Novel OX40 and 4-1BB Derived Spacers Enhance CD30 CAR Activity and Safety in CD30 Positive Lymphoma Models.

The chimeric antigen receptor (CAR) derived from the CD30 specific murine antibody, HRS-3, has produced promising clinical efficacy with a favorable safety profile in the treatment of relapsed or refractory CD30-positive lymphomas. However, persistence of the autologous CAR T cells was brief, and many patients relapsed a year after treatment. The lack of persistence may be attributed to the use of a wildtype IgG1 spacer that can associate with Fc receptors. We first identified the cysteine rich domain (CRD) 5 of CD30 as the primary binding epitope of HRS-3 and armed with this insight, attempted to improve the HRS-3 CAR functionality with a panel of novel spacer designs. We demonstrate that HRS-3 CARs with OX40 and 4-1BB derived spacers exhibited similar anti-tumor efficacy, circumvented interactions with Fc receptors and secreted lower levels of cytokines in vitro than a CAR employing the IgG1 spacer. Humanization of the HRS-3 scFv coupled with the 4-1BB spacer preserved potent on-target, on-tumor efficacy, and on-target, off-tumor safety. In a lymphoma mouse model of high tumor burden, T cells expressing a humanized HRS-3 CD30.CARs with the 4-1BB spacer potently killed tumors with low levels of circulating inflammatory cytokines, providing a promising candidate for future clinical development in the treatment of CD30-positive malignancies.

Phase I Trial of GD2.CART Cells Augmented With Constitutive Interleukin-7 Receptor for Treatment of High-Grade Pediatric CNS Tumors.

PURPOSE: T cells modified with chimeric antigen receptors (CARTs) have demonstrated efficacy for hematologic malignancies; however, benefit for patients with CNS tumors has been limited. To enhance T cell activity against GD2+ CNS malignancies, we modified GD2-directed CART cells (GD2.CARTs) with a constitutively active interleukin (IL)-7 receptor (C7R-GD2.CARTs). METHODS: Patients age 1-21 years with H3K27-altered diffuse midline glioma (DMG) or other recurrent GD2-expressing CNS tumors were eligible for this phase I trial (ClinicalTrials.gov identifier: NCT04099797). All subjects received standard-of-care adjuvant radiation therapy or chemotherapy before study enrollment. The first treatment cohort received GD2.CARTs alone (1 × 107 cells/m2), and subsequent cohorts received C7R-GD2.CARTs at two dose levels (1 × 107 cells/m2; 3 × 107 cells/m2). Standard lymphodepletion with cyclophosphamide and fludarabine was included at all dose levels. RESULTS: Eleven patients (age 4-18 years) received therapy without dose-limiting toxicity. The GD2.CART cohort did not experience toxicity, but had disease progression after brief improvement of residual neurologic deficits (≤3 weeks). The C7R-GD2.CART cohort developed grade 1 tumor inflammation-associated neurotoxicity in seven of eight (88%) cases, controllable with anakinra. Cytokine release syndrome was observed in six of eight (75%, grade 1 in all but one patient) and associated with increased circulating IL-6 and IP-10 (P < .05). Patients receiving C7R-GD2.CARTs experienced temporary improvement from baseline neurologic deficits (range, 2 to >12 months), and seven of eight (88%) remained eligible for additional treatment cycles (range 2-4 cycles). Partial responses by iRANO criteria were observed in two of seven (29%) patients with DMG treated by C7R-GD2.CARTs. CONCLUSION: Intravenous GD2.CARTs with and without C7R were well tolerated. Patients treated with C7R-GD2.CARTs exhibited transient improvement of neurologic deficits and increased circulating cytokines/chemokines. Treatment with C7R-GD2.CARTs represents a novel approach warranting further investigation for children with these incurable CNS cancers.

Cotargeting EBV lytic as well as latent cycle antigens increases T-cell potency against lymphoma.

