A dual-luciferase bioluminescence system for the assessment of cellular therapies.

Bioluminescence imaging is a well-established platform for evaluating engineered cell therapies in preclinical studies. However, despite the discovery of new luciferases and substrates, optimal combinations to simultaneously monitor two cell populations remain limited. This makes the functional assessment of cellular therapies cumbersome and expensive, especially in preclinical in vivo models. In this study, we explored the potential of using a green bioluminescence-emitting click beetle luciferase, CBG99, and a red bioluminescence-emitting firefly luciferase mutant, Akaluc, together to simultaneously monitor two cell populations. Using various chimeric antigen receptor T cells and tumor pairings, we demonstrate that these luciferases are suitable for real-time tracking of two cell types using 2D and 3D cultures in vitro and experimental models in vivo. Our data show the broad compatibility of this dual-luciferase (duo-luc) system with multiple bioluminescence detection equipment ranging from benchtop spectrophotometers to live animal imaging systems. Although this study focused on investigating complex CAR T cells and tumor cell interactions, this duo-luc system has potential utility for the simultaneous monitoring of any two cellular components-for example, to unravel the impact of a specific genetic variant on clonal dominance in a mixed population of tumor cells.

Final Outcomes from a Phase 2 Trial of Posoleucel in Allogeneic Hematopoietic Cell Transplant Recipients.

Allogeneic hematopoietic cell transplantation (allo-HCT) recipients are susceptible to viral infections. We conducted a phase 2 trial evaluating the safety and rate of clinically significant infections (CSIs; viremia requiring treatment or end-organ disease) following infusion of posoleucel, a partially HLA-matched, allogeneic, off-the-shelf, multivirus-specific T cell investigational product for preventing CSIs with adenovirus, BK virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus-6, or JC virus. This open-label trial enrolled high-risk allo-HCT recipients based on receiving grafts from umbilical cord blood, haploidentical, mismatched, or matched unrelated donors; post-HCT lymphocytes <180/mm3; or use of T cell depletion. Posoleucel dosing was initiated within 15-49 days of allo-HCT and subsequently every 14 days for up to seven doses. The primary endpoint was the number of CSIs due to the six target viruses by week 14. Of the 26 patients enrolled just three (12%) had a CSI by week 14, each with a single target virus. In vivo expansion of functional virus-specific T cells detected via interferon-γ ELISpot assay was associated with viral control. Persistence of posoleucel-derived T cell clones for up to 14 weeks after the last infusion was confirmed by T cell receptor deep-sequencing. Five patients (19%) had acute GVHD grade II-IV. No patient experienced cytokine release syndrome. All six deaths were due to relapse or disease progression. High-risk allo-HCT patients who received posoleucel had low rates of CSIs from six targeted viruses. Repeat posoleucel dosing was generally safe and well tolerated and associated with functional immune reconstitution. www.clinicaltrials.gov NCT04693637.

Phenotypic and functional characterization of posoleucel, a multivirus-specific T cell therapy for the treatment and prevention of viral infections in immunocompromised patients.

BACKGROUND: Deficits in T cell immunity translate into increased risk of severe viral infection in recipients of solid organ and hematopoietic cell transplants. Thus, therapeutic strategies that employ the adoptive transfer of virus-specific T cells are being clinically investigated to treat and prevent viral diseases in these highly immunocompromised patients. Posoleucel is an off-the-shelf multivirus-specific T cell investigational product for the treatment and prevention of infections due to adenovirus, BK virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6 or JC virus. METHODS: Herein we perform extensive characterization of the phenotype and functional profile of posoleucel to illustrate the cellular properties that may contribute to its in vivo activity. RESULTS AND CONCLUSIONS: Our results demonstrate that posoleucel is enriched for central and effector memory CD4+ and CD8+ T cells with specificity for posoleucel target viruses and expressing a broad repertoire of T cell receptors. Antigen-driven upregulation of cell-surface molecules and production of cytokine and effector molecules indicative of proliferation, co-stimulation, and cytolytic potential demonstrate the specificity of posoleucel and its potential to mount a broad, polyfunctional, and effective Th1-polarized antiviral response upon viral exposure. We also show the low risk for off-target and nonspecific effects as evidenced by the enrichment of posoleucel in memory T cells, low frequency of naive T cells, and lack of demonstrated alloreactivity in vitro. The efficacy of posoleucel is being explored in four placebo-controlled clinical trials in transplant recipients to treat and prevent viral infections (NCT05179057, NCT05305040, NCT04390113, NCT04605484).

