Antitumor Effects In Murine Models Research Articles

Boosting Interleukin-12 Antitumor Activity and Synergism with Immunotherapy by Targeted Delivery with isoDGR-Tagged Nanogold.

The clinical use of interleukin-12 (IL12), a cytokine endowed with potent immunotherapeutic anticancer activity, is limited by systemic toxicity. The hypothesis is addressed that gold nanoparticles tagged with a tumor-homing peptide containing isoDGR, an αvβ3-integrin binding motif, can be exploited for delivering IL12 to tumors and improving its therapeutic index. To this aim, gold nanospheres are functionalized with the head-to-tail cyclized-peptide CGisoDGRG (Iso1) and murine IL12. The resulting nanodrug (Iso1/Au/IL12) is monodispersed, stable, and bifunctional in terms of αvβ3 and IL12-receptor recognition. Low-dose Iso1/Au/IL12, equivalent to 18-75 pg of IL12, induces antitumor effects in murine models of fibrosarcomas and mammary adenocarcinomas, with no evidence of toxicity. Equivalent doses of Au/IL12 (a nanodrug lacking Iso1) fail to delay tumor growth, whereas 15 000 pg of free IL12 is necessary to achieve similar effects. Iso1/Au/IL12 significantly increases tumor infiltration by innate immune cells, such as NK and iNKT cells, monocytes, and neutrophils. NK cell depletion completely inhibits its antitumor effects. Low-dose Iso1/Au/IL12 can also increase the therapeutic efficacy of adoptive T-cell therapy in mice with autochthonous prostate cancer. These findings indicate that coupling IL12 to isoDGR-tagged nanogold is a valid strategy for enhancing its therapeutic index and sustaining adoptive T-cell therapy.

Abstract P2-09-16: CD8 T cells induced by novel alphaviral vector predict improved progression free survival in advanced HER2+ breast cancer patients

Abstract Background: Immune-based therapy for metastatic breast cancer has had limited success. Strategies to augment adaptive immunity include vaccines targeting genomic amplifications like Human Epidermal Growth Factor Type 2 (HER2), an established driver of malignancy. Using a novel alphaviral vector, we constructed a vaccine encoding a portion of HER2 (VRP-HER2). Methods: In preclinical studies, mice were immunized before or after implantation of hHER2+ tumor cells and HER2-specific immune responses and anti-tumor function were assessed. We then translated this vaccine into a phase I clinical trial in which subjects with advanced HER2-overexpressing breast cancers received VRP-HER2 every 2 weeks for a total of three doses (cohort 1). In cohort 2, subjects received the same dose of VRP-HER2 along with a standard HER2 targeted therapy. Results: VRP-HER2 induced HER2-specific T cell and antibody responses while controlling tumor growth in murine models. Vaccination with VRP-HER2 was well tolerated in both patient cohorts. PFS was modest, while median OS was 50.2 months in cohort 1 and 32.7 months in cohort 2. In cohort 2, there is one partial response and two patients with continued stable disease. Vaccine induced anti-HER2 antibodies and T cells were identified. Increased perforin expression by memory CD8 T cells post vaccination significantly correlated with improved PFS. Conclusions: VRP-HER2 led to an increase in perforin expressing HER2-specific memory CD8 T cells in preclinical and clinical studies, and had profound antitumor effects in murine models. The generation of HER2-specific memory CD8 T cells was significantly correlated with increased PFS in patients. Subsequent studies will seek to enhance T cell activity by combination with anti-PD-1/PD-L1 antibodies. Citation Format: Crosby EJ, Gwin WR, Chang S, Maecker HT, Lubkov V, Snyder JC, Broadwater G, Hyslop T, Osada T, Hobeika AC, Hartman ZC, Morse MA, Lyerly HK. CD8 T cells induced by novel alphaviral vector predict improved progression free survival in advanced HER2+ breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-09-16.

Preclinical antitumor activity of SST0116CL1: a novel heat shock protein 90 inhibitor.

