Generation Of Tumor-specific Immune Response Research Articles

LTX-315 triggers anticancer immunity by inducing MyD88-dependent maturation of dendritic cells.

LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer activity but limited toxicity for non-malignant cells. LTX-315 induces both immunogenic tumor cell death and generation of tumor-specific immune responses in multiple experimental tumor models. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immunity, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. We found that LTX-315 treatment induces the maturation of DCs, both indirectly through the release of cancer cell-derived damage-associated molecular patterns (DAMPs)/alarmins and nucleic acids (DNA and RNA) capable of triggering distinct Toll-like receptor (TLR) signaling, and, directly by activating TLR7. The latter results in the ignition of multiple intracellular signaling pathways that promotes DC maturation, including NF-κB, mitogen activated protein kinases (MAPKs), and inflammasome signaling, as well as increased type 1 interferon production. Critically, the effects of LTX-315 on DCs the consequent promotion of anti-melanoma immunity depend on the cytosolic signal transducer myeloid differentiation response gene 88 (MyD88). These results cast light on the mechanisms by which LTX-315 induces DC maturation and hence elicits anticancer immunity, with important implications for the use of LTX-315 as an anticancer immunotherapeutic.

Oncolytic peptide LTX-315 enhances anti-melanoma immunity by inducing MyD88-dependent maturation of dendritic cells

Abstract LTX-315 is a synthetic cationic oncolytic peptide with potent anticancer cytotoxicity. LTX-315 induces both immunogenic tumor cell death and the generation of tumor-specific immune response. Given the central role of dendritic cell (DC) maturation in the induction of antigen-specific immune responses, we investigated the effect of LTX-315 treatment on the maturation of tumor-infiltrating DCs (TiDCs) and the generation of anti-melanoma immunity. The results reveal that LTX-315 induces the maturation of DCs through three distinct mechanisms: 1) release of DAMPs/alarmins; 2) complexes of LTX-315 with nucleic acids that act immunostimulatory; and 3) direct induction of the maturation of both conventional and plasmacytoid DCs by triggering TLR7. Importantly, LTX-315-induced maturation of DCs in vitro and in vivo was dependent on the presence of the signal transducer MyD88 as well as distinct TLRs. Our data also substantiate the notion that reagents like LTX-315 with both tumor cell cytotoxic and immunostimulaing capacities are more effective immunotherapeutics.

A novel systemically administered Toll-like receptor 7 agonist potentiates the effect of ionizing radiation in murine solid tumor models.

Although topical TLR7 therapies such as imiquimod have proved successful in the treatment of dermatological malignancy, systemic delivery may be required for optimal immunotherapy of nondermatological tumors. We report that intravenous delivery of the novel small molecule TLR7 agonist, DSR-6434, leads to the induction of type 1 interferon and activation of T and B lymphocytes, NK and NKT cells. Our data demonstrate that systemic administration of DSR-6434 enhances the efficacy of ionizing radiation (IR) and leads to improved survival in mice bearing either CT26 or KHT tumors. Of the CT26 tumor-bearing mice that received combined therapy, 55% experienced complete tumor resolution. Our data reveal that these long-term surviving mice have a significantly greater frequency of tumor antigen specific CD8+ T cells when compared to age-matched tumor-naïve cells. To evaluate therapeutic effects on spontaneous metastases, we showed that combination of DSR-6434 with local IR of the primary tumor significantly reduced metastatic burden in the lung, when compared to time-matched cohorts treated with IR alone. The data demonstrate that systemic administration of the novel TLR7 agonist DSR-6434 in combination with IR primes an antitumor CD8+ T-cell response leading to improved survival in syngeneic models of colorectal carcinoma and fibrosarcoma. Importantly, efficacy extends to sites outside of the field of irradiation, reducing metastatic load. Clinical evaluation of systemic TLR7 therapy in combination with IR for the treatment of solid malignancy is warranted.What's new?Recent evidence suggests that damage from ionizing radiation (IR) can render tumor cells immunogenic. Unfortunately, established tumors often suppress this anti-tumor immune response. Combination therapy with IR and immune-modulators such as Toll-like-receptor (TLR) family agonists may overcome this problem. In this proof-of-concept study, the authors examined one such small-molecule drug, called DSR-6434. They found that systemic administration of DSR-6434 can enhance the effectiveness of radiotherapy in mice, and that this occurs via the generation of tumor-specific immune responses. Easily delivered drugs that activate TLR-family molecules may thus offer a promising therapeutic approach.