ABSTRACT: The remarkable efficacy of Epstein-Barr virus (EBV)-specific T cells for the treatment of posttransplant lymphomas has not been reproduced for EBV-positive (EBV+) malignancies outside the transplant setting. This is because of, in part, the heterogeneous expression and poor immunogenicity of the viral antigens expressed, namely latent membrane proteins 1 and 2, EBV nuclear antigen 1, and BamHI A rightward reading frame 1 (type-2 [T2] latency). However, EBV lytic cycle proteins are also expressed in certain EBV+ malignancies and, because several EBV lytic cycle proteins are abundantly expressed, have oncogenic activity, and likely contribute to malignancy, we sought and identified viral lytic-cycle transcripts in EBV+ Hodgkin lymphoma biopsies. This provided the rationale for broadening the target antigen-specific repertoire of EBV-specific T cells (EBVSTs) for therapy. We stimulated, peripheral blood mononuclear cells from healthy donors and patients with EBV+ lymphoma with both lytic and latent cycle proteins to produce broad repertoire (BR) EBVSTs. Compared with T2 antigen-specific EBVSTs, BR-EBVSTs more rapidly cleared autologous EBV+ tumors in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and produced higher levels of proinflammatory cytokines that should reactivate the immunosuppressive tumor microenvironment leading to epitope spreading. Our results confirm that lytic cycle antigens are clinically relevant targets for EBV+ lymphoma and underpin the rationale for integrating BR-EBVSTs as a therapeutic approach for relapsed/refractory EBV+ lymphoma (www.clinicaltrials.gov identifiers: #NCT01555892 and #NCT04664179), as well as for other EBV-associated malignancies.

Eighteen-year survival after GD2-directed Chimeric Antigen Receptor-Modified Immune Effector Cell Treatment for Neuroblastoma.

We report long-term outcomes up to 18 years of a clinical trial treating children with neuroblastoma with EBV-specific T lymphocytes and CD3-activated T cells - each expressing a first-generation chimeric antigen receptor targeting GD2 with barcoded transgenes to allow tracking of each population. Of 11 patients with active disease at infusion, three patients achieved a complete response that was sustained in 2, one for 8 years until lost to follow up and one for 18+ years. Of eight patients with a history of relapse or at high risk of recurrence, five are disease-free at their last follow-up between 10-14 years post-infusion. Intermittent low levels of transgene were detected during the follow up period with significantly greater persistence in those who were long-term survivors. In conclusion, patients with relapsed/refractory neuroblastoma achieved long-term disease control after receiving GD2 CAR-T cell therapy including one patient now in remission of relapsed disease for >18 years.

Constitutive Interleukin-7 Cytokine Signaling Enhances the Persistence of Epstein-Barr Virus-Specific T-Cells.

The efficacy of therapeutic T-cells is limited by a lack of positive signals and excess inhibitory signaling in tumor microenvironments. We previously showed that a constitutively active IL7 receptor (C7R) enhanced the persistence, expansion, and anti-tumor activity of T-cells expressing chimeric antigen receptors (CARs), and C7R-modified GD2.CAR T-cells are currently undergoing clinical trials. To determine if the C7R could also enhance the activity of T-cells recognizing tumors via their native T-cell receptors (TCRs), we evaluated its effects in Epstein-Barr virus (EBV)-specific T-cells (EBVSTs) that have produced clinical benefits in patients with EBV-associated malignancies. EBVSTs were generated by stimulation of peripheral blood T-cells with overlapping peptide libraries spanning the EBV lymphoma antigens, LMP1, LMP2, and EBNA 1, followed by retroviral vector transduction to express the C7R. The C7R increased STAT5 signaling in EBVSTs and enhanced their expansion over 30 days of culture in the presence or absence of exogenous cytokines. C7R-EBVSTs maintained EBV antigen specificity but were dependent on TCR stimulation for continued expansion. C7R-EBVSTs produced more rapid lymphoma control in a murine xenograft model than unmodified EBVSTs and persisted for longer. The findings have led to a clinical trial, evaluating C7R-EBVSTs for the treatment of refractory or relapsed EBV-positive lymphoma (NCT04664179).

