Combining synthetic carbohydrate vaccines with cancer cell glycoengineering for effective cancer immunotherapy

Tumor-associated carbohydrate antigens (TACAs) are useful targets for the development of cancer vaccines or immunotherapies. However, a major obstacle in this application of TACAs is their poor immunogenicity. To overcome the problem, a new immunotherapeutic strategy combining synthetic vaccines made of artificial TACA derivatives and metabolic glycoengineering of cancer cells to express the artificial TACA derivatives was explored. Using a murine leukemia model FBL3 with GM3 antigen as the target, it was shown that artificial GM3?N-phenylacetyl derivative (GM3NPhAc) elicited robust antigen-specific T cell-dependent immunity and that N-phenylacetyl-d-mannosamine (ManNPhAc) as the biosynthetic precursor of GM3NPhAc selectively glycoengineered cancer cells to express GM3NPhAc both in vitro and in vivo. It was also demonstrated that GM3NPhAc-specific antisera and antibodies mediated strong cytotoxicity to ManNPhAc-treated FBL3 cell. Furthermore, vaccination with a conjugate vaccine made of GM3NPhAc followed by ManNPhAc treatment could significantly suppress tumor growth and prolong the survival of tumor-bearing mouse. These results have proved the feasibility of the new cancer immunotherapeutic strategy, as well as its efficacy to cure cancer, which is of general significance.     

Improving the antigenicity of sTn antigen by modification of its sialic acid residue for development of glycoconjugate cancer vaccines

Sialyl Tn (sTn) antigen is a sialylated disaccharide abundantly expressed by many tumors. To search for effective cancer immunotherapies based on sTn antigen, we designed and synthesized a series of unnatural N-acyl derivatives of sTn and studied their immunological properties. For this purpose, an efficient method was developed to synthesize the natural and unnatural forms of sTn antigen and their protein conjugates. The resultant glycoconjugates were used to immunize C57BL/6 mice, and the immune response was assessed by enzyme-linked immunosorbent assay (ELISA). Whereas the keyhole limpet hemocyanin (KLH) conjugate of sTn elicited low levels of IgM antibodies, the KLH conjugates of N-iso-butanoyl sTn and N-phenylacetyl sTn, especially the latter, induced high titers of antigen-specific IgG antibodies, showing a T-cell-dependent response that is critical for the antitumor activity. The results suggest that the modified forms of sTn, especially N-phenylacetyl sTn, have improved antigenicity and promising immunological properties for use as cancer vaccines.     

CpG-conjugated apoptotic tumor cells elicit potent tumor-specific immunity

The primary goal of cancer immunotherapy is to elicit an immune response capable of eradicating established tumors and preventing tumor metastasis. One strategy to achieve this goal utilizes whole killed tumor cells as the primary immunogen. Killed tumor cells provide a comprehensive source of tumor-associated antigens (TAAs), thereby eliminating the need to identify individual antigens. Unfortunately, killed tumor cells tend to be poorly immunogenic. To overcome this limitation, we covalently conjugated immunostimulatory CpG oligodeoxynucleotides (ODN) to apoptotic tumor cells and examined their ability to induce TAA-specific immune responses. Results indicate that CpG conjugation enhances the uptake of cell-based vaccines by dendritic cells (DCs), up-regulates co-stimulatory molecule expression, and promotes the production of immunostimulatory cytokines. Vaccination with CpG-conjugated tumor cells triggers the expansion of tumor-specific cytotoxic T lymphocytes (CTL) that reduce the growth of established tumors and prevents their metastatic spread. Thus, conjugating CpG ODN to cell-based tumor vaccines is an important step toward improving cancer immunotherapy.     

Carbohydrate-based cancer vaccines: target cancer with sugar bullets

With the booming development of glycobiology and glycochemistry, more and more structures of tumor-associated carbohydrate antigens (TACAs) are identified. Their broad expression and high specificity in cancer make them important targets to develop cancer vaccines or immunotherapies. However, most of the TACAs are T cell-independent antigens, they cannot elicit a powerful enough immune response to prevent or treat cancer. Immunotolerance and immunosuppression are more easily induced due to their endogenous properties and the declining immunity of the patients. This review summarizes the recent efforts to overcome these obstacles: coupling the carbohydrate antigens to proper carriers such as proteins or some small molecule carriers, and chemically modifying the structures of the TACAs to enhance the immunogenicity of TACAs and break the immunotolerance.     