846. Enhanced Antitumor Effect of Oncolytic Adenovirus Expressing Interleukin-12 and B7.1 in an Immuneocompetenet Mouse Model

Interleukin (IL)-12 is reported to exhibit potent anti-tumor effect by promoting NK cell and cytotoxic T cell activities. The B7 family has been shown to take an important role in stimulating anti-tumor response, and synergy between IL-12 and B7-1 for cancer immunotherapy has previously been demonstrated. To increase the potential antitumor activity of the oncolytic adenovirus, we have constructed an E1B 55kDa deleted oncolytic adenoviral vector, YKL-IL12/B, which expresses murine IL-12 and B7.1. The therapeutic efficacy of YKL-IL12 and YKL-IL12/B was evaluated in an immunocompetent mouse bearing murine melanoma B16F10 tumor. The results showed significant inhibition of tumor growth following YKL-IL12/B treatment compared to PBS treated tumors. Moreover, YKL-IL12/B adenoviral vector elicited enhanced antitumor activity and a higher incidence of complete tumor regression compared to tumors treated with the analogous vector (YKL-1) that lacks IL-12 and B7.1. Survival was also significantly prolonged in YKL-IL12/B-treated mice, and immunohistochemistry analysis demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the YKL-1 subjects. In addition, we observed that YKL-IL12/B induced significantly higher CTL activity than YKL-1, demonstrating that anti-tumor effect is associated with the generation of tumor-specific immune response. In summary, these data indicate that local expression of IL-12 and B7.1 in tumor bed may be an attractive alternative treatment against metastatic carcinoma. Interleukin (IL)-12 is reported to exhibit potent anti-tumor effect by promoting NK cell and cytotoxic T cell activities. The B7 family has been shown to take an important role in stimulating anti-tumor response, and synergy between IL-12 and B7-1 for cancer immunotherapy has previously been demonstrated. To increase the potential antitumor activity of the oncolytic adenovirus, we have constructed an E1B 55kDa deleted oncolytic adenoviral vector, YKL-IL12/B, which expresses murine IL-12 and B7.1. The therapeutic efficacy of YKL-IL12 and YKL-IL12/B was evaluated in an immunocompetent mouse bearing murine melanoma B16F10 tumor. The results showed significant inhibition of tumor growth following YKL-IL12/B treatment compared to PBS treated tumors. Moreover, YKL-IL12/B adenoviral vector elicited enhanced antitumor activity and a higher incidence of complete tumor regression compared to tumors treated with the analogous vector (YKL-1) that lacks IL-12 and B7.1. Survival was also significantly prolonged in YKL-IL12/B-treated mice, and immunohistochemistry analysis demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the YKL-1 subjects. In addition, we observed that YKL-IL12/B induced significantly higher CTL activity than YKL-1, demonstrating that anti-tumor effect is associated with the generation of tumor-specific immune response. In summary, these data indicate that local expression of IL-12 and B7.1 in tumor bed may be an attractive alternative treatment against metastatic carcinoma.

702. Concurrent Delivery of GM-CSF and B7-1 Using an Oncolytic Adenovirus Elicits Potent Anti-Tumor Effect on the Growth Established B16-F10 Tumor Model

Oncolytic adenoviral vectors are currently being developed as biological anti-cancer therapeutic agents. Coupling the lytic function of oncolytic adenovirus with virus-mediated transgene delivery represents a powerful extension of this methodolgy. To increase the potential anti-tumor activity of oncolytic adenovirus, we constructed an E1B 55kDa deleted oncolytic adenoviral vector, YKL-GB, which expresses both GM-CSF and B7-1. The therapeutic efficacy of YKL-GB adenovirus was evaluated in immunocompetent mice bearing murine melanoma B16-F10 tumors. Significant inhibition of tumor growth was seen in mice treated with YKL-GB compared to those treated with an analogous vector, YKL-1, that lacks GM-CSF and B7-1. Moreover, YKL-GB oncolytic adenovirus demonstrated enhanced anti-tumor activity and a higher incidence of complete tumor regression compared to a replication-incompetent adenovirus, dl-GB, which co-expresses the GM-CSF and B7-1. Localized GM-CSF and B7-1 gene transfer also conferred long-lasting immunity against tumor rechallenge. To establish that anti-tumor effect is associated with the generation of tumor-specific immune response, we examined the cytolytic activity by IFN-|[gamma]| ELISpot assay. We observed that YKL-GB induced significantly higher T cell-mediated antitumor activity than YKL-1. Furthermore, immunohistochemical studies demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the YKL-1 groups. In agreement with these results, splenocytes from tumor-bearing mice treated with YKL-GB expressed high levels of costimulatory and activation molecules. In conclusion, the findings of this study demonstrate the effectiveness of augmenting the immune response against tumors with oncolytic adenovirus expressing both GM-CSF and B7-1, which provide a potential therapeutic strategy for the management of neoplasia.

