The future of affordable cancer immunotherapy.

The treatment of cancer was revolutionized within the last two decades by utilizing the mechanism of the immune system against malignant tissue in so-called cancer immunotherapy. Two main developments boosted cancer immunotherapy: 1) the use of checkpoint inhibitors, which are characterized by a relatively high response rate mainly in solid tumors; however, at the cost of serious side effects, and 2) the use of chimeric antigen receptor (CAR)-T cells, which were shown to be very efficient in the treatment of hematologic malignancies, but failed to show high clinical effectiveness in solid tumors until now. In addition, active immunization against individual tumors is emerging, and the first products have reached clinical approval. These new treatment options are very cost-intensive and are not financially compensated by health insurance in many countries. Hence, strategies must be developed to make cancer immunotherapy affordable and to improve the cost-benefit ratio. In this review, we discuss the following strategies: 1) to leverage the antigenicity of "cold tumors" with affordable reagents, 2) to use microbiome-based products as markers or therapeutics, 3) to apply measures that make adoptive cell therapy (ACT) cheaper, e.g., the use of off-the-shelf products, 4) to use immunotherapies that offer cheaper platforms, such as RNA- or peptide-based vaccines and vaccines that use shared or common antigens instead of highly personal antigens, 5) to use a small set of predictive biomarkers instead of the "sequence everything" approach, and 6) to explore affordable immunohistochemistry markers that may direct individual therapies.

Preventing skin toxicities induced by EGFR inhibitors by topically blocking drug-receptor interactions.

Epidermal growth factor receptor (EGFR) inhibitors are used to treat many advanced-stage epithelial cancers but induce severe skin toxicities in most treated patients. These side effects lead to a deterioration in the quality of life of the patients and compromise the anticancer treatment. Current treatment strategies for these skin toxicities focus on symptom reduction rather than preventing the initial trigger that causes the toxicity. In this study, we developed a compound and method for treating "on-target" skin toxicity by blocking the drug at the site of toxicity without reducing the systemic dose reaching the tumor. We first screened for small molecules that effectively blocked the binding of anti-EGFR monoclonal antibodies to EGFR and identified a potential candidate, SDT-011. In silico docking predicted that SDT-011 interacted with the same residues on EGFR found to be important for the binding of EGFR inhibitors cetuximab and panitumumab. Binding of SDT-011 to EGFR reduced the binding affinity of cetuximab to EGFR and could reactivate EGFR signaling in keratinocyte cell lines, ex vivo cetuximab-treated whole human skin, and A431-injected mice. Specific small molecules were topically applied and were delivered via a slow-release system derived from biodegradable nanoparticles that penetrate the hair follicles and sebaceous glands, within which EGFR is highly expressed. Our approach has the potential to reduce skin toxicity caused by EGFR inhibitors.

The role of immune checkpoint receptors in the malignant phenotype of cutaneous T cell lymphoma.

Immune checkpoint receptors (ICR) modulate the immune response and are critical hubs for immunotherapy. However, data on their role in T lymphoid malignancies, such as cutaneous T cell lymphoma (CTCL), is sparse. We aimed to explore the role of ICR in the malignant features of transformed T lymphocytes and evaluate the effect of ICR-targeting monoclonal antibodies, often used as immunotherapy for solid tumors. We used the CTCL cell line HH and the Sézary cell line Hut78 to examine ICR expression and the effects of ICR inhibition on cell viability and proliferation. Despite their shared T cell progeny, the different CTCL cell lines exhibit markedly different ICR expression profiles. Programmed cell death-ligand 1 (PD-L1) was expressed by both cell lines, while programmed death-1 (PD-1) was expressed only by the HH cell line. Common to all malignant T cells was an autonomous hyper-proliferative state that did not require T cell receptor stimulation. A monoclonal antibody blocking PD-1 had a small but statistically significant augmenting effect on T cell proliferation. Of note, when the cells were exposed to ionizing radiation, healthy lymphocytes and those derived from the HH cell line were salvaged by anti-PD-L1. We show a regulatory role of ICR, mainly PD-1 and its ligand PD-L1, on cutaneous T cell malignancy.

PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR.

Cancer immunotherapy focuses on inhibitors of checkpoint proteins, such as programmed death ligand 1 (PD-L1). Unlike RAS-mutated lung cancers, EGFR mutant tumors have a generally low response to immunotherapy. Because treatment outcomes vary by EGFR allele, intrinsic and microenvironmental factors may be involved. Among all non-immunological signaling pathways surveyed in patients' datasets, EGFR signaling is best associated with high PD-L1. Correspondingly, active EGFRs stabilize PD-L1 transcripts and depletion of PD-L1 severely inhibits EGFR-driven tumorigenicity and metastasis in mice. The underlying mechanisms involve the recruitment of phospholipase C-γ1 (PLC-γ1) to a cytoplasmic motif of PD-L1, which enhances PLC-γ1 activation by EGFR. Once stimulated, PLC-γ1 activates calcium flux, Rho GTPases, and protein kinase C, collectively promoting an aggressive phenotype. Anti-PD-L1 antibodies can inhibit these intrinsic functions of PD-L1. Our results portray PD-L1 as a molecular amplifier of EGFR signaling and improve the understanding of the resistance of EGFR+ tumors to immunotherapy.

Alternative Splicing of the Inhibitory Immune Checkpoint Receptor SLAMF6 Generates a Dominant Positive Form, Boosting T-cell Effector Functions.

SLAMF6 is a homotypic receptor of the Ig-superfamily associated with progenitor-exhausted T cells. Here we show that in humans, SLAMF6 has three splice isoforms involving its V-domain. Although the canonical receptor inhibited T-cell activation through SAP recruitment, the short isoform SLAMF6Δ17-65 had a strong agonistic effect. The costimulatory action depended on protein phosphatase SHP1 and led to a cytotoxic molecular profile mediated by the expression of TBX21 and RUNX3. Patients treated with immune checkpoint blockade showed a shift toward SLAMF6Δ17-65 in peripheral blood T cells. We developed splice-switching antisense oligonucleotides (ASO) designed to target the relevant SLAMF6 splice junction. Our ASOs enhanced SLAMF6Δ17-65 expression in human tumor-infiltrating lymphocytes and improved their capacity to inhibit human melanoma in mice. The yin-yang relationship of SLAMF6 splice isoforms may represent a balancing mechanism that could be exploited to improve cancer immunotherapy.

Immunotherapy Potentiates the Effect of Chemotherapy in Metastatic Melanoma-A Retrospective Study.

Melanoma survival increased with targeted- and immunotherapy agents, yet most patients ultimately progress and require salvage therapy. In our experience, some progressive disease patients on immune-checkpoint inhibitors (ICIs) demonstrate deep and sustained responses to chemotherapy. We hypothesized that ICIs improve the response to subsequent chemotherapy in metastatic melanoma. We conducted a retrospective study to compare the efficacy of chemotherapy given with prior immunotherapy, to its efficacy given without it. We measured progression free survival (PFS), overall survival, and response rate. Immune-monitoring was performed on sequential peripheral blood mononuclear cell samples taken from a chemotherapy-responsive patient. The chemotherapy post-immunotherapy group (CpI) included 11 patients, the chemotherapy without prior immunotherapy (CNPI) group included 24 patients. Median PFS was 5.2 months in the CpI vs. 2.5 months in the CNPI groups; HR 0.37 [95% Confidence interval (CI) 0.144-0.983], P = 0.046. Immune-monitoring showed an increased proportion of CD8+ cells, with elevated PD-1 and CD69 expression, while on chemotherapy, as compared with all-time points on ICIs, suggesting immune-activation. Immunotherapy potentiates the effect of chemotherapy in metastatic melanoma possibly through activation of CD8+ T cells.