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine

Mammary cancer is among the most prevalent canine tumors and frequently resulting in death due to metastatic disease that is highly homologous to human breast cancer. Most canine tumors fail to raise effective immune reactions yet, some spontaneous remissions do occur. Hybrid canine dendritic cell–tumor cell fusion vaccines were designed to enhance antigen presentation and tumor immune recognition. Peripheral blood-derived autologous dendritic cell enriched populations were isolated from dogs based on CD11c⁺ expression and fused with canine mammary tumor (CMT) cells for vaccination of laboratory Beagles. These hybrid cells were injected into popliteal lymph nodes of normal dogs, guided by ultrasound, and included CpG-oligonucleotide adjuvants. Three rounds of vaccination were delivered. Significant IgG responses were observed in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized shared CMT antigens as was demonstrated by IgG-recognition of three unrelated/independently derived CMT cell lines, and recognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype response was observed after two vaccinations in most dogs. Neither significant cytotoxic T cell responses were detected, nor adverse or side-effects due to vaccination or due to the induced immune responses noted. These data provide proof-of-principle for this cancer vaccine strategy and demonstrate the presence of shared CMT antigens that promote immune recognition of mammary cancer.     

TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction

Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.     

Synthesis and Evaluation of Protein Conjugates of GM3 Derivatives Carrying Modified Sialic Acids as Highly Immunogenic Cancer Vaccine Candidates

GM3, a sialylated trisaccharide antigen expressed by a number of tumors, is an attractive target in the design of therapeutic cancer vaccines. However, a serious problem associated with GM3 is that it is poorly immunogenic. To overcome this problem for the development of GM3-based cancer vaccines, four GM3 derivatives, including 5'-N-p-methylphenylacetyl, 5'-N-p-methoxyphenylacetyl, 5'-N-p-acetophenylacetyl and 5'-N-p-chlorophenylacetyl GM3, were synthesized and then coupled to a carrier protein, keyhole limpet haemocyanin (KLH). The resultant glycoconjugates were evaluated as vaccines in mouse and compared to the KLH conjugate of 5'-N-phenylacetyl GM3 (GM3NPhAc), a highly immunogenic GM3 derivative that was previously investigated as a vaccine candidate. All of the four new GM3 derivatives were proved to be more immunogenic than GM3NPhAc and elicit very strong T cell-dependent immune responses desirable for cancer immunotherapy. It was concluded that the new GM3 derivatives can form promising vaccine candidates that may be used to combine with cell glycoengineering for cancer immunotherapy.     

Immunotherapeutic strategies employing RNA interference technology for the control of cancers

Summary The human immune system is comprised of several types of cells that have the potential to eradicate tumors without inflicting damage on normal tissue. Over the past decade, progress in the understanding of tumor biology and immunology has offered the exciting possibility of treating malignant disease with vaccines that exploit the capacity of T cells to effectively and selectively kill tumor cells. However, the immune system frequently fails to mount a successful defense against cancers despite vaccination with tumor-associated antigens. The ability of these vaccines to generate an abundant supply of armed effector T cells is often limited by immunoregulatory signaling pathways that suppress T cell activation. In addition, many tumors create a local microenvironment that inhibits the function of T cells. The attenuation of these pathways, which facilitate the evasion of tumors from immune surveillance, thus represents a potentially effective approach for cancer immunotherapy. Specifically, it may be of interest to modify the properties of dendritic cells, T cells, and tumor cells to downregulate the expression of proteins that diminish the immune response to cancers. RNA interference (RNAi) techniques have developed into a highly effective means of intracellular gene ‘knockdown’ and may be successfully employed in this way to improve cancer immunotherapies. This strategy has recently been explored both in vitro and in vivo, and has generated significantly enhanced antitumor immunity in numerous studies. Nevertheless, several practical concerns remain to be resolved before RNAi technology can be implemented safely and efficiently in humans. As novel developments and discoveries in molecular biology rapidly continue to unfold, it is likely that this technology may soon translate into a potent form of gene silencing in the clinic with profound applications to cancer immunotherapy.     