581. Enhanced Antitumor Effect of Interleukin-12 and B7.1 Expressing Oncolytic Adenovirus in Immunocompetent Mouse

Genetically modified replication-selective adenoviruses are currently under human clinical trials. These anticancer agents infect host cells through their lytic nature as part of their viral life cycle to eradicate tumor mass. The lytic property of the viral infection is coupled with virus-mediated delivery of therapeutic transgenes, enhancing the potency of the virus-based therapy against a complex array of human tumors. Interleukin (IL)-12 is reported to exhibit potent anti-tumor effect by promoting NK cell and cytotoxic T cell activities. The B7 family has been shown to take an important role in stimulating anti-tumor response, and synergy between IL-12 and B7-1 for cancer immunotherapy has previously been demonstrated. To increase the potential antitumor activity of the oncolytic adenovirus, we have constructed an E1B 55kDa deleted oncolytic adenoviral vector, Y/mIL-12/mB, which expresses murine IL-12 and B7.1. The therapeutic efficacy of Y/mIL-12/mB and Y/mIL-12 was evaluated in an immunocompetent mouse bearing murine melanoma B16F10 tumor. The results showed significant inhibition of tumor growth following Y/mIL-12/mB treatment compared to PBS treated tumors. Moreover, the oncolytic adenovirus expressing IL-12 and B7.1 demonstrated enhanced antitumor activity and a higher incidence of complete tumor regression compared to tumors treated with the analogous vector (YKL-1) that lacks IL-12 and B7.1. Survival was also significantly prolonged in Y/mIL-12/mB-treated mice, and immunohistochemistry experiments demonstrated robust CD4+ and CD8+ T-cell infiltration in these mice compared to the YKL-1 subjects. In addition, we observed that Y/mIL-12/mB induced significantly higher CTL activity than YKL-1, demonstrating that anti-tumor effect is associated with the generation of tumor-specific immune response. These data indicate that local expression of IL-12 and B7.1 in tumor bed may be an attractive alternative treatment against metastatic carcinoma.

4-1BB-specific monoclonal antibody promotes the generation of tumor-specific immune responses by direct activation of CD8 T cells in a CD40-dependent manner.

4-1BB (CD137) is a member of the TNFR superfamily (TNFRSF9). T cell expression of 4-1BB is restricted to activated cells, and cross-linking has been shown to deliver a costimulatory signal. Here we have shown that treatment of tumor-bearing mice with agonistic 4-1BB-specific Abs can lead to T cell-mediated tumor rejection. In vivo mAb depletion experiments demonstrated that this rejection requires CD8(+) cells but not CD4(+) or NK cells. Both IFN-gamma- and CD40-mediated signals were also required, because no benefit was observed on treatment with 4-1BB mAb in mice in which the genes for these molecules had been knocked out. Interestingly, 4-1BB-mediated stimulation of immune responses in CD40L(-/-) mice is effective (although at a reduced level), and may suggest the existence of an alternative ligand for CD40. Additional experiments in IL-15(-/-) mice indicate that IL-15 is not required for either the generation of the primary tumor-specific immune response or the maintenance of the memory immune response. In contrast, the presence of CD4 cells during the primary immune response appears to play a significant role in the maintenance of effective antitumor memory. Finally, in mice in which the number of dendritic cells had been expanded by Fms-like tyrosine kinase3 ligand treatment, the antitumor effects of 4-1BB ligation were enhanced.

Recombinant DNA vaccines protect against tumors that are resistant to recombinant vaccinia vaccines containing the same gene

