Secreting Tumor Vaccine Research Articles

Effects of tumor vaccine expressing Granulocyte–Macrophage Colony Stimulating Factor and interleukin-18 fusion on cancer cells and its possible application for cancer immunotherapy

To access antitumor effects of a combined Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) and interleukin-18 (IL-18), cDNA fusion of murine GM-CSF and mature IL-18 (GMIL-18) was constructed and transfected in mammalian cells. GMIL-18 fusion protein was highly secreted and displayed bifunctional activities, possessing immune response initiation and cytokine roles, including IFN-γ induction in mouse splenocytes and increased proliferation of GM-CSF-dependent cells, M-NSF-60. The GMIL-18 secreting tumor vaccine was generated and it strongly stimulated differentiation of dendrite cells (DCs) and effusive CD8+ and CD4+ cell infiltration into tumor mice. Moreover, growth of CT26 mouse colon cancer cells was significantly retarded by GMIL-18 (CT26GMIL-18), but not by CT26GM-CSF- or CT26IL-18. The efficiency of prophylactic vaccination was greater than that of therapeutic vaccination in terms of tumor size and its inhibitory role in proliferation. In micrometastasis analysis of tumor models, γ-ray irradiated GMIL-18 tumor vaccine showed a smaller number of liver-meta tumor nodules in mouse liver cells. We concluded that bifunctional GMIL-18 fusion protein could be applied as an immune therapy for cancer treatments.

Construction and identification of a recombinant adenovirus expressing bioactive GM-CSF: Implication for GM-CSF secreting tumor vaccines design

IntroductionGranulocyte-monocyte colony stimulating factor (GM-CSF) has been reported to to mediate antitumor effects through stimulation of various antigen presentation cells. Clearly, these properties make GM-CSF a potent adjuvant. Therefore, an oncolytic adenovirus coding for GMCSF was engineered. ObjectiveContraction, identification and characterization of recombinant adenovirus encoding GM-CSF. MethodsIn this study, a recombinant adenovirus expressing GM-CSF (rAd-GM) was constructed and characterized. Homologous recombination in bacteria followed by transfection of recombinant plasmid into a mammalian packaging cell line, viral production was conveniently followed by the gene incorporated into the viral backbone. Results & DiscussionWe also showed that the construct produced a 12 kDa recombinant protein which stimulated colony formation in bone marrow cells in vitro, indicating that the recombinant GM-CSF was biologically active. ConclusionFuture work will demonstrate the rAd-GM mediated antitumor immunity in rats with experimentally induced breast cancer.

Multicenter, randomized phase II trial of GM-CSF (GM) plus ipilimumab (Ipi) versus Ipi alone in metastatic melanoma: E1608.

CRA9007 Background: CTLA-4 blockade and GM secreting tumor vaccine combinations demonstrate therapeutic synergy in multiple preclinical models. GM has activity in prostate and ovarian carcinoma and is being evaluated in phase III adjuvant trials for melanoma and lymphoma. GM enhances dendritic cell activation and potentiates antitumor T and B cell responses. GM may induce regulatory immune responses. A key issue is whether systemic GM might synergize with CTLA-4 blockade. Methods: Eligibility: measureable disease, ≤1 prior therapy, no CNS mets, ECOG PS 0-1, > 4 wks prior therapy, adequate end organ function, no autoimmune disease, no prior CTLA-4 blockade/CD137 agonist. OS was primary endpoint. Pts randomized to Arm A Ipi 10 mg/kg q3 wks IV x 4 then q12 wks plus GM 250 μg SC days 1-14 of 21 day cycles vs. Arm B Ipi 10 mg/kg as in Arm A alone. Due to known inflammatory effects of treatments, pts were permitted to continue up to 100% increase in SPD and four new lesions in absence of declining performance status and discretion of treating physician. Drug supply, funding from Sanofi/Bristol-Myers Squibb. Results: 245 pts were enrolled. Arms were balanced for demographics. Median follow up 13.3 mos. RR Arm A 11.3 % (6.1, 18.6), Arm B 14.7% (8.6,22.8) (not significant;NS). PFS Arm A 3 mos (2.9,4.3), Arm B 3.2 mos (3,4) (NS). Median OS Arm A not reached, Arm B 12.6 mos (9.2,-). One year OS for Arm A was 67.9% (59%,76%), Arm B 51.2% (42.6%, 61.3%) (stratified log rank p1=0.016, p2=0.033). HR for mortality on Ipi + GM/Ipi=0.65. Per study design (overall one-sided type I error 0.10), OS interim analysis was conducted at 69% info time. O’Brien-Fleming boundary was crossed for OS. Toxicity assessed for all cases regardless of eligibility. Gr 3-5 AEs 45% Arm A, 57% Arm B (p2=0.078). Gr 5 AEs: Arm A colonic perforation (1), cardiac arrest (1); Arm B multiorgan failure (2), colonic perforation (2), hepatic failure (1), respiratory failure (2). Conclusions: Ipi plus GM significantly improves OS over Ipi alone. No significant differences in toxicity were observed. A trend toward improved tolerability is noted in the GM arm. Clinical trial information: NCT01134614.

