Generating CAR T cells from tumor-infiltrating lymphocytes.

Background: Tumor-infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T-cell therapies have demonstrated promising, though limited, efficacy against melanoma. Methods: We designed a model system to explore the efficacy of dual specific T cells derived from melanoma patient TILs by transduction with a Her2-specific CAR. Results: Metastatic melanoma cells in our biobank constitutively expressed Her2 antigen. CAR-TIL produced greater amounts of IFN compared with parental TIL, when co-cultured with Her2 expressing tumor lines, including autologous melanoma tumor lines, although no consistent increase in cytotoxicity by TIL was afforded by expression of a CAR. Results of an in vivo study in NSG mice demonstrated tumor shrinkage when CAR-TILs were used in an adoptive cell therapy protocol. Conclusion: Potential limitations of transduced TIL in our study included limited proliferative potential and a terminally differentiated phenotype, which would need addressing in further work before consideration of clinical translation.

Mobile Health Intervention to Increase Oral Cancer Therapy Adherence in Patients With Chronic Myeloid Leukemia (The REMIND System): Clinical Feasibility and Acceptability Assessment.

BACKGROUND: Optimal dosing of oral tyrosine kinase inhibitor therapy is critical to treatment success and survival of patients with chronic myeloid leukemia (CML). Drug intolerance secondary to toxicities and nonadherence are significant factors in treatment failure. OBJECTIVE: The objective of this study was to develop and pilot-test the clinical feasibility and acceptability of a mobile health system (REMIND) to increase oral drug adherence and patient symptom self-management among people with CML (chronic phase). METHODS: A multifaceted intervention was iteratively developed using the intervention development framework by Schofield and Chambers, consisting of defining the patient problem and iteratively refining the intervention. The clinical feasibility and acceptability were examined via patient and intervention nurse interviews, which were audiotaped, transcribed, and deductively content analyzed. RESULTS: The intervention comprised 2 synergistically operating elements: (1) daily medication reminders and routine assessment of side effects with evidence-based self-care advice delivered in real time and (2) question prompt list (QPL) questions and routinely collected individual patient adherence and side effect profile data used to shape nurses' consultations, which employed motivational interviewing to support adoption of self-management behaviors. A total of 4 consultations and daily alerts and advice were delivered over 10 weeks. In total, 58% (10/17) of patients and 2 nurses participated in the pilot study. Patients reported several benefits of the intervention: help in establishing medication routines, resolution of symptom uncertainty, increased awareness of self-care, and informed decision making. Nurses also endorsed the intervention: it assisted in establishing pill-taking routines and patients developing effective solutions to adherence challenges. CONCLUSIONS: The REMIND system with nurse support was usable and acceptable to both patients and nurses. It has the potential to improve adherence and side-effect management and should be further evaluated.

An ultrastructural investigation of tumors undergoing regression mediated by immunotherapy.

While immunotherapy employing chimeric antigen receptor (CAR) T cells can be effective against a variety of tumor types, little is known about what happens within the tumor at an ultrastructural level during tumor regression. Here, we used transmission electron microscopy to investigate morphologic and cellular features of tumors responding to immunotherapy composed of adoptive transfer of dual-specific CAR T cells and a vaccine, supported by preconditioning irradiation and interleukin-2. Tumors responded rapidly, and large areas of cell death were apparent by 4 days after treatment. The pleomorphic and metabolically active nature of tumor cells and phagocytic activity of macrophages were apparent in electron microscopic images of tumors prior to treatment. Following treatment, morphologic features of various types of tumor cell death were observed, including apoptosis, paraptosis and necrosis. Large numbers of lipid droplets were evident in tumor cells undergoing apoptosis. Macrophages were the predominant leukocyte type infiltrating tumors before treatment. Macrophages decreased in frequency and number after treatment, whereas an increasing accumulation of neutrophils and T lymphocytes was observed following treatment. Phagocytic activity of macrophages and neutrophils was apparent, while T cells could be observed in close association with tumor cells with potential immunological synapses present. These observations highlight the cellular composition and ultrastructural appearance of tumors undergoing regression mediated by immunotherapy.

Dual-specific Chimeric Antigen Receptor T Cells and an Indirect Vaccine Eradicate a Variety of Large Solid Tumors in an Immunocompetent, Self-antigen Setting.

