Aude G. Chapuis

Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients

Donor-derived WT1-specific CD8+ T cells transferred after hematopoietic cell transplantation mediate antileukemic activity and can establish persistent responses without toxicity to normal tissues in high-risk leukemia patients. Targeting Leukemic Cells for Destruction After a patient is diagnosed with leukemia, the first line of treatment is generally chemotherapy. If it doesn’t work, the patient can get a bone marrow transplant, which can sometimes cure otherwise untreatable leukemia. However, for patients who have already relapsed after a transplant, or whose cancer has particularly unfavorable characteristics, few options remain. Such patients’ prognosis is generally very poor, with a high risk of relapse and death from leukemia. Immune cells derived from the donor bone marrow can help fight the cancer by attacking malignant cells inside the graft recipient. At the same time, however, they often attack the recipient’s healthy cells as well and cause graft-versus-host disease (GVHD), which can itself be lethal. Now, Chapuis and coauthors report a way to harness the power of the donors’ immune cells against some leukemias, without triggering GVHD in the bone marrow recipients. In this pilot trial, the authors enrolled 11 patients who had leukemia with poor prognostic characteristics and who had each undergone a bone marrow transplant. The patients then received T cells that were derived from their respective donors and selected for their ability to recognize Wilms tumor antigen 1 (WT1). Very small amounts of WT1 are present in some healthy cells, but its expression in malignant cells is much greater and corresponds to the aggressiveness of the cancer. In the first seven patients, the authors found that T cells that recognized WT1 could suppress the leukemia temporarily, but the new cells disappeared within 14 days, and the cancer rebounded. In the last four patients, the authors tried a modified protocol, pretreating the cells with interleukin-21 (IL-21) before infusion into the recipients. T cells pretreated with IL-21 developed characteristics of memory cells, including a greatly extended life span. At this time, all four patients who received IL-21–treated T cells are still alive, with no recurrence of their cancer up to 30 months after the T cell infusion. Notably, none of the patients who received WT1-specific T cells in this study have developed GVHD, supporting the idea that WT1 targeting is specific to the tumor cells and safe for patient use. The study by Chapuis et al. is a small pilot trial, and its results must be replicated with larger groups of patients before this protocol can become standard practice. Nevertheless, the combination of targeting WT1 in leukemia and pretreating leukemia-targeting T cells with IL-21 to extend their life span looks very promising and offers a potential safe and effective treatment for patients who have few other options. Relapse remains a leading cause of death after allogeneic hematopoietic cell transplantation (HCT) for patients with high-risk leukemias. The potentially beneficial donor T cell–mediated graft-versus-leukemia (GVL) effect is often mitigated by concurrent graft-versus-host disease (GVHD). Providing T cells that can selectively target Wilms tumor antigen 1 (WT1), a transcription factor overexpressed in leukemias that contributes to the malignant phenotype, represents an opportunity to promote antileukemic activity without inducing GVHD. HLA-A*0201–restricted WT1-specific donor-derived CD8+ cytotoxic T cell (CTL) clones were administered after HCT to 11 relapsed or high-risk leukemia patients without evidence of on-target toxicity. The last four treated patients received CTL clones generated with exposure to interleukin-21 (IL-21) to prolong in vivo CTL survival, because IL-21 can limit terminal differentiation of antigen-specific T cells generated in vitro. Transferred cells exhibited direct evidence of antileukemic activity in two patients: a transient response in one patient with advanced progressive disease and the induction of a prolonged remission in a patient with minimal residual disease (MRD). Additionally, three treated patients at high risk for relapse after HCT survive without leukemia relapse, GVHD, or additional antileukemic treatment. CTLs generated in the presence of IL-21, which were transferred in these latter three patients and the patient with MRD, all remained detectable long term and maintained or acquired in vivo phenotypic and functional characteristics associated with long-lived memory CD8+ T cells. This study supports expanding efforts to immunologically target WT1 and provides insights into the requirements necessary to establish potent persistent T cell responses.

Transferred melanoma-specific CD8+ T cells persist, mediate tumor regression, and acquire central memory phenotype

Adoptively transferred tumor-specific T cells offer the potential for non–cross-resistant therapy and long-term immunoprotection. Strategies to enhance in vivo persistence of transferred T cells can lead to improved antitumor efficacy. However, the extrinsic (patient conditioning) and intrinsic (effector cell) factors contributing to long-term in vivo persistence are not well-defined. As a means to enhance persistence of infused T cells in vivo and limit toxicity, 11 patients with refractory, progressive metastatic melanoma received cyclophosphamide alone as conditioning before the infusion of peripheral blood mononuclear cell-derived, antigen-specific, CD8+ cytotoxic T-lymphocyte (CTL) clones followed by low-dose or high-dose IL-2. No life-threatening toxicities occurred with low-dose IL-2. Five of 10 evaluable patients had stable disease at 8 wk, and 1 of 11 had a complete remission that continued for longer than 3 y. On-target autoimmune events with the early appearance of skin rashes were observed in patients with stable disease or complete remission at 4 wk or longer. In vivo tracking revealed that the conditioning regimen provided a favorable milieu that enabled CTL proliferation early after transfer and localization to nonvascular compartments, such as skin and lymph nodes. CTL clones, on infusion, were characterized by an effector memory phenotype, and CTL that persisted long term acquired phenotypic and/or functional qualities of central memory type CTLs in vivo. The use of a T-cell product composed of a clonal population of antigen-specific CTLs afforded the opportunity to demonstrate phenotypic and/or functional conversion to a central memory type with the potential for sustained clinical benefit.

