Bilal Omer

Off-the-Shelf Virus-Specific T Cells to Treat BK Virus, Human Herpesvirus 6, Cytomegalovirus, Epstein-Barr Virus, and Adenovirus Infections After Allogeneic Hematopoietic Stem-Cell Transplantation.

Purpose Improvement of cure rates for patients treated with allogeneic hematopoietic stem-cell transplantation (HSCT) will require efforts to decrease treatment-related mortality from severe viral infections. Adoptively transferred virus-specific T cells (VSTs) generated from eligible, third-party donors could provide broad antiviral protection to recipients of HSCT as an immediately available off-the-shelf product. Patient and Methods We generated a bank of VSTs that recognized five common viral pathogens: Epstein-Barr virus (EBV), adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), and human herpesvirus 6 (HHV-6). The VSTs were administered to 38 patients with 45 infections in a phase II clinical trial. Results A single infusion produced a cumulative complete or partial response rate of 92% (95% CI, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKV (n = 16), 94% for CMV (n = 17), 71% for AdV (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). Clinical benefit was achieved in 31 patients treated for one infection and in seven patients treated for multiple coincident infections. Thirteen of 14 patients treated for BKV-associated hemorrhagic cystitis experienced complete resolution of gross hematuria by week 6. Infusions were safe, and only two occurrences of de novo graft-versus host disease (grade 1) were observed. VST tracking by epitope profiling revealed persistence of functional VSTs of third-party origin for up to 12 weeks. Conclusion The use of banked VSTs is a feasible, safe, and effective approach to treat severe and drug-refractory infections after HSCT, including infections from two viruses (BKV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the VSTs ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.

Treatment of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type Lectin-like Molecule 1

The successful immunotherapy of acute myeloid leukemia (AML) has been hampered because most potential antigenic targets are shared with normal hematopoietic stem cells (HSCs), increasing the risk of sustained and severe hematopoietic toxicity following treatment. C-type lectin-like molecule 1xa0(CLL-1) is a membrane glycoprotein expressed by >80% of AML but is absent on normal HSCs. Here we describe the development and evaluation of CLL-1-specific chimeric antigen receptor Txa0cells (CLL-1.CAR-Ts) and we demonstrate their specificxa0activity against CLL-1+ AML cell lines as well as primary AML patient samples inxa0vitro. CLL-1.CAR-Ts selectively reduced leukemic colony formation in primary AML patient peripheral blood mononuclear cells compared to control Txa0cells. In a human xenograft mouse model, CLL-1.CAR-Ts mediated anti-leukemic activity against disseminated AML and significantly extended survival. By contrast, the colony formationxa0of normal progenitor cells remained intact following CLL-1.CAR-T treatment. Although CLL-1.CAR-Ts are cytotoxic to mature normal myeloid cells, the selective sparing of normal hematopoietic progenitor cells should allow full myeloid recovery once CLL-1.CAR-T activity terminates. To enable elective ablation of the CAR-T, we therefore introduced the inducible caspase-9 suicide gene system and we show that exposure to the activating drug rapidly induced a controlled decrease of unwanted CLL-1.CAR-T activity against mature normal myeloid cells.

Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer.Significance: The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. Cancer Discov; 7(11); 1238-47. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

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.

Wormwood (Artemisia absinthium) for poorly responsive early-stage IgA nephropathy: a pilot uncontrolled trial.

BACKGROUNDnInhibition of the renin-angiotensin system is a widely accepted approach to treat immunoglobulin A (IgA) nephropathy, whereas the role of fish oils as a supplement is controversial. Tumor necrosis factor α (TNF-α) is considered to be involved in the pathophysiologic process of this disorder. Recent in vitro and clinical observations that wormwood can decrease TNF-α levels has led us to investigate the effect of wormwood as a supplement in patients with IgA nephropathy.nnnSTUDY DESIGNnPilot uncontrolled trial.nnnSETTING & PARTICIPANTSn10 patients with biopsy-proven IgA nephropathy, normal kidney function, and a history of at least 3 months of proteinuria with protein excretion > 500 mg/d and < 3,500 mg/d despite ongoing dual renin-angiotensin system blockade.nnnINTERVENTIONnThe selected patients were given supplements of 1.8 g/d of thujone-free wormwood preparation for 6 months without discontinuing their renin-angiotensin system blockade.nnnOUTCOMESnProteinuria and blood pressure after intervention compared with baseline values.nnnMEASUREMENTSnMonthly assessment of urine protein-creatinine ratio and blood pressure during the observation period.nnnRESULTSnUrine protein-creatinine ratio decreased significantly from 2,340 ± 530 to 315 ± 200 mg/g at the end of the supplementation period (P < 0.001) and was stable during the supplement-free follow-up of another 6 months. Estimated glomerular filtration rate and endogenous creatinine clearance were unchanged during the entire study period. There was a moderate, but significant, decrease (P < 0.002) in mean arterial blood pressure.nnnLIMITATIONSnOpen uncontrolled trial including a small number of patients.nnnCONCLUSIONSnThujone-free wormwood with its favorable safety profile can be an alternative supplement to manage proteinuria in patients with IgA nephropathy.

