Padmasini Kumar

Lipopolysaccharides from Distinct Pathogens Induce Different Classes of Immune Responses In Vivo

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4+ Th and CD8+ T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4+ and CD8+ T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-γ, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-γ. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8α+ dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-α. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.

Phase II study of belagenpumatucel-L, a transforming growth factor beta-2 antisense gene-modified allogeneic tumor cell vaccine in non-small-cell lung cancer.

PURPOSE Belagenpumatucel-L is a nonviral gene-based allogeneic tumor cell vaccine that demonstrates enhancement of tumor antigen recognition as a result of transforming growth factor beta-2 inhibition. PATIENTS AND METHODS We performed a randomized, dose-variable, phase II trial involving stages II, IIIA, IIIB, and IV non-small-cell lung cancer patients. Each patient received one of three doses (1.25, 2.5, or 5.0 x 10(7) cells/injection) of belagenpumatucel-L on a monthly or every other month schedule to a maximum of 16 injections. Immune function, safety, and anticancer activity were monitored. RESULTS Seventy-five patients (two stage II, 12 stage IIIA, 15 stage IIIB, and 46 stage IV patients) received a total of 550 vaccinations. No significant adverse events were observed. A dose-related survival difference was demonstrated in patients who received > or = 2.5 x 10(7) cells/injection (P = .0069). Focusing on the 61 late-stage (IIIB and IV) assessable patients, a 15% partial response rate was achieved. The estimated probabilities of surviving 1 and 2 years were 68% and 52%, respectively for the higher dose groups combined and 39% and 20%, respectively, for the low-dose group. Immune function was explored in the 61 advanced-stage (IIIB and IV) patients. Increased cytokine production (at week 12 compared with patients with progressive disease) was observed among clinical responders (interferon gamma, P = .006; interleukin [IL] -6, P = .004; IL-4, P = .007), who also displayed an elevated antibody-mediated response to vaccine HLAs (P = .014). Furthermore, positive enzyme-linked immunospot reactions to belagenpumatucel-L showed a correlation trend (P = .086) with clinical responsiveness in patients achieving stable disease or better. CONCLUSION Belagenpumatucel-L is well tolerated, and the survival advantage justifies further phase III evaluation.

Phase I Trial of “bi-shRNAifurin/GMCSF DNA/Autologous Tumor Cell” Vaccine (FANG) in Advanced Cancer

We performed a phase I trial of FANG vaccine, an autologous tumor-based product incorporating a plasmid encoding granulocyte-macrophage colony-stimulating factor (GMCSF) and a novel bifunctional short hairpin RNAi (bi-shRNAi) targeting furin convertase, thereby downregulating endogenous immunosuppressive transforming growth factors (TGF) β1 and β2. Patients with advanced cancer received up to 12 monthly intradermal injections of FANG vaccine (1 × 107 or 2.5 × 107 cells/ml injection). GMCSF, TGFβ1, TGFβ2, and furin proteins were quantified by enzyme-linked immunosorbent assay (ELISA). Safety and response were monitored. Vaccine manufacturing was successful in 42 of 46 patients of whom 27 received ≥1 vaccine. There were no treatment-related serious adverse events. Most common grade 1, 2 adverse events included local induration (n = 14) and local erythema (n = 11) at injection site. Post-transfection mean product expression GMCSF increased from 7.3 to 1,108 pg/106 cells/ml. Mean TGFβ1 and β2 effective target knockdown was 93.5 and 92.5% from baseline, respectively. Positive enzyme-linked immunospot (ELISPOT) response at month 4 was demonstrated in 9 of 18 patients serially assessed and correlated with survival duration from time of treatment (P = 0.025). Neither dose-adverse event nor dose-response relationship was noted. In conclusion, FANG vaccine was safe and elicited an immune response correlating with prolonged survival. Phase II assessment is justified.We performed a phase I trial of FANG vaccine, an autologous tumor-based product incorporating a plasmid encoding granulocyte-macrophage colony-stimulating factor (GMCSF) and a novel bifunctional short hairpin RNAi (bi-shRNAi) targeting furin convertase, thereby downregulating endogenous immunosuppressive transforming growth factors (TGF) β1 and β2. Patients with advanced cancer received up to 12 monthly intradermal injections of FANG vaccine (1 × 10(7) or 2.5 × 10(7) cells/ml injection). GMCSF, TGFβ1, TGFβ2, and furin proteins were quantified by enzyme-linked immunosorbent assay (ELISA). Safety and response were monitored. Vaccine manufacturing was successful in 42 of 46 patients of whom 27 received ≥1 vaccine. There were no treatment-related serious adverse events. Most common grade 1, 2 adverse events included local induration (n = 14) and local erythema (n = 11) at injection site. Post-transfection mean product expression GMCSF increased from 7.3 to 1,108 pg/10(6) cells/ml. Mean TGFβ1 and β2 effective target knockdown was 93.5 and 92.5% from baseline, respectively. Positive enzyme-linked immunospot (ELISPOT) response at month 4 was demonstrated in 9 of 18 patients serially assessed and correlated with survival duration from time of treatment (P = 0.025). Neither dose-adverse event nor dose-response relationship was noted. In conclusion, FANG vaccine was safe and elicited an immune response correlating with prolonged survival. Phase II assessment is justified.