LOAd703, an oncolytic virus-based immunostimulatory gene therapy, combined with chemotherapy for unresectable or metastatic pancreatic cancer (LOKON001): results from arm 1 of a non-randomised, single-centre, phase 1/2 study.

BACKGROUND: Pancreatic ductal adenocarcinoma is characterised by low immunogenicity and an immunosuppressive tumour microenvironment. LOAd703, an oncolytic adenovirus with transgenes encoding TMZ-CD40L and 4-1BBL, lyses cancer cells selectively, activates cytotoxic T cells, and induces tumour regression in preclinical models. The aim of this study was to evaluate the safety and feasibility of combining LOAd703 with chemotherapy for advanced pancreatic ductal adenocarcinoma. METHODS: LOKON001 was a non-randomised, phase 1/2 study conducted at the Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA, and consisted of two arms conducted sequentially; the results of arm 1 are presented here. In arm 1, patients 18 years or older with previously treated or treatment-naive unresectable or metastatic pancreatic ductal adenocarcinoma were treated with standard 28-day cycles of intravenous nab-paclitaxel 125 mg/m2 plus gemcitabine 1000 mg/m2 (up to 12 cycles) and intratumoural injections of LOAd703 every 2 weeks. Patients were assigned using Bayesian optimal interval design to receive 500 µL of LOAd703 at 5 × 1010 (dose 1), 1 × 1011 (dose 2), or 5 × 1011 (dose 3) viral particles per injection, injected endoscopically or percutaneously into the pancreatic tumour or a metastasis for six injections. The primary endpoints were safety and treatment-emergent immune response in patients who received at least one dose of LOAd703, and antitumour activity was a secondary endpoint. This study was registered with ClinicalTrials.gov, NCT02705196, arm 2 is ongoing and open to new participants. FINDINGS: Between Dec 2, 2016, and Oct 17, 2019, 23 patients were assessed for eligibility, leading to 22 patients being enrolled. One patient withdrew consent, resulting in 21 patients (13 [62%] men and eight [38%] women) assigned to a dose group (three to dose 1, four to dose 2, and 14 to dose 3). 21 patients were evaluable for safety. Median follow-up time was 6 months (IQR 4-10), and data cutoff was Jan 5, 2023. The most common treatment-emergent adverse events overall were anaemia (96 [8%] of 1237 events), lymphopenia (86 [7%] events), hyperglycaemia (70 [6%] events), leukopenia (63 [5%] events), hypertension (62 [5%] events), and hypoalbuminaemia (61 [5%] events). The most common adverse events attributed to LOAd703 were fever (14 [67%] of 21 patients), fatigue (eight [38%]), chills (seven [33%]), and elevated liver enzymes (alanine aminotransferase in five [24%], alkaline phosphatase in four [19%], and aspartate aminotransferase in four [19%]), all of which were grade 1-2, except for a transient grade 3 aminotransferase elevation occurring at dose 3. A maximum tolerated dose was not reached, thereby establishing dose 3 as the highest-evaluated safe dose when combined with nab-paclitaxel plus gemcitabine. Proportions of CD8+ effector memory cells and adenovirus-specific T cells increased after LOAd703 injections in 15 (94%) of 16 patients for whom T-cell assays could be performed. Eight (44%, 95% CI 25-66) of 18 patients evaluable for activity had an objective response. INTERPRETATION: Combining LOAd703 with nab-paclitaxel plus gemcitabine in patients with advanced pancreatic ductal adenocarcinoma was feasible and safe. To build upon this novel chemoimmunotherapeutic approach, arm 2 of LOKON001, which combines LOAd703, nab-paclitaxel plus gemcitabine, and atezolizumab, is ongoing. FUNDING: Lokon Pharma, the Swedish Cancer Society, and the Swedish Research Council.