Structural surfaceomics reveals an AML-specific conformation of integrin ß2 as a CAR T cellular therapy target.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

Naive T cells inhibit the outgrowth of intractable antigen-activated memory T cells: implications for T-cell immunotherapy.

BACKGROUND: The wider application of T cells targeting viral tumor-antigens via their native receptors is hampered by the failure to expand potent tumor-specific T cells from patients. Here, we examine reasons for and solutions to this failure, taking as our model the preparation of Epstein-Barr virus (EBV)-specific T cells (EBVSTs) for the treatment of EBV-positive lymphoma. EBVSTs could not be manufactured from almost one-third of patients, either because they failed to expand, or they expanded, but lacked EBV specificity. We identified an underlying cause of this problem and established a clinically feasible approach to overcome it. METHODS: CD45RO+CD45RA- memory compartment residing antigen-specific T cells were enriched by depleting CD45RA positive (+) peripheral blood mononuclear cells (PBMCs) that include naïve T cells, among other subsets, prior to EBV antigen stimulation. We then compared the phenotype, specificity, function and T-cell receptor (TCR) Vß repertoire of EBVSTs expanded from unfractionated whole (W)-PBMCs and CD45RA-depleted (RAD)-PBMCs on day 16. To identify the CD45RA component that inhibited EBVST outgrowth, isolated CD45RA+ subsets were added back to RAD-PBMCs followed by expansion and characterization. The in vivo potency of W-EBVSTs and RAD-EBVSTs was compared in a murine xenograft model of autologous EBV+ lymphoma. RESULTS: Depletion of CD45RA+ PBMCs before antigen stimulation increased EBVST expansion, antigen-specificity and potency in vitro and in vivo. TCR sequencing revealed a selective outgrowth in RAD-EBVSTs of clonotypes that expanded poorly in W-EBVSTs. Inhibition of antigen-stimulated T cells by CD45RA+ PBMCs could be reproduced only by the naïve T-cell fraction, while CD45RA+ regulatory T cells, natural killer cells, stem cell memory and effector memory subsets lacked inhibitory activity. Crucially, CD45RA depletion of PBMCs from patients with lymphoma enabled the outgrowth of EBVSTs that failed to expand from W-PBMCs. This enhanced specificity extended to T cells specific for other viruses. CONCLUSION: Our findings suggest that naïve T cells inhibit the outgrowth of antigen-stimulated memory T cells, highlighting the profound effects of intra-T-cell subset interactions. Having overcome our inability to generate EBVSTs from many patients with lymphoma, we have introduced CD45RA depletion into three clinical trials: NCT01555892 and NCT04288726 using autologous and allogeneic EBVSTs to treat lymphoma and NCT04013802 using multivirus-specific T cells to treat viral infections after hematopoietic stem cell transplantation.

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).

Banking on virus-specific T cells to fulfill the need for off-the-shelf cell therapies.

Adoptively transferred virus-specific T cells (VSTs) have shown remarkable safety and efficacy for the treatment of virus-associated diseases and malignancies in hematopoietic stem cell transplant (HSCT) recipients, for whom VSTs are derived from the HSCT donor. Autologous VSTs have also shown promise for the treatment of virus-driven malignancies outside the HSCT setting. In both cases, VSTs are manufactured as patient-specific products, and the time required for procurement, manufacture, and release testing precludes their use in acutely ill patients. Further, Good Manufacturing Practices-compliant products are expensive, and failures are common in virus-naive HSCT donors and patient-derived VSTs that are rendered anergic by immunosuppressive tumors. Hence, highly characterized, banked VSTs (B-VSTs) that can be used for multiple unrelated recipients are highly desirable. The major challenges facing B-VSTs result from the inevitable mismatches in the highly polymorphic and immunogenic human leukocyte antigens (HLA) that present internally processed antigens to the T-cell receptor, leading to the requirement for partial HLA matching between the B-VST and recipient. HLA mismatches lead to rapid rejection of allogeneic T-cell products and graft-versus-host disease induced by alloreactive T cells in the infusion product. Here, we summarize the clinical outcomes to date of trials of B-VSTs used for the treatment of viral infections and malignancies and their potential as a platform for chimeric antigen receptors targeting nonviral tumors. We will highlight the properties of VSTs that make them attractive off-the-shelf cell therapies, as well as the challenges that must be overcome before they can become mainstream.

