doi:10.1182/blood-2009-03-211714 Prepublished online May 18, 2009;2009 114: 535-546€€€€Ripley, Debbie A. Nathan, Carolyn M. Laurencot and Steven A. Rosenberg Carmen C. Brewer, Susan F. Rudy, Carter VanWaes, Jeremy L. Davis, Aarti Mathur, Robert T. Lee, Nicholas P. Restifo, Susan L. Schwarz, Alexandria P. Cogdill, Rachel J. Bishop, Hung Kim,Hughes, Udai S. Kammula, Richard E. Royal, Richard M. Sherry, John R. Wunderlich, Chyi-Chia R. Laura A. Johnson, Richard A. Morgan, Mark E. Dudley, Lydie Cassard, James C. Yang, Marybeth S.€

destroy, cancer. -derived cells can not only seek out, but also successfully CM need to be performed to determine whether these T already being used in the clinic, and future studies combining transfer of these persistent cells with the available drugs engrafted cells expressed high levels of inhibitory receptors. Drugs that block some of these inhibitory receptors are the number is small, the authors did not observe tumor regression with this therapy. Subsequent analysis showed that characteristics. However, although the patient CM skin melanocytes but also persisted long term and reacquired T into lytic effector cells, and transferred into five patients with metastatic melanoma. These cells not only trafficked to phenotype, then expanded, differentiated CM interleukin-2. The resulting cell population was confirmed to express a T and γ interferon−− manner. The authors sorted antigen-stimulated T cells on the basis of the ratio of two cytokines need to be identified and isolated in a high-throughput CM For transplant to be practical at the clinical level, T -derived cells into human patients. CM T now translate these studies by transferring melanoma-specific et al. able to survive long term after transplant. Wang ) are CM of the transferred T cells to persist and engraft after transplant. In animal studies, central memory T cells (T tumors but not healthy tissue into patients. Early trials suggest that the success of this therapy depends on the ability Researchers have long hoped to leverage these cells to treat cancer by transferring cells that specifically target deadly assassins that seek out and destroy invading organisms. −− Antigen-specific T cells are targeted killers The Right Cells for the Job

cDNAs encoding functional T cell receptor (TCR) α and β chains from a CD4+ T cell line (SG6) generated by repeated stimulation of a melanoma patient's peripheral blood mononuclear cells with HLA-DP4–restricted, NY-ESO-1–specific peptide p161-180 were cloned using a 5′rapid amplification of cDNA end method. Three different TCR α chains and 7 TCR β chains were found among the 84 α and 162 β cDNA clones tested. By screening different combination of the α/β chains using RNA electroporation, TRAV9-1 (Vα22.1) and TRBV20-1 (Vβ2) were found to be the functional pair in line SG6. Antibody blocking experiments confirmed that the specificity of TRAV9-1/TRBV20-1 mRNA-transfected T cells were CD4 dependent and HLA-DP4 restricted. A retroviral vector expressing both TRAV9-1 and TRBV20-1 was constructed and used for transduction of OKT3-stimulated peripheral blood lymphocytes from melanoma patients. TCR-transduced CD4 T cells were capable of recognizing peptide-pulsed antigen-presenting cells (Epstein-Barr virus transformed B-cells, dendritic cells, and peripheral blood mononuclear cells), and protein-pulsed dendritic cells. Transduced cells were also capable of proliferation upon peptide stimulation and recognized peptide concentrations that were recognized by the parental line (0.2 μM). In contrast to SG6, which could not recognize human tumors, TCR-transduced CD4 T cells could specifically recognize NY-ESO-1/HLA-DP4–expressing melanoma cells. Major histocompatibility complex class II TCR-transduced CD4 T cells provides an alternative source of tumor antigen-specific T cells for adoptive immunotherapy of cancer patients.

With advances in surgical techniques and chemotherapeutic agents, mortality rates from epithelial ovarian cancer (EOC) have slightly decreased over the last 30 years. However, EOC still ranks as the most deadly gynecologic cancer with an overall 5-year survival rate of 45%. Prognosis is especially disappointing for women with platinum-resistant disease, where 80% of patients will fail to respond to available therapies. Emerging treatment strategies have subsequently focused on targets which are integral to tumor growth and metastasis. In this review, we will focus on those innovative agents currently under investigation in clinical trials.