Targeting IDH2R140Q and other neoantigens in acute myeloid leukemia.

ABSTRACT: For patients with high-risk or relapsed/refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (allo-HSCT) and the graft-versus-leukemia effect mediated by donor T cells, offer the best chance of long-term remission. However, the concurrent transfer of alloreactive T cells can lead to graft-versus-host disease that is associated with transplant-related morbidity and mortality. Furthermore, ∼60% of patients will ultimately relapse after allo-HSCT, thus, underscoring the need for novel therapeutic strategies that are safe and effective. In this study, we explored the feasibility of immunotherapeutically targeting neoantigens, which arise from recurrent nonsynonymous mutations in AML and thus represent attractive targets because they are exclusively present on the tumor. Focusing on 14 recurrent driver mutations across 8 genes found in AML, we investigated their immunogenicity in 23 individuals with diverse HLA profiles. We demonstrate the immunogenicity of AML neoantigens, with 17 of 23 (74%) reactive donors screened mounting a response. The most immunodominant neoantigens were IDH2R140Q (n = 11 of 17 responders), IDH1R132H (n = 7 of 17), and FLT3D835Y (n = 6 of 17). In-depth studies of IDH2R140Q-specific T cells revealed the presence of reactive CD4+ and CD8+ T cells capable of recognizing distinct mutant-specific epitopes restricted to different HLA alleles. These neo-T cells could selectively recognize and kill HLA-matched AML targets endogenously expressing IDH2R140Q both in vitro and in vivo. Overall, our findings support the clinical translation of neoantigen-specific T cells to treat relapsed/refractory AML.

Incongruity between T cell receptor recognition of breast cancer hotspot mutations ESR1 Y537S and D538G following exogenous peptide loading versus endogenous antigen processing.

T cell receptor engineered T cell (TCR T) therapies have shown recent efficacy against certain types of solid metastatic cancers. However, to extend TCR T therapies to treat more patients across additional cancer types, new TCRs recognizing cancer-specific antigen targets are needed. Driver mutations in AKT1, ESR1, PIK3CA, and TP53 are common in patients with metastatic breast cancer (MBC) and if immunogenic could serve as ideal tumor-specific targets for TCR T therapy to treat this disease. Through IFN-γ ELISpot screening of in vitro expanded neopeptide-stimulated T cell lines from healthy donors and MBC patients, we identified reactivity towards 11 of 13 of the mutations. To identify neopeptide-specific TCRs, we then performed single-cell RNA sequencing of one of the T cell lines following neopeptide stimulation. Here, we identified an ESR1 Y537S specific T cell clone, clonotype 16, and an ESR1 Y537S/D538G dual-specific T cell clone, clonotype 21, which were HLA-B*40:02 and HLA-C*01:02 restricted, respectively. TCR Ts expressing these TCRs recognized and killed target cells pulsed with ESR1 neopeptides with minimal activity against ESR1 WT peptide. However, these TCRs failed to recognize target cells expressing endogenous mutant ESR1. To investigate the basis of this lack of recognition we performed immunopeptidomics analysis of a mutant-overexpressing lymphoblastoid cell line and found that the ESR1 Y537S neopeptide was not endogenously processed, despite binding to HLA-B*40:02 when exogenously pulsed onto the target cell. These results indicate that stimulation of T cells that likely derive from the naïve repertoire with pulsed minimal peptides may lead to the expansion of clones that recognize non-processed peptides, and highlights the importance of using methods that selectively expand T cells with specificity for antigens that are efficiently processed and presented.