Targeting amino acid metabolism in cancer growth and anti-tumor immune response

Recent advances in amino acid metabolism have revealed that targeting amino acid metabolic enzymes in cancer therapy is a promising strategy for the development of novel therapeutic agents. There are currently several drugs in clinical trials that specifically target amino acid metabolic pathways in tumor cells. In the context of the tumor microenvironment,however,tumor cells form metabolic relationships with immune cells,and they oftencompete for common nutrients. Many tumors evolved to escape immune surveillance by taking advantage of their metabolic flexibility and redirecting nutrients for their own advantage. This review outlines the most recent advances in targeting amino acid metabolic pathways in cancer therapy while giving consideration to the impact these pathways may have on the anti-tumor immune response.     

Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines

Background

The production of cell-based cancer vaccines by gene vectors encoding proteins that stimulate the immune system has advanced rapidly in model systems. We sought to develop non-viral transfection methods that could transform patient tumor cells into cancer vaccines, paving the way for rapid production of autologous cell-based vaccines.

Methods

As the extended culture and expansion of most patient tumor cells is not possible, we sought to first evaluate a new technology that combines electroporation and chemical transfection in order to determine if plasmid-based gene vectors could be instantaneously delivered to the nucleus, and to determine if gene expression was possible in a cell-cycle independent manner. We tested cultured cell lines, a primary murine tumor, and primary human leukemia cells from diagnostic work-up for transgene expression, using both RFP and CD137L expression vectors.

Results

Combined electroporation-transfection directly delivered plasmid DNA to the nucleus of transfected cells, as demonstrated by confocal microscopy and real-time PCR analysis of isolated nuclei. Expression of protein from plasmid vectors could be detected as early as two hours post transfection. However, the kinetics of gene expression from plasmid-based vectors in tumor cell lines indicated that optimal gene expression was still dependent on cell division. We then tested to see if pediatric acute lymphocytic leukemia (ALL) would also display the rapid gene expression kinetics of tumor cells lines, determining gene expression 24 hours after transfection. Six of 12 specimens showed greater than 17% transgene expression, and all samples showed at least some transgene expression.

Conclusion

Given that transgene expression could be detected in a majority of primary tumor samples analyzed within hours, direct electroporation-based transfection of primary leukemia holds the potential to generate patient-specific cancer vaccines. Plasmid-based gene therapy represents a simple means to generate cell-based cancer vaccines and does not require the extensive infrastructure of a virus-based vector system.     

Immunization with a GM3 ganglioside nanoparticulated vaccine confers an effector CD8<Superscript>+</Superscript> T cells-mediated protection against melanoma B16 challenge

Preventive immunotherapy is an attractive strategy for patients at a high risk of having cancer. The success of prophylactic cancer vaccines would depend on the selection of target antigens that are essential for tumour growth and progression. The overexpression of GM3 ganglioside in murine and human melanomas and its important role in tumour progression makes this self antigen a potential target for preventive immunotherapy of this neoplasm. We have previously shown that preventive administration of a GM3-based vaccine to C57BL/6 mice elicited the rejection of the GM3 positive-B16 melanoma cells in most of the animals. Despite the crucial role of cellular immune response in tumour protection, the involvement of T cells in anti-tumour immunity of ganglioside vaccines is not described. Here, we examined the mechanisms by which this immunogen confers tumour protection. We have found that induction of anti-GM3 IgG antibodies correlated with tumour protection. Surprisingly, CD8⁺ T cells, but not NK1.1⁺ cells, are required in the effector phase of the antitumour immune response. The depletion of CD4⁺ T cells during immunization phase did not affect the anti-tumour activity. In addition, T cells from surviving-immunized animals secreted IFNγ when were co-cultured with IFNα-treated B16 melanoma cells or DCs pulsed with melanoma extract. Paradoxically, in spite of the glycolipidic nature of this antigen, these findings demonstrate the direct involvement of the cellular immune response in the anti-tumour protection induced by a ganglioside-based vaccine. Grant support: Center of Molecular Immunology, Elea Laboratories and Recombio.     