SLAMF6​ deficiency augments tumor killing and skews toward an effector phenotype revealing it as a novel T cell checkpoint.

SLAMF6 is a homotypic receptor of the Ig-superfamily whose exact role in immune modulation has remained elusive. Its constitutive expression on resting and activated T cells precludes it from being a bona fide exhaustion marker. By breeding Pmel-1 mice with SLAMF6 -/- mice, we generated donors for T cells lacking SLAMF6 and expressing a transgenic TCR for gp100-melanoma antigen. Activated Pmel-1xSLAMF6 -/- CD8+ T cells displayed improved polyfunctionality and strong tumor cytolysis. T-bet was the dominant transcription factor in Pmel-1 x SLAMF6 -/- cells, and upon activation, they acquired an effector-memory phenotype. Adoptive transfer of Pmel-1 x SLAMF6 -/- T cells to melanoma-bearing mice resulted in lasting tumor regression in contrast to temporary responses achieved with Pmel-1 T cells. LAG-3 expression was elevated in the SLAMF6 -/- cells, and the addition of the LAG-3-blocking antibody to the adoptive transfer protocol improved the SLAMF6 -/- T cells and expedited the antitumor response even further. The results from this study support the notion that SLAMF6 is an inhibitory immune receptor whose absence enables powerful CD8+ T cells to eradicate tumors.

The immune system helps to protect our bodies from illnesses and infections. Immunotherapies are medicines designed to treat diseases, such as cancer, by boosting the immune system against the condition. This is a powerful approach but so far immunotherapies have only had partial success and there is a need for further improvements. One protein called SLAMF6 is found on cells from the immune system that attack and kill cancer cells. Immunotherapies that suppress SLAMF6 on immune cells called killer T cells could increase immune system activity helping to treat cancers, particularly melanoma skin cancers. So far the potential for SLAMF6 as a target for immunotherapy has not been fully explored. Hajaj et al. created mice with killer T cells that recognized skin cancer cells and lacked SLAMF6. These modified cells were better at fighting cancer, producing more anti-cancer chemicals called cytokines and killing more cancer cells. The modified cells had a lasting effect on tumors and helped the mice to live longer. The effects could be further boosted by treating the mice in combination with other immunotherapies. SLAMF6 is a possible new target for skin cancer immunotherapy that could help more people to live longer following cancer diagnosis. The next step is to create a drug to target SLAMF6 in humans and to test it in clinical trials.

Combined Expression of Genetic Adjuvants Via mRNA Electroporation Exerts Multiple Immunostimulatory Effects on Antitumor T Cells.

Adoptive transfer of tumor-infiltrating lymphocytes (TILs) or gene-modified T cells expressing antitumor TCRs or chimeric antigen receptors often yields a high rate of clinical response in several types of cancer. New approaches for enhancing the functional properties of antitumor T cells could improve the clinical outcome of these treatments. To this end, we created 3 classes of genes, each designed to operate autonomously upon expression in T cells. We recently reported on the enhancing effects of constitutively active toll-like receptor 4 (caTLR4), membrane (mem) interleukin-2, memIL-12, and memIL-15, and self-oligomerizing, constitutively active CD40 (caCD40). Here, we evaluated their combined effects on peripheral blood CD8 T cells and different antimelanoma TIL cultures following mRNA electroporation. Expression in CD8 T cells induced transient production of interferon-γ and prolonged and robust upregulation of CD25, CD69, 4-1BB, and OX40. The adjuvants enhanced cytolytic activity of TILs and production of interferon-γ and TNF-α in the presence of autologous, but not mismatched, melanoma for at least 3 days after electroporation. Expression of the 3 adjuvants in young TILs from different patients markedly increased the expression of CD25, OX40, 4-1BB, CD127, and CD28 and exhibited cooperative and, at times, synergistic effects. Furthermore, predefined mixtures of mRNA encoding these adjuvants markedly enhanced the specific antitumor response of selected TILs and killing of autologous melanoma cells by young TILs. Our findings suggest that combinations of these new genetic adjuvants can substantially improve the functional properties of antitumor T cells, offering a new tool of unique versatility in adoptive cell therapy.