T cell immune profiling of respiratory syncytial virus for the development of a targeted immunotherapy.

Respiratory syncytial virus (RSV)-associated viral infections are a major public health problem affecting the immunologically naïve/compromised populations. Given the RSV-associated morbidity and the limited treatment options, we sought to characterize the cellular immune response to RSV to develop a targeted T cell therapy for off-the-shelf administration to immunocompromised individuals. Here we report on the immunological profiling, as well as manufacturing, characterization and antiviral properties of these RSV-targeted T cells. A randomized, phase 1/2 clinical trial evaluating their safety and activity in haematopoietic stem cell transplant recipients as an off-the-shelf multi-respiratory virus-directed product is currently underway (NCT04933968, https://clinicaltrials.gov).

Therapy With Allogeneic Severe Acute Respiratory Syndrome Coronavirus-2-Specific T Cells for Persistent Coronavirus Disease 2019 in Immunocompromised Patients.

We administered severe acute respiratory syndrome coronavirus-2 viral-specific T cells (VSTs) under emergency investigational new drug applications to 6 immunocompromised patients with persistent coronavirus disease 2019 (COVID-19) and characterized clinical and virologic responses. Three patients had partial responses after failing other therapies but then died. Two patients completely recovered, but the role of VSTs in recovery was unclear due to concomitant use of other antivirals. One patient had not responded to 2 courses of remdesivir and experienced sustained recovery after VST administration. The use of VSTs in immunocompromised patients with persistent COVID-19 requires further study.

Posoleucel, an Allogeneic, Off-the-Shelf Multivirus-Specific T-Cell Therapy, for the Treatment of Refractory Viral Infections in the Post-HCT Setting.

PURPOSE: Viral infections are a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (allo-HCT). In the absence of safe and effective antiviral treatments, virus-specific T cells have emerged as a promising therapeutic option. Posoleucel is a multivirus-specific T-cell therapy for off-the-shelf use against six viral infections that commonly occur in allo-HCT recipients: adenovirus, BK virus (BKV), cytomegalovirus, Epstein-Barr virus, human herpes virus-6, and JC virus. PATIENTS AND METHODS: We conducted an open-label, phase II trial to determine the feasibility and safety of posoleucel in allo-HCT recipients infected with one or more of these viruses. Infections were either unresponsive to or patients were unable to tolerate standard antiviral therapies. Fifty-eight adult and pediatric patients were enrolled and treated. RESULTS: Posoleucel was well tolerated, with no cytokine release syndrome or other infusion-related toxicities; two patients (3.4%) developed Grade 2 and one patient (1.7%) Grade 3 GvHD during the trial. The overall response rate 6 weeks after the first posoleucel infusion was 95%, with a median plasma viral load reduction of 97%. Of the 12 patients who had two or more target viral infections identified at study entry, 10 (83%) had a clinical response for all evaluable viruses. Of the 23 patients treated for refractory BKV-associated hemorrhagic cystitis, 74% had resolution of symptoms and macroscopic hematuria by 6 weeks post-infusion. CONCLUSIONS: In this open-label trial, treatment of refractory viral infections/disease in allo-HCT recipients with posoleucel was feasible, safe, and effective.

Allogeneic, off-the-shelf, SARS-CoV-2-specific T cells (ALVR109) for the treatment of COVID-19 in high-risk patients.