Antigen-specific cancer immunotherapy involves the delivery of tumor-associated antigen to the host for the generation of tumor-specific immune responses and antitumor effects. We hypothesized that different delivery systems may influence the pattern of antigen-specific immune response and the outcome of antitumor effect. We therefore evaluated recombinant vaccinia virus and naked DNA for the generation of antigen-specific immune responses and antitumor effects. We previously found that recombinant vaccinia and naked DNA vaccines containing the chimeric Sig/E7/LAMP-1 gene were capable of controlling the growth of HPV-16 E7-expressing tumor cells (TC-1). In this study, we performed a head-to-head comparison of optimized delivery of Sig/E7/LAMP-1 vaccinia and DNA vaccines using dose-escalating tumor challenge. At a dose of 1 x 10(6) TC-1 cells per mouse, Sig/E7/LAMP-1 DNA provided 100% protection against subcutaneous growth of tumors, while Vac-Sig/E7/LAMP-1 protected only 40% of the mice. Furthermore, Sig/E7/LAMP-1 DNA vaccines are capable of protecting against challenge with a more stringent subclone of TC-1 (TC-1 P2) established from TC-1 tumors that survived initial Sig/E7/LAMP-1 vaccinia vaccination. Immunological assays revealed that both vaccines induced comparable levels of CD8(+) T cell precursors and anti-E7 antibody titers. Interestingly, Sig/E7/LAMP-1 vaccinia induced both E7-specific IFN-gamma- and IL4-secreting CD4(+) T cell precursors while Sig/E7/LAMP-1 DNA induced only E7-specific IFN-gamma-secreting CD4(+) T cell precursors. We also found that IL-4 knockout C57BL/6 mice vaccinated with Sig/E7/LAMP-1 vaccinia exhibited a more potent antitumor effect than vaccinated wild-type C57BL/6 mice in our tumor protection experiments. These results suggest that IL-4 may play a detrimental role in the antitumor effect mediated by vaccinia vaccines. Our findings suggested that DNA vaccines may provide better tumor protection than vaccinia vaccines employing the same gene, which may have implications in the future design of antigen-specific cancer immunotherapy.

Idiotype Vaccination in Human Myeloma: Generation of Tumor-Specific Immune Responses After High-Dose Chemotherapy

Igs contain unique portions, collectively termed idiotypes (Id), that can be recognized by the immune system. Id expressed by tumor cells in B-cell malignancies can be regarded as tumor-specific antigens and a target for vaccine immunotherapy. We have started a vaccination trial in multiple myeloma (MM) using Id-specific proteins conjugated to keyhole limpet hemocyanin (KLH) as immunogens and low doses of subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2 (IL-2) as immunoadjuvants. Twelve patients who had previously been treated with high-dose chemotherapy followed by peripheral blood progenitor cell (PBPC) transplantation entered this study from August 1995 to January 1998. All patients were in first remission at the time of vaccination. They received subcutaneous injections of Id vaccines and immunoadjuvants in an outpatient setting. The generation of Id-specific T-cell proliferative responses was documented in 2 patients, whereas a positive Id-specific delayed-type hypersensitivity (DTH) reaction was observed in 8 of the 10 patients studied. DTH specificity was confirmed in 1 patient by investigating the reactivity to synthetic peptides derived from the VDJ sequence of the tumor-specific Ig heavy chain. None of the patients generated soluble immune responses to Id, whereas the generation of soluble and cellular immune responses to KLH was observed in 100% and 80%, respectively. Eleven patients completed the treatment, whereas 1 patient failed to finish owing to progression of disease. Freedom from disease progression (FFDP), measured from the date of first Id/KLH injection to the date of first treatment after vaccination or last follow-up, ranged from 9 to 36 months. These data indicate that the immune competence status of MM patients is still susceptible to specific immunization after high-dose chemotherapy and PBPC transplantation. It remains to be determined whether generation of Id-specific immune responses can reduce the relapse rate of patients with minimal residual disease.

Idiotype Vaccination in Human Myeloma: Generation of Tumor-Specific Immune Responses After High-Dose Chemotherapy

AbstractIgs contain unique portions, collectively termed idiotypes (Id), that can be recognized by the immune system. Id expressed by tumor cells in B-cell malignancies can be regarded as tumor-specific antigens and a target for vaccine immunotherapy. We have started a vaccination trial in multiple myeloma (MM) using Id-specific proteins conjugated to keyhole limpet hemocyanin (KLH) as immunogens and low doses of subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2 (IL-2) as immunoadjuvants. Twelve patients who had previously been treated with high-dose chemotherapy followed by peripheral blood progenitor cell (PBPC) transplantation entered this study from August 1995 to January 1998. All patients were in first remission at the time of vaccination. They received subcutaneous injections of Id vaccines and immunoadjuvants in an outpatient setting. The generation of Id-specific T-cell proliferative responses was documented in 2 patients, whereas a positive Id-specific delayed-type hypersensitivity (DTH) reaction was observed in 8 of the 10 patients studied. DTH specificity was confirmed in 1 patient by investigating the reactivity to synthetic peptides derived from the VDJ sequence of the tumor-specific Ig heavy chain. None of the patients generated soluble immune responses to Id, whereas the generation of soluble and cellular immune responses to KLH was observed in 100% and 80%, respectively. Eleven patients completed the treatment, whereas 1 patient failed to finish owing to progression of disease. Freedom from disease progression (FFDP), measured from the date of first Id/KLH injection to the date of first treatment after vaccination or last follow-up, ranged from 9 to 36 months. These data indicate that the immune competence status of MM patients is still susceptible to specific immunization after high-dose chemotherapy and PBPC transplantation. It remains to be determined whether generation of Id-specific immune responses can reduce the relapse rate of patients with minimal residual disease.

