AIM Platform: A Novel Nano Artificial Antigen-Presenting Cell-Based Clinical System Designed to Consistently Produce Multi-Antigen-Specific T-Cell Products with Potent and Durable Anti-Tumor Properties.

Over the last decade, tremendous progress has been made in the field of adoptive cell therapy. The two prevailing modalities include endogenous non-engineered approaches and genetically engineered T-cell approaches. Endogenous non-engineered approaches include dendritic cell-based systems and tumor-infiltrating lymphocytes (TIL) that are used to produce multi-antigen-specific T-cell products. Genetically engineered approaches, such as T-cell receptor engineered cells and chimeric antigen receptor T cells are used to produce single antigen-specific T-cell products. It is noted by the authors that there are alternative methods to sort for antigen-specific T cells such as peptide multimer sorting or cytokine secretion assay-based sorting, both of which are potentially challenging for broad development and commercialization. In this review, we are focusing on a novel nanoparticle technology that generates a non-engineered product from the endogenous T-cell repertoire. The most common approaches for ex vivo activation and expansion of endogenous, non-genetically engineered cell therapy products rely on dendritic cell-based systems or IL-2 expanded TIL. Hurdles remain in developing efficient, consistent, controlled processes; thus, these processes still have limited access to broad patient populations. Here, we describe a novel approach to produce cellular therapies at clinical scale, using proprietary nanoparticles combined with a proprietary manufacturing process to enrich and expand antigen-specific CD8+ T-cell products with consistent purity, identity, and composition required for effective and durable anti-tumor response.

Cord blood-derived T cells allow the generation of a more naïve tumor-reactive cytotoxic T-cell phenotype.

BACKGROUND: Transplantation of hematopoietic stem cells (HSCs) from peripheral blood (PB) or cord blood (CB) is well established. HSCs from CB are associated with a lower risk of graft-versus-host disease (GVHD), but antigen-independent expanded CB- and PB-derived T cells can induce GVHD in allo-HSC recipients. CB-derived cells might be more suitable for adoptive immunotherapy as they have unique T-cell characteristics. Here, we describe functional differences between CB and PB T cells stimulated with different cytokine combinations involved in central T-cell activation. STUDY DESIGN AND METHODS: Isolated CD8+ T cells from CB and PB were stimulated antigen independently with anti-CD3/CD28 stimulator beads or in an antigen-dependent manner with artificial antigen-presenting cells loaded with the HLA-A*02:01-restricted peptide of tumor-associated melanoma antigen recognized by T cells 1 (MART1). CB and PB T cells cultured in the presence of interleukin (IL)-7, IL-15, IL-12, and IL-21 were characterized for T-cell phenotype and specificity, that is, by CD107a, interferon-γ, tumor necrosis factor-α, and IL-2 expression. RESULTS: After antigen-independent stimulation, activated CD8+ CB T cells exhibited stronger proliferation and function than those from PB. After antigenic stimulation, MART1-reactive CB T cells were naïve (CD45RA+CCR7+), cytotoxic, and highly variable in expressing homing marker CD62L. Addition of IL-21 resulted in increased T-cell proliferation, whereas supplementation with IL-12 decreased IL-21-induced expansion, but increased the functionality and cytotoxicity of CB and PB T cells. CONCLUSION: MART1-reactive CB T cells with a more naïve phenotype and improved properties for homing can be generated. The results contribute to better understanding the effects on GVHD and graft versus tumor.

Telomere Length as an Indicator of the Robustness of B- and T-Cell Response to Influenza in Older Adults.

BACKGROUND: Telomeres provide a key mechanism for protecting the integrity of chromosomes and their attrition after cell division and during aging are evident in lymphocytes. However, the significance of telomere shortening in age-associated decline of immune function is unknown. METHODS: We selected 22 HLA-A2-positive healthy older adults who have relatively short or long telomere lengths to compare their antibody response against the influenza vaccine, and their CD8(+) T-cell response against an influenza antigen. RESULTS: B cells from individuals with a robust antibody response to the influenza vaccine had significantly longer telomeres than those with a poor antibody response. Monocyte-derived antigen-presenting cells of both short and long telomere groups induced similar expansions of influenza M1-specific CD8(+) T cells. Vaccination did not increase M1-specific CD8(+) T cells in blood, but M1-specific CD8(+) T cells from the long telomere group exhibited significantly greater expansion in vitro than those from the short telomere group. Finally, M1-specific CD8(+) T cells that underwent more expansions had significantly longer telomeres than cells with fewer divisions. CONCLUSIONS: Telomere length is positively associated with a robust lymphocyte response, and telomere attrition may contribute to the age-associated decline of adaptive immunity.

IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells.

The glycoprotein CD86 is an important costimulatory molecule that has been shown to be predominantly expressed on APCs, such as dendritic cells, macrophages, and B cells. More recently, CD86 was also detected on T cells in specific pathological conditions. The mechanisms of how CD86 might be induced and its functional role in T cells are not well understood. In the present study, we showed that treatment with IL-2 markedly upregulated CD86, but not CD80, in human CD4(+) and CD8(+) T cells. This upregulation occurred in the absence of bystander cells, and isolated naive CD4(+) or CD8(+) T cells exhibited different time-dependent CD86-expression patterns in response to IL-2. Upregulation of CD86 on activated T cells was reduced by Abs that block IL-2 and IL-2Rα (CD25), indicating a receptor-mediated mechanism. IL-2-dependent CD86 upregulation was blocked by pharmacological inhibitors of the NFAT and mammalian target of rapamycin pathways and was largely reduced by simultaneous exposure to IFN-α. Importantly, a marked increase in CD86 on T cells was also observed in vivo in IL-2-treated patients. In conclusion, IL-2 upregulates CD86 expression on human CD4(+) and CD8(+) T cells via a receptor-dependent mechanism that involves the NFAT and mammalian target of rapamycin pathways.

Nanoscale artificial antigen presenting cells for T cell immunotherapy.

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. FROM THE CLINICAL EDITOR: Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.

Dynamic regulation of functionally distinct virus-specific T cells.

The functional capacities of CD8(+) T cells important for virus clearance are influenced by interactions with antigen presenting cells (APCs) and CD4(+) T cells during initial selection, subsequent expansion, and development of memory. Recently, investigators have shown that polyfunctional T cells correlate best with long-term protection, however, it is still unknown how to stimulate T cells to achieve these responses. To study this, we examined the phenotypes and functions of CD8(+) T cells specific for two different virus antigens stimulated ex vivo using either autologous monocyte-derived dendritic cells (moDCs) or HLA-A2-Ig-based artificial APCs (aAPCs). Although similar numbers of influenza virus and measles virus tetramer-positive cells were generated by stimulation with peptide-loaded moDCs and aAPCs, T cell function, assessed by expression of IL-2, IFN-gamma, TNF-alpha, MIP1beta, and CD107a, showed that aAPC-generated CD8(+) T cells were multifunctional, whereas moDC-generated cells were mostly monofunctional. aAPC-generated cells also produced more of each cytokine per cell than CD8(+) T cells generated with moDCs. These phenotypes were not fixed, as changing the culture conditions of expanding T cells from aAPCs to moDCs, and moDCs to aAPCs, reversed the phenotypes. We conclude that CD8(+) T cells are heterogeneous in their functionality and that this is dependent, in a dynamic way, on the stimulating APC. These studies will lead to understanding the factors that influence induction of optimal CD8(+) T cell function.

AIM™ platform: A new immunotherapy approach for viral diseases.