When performing an exploratory post hoc subgroup analysis, it is important to understand and try to mitigate the pitfalls of such an analysis. In a recent issue of the Journal, huber et al. (1) reported an exploratory analysis of the ImPACt trial (2) at an arbitrary age cutoff of 65 years, which was not used in the published primary analysis (2). Although this age cutoff makes sense for a Center for medicare and medicaid Services analysis because of the age of medicare eligibility, the asymmetry (~2:1) of patients above and below that cutoff, as well as the small subset of patients younger than age 65 in the placebo group (66 of 737 or 9.0%), amplifies the potential for small changes in the subgroup to result in misleading data. In the absence of a compelling scientific rationale, use of a median age would limit the potential imbalance. the analysis of the ImPACt study (2) showed that patients above and below the median age of 71 years had a similar hazard ratio (measure of effectiveness of sipuleucel-t) in which overall survival favored patients treated with vaccine compared with those who received the control. the authors also suggested that older patients in the placebo group may have a shorter survival than can be expected in other studies. In fairness, it should be mentioned that comparing asymmetric, small post hoc subsets from one trial with small post hoc subsets from another trial with different enrollment criteria is fraught with numerous obvious perils that could lead to imbalanced comparisons. In their final argument, huber et al. (1) stated that overall survival of patients in the placebo arm was potentially negatively affected by the placebo treatment. the assertion that removing a large proportion of circulating lymphocytes may negatively impact the immune system may seem persuasive superficially. however, no evaluable data support this assertion. We have previously reported the National Institutes of health experience on lymphocyte levels in more than 400 healthy subjects following serial leukapheresis procedures (4957 donations with a median of 6.8 L processed per procedure) (3). Although there were decreases in lymphocyte count over time in subjects undergoing repeated leukapheresis, after 2–9 procedures the median lymphocyte count decreased by less than 10%, which is not clinically significant. In the majority of subjects who had a subsequent leukapheresis within 56 days of the previous procedure (n = 3370), the median change in absolute lymphocyte count (a count before leukapheresis compared with a count before the next leukapheresis [median = 23 days]) was −3.6%. most importantly, for each of the more than 400 subjects who underwent serial leukapheresis, we took careful histories and physical exams and observed no susceptibility to infectious diseases or cancer. When one considers that circulating lymphocytes are only a fraction of the total number of lymphocytes in the human body (~2%) (4), and that lymphocytes can readily traffic from the organs to the blood, even removal of the total circulating lymphocyte pool without any reinfusion of lymphocytes would still leave 98% of the total body lymphocytes within the patient to perform immune surveillance and protect against disease. Finally, if patients in the placebo group suffered a clinically significant disruption of their immune systems, there would likely be a resulting increase in infections, which was thoroughly investigated and not observed (5).

We reported previously that vaccination of reconstituted, lymphopenic mice resulted in a higher frequency of tumor‐specific effector T cells with therapeutic activity than vaccination of normal mice. Here, we show that lymphopenic mice reconstituted with spleen cells from tumor‐bearing mice (TBM), a situation that resembles the clinical condition, failed to generate tumor‐specific T cells with therapeutic efficacy. However, depletion of CD25+ Treg from the spleen cells of TBM restored tumor‐specific priming and therapeutic efficacy. Adding back TBM CD25+ Treg to CD25− naïve and TBM donor T cells prior to reconstitution confirmed their suppressive role. CD25+ Treg from TBM prevented priming of tumor‐specific T cells since subsequent depletion of CD4+ T cells did not restore therapeutic efficacy. This effect may not be antigen‐specific as three histologically distinct tumors generated CD25+ Treg that could suppress the T‐cell immune response to a melanoma vaccine. Importantly, since ex vivo depletion of CD25+ Treg from TBM spleen cells prior to reconstitution and vaccination fully restored the generation of therapeutic effector T cells, even in animals with established tumor burden, we have initiated a translational clinical trial of this strategy in patients with metastatic melanoma.

We propose a freely accessible web-based pipeline, which processes raw microarray scan data to obtain experimentally consolidated gene expression values. The tool MADSCAN, which stands for MicroArray Data Suites of Computed ANalysis, makes a practical choice among the numerous methods available for filtering, normalizing and scaling of raw microarray expression data in a dynamic and automatic way. Different statistical methods have been adapted to extract reliable information from replicate gene spots as well as from replicate microarrays for each biological situation under study. A carefully constructed experimental design thus allows to detect outlying expression values and to identify statistically significant expression values, together with a list of quality controls with proposed threshold values. The integrated processing procedure described here, based on multiple measurements per gene, is decisive for reliably monitoring subtle gene expression changes typical for most biological events.

