cells pulsed with peptides representing Gag, Pol, and Nef. All but two lines were multispecific for all three antigens. HXTCs demonstrated efficacy as shown by release of proinflammatory cytokines, specific lysis of antigen-pulsed targets, and the ability to suppress HIV replication in vitro. In conclusion, we are able to generate broadly-specific cytotoxic T cell lines that simultaneously target multiple HIV antigens and show robust antiviral function.

Complementation of antigen-presenting cells to generate T lymphocytes with broad target specificity.

Antigen-specific T cells provide a therapy for cancer that is highly specific, self-replicating, and potentially devoid of toxicity. Ideally, tumor-specific T cells should recognize multiple epitopes on multiple antigens to prevent tumor immune escape. However the large-scale expansion of such broad-spectrum T cells has been limited by the availability of potent autologous antigen-presenting cells that can present antigens on the polymorphic array of each patient’s HLA allotype. We evaluated a novel antigen-presenting complex (KATpx) in which antigens in the form of peptide libraries can be presented by autologous activated T cells, whereas costimulation is complemented in trans by an HLA-negative K562 cell line genetically modified to express CD80, CD83, CD86, and 4-1BBL (K562cs). The additional costimulation provided by K562cs significantly enhanced T-cell expansion in culture over autologous activated T cells alone while maintaining antigen specificity. We validated this antigen-presenting system by generating Epstein-Barr virus (EBV) antigen-specific T cells from healthy donors and from patients with EBV-positive malignancies including nasopharyngeal carcinoma and multiply relapsed EBV-positive lymphoma. These T cells were specific for EBNA1, LMP1, and LMP2, the viral antigens expressed in these type 2 latency EBV-associated malignancies. The KATpx system consistently activated and expanded antigen-specific T cells both from healthy donors and from 5 of 6 patients with lymphoma and 6 of 6 with nasopharyngeal carcinoma, while simplifying the process for generating APCs by eliminating the need for live virus (EBV) or viral vectors to force expression of transgenic EBV antigens. Hence, KATpx provides a robust, reliable, and scalable process to expand tumor-directed T cells for the treatment of virus-associated cancers.

Vaccination Targeting Native Receptors to Enhance the Function and Proliferation of Chimeric Antigen Receptor (CAR)-Modified T Cells

Purpose: The multiple mechanisms used by solid tumors to suppress tumor-specific immune responses are a major barrier to the success of adoptively transferred tumor-specific T cells. As viruses induce potent innate and adaptive immune responses, we hypothesized that the immunogenicity of viruses could be harnessed for the treatment of solid tumors if virus-specific T cells (VST) were modified with tumor-specific chimeric antigen receptors (CAR). We tested this hypothesis using VZV-specific T cells (VZVST) expressing a CAR for GD2, a disialoganglioside expressed on neuroblastoma and certain other tumors, so that the live-attenuated VZV vaccine could be used for in vivo stimulation. Experimental Design: We generated GMP-compliant, GD2.CAR-modified VZVSTs from healthy donors and cancer patients by stimulation of peripheral blood mononuclear cells with overlapping peptide libraries spanning selected VZV antigens, then tested their ability to recognize and kill GD2- and VZV antigen–expressing target cells. Results: Our choice of VZV antigens was validated by the observation that T cells specific for these antigens expanded in vivo after VZV vaccination. VZVSTs secreted cytokines in response to VZV antigens, killed VZV-infected target cells and limited infectious virus spread in autologous fibroblasts. However, while GD2.CAR–modified VZVSTs killed neuroblastoma cell lines on their first encounter, they failed to control tumor cells in subsequent cocultures. Despite this CAR-specific dysfunction, CAR-VZVSTs retained functional specificity for VZV antigens via their TCRs and GD2.CAR function was partially rescued by stimulation through the TCR or exposure to dendritic cell supernatants. Conclusions: Vaccination via the TCR may provide a means to reactivate CAR-T cells rendered dysfunctional by the tumor microenvironment (NCT01953900). Clin Cancer Res; 23(14); 3499–509. ©2017 AACR.

