Autologous Immune Cells Research Articles

IM-01 * ANTI-TUMOR EFFECT OF TRASTUZUMAB, GM-CSF AND IL-2 COMBINATORIAL THERAPY IN PEDIATRIC EPENDYMOMA

We have previously shown that (i) the amount of tumor-infiltrating microglia/macrophage (M/M) in pediatric brain tumor ependymoma (EPN) is positively correlated with survival and (ii) identified an immunosuppressive phenotype in poor prognosis subgroup A of EPN. These data support the development of immunotherapy for EPN, a tumor in which approximately 50% of patients suffer recurrence and for which chemotherapy has not yet shown any benefit. In these preclinical and clinical studies we evaluated the combination of therapeutic antibody trastuzumab, GM-CSF and IL2 for the treatment of EPN. Transcriptomic analysis identified ERBB2, targetable by therapeutic antibody trastuzumab, as overexpressed in EPN. Flow cytometric analysis showed that trastuzumab was bound to ErbB2-expressing EPN tumor and infiltrating M/M. Tumor infiltrating M/M express high levels of high-affinity Fc-gamma receptor CD64 which we suspect are binding the Fc of trastuzumab. Allogeneic and autologous EPN cell line immune cell co-cultures demonstrated significantly increased ADCC with combined trastuzumab and GM-CSF. Primary human EPN organotype culture studies demonstrated decreased tumor proliferation and increased immune cell proliferation in response to combined trastuzumab, GM-CSF and IL2. To date we have recruited 4 patients to the first pilot clinical study of GM-CSF delivered prior to surgery in children with relapsed EPN. Transcriptomic analysis of post-treatment tumor samples demonstrated that GM-CSF upregulated antigen processing and presentation genes, a hallmark of an activated immunophenotype. These results demonstrate that combined trastuzumab/GM-CSF/IL2 is effective at not only priming the newly recruited immune cells to target the tumor but may also reverse the immunosuppressive phenotype. The potential of complementary combinatorial immunotherapeutic regimens in EPN warrants expedited investigation.

Harnessing oncolytic virus-mediated anti-tumor immunity.

Harnessing oncolytic virus-mediated anti-tumor immunity.

Multimodal Cancer Therapy Involving Oncolytic Newcastle Disease Virus, Autologous Immune Cells, and Bi-Specific Antibodies

This paper focuses on oncolytic Newcastle disease virus (NDV). This paper summarizes (i) the peculiarities of this virus as an anti-cancer and immune stimulatory agent and (ii) the approaches to further harness this virus as a vector to combat cancer. Special emphasis is given on combining virus therapy with cell therapy and on improving tumor targeting. The review will include some of the authors work on NDV, bi-specific antibodies, and cell therapy as building blocks for a new perspective of multimodal cancer therapy. The broad anti-tumor immune reactivation includes innate and adaptive, tumor antigen (TA) specific and TA independent activities

Human Regulatory T Cells Kill Tumor Cells through Granzyme-Dependent Cytotoxicity upon Retargeting with a Bispecific Antibody

A major mechanism by which human regulatory T cells (T(regs)) have been shown to suppress and kill autologous immune cells is through the granzyme-perforin pathway. However, it is unknown whether T(regs) also possess the capacity to kill tumor cells using similar mechanisms. Bispecific antibodies (bscAbs) have emerged as a promising class of therapeutics that activate T cells against tumor antigens without the need for classical MHC-restricted TCR recognition. Here, we show that a bscAb targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, redirects human CD4(+)CD25(+)FoxP3(+) T(regs) to kill glioblastoma (GBM) cells. This activity was significantly abrogated by inhibitors of the granzyme-perforin pathway. Notably, analyses of human primary GBM also displayed diffuse infiltration of granzyme-expressing FoxP3(+) T cells. Together, these data suggest that despite their known suppressive functions, tumor-infiltrating T(regs) possess potent cytotoxic mechanisms that can be co-opted for efficient tumor cell lysis.