Phase I Trial of TGF-β2 Antisense GM-CSF Gene-Modified Autologous Tumor Cell (TAG) Vaccine

Purpose: On the basis of the hypothesis that the combined expression of immunostimulatory granulocyte macrophage colony stimulating factor (GM-CSF) and antitumor suppressor TGF-β2 antisense (AS) transgenes can break tolerance and stimulate immune responses to cancer-associated antigens, we constructed an expression plasmid [the tumor-associated glycoprotein (TAG) plasmid] that coexpresses GM-CSF and TGF-β2 AS nucleotide sequences and which was incorporated into an autologous whole-cell vaccine. Experimental Design: Patients undergoing resection were enrolled. Freshly harvested autologous tumor cells were mechanically and enzymatically disaggregated, then electroporated with the TAG vector. The resulting vaccine was irradiated, then aliquoted and cryopreserved until the time of injection. Patients received a minimum of 5 to a maximum of 12 monthly intradermal injections. Immune function was monitored at baseline and at months 3 and 6. Results: Vaccine manufacturing efficiency was 84% (32/38). Twenty-three patients received at least 1 vaccination. There were no grade 3 or 4 toxicities, and grade 1 and 2 events were local in nature. Seventeen of 21 patients had stable disease (SD) at month 2 or later as their best response, and 1 patient with stage IVa malignant melanoma achieved a complete response (CR) following 11 vaccinations and remains without evidence of disease 2 years following initiation of therapy. Six of 13 patients displayed a positive enzyme-linked immunospot (ELISPOT) response to autologous TAG vaccine at week 12 including 3 patients with prolonged SD or CR. The 3 other patients survived through week 24, as compared with none of the 7 ELISPOT-negative patients. Conclusions: On the basis of safety and clinical and immunologic results, further evaluation of bifunctional vaccines is warranted. Clin Cancer Res; 17(1); 183–92. ©2011 AACR.

In vivo Safety and Antitumor Efficacy of Bifunctional Small Hairpin RNAs Specific for the Human Stathmin 1 Oncoprotein

Bifunctional small hairpin RNAs (bi-shRNAs) are functional miRNA/siRNA composites that are optimized for posttranscriptional gene silencing through concurrent mRNA cleavage-dependent and -independent mechanisms (Rao et al., 2010 ). We have generated a novel bi-shRNA using the miR30 scaffold that is highly effective for knockdown of human stathmin (STMN1) mRNA. STMN1 overexpression well documented in human solid cancers correlates with their poor prognosis. Transfection with the bi-shSTMN1-encoding expression plasmid (pbi-shSTMN1) markedly reduced CCL-247 human colorectal cancer and SK-Mel-28 melanoma cell growth in vitro (Rao et al., 2010 ). We now examine in vivo the antitumor efficacy of this RNA interference-based approach with human tumor xenografted athymic mice. A single intratumoral (IT) injection of pbi-shSTMN1 (8 μg) reduced CCL-247 tumor xenograft growth by 44% at 7 days when delivered as a 1,2-dioleoyl-3-trimethyl-ammoniopropane:cholesterol liposomal complex. Extended growth reductions (57% at day 15; p < 0.05) were achieved with three daily treatments of the same construct. STMN1 protein reduction was confirmed by immunoblot analysis. IT treatments with pbi-shSTMN1 similarly inhibited the growth of tumorgrafts derived from low-passage primary melanoma (≥70% reduction for 2 weeks) and abrogated osteosarcoma tumorgraft growth, with the mature bi-shRNA effector molecule detectable for up to 16 days after last injection. Antitumor efficacy was evident for up to 25 days posttreatment in the melanoma tumorgraft model. The maximum tolerated dose by IT injection of >92 μg (Human equivalent dose [HED] of >0.3 mg/kg) in CCL-247 tumor xenograft-bearing athymic mice was ∼10-fold higher than the extrapolated IC(50) of 9 μg (HED of 0.03 mg/kg). Healthy, immunocompetent rats were used as biorelevant models for systemic safety assessments. The observed maximum tolerated dose of <100 μg for intravenously injected pbi-shSTMN1 (mouse equivalent of <26.5 μg; HED of <0.09 mg/kg) confirmed systemic safety of the therapeutic dose, hence supporting early-phase assessments of clinical safety and preliminary efficacy.