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.

Long-term follow-up for the development of subsequent malignancies in patients treated with genetically modified IECs.

Subsequent malignancies are well-documented complications in long-term follow-up of cancer patients. Recently, genetically modified immune effector (IE) cells have shown benefit in hematologic malignancies and are being evaluated in clinical trials for solid tumors. Although the short-term complications of IE cells are well described, there is limited literature summarizing long-term follow-up, including subsequent malignancies. We retrospectively reviewed data from 340 patients treated across 27 investigator-initiated pediatric and adult clinical trials at our center. All patients received IE cells genetically modified with γ-retroviral vectors to treat relapsed and/or refractory hematologic or solid malignancies. In a cumulative 1027 years of long-term follow-up, 13 patients (3.8%) developed another cancer with a total of 16 events (4 hematologic malignancies and 12 solid tumors). The 5-year cumulative incidence of a first subsequent malignancy in the recipients of genetically modified IE cells was 3.6% (95% confidence interval, 1.8% to 6.4%). For 11 of the 16 subsequent tumors, biopsies were available, and no sample was transgene positive by polymerase chain reaction. Replication-competent retrovirus testing of peripheral blood mononuclear cells was negative in the 13 patients with subsequent malignancies tested. Rates of subsequent malignancy were low and comparable to standard chemotherapy. These results suggest that the administration of IE cells genetically modified with γ retroviral vectors does not increase the risk for subsequent malignancy.

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.

A Costimulatory CAR Improves TCR-based Cancer Immunotherapy.

T-cell receptors (TCR) recognize intracellular and extracellular cancer antigens, allowing T cells to target many tumor antigens. To sustain proliferation and persistence, T cells require not only signaling through the TCR (signal 1), but also costimulatory (signal 2) and cytokine (signal 3) signaling. Because most cancer cells lack costimulatory molecules, TCR engagement at the tumor site results in incomplete T-cell activation and transient antitumor effects. To overcome this lack of signal 2, we genetically modified tumor-specific T cells with a costimulatory chimeric antigen receptor (CoCAR). Like classical CARs, CoCARs combine the antigen-binding domain of an antibody with costimulatory endodomains to trigger T-cell proliferation, but CoCARs lack the cytotoxic CD3ζ chain to avoid toxicity to normal tissues. We first tested a CD19-targeting CoCAR in combination with an HLA-A*02:01-restricted, survivin-specific transgenic TCR (sTCR) in serial cocultures with leukemia cells coexpressing the cognate peptide-HLA complex (signal 1) and CD19 (signal 2). The CoCAR enabled sTCR+ T cells to kill tumors over a median of four additional tumor challenges. CoCAR activity depended on CD19 but was maintained in tumors with heterogeneous CD19 expression. In a murine tumor model, sTCR+CoCAR+ T cells improved tumor control and prolonged survival compared with sTCR+ T cells. We further evaluated the CoCAR in Epstein-Barr virus-specific T cells (EBVST). CoCAR-expressing EBVSTs expanded more rapidly than nontransduced EBVSTs and delayed tumor progression in an EBV+ murine lymphoma model. Overall, we demonstrated that the CoCAR can increase the activity of T cells expressing both native and transgenic TCRs and enhance antitumor responses.

Epstein Barr virus-positive B-cell lymphoma is highly vulnerable to MDM2 inhibitors in vivo.