Preparation of dendritic cells for cancer immunotherapy

Development of new effective method for cancer therapy is one of the most important trends in the modern medicine. Along with surgery, chemotherapy and radiotherapy, induction of an immune response against the tumor cells is a promising approach for therapy of cancer, particularly metastatic, slowly dividing tumors and cancer stem cells. Induction of the antitumor T-cell immune response involves activation of antigen-presenting cells, which can efficiently present the cancer antigens and activate T-lymphocytes. The immune response may be activated by dendritic cells (DC) loaded with tumor antigens, such as tumor-specific proteins, tumor cell lysates, apoptotic or necrotic tumor cells, as well as nucleic acids encoding tumor antigens. Regardless of the selected source of the tumor antigen, preparation of mature DC is a principal step in the development of anticancer vaccines aimed at the induction of the cytotoxic T-cell immune response. Recently, various research groups have proposed several strategies for producing mature DC, differed by the set of agents used. It has been shown that the maturation strategy influences both their phenotype and the ability to induce the immune response. In this review we have analyzed the results of studies on the various strategies of preparation of mature DCs.     

Intracellular retention of the NKG2D ligand MHC class I chain-related gene A in human melanomas confers immune privilege and prevents NK cell-mediated cytotoxicity

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However, their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice, these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors, given that this effect was not observed in NK cell-depleted mice. Also, mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum, an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA, retrograde transport to the cytoplasm, and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore, this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells.     

The anti-tumor efficacy of a GM-CSF-secreting tumor cell vaccine is not inhibited by docetaxel administration

Docetaxel has demonstrated therapeutic efficacy against breast, prostate, and ovarian cancer and other solid tumors. The tumoricidal activity of docetaxel is mainly attributed to its ability to block microtubule depolymerization, thus inducing G₂-M arrest and apoptosis. Mounting evidence indicates that docetaxel also possesses immunomodulatory activity such as augmenting macrophage and lymphokine activated killer activity and inducing pro-inflammatory cytokines, suggesting that docetaxel may be a good chemotherapeutic agent to combine with cancer immunotherapies, assuming that it does not inhibit the vaccine-induced immune response. The anti-tumor activity of the combination of docetaxel and a GM-CSF-secreting B16F10 tumor cell vaccine (B16.GM) was evaluated in the murine B16 melanoma model. Dose levels of docetaxel and the B16.GM vaccine known to be ineffective when used as single agents were selected. Three iv treatments of 6 mg/kg docetaxel per injection given on days 5, 9, and 13 after tumor challenge or a single vaccination with 2–3×10⁶ B16.GM cells on day 3 were ineffective at inhibiting tumor growth when used as single agents [median survival time (MST)=24 days in both treatment groups and in control animals]. However, combination of docetaxel and B16.GM vaccine significantly delayed tumor growth, increasing MST to 45 days. A similar improvement in anti-tumor efficacy was observed using multiple treatment cycles of the B16.GM vaccine/docetaxel combination. Administration of docetaxel every 4 days between bi-weekly B16.GM vaccinations increased the median survival of tumor-bearing mice from 31 to 52 days compared to multiple B16.GM vaccinations alone. In summary, these data demonstrate that rather than inhibiting the anti-tumor effects of a GM-CSF-secreting tumor cell vaccine, docetaxel combined with a whole cell vaccine significantly inhibits tumor growth, increases survival time and does not impede T-cell activation in the murine B16F10 melanoma tumor model. GM-secreting tumor cell vaccines in combination with docetaxel may represent a new strategy for combining chemo and immunotherapy for cancer.     

Mapping the immunosuppressive environment in uterine tumors: implications for immunotherapy