Granulocyte/macrophage-colony-stimulating factor released by adenovirally transduced CT26 cells leads to the local expression of macrophage inflammatory protein 1alpha and accumulation of dendritic cells at vaccination sites in vivo.

Antigen presenting cells (APC) play an essential role in the generation of tumor-specific immune responses. Dendritic cells are the most potent of APC, capable of activating both antigen-specific CD4+ and CD8+ T cells. Previously, we have described how vaccination of mice with irradiated tumor cells producing granulocyte/macrophage-colony-stimulating factor (GM-CSF) induces tumor-specific immunity capable of protecting mice from a subsequent tumor challenge. The present study extends these findings to examine the types of APC infiltrating vaccination sites and the chemokines responsible for their recruitment. GM-CSF released from genetically engineered tumor cells led to the local accumulation of dendritic cells in and around the vaccination site. Quantification revealed a significant ten-fold increase in the number of dendritic cells infiltrating GM-CSF-producing as opposed to beta-galactosidase-producing (control) vaccination sites. Reverse transcription/polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical analysis of vaccination sites revealed that MIP-1alpha may be responsible for dendritic cell infiltration into GM-CSF-producing tissues. These findings suggest that GM-CSF may indirectly recruit dendritic cells into vaccination sites through the local production of MIP-1alpha.

Induction of dendritic cells (DC) by Flt3 Ligand (FL) promotes the generation of tumor-specific immune responses in vivo.

Daily treatment of mice with human Flt3 ligand (FL) induces the production of large numbers of MHC-class II+, CD11c+, DEC205+ dendritic cells (DC) in both lymphoid and nonlymphoid tissues. Because DC play a pivotal role in the induction of immune responses, we evaluated the effects of FL in the augmentation of anti-tumor immune responses in vivo. Treatment with daily subcutaneous injections of FL not only caused complete regression of tumors in a significant proportion of mice challenged with a syngeneic methylcholanthrene-induced fibrosarcoma, but also caused a significant decrease in the tumor growth rate in the remaining mice. The effects of FL were both dose and schedule dependent, induced regression of tumors established as much as 7 days prior to beginning cytokine treatment, and was mediated by both T-cell mediated and non-T cell-mediated mechanisms. Histopathologic evaluation of tumors during and after systemic treatment with FL revealed a dense infiltration of mononuclear and myeloid precursor cells into both the tumor and tissue surrounding the tumor that generally correlated with the degree of regression and resolved after rejection of the tumor. Collectively, the data suggest that FL may be an important cytokine in the generation of effective anti-tumor immune responses in vivo and the treatment of cancer in situ.

New antigens presented on tumor cells can cause immune rejection without influencing the frequency of tumor-specific cytolytic T cells

The specific immune response against syngeneic tumors by T cells is dependent on the existence of tumor-associated transplantation antigens (TATA). In the case of the chemically induced DBA/2-derived lymphoma Eb and its highly metastatic variant ESb the immunogenicity of these antigens is not sufficient to prevent tumor growth. Therefore we tested in two systems the influence of additional antigens as possible helper determinants for the generation of tumor-specific immune responses. In the Eb tumor system additional antigens were induced by mutagenization. The frequency of cytotoxic T lymphocytes (CTL) in response to mutagenized Eb cells was higher than that in response to untreated Eb cells. Fine specificity analysis revealed there there was no increase in the CTL response against the original TATA, but an activation of additional CTL clones responding to mutagen-induced antigens. In the ESb tumor system we tested the effect of additional recognition of minor histocompatibility antigens on the frequency of TATA-specific CTL. Transplantation of ESb tumor cells into B10.D2 mice, which are H-2-identical but differ in minor antigens, results in strong tumor rejection responses. In a limiting dilution mixed-leukocyte-tumor microculture system it was found that the minor antigens are recognized at the clonal level as independent antigens. The overall frequency of anti-tumor CTL in ESb-immunized B10.D2 mice was about 1 3000 . Among these, the frequency of TATA-specific CTL was 1 16,709 and thus not significantly different from that of syngeneic DBA/2 mice. Thus neither minor antigens nor mutagen-induced antigens acted in the Eb/ESb tumor system as helper determinants and did not increase the frequency of tumor-specific CTLs.