NK Cells in Central Nervous System Disorders

NK cells are important players in immunity against pathogens and neoplasms. As a component of the innate immune system, they are one of the first effectors on sites of inflammation. Through their cytokine production capacities, NK cells participate in the development of a potent adaptive immune response. Furthermore, NK cells were found to have regulatory functions to limit and prevent autoimmunity via killing of autologous immune cells. These paradoxical functions of NK cells are reflected in CNS disorders. In this review, we discuss the phenotypes and functional features of peripheral and brain NK cells in brain tumors and infections, neurodegenerative diseases, acute vascular and traumatic damage, as well as mental disorders. We also discuss the implication of NK cells in neurotoxicity and neuroprotection following CNS pathology, as well as the crosstalk between NK cells and brain-resident immune cells.

Cancer Vaccination, Will You Have To Pay The Toll?

Cancer vaccines based on patient-derived, autologous immune cells are actively being pursued as a novel strategy to utilize the body’s natural defenses against malignancy. Harnessing the ability of the immune system to fight cancer involves overcoming many obstacles including tumor-specific targeting, overcoming tolerance, and generating effective tumoricidal responses. Co-administration of immune activating adjuvants may hold the key to breaking through several of these barriers. Toll-like receptors (TLR) are pathogen sensors of the innate immune system that activate proinflammatory responses to fight infection and initiate adaptive immune responses. TLRs are increasingly being explored in combination with cancer vaccine strategies since they may have the potential to enhance immunotherapy by promoting tumor-specific immunity. This review will focus on recent basic and clinical research on the use of TLR agonists in cancer therapy.

Dendritic cell immunotherapy in ovarian cancer

Ovarian cancer is one of the most frequent gynecological malignancies. However, as there is no effective screening method to detect early disease, it is usually only diagnosed when already widespread in the abdomen. The majority of patients diagnosed with advanced-stage disease will relapse and require additional therapy. In the search for additional effective treatments for the management of recurrent disease, researchers have focused on the potential usefulness of immunotherapeutic modulation by administering autologous immune cells, such as dendritic cells (DCs), to stimulate antitumor host responses. With the ultimate goal of improved survival, this review addresses mechanisms in ovarian cancer that may limit the expansion of antitumor immunity, discusses the parameters to be considered for optimal DC immunotherapy, outlines evaluation methodology used to monitor the success of treatment regimens and reviews reported DC immunotherapy trials in ovarian cancer.

Development of a Novel in Vivo Model for Human Myeloma Via Humanization of the Bone Marrow Niche

Abstract 325Multiple Myeloma (MM) is a human hematopoietic malignancy of the B cell lineage with 5 year survival of only 40%. Most MM patients succumb to progressive disease and therefore novel therapies are urgently needed. MM is characterized by growth of malignant plasma cells in the bone marrow microenvironment (ME) leading to bone erosion and fracture. Interactions between tumor and host ME play a critical role in the biology of MM and are a target of most new therapies against this tumor. The dependence of human MM on ME is further illustrated by the preferential requirement for growth of primary MM cells in implanted human fetal bone, but not the murine bone in immune-deficient mice in the scid-hu model. This model is however suboptimal for preclinical testing of novel therapies and there is a major unmet need for development of newer models to study MM and the interactions between human MM and its microenvironment.Some of the barriers to growth and xenotransplantation of human cells in immune-deficient Rag2−/−Il2rg−/− mice include macrophage-mediated innate immune rejection, as well as non-cross reactive growth factors. In order to overcome these limitations, Rag2−/−Il2rg−/− mice were genetically engineered to express human versions of the macrophage receptor signal regulatory protein-alpha (SIRPa), as well as several non-cross-reactive growth factors. The combination of these growth factors leads to synergistic enhancement of growth of transplanted human cells in these mice. In order to facilitate the potential ability of these mice to serve as hosts for human MM, these mice were further modified to express a human growth factor that is a critical MM-related cytokine and that is also non-cross reactive between humans and mice.INA-6 is a human IL6 dependent MM cell line that is unable to grow in standard immunodeficient mice such as NOD/scid/Il2rg−/−, but instead grows only in the implanted fetal bone in the scid-hu model. Injection of genetically humanized mice with INA6 cells led to facile growth of MM cells in the bone, leading to lytic bone disease. Next we tested whether primary tumor cells from MM patients could grow in these genetically engineered mice. Bone marrow mononuclear cells were separated into CD138+ and T cell depleted-CD138- fractions. Injection of either fraction led to growth of MM cells in these mice. These data therefore indicate that the prior inability to reliably grow primary MM cells in mice outside of the scid-hu model was primarily related to the non-cross-reactive cytokines and innate immune barriers mediated by the CD47-SIRPa axis.These data demonstrate that the genetic humanization of immune-deficient mice to modify the bone marrow niche to express non-cross reactive growth factors leads to facile growth of primary human MM cells in vivo. These mice can therefore serve as a valuable tool to test new patient-specific therapies depending on the genetic makeup of the tumor. Further advancement of this model to include growth of autologous immune cells would lead to humanized immune-competent models much needed for patient-specific preclinical testing of novel targets. Similar approaches can also be extended to develop in vivo models for other hematologic malignancies as well. Disclosures:Dhodapkar:Celgene: Research Funding; KHK: Research Funding.