In addition to complications of acute diseases, chronic viral infections are linked to both malignancies and autoimmune disorders. Lack of adequate treatment options for Epstein-Barr virus (EBV), Human T-lymphotropic virus type 1 (HTLV-1), and human papillomavirus (HPV) remains. The NexImmune Artificial Immune Modulation (AIM) nanoparticle platform can be used to direct T cell responses by mimicking the dendritic cell function. In one application, AIM nanoparticles are used ex vivo to enrich and expand (E+E) rare populations of multi-antigen-specific CD8+ T cells for use of these cells as an AIM adoptive cell therapy. This study has demonstrated using E+E CD8+ T cells, the functional relevance of targeting EBV, HTLV-1, and HPV. Expanded T cells consist primarily of effector memory, central memory, and self-renewing stem-like memory T cells directed at selected viral antigen peptides presented by the AIM nanoparticle. T cells expanded against either EBV- or HPV-antigens were highly polyfunctional and displayed substantial in vitro cytotoxic activity against cell lines expressing the respective antigens. Our initial work was in the context of exploring T cells expanded from healthy donors and restricted to human leukocyte antigen (HLA)-A*02:01 serotype. AIM Adoptive Cell Therapies (ACT) are also being developed for other HLA class I serotypes. AIM adoptive cell therapies of autologous or allogeneic T cells specific to antigens associated with acute myeloid leukemia and multiple myeloma are currently in the clinic. The utility and flexibility of the AIM nanoparticle platform will be expanded as we advance the second application, an AIM injectable off-the-shelf nanoparticle, which targets multiple antigen-specific T cell populations to either activate, tolerize, or destroy these targeted CD8+ T cells directly in vivo, leaving non-target cells alone. The AIM injectable platform offers the potential to develop new multi-antigen specific therapies for treating infectious diseases, cancer, and autoimmune diseases.

Heat shock protein 70/peptide complexes: potent mediators for the generation of antiviral T cells particularly with regard to low precursor frequencies.

BACKGROUND: Heat shock protein 70 (HSP70) has gained major attention as an adjuvant capable of inducing antigen-specific CD8(+) and CD4(+) T-cell responses. The ability of HSP70/peptide complexes to elicit cytotoxic T-cell (CTL) responses by cross-presentation of exogenous antigens via HLA class I molecules is of central interest in immunotherapy. We examined the role of HSP70/CMVpp65(495-503)-peptide complex (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for ex vivo expansion of CMV-specific CTLs. METHODS: CMV-specific T cells generated from PBMCs of HLA-A*02:01/CMV-seropositive donors were stimulated for 21 days with HSP70/CMV-PC and analyzed in functional assays. As a control PBMCs were cultured in the presence of CMVpp65(495-503) peptide or HSP70. Increase of CMV-specific CTLs was visualized by pentameric HLA-A*02:01/CMVpp65(495-503) complex. RESULTS: About 90% of HSP70/CMV-PC generated T cells were CMV-specific and exhibited significantly higher IFN-γ secretion, cytotoxic activity, and an increased heme oxygenase 1 (HO-1) gene expression as compared to about 69% of those stimulated with CMVpp65(495-503) peptide. We decided to classify the HLA-A*02:01/CMV-seropositive donors as weak, medium, and strong responder according to the frequency of generated A2/CMV-pentamer-positive CD8(+) T cells. HSP70/CMV-PC significantly induces strong antiviral T-cell responses especially in those donors with low memory precursor frequencies. Blockage of CD91 with α2-macroglobulin markedly reduced proliferation of antiviral T cells suggesting a major role of this receptor in the uptake of HSP70/CMV-PC. CONCLUSION: This study clearly demonstrates that HSP70/CMV-PC is a potent mediator to induce stronger T-cell responses compared to antiviral peptides. This simple and efficient technique may help to generate significant quantities of antiviral CTLs by cross-presentation. Thus, we propose HSP70 for chaperoning peptides to reach an efficient level of cross-presentation. HSP70/peptide complexes may be particularly useful to generate stronger T-cell responses in cases of low precursor frequencies and may help to improve the efficiency of antigen-specific T-cell therapy for minor antigens.

Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells.

Adoptive immunotherapy holds promise as a treatment for cancer and infectious diseases, but its development has been impeded by the lack of reproducible methods for generating therapeutic numbers of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs). As a result, there are only limited reports of expansion of antigen-specific CTLs to the levels required for clinical therapy. To address this issue, artificial antigen-presenting cells (aAPCs) were made by coupling a soluble human leukocyte antigen-immunoglobulin fusion protein (HLA-Ig) and CD28-specific antibody to beads. HLA-Ig-based aAPCs were used to induce and expand CTLs specific for cytomegalovirus (CMV) or melanoma. aAPC-induced cultures showed robust antigen-specific CTL expansion over successive rounds of stimulation, resulting in the generation of clinically relevant antigen-specific CTLs that recognized endogenous antigen-major histocompatibility complex complexes presented on melanoma cells. These studies show the value of HLA-Ig-based aAPCs for reproducible expansion of disease-specific CTLs for clinical approaches to adoptive immunotherapy.