We identified a polyclonal CD8+ T-cell response against mutant KRAS G12D in tumor-infiltrating lymphocytes obtained from a patient with metastatic colorectal cancer. We observed objective regression of all seven lung metastases after the infusion of approximately 1.11×1011 HLA-C*08:02-restricted tumor-infiltrating lymphocytes that were composed of four different T-cell clonotypes that specifically targeted KRAS G12D. However, one of these lesions had progressed on evaluation 9 months after therapy. The lesion was resected and found to have lost the chromosome 6 haplotype encoding the HLA-C*08:02 class I major histocompatibility complex (MHC) molecule. The loss of expression of this molecule provided a direct mechanism of tumor immune evasion. Thus, the infusion of CD8+ cells targeting mutant KRAS mediated effective antitumor immunotherapy against a cancer that expressed mutant KRAS G12D and HLA-C*08:02.

We demonstrated in previous studies that interleukin (IL) -2 supports in vitro cell proliferation of donor-derived cytotoxic T lymphocyte (CTL) lines directed against different types of leukemia blasts. The aim of this study was to compare the capacity of IL-15 with that of IL-2 in supporting the proliferation and cytotoxic activity of antileukemia CTL cultures, and their influence on T-cell memory compartment differentiation. Antileukemia CTL lines were generated using donor-derived dendritic cells pulsed with apoptotic leukemia blasts, in the presence of IL-12 and IL-7, during the primary culture, and expanded through 2 rounds of leukemia-specific stimulation and 1 round of antigen-independent expansion, each supplemented with either IL-2 or IL-15. Both IL-2–supplemented (IL-2–CTLs) and IL-15–supplemented (IL-15–CTLs) lines contained predominant numbers of CD45RA−/CCR7− effector memory (TEM) and CD45RA+/CCR7− (TEMRA+) T cells. Significantly higher numbers (P<0.05) of CD8-positive central memory T cells (TCM), and higher expansion rate, together with comparable cytotoxic activity, were observed in IL-15–CTLs compared with IL-2–CTLs. Altogether, these results demonstrate that IL-15 enhances recovery of CTL activity, without loss of leukemia-directed specificity, and favors expansion of TCM CD8-positive cells, expected to exhibit long-term survival and differentiation capacity in vivo in the presence of a limited amount of antigen.

T‐cell immunotherapy is a promising approach to treat disseminated cancer. However, it has been limited by the ability to isolate and expand T cells restricted to tumour‐associated antigens. Using ex vivo gene transfer, T cells from patients can be genetically engineered to express a novel T cell receptor or chimeric antigen receptor to specifically recognize a tumour‐associated antigen and thereby selectively kill tumour cells. Indeed, genetically engineered T cells have recently been successfully used for cancer treatment in a small number of patients. Here we review the recent progress in the field, and summarize the challenges that lie ahead and the strategies being used to overcome them.

Tumour angiogenesis is a fast growing domain in tumour biology. Many growth factors and mechanisms have been unravelled. For almost 30 years, the sprouting of new vessels out of existing ones was considered as an exclusive way of tumour vascularisation. However, over the last years several additional mechanisms have been identified. With the discovery of the contribution of intussusceptive angiogenesis, recruitment of endothelial progenitor cells, vessel co‐option, vasculogenic mimicry and lymphangiogenesis to tumour growth, anti‐tumour targeting strategies will be more complex than initially thought. This review highlights these processes and intervention as an potential application in cancer therapy. It is concluded that future anti‐vascular therapies might be most beneficial when based on multimodal anti‐angiogenic, anti‐ vasculogenic mimicry and anti‐lymphangiogenic strategies. Introduction Tumours can grow to a size of approximately 1‐2 mm3 before their metabolic demands are restricted due to the diffusion limit of oxygen and nutrients. In order to grow beyond this size, the tumour switches to an angiogenic phenotype and attracts blood vessels from the surrounding stroma. This process is regulated by a variety of pro‐ and anti‐angiogenic factors, and is a prerequisite for further outgrowth of the tumour 1. Next to sprouting angiogenesis, the process by which new vessels are formed from pre‐ existing vasculature, several other mechanisms of neovascularization have been identified in tumours, including intussusceptive angiogenesis, the recruitment of endothelial progenitor cells, vessel co‐option, vasculogenic mimicry and lymphangiogenesis (figure 1). Due to application for treatment of disease, these processes gained a lot of interest over the last years. This review summarizes the different mechanisms of tumour vascularization, the molecular players that are involved and their relevance in clinical practice. Sprouting angiogenesis Sprouting angiogenesis is the growth of new capillary vessels out of pre‐existing ones. These blood vessels will provide expanding tissues and organs with oxygen and nutrients, and remove the metabolic waste. Angiogenesis takes place in physiological situations, such as embryonic development, wound healing and reproduction. It also plays an important role in many pathologies, like diabetes 2, rheumatoid arthritis 3, cardiovascular ischemic complications 4, and cancer 5. In cancer, sprouting angiogenesis is not only important in primary tumours, it is also involved in metastasis formation and further outgrowth of metastases 6.