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol

We have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safe. Five patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2-3 weeks in culture, a median of 4.65 × 10(6) immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 days. ITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3(+) CD16(+) CD56(+) CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10(6) CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1-13) from the first infusion of CIK cells. This study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.

Recruitment and interaction of human dendritic and T cells in autologous liver slices experimentally infected with HCV produced in cell culture

Studying the immunological processes taking place during the initial steps of acute hepatitis C virus (HCV) infection has been a challenge in patients. Shin et al. have recently reported that delayed induction, not impaired recruitment of specific CD8+ T cells, causes the late onset of acute hepatitis C in chimpanzees (Gastroenterology, 2011). However, further elucidation of the underlying mechanisms is difficult in vivo. We made observations consistent with their conclusions in human liver slices inoculated ex vivo with HCV produced in cell culture (HCVcc). Autologous immune cells were purified from blood and differentially stained prior to their incubation with the slices for 2hours. A two-photon confocal microscopic analysis revealed that many more stained dendritic and T cells contracted interactions within two-day infected slices than non-inoculated ones (p<0.001). While in the first instance some dendritic and T cells entered into closer interactions, they never did in the latter case. These results suggest that ex vivo infection of human liver slices with HCVcc may be useful for gaining experimental insight regarding the immunological processes taking place at early steps of HCV infections.

Immunotherapy in human glioblastoma

Glioblastoma patients spontaneously develop anti-tumour immune responses. However, the tumour itself develops several mechanisms that allow the tumor to escape the immune system. Clinical trials using infusion of activated autologous immune cells, or active immunotherapy with tumor antigens and dendritic cells have successfully induced anti-tumour immunity and some radiological responses. More recently, approaches targeting the mechanisms of tolerance have shown promising data in melanoma, and are currently under investigations in gliomas. However, large randomised trials are still needed to prove the usefulness of cancer vaccines in brain tumors.

Immunotherapy of Kidney Cancer

Renal cell carcinoma (RCC) accounts for 4% of all new cancer cases in males and 3% in females in the US. Compared to other solid tumors, it does not respond to traditional management modalities, such as chemotherapy and radiation therapy. However, it appears to be an immune-responsive tumor and several immunotherapeutic strategies have been investigated in the management of RCC with variable degrees of success. Active immunotherapy refers mainly to the use of vaccines, while adoptive (passive) immunotherapy includes the use of autologous immune cells, allogeneic immune cells (stem cell transplantation, donor lymphocyte infusion), as well as antibody delivery. Cytokine delivery with IL-2 has resulted in long-term disease-free survival in a small proportion of patients with metastatic disease. The continuous understanding of the mechanisms that underlie the immune complex networks has led to the identification of key molecules that play a major role in the immune response process. A panel of immuno-modulatory compounds that target such molecules has been tested in the preclinical and clinical setting. At the post-genomic era, the development of novel biomarkers can contribute to more accurate patient selection, resulting in higher responses and less toxicity of immunotherapeutic approaches.