Purpose: While adoptive transfer of T cells bearing a chimeric antigen receptor (CAR) can eliminate substantial burdens of some leukemias, the ultimate challenge remains the eradication of large solid tumors for most cancers. We aimed to develop an immunotherapy approach effective against large tumors in an immunocompetent, self-antigen preclinical mouse model.Experimental Design: In this study, we generated dual-specific T cells expressing both a CAR specific for Her2 and a TCR specific for the melanocyte protein (gp100). We used a regimen of adoptive cell transfer incorporating vaccination (ACTIV), with recombinant vaccinia virus expressing gp100, to treat a range of tumors including orthotopic breast tumors and large liver tumors.Results: ACTIV therapy induced durable complete remission of a variety of Her2+ tumors, some in excess of 150 mm2, in immunocompetent mice expressing Her2 in normal tissues, including the breast and brain. Vaccinia virus induced extensive proliferation of T cells, leading to massive infiltration of T cells into tumors. Durable tumor responses required the chemokine receptor CXCR3 and exogenous IL2, but were independent of IFNγ. Mice were resistant to tumor rechallenge, indicating immune memory involving epitope spreading. Evidence of limited neurologic toxicity was observed, associated with infiltration of cerebellum by T cells, but was only transient.Conclusions: This study supports a view that it is possible to design a highly effective combination immunotherapy for solid cancers, with acceptable transient toxicity, even when the target antigen is also expressed in vital tissues. Clin Cancer Res; 23(10); 2478-90. ©2016 AACR.

Expression of a Chimeric Antigen Receptor in Multiple Leukocyte Lineages in Transgenic Mice.

Genetically modified CD8+ T lymphocytes have shown significant anti-tumor effects in the adoptive immunotherapy of cancer, with recent studies highlighting a potential role for a combination of other immune subsets to enhance these results. However, limitations in present genetic modification techniques impose difficulties in our ability to fully explore the potential of various T cell subsets and assess the potential of other leukocytes armed with chimeric antigen receptors (CARs). To address this issue, we generated a transgenic mouse model using a pan-hematopoietic promoter (vav) to drive the expression of a CAR specific for a tumor antigen. Here we present a characterization of the immune cell compartment in two unique vav-CAR transgenic mice models, Founder 9 (F9) and Founder 38 (F38). We demonstrate the vav promoter is indeed capable of driving the expression of a CAR in cells from both myeloid and lymphoid lineage, however the highest level of expression was observed in T lymphocytes from F38 mice. Lymphoid organs in vav-CAR mice were smaller and had reduced cell numbers compared to the wild type (WT) controls. Furthermore, the immune composition of F9 mice differed greatly with a significant reduction in lymphocytes found in the thymus, lymph node and spleen of these mice. To gain insight into the altered immune phenotype of F9 mice, we determined the chromosomal integration site of the transgene in both mouse strains using whole genome sequencing (WGS). We demonstrated that compared to the 7 copies found in F38 mice, F9 mice harbored almost 270 copies. These novel vav-CAR models provide a ready source of CAR expressing myeloid and lymphoid cells and will aid in facilitating future experiments to delineate the role for other leukocytes for adoptive immunotherapy against cancer.

Cross-talk between tumors can affect responses to therapy.

Advanced stages of cancer often involve multiple tumors in different locations in the body. These tumors are associated with a microenvironment that can influence tumor responses to immunotherapy. Whether tumors and their disparate microenvironment can interact together at distance in a multiple tumor setting, through a form of cross-talk, and affect their responses to immunotherapy has never been described. Our study investigated the cross-talk between two tumors with disparate microenvironments in a mouse model. We demonstrated that immunosuppressive visceral tumors could influence distant subcutaneous (SC) tumors to render them resistant to immunotherapy. We observed distinct modifications in the SC tumor microenvironment following cross-talk with kidney tumors that exhibit a type-2 macrophage-related immunosuppressive microenvironment. Indeed, when a concomitant kidney tumor was present in the mouse, the SC tumors were highly infiltrated with M2 macrophages and had a reduced T cell and NK cell effector immune profile. Finally, blocking the M2-associated chemokine CCL2 or depleting macrophages, significantly improved the effect of immunotherapy on growth of SC tumors in the presence of concomitant kidney tumors. This work emphasizes the potential negative influence that a tumor, with a strong immunosuppressive microenvironment, can exert on distant tumors that would normally be treatment-responsive. This report may lead to a new vision of the prioritization in the treatment of advanced metastatic cancer.