4-Amino substituted resorcino-isoxazole (SST0116CL1) (property of Sigma-Tau Research Switzerland S.A.) is a potent, second generation, small-molecule heat shock protein 90 inhibitor (Hsp90i). SST0116CL1 binds to the ATP binding pocket of Hsp90, and interferes with Hsp90 chaperone function thus resulting in client protein degradation and tumor growth inhibition. The aim of the study was to assess SST0116CL1 in various solid and haematological tumors. The antitumor properties of SST0116CL1 were assessed using in vitro cell proliferation and client protein degradation assays and in vivo different tumor xenograft models. Pharmacokinetic (PK) data were also generated in tumor-bearing mice to gain an understanding of optimal dosing schedules and regimens. SST0116CL1 was shown to inhibit recombinant Hsp90α and to induce the destabilization of different client proteins, often overexpressed and constitutively activated in different types of hematological or solid human tumors. In preclinical in vivo studies, it was revealed to induce antitumor effects in murine models of leukemia and of gastric and ovarian carcinoma. A modulation of PD biomarkers in terms of downregulation of Hsp90 client proteins in tumor-bearing mice was found. SST0116CL1 is a new clinical candidate for cancer therapy. The antitumor property of SST0116CL1, likely due to direct inhibition of the Hsp90 enzymatic activity, may prove to be a critical attribute as the compound enters phase I clinical trials.

Locally delivered CD40 agonist antibody accumulates in secondary lymphoid organs and eradicates experimental disseminated bladder cancer.

Immunotherapy with intratumoral injection of adenoviral vectors expressing CD40L has yielded positive results in experimental and clinical bladder cancer. We therefore hypothesized that anti-CD40 antibody would be effective in this setting. Agonistic CD40 antibodies were developed as vaccine adjuvants but have later been used as treatment of advanced solid tumors and hematologic cancers. Systemic anti-CD40 therapy has been associated with immune-related adverse events, such as cytokine release syndrome and liver toxicity, and local delivery is an attractive approach that could reduce toxicity. Herein, we compared local and systemic anti-CD40 antibody delivery to evaluate efficacy, toxicity, and biodistribution in the experimental MB49 bladder cancer model. Antitumor effects were confirmed in the B16 model. In terms of antitumor efficacy, local anti-CD40 antibody stimulation was superior to systemic therapy at an equivalent dose and CD8 T cells were crucial for tumor growth inhibition. Both administration routes were dependent on host CD40 expression for therapeutic efficacy. In vivo biodistribution studies revealed CD40-specific antibody accumulation in the tumor-draining lymph nodes and the spleen, most likely reflecting organs with frequent target antigen-expressing immune cells. Systemic administration led to higher antibody concentrations in the liver and blood compared with local delivery, and was associated with elevated levels of serum haptoglobin. Despite the lack of a slow-release system, local anti-CD40 therapy was dependent on tumor antigen at the injection site for clearance of distant tumors. To summarize, local low-dose administration of anti-CD40 antibody mediates antitumor effects in murine models with reduced toxicity and may represent an attractive treatment alternative in the clinic.

Ad5 immunity after multiple safe, homologous immunizations against tumor-associated antigens with new recombinant Ad5 vector.

85 Background: The major limitation for the use of Ad5 and other vectored vaccines is the inability to be effective in the presence of pre-existing or concomitant vector immunity. An Ad5-based vector with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), by virtue of diminished late phase viral protein expression, avoids immunological clearance and induces immune responses. Methods: The Ad5 [E1-, E2b-] platform encoding tumor associated antigen(s) (TAA) such as a modified carcinoembryonic antigen (CEA(6D)), HER2 and HPV E6/E7, were evaluated for the induction of TAA specific immune responses and anti-tumor effects in murine models. In a phase I/II clinical trial, cohorts of patients (n=25 total) with advanced colorectal cancer, refractory to prior therapies, received escalating doses of Ad5 [E1-, E2b-]-CEA(6D) (109 to 1011 vp) subcutaneously every 3 weeks for 3 immunizations. CEA-specific cell mediated immunity was measured by ELISPOT. Results: In murine immunotherapy studies, mice implanted with tumors expressing TAA and subsequently treated with the Ad5 [E1-, E2b-] platform expressing that TAA had significant inhibition of tumor progression. Pre-vaccination against the TAA utilizing the Ad5 [E1-, E2b-]-TAA resulted in inhibition of tumor establishment. In a clinical trial, patients who received the highest dose of Ad5 [E1-, E2b-]-CEA(6D) exhibited the highest levels of CEA-specific CMI responses. The induction of CEA-specific CMI responses increased over the course of the 3 injections despite the presence of pre-existing Ad5 immunity in the majority (75%) of patients. There were no drug related grade 3/4 toxicities. Conclusions: The results demonstrate that the novel Ad5 [E1-, E2b-] gene delivery platform can both break tolerance and generate significant CMI responses to the TAA CEA in the setting of both naturally acquired Ad5-specific immunity and/or immunization-induced Ad5 immunity.