Decline of influenza-specific CD8+ T cell repertoire in healthy geriatric donors.

BACKGROUND: While influenza vaccination results in protective antibodies against primary infections, clearance of infection is primarily mediated through CD8+ T cells. Studying the CD8+ T cell response to influenza epitopes is crucial in understanding the disease associated morbidity and mortality especially in at risk populations such as the elderly. We compared the CD8+ T cell response to immunodominant and subdominant influenza epitopes in HLA-A2+ control, adult donors, aged 21-42, and in geriatric donors, aged 65 and older. RESULTS: We used a novel artificial Antigen Presenting Cell (aAPC) based stimulation assay to reveal responses that could not be detected by enzyme-linked immunosorbent spot (ELISpot). 14 younger control donors and 12 geriatric donors were enrolled in this study. The mean number of influenza-specific subdominant epitopes per control donor detected by ELISpot was only 1.4 while the mean detected by aAPC assay was 3.3 (p = 0.0096). Using the aAPC assay, 92% of the control donors responded to at least one subdominant epitopes, while 71% of control donors responded to more than one subdominant influenza-specific response. 66% of geriatric donors lacked a subdominant influenza-specific response and 33% of geriatric donors responded to only 1 subdominant epitope. The difference in subdominant response between age groups is statistically significant (p = 0.0003). CONCLUSION: Geriatric donors lacked the broad, multi-specific response to subdominant epitopes seen in the control donors. Thus, we conclude that aging leads to a decrease in the subdominant influenza-specific CTL responses which may contribute to the increased morbidity and mortality in older individuals.

Dietary fatty acids modulate antigen presentation to hepatic NKT cells in nonalcoholic fatty liver disease.

Dietary fatty acids are major contributors to the development and progression of insulin resistance and nonalcoholic fatty liver disease (NAFLD). Dietary fatty acids also alter hepatic NKT cells that are activated by antigens presented by CD1d. In the current study, we examine the mechanism of dietary fatty acid induced hepatic NKT cell deficiency and its causal relationship to insulin resistance and NAFLD. We discover that dietary saturated fatty acids (SFA) or monounsaturated fatty acids (MUFA), but not polyunsaturated fatty acids (PUFA), cause hepatic NKT cell depletion with increased apoptosis. Dietary SFA or MUFA also impair hepatocyte presentation of endogenous, but not exogenous, antigen to NKT cells, indicating alterations of the endogenous antigen processing or presenting pathway. In vitro treatment of normal hepatocytes with fatty acids also demonstrates impaired ability of CD1d to present endogenous antigen by dietary fatty acids. Furthermore, dietary SFA and MUFA activate the NFkappaB signaling pathway and lead to insulin resistance and hepatic steatosis. In conclusion, both dietary SFA and MUFA alter endogenous antigen presentation to hepatic NKT cells and contribute to NKT cell depletion, leading to further activation of inflammatory signaling, insulin resistance, and hepatic steatosis.

Rapid Expansion of Highly Functional Antigen-Specific T Cells from Patients with Melanoma by Nanoscale Artificial Antigen-Presenting Cells.