Psychological distress, quality of life, symptoms and unmet needs of colorectal cancer survivors near the end of treatment.

PURPOSE: This study investigated psychological morbidity, quality of life (QoL), colorectal cancer (CRC)-specific symptoms and supportive care needs in a CRC population at the end of treatment (EOT). METHODS: CRC survivors (n = 152) completed a post-treatment baseline questionnaire as part of a multisite supportive care randomised controlled trial (SurvivorCare). CRC survivors had completed treatment with curative intent within 0 to 6 months. Measures are as follows: Brief Symptom Inventory 18 (BSI-18) (psychological morbidity), EORTC QLQ-C30 and QLQ-CR29 (QoL and CRC-specific symptoms and problems) and Cancer Survivors' Unmet Needs (CaSUN) measure with a simplified response format (unmet needs). Linear regression models were used to compare participants' QoL with a general population sample. Correlation analysis examined associations between psychological morbidity, QoL and CRC-specific symptoms and problems. RESULTS: Average participant age was 64 years, and 51% were male. The majority (68%) had stage 3 disease. In comparison to population norms, CRC survivors had lower depression and anxiety scores (47.4 and 45.6, respectively) but higher somatisation, and lower role, cognitive and social functioning (p < 0.001). CRC survivors had higher fatigue, nausea/vomiting, appetite loss, diarrhoea and financial problems (all p < 0.001), as well as pain (p = 0.002) and constipation (p = 0.019). CRC-specific psychological scores were positively correlated with all three BSI domain scores, and pain and fatigue symptom scores on the QLQ-C30 while negatively correlated with all five functional scales of the QLQ-C30. CONCLUSIONS: CRC survivors reported good mental health at EOT. Role and social functioning were impaired compared to population norms, possibly related to physical symptoms. IMPLICATIONS FOR CANCER SURVIVORS: Findings may help guide consultations with patients and inform the design of more tailored supportive care interventions. TRIAL REGISTRATION: ACTRN12610000207011.

Clinical application of genetically modified T cells in cancer therapy.

Immunotherapies are emerging as highly promising approaches for the treatment of cancer. In these approaches, a variety of materials are used to boost immunity against malignant cells. A key component of many of these approaches is functional tumor-specific T cells, but the existence and activity of sufficient T cells in the immune repertoire is not always the case. Recent methods of generating tumor-specific T cells include the genetic modification of patient lymphocytes with receptors to endow them with tumor specificity. These T cells are then expanded in vitro followed by infusion of the patient in adoptive cell transfer protocols. Genes used to modify T cells include those encoding T-cell receptors and chimeric antigen receptors. In this review, we provide an introduction to the field of genetic engineering of T cells followed by details of their use against cancer in the clinic.

Foxp3 expression in macrophages associated with RENCA tumors in mice.

The transcription factor Foxp3 represents the most specific functional marker of CD4+ regulatory T cells (TRegs). However, previous reports have described Foxp3 expression in other cell types including some subsets of macrophages, although there are conflicting reports and Foxp3 expression in cells other than Treg is not well characterized. We performed detailed investigations into Foxp3 expression in macrophages in the normal tissue and tumor settings. We detected Foxp3 protein in macrophages infiltrating mouse renal cancer tumors injected subcutaneously or in the kidney. Expression was demonstrated using flow cytometry and Western blot with two individual monoclonal antibodies. Further analyses confirmed Foxp3 expression in macrophages by RT PCR, and studies using ribonucleic acid-sequencing (RNAseq) demonstrated a previously unknown Foxp3 messenger (m)RNA transcript in tumor-associated macrophages. In addition, depletion of Foxp3+ cells using diphtheria toxin in Foxp3DTR mice reduced the frequency of type-2 macrophages (M2) in kidney tumors. Collectively, these results indicate that tumor-associated macrophages could express Foxp3.

Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers.