A feasibility study of combination therapy with trastuzumab (T), cyclophosphamide (CY), and an allogeneic GM-CSF-secreting breast tumor vaccine for the treatment of HER2+ metastatic breast cancer.

2535 Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccines are bioactive, but limited by disease burdens and immune tolerance. Cyclophosphamide (CY) augments the activity of a GM-CSF-secreting breast tumor vaccine in ER+ metastatic breast cancer (MBC) patients. HER-2-specific monoclonal antibodies given with GM-CSF-secreting HER-2-targeted vaccination enhance CD8+ effector T cell function and tumor-free survival in immune tolerant neu transgenic mice. Here, we test the hypothesis that CY-modulated vaccination with weekly Trastuzumab (T) can induce clinically-relevant HER-2-specific immunity in HER-2+ MBC patients. Methods: We conducted a single arm, open label, two-stage feasibility study of CY, an allogeneic HER-2+ GM-CSF-secreting breast tumor vaccine, and standard T in patients with measurable/evaluable HER-2+ MBC. Vaccinations were given every 4-6 weeks for 3 cycles, with a 4th vaccination given 6-8 months from trial entry. Primary objectives included safety, bioactivity, and clinical benefit (CB=complete response+partial response+stable disease). A secondary outcome was delayed type hypersensitivity (DTH) to the MHC Class II HER-2 epitopes p369 and p776. This study has a power of 0.86 to detect a CB rate of 0.45 from a null hypothesis rate of 0.20 at an alpha level of 0.08. Results: Twenty patients were vaccinated. 75% received at least 3 vaccinations, and 40% completed all 4 vaccinations. Toxicities were limited to low grade fever, myalgias, injection site reactions, and urticaria. No dose limiting toxicities were observed. The CB rates at 6 months and 1 year were 10/20 (50%, 95% CI:27-72%) and 7/20 (35%, 95%CI:15-59%) respectively. HER-2-specific DTH developed in 7/20 subjects (35%); four (57%, 95% CI:18-90%) of these had a CB. Conclusions: CY-modulated immunotherapy with an allogeneic, HER-2+ GM-CSF-secreting breast tumor vaccine and weekly T is safe, bioactive, and induces new HER-2-specific DTH in HER-2+ MBC patients. The 50% CB rate at 6 months supports further investigation of CY-modulated vaccination with standard T. The GM-CSF-secreting vaccine platform is licensed from Johns Hopkins University to BioSante. The Johns Hopkins University has the potential to receive royalties in the future.

Timed Sequential Treatment With Cyclophosphamide, Doxorubicin, and an Allogeneic Granulocyte-Macrophage Colony-Stimulating Factor–Secreting Breast Tumor Vaccine: A Chemotherapy Dose-Ranging Factorial Study of Safety and Immune Activation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) -secreting tumor vaccines have demonstrated bioactivity but may be limited by disease burdens and immune tolerance. We tested the hypothesis that cyclophosphamide (CY) and doxorubicin (DOX) can enhance vaccine-induced immunity in patients with breast cancer. We conducted a 3 x 3 factorial (response surface) dose-ranging study of CY, DOX, and an HER2-positive, allogeneic, GM-CSF-secreting tumor vaccine in 28 patients with metastatic breast cancer. Patients received three monthly immunizations, with a boost 6 to 8 months from study entry. Primary objectives included safety and determination of the chemotherapy doses that maximize HER2-specific immunity. Twenty-eight patients received at least one immunization, and 16 patients received four immunizations. No dose-limiting toxicities were observed. HER2-specific delayed-type hypersensitivity developed in most patients who received vaccine alone or with 200 mg/m(2) CY. HER2-specific antibody responses were enhanced by 200 mg/m(2) CY and 35 mg/m(2) DOX, but higher CY doses suppressed immunity. Analyses revealed that CY at 200 mg/m(2) and DOX at 35 mg/m(2) is the combination that produced the highest antibody responses. First, immunotherapy with an allogeneic, HER2-positive, GM-CSF-secreting breast tumor vaccine alone or with CY and DOX is safe and induces HER2-specific immunity in patients with metastatic breast cancer. Second, the immunomodulatory activity of low-dose CY has a narrow therapeutic window, with an optimal dose not exceeding 200 mg/m(2). Third, factorial designs provide an opportunity to identify the most active combination of interacting drugs in patients. Further investigation of the impact of chemotherapy on vaccine-induced immunity is warranted.