Re‐adapting T cells for cancer therapy: from mouse models to clinical trials

Adoptive T‐cell therapy involves the isolation, expansion, and reinfusion of T lymphocytes with a defined specificity and function as a means to eradicate cancer. Our research has focused on specifying the requirements for tumor eradication with antigen‐specific T cells and T cells transduced to express a defined T‐cell receptor (TCR) in mouse models and then translating these strategies to clinical trials. Our design of T‐cell‐based therapy for cancer has reflected efforts to identify the obstacles that limit sustained effector T‐cell activity in mice and humans, design approaches to enhance T‐cell persistence, develop methods to increase TCR affinity/T‐cell functional avidity, and pursue strategies to overcome tolerance and immunosuppression. With the advent of genetic engineering, a highly functional population of T cells can now be rapidly generated and tailored for the targeted malignancy. Preclinical studies in faithful and informative mouse models, in concert with knowledge gained from analyses of successes and limitations in clinical trials, are shaping how we continue to develop, refine, and broaden the applicability of this approach for cancer therapy.

Regression of metastatic Merkel cell carcinoma following transfer of polyomavirus-specific T cells and therapies capable of re-inducing HLA class-I

Chapuis, Afanasiev, and colleagues show that the combined regimen of local tumor-targeted preconditioning and systemic immune therapies elicited a durable complete response in two of three lesions with a prolonged period without development of additional distant metastasis. Merkel cell carcinoma (MCC) is an aggressive skin cancer that typically requires the persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins that can serve as ideal immunotherapeutic targets. Several immune evasion mechanisms are active in MCC, including downregulation of HLA class-I expression on tumor cells and dysfunctional endogenous MCPyV-specific CD8 T-cell responses. To overcome these obstacles, we combined local and systemic immune therapies in a 67-year-old man, who developed metastatic MCPyV-expressing MCC. Intralesional IFN-β-1b or targeted single-dose radiation was administered as a preconditioning strategy to reverse the downregulation of HLA-I expression noted in his tumors and to facilitate the subsequent recognition of tumor cells by T cells. This was followed by the adoptive transfer of ex vivo expanded polyclonal, polyomavirus-specific T cells as a source of reactive antitumor immunity. The combined regimen was well tolerated and led to persistent upregulation of HLA-I expression in the tumor and a durable complete response in two of three metastatic lesions. Relative to historical controls, the patient experienced a prolonged period without development of additional distant metastases (535 days compared with historic median of 200 days; 95% confidence interval, 154–260 days). The transferred CD8+ T cells preferentially accumulated in the tumor tissue, remained detectable and functional for more than 200 days, persisted with an effector phenotype, and exhibited evidence of recent in vivo activation and proliferation. The combination of local and systemic immune stimulatory therapies was well tolerated and may be a promising approach to overcome immune evasion in virus-driven cancers. Cancer Immunol Res; 2(1); 27–36. ©2013 AACR.

HIV-specific CD8+ T cells from HIV+ individuals receiving HAART can be expanded ex vivo to augment systemic and mucosal immunity in vivo

Most HIV+ individuals require lifelong highly active antiretroviral therapy (HAART) to suppress HIV replication, but fail to eliminate the virus in part because of residual replication in gut-associated lymphoid tissues (GALT). Naturally elicited HIV-specific CD8+ T cells generated in the acute and chronic infectious phases exhibit antiviral activity, but decrease in number after HAART. Therapeutic vaccines represent a potential strategy to expand cellular responses, although previous efforts have been largely unsuccessful, conceivably because of a lack of responding HIV-specific central-memory CD8+ T cells (Tcm). To determine whether patients receiving HAART possess CD8+ T cells with Tcm qualities that are amenable to augmentation, HIV-specific CD8+ T-cell clones were derived from HIV-reactive CD28+CD8+ T-cell lines isolated from 7 HIV+ HAART-treated patients, expanded ex vivo, and reinfused into their autologous host. Tracking of the cells in vivo revealed that clones could persist for ≥ 84 days, maintain expression and/or re-express CD28, up-regulate CD62L, secrete IL-2, proliferate on cognate Ag encounter and localize to the rectal mucosa. These results suggest some infused cells exhibited phenotypic and functional characteristics shared with Tcm in vivo, and imply that more effective therapeutic vaccination strategies targeting CD8+ Tcm in patients on HAART might provide hosts with expanded, long-lasting immune responses not only systemically but also in GALT.