Potent Activation of Human T Cells by mRNA Encoding Constitutively Active CD40.

New strategies for augmenting the actual performance of therapeutic T cells in vivo are needed for improving clinical outcome of adoptive cell therapy. Cumulative findings suggest that CD40 plays an intrinsic role in T cell costimulation. Recently, we demonstrated the ability of truncated, auto-oligomerizing CD40 derivatives to induce strong activation of APCs in a ligand-independent manner. We reasoned that constitutively active CD40 (caCD40) can similarly exert enhancing effects on human antitumor T cells. To test this assumption, we transfected human T cells with in vitro-transcribed caCD40 mRNA. In polyclonal T cells, caCD40 triggered IFN-γ secretion and upregulated CD25 and 4-1BB. In antimelanoma tumor-infiltrating lymphocytes (TILs), caCD40 induced massive production of IFN-γ, exerting a pronounced synergistic effect when coexpressed with constitutively active TLR4 devoid of its extracellular ligand binding. In unselected "young" TILs, caCD40 reproducibly increased surface expression of CD25, OX40, 4-1BB, CD127, and CD28. Three days post-mRNA electroporation of CD8 TILs, caCD40 elevated IFN-γ and TNF-α production and cytolytic activity in the presence of autologous but not HLA-I-mismatched melanoma. Enhanced killing of autologous melanoma by young TILs was observed 4 d posttransfection. These findings suggest that caCD40 can function as a potent T cell adjuvant and provide essential guidelines for similar manipulation of other key members of the TNFR family.

Soluble SLAMF6 Receptor Induces Strong CD8+ T-cell Effector Function and Improves Anti-Melanoma Activity In Vivo.

SLAMF6, a member of the SLAM (signaling lymphocyte activation molecules) family, is a homotypic-binding immune receptor expressed on NK, T, and B lymphocytes. Phosphorylation variance between T-cell subclones prompted us to explore its role in anti melanoma immunity. Using a 203-amino acid sequence of the human SLAMF6 (seSLAMF6) ectodomain, we found that seSLAMF6 reduced activation-induced cell death and had an antiapoptotic effect on tumor-infiltrating lymphocytes. CD8+ T cells costimulated with seSLAMF6 secreted more IFNγ and displayed augmented cytolytic activity. The systemic administration of seSLAMF6 to mice sustained adoptively transferred transgenic CD8+ T cells in comparable numbers to high doses of IL2. In a therapeutic model, lymphocytes activated by seSLAMF6 delayed tumor growth, and when further supported in vivo with seSLAMF6, induced complete tumor clearance. The ectodomain expedites the loss of phosphorylation on SLAMF6 that occurs in response to T-cell receptor triggering. Our findings suggest that seSLAMF6 is a costimulator that could be used in melanoma immunotherapy. Cancer Immunol Res; 6(2); 127-38. ©2018 AACR.

Spontaneous Activation of Antigen-presenting Cells by Genes Encoding Truncated Homo-Oligomerizing Derivatives of CD40.