Defects in T-cell immunity to SARS-CoV-2 have been linked to an increased risk of severe COVID-19 (even after vaccination), persistent viral shedding and the emergence of more virulent viral variants. To address this T-cell deficit, we sought to prepare and cryopreserve banks of virus-specific T cells, which would be available as a partially HLA-matched, off-the-shelf product for immediate therapeutic use. By interrogating the peripheral blood of healthy convalescent donors, we identified immunodominant and protective T-cell target antigens, and generated and characterized polyclonal virus-specific T-cell lines with activity against multiple clinically important SARS-CoV-2 variants (including 'delta' and 'omicron'). The feasibility of making and safely utilizing such virus-specific T cells clinically was assessed by administering partially HLA-matched, third-party, cryopreserved SARS-CoV-2-specific T cells (ALVR109) in combination with other antiviral agents to four individuals who were hospitalized with COVID-19. This study establishes the feasibility of preparing and delivering off-the-shelf, SARS-CoV-2-directed, virus-specific T cells to patients with COVID-19 and supports the clinical use of these products outside of the profoundly immune compromised setting (ClinicalTrials.gov number, NCT04401410).

Cord blood transplantation for nonmalignant disorders: early functional immunity and high survival.

There is no consensus on the best donor for children with nonmalignant disorders and immune deficiencies in the absence of a matched related donor (MRD). We evaluated the 2-year overall survival (OS) after umbilical cord blood transplantation (UCBT) in patients with nonmalignant disorders from 2009 to 2020 enrolled in a prospective clinical trial using either 5/6 or 6/6 UCB as the cell source. Patients receive a fully ablative busulfan, cyclophosphamide, and fludarabine without serotherapy. Fifty-five children were enrolled, median age 5 months (range, 1-111 months); primary immune deficiency (45), metabolic (5), hemophagocytic lymphohistiocytosis (1), and hematologic disorders (4). Twenty-six patients had persistent infections before transplant. Nineteen of them (34%) were 6/6 matched, and 36 (66%) were 5/6 human leukocyte antigen-matched. The OS at 2 years was 91% (95% cumulative incidence, 79-96), with a median follow-up of 4.3 years. The median time to neutrophil and platelet recovery were 17 days (range, 5-39 days) and 37 days (range, 20-92 days), respectively. All but one evaluable patient achieved full donor chimerism. The cumulative incidence of acute GVHD grades 2-4 on day 100 was 16% (n = 9). All patients with viral infections at the time of transplant cleared the infection at a median time of 54 days (range, 44-91 days). All evaluable patients underwent correction of their immune or metabolic defects. We conclude that in the absence of MRD, UCBT following myeloablative conditioning without serotherapy is an excellent curative option in young children with nonmalignant disorders. This trial has been registered at www.clinicaltrials.gov as NCT00950846.

Assessment of the cytolytic potential of a multivirus-targeted T cell therapy using a vital dye-based, flow cytometric assay.

Reliable and sensitive characterization assays are important determinants of the successful clinical translation of immunotherapies. For the assessment of cytolytic potential, the chromium 51 (51Cr) release assay has long been considered the gold standard for testing effector cells. However, attaining the approvals to access and use radioactive isotopes is becoming increasingly complex, while technical aspects [i.e. sensitivity, short (4-6 hours) assay duration] may lead to suboptimal performance. This has been the case with our ex vivo expanded, polyclonal (CD4+ and CD8+) multivirus-specific T cell (multiVST) lines, which recognize 5 difficult-to-treat viruses [Adenovirus (AdV), BK virus (BKV), cytomegalovirus (CMV), Epstein Barr virus (EBV), and human herpes virus 6 (HHV6)] and when administered to allogeneic hematopoietic stem cell (HCT) or solid organ transplant (SOT) recipients have been associated with clinical benefit. However, despite mediating potent antiviral effects in vivo, capturing in vitro cytotoxic potential has proven difficult in a traditional 51Cr release assay. Now, in addition to cytotoxicity surrogates, including CD107a and Granzyme B, we report on an alternative, vital dye -based, flow cytometric platform in which superior sensitivity and prolonged effector:target co-culture duration enabled the reliable detection of both CD4- and CD8-mediated in vitro cytolytic activity against viral targets without non-specific effects.

Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand-Expressing Solid Tumors.