Autoimmune response induced by dendritic cells exerts anti-tumor effect in murine model of leukemia

An autoimmune response can be induced with the application of dendritic cells (DCs), offering a viable tumor vaccine for cancer immunotherapy. Previous studies have shown that DC-based tumor vaccines in animal tumor models can inhibit tumor growth and induce autoantibodies transiently without clinical or histological features of autoimmunity. The present study aimed to investigate the role of immune response induced by dendritic cells-based therapy, especially anti-ds DNA antibodies in tumor inhibition. In this study, high titers of anti-ds DNA antibodies were induced after injecting syngeneic dendritic cells into BALB/c mice. In addition, mice immunized with DCs showed the inhibition of RL ♂ 1, BALB/c leukemia cell line, tumor growth, and prolonged survival times of tumor mice but no significant difference was found in specific CTL response and NK cell activity when compared to those of the control group. Anti-ds DNA monoclonal antibodies can recognize RL ♂ 1 cells but not normal cells by FACS analysis. Monoclonal anti-ds DNA antibody was demonstrated to be able to lyse tumor cells via complement mediated reaction in vitro and also exhibits the anti-tumor effects when the antibody was injected into tumor-implanted mice. The data suggested that immunization with dendritic cells can induce autoimmune response, which might exert anti-tumor activity in vivo.

A Therapeutic Cancer Vaccine Targeting Carcinoembryonic Antigen in Intestinal Carcinomas

A genetic vaccine platform based on DNA electroporation (DNA-EP) and adenovirus (Ad) was used to generate immune response against human carcinoembryonic antigen (CEA) and antitumor effects in murine models with spontaneous tumors arising in an orthotopic location. CEA transgenic (CEA.Tg) mice treated with the carcinogen 1,2-dimethylhydrazine developed CEA-overexpressing tumors that resembled human sporadic colorectal cancer. APC1638N/CEA hybrid mice, generated by crossing mice carrying the adenomatous polyposis coli (Apc1638N) gene mutation with CEA.Tg mice, are representative of human familial polyposis and develop polyps that overexpress the antigen. In both models, the DNA-EP/Ad vaccine succeeded in breaking immune tolerance and achieved significant antitumor effects in therapeutic settings. Our data suggest that genetic vaccines targeting CEA may be feasible strategies against gut tumors that overexpress the antigen. In addition, these models are powerful systems for evaluating antigen-specific tumor immunity and assessing therapeutic vaccine strategies for human colorectal cancer.

Optimal amount of monocyte chemoattractant protein‐1 enhances antitumor effects of suicide gene therapy against hepatocellular carcinoma by M1 macrophage activation

Suicide gene therapy combined with chemokines provides significant antitumor efficacy. Coexpression of suicide gene and monocyte chemoattractant protein-1 (MCP-1) increases antitumor effects in murine models of hepatocellular carcinoma (HCC) and colon cancer. However, it is unclear whether the doses administered achieved the maximum antitumor effects. We evaluated antitumor effects of various amounts of recombinant adenovirus vector (rAd) expressing MCP-1 in the presence of a suicide gene in a murine model of HCC. HCC cells were transplanted subcutaneously into BALB/c nude mice, and transduced with a fixed amount of Ad-tk harboring the suicide gene, HSV-tk, and various doses of Ad-MCP1 harboring MCP-1 (ratios of 1:1, 0.1:1, and 0.01:1 relative to Ad-tk). Growth of primary tumors was suppressed when treated with Ad-tk plus Ad-MCP1 (1:1 and 1:0.1) as compared with Ad-tk alone. The antitumor effects against tumor rechallenge tended to be high in the Ad-tk plus Ad-MCP1 group (1:0.1). The effects were dependent on production of Th1 type-cytokines. Delivery of an optimal amount of rAd expressing MCP-1 enhanced the antitumor effects of suicide gene therapy against HCC by M1 macrophage activation, suggesting that this is a plausible form of cancer gene therapy to prevent HCC progression and recurrence.