Summary of bi-shRNAfurin/GM-CSF Augmented Autologous Tumor Cell Immunotherapy (FANG™) in Advanced Cancer of the Liver

Therapies for advanced hepatocellular carcinoma (HCC) are limited. We carried out a phase I trial of a novel autologous whole-cell tumor cell immunotherapy (FANG™), which incorporates a dual granulocyte macrophage colony-stimulating factor (GM-CSF) expressive/bifunctional small hairpin RNA interference (bi-shRNAi) vector. The bi-shRNAi DNA targets furin, which is a proconvertase of transforming growth factors beta (TGFβ) 1 and 2. Safety, mechanism, immunoeffectiveness, and suggested benefit were previously shown [Senzer et al.: Mol Ther 2012;20:679-689; Senzer et al.: J Vaccines Vaccin 2013;4:209]. We now provide further follow-up of a subset of 8 HCC patients. FANG manufacturing was successful in 7 of 8 attempts (one failure due to insufficient cell yield). Median GM-CSF expression was 144 pg/106 cells, TGFβ1 knockdown was 100%, and TGFβ2 knockdown was 93% of the vector-transported cells. Five patients were vaccinated (1 or 2.5 × 107 cells/intradermal injection, 6-11 vaccinations). No FANG toxicity was observed. Three of these patients demonstrated evidence of an immune response to the autologous tumor cell sample. Long-term follow-up demonstrated survival of 319, 729, 784, 931+, and 1,043+ days of the FANG-treated patients. In conclusion, evidence supports further assessment of the FANG immunotherapy in HCC.

Pilot Trial of FANG Immunotherapy in Ewing's Sarcoma

We report on 12 consecutive patients with advanced/metastatic Ewings sarcoma who were treated as a separate cohort of a phase 1 trial of FANG autologous immunotherapy (1 × 106–2.5 × 107 cells/intradermal injection each month for minimum 4 months). Safety and clinical response were monitored. Patient immune response to unmodified autologous tumor cells was assessed by gamma interferon-enzyme-linked immunospot (γIFN-ELISPOT) assay using peripheral blood mononuclear cells from baseline (pretreatment) and multiple postvaccination time points. None of the 12 patients (47 vaccinations) developed grade 2/3/4 drug-related toxicity. Median product release granulocyte-macrophage colony-stimulating factor expression was 1,941 pg/106 cells, and TGFβ1and TGFβ2 knockdown were 99 and 100%, respectively. Eight patients were assessed for ELISPOT response to autologous tumor cells at baseline and all (100%) were negative. In contrast, follow-up ELISPOT response at month 1 or month 4 (one patient) after FANG was positive in all eight patients. One patient achieved a partial tumor response (38% tumor reduction, RECIST 1.1). The Kaplan–Meier estimated survival of these 12 patients at 1 year was 75%. In this phase 1 study in patients with Ewings sarcoma, FANG immunotherapy was well tolerated, elicited a tumor-specific systemic immune response in all patients, and was associated with favorable 1-year survival. Further clinical testing is indicated.

Long Term Follow Up: Phase I Trial of "bi-shRNA furin/GMCSF DNA/ Autologous Tumor Cell" Immunotherapy (FANG™) in Advanced Cancer

Study Background: Previously, we demonstrated safety and correlated induced immune response with survival in a Phase I study of FANG immunotherapy in advanced cancer patients. We now report long term follow-up (FU) of Phase I treated patients including assessment of relationships of dose, γIFN-ELISPOT response, and patient demographics to safety and survival. Methods: Safety, γIFN-ELISPOT response, and survival have been followed through 3+ years in advanced cancer patients who received ≥ 2-12 intradermal monthly injections of 1×107 or 2.5×107 cells/injection. Clinical and serological assessments were performed monthly, radiographic evaluations bimonthly, and γ-IFN-ELISPOT at baseline, and start of Cycle 2, 4, 6, 9, 12 then sequentially at FU. Results: Previously, we reported results on 45 patients with successful FANG construction followed for 1 year (28 treated (designated FANG); 17 not treated based on availability of other alternative treatments or failed manufacturing (designated No FANG)). We now report FU results through year 3 on those patients and an additional 29 patients (7 FANG, 22 No FANG) subsequently entered into Phase I study (total N=35 FANG; total N=39 No FANG). The median survival of the current expanded Phase I trial population is 562 days vs. 122 days (p=0.00001). This is similar to the originally published data from two years earlier. The γ-IFN-ELISPOT reaction was positive in 14 of the current FANG treated patients and negative in 12 FANG treated patients at Month 3 or less post first injection. Survival correlated with γ-IFN-ELISPOT reaction; median 836 days vs. 440 days with positive and negative ELISPOT respectively, (p=0.04). No long term adverse toxicity has been seen and there was no significant correlation of immune response or survival with either dose or demographics. Conclusions: Treatment with FANG vaccine continues to show long term safety and evidence of benefit in patients with many types of advanced cancer thereby justifying further efficacy testing.