T-cell immunotherapy has demonstrated remarkable clinical outcomes in certain hematologic malignancies. However, efficacy in solid tumors has been suboptimal, partially due to the hostile tumor microenvironment composed of immune-inhibitory molecules. One such suppressive agent abundantly expressed in solid tumors is Fas ligand (FasL), which can trigger apoptosis of Fas-expressing effector cells such as T cells and natural killer (NK) cells. To alleviate this FasL-induced suppression of tumor-specific immune cells in solid tumors, we describe here the development of a Fas decoy that is secreted by engineered cells upon activation and sequesters the ligand, preventing it from engaging with Fas on the surface of effector cells. We further improved the immune-stimulatory effects of this approach by creating a Fas decoy and IL15 cytokine fusion protein, which enhanced the persistence and antitumor activity of decoy-engineered as well as bystander chimeric-antigen receptor (CAR) T cells in xenograft models of pancreatic cancer. Our data indicate that secreted Fas decoys can augment the efficacy of both adoptively transferred and endogenous tumor-specific effector cells in FasL-expressing solid tumors.

Multi-antigen-targeted T-cell therapy to treat patients with relapsed/refractory breast cancer.

Purpose: Adoptively transferred, ex vivo expanded multi-antigen-targeted T cells (multiTAA-T) represent a new, potentially effective, and nontoxic therapeutic approach for patients with breast cancer (BC). In this first-in-human trial, we investigated the safety and clinical effects of administering multiTAA T cells targeting the tumor-expressed antigens, Survivin, NY-ESO-1, MAGE-A4, SSX2, and PRAME, to patients with relapsed/refractory/metastatic BC. Materials and methods: MultiTAA T-cell products were generated from the peripheral blood of heavily pre-treated patients with metastatic or locally recurrent unresectable BC of all subtypes and infused at a fixed dose level of 2 × 107/m2. Patients received two infusions of cells 4 weeks apart and safety and clinical activity were determined. Cells were administered in an outpatient setting and without prior lymphodepleting chemotherapy. Results: All patients had estrogen receptor/progesterone receptor positive BC, with one patient also having human epidermal growth factor receptor 2-positive. There were no treatment-related toxicities and the infusions were well tolerated. Of the 10 heavily pre-treated patients enrolled and infused with multiTAA T cells, nine had disease progression while one patient with 10 lines of prior therapies experienced prolonged (5 months) disease stabilization that was associated with the in vivo expansion and persistence of T cells directed against the targeted antigens. Furthermore, antigen spreading and the endogenous activation of T cells directed against a spectrum of non-targeted tumor antigens were observed in 7/10 patients post-multiTAA infusion. Conclusion: MultiTAA T cells were well tolerated and induced disease stabilization in a patient with refractory BC. This was associated with in vivo T-cell expansion, persistence, and antigen spreading. Future directions of this approach may include additional strategies to enhance the therapeutic benefit of multiTAA T cells in patients with BC.

Donor-derived multiple leukemia antigen-specific T-cell therapy to prevent relapse after transplant in patients with ALL.

Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk acute lymphoblastic leukemia (ALL), but relapse remains a major cause of treatment failure. To prevent disease relapse, we prepared and infused donor-derived multiple leukemia antigen-specific T cells (mLSTs) targeting PRAME, WT1, and survivin, which are leukemia-associated antigens frequently expressed in B- and T-ALL. Our goal was to maximize the graft-versus-leukemia effect while minimizing the risk of graft-versus-host disease (GVHD). We administered mLSTs (dose range, 0.5 × 107 to 2 × 107 cells per square meter) to 11 patients with ALL (8 pediatric, 3 adult), and observed no dose-limiting toxicity, acute GVHD or cytokine release syndrome. Six of 8 evaluable patients remained in long-term complete remission (median: 46.5 months; range, 9-51). In these individuals we detected an increased frequency of tumor-reactive T cells shortly after infusion, with activity against both targeted and nontargeted, known tumor-associated antigens, indicative of in vivo antigen spreading. By contrast, this in vivo amplification was absent in the 2 patients who experienced relapse. In summary, infusion of donor-derived mLSTs after allogeneic HSCT is feasible and safe and may contribute to disease control, as evidenced by in vivo tumor-directed T-cell expansion. Thus, this approach represents a promising strategy for preventing relapse in patients with ALL.

