- Email: [email protected]
623. HLA Typing Identity Test for Verification of Patient to their FANG™ Autologous Cancer Vaccine
CANCER-IMMUNOTHERAPY III An aliquot of cells from before and after transfection with FANG™ plasmid were placed in culture and incubated at 37ºC. Cells and supernatants were collected on Days 0, 1, 2, 3, 4, 7, 10 and 14. GMCSF, TGFβ1, TGFβ2 (R&D Systems) and Furin (USCN Life Sciences) proteins were quantified by enzyme-linked immunosorbent assay (ELISA) kits. We demonstrated earlier an average increase (n=85) in the expression of GMCSF from 7.3 to 1,108 pg/106cells/ ml at Day 4. Mean TGFβ1 and β2 effective target knockdown was 93.5 and 92.5% from baseline, respectively (Senzer et.al. Mol. Ther 2011). Furin knockdown was 85.09%. The percentage knockdown of TGFβ1 and TGFβ2 were significant with p <0.001 and consistent with the Furin knockdown also with p<0.001 results. The number of vaccines manufactured in Phase I is currently at 79. In addition we have manufactured 81 vaccines for Phase II Ovarian Cancer, 10 vaccines for Phase II Melanoma and 3 vaccines for Phase II Colorectal cancers with liver metastasis. A total of 173 FANG vaccines all together were manufactured todate. We have reduced the number of time points the assay samples were collected from 8 to 5 time points with Day 0, 3, 4, 7 and 10 only for the supernatants collected for cytokine expression. We show that this reduction in assay time points does not alter the outcome of the QC release criteria. Updated cytokine evaluation for Phase I and Phase II studies will be presented in ASGCT 2014. An increase in the expression of GMCSF and knockdown of TGFβ1, TGFβ2 and FURIN are consistently achieved in all tumor cells transfected with FANG plasmid.
623. HLA Typing Identity Test for Verification of Patient to their FANGTM Autologous Cancer Vaccine
Yang Yu,1 Nicolas Taquet,1 John Nemunaitis,1,2 Phillip B. Maples.1 1 Gradalis, Inc., Dallas, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX.
In Phase II clinical development of the FANGTM autologous cancer vaccine, Gradalis receives tumor sample deliveries from multiple clinics and hospitals. Identity match of the tumor sample and the vaccine manufactured with the patient is an essential QA component. We have taken a number of steps to ensure the integrity of Patient – Vaccine tumor collection / cGMP manufacturing / clinical treatment continuum. A most reliable method for specimen identification is DNA identity testing, which will verify the origin of two samples. We adopted the HLA (Human Leukocyte Antigen) molecular genotyping by SSP-PCR (sequence-specific primer polymerase chain reaction) as our identity method. HLA testing is a widely accepted method of identity testing commonly used in solid organ and bone marrow transplantation and paternity testing. In comparison to the comprehensive STR (Short Tandem Repeat) DNA profiling, HLA typing is reliable, cost effective, and has a short turnaround time. Genomic DNA isolated from the peripheral white blood cells of the patient is considered “correct” or “known”. This is compared to the tumor cells which are obtained on day-1 of vaccine manufacturing before plasmid transfection. The DNA extraction was performed by the DNeasy kit (Qiagen) and DNA quantity and quality is subsequently determined by Nano-Drop. The ABDRDQ SSP-PCR HLA Typing Kit which include Class I A Locus, B Locus and the Class II DR/DQ loci from One Lambda, and Taq polymerase from Promega were utilized for the HLA typing. The positive HLA antigens are identified by post-PCR gel electrophoresis. The confirmation of the tumor tissue origin from the patient is determined by the complete molecular HLA typing match between the tumor tissue and the blood. We previously reported an analysis of 58 vaccine-blood sample pairs on Phase I and Phase II FANG vaccine trials. Since that time another 23 Phase-II FANG vaccine-blood sample pairs have been
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
tested and analyzed. All vaccine-blood sample paired HLA typing results are complete matches. These results support HLA typing as an identity verification test for FANG™ vaccine.