Treatment of metastatic melanoma patients with adoptively transferred tumor infiltrating lymphocytes (TIL) has developed into an effective therapy. Various studies reported objective responses of 50% and more. The use of unselected, minimally cultured, bulk TIL (Young-TIL) has simplified the TIL production process and may therefore, allow the accessibility of this approach to cancer centers worldwide. This article describes the precise process leading to the large-scale production of Young-TIL for therapy. We have enrolled 55 melanoma patients and optimized their Young-TIL generation process. Young-TIL cultures were successfully established for 51 of 55 (93%) patients in 16.7±5.5 days. In a large-scale expansion procedure Young-TIL of 32 patients were further expanded to treatment levels, resulting in a final number of 4.5×1010 ±2.0×1010 TIL. Fifteen of 31 (48%) patients, who were evaluated, achieved a clinical response, including 4 complete and 11 partial responses. We confirmed the significant correlation between short culture duration, high number of infused cells, and tumor regression. A high percentage of CD8+ T cells in the infusion product was beneficial to achieve an objective response. All responding patients were treated with Young-TIL cultures established in <20 days. In summary, we describe here an efficient and reliable method to generate Young-TIL for adoptive transfer therapy, which may easily be adopted by other cancer centers and can lead to objective responses in 50% of refractory melanoma patients. In the future this approach may be used also in other types of malignancies.

Treatment of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of autologous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at (http://www.clinialtrials.gov) as NCT00608257.

Treatment of C57BL/6 mice with cyclophosphamide (100 mg/kg) and fludarabine (200 mg/kg) induced nonmyeloablative lymphodepletion without inhibiting D5 melanoma tumor growth. Using this model, we found that induction of lymphopenia before adoptive transfer of ex vivo anti-CD3/CD28 activated and interleukin-2 expanded D5-G6 tumor draining lymph node cells enhanced the antitumor efficacy of the infused cells in both pulmonary metastases and subcutaneous D5 bearing mice. However, induction of lymphopenia did not promote intratumoral or extratumoral proliferation or accumulation of the infused cells. We have previously shown that radiotherapy enhances the therapeutic efficacy of intratumoral unpulsed dendritic cell vaccination in subcutaneous murine tumor models by augmenting the induction of antitumor cellular immune responses. Here, we confirmed this finding in a murine metastatic melanoma liver tumor model. Furthermore, local tumor irradiation combined with intratumoral dendritic cell administration significantly enhanced the therapeutic efficacy of tumor-reactive T cell adoptive transfer in this lymphodepleted liver tumor model. This was evident by reduced liver tumor size, decreased incidence of spontaneous intra-abdominal metastasis, and prolonged survival, resulting in 46% of mice cured. This enhanced antitumor activity was associated with a selective increase in proliferation, accumulation, and function of CD4+ rather than CD8+ infused cells. This multimodality regimen may have translational applications for the treatment of human cancers.