Epstein-Barr virus-positive (EBV-positive) B-cell lymphomas are common in immunocompromised patients and remain an unmet medical need. Here we report that MDM2 inhibitors (MDM2is) navtemadlin and idasanutlin have potent in vivo activity in EBV-positive B-cell lymphoma established in immunocompromised mice. Tumor regression was observed in all 5 EBV-positive xenograft-associated B-cell lymphomas treated with navtemadlin or idasanutlin. Molecular characterization showed that treatment with MDM2is resulted in activation of p53 pathways and downregulation of cell cycle effectors in human lymphoma cell lines that were either EBV-positive or had undetectable expression of BCL6, a transcriptional inhibitor of the TP53 gene. Moreover, treatment with navtemadlin resulted in tumor regression and prevented systemic dissemination of EBV-positive lymphoma derived from 2 juvenile patients with posttransplant lymphoproliferative diseases, including 1 whose tumor was resistant to virus-specific T-cell therapy. These results provide proof-of-concept for targeted therapy of EBV-positive lymphoma with MDM2is and the feasibility of using EBV infection or loss of BCL6 expression to identify responders to MDM2is.

Autologous EBV-specific T cell treatment results in sustained responses in patients with advanced extranodal NK/T lymphoma: results of a multicenter study.

We conducted a phase II clinical trial to develop an autologous EBV-specific T cell product (baltaleucel T) for advanced, relapsed ENKTL. Among 47 patients who provided whole blood starting material for manufacturing the product, 15 patients received a median of 4 doses of baltaleucel T. Thirty-two (68%) patients did not receive baltaleucel-T due to manufacturing failure, rapid disease progression, and death. Of the 15 patients, 10 patients had measurable disease at baseline (salvage cohort), and 5 patients had no disease at baseline assessment (adjuvant cohort). In the 15 patients, the median follow-up duration was 10.2 months (range 2.0-23.5 months), median progression-free survival (PFS) was 3.9 months, and the median overall survival (OS) was not reached. Patients in the salvage cohort achieved a 30% complete response (CR) and a 50% overall response rate (ORR). In the adjuvant cohort, disease progression was reported in three patients and two patients did not relapse during study follow-up. When we compared survival outcomes of seven responders and eight non-responders, the PFS (P = 0.001) and OS (P = 0.014) of responders proved statistically superior to that of non-responders. Baltaleucel-T was well tolerated. We have performed a phase II clinical trial of autologous EBV-specific T cell treatment (baltaleucel-T) in R/R ENKTL. Autologous EBV-specific T cells were well tolerated and demonstrated single-agent activity in R/R ENTKL.

CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity.

The prognosis of patients with acute myeloid leukemia (AML) remains dismal, highlighting the need for novel innovative treatment strategies. The application of chimeric antigen receptor (CAR) T-cell therapy to patients with AML has been limited, in particular by the lack of a tumor-specific target antigen. CD70 is a promising antigen to target AML, as it is expressed on most leukemic blasts, whereas little or no expression is detectable in normal bone marrow samples. To target CD70 on AML cells, we generated a panel of CD70-CAR T cells that contained a common single-chain variable fragment (scFv) for antigen detection, but differed in size and flexibility of the extracellular spacer and in the transmembrane and the costimulatory domains. These CD70scFv CAR T cells were compared with a CAR construct that contained human CD27, the ligand of CD70 fused to the CD3ζ chain (CD27z). The structural composition of the CAR strongly influenced expression levels, viability, expansion, and cytotoxic capacities of CD70scFv-based CAR T cells, but CD27z-CAR T cells demonstrated superior proliferation and antitumor activity in vitro and in vivo, compared with all CD70scFv-CAR T cells. Although CD70-CAR T cells recognized activated virus-specific T cells (VSTs) that expressed CD70, they did not prevent colony formation by normal hematopoietic stem cells. Thus, CD70-targeted immunotherapy is a promising new treatment strategy for patients with CD70-positive AML that does not affect normal hematopoiesis but will require monitoring of virus-specific T-cell responses.

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.

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.