The interaction between the CD40 receptor on antigen-presenting cells (APCs) and its trimeric ligand on CD4 T cells is essential for the initiation and progression of the adaptive immune response. Here we undertook to endow CD40 with the capacity to trigger spontaneous APC activation through ligand-independent oligomerization. To this end we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif that induces homophilic interactions. We incorporated GCN4 variants forming homodimers, trimers, or tetramers at the intracellular domain of human and mouse CD40 and replaced the extracellular portion with peptide-ß2m or other peptide tags. In parallel we examined similarly truncated CD40 monomers lacking a GCN4 motif. The oligomeric products appeared to arrange in high-molecular-weight aggregates and were considerably superior to the monomer in their ability to trigger nuclear factor kB signaling, substantiating the anticipated constitutively active (ca) phenotype. Cumulative results in human and mouse APC lines transfected with caCD40 mRNA revealed spontaneous upregulation of CD80, IL-1ß, TNFα, IL-6, and IL-12, which could be further enhanced by caTLR4 mRNA. In mouse bone-marrow-derived dendritic cells caCD40 upregulated CD80, CD86, MHC-II, and IL-12 and in human monocyte-derived dendritic cells it elevated surface CD80, CD83 CD86, CCR7, and HLA-DR. Oligomeric products carrying the peptide-ß2m extracellular portion could support MHC-I presentation of the linked peptide up to 4 days post-mRNA transfection. These findings demonstrate that the expression of a single caCD40 derivative in APCs can exert multiple immunostimulatory effects, offering a new powerful tool in the design of gene-based cancer vaccines.

Immune Monitoring of Patients Treated With a Whole-Cell Melanoma Vaccine Engineered to Express 4-1BBL.

CD8 lymphocytes are mandatory mediators of tumor regression. To enhance their specific antitumor activity, we aimed to improve a melanoma cell-based vaccine by transfecting it with 4-1BB ligand, a costimulatory and immune modulatory molecule. Thirty-four American Joint Committee on Cancer (AJCC) stage IIB-IV patients were vaccinated with a melanoma antigen-rich cell line engineered to express HLA-A2 and 4-1BBL (M20/A2/BBL). Twelve serially recruited patients were monitored for interferon γ expression and CD107a mobilization before and after vaccination. Thirty-three patients remained alive, with an estimated mean overall survival of 26.2 months. No grade 3-4 adverse events were encountered. Immune monitoring detected an increase in circulating antimelanoma CD8 T cells in 9 of 12 patients, which were significantly stimulated by the parental melanoma, reflecting a relevant antitumor response. The results from this study show that the costimulatory 4-1BB ligand fortifies an antigen-rich melanoma cell line with enhanced antigen-specific stimulation of CD8 T cells. The use of a costimulatory molecule as part of a vaccine confers a selective increase of T-cell subsets with antimelanoma reactivity, which in some cases were characterized for their epitope specificity.

Adjuvant Autologous Melanoma Vaccine for Macroscopic Stage III Disease: Survival, Biomarkers, and Improved Response to CTLA-4 Blockade.

Background. There is not yet an agreed adjuvant treatment for melanoma patients with American Joint Committee on Cancer stages III B and C. We report administration of an autologous melanoma vaccine to prevent disease recurrence. Patients and Methods. 126 patients received eight doses of irradiated autologous melanoma cells conjugated to dinitrophenyl and mixed with BCG. Delayed type hypersensitivity (DTH) response to unmodified melanoma cells was determined on the vaccine days 5 and 8. Gene expression analysis was performed on 35 tumors from patients with good or poor survival. Results. Median overall survival was 88 months with a 5-year survival of 54%. Patients attaining a strong DTH response had a significantly better (p = 0.0001) 5-year overall survival of 75% compared with 44% in patients without a strong response. Gene expression array linked a 50-gene signature to prognosis, including a cluster of four cancer testis antigens: CTAG2 (NY-ESO-2), MAGEA1, SSX1, and SSX4. Thirty-five patients, who received an autologous vaccine, followed by ipilimumab for progressive disease, had a significantly improved 3-year survival of 46% compared with 19% in nonvaccinated patients treated with ipilimumab alone (p = 0.007). Conclusion. Improved survival in patients attaining a strong DTH and increased response rate with subsequent ipilimumab suggests that the autologous vaccine confers protective immunity.

Human T cell crosstalk is induced by tumor membrane transfer.