Selectively targeting myeloid-derived suppressor cells through TRAIL receptor 2 to enhance the efficacy of CAR T cell therapy for treatment of breast cancer.

BACKGROUND: Successful targeting of solid tumors such as breast cancer (BC) using chimeric antigen receptor (CAR) T cells has proven challenging, largely attributed to the immunosuppressive tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSCs) inhibit CAR T cell function and persistence within the breast TME. To overcome this challenge, we have developed CAR T cells targeting tumor-associated mucin 1 (MUC1) with a novel chimeric costimulatory receptor that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TR2) expressed on MDSCs. METHODS: The function of the TR2.41BB costimulatory receptor was assessed by exposing non-transduced (NT) and TR2.41BB transduced T cells to recombinant TR2, after which nuclear translocation of NFκB was measured by ELISA and western blot. The cytolytic activity of CAR.MUC1/TR2.41BB T cells was measured in a 5-hour cytotoxicity assay using MUC1+ tumor cells as targets in the presence or absence of MDSCs. In vivo antitumor activity was assessed using MDSC-enriched tumor-bearing mice treated with CAR T cells with or without TR2.41BB. RESULTS: Nuclear translocation of NFκB in response to recombinant TR2 was detected only in TR2.41BB T cells. The presence of MDSCs diminished the cytotoxic potential of CAR.MUC1 T cells against MUC1+ BC cell lines by 25%. However, TR2.41BB expression on CAR.MUC1 T cells induced MDSC apoptosis, thereby restoring the cytotoxic activity of CAR.MUC1 T cells against MUC1+ BC lines. The presence of MDSCs resulted in an approximately twofold increase in tumor growth due to enhanced angiogenesis and fibroblast accumulation compared with mice with tumor alone. Treatment of these MDSC-enriched tumors with CAR.MUC1.TR2.41BB T cells led to superior tumor cell killing and significant reduction in tumor growth (24.54±8.55 mm3) compared with CAR.MUC1 (469.79±81.46 mm3) or TR2.41BB (434.86±64.25 mm3) T cells alone. CAR.MUC1.TR2.41BB T cells also demonstrated improved T cell proliferation and persistence at the tumor site, thereby preventing metastases. We observed similar results using CAR.HER2.TR2.41BB T cells in a HER2+ BC model. CONCLUSIONS: Our findings demonstrate that CAR T cells that coexpress the TR2.4-1BB receptor exhibit superior antitumor potential against breast tumors containing immunosuppressive and tumor promoting MDSCs, resulting in TME remodeling and improved T cell proliferation at the tumor site.

T-Cell Therapy for Lymphoma Using Nonengineered Multiantigen-Targeted T Cells Is Safe and Produces Durable Clinical Effects.

PURPOSE: Patients with relapsed lymphomas often fail salvage therapies including high-dose chemotherapy and mono-antigen-specific T-cell therapies, highlighting the need for nontoxic, novel treatments. To that end, we clinically tested an autologous T-cell product that targets multiple tumor-associated antigens (TAAs) expressed by lymphomas with the intent of treating disease and preventing immune escape. PATIENTS AND METHODS: We expanded polyclonal T cells reactive to five TAAs: PRAME, SSX2, MAGEA4, SURVIVIN, and NY-ESO-1. Products were administered to 32 patients with Hodgkin lymphomas (n = 14) or non-Hodgkin lymphomas (n = 18) in a two-part phase I clinical trial, where the objective of the first phase was to establish the safety of targeting all five TAAs (fixed dose, 0.5 × 107 cells/m2) simultaneously and the second stage was to establish the maximum tolerated dose. Patients had received a median of three prior lines of therapy and either were at high risk for relapse (adjuvant arm, n = 17) or had chemorefractory disease (n = 15) at enrollment. RESULTS: Infusions were safe with no dose-limiting toxicities observed in either the antigen- or dose-escalation phases. Although the maximum tolerated dose was not reached, the maximum tested dose at which efficacy was observed (two infusions, 2 × 107 cells/m2) was determined as the recommended phase II dose. Of the patients with chemorefractory lymphomas, two (of seven) with Hodgkin lymphomas and four (of eight) with non-Hodgkin lymphomas achieved durable complete remissions (> 3 years). CONCLUSION: T cells targeting five TAAs and administered at doses of up to two infusions of 2 × 107 cells/m2 are well-tolerated by patients with lymphoma both as adjuvant and to treat chemorefractory lymphoma. Preliminary indicators of antilymphoma activity were seen in the chemorefractory cohort across both antigen- and dose-escalation phases.