Interleukin-21: biology and application to cancer therapy

Background: IL-21, a recently described common γ-chain cytokine, can induce the maturation and enhanced cytotoxicity of natural killer (NK) and CD8+ T cells and proliferation of CD40-stimulated B cells. Exogenous IL-21 has antitumor effects in murine models via immunological mechanisms. In addition, IL-21 can also directly induce apoptosis in chronic lymphocytic leukemia cells and other B cell lymphomas. Objective/methods: We examine preclinical and clinical data regarding anticancer therapy with IL-21. Published original research, abstracts and ongoing clinical trials are reviewed. A brief summary of IL-21 biology is also provided. Conclusion: Three Phase I and II clinical trials with recombinant IL-21 have been completed, providing data on the safety and efficacy in subjects with advanced melanoma, renal cell carcinoma and non-Hodgkin's B cell lymphoma. Numerous additional single-agent and combination therapy clinical trials are ongoing for a variety of human malignancies. B cell malignancies in particular warrant further clinical investigation.

Interferon-α Gene Therapy by Lentiviral Vectors Contrasts Ovarian Cancer Growth Through Angiogenesis Inhibition

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.

Intratumoral injection of interferon-gamma gene-modified dendritic cells elicits potent antitumor effects: effective induction of tumor-specific CD8+ CTL response

To examine the antitumor efficacy of intratumoral injection of interferon-gamma gene-modified dendritic cells (DC-IFN-gamma) in a B16 melanoma model and to investigate its related immunological mechanisms. C57BL/6 mice-derived DC were transfected with adenovirus encoding IFN-gamma or beta-galactosidase (DC-LacZ). Secretion of IFN-gamma and TNF-alpha by DC was detected by ELISA. Nitric oxide (NO) production was measured by Griess reaction. Cytotoxicity of DC against tumor cell lines and activity of cytotoxic T lymphocytes (CTLs) were determined by 51Cr-release assay. TRP-2aa180-188-specific CD8+ CTLs in tumor-bearing mice with different treatment were determined by ELISPOT. DC-IFN-gamma could secrete high levels of IFN-gamma, NO and TNF-alpha. DC-IFN-gamma were cytolytic to B16 melanoma cells in vitro, but DC-LacZ and DC were not. Significant inhibition of tumor growth and prolonged survival were achieved in tumor-bearing mice intratumorally injected with DC-IFN-gamma when compared with those in tumor-bearing mice intratumorally injected with DC, DC-LacZ, fibroblasts, IFN-gamma gene-modified fibroblasts or PBS. After treatment with DC-IFN-gamma, enhanced Th1 and decreased Th2 responses were observed, and B16 melanoma antigen TRP-2aa180-188-specific CD8+ CTLs were induced significantly in the tumor-bearing mice. Intratumorally injected DC-IFN-gamma can uptake tumor antigens in situ and cross-present tumor antigens to specific CD8+ T cells, hereby eliciting effective antitumor effects in murine model with preestablished B16 melanoma.

A Phase I trial of the sulfonylhydrazine alkylator, VNP40101M (101M), administered weekly in patients (pts) with metastatic cancer