PURPOSE: Generation of antigen-specific T cells from patients with cancer employs large numbers of peripheral blood cells and/or tumor-infiltrating cells to generate antigen-presenting and effector cells commonly requiring multiple rounds of restimulation ex vivo. We used a novel paramagnetic, nanoparticle-based artificial antigen-presenting cell (nano-aAPC) that combines anti-CD28 costimulatory and human MHC class I molecules that are loaded with antigenic peptides to rapidly expand tumor antigen-specific T cells from patients with melanoma. EXPERIMENTAL DESIGN: Nano-aAPC-expressing HLA-A*0201 molecules and costimulatory anti-CD28 antibody and HLA-A*0201 molecules loaded with MART-1 or gp100 class I-restricted peptides were used to stimulate CD8 T cells purified from the peripheral blood of treatment-naïve or PD-1 antibody-treated patients with stage IV melanoma. Expanded cells were restimulated with fresh peptide-pulsed nano-aAPC at day 7. Phenotype analysis and functional assays including cytokine release, cytolysis, and measurement of avidity were conducted. RESULTS: MART-1-specific CD8 T cells rapidly expanded up to 1,000-fold by day 14 after exposure to peptide-pulsed nano-aAPC. Expanded T cells had a predominantly stem cell memory CD45RA+/CD62L+/CD95+ phenotype; expressed ICOS, PD-1, Tim3, and LAG3; and lacked CD28. Cells from patients with melanoma were polyfunctional; highly avid; expressed IL2, IFNγ, and TNFα; and exhibited cytolytic activity against tumor cell lines. They expanded 2- to 3-fold after exposure to PD-1 antibody in vivo, and expressed a highly diverse T-cell receptor V beta repertoire. CONCLUSIONS: Peptide-pulsed nano-aAPC rapidly expanded polyfunctional antigen-specific CD8 T cells with high avidity, potent lytic function, and a stem cell memory phenotype from patients with melanoma.

In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC).

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.

Ascites specific inhibition of CD1d-mediated activation of natural killer T cells.

PURPOSE: Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate antitumor immunity. It has been shown, however, that tumor-associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer-associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor-associated and other ascites on CD1d-mediated antigen presentation to NKT cells. EXPERIMENTAL DESIGN: To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-immunoglobulin-based artificial antigen presenting cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classic CD8+ T cells. RESULTS: Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the ability of the cells to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of alpha-galactosylceramide-loaded CD1d-immunoglobulin-based aAPC to activate NKT cells. In addition, our data show that treatment with ascites does not inhibit HLA-A2-mediated activation of classic CD8+ T cells. CONCLUSIONS: Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system.

Soluble MHC class I complexes for targeted immunotherapy.

Major histocompatibility complexes (MHC) have been used for more than two decades in clinical and pre-clinical approaches of tumor immunotherapy. They have been proven efficient for detecting anti-tumor-specific T cells when utilized as soluble multimers, immobilized on cells or artificial structures such as artificial antigen-presenting cells (aAPC) and have been shown to generate effective anti-tumor responses. In this review we summarize the use of soluble MHC class I complexes in tumor vaccination studies, highlighting the different strategies and their contradicting results. In summary, we believe that soluble MHC class I molecules represent an exciting tool with great potential to impact the understanding and development of immunotherapeutic approaches on many levels from monitoring to treatment.

Correction: Antigen-specific T cell Redirectors: a nanoparticle based approach for redirecting T cells.

[This corrects the article DOI: 10.18632/oncotarget.11785.].

Selective Effects of mTOR Inhibitor Sirolimus on Naïve and CMV-Specific T Cells Extending Its Applicable Range Beyond Immunosuppression.

Cytomegalovirus (CMV) infection/reactivation remains among the most important complications of immunosuppression after transplantation. However, recent clinical observations indicate that mammalian target of rapamycin (mTOR) inhibition with sirolimus may improve the outcome of CMV complications. Underlying mechanisms of this observation, particularly the effect of sirolimus on naïve- and CMV-specific cytotoxic CD8+ T-cell (CMV-CTL) functionality is still undiscovered. Here, the influence of sirolimus on naïve and memory CMV-CTLs was determined by CD3/CD28 crosslinking and alloreactivity assays. After stimulating CMV-CTL with HLA-A*02:01-restricted CMVpp65-peptide loaded artificial antigen-presenting cells (aAPCs), we measured the effect of sirolimus on T-cell proliferation, phenotype, and functionality. Sirolimus significantly improved CMV-specific effector memory T-cell function and negatively influenced naïve T cells. This unique mechanism of action was further characterized by increased secretion of interferon-gamma (IFN-γ), granzyme B (GzB) and enhanced target-cell-dependent cytotoxic capacity of activated CMV-CTLs. Next-generation-sequencing (NGS) was applied to monitor T-cell receptor (TCR)-repertoire dynamics and to verify, that the increased functionality was not related to sirolimus-resistant CTL-clones. Instead, modulation of environmental cues during CMV-CTL development via IL-2 receptor (IL-2R)-driven signal transducer and activator of transcription-5 (STAT-5) signaling under mTOR inhibition allowed fine-tuning of T-cell programming for enhanced antiviral response with stable TCR-repertoire dynamics. We show for the first time that sirolimus acts selectively on human naïve and memory T cells and improves CMV-specific T-cell function via modulation of the environmental milieu. The data emphasize the importance to extend immune monitoring including cytokine levels and T-cell functionality which will help to identify patients who may benefit from individually tailored immunosuppression.