Trogocytosis is a contact-dependent unidirectional transfer of membrane fragments between immune effector cells and their targets, initially detected in T cells following interaction with professional antigen presenting cells (APC). Previously, we have demonstrated that trogocytosis also takes place between melanoma-specific cytotoxic T lymphocytes (CTLs) and their cognate tumors. In the present study, we took this finding a step further, focusing on the ability of melanoma membrane-imprinted CD8+ T cells to act as APCs (CD8+ T-APCs). We demonstrate that, following trogocytosis, CD8+ T-APCs directly present a variety of melanoma derived peptides to fraternal T cells with the same TCR specificity or to T cells with different TCRs. The resulting T cell-T cell immune synapse leads to (1) Activation of effector CTLs, as determined by proliferation, cytokine secretion and degranulation; (2) Fratricide (killing) of CD8+ T-APCs by the activated CTLs. Thus, trogocytosis enables cross-reactivity among CD8+ T cells with interchanging roles of effectors and APCs. This dual function of tumor-reactive CTLs may hint at their ability to amplify or restrict reactivity against the tumor and participate in modulation of the anti-cancer immune response.

Imprinting of lymphocytes with melanoma antigens acquired by trogocytosis facilitates identification of tumor-reactive T cells.

Trogocytosis is a contact-dependent intercellular transfer of membrane fragments and associated molecules from APCs to effector lymphocytes. We previously demonstrated that trogocytosis also occurs between tumor target and cognate melanoma Ag-specific cytotoxic T cells (CTL). In this study, we show that, following trogocytosis, immune effector cells acquire molecular components of the tumor, including surface Ags, which are detectable by specific mAbs. We demonstrate that CD8(+) and CD4(+) T cells from melanoma patients' PBMC and tumor-infiltrating lymphocytes (TIL) capture melanoma Ags, enabling identification of trogocytosing lymphocytes by staining with Ag-specific Abs. This finding circumvents the necessity of tumor prelabeling, which in the past was mandatory to detect membrane-capturing T cells. Through the detection of melanoma Ags on TIL, we sorted trogocytosing T cells and verified their preferential reactivity and cytotoxicity. Furthermore, tumor Ag-imprinted T cells were detected at low frequency in fresh TIL cultures shortly after extraction from the tumor. Thus, T cell imprinting by tumor Ags may allow the enrichment of melanoma Ag-specific T cells for research and potentially even for the adoptive immunotherapy of patients with cancer.

Trogocytosis is a gateway to characterize functional diversity in melanoma-specific CD8+ T cell clones.

Trogocytosis, the transfer of membrane patches from target to immune effector cells, is a signature of tumor-T cell interaction. In this study, we used the trogocytosis phenomenon to study functional diversity within tumor-specific T cell clones with identical TCR specificity. MART-1(26-35)-specific CD8 T cell clones, which differed in their trogocytosis capacity (low [2D11], intermediate [2G1], high [2E2]), were generated from melanoma patients. Functional evaluation of the clones showed that the percentage of trogocytosis-capable T cells closely paralleled each clone's IFN-γ and TNF-α production, lysosome degranulation, and lysis of peptide-pulsed targets and unmodified melanoma. The highly cytotoxic 2E2 clone displayed the highest TCR peptide binding affinity, whereas the low-activity 2D11 clone showed TCR binding to peptide-MHC in a CD8-dependent manner. TCR analysis revealed Vß16 for clones 2E2 and 2G1 and Vß14 for 2D11. When peptide-affinity differences were bypassed by nonspecific TCR stimulation, clones 2E2 and 2D11 still manifested distinctive signaling patterns. The high-activity 2E2 clone displayed prolonged phosphorylation of ribosomal protein S6, an integrator of MAPK and AKT activation, whereas the low-activity 2D11 clone generated shorter and weaker phosphorylation. Screening the two clones with identical TCR Vß by immunoreceptor array showed higher phosphorylation of NK, T, and B cell Ag (NTB-A), a SLAM family homophilic receptor, in clone 2E2 compared with 2G1. Specific blocking of NTB-A on APCs markedly reduced cytokine production by CD8 lymphocytes, pointing to a possible contribution of NTB-A costimulation to T cell functional diversity. This finding identifies NTB-A as a potential target for improving anti-cancer immunotherapy.