Current Status and Future Applications of Cellular Therapies for Cancer

Therapies based on the use of autologous immune cells are among the best candidates for cancer immunotherapy. Dendritic cell vaccines have demonstrated very encouraging responses for some solid tumors, while in melanoma autologous T-cell therapies have exceeded 70% objective response rates in selected Phase I trials. However, it is clear that a number of barriers exist to the effective, practical application of these therapies. The aim of this article is to consider modifications to such strategies over the last 3 years and the resultant clinical research in autologous dendritic cell vaccines, T-cell therapy and γδ T-cell therapy for cancer.

Novel mobilization strategies to enhance autologous immune effector cells in multiple myeloma

The immune system plays a critical role determining the outcomes in transplanted multiple myeloma patients, since enhanced lymphocyte recovery results in improved survival. Since mobilization regimens influence the cellular subsets collected and infused for transplant, these regimens may determine immune recovery following transplant. We hypothesized that a mobilized stem cell product harboring an increased number of lymphocytes would enhance immune recovery following autologous stem cell infusion, increase lymphocyte recovery, and improve clinical outcomes. We designed a phase I immune mobilization trial using IL-2 and growth factors to increase the number of lymphocytes within the stem cell product. This regimen efficiently mobilized CD34+ progenitor cells (median: 3.6 x 10(6) cells/kg; range 1.9-6.6 x 10(6) cells/kg) and improved the immune properties of the mobilized stem cells, including an increase in CD8+ T cells expressing an NK activating receptor called NKG2D (P less than 0.004), cells that are extremely potent at killing myeloma cells using non-MHC-I restricted and TCR-independent mechanisms. Novel mobilization techniques can improve the mobilized graft and may improve clinical outcomes in myeloma patients.

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-Scid, IL-2Rγnull Mice In Vivo.

Abstract 726There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities, and it is a current crucial problem in the field of human ADCC research. To overcome this, we have established “humanized mice,” in which human immune cells from healthy individuals function as ADCC effector cells against allogeneic tumor cell lines, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients. In this model, the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to highly enhance ADCC, showed potent antitumor activity by human ADCC against CCR4 expressing tumor cell lines. In addition, KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in the tumor bearing humanized mice. These observations indicate that KM2760 could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. Using this humanized mouse model, we now have the opportunity to perform more appropriate preclinical evaluation of many types of mAb based immunotherapy, although in the initial study, we could not completely exclude nonspecific allogeneic immune responses because target and effector cells were obtained from different individuals. In addition, susceptibility to immunotherapy is likely to be different in established cell lines and primary tumor cells isolated directly ex vivo from patients, with the latter certainly being more relevant for evaluation of immunotherapeutic agents. To overcome the subsequent problems, we have established a primary human tumor bearing NOG mouse model, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T-cell leukemia/lymphoma (ATLL) cells. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in mAb based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future. Disclosures:No relevant conflicts of interest to declare.

Immune System Cooperatively Supports Endocrine System-Primed Embryo Implantation

Attachment to endometrial epithelium is an essential process for human embryos. Although it is widely accepted that this process is largely regulated by the endocrine system, the precise molecular mechanism(s) remains unclear. Recent evidence suggests that immune cells actively contribute to the establishment of embryo implantation. In accordance with this, we found that peripheral blood immune cells positively affect the differentiation of maternal endometrium to facilitate embryo implantation during early pregnancy. From these findings, we propose a novel concept that circulating immune cells are important regulators of embryo implantation. Lately, implantation failure in patients treated with in vitro fertilization and embryo transfer has received increasing attention. Based on our hypothesis, we have successfully developed a new therapy for implantation failure using autologous peripheral blood immune cells. These findings suggest that supportive mechanisms via the immune system facilitate embryo implantation and will be useful in the field of assisted reproductive technology.