Transfer of autologous tumor-specific tumor infiltrating lymphocytes (TILs) in adoptive immunotherapy can mediate the regression of tumor in patients with metastatic melanoma. In this procedure, TILs from resected tumors are expanded in vitro, then administered to patients and further stimulated to proliferate in vivo by the administration of high dose IL-2. After in vitro expansion, TILs are often dominated by a few specific clonotypes, and recently it was reported that the persistence in vivo of one or more of these clonotypes correlated with positive therapeutic response. We and others have previously shown that repeated in vitro stimulation and clonal expansion of normal human T lymphocytes results in progressive decrease in telomerase activity and shortening of telomeres, ultimately resulting in replicative senescence. In the studies reported here, we therefore compared telomerase activity and telomere length in persistent and nonpersistent TIL clonotypes before transfer in vivo, and found a correlation between telomere length and clonal persistence. We also observed that TILs proliferate extensively in vivo in the days after transfer, but fail to induce substantial telomerase activity, and undergo rapid decreases in telomere length within days after transfer. Thus, in vivo loss of telomeres by clonotypes that have the shortest telomeres at the time of administration may drive these clones to replicative senescence, whereas cells with longer telomeres are able to persist and mediate antitumor effects. These findings are relevant both to predicting effectiveness of adoptive immunotherapy and in deriving strategies for improving effectiveness by sustaining telomere length.

Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology, that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. While successful, complete editing requires extensive cell manipulation and four transduction procedures. Here we propose a novel and clinically feasible ‘single TCR editing’ (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165 specific TCR. SE allowed the rapid production of high numbers of tumor specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TR), single edited T cells killed efficiently NY-ESO-1 targets; however, while TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft versus host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach, able to increase the safety and efficacy of cancer adoptive immunotherapy. For personal use only. on June 24, 2017. by guest www.bloodjournal.org From

Tolerogenic dendritic cells in the tumor microenvironment can inhibit the generation and maintenance of robust anti-tumor T cell responses. Here, we investigated the effects of local delivery of CD40 ligand (CD40L) by tumor-reactive CD8 + T cells on dendritic cell activation and anti-tumor T cell responses in the TRansgenic Adenocarcimona of the Mouse Prostate (TRAMP) model. To increase the immunostimulatory signal, CD40L was engineered, by deleting the majority of the cytoplasmic domain, to increase both its level of expression and duration on the surface of CD8 + T cells. Tumor-reactive CD8 + T cells expressing the truncated form of CD40L stimulated maturation of dendritic cells in vitro and in the prostate draining lymph nodes (PDLN) in vivo. Following dendritic cell maturation, a significantly higher fraction of adoptively transferred, tumor-reactive (reporter) CD8 + T cells was stimulated to express IFN- γ and infiltrate the prostate tissue. The antitumor CD8 + T cell response was further enhanced if TRAMP mice were also immunized with a tumor-specific antigen. These findings demonstrate that augmented T cell responses can be achieved by engineering tumor-reactive T cells to deliver stimulatory signals to dendritic cells in the tumor microenvironment. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI , holds the copyright to this manuscript.

Through the adoptive transfer of lymphocytes after host immunodepletion, it is possible to mediate objective cancer regression in human patients with metastatic melanoma. However, the generation of tumor-specific T cells in this mode of immunotherapy is often limiting. Here we report the ability to specifically confer tumor recognition by autologous lymphocytes from peripheral blood by using a retrovirus that encodes a T cell receptor. Adoptive transfer of these transduced cells in 15 patients resulted in durable engraftment at levels exceeding 10% of peripheral blood lymphocytes for at least 2 months after the infusion. We observed high sustained levels of circulating, engineered cells at 1 year after infusion in two patients who both demonstrated objective regression of metastatic melanoma lesions. This study suggests the therapeutic potential of genetically engineered cells for the biologic therapy of cancer.

This review provides a review of the basic biology of the major cytokines under consideration for use in tumor immunotherapy. The authors also describe the clinical role of cytokine therapy for human tumors, with a focus on melanoma and renal cell carcinoma. Finally, they present details of new cytokines and suggest possible direction for future clinical investigation using new cytokines or combinations of biologic agents. A better understanding of cytokine biology, coupled with new insights into the role of immunoregulation and the microenvironment in patients who have cancer, has provided new therapeutic targets for the treatment of human cancer.

The transfusion of lymphocytes, referred to as adoptive T cell therapy, is being tested for the treatment of cancer and chronic infections. Adoptive T cell therapy has the potential to enhance antitumor immunity, augment vaccine efficacy, and limit graft-versus-host disease. This form of personalized medicine is now in various early- and late-stage clinical trials. These trials are currently testing strategies to infuse tumor-infiltrating lymphocytes, CTLs, Th cells, and Tregs. Improved molecular biology techniques have also increased enthusiasm and feasibility for testing genetically engineered T cells. The current status of the field and prospects for clinical translation are reviewed herein.