624. Randomized Phase II Trial of Adjuvant Autologous Tumor Cell Vaccine (FANG™) for High Risk Stage III/IV Ovarian Cancer: Preliminary Results
J. Oh,3 M. Barve,1,3 C. Bedell,1 J. Kuhn,5 B. Fine,3 T. P. Heffernan,6 C. M. Matthews,3 C. A. Stringer,3 E. C. Koon,3 H. Goodman,7 E. L. Fleming,8 L. R. DeMars,8 M. K. Bergman,9 B. O. Pappen,4 P. Kumar,4 P. B. Maples,4 N. Senzer,1,4 J. Nemunaitis.1,2,3,4 1 Mary Crowley Cancer Research Centers, Dallas; 2Medical City Dallas Hospital, Dallas; 3Texas Oncology, PA, Dallas; 4Gradalis, Inc, Dallas; 5WLS Surgery Associates, PA, Dallas; 6North Texas Gyn Onc, Dallas; 7Florida Cancer Specialists, West Palm Beach; 8 Dartmouth-Hitchcock Medical Ctr, Lebanon; 9Cancer Care Northwest, Spokane. Introduction: Phase I evaluation of FANG™ vaccine (GMCSF/ bi-shRNAi furin vector transfected autologous tumor) demonstrated safety; confirmed GMCSF transgene expression and knockdown of furin and consequent TGFβ1 and β2 expression; showed T-cell activation per gamma IFN-ELISPOT and resulted in a longer than expected survival duration thereby justifying Phase II testing [Senzer et al Mol Ther 2012; Senzer et al Vaccines and Vaccin in press]. The majority of women with Stage III/IV ovarian cancer who achieve a complete clinical response with standard of care management involving debulking surgery and consolidation chemotherapy will relapse within 2 years. Methods: This is a 2:1 randomized Phase II open-label trial of FANG using tumor harvested at the time of surgical debulking. Sixty patients achieving clinical complete response following primary surgical debulking and chemotherapy will undergo vaccine construction. Given the high recurrence rate and subsequent progression seen with frontline standard of care treatment of advanced stage ovarian cancer, we initiated a randomized Phase II study of adjuvant FANG vaccination following initial debulking/chemotherapy. Forty patients will receive 1.0x107 cells/intradermal injection once a month for up to 12 doses and 20 will be followed as per standard of care without maintenance therapy. Key endpoints include safety, immune response and progression free survival. Preliminary results: Thus far, 23 patients have been randomized, 15 of whom have received at least 1 FANG injection (103 total injections) and 8 have been randomized to No FANG treatment. This trial design allows for control group cross over at time of progression. No toxic effect has been observed. A higher rate of ELISPOT response has been elicited in this minimum disease population in comparison to that achieved in Phase I trial (92% vs 50%). Mean PFS internal difference of the FANG vs No FANG patients was 491 vs 337 days. ELISPOT positivity at baseline, although rare after debulking surgery, may be identified as a positive prognostic factor for PFS independent of FANG vaccine. Preliminary PFS of patients receiving FANG with negative ELISPOT (n=20) prior to randomization is shown in figure. In conclusion, Phase II accrual continues. Updated results will be presented.
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623. HLA Typing Identity Test for Verification of Patient to their FANGTM Autologous Cancer Vaccine
Yang Yu,1 Nicolas Taquet,1 John Nemunaitis,1,2 Phillip B. Maples.1 1 Gradalis, Inc., Dallas, TX; 2Mary Crowley Cancer Research Centers, Dallas, TX.