Artificial antigen-presenting cells: artificial solutions for real diseases.

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat a variety of life-threatening diseases. Unfortunately, current approaches for generating sufficient numbers of antigen-specific T cells lack the ability to serve as reproducible and economically viable methods. This has spurred the development of both cell- and non-cell-based artificial antigen-presenting cells to alleviate problems associated with peptide-loaded dendritic cells in current approaches to adoptive immunotherapy. Here, we review new strategies for the ex vivo generation of antigen-specific T cells and their clinical application. These new approaches have the potential to spearhead a new era of successful adoptive immunotherapy for cancer and infectious diseases.

Antigen-specific T cell Redirectors: a nanoparticle based approach for redirecting T cells.

Redirection of T cells to target and destroy tumors has become an important clinical tool and major area of research in tumor immunology. Here we present a novel, nanoparticle-based approach to selectively bind antigen-specific cytotoxic T cells (CTL) and redirect them to kill tumors, termed ATR (Antigen-specific T cell Redirectors). ATR were generated by decorating nanoparticles with both an antigen-specific T cell binding moiety, either peptide loaded MHC-Ig dimer or clonotypic anti-TCR antibody, and a model tumor cell binding moiety, anti-CD19 antibody to engage CD19+ tumor cells. ATR stably bind tumor cells and CTL in a dose dependent fashion and stimulate antigen-specific conjugate formation between those cells. ATR induced redirected lysis of tumor cells in vitro, as demonstrated by 51Cr-release killing. In vivo ATR administration led to reduced tumor growth in a SCID/beige human lymphoma treatment model. In summary, ATR represent a novel, nanoparticle based approach for redirecting antigen-specific CTL to kill tumors.

VEGF Potentiates GD3-Mediated Immunosuppression by Human Ovarian Cancer Cells.

PURPOSE: Natural killer T (NKT) cells are important mediators of antitumor immune responses. We have previously shown that ovarian cancers shed the ganglioside GD3, which inhibits NKT-cell activation. Ovarian cancers also secrete high levels of VEGF. In this study, we sought to test the hypothesis that VEGF production by ovarian cancers suppresses NKT-cell-mediated antitumor responses. EXPERIMENTAL DESIGN: To investigate the effects of VEGF on CD1d-mediated NKT-cell activation, a conditioned media model was established, wherein the supernatants from ovarian cancer cell lines (OV-CAR-3 and SK-OV-3) were used to treat CD1d-expressing antigen-presenting cells (APC) and cocultured with NKT hybridomas. Ovarian cancer-associated VEGF was inhibited by treatment with bevacizumab and genistein; conditioned medium was collected, and CD1d-mediated NKT-cell responses were assayed by ELISA. RESULTS: Ovarian cancer tissue and ascites contain lymphocytic infiltrates, suggesting that immune cells traffic to tumors, but are then inhibited by immunosuppressive molecules within the tumor microenvironment. OV-CAR-3 and SK-OV-3 cell lines produce high levels of VEGF and GD3. Pretreatment of APCs with ascites or conditioned medium from OV-CAR-3 and SK-OV-3 blocked CD1d-mediated NKT-cell activation. Inhibition of VEGF resulted in a concomitant reduction in GD3 levels and restoration of NKT-cell responses. CONCLUSIONS: We found that VEGF inhibition restores NKT-cell function in an in vitro ovarian cancer model. These studies suggest that the combination of immune modulation with antiangiogenic treatment has therapeutic potential in ovarian cancer. Clin Cancer Res; 22(16); 4249-58. ©2016 AACR.