In order to stimulate antigen presentation and T cell activity against cancer, we treated three different tumor models in mice with the monoclonal antibodies anti-CD40 plus anti-CD137 (BiMab). In a subcutaneous transplantable MC38 colon cancer model, there was significant enhancement in the survival of mice following BiMab treatment. Anti-CD40 has shown considerable success against lymphoma in previous studies by other investigators, and we also showed in this study that, in a model of Eµ-Myc lymphoma, there was a statistically significant enhancement of survival of mice following BiMab treatment. Following the success of the BiMab treatment in the previous two models, we wished to determine if it would be successful in a mouse model of multiple myeloma. Firstly, we tested a transplantable model of disease in which multiple myeloma cells derived from Vk*MYC mice were injected intravenously. A minor proportion of anti-CD137 and BiMab treated mice experienced prolongation of life beyond 250 days. Then we tested the therapy in a spontaneously occurring multiple myeloma model, in Vk*MYC transgenic mice. The majority of mice treated survived longer than control mice, although statistical significance was not demonstrated.

Routes of delivery for CpG and anti-CD137 for the treatment of orthotopic kidney tumors in mice.

We have found previously that the tumor cell lines, Renca (a renal cancer) and MC38 (a colon tumor) which had been injected subcutaneously in mice, could be successfully treated with a combination therapy of an oligodeoxynucleotide (CpG1826) (injected intratumorally) and anti-CD137 antibody (injected intraperitoneally). Thus the combination treatment was expected to initiate a "danger" signal via TLR9 on immune cells, and the anti-CD137 was expected to further activate T cells. In the present study, we found that several other tumor types injected subcutaneously could also be successfully treated with this combination therapy. In addition, we wished to determine if the treatment could work as effectively in an orthotopic metastatic model, which is more physiologically relevant to cancer in humans. Renca was selected as we were familiar with injecting this orthotopically into the outer cortex of the kidney in mice, and it spontaneously metastasizes to lung and abdominal sites. We tested various routes of delivery of CpG combined with intraperitoneal delivery of anti-CD137. Orthotopic tumors were injected with CpG intratumorally, using ultrasound-guided delivery on multiple occasions, combined with anti-CD137 intraperitoneally. A reduction in primary tumor size was observed following intratumoral injection of CpG compared to other treatments. We found that there was a statistically significant increase in survival of mice with orthotopic Renca tumor following intratumoral injection of CpG. However, we determined that the most effective route of delivery of CpG was intravenous, which led to further significantly enhanced survival of mice when combined with anti-CD137 intraperitoneally, likely due to inhibition of metastatic disease. Our data supports future development of this combination therapy for cancer.

Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy.

The tumor microenvironment can promote tumor growth and reduce treatment efficacy. Tumors can occur in many sites in the body, but how surrounding normal tissues at different anatomical sites affect tumor microenvironments and their subsequent response to therapy is not known.We demonstrated that tumors from renal, colon, or prostate cell lines in orthotopic locations responded to immunotherapy consisting of three agonist antibodies, termed Tri-mAb, to a much lesser extent than the same tumor type located subcutaneously. A tissue-specific response to Tri-mAb was confirmed by ex vivo separation of subcutaneous (SC) or orthotopic tumor cells from stromal cells, followed by reinjection of tumor cells into the opposite site. Compared with SC tumors, orthotopic tumors had a microenvironment associated with a type 2 immune response, related to immunosuppression, and an involvement of alternatively activated macrophages in the kidney model. Orthotopic kidney tumors were more highly vascularized than SC tumors. Neutralizing the macrophage- and Th2-associated molecules chemokine (C-C motif) ligand 2 or interleukin-13 led to a significantly improved therapeutic effect. This study highlights the importance of the tissue of implantation in sculpting the tumor microenvironment. These are important fundamental issues in tumor biology and crucial factors to consider in the design of experimental models and treatment strategies.

The potential impact of mouse model selection in preclinical evaluation of cancer immunotherapy.

This commentary provides the authors' perspective on the article "Routes of delivery for CpG and anti-CD137 for the treatment of orthotopic kidney tumors in mice", published in PLoS ONE. It also discusses the caveats of using subcutaneous tumors to model the treatment of human cancers versus orthotopic mouse models that more closely mimic human disease.

Differential potency of regulatory T cell-mediated immunosuppression in kidney tumors compared to subcutaneous tumors.

