Sharon Lam

Infusion of donor-derived CD19-redirected virus-specific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study

Autologous T cells expressing a CD19-specific chimeric antigen receptor (CD19.CAR) are active against B-cell malignancies, but it is unknown whether allogeneic CD19.CAR T cells are safe or effective. After allogeneic hematopoietic stem cell transplantation (HSCT), infused donor-derived virus-specific T cells (VSTs) expand in vivo, persist long term, and display antiviral activity without inducing graft-vs-host disease; therefore, we determined whether donor VSTs, engineered to express CD19.CAR, retained the characteristics of nonmanipulated allogeneic VSTs while gaining antitumor activity. We treated 8 patients with allogeneic (donor-derived) CD19.CAR-VSTs 3 months to 13 years after HSCT. There were no infusion-related toxicities. VSTs persisted for a median of 8 weeks in blood and up to 9 weeks at disease sites. Objective antitumor activity was evident in 2 of 6 patients with relapsed disease during the period of CD19.CAR-VST persistence, whereas 2 patients who received cells while in remission remain disease free. In 2 of 3 patients with viral reactivation, donor CD19.CAR-VSTs expanded concomitantly with VSTs. Hence CD19.CAR-VSTs display antitumor activity and, because their number may be increased in the presence of viral stimuli, earlier treatment post-HSCT (when lymphodepletion is greater and the incidence of viral infection is higher) or planned vaccination with viral antigens may enhance disease control.

Expanded Cytotoxic T-cell Lymphocytes Target the Latent HIV Reservoir

Enhanced human immunodeficiency virus (HIV)-specific immunity may be required for HIV eradication. Administration of autologous, ex vivo expanded, virus-specific, cytotoxic T-lymphocytes derived from HIV-infected patients on suppressive antiretroviral therapy (HXTCs) are a powerful tool for proof-of-concept studies. Broadly specific, polyclonal HXTCs resulting from ex vivo expansion demonstrated improved control of autologous reservoir virus compared to bulk CD8(+) T cells in viral inhibition assays. Furthermore, patient-derived HXTCs were able to clear latently infected autologous resting CD4(+) T cells following exposure to the latency-reversing agent, vorinostat. HXTCs will be ideal reagents to administer with precise control in future in vivo studies in combination with latency-reversing agents.

Expanding cytotoxic T lymphocytes from umbilical cord blood that target cytomegalovirus, Epstein-Barr virus, and adenovirus.

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection. We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective. However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. In an effort to better mimic the in vivo priming conditions of naïve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus. At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15. At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. From 50x10(6) CB mononuclear cells we are able to generate upwards of 150 x 10(6) virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation. These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

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.

Functionally Active HIV-Specific T Cells that Target Gag and Nef Can Be Expanded from Virus-Naïve Donors and Target a Range of Viral Epitopes: Implications for a Cure Strategy after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) can potentially cure human immunodeficiency virus (HIV) by eliminating infected recipient cells, particularly in the context of technologies that may confer HIV resistance to these stem cells. But, to date, the Berlin patient remains the only case of HIV cure despite multiple attempts to eradicate infection with HSCT. One approach to improve this is to administer virus-specific T cells, a strategy that has proven success in preventing other infections after transplantation. Although we have reported that broadly HIV-specific T cells can be expanded from HIV+ patients, allogeneic transplantations only contain virus-naïve T cells. Modifying this approach for the allogeneic setting requires a robust, reproducible platform that can expand HIV-specific cells from the naïve pool. Hence, we hypothesized that HIV-specific T cells could be primed ex vivo from seronegative individuals to effectively target HIV. Here, we show that ex vivo-primed and expanded HIV-specific T cells released IFNγ in response to HIV antigens and that these cells have enhanced ability to suppress replication in vitro. This is the first demonstration of ex vivo priming and expansion of functional, multi-HIV antigen-specific T cells from HIV-negative donors, which has implications for use of allogeneic HSCT as a functional HIV cure.

763. HIV-Specific T Cells Can Be Expanded from Virus-Naive Donors to Target a Range of Viral Epitopes: Implications for a Cure Strategy After Allogeneic HSCT

Background: Adoptive T cell therapy has been successful in boosting viral-specific immunity post-hematopoietic stem cell transplant (HSCT), preventing viral rebound of CMV and EBV. However, the therapeutic use of T cells to boost HIV-specific T cell immunity in HIV+ patients has been met with limited success. Despite multiple attempts to eradicate HIV infection with allogeneic HSCT, the Berlin patient remains the only case of functional HIV cure. Previous infusions of HIV-specific T cells have resulted in immune escape from single epitope specificity and limited persistence of the T cell product. Our approach to address these limitations is to expand HIV-specific T cells derived from virus-naive donors including umbilical cord blood, employing a non-HLA restricted approach for HIV+ patients receiving allogeneic HSCT for HIV-associated hematologic malignancies. Design: We have developed a robust, reproducible platform that can expand HIV-specific T cells (HXTCs) from the naive pool in the allogeneic setting. Peripheral blood mononuclear cells isolated from virus-naive donors are used to generate dendritic cells and T cells. T cells are stimulated with antigen presenting cells pulsed with HIV-pepmix and a combination of cytokines that promote proliferation and differentiation. T cells were tested for: (1) specificity against HIV antigens and individual peptides, (2) pro-inflammatory cytokine secretion in response to stimulation with HIV peptides, and (3) ability to suppress HIV replication in vitro. Results: We successfully expanded (75.705 mean fold expansion) HXTCs recognizing HIV antigens from virus naive donors. IFNg ELISPOT showed HXTCs (n=8) were specific against Gag (mean=331.25 SFC/1e5 cells) and Nef (mean=242.63 SFC/1e5 cells) vs Irrelevant (mean=13 SFC/1e5 cells). HXTCs produced significantly pro-inflammatory responses (p less than 0.05) to stimulation by gag/nef, as determined by levels of TNF-alpha, IL-2, IL-6, IL-8, and perforin (n=3). Importantly, HXTCs (n=4) were able to suppress HIV replication more than non-specific CD8+ T cells when co-cultured with autologous CD4+ T cells infected with HIV SF162 (HXTC 78.62% viral suppression compared to CD8+ T cell 34.19% viral suppression). HXTCs showed both HLA Class I or II specificity for individual HIV epitopes, as determined by HLA blocking and IFNg ELISPOT. Conclusion: This is the first report demonstrating generation of functional, multi-HIV antigen specific T-cells from HIV-negative donors, which has implications for using allogeneic HSCT as a functional HIV cure. The low frequency of circulating HXTCs post-infusion suggests these HXTCs could have a significant effect on preventing viral rebound. The generation of HXTCs from cord blood could provide a further advantage to increase the donor pool.