Clinical effects of administering leukemia-specific donor T cells to patients with AML/MDS after allogeneic transplant.

Relapse after allogeneic hematopoietic stem cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). Infusion of unselected donor lymphocytes (DLIs) enhances the graft-versus-leukemia (GVL) effect. However, because the infused lymphocytes are not selected for leukemia specificity, the GVL effect is often accompanied by life-threatening graft-versus-host disease (GVHD), related to the concurrent transfer of alloreactive lymphocytes. Thus, to minimize GVHD and maximize GVL, we selectively activated and expanded stem cell donor-derived T cells reactive to multiple antigens expressed by AML/MDS cells (PRAME, WT1, Survivin, and NY-ESO-1). Products that demonstrated leukemia antigen specificity were generated from 29 HCT donors. In contrast to DLIs, leukemia-specific T cells (mLSTs) selectively recognized and killed leukemia antigen-pulsed cells, with no activity against recipient's normal cells in vitro. We administered escalating doses of mLSTs (0.5 to 10 × 107 cells per square meter) to 25 trial enrollees, 17 with high risk of relapse and 8 with relapsed disease. Infusions were well tolerated with no grade >2 acute or extensive chronic GVHD seen. We observed antileukemia effects in vivo that translated into not-yet-reached median leukemia-free and overall survival at 1.9 years of follow-up and objective responses in the active disease cohort (1 complete response and 1 partial response). In summary, mLSTs are safe and promising for the prevention and treatment of AML/MDS after HCT. This trial is registered at www.clinicaltrials.com as #NCT02494167.

Identification of protective T-cell antigens for smallpox vaccines.

Background aims: E3L is an immediate-early protein of vaccinia virus (VV) that is detected within 0.5 h of infection, potentially before the many immune evasion genes of vaccinia can exert their protective effects. E3L is highly conserved among orthopoxviruses and hence could provide important protective T-cell epitopes that should be retained in any subunit or attenuated vaccine. We have therefore evaluated the immunogenicity of E3L in healthy VV-vaccinated donors. Methods: Peripheral blood mononuclear cells from healthy volunteers (n = 13) who had previously received a smallpox vaccine (Dryvax) were activated and expanded using overlapping E3L peptides and their function, specificity and antiviral activity was analyzed. E3L-specific T cells were expanded from 7 of 12 (58.3%) vaccinated healthy donors. Twenty-five percent of these produced CD8+ T-cell responses and 87.5% produced CD4+ T cells. We identified epitopes restricted by HLA-B35 and HLA-DR15. Results: E3L-specific T cells killed peptide-loaded target cells as well as vaccinia-infected cells, but only CD8+ T cells could prevent the spread of infectious virus in virus inhibition assays. The epitopes recognized by E3L-specific T cells were shared with monkeypox, and although there was a single amino acid change in the variola epitope homolog, it was recognized by vaccinia-specific T-cells. Conclusions: It might be important to include E3L in any deletion mutant or subunit vaccine and E3L could provide a useful antigen to monitor protective immunity in humans.