2060 Background: 101M demonstrated broad anti-tumor effects in murine models and was active against selected alkylator-resistant cell lines. In an initial phase I trial, the MTD was 305 mg/m2 IV q6 weeks, and the DLT was delayed grade (gr) 3 thrombocytopenia. This phase I trial was designed to determine the safety and MTD of 101M administered by short IV infusion weekly x 3. A secondary objective was to measure effects on alkylguanine transferase (AGT) in PBMC. Methods: A cycle was 3 consecutive weeks of dosing. New cycles could begin on day 29 if neutrophils ≥ 1500/μL and platelets ≥ 75k/μL. The dose of 101M was escalated in cohorts of 3–6 pts. Results: Four, 6, 5, and 6 pts were entered to the following dose levels (DL): 80, 100, 125, and 155 mg/m2/week. Thirteen were male; median age was 54 (range 18–72); and ECOG performance status was 0 in 11 pts and 1 in 10 pts. No tumor type was represented by more than 3 pts. Eighteen pts had received ≥ 2 prior chemoRx regimens; 20 had received an alkylator or platinum; 14 had prior radiation. No gr 2–4 non-heme toxicities were observed. Hematologic toxicities for the first 2 cycles are shown in the following table: Platelet and neutrophil nadirs occurred at a median of 33 and 52 days, respectively. Cumulative but mild and reversible thrombocytopenia/neutropenia was observed in the pt receiving 5 cycles w x 3 q4w. PBMC AGT depletion was not observed at any DL. Conclusions: The MTD is 100–125 mg/m2weekly x 3. Most patients can receive 2 or more cycles on a q4w schedule. Dose intensity appears increased compared to the single dose q6w schedule. Some evidence of anti-tumor effect was observed. Phase II trials with the weekly schedule are warranted. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Vion Pharmaceuticals Vion Pharmaceuticals Vion Pharmaceuticals Vion Pharmaceuticals

A Phase I trial of the sulfonylhydrazine alkylator, VNP40101M (101M), administered weekly in patients (pts) with metastatic cancer

2060 Background: 101M demonstrated broad anti-tumor effects in murine models and was active against selected alkylator-resistant cell lines. In an initial phase I trial, the MTD was 305 mg/m2 IV q6 weeks, and the DLT was delayed grade (gr) 3 thrombocytopenia. This phase I trial was designed to determine the safety and MTD of 101M administered by short IV infusion weekly x 3. A secondary objective was to measure effects on alkylguanine transferase (AGT) in PBMC. Methods: A cycle was 3 consecutive weeks of dosing. New cycles could begin on day 29 if neutrophils ≥ 1500/μL and platelets ≥ 75k/μL. The dose of 101M was escalated in cohorts of 3–6 pts. Results: Four, 6, 5, and 6 pts were entered to the following dose levels (DL): 80, 100, 125, and 155 mg/m2/week. Thirteen were male; median age was 54 (range 18–72); and ECOG performance status was 0 in 11 pts and 1 in 10 pts. No tumor type was represented by more than 3 pts. Eighteen pts had received ≥ 2 prior chemoRx regimens; 20 had received an alkylator or pla...

The use of interleukin-6 to generate tumor-infiltrating lymphocytes with enhanced in vivo antitumor activity.

Tumor-infiltrating lymphocytes (TIL) are cytotoxic T cells isolated from solid tumors and expanded in vitro in recombinant interleukin-2 (rIL-2). TIL have antitumor effects in murine models and in some patients with melanoma. In an effort to generate murine TIL with enhanced in vivo therapeutic efficacy, viable tumor cells were coinjected with a collagen matrix plus recombinant human IL-6 (rIL-6) subcutaneously into syngeneic mice to achieve sustained local concentrations of rIL-6 at the tumor site from which TIL were derived. In five separate experiments, single cell suspensions of tumors were admixed with either (a) Hanks' balanced salt solution (HBSS), (b) 2% (20 mg/ml) collagen matrix only, (c) 250 micrograms rIL-6 only, or (d) 250 micrograms rIL-6 in a 2% collagen matrix (prolonged release) before subcutaneous inoculation. These tumors were subsequently resected and TIL were isolated and expanded in vitro. TIL generated from tumors admixed with matrix plus rIL-6 were significantly more effective than TIL expanded from tumors admixed with HBSS (four of five experiments), TIL from tumors admixed with matrix only (five of five experiments), and TIL from tumors admixed with rIL-6 only (three of four experiments) in an established tumor treatment model. In no experiment was any other TIL culture superior to TIL grown from tumors augmented with collagen matrix plus rIL-6. These results suggest that strategies designed to increase the local concentrations of cytokines at tumor sites may lead to the generation of more potent TIL for clinical administration.