Increasing doses of cyclophosphamide suppress antigen-specific T helper-dependent immunity induced by a GM-CSF-secreting breast tumor vaccine

3009 Background: Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccines have shown promise, but their activity is limited by immune tolerance. We tested the hypothesis that integrating vaccination with cyclophosphamide (CY) and doxorubicin (DOX) can enhance vaccine activity. Methods: We conducted a 3-by-3 factorial dose-ranging study of CY and DOX with an allogeneic, HER-2+ GM-CSF-secreting breast tumor vaccine in patients with stable metastatic breast cancer. CY was given at doses of 200, 250, and 350 mg/m2, and DOX at 15, 25, and 35 mg/m2. Subjects received three monthly immunizations, and a boost 6 to 8 months later. CY was given Day -1, vaccination Day 0, and DOX Day +7. Vaccine-induced HER-2-specific immunity was measured by delayed type hypersensitivity (DTH) to the HER-2-derived MHC Class II peptide epitopes p369 and p776. HER-2-specific antibody was measured by ELISA. Results: Twenty-seven individuals received at least 1 immunization with vaccine alone (6 subjects), or combined with low dose chemotherapy (21 subjects); 14 individuals received 4 vaccinations. Four of 6 subjects (66%) receiving vaccine alone developed HER-2-specific DTH, whereas 4 of 7 (57%) subjects receiving it with 200 mg/m2 CY developed a post- vaccination DTH (p=0.587, 1-sided Fisher’s exact test). However, higher doses of CY (250 or 350 mg/m2) one day before vaccination completely suppressed vaccine-induced DTH compared to vaccine alone or with 200 mg/m2 CY (p=0.007). Significant HER-2-specific humoral immunity developed in 3 of 5 (60%) evaluated subjects who received vaccine with 200 mg/m2 CY, but only 1 subject receiving higher CY doses. DOX did not impact the development of DTH or humoral immunity. Conclusions: Vaccination with an allogeneic, GM-CSF-secreting tumor vaccine with or without CY and DOX induced HER-2-specific DTH in patients with metastatic breast cancer. CY at 200 mg/m2 was associated with the development of humoral immunity. Doses of CY higher than 200 mg/m2 suppressed vaccine-induced HER-2-specific DTH, whereas DOX had no influence. Further investigation of the impact of chemotherapy on distinct measures of vaccine-induced antigen-specific immunity is warranted. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Cell Genesys, Incorporated

Establishment and characterization of a rectal cancer model in mice: application to cytokine gene therapy.

We established an orthotopic animal model of rectal cancer in mice and applied this model to the study of the antitumor effects of cytokine-assisted tumor vaccine. The CT-26 murine colon adenocarcinoma cells were inoculated into the submucosa of the rectum of the mice to induce the rectal tumor. The tumor growth rate and the survival time of the mice were observed. The cDNA of granulocyte-macrophage colony-stimulating factor (GM-CSF) was transduced to the CT-26 cell line via a retroviral vector, and the therapeutic effects of irradiated GM-CSF secreting tumor vaccine on the rectal tumor were investigated. All the mice implanted with the wild-type tumor cells had tumor growth in the rectum and died. The mean survival time of the mice was 28.9 days. Two doses of irradiated GM-CSF secreting tumor vaccine administered on days 0 and 3 after tumor cell implantation significantly prolonged the survival of the mice with rectal tumor compared with that of the control groups ( P<0.0001). In contrast, no antitumor effect was observed when the treatment with GM-CSF secreting tumor vaccine was delayed to 3 days after tumor cell implantation ( P>0.17). The results suggest that cytokine gene therapy exerts an antitumor effect on small tumors and may be considered as an adjuvant immunotherapy of rectal cancers and prevention of reimplantation of tumor cells disseminated during or following surgery. The orthotopic animal model of the rectal cancer in mice could be applied to the in vivo experimental studies of rectal cancer.

Tumor-induced immunosuppression: a barrier to immunotherapy of large tumors by cytokine-secreting tumor vaccine.

An active immunotherapy strategy with cytokine-assisted tumor vaccine, although often effective for small tumor burdens, is much less so for large tumor burdens. This study examines how large tumors might suppress the T cell functions and escape from the immune responses elicited by a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccine. According to our results, the T cells isolated from the tumor-bearing mice treated late with the vaccine failed to confer protective activity on naive mice against a wild-type tumor challenge, unlike those isolated from the early-treated group. Nevertheless, the antitumor activity of the inactive T cells could be restored on in vitro stimulation. Expression of transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10), the potent immunosuppressive factors, was detected in the parental tumor cell line RLmale 1 (a murine T leukemia cell line), as well as in the tumor region, the levels of which correlated with tumor progression. An in vitro assay of T cell functions revealed that the TGF-beta in the conditioned medium of RLmale 1 cells mainly affected the activation, whereas the IL-1male affected the activation to a lesser extent, but significantly affected the cytolytic activity, of tumor-specific T cells. The immunosuppressive activity of IL-10 was also signified by the findings that administration of the conditioned medium of RLmale 1 cultured in a serum-free medium, in which the TGF-beta activity was then lost while the IL-10 activity still remained, or of recombinant IL-10 to the early-treated group of mice abrogated the known efficacy of tumor vaccine on the small tumors. These data suggested that the efficacy of cytokine-secreting tumor vaccine was blocked by the immunosuppressive factors secreted from the large tumors. The results have important implications for the clinical design of immunotherapeutic strategies for advanced cancer patients.