722. Overcoming EBV Tumor Specific T-Cell Anergy in Rapidly-Generated EBVST-Cells for Adoptive Transfer Therapy

Up to 30% of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express the viral latency proteins EBNA1, LMP1, LMP2 and BARF1; a type 2 latency pattern of EBV gene expression. We previously reported that EBV-specific T-cells (EBVSTs) directed to LMP1 and LMP2 expanded from the blood of lymphoma patients produced complete tumor responses in over 50% of patients. To shorten and simplify the EBVST production time, we removed viral-vector components from our manufacturing process, replacing these components with dendritic cells pulsed with peptide libraries (pepmixes) spanning type 2 latency antigens. Responder T-cells are then expanded by restimulation with pepmix-pulsed, autologous, activated T-cells and HLA-negative K562 costimulatory cells in the presence of IL-4 and IL-7. Despite enhanced antigen specificity from EBVSTs generated from healthy donors, we were unable to consistently generate patient-derived EBVSTs with significant activity against the Type 2 antigens. We hypothesized that patient T-cells were anergized by their immunosuppressive tumors. IL-15 has been shown to rescue tolerant or anergized CD8+ T-cells and we found that substitution of IL-15 (5ng/mL) for IL-4 (in combination with IL-7 (10ng/mL)) improved CD4+ and CD8+ T-cells’ specificity for the EBV Type 2 latency antigens by up to 10-fold. By increasing the concentration of IL-15, we achieved significantly higher fold expansion (3 fold mean increase in absolute EBVST number) and further enhanced specificity for type 2 latency EBV antigens (high vs. low IL-15 concentration: EBNA1: 216±273 vs. 29±48, LMP1: 145±253 vs. 44±63, LMP2: 636±548 vs. 106±74 and BARF1: 80±100 vs. 29±22; SFC/105 cells; n=5). There was no increase in the absolute numbers of NK-cells despite high doses of IL-15. Enhanced antigen-specificity correlated with increased cytotoxic effect, with an increase in mean % specific lysis of EBV pepmix-pulsed autologous activated T-cell targets from 5 to 66% at a 20:1 E:T ratio in the low vs. high dose IL-15 conditions (n=5). We have infused EBVSTs manufactured using all three conditions into 17 patients with multiply-relapsed, EBV-positive lymphoma as adjuvant therapy after stem cell transplantation or chemotherapy in 8 patients and as treatment for disease in 9 patients. Of patients in remission at the time of infusion, two with IL-4/7-grown EBVSTs and 6 with IL-15/7 grown EBVSTs remain in remission. Of patients with disease at the time of infusion, one receiving IL-4/7-grown EBVSTs had stable disease and 3 had progressive disease, while of 5 patients with IL-15/7-grown EBVSTs, one had stable disease, one had a partial response, one had a complete response and two are too early to assess. We will continue to modify our manufacturing process in an attempt to further increase the specificity and function of EBVSTs from patients with relapsed or refractory lymphoma.

Increasing the purity, potency and specificity of ebv-specific T cells to improve the treatment of EBV-positive lymphoma

Meeting abstracts Up to ~40 % of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express type 2 viral latency proteins (T2LPs) EBNA-1, LMP-1, LMP-2 and BARF-1. EBV specific T cells (EBVSTs) can be generated from the blood of EBV+ individuals, but are usually

Abstract IA21: Using viruses to advance T cell therapy for malignancy.

Virus-specific T cells effectively prevent and treat viral diseases after stem cell transplantation and have produced complete remissions (CRs) in patients with EBV-positive malignancy outside the transplant setting. The success of EBV-specific T cells is thought to be due to the strong viral antigens expressed by these tumors and the vaccine effect of endogenous EBV. We tested if this endogenous viral vaccine effect could be exploited for the treatment of non-viral malignancy by engrafting chimeric antigen receptors specific for the disialoganglioside,GD2 (GD2.CAR) on EBV-specific T cells and comparing their in vivo expansion with that of GD2.CAR-engrafted CD3-activated T cells (ATCs) in patients with relapsed neuroblastoma. Transgenic EBV-CTLs were detected at higher levels than GD2.CAR-ATCs and 6 of 8 patients without disease at infusion remain tumor-free, while 6 of 11 with disease had tumor responses including 3 CRs. However the expected increase in frequency of EBV-specific T cells in patient blood was not observed, perhaps because EBV was well-controlled in these patients.1,2 To improve this vaccine effect, we looked to VZV, for which a potent, but safe commercially-available vaccine exists. We identified five VZV antigens that we predicted would be presented to the immune system early after infection, generated overlapping peptide libraries (pepmixes) for each and used them to stimulate T- cells from both naturally-infected and vaccinated individuals. T cells expanded in vitro and were antigen-specific as measured on day 9 using γ-IFN ELIspot analysis. T cells specific for all five antigens increased in frequency in response to VZV vaccination. VZV-specific T cells transduced with a GD2.CAR retained their ability to respond to VZV antigens and acquired the ability to eliminate GD2+ tumor cells. Ultimately, we plan to combine GD2.CAR-engrafted, VZV-specific T cells with VZV vaccination for the treatment of patients with relapsed GD2+ tumors. References: 1 Pule et al. Nat Med. 2008 Nov;14(11):1264-70 2 Louis et al. Blood. 2011 Dec 1;118(23):6050-6 Citation Format: Miyuki Yanagisawa, Minhtran Ngo, Cliona M. Rooney, Ann Marie Leen, Juan Vera, Helen E. Heslop, Malcolm K. Brenner. Using viruses to advance T cell therapy for malignancy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr IA21.