Transcutaneous immunization with hydrophilic recombinant gp100 protein induces antigen-specific cellular immune response.

The objective of this study was to evaluate the potential of transcutaneous immunization with tumor antigen to induce cell-mediated immunity. For this purpose, hydrophilic recombinant gp100 protein (HR-gp100) was topically applied on human intact skin in vitro, and used as a vaccine in a mouse model. We demonstrate that HR-gp100 permeates into human skin, and is processed and presented by human dendritic cells. In a mouse model, an HR-gp100-based vaccine triggered antigen-specific T cell responses, as shown by proliferation assays, ELISA and intracellular staining for IFN-γ. Transcutaneous antigen delivery may provide a safe, simple and effective method to elicit cell-mediated immunity.

Physicochemical parameters affecting liposomal bisphosphonates bioactivity for restenosis therapy: internalization, cell inhibition, activation of cytokines and complement, and mechanism of cell death.

Partial inactivation and transient depletion of monocytes/macrophages by liposomal bisphosphonates (LIP-BPs) is widely experimented in various inflammatory disorders including restenosis. Previous studies on activation of cytokines by LIP-BPs are limited to certain cell lines. Moreover, the correlation between in vitro and in vivo studies and complement (C) activation has not been reported. We report here a comprehensive study on the bioactivity of LIP-BPs on various cells' internalization and proliferation, mechanism of cell death, cytokines (in vitro and in vivo) and C activation (in the rat, rabbit and pig). The role of the following parameters has been determined i) drug type (clodronate/alendronate); ii) vesicles size (60-800nm); iii) charge (neutral/negative/ positive); and iv) cell culture type (various cell lines and primary cultures). It was found that monocyte/macrophage inhibition and cytokine activation depend on the cell type, with a limited correlation to the bioactivity obtained in the rat and rabbit models of restenosis. Negatively charged liposomes (85+/-20nm) effectively depleted rabbit's monocytes (67% depletion), with a minor activation of cytokines and no C activation. It is concluded that cell culture studies are insufficient for assessing cytokine activation, and that by controlling LIP-BP properties (size, charge and drug type) optimal bioactivity could be achieved.

Capture of tumor cell membranes by trogocytosis facilitates detection and isolation of tumor-specific functional CTLs.

The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8(+) T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8(+) lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8(+) T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigen-specific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.

Nanosuspensions of alendronate with gallium or gadolinium attenuate neointimal hyperplasia in rats.

Monocytes/macrophages play a pivotal role in the formation of neointinal hyperplasia following vascular injury. Transient depletion of circulating monocytes by particulate delivery systems containing bisphosphonates, such as alendronate, results in restenosis inhibition. We hypothesized that a self-suspendable nanoparticulate dosage form, with a minimum amount of expients, could be formulated by complexing the negatively charged alendronate with gallium or gadolinium. We further hypothesized that a synergistic biological effect could be obtained by nanosuspensions of alendronate with these counter ions. Nanosuspensions (150-250 nm) of alendronate-gallium and alendronate-gadolinium were successfully formulated with no additives except for the active agents and HCl for pH adjustment. Both nanosuspensions exhibited macrophage cell line growth inhibition in a dose-response relationship in comparison to the various agents in solution and in liposomes. A synergistic effect of the nanosuspensions was observed in the inhibition of raw264 macrophages, and in reducing IL-1beta and TNF-alpha secretion in cell culture. Single IV administration at the time of injury, of alendronate-gallium or alendronate-gadolinium nanosuspensions resulted in inhibition of neointimal hyperplasia and stenosis in the rat model of vascular injury. The results correlated with the significant reduction of circulating monocytes. The nanosuspensions possess the advantages of no additives for minimal provocation of side effects, and the potential of immunomodulating inflammatory disorders.