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-scid, IL-2Rγnull Mice In Vivo

There is a lack of suitable small animal models to evaluate human Ab-dependent cellular cytotoxicity (ADCC) in vivo, because of the species incompatibility between humans and animals or due to nonspecific allogeneic immune reactions. To overcome these problems, we established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice as recipients, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T cell leukemia/lymphoma (ATLL) cells and a therapeutic chimeric anti-CCR4 mAb, the Fc region of which is defucosylated to enhance ADCC. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in Ab-based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future.

The Immunomodulatory Role of Angiocidin, a Novel Angiogenesis Inhibitor

The observation that many tumors exist in a microenvironment comprised of immune cells has led to the hypothesis that the immune system may play a significant role in the suppression of tumor growth. It is now clear that immune effector cells are capable of recognizing and destroying some cancer cells. However, tumors have developed numerous mechanisms by which they avoid immune recognition and death. Cancer immunotherapy attempts to harness the power of the immune system and direct it against tumor growth, while circumventing the immune-evasion strategies utilized by tumors. Many approaches are currently being investigated, including the re-infusion of autologous immune effector cells (i.e. cytotoxic T lymphocytes and macrophages) back into hosts after ex vivo expansion and activation. The therapeutic effects of specific cytokines are also being evaluated for their impact on tumor growth. Our lab has discovered a novel thrombospondin-1 (TSP-1) binding protein, termed "angiocidin", with potent anti-tumor and anti-angiogenic capabilities. To further investigate the anti-tumor activity of angiocidin, we examined whether angiocidin could play a role in immune system modulation. We have found that the monocytic leukemia cell line THP-1, as well as freshly isolated human peripheral blood monocytes, differentiate into macrophage-like cells when treated with angiocidin. These cells underwent dramatic morphological changes and became more phagocytic. Angiocidin-treated monocytes also activated T lymphocytes in co-culture conditions. Angiocidin-treated THP-1 cells upregulated cytokine mRNA expression and secretion via NF-kappaB, MAPK, and PI3-K. Based on these data, we hypothesize that angiocidin's ability to elicit tumor cell death may be mediated in part by it's pro-inflammatory effects on immune cells in the tumor microenvironment.

Interleukin‐4 suppresses the cytotoxic potential of in vitro generated, adaptive regulatory CD4+ T cells by down‐regulation of granzyme B

Regulatory CD4+ T cells (Tregs) control immune responses using secretion of anti-inflammatory cytokines and/or cytotoxic mechanisms and play a central role in the outcomes of several immune pathologies. Previous studies suggest an impaired function of Tregs in allergy, especially during allergen seasons, but the underlying mechanism is not known. Therefore, we analysed the impact of the T helper type 2 cytokine interleukin (IL)-4 on in vitro generated adaptive Tregs (aTregs), which have been reported to use the granzyme B (GrB)/perforin pathway to kill autologous immune cells. aTregs were generated by co-ligation of CD3 and CD46 on CD4+ T lymphocytes and granzyme expression was analysed using flow cytometry. To quantify GrB and perforin expression as well as IL-10 secretion in response to IL-4, specific enzyme-linked immunosorbent assays were performed in cell lysates and/or culture supernatants. Using a flow cytometry-based cytotoxicity assay the impact of IL-4 on the cytotoxic potential of aTregs was investigated. While IL-4 did not affect IL-10 secretion and perforin expression in aTregs, a significant suppression of GrB synthesis was detected in the presence of IL-4. In addition, IL-4-mediated suppression of GrB led to impaired cytotoxicity of aTregs against K562 target cells. In conclusion, our data suggest that IL-4 might play a role in impaired aTreg function in allergy.

Novel mobilization strategies to enhance autologous immune effector cells in multiple myeloma

Novel mobilization strategies to enhance autologous immune effector cells in multiple myeloma