Comprehensive Approach for Identifying the T Cell Subset Origin of CD3 and CD28 Antibody–Activated Chimeric Antigen Receptor–Modified T Cells

The outcome of therapy with chimeric Ag receptor (CAR)-modified T cells is strongly influenced by the subset origin of the infused T cells. However, because polyclonally activated T cells acquire a largely CD45RO+CCR7− effector memory phenotype after expansion, regardless of subset origin, it is impossible to know which subsets contribute to the final T cell product. To determine the contribution of naive T cell, memory stem T cell, central memory T cell, effector memory T cell, and terminally differentiated effector T cell populations to the CD3 and CD28–activated CAR-modified T cells that we use for therapy, we followed the fate and function of individually sorted CAR-modified T cell subsets after activation with CD3 and CD28 Abs (CD3/28), transduction and culture alone, or after reconstitution into the relevant subset-depleted population. We show that all subsets are sensitive to CAR transduction, and each developed a distinct T cell functional profile during culture. Naive-derived T cells showed the greatest rate of proliferation but had more limited effector functions and reduced killing compared with memory-derived populations. When cultured in the presence of memory T cells, naive-derived T cells show increased differentiation, reduced effector cytokine production, and a reduced reproliferative response to CAR stimulation. CD3/28-activated T cells expanded in IL-7 and IL-15 produced greater expansion of memory stem T cells and central memory T cell–derived T cells compared with IL-2. Our strategy provides a powerful tool to elucidate the characteristics of CAR-modified T cells, regardless of the protocol used for expansion, reveals the functional properties of each expanded T cell subset, and paves the way for a more detailed evaluation of the effects of manufacturing changes on the subset contribution to in vitro–expanded T cells.

Abstract IA22: Immunotherapy with virus-specific T cells

Epstein-Barr virus (EBV) reactivation post allogeneic hematopoietic stem cell transplantation (HSCT) can lead to the outgrowth of EBV-infected B cells and the development of post-transplant lymphoproliferative disease (EBV-PTLD). Since 1993 our group has used adoptive transfer of in vitro expanded EBV-specific T cells as a means to prevent or treat these EBV-driven lymphomas. In a series of Phase I and II clinical trials we have assessed the safety and clinical benefit associated with these transferred cells in allogeneic HSCT recipients. The T cell products infused were generated using 3 different manufacturing methodologies - [(i) EBV-transformed lymphoblastoid cell lines (EBV-LCLs) (~12 weeks manufacturing time), (ii) plasmid-nucloefected dendritic cells (DCs) (17 day manufacturing) and (iii) direct stimulation of PBMCs using overlapping peptide libraries (10 day manufacturing)] and were administered to either prevent (n=162) or treat (n=47) EBV reactivation/disease in a total of 209 allogeneic HSCT recipients ranging in age from 6 months to 63 years. Of 162 patients infused prophylactically only 1 (0.6%) developed EBV-PTLD, which occurred following the administration of steroids 3 weeks-post VST infusion. However, this patient responded to a second VST infusion after the steroids. 31 of 36 (86%) patients with elevated viral load (n=21) or biopsy-proven/probable LPD (n=15) achieved durable complete remissions and the infused cells persisted long term as demonstrated in 26 patients who received gene-marked VSTs that were detectable for up to 9 years post-infusion. To extend the approach to additional viruses we initially developed methodology for using genetically modified antigen presenting cells approach to generate VSTs from donor peripheral blood that target CMV, EBV and adenovirus and showed that adoptively transferred donor-derived VSTs can reconstitute antiviral immunity to all three viruses and effectively treat established infections. However, the time taken to prepare patient-specific products and the lack of virus-specific memory T cells in cord blood and seronegative donors restricts application. More recently we have evaluated whether T-cell lines manufactured using overlapping peptide pools and extended the specificity to include HHV6 and BK. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term. Another means of avoiding growing CTLs for individual patients is to bank lines that are then available as an off the shelf product of most closely HLA-matched allogeneic cytotoxic T lymphocyte lines. We evaluated this strategy in a multicenter study through the NHLBI Specialized Centers for Cell-Based Therapy (SCCT) program in HSCT recipients who had viral reactivation or infection refractory to standard therapy. The overall cumulative incidence of first CR/PR in 50 patients based on viral load by day 42 was 74.0% (73.9% for CMV, 66.7% for EBV and 77.8% for adenovirus). In a follow up study using the peptide induced VSTs that recognize 5 viruses, based on viral load measurements by quantitative PCR a single VST infusion successfully controlled active infections in 19/21 evaluable patients. These results demonstrate the feasibility and safety of 3rd party multivirus-directed VSTs, generated by direct stimulation of PBMCs with synthetic peptides and administered as an off the shelf product. Citation Format: Helen E. Heslop, Ifigenia Tzannou, Bilal Omer, Malcolm K. Brenner, Ann M. Leen, Cliona M. Rooney. Immunotherapy with virus-specific T cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr IA22.