Phase II study of Vigil® DNA engineered immunotherapy as maintenance in advanced stage ovarian cancer

OBJECTIVES The majority of women with Stage III/IV ovarian cancer who achieve clinical complete response with frontline standard of care will relapse within 2years. Vigil immunotherapy, a GMCSF/bi-shRNA furin DNA engineered autologous tumor cell (EATC) product, demonstrated safety and induction of circulating activated T-cells against autologous tumor in Phase I trial Senzer et al. (2012, 2013) . Our objectives for this study include evaluation of safety, immune response and recurrence free survival (RFS). METHODS This is a Phase II crossover trial of Vigil (1.0×107 cells/intradermal injection/month for 4 to 12 doses) in Stage III/IV ovarian cancer patients achieving cCR (normal imaging, CA-125≤35units/ml, physical exam, and no symptoms suggestive of the presence of active disease) following primary surgical debulking and carboplatin/paclitaxel adjuvant or neoadjuvant chemotherapy. Patients received Vigil or standard of care during the maintenance period. RESULTS Forty-two patients were entered into trial, 31 received Vigil and 11 received standard of care. No≥Grade 3 toxicity related to product was observed. A marked induction of circulating activated T-cell population was observed against individual, pre-processed autologous tumor in the Vigil arm as compared to pre-Vigil baseline using IFNγ ELISPOT response (30/31 negative ELISPOT pre Vigil to 31/31 positive ELISPOT post Vigil, median 134 spots). Moreover, in correlation with ELISPOT response, RFS from time of procurement was improved (mean 826days/median 604days in the Vigil arm from mean 481days/median 377days in the control arm, p=0.033). CONCLUSION In conjunction with the demonstrated safety, the high rate of induction of T-cell activation and correlation with improvement in RFS justify further Phase II/III assessment of Vigil.

Preclinical Biodistribution and Safety Evaluation of a pbi-shRNA STMN1 Lipoplex after Subcutaneous Delivery.

Stathmin-1 (STMN1) is a microtubule-destabilizing protein which is overexpressed in cancer. Its overexpression is associated with poor prognosis and also serves as a predictive marker to taxane therapy. We have developed a proprietary bi-functional shRNA (bi-shRNA) platform to execute RNA interference (RNAi)-mediated gene silencing and a liposome–carrier complex to systemically deliver the pbi-shRNA plasmids. In vitro and in vivo testing demonstrated efficacy and specificity of pbi-shRNA plasmid in targeting STMN1 (Phadke, A. P., Jay, C. M., Wang, Z., Chen, S., Liu, S., Haddock, C., Kumar, P., Pappen, B. O., Rao, D. D., Templeton, N. S., et al. (2011). In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein. DNA Cell Biol. 30, 715–726.). Biodistribution and toxicology studies in bio-relevant Sprague Dawley rats with pbi-shRNA STMN1 lipoplex revealed that the plasmid DNA was delivered to a broad distribution of organs after a single subcutaneous injection. Specifically, plasmid was detected within the first week using QPCR (threshold 50 copies plasmid/1 µg genomic DNA) at the injection site, lung, spleen, blood, skin, ovary (limited), lymph nodes, and liver. It was not detected in the heart, testis or bone marrow. No plasmid was detected from any organ 30 days after injection. Treatment was well tolerated. Minimal inflammation/erythema was observed at the injection site. Circulating cytokine response was also examined by ELISA. The IL-6 levels were induced within 6 h then declined to the vehicle control level 72 h after the injection. TNF&agr; induction was transiently observed 4 days after the DNA lipoplex treatment. In summary, the pbi-shRNA STMN1 lipoplex was well tolerated and displayed broad distribution after a single subcutaneous injection. The pre-clinical data has been filed to FDA and the pbi-shRNA STMN1 lipoplex is being investigated in a phase I clinical study.