The major hurdle for cancer vaccines to be effective is posed by tumor immune evasion. Several common immune mechanisms and mediators are exploited by tumors to avoid immune destruction. In an attempt to shed more light on the immunosuppressive environment in uterine tumors, we analyzed the presence of PD-L1, PD-L2, B7-H4, indoleamine 2,3-dioxygenase (IDO), galectin-1, galectin-3, arginase-1 activity and myeloid-derived suppressor cell (MDSC) infiltration. IDO, PD-L1, PD-L2 and B7-H4 were analyzed by immunohistochemistry. PD-L2 was mostly expressed at low levels in these tumors. We found high IDO expression in 21 % of endometrial carcinoma samples and in 14 % of uterine sarcoma samples. For PD-L1 and B7-H4, we found high expression in 92 and 90 % of endometrial cancers, respectively, and in 100 and 92 % of the sarcomas. Galectin-1 and 3 were analyzed in tissue lysates by ELISA, but we did not find an increase in both molecules in tumor lysates compared with benign tissues. We detected expression of galectin-3 by fibroblasts, immune cells and tumor cells in single-cell tumor suspensions. In addition, we noted a highly significant increase in arginase-1 activity in endometrial carcinomas compared with normal endometria, which was not the case for uterine sarcomas. Finally, we could demonstrate MDSC infiltration in fresh tumor suspensions from uterine tumors. These results indicate that the PD-1/PD-L1 interaction and B7-H4 could be possible targets for immune intervention in uterine cancer patients as well as mediation of MDSC function. These observations are another step toward the implementation of inhibitors of immunosuppression in the treatment of uterine cancer patients.     

Liposomal vaccines--targeting the delivery of antigen

Vaccines that can prime the adaptive immune system for a quick and effective response against a pathogen or tumor cells, require the generation of antigen (Ag)-specific memory T and B cells. The unique ability of dendritic cells (DCs) to activate na?ve T cells, implies a key role for DCs in this process. The generation of tumor-specific CD8(+) cytotoxic T cells (CTLs) is dependent on both T cell stimulation with Ag (peptide-MHC-complexes) and costimulation. Interestingly, tumor cells that lack expression of T cell costimulatory molecules become highly immunogenic when transfected to express such molecules on their surface. Adoptive immunotherapy with Ag-pulsed DCs also is a strategy showing promise as a treatment for cancer. The use of such cell-based vaccines, however, is cumbersome and expensive to use clinically, and/or may carry risks due to genetic manipulations. Liposomes are particulate vesicular lipid structures that can incorporate Ag, immunomodulatory factors and targeting molecules, and hence can serve as potent vaccines. Similarly, Ag-containing plasma membrane vesicles (PMV) derived from tumor cells can be modified to incorporate a T cell costimulatory molecule to provide both TCR stimulation, and costimulation. PMVs also can be modified to contain IFN-gamma and molecules for targeting DCs, permitting delivery of both Ag and a DC maturation signal for initiating an effective immune response. Our results show that use of such agents as vaccines can induce potent anti-tumor immune responses and immunotherapeutic effects in tumor models, and provide a strategy for the development of effective vaccines and immunotherapies for cancer and infectious diseases.     

Vaccination against angiogenesis-associated antigens: a novel cancer immunotherapy strategy

Therapeutic vaccines represent an attractive approach to cancer treatment. Traditionally, cancer immunotherapy targets antigens expressed by the tumor cells. Although numerous clinical trials studying different cancer vaccines have been conducted during the past twenty years, very limited clinical responses have been observed. The inefficient anti-tumor immunity is thought to be due, in major part, to the escape mechanisms exerted by the genetically unstable tumor cells, e.g., emergence of antigen-loss mutants, downregulation of MHC molecules and lack of expression of costimulatory molecules. Recently, a novel vaccine strategy has been developed to circumvent these obstacles. Taking advantage of the importance of angiogenesis in tumor growth and the genetic stability of endothelial cells, this immunotherapy strategy targets antigens (e.g., angiogenic growth factor receptors) overexpressed by the tumor neo-vasculature rather than the tumor cells per se. For example, active immunization against vascular endothelial growth factor receptor-2 (VEGFR-2) has been shown to generate strong cellular and humoral immune responses, which lead to the inhibition of angiogenesis and tumor growth and metastasis. This review provides an outline of this emerging field and discusses the advantages and potential pitfalls of such a vaccine strategy.     

Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death

One particular strategy to render anticancer therapies efficient consists of converting the patient's own tumor cells into therapeutic vaccines, via the induction of immunogenic cell death (ICD). One of the hallmarks of ICD dwells in the active release of ATP by cells committed to undergo, but not yet having succumbed to, apoptosis. We observed that the knockdown of essential autophagy-related genes (ATG3, ATG5, ATG7 and BECN1) abolishes the pre-apoptotic secretion of ATP by several human and murine cancer cell lines undergoing ICD. Accordingly, autophagy-competent, but not autophagy-deficient, tumor cells treated with ICD inducers in vitro could induce a tumor-specific immune response in vivo. Cancer cell lines stably depleted of ATG5 or ATG7 normally generate tumors in vivo, and such autophagy-deficient neoplasms, upon systemic treatment with ICD inducers, exhibit the same levels of apoptosis (as monitored by nuclear shrinkage and caspase-3 activation) and necrosis (as determined by following the kinetics of HMGB1 release) as their autophagy-proficient counterparts. However, autophagy-incompetent cancers fail to release ATP, to recruit immune effectors into the tumor bed and to respond to chemotherapy in conditions in which autophagy-competent tumors do so. The intratumoral administration of ecto-ATPase inhibitors increases extracellular ATP concentrations, re-establishes the therapy-induced recruitment of dendritic cells and T cells into the tumor bed, and restores the chemotherapeutic response of autophagy-deficient cancers. Altogether, these results suggest that autophagy-incompetent tumor cells escape from chemotherapy-induced (and perhaps natural?) immunosurveillance because they are unable to release ATP.     

Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas

Cancer vaccines have been developed to instruct the endogenous immune responses to autologous tumors and to generate durable clinical responses. However, the therapeutic benefits of cancer vaccines remain insufficient due to the multiple immunosuppressive signals delivered by tumors. Thus, to improve the clinical efficacy of cancer immunotherapy, it is important to develop new modalities to overcome immunosuppressive tumor microenvironments and elicit effective antitumor immune responses. In this study, we show that novel monoclonal antibodies (mAbs) specifically targeting either T cell immunoglobulin mucin protein-3 (TIM-3) or T cell immunoglobulin mucin protein-4 (TIM-4) enhance the therapeutic effects of vaccination against established B16 murine melanomas. This is true for vaccination with irradiated B16 melanoma cells engineered to express the flt3 ligand gene (FVAX). More importantly, combining anti-TIM-3 and anti-TIM-4 mAbs markedly increased vaccine-induced antitumor responses against established B16 melanoma. TIM-3 blockade mainly stimulated antitumor effector activities via natural killer cell-dependent mechanisms, while CD8⁺ T cells served as the main effectors induced by anti-TIM-4 mAb. Our findings reveal that therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy for improving the clinical efficacy of cancer immunotherapy.     

Immune modulation by melanoma and ovarian tumor cells through expression of the immunosuppressive molecule CD200

Background and Objective Immune escape by tumors can occur by multiple mechanisms, each a significant barrier to immunotherapy. We previously demonstrated that upregulation of the immunosuppressive molecule CD200 on chronic lymphocytic leukemia cells inhibits Th1 cytokine production required for an effective cytotoxic T cell response. CD200 expression on human tumor cells in animal models prevents human lymphocytes from rejecting the tumor; treatment with an antagonistic anti-CD200 antibody restored lymphocyte-mediated tumor growth inhibition. The current study evaluated CD200 expression on solid cancers, and its effect on immune response in vitro. Methods and Results CD200 protein was expressed on the surface of 5/8 ovarian cancer, 2/4 melanoma, 2/2 neuroblastoma and 2/3 renal carcinoma cell lines tested, but CD200 was absent on prostate, lung, breast, astrocytoma, or glioblastoma cell lines. Evaluation of patient samples by immunohistochemistry showed strong, membrane-associated CD200 staining on malignant cells of melanoma (4/4), ovarian cancer (3/3) and clear cell renal cell carcinoma (ccRCC) (2/3), but also on normal ovary and kidney. CD200 expression on melanoma metastases was determined by RT-QPCR, and was found to be significantly higher in jejunum metastases (2/2) and lung metastases (2/6) than in normal samples. Addition of CD200-expressing, but not CD200-negative solid tumor cell lines to mixed lymphocyte reactions downregulated the production of Th1 cytokines. Inclusion of antagonistic anti-CD200 antibody restored Th1 cytokine responses. Conclusion These data suggest that melanoma, ccRCC and ovarian tumor cells can express CD200, thereby potentially suppressing anti-tumor immune responses. CD200 blockade with an antagonistic antibody may permit an effective anti-tumor immune response in these solid tumor types.