In many cancers, regulatory T cells (Treg) play a crucial role in suppressing the effector immune response thereby permitting tumor development. Indeed, in mouse models, their depletion can promote the regression of tumors of various origins, including renal cell carcinoma when located subcutaneous (SC). In the present study, we aimed to assess the importance of Treg immunosuppression in the physiologic context of metastatic renal carcinoma (Renca) disease. To that purpose we inoculated renal tumors orthotopically, intra-kidney (IK), in mice. Treg depletions were performed using anti-CD4 antibody in wild type mice or diphtheria toxin (DT) in Foxp3DTR transgenic mice. Our main observation was that Treg were not the key immunosuppressive component of the IK tumoral microenvironment, compared to the same tumors located SC. We demonstrated that the CD8+ effector immune response was still suppressed in IK tumors when compared to SC tumors, following Treg depletion. Furthermore, the level of program cell death protein (PD)-1 was increased on the surface of CD4+ T cells infiltrating IK tumors compared to SC tumors. Finally, the Treg-independent immunosuppression, occurring in IK tumors, was potent enough to inhibit regression of concomitant SC tumors, normally responsive to Treg depletion. Our findings provide further insight into the immunosuppressive nature of the immune response generated in the kidney microenvironment, suggesting that it can have additional mechanisms in addition to Treg. These observations might help to identify better targets from the kidney tumor microenvironment for future cancer therapies.

Enhancing immunotherapy using chemotherapy and radiation to modify the tumor microenvironment.

The tumor microenvironment is a complex assortment of cells that includes a variety of leukocytes. The overall effect of the microenvironment is to support the growth of tumors and suppress immune responses. Immunotherapy is a highly promising form of cancer treatment, but its efficacy can be severely compromised by an immunosuppressive tumor microenvironment. Chemotherapy and radiation treatment can mediate tumor reduction through cytotoxic effects, but it is becoming increasingly clear that these forms of treatment can be used to modify the tumor microenvironment to liberate tumor antigens and decrease immunosuppression. Chemotherapy and radiotherapy can be used to modulate the tumor microenvironment to enhance immunotherapy.

Immune modulation of the tumor microenvironment for enhancing cancer immunotherapy.

There is much promise in the use of immunotherapy for the treatment of cancer. Approaches such as those using antibodies or adoptive cell transfer can mediate complete tumor regression in a proportion of patients. However, the tumor microenvironment can inhibit immune responses leading to ineffective or suboptimal responses of tumors to immunotherapy in the majority of cases. As our knowledge of the tumor microenvironment increases, many strategies are emerging for changing the immunosuppressive nature of the tumor toward a microenvironment able to support immunity. These strategies aim to enhance the ability of immunotherapies to initiate effective immune responses able to destroy tumors. In this article, we review approaches that use immunomodulators specifically to modify the tumor microenvironment, and their use in combination with other immune-based strategies for cancer therapy.

Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia.

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence.

Gene-engineered T cells for cancer therapy.

T cells have the capacity to eradicate diseased cells, but tumours present considerable challenges that render T cells ineffectual. Cancer cells often make themselves almost 'invisible' to the immune system, and they sculpt a microenvironment that suppresses T cell activity, survival and migration. Genetic engineering of T cells can be used therapeutically to overcome these challenges. T cells can be taken from the blood of cancer patients and then modified with genes encoding receptors that recognize cancer-specific antigens. Additional genes can be used to enable resistance to immunosuppression, to extend survival and to facilitate the penetration of engineered T cells into tumours. Using genetic modification, highly active, self-propagating 'slayers' of cancer cells can be generated.

Chimeric antigen receptor-redirected T cells display multifunctional capacity and enhanced tumor-specific cytokine secretion upon secondary ligation of chimeric receptor.

AIM: The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR). MATERIAL & METHODS: In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor. RESULTS: Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation. A multifunctional capacity of these CAR T cells was also demonstrated, where 13.7% of cells were found to simultaneously express IFN-γ and CD107a, indicative of cytolytic granule release. In addition, the CAR T cells were able to respond to a greater degree on the second ligation of CAR, which has not been previously shown. IFN-γ secretion levels were significantly higher on second ligation than those secreted following initial ligation. CAR-expressing T cells were also demonstrated to lyze erbB2-expressing tumor cells in the absence of activity against bystander cells not expressing the erbB2 antigen, thereby demonstrating exquisite specificity. CONCLUSION: This study demonstrates the specificity of CAR gene-engineered T cells and their capacity to deliver a wide range of functions against tumor cells with an enhanced response capability after initial receptor engagement.