Combined IL-21–primed polyclonal CTL plus CTLA4 blockade controls refractory metastatic melanoma in a patient

Chapuis et al. demonstrate that the combination of adoptive cellular therapy with CTLA4 blockade induces long-term remission in a melanoma patient resistant to both modalities administered serially and individually.

T-Cell Therapy Using Interleukin-21–Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.

New Strategies in Engineering T-cell Receptor Gene-Modified T cells to More Effectively Target Malignancies

The immune system, T cells in particular, have the ability to target and destroy malignant cells. However, antitumor immune responses induced from the endogenous T-cell repertoire are often insufficient for the eradication of established tumors, as illustrated by the failure of cancer vaccination strategies or checkpoint blockade for most tumors. Genetic modification of T cells to express a defined T-cell receptor (TCR) can provide the means to rapidly generate large numbers of tumor-reactive T cells capable of targeting tumor cells in vivo. However, cell-intrinsic factors as well as immunosuppressive factors in the tumor microenvironment can limit the function of such gene-modified T cells. New strategies currently being developed are refining and enhancing this approach, resulting in cellular therapies that more effectively target tumors and that are less susceptible to tumor immune evasion. Clin Cancer Res; 21(23); 5191–7. ©2015 AACR.

Tracking the fate and origin of clinically relevant adoptively transferred CD8+ T cells in vivo

Long-term tumor control originates from very rare T cells with proliferative and/or survival advantages. Tracking tumor immunity Tumor-specific immune cells can lead to tumor regression in some cancer patients who do not respond to other therapies; however, the ability to track these cells after transfer has remained limited. Now, Chapuis et al. use high-throughput T cell receptor Vβ sequencing (HTTCS) to track individual clonotypes after transfer. They find that very low frequency clonotypes with proliferative and survival advantages can contribute to long-term tumor control. These data will help guide the selection of cell populations for immunotherapy. Adoptively transferred tumor-specific cells can mediate tumor regression in cancers refractory to conventional therapy. Autologous polyclonal tumor-specific cytotoxic T cells (CTLs) generated from peripheral blood and infused into patients with metastatic melanoma show enhanced persistence, compared with equivalent numbers of more extensively expanded monoclonal CTLs, and are associated with complete remissions (CRs) in select patients. We applied high-throughput T cell receptor Vβ sequencing (HTTCS) to identify individual clonotypes within CTL products, track them in vivo after infusion, and then deduce the preadoptive transfer (endogenous) frequencies of cells ultimately responsible for tumor regression. The summed in vivo posttransfer frequencies of the top 25 HTTCS-defined clonotypes originally detected in the infused CTL population were comparable with enumeration by binding of antigen peptide–human leukocyte antigen multimers, revealing that quantitative HTTCS is a reliable, multimer-independent alternative. The polyclonal CTL products were composed predominantly of clonotypes that were of very low frequency (VLF) in the endogenous samples, often below the limit of HTTCS detection (0.001%). In patients who achieved durable CRs, the composition of transferred CTLs was dominated (57 to 90%) by cells derived from a single VLF clonotype. Thus, HTTCS now reveals that tumor-specific CTLs enabling long-term tumor control originate from endogenous VLF populations that exhibit proliferative or survival advantages. Along with results indicating that naïve cell populations are most likely to contain cells that exist at VLF within the repertoire, our results provide a strong rationale for favoring T cells arising from VLF populations and with early differentiation phenotypes when selecting subset populations for adoptive transfer.

Merkel cell carcinoma: Current US incidence and projected increases based on changing demographics

Background Merkel cell carcinoma (MCC) incidence rates are rising and strongly age‐associated, relevant for an aging population. Objective Determine MCC incidence in the United States and project incident cases through the year 2025. Methods Registry data were obtained from the SEER‐18 Database, containing 6600 MCC cases. Age‐ and sex‐adjusted projections were generated using US census data. Results During 2000‐2013, the number of reported solid cancer cases increased 15%, melanoma cases increased 57%, and MCC cases increased 95%. In 2013, the MCC incidence rate was 0.7 cases/100,000 person‐years in the United States, corresponding to 2488 cases/year. MCC incidence increased exponentially with age, from 0.1 to 1.0 to 9.8 (per 100,000 person‐years) among age groups 40‐44 years, 60‐64 years, and ≥85 years, respectively. Due to aging of the Baby Boomer generation, US MCC incident cases are predicted to climb to 2835 cases/year in 2020 and 3284 cases/year in 2025. Limitations We assumed that the age‐adjusted incidence rate would stabilize, and thus, the number of incident cases we projected might be an underestimate. Conclusion An aging population is driving brisk increases in the number of new MCC cases in the United States. This growing impact combined with the rapidly evolving therapeutic landscape warrants expanded awareness of MCC diagnosis and management.