In Phase II clinical development of the FANGTM autologous cancer vaccine, Gradalis receives tumor sample deliveries from multiple clinics and hospitals. Identity match of the tumor sample and the vaccine manufactured with the patient is an essential QA component. We have taken a number of steps to ensure the integrity of Patient – Vaccine tumor collection / cGMP manufacturing / clinical treatment continuum. A most reliable method for specimen identification is DNA identity testing, which will verify the origin of two samples. We adopted the HLA (Human Leukocyte Antigen) molecular genotyping by SSP-PCR (sequence-specific primer polymerase chain reaction) as our identity method. HLA testing is a widely accepted method of identity testing commonly used in solid organ and bone marrow transplantation and paternity testing. In comparison to the comprehensive STR (Short Tandem Repeat) DNA profiling, HLA typing is reliable, cost effective, and has a short turnaround time. Genomic DNA isolated from the peripheral white blood cells of the patient is considered “correct” or “known”. This is compared to the tumor cells which are obtained on day-1 of vaccine manufacturing before plasmid transfection. The DNA extraction was performed by the DNeasy kit (Qiagen) and DNA quantity and quality is subsequently determined by Nano-Drop. The ABDRDQ SSP-PCR HLA Typing Kit which include Class I A Locus, B Locus and the Class II DR/DQ loci from One Lambda, and Taq polymerase from Promega were utilized for the HLA typing. The positive HLA antigens are identified by post-PCR gel electrophoresis. The confirmation of the tumor tissue origin from the patient is determined by the complete molecular HLA typing match between the tumor tissue and the blood. We previously reported an analysis of 58 vaccine-blood sample pairs on Phase I and Phase II FANG vaccine trials. Since that time another 23 Phase-II FANG vaccine-blood sample pairs have been
Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy
tested and analyzed. All vaccine-blood sample paired HLA typing results are complete matches. These results support HLA typing as an identity verification test for FANG™ vaccine.
624. Randomized Phase II Trial of Adjuvant Autologous Tumor Cell Vaccine (FANG™) for High Risk Stage III/IV Ovarian Cancer: Preliminary Results
J. Oh,3 M. Barve,1,3 C. Bedell,1 J. Kuhn,5 B. Fine,3 T. P. Heffernan,6 C. M. Matthews,3 C. A. Stringer,3 E. C. Koon,3 H. Goodman,7 E. L. Fleming,8 L. R. DeMars,8 M. K. Bergman,9 B. O. Pappen,4 P. Kumar,4 P. B. Maples,4 N. Senzer,1,4 J. Nemunaitis.1,2,3,4 1 Mary Crowley Cancer Research Centers, Dallas; 2Medical City Dallas Hospital, Dallas; 3Texas Oncology, PA, Dallas; 4Gradalis, Inc, Dallas; 5WLS Surgery Associates, PA, Dallas; 6North Texas Gyn Onc, Dallas; 7Florida Cancer Specialists, West Palm Beach; 8 Dartmouth-Hitchcock Medical Ctr, Lebanon; 9Cancer Care Northwest, Spokane. Introduction: Phase I evaluation of FANG™ vaccine (GMCSF/ bi-shRNAi furin vector transfected autologous tumor) demonstrated safety; confirmed GMCSF transgene expression and knockdown of furin and consequent TGFβ1 and β2 expression; showed T-cell activation per gamma IFN-ELISPOT and resulted in a longer than expected survival duration thereby justifying Phase II testing [Senzer et al Mol Ther 2012; Senzer et al Vaccines and Vaccin in press]. The majority of women with Stage III/IV ovarian cancer who achieve a complete clinical response with standard of care management involving debulking surgery and consolidation chemotherapy will relapse within 2 years. Methods: This is a 2:1 randomized Phase II open-label trial of FANG using tumor harvested at the time of surgical debulking. Sixty patients achieving clinical complete response following primary surgical debulking and chemotherapy will undergo vaccine construction. Given the high recurrence rate and subsequent progression seen with frontline standard of care treatment of advanced stage ovarian cancer, we initiated a randomized Phase II study of adjuvant FANG vaccination following initial debulking/chemotherapy. Forty patients will receive 1.0x107 cells/intradermal injection once a month for up to 12 doses and 20 will be followed as per standard of care without maintenance therapy. Key endpoints include safety, immune response and progression free survival. Preliminary results: Thus far, 23 patients have been randomized, 15 of whom have received at least 1 FANG injection (103 total injections) and 8 have been randomized to No FANG treatment. This trial design allows for control group cross over at time of progression. No toxic effect has been observed. A higher rate of ELISPOT response has been elicited in this minimum disease population in comparison to that achieved in Phase I trial (92% vs 50%). Mean PFS internal difference of the FANG vs No FANG patients was 491 vs 337 days. ELISPOT positivity at baseline, although rare after debulking surgery, may be identified as a positive prognostic factor for PFS independent of FANG vaccine. Preliminary PFS of patients receiving FANG with negative ELISPOT (n=20) prior to randomization is shown in figure. In conclusion, Phase II accrual continues. Updated results will be presented.
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