- Email: [email protected]
785. Targeting of Myeloid Leukemia Stem Cells through Genetic Redirection of T Cells Against CD44 Variant 6
CANCER - IMMUNOTHERAPY III 6 patients, respectively). Up to 5 extra doses were administered every 8 weeks, or until disease progression. 24 hours after each injection, AP1903 (0.4 mg/kg) was administered i.v. Serum for multiplex cytokine testing was obtained weekly during the acute phase. PSA and radiological testing was performed at regular intervals. Injection site biopsies were obtained for antigen-specific immunological testing following the 4th vaccination. Results: Planned enrollment was completed and vaccine successfully manufactured for all patients. Most (10/12) completed the acute phase and 2 went off protocol due to progression. Toxicities were mild and primarily confined to injection-site DTH responses, which were common and expected. Most (5/7) patients with evaluable T cells grown out from injection sites revealed PSMA-specific immunity, biased towards Th1 (3/5) or Th2 (2/5) cytokine production. 9/12 patients revealed significant (10 to 10,000-fold) but asymptomatic cytokine spikes 1 week after each vaccination that returned to baseline within 7 days. At 12 weeks, PSA doubling times (PSADT) increased on most (8/12) patients. Notably, 1 post-docetaxel, low-dose patient achieved a RECIST PR, and 1 high-dose patient underwent near complete elimination of multi-focal lung metastases with SD following the induction phase. Remarkably, 1 mid-dose patient with widespread visceral and bone metastasis achieved a RECIST CR with docetaxel-based chemotherapy after the induction phase and remains stable with undetectable PSA 1 year after enrollment. Conclusions: Vaccination with BPX-101 followed by AP1903 appears safe and can elicit strong PSMA-specific and other immune responses that appear to correlate with clinical responses. Combination therapy with chemotherapy may further expand efficacy. Finally, we will discuss observed improvements from “unified” vectors under development, comprising both iCD40 and TLR signaling along with antigen expression.
784. Pre-Clinical Safety and Toxicity DoseFinding of a Novel Bi-Cistronic High-Capacity Adenoviral Vector Encoding Flt3L and TK: Identification of a Maximally Tolerated Dose in the Brain with Restricted Biodistribution of Vector Genomes
Mariana Puntel,1 A. K. M. G. Muhammad,1 Weidong Xiong,1 Kyle Kelson,1 Alireza Salem,1 Kurt M. Kroeger,1 Chunyan Liu,1 Catherine Farrokhi,1 Liliana Lacayo,1 Robert N. Pechnick,1 Donna Palmer,2 Philip Ng,2 Maria G. Castro,1 Pedro R. Lowenstein.1 1 Cedars-Sinai Medical Center/UCLA, Los Angeles, CA; 2Baylor College of Medicine, Houston, TX. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, is highly invasive, and invariably recurs killing the patient. Using a combined immunotherapy/conditional cytotoxic approach, we have previously demonstrated that intratumoral delivery of first generation adenoviral vectors (Ad) encoding the conditional cytotoxic gene, herpes simplex virus type 1-thimidine kinase (TK) and Fms-like Tyrosine Kinase 3 ligand (Flt3L) generates a tumor antigen specific anti-GBM immune response resulting in s long-term survival and immunological memory in over eight orthotopic rat and mouse models of GBM. The large cloning capacity of HC-Ads has allowed us to develop a single novel, bi-cistronic HC-Ad vector engineered for inducible expression of Flt3L using the TetOn regulatory system and constitutive expression of HSV1-TK; this simplifies downstream process development, improves the immunological safety profile, and facilitates clinical implementation. We assessed the safety profile of this novel dual HC-Ad vector in naive Lewis rats at three doses of HCAd vector, at short-term (5 days), medium-term (1 month) and longterm (12 months) time points. To identify the maximally tolerated dose in the brain, we assessed safety and toxicity as determined by biodistribution of vector genomes using real-time quantitative PCR (qPCR), levels of Flt3L in the serum, Ad-specific neutralizing antibodies, TK-specific serum antibodies, serum biochemistry, and S300
a comprehensive panel of behavioral tests. HC-Ad vector genomes were below detectable limits in all peripheral organs tested and restricted to the tumor bearing brain hemisphere. At the highest dose tested (1x10^10 vp), neuropathological analysis of brain sections reveled considerable disruption of the neuronal architecture and transient short-term inflammation. Levels of Flt3L in serum were only detectable at 5 days, but not at 1 month and 12 months post treatment. Biochemical laboratory parameters indicated normal liver and renal function for all doses. Analysis of amphetamine-induced rotational behavior, total locomotor activity and rearing activity did not reveal any behavioral abnormalities compared to age matched control animals, even at the high dose that caused neuropathology. These data reveal that 1x10^9 vp is the MTD in the naïve Lewis rat brain, thus further demonstrating the high safety profile of the dual HC-Ad vector administration, and supports further downstream process development for eventual implementation in human clinical trials.
785. Targeting of Myeloid Leukemia Stem Cells through Genetic Redirection of T Cells Against CD44 Variant 6
Monica Casucci,1 Laura Falcone,1 Barbara Camisa,1 Zulma Magnani,1 Claudio Bordignon,2 Barbara Savoldo,3 Gianpietro Dotti,3 Chiara Bonini,1 Attilio Bondanza.1 1 Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Experimental Hematology Unit, San Raffaele Scientific Institute, Milano, Italy; 2MolMed S.p.A, Milano, Italy; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Acute myeloid leukemia (AML) is organized as a hierarchy dominated by leukemia stem cells (LSC). LSC are resistant to radiochemotherapy and ultimately responsible for disease relapse. The results of allogeneic hematopoietic stem cell transplantation (allo-SCT) suggest that LSC are sensitive to adoptive T-cell therapy. Nevertheless, the frequent occurrence of HLA-loss variants after transplantation recommends the targeting of antigens crucial for LSC. LSC self-renewal depends on signals provided within the SC-niche, among which those mediated by CD44 and its variant isoforms. Accordingly, CD44-specific mAb have been shown to specifically eradicate LSC in immunodeficient mouse models. With the aim of targeting LSC, we constructed a second-generation chimeric antigen receptor (CAR) specific for CD44 variant 6 (CD44v6-CAR.28z). Retroviral-mediated gene transfer after stimulation with cell-sized beads coupled to anti-CD3 and anti-CD28 antibodies allowed robust transgene expression. Culture with IL-7 and IL-15 efficiently expanded CD44v6-CAR28z+ T cells, while preserving a central memory T-cell phenotype. In co-culture experiments, CD44v6-CAR28z+ T cells specifically cleared AML cell lines expressing CD44v6. CD44v6CAR28z+ T cells were also effective against a panel of primary AML cells isolated from patients and expressing CD44v6, but not against healthy CD34+ stem cells. Moreover, CD44v6 expression on primary AML and recognition by CD44v6-CAR28z+ T cells was enhanced upon culture on bone marrow-derived stromal cells. Interestingly, similar results were observed on multiple myeloma (MM) cell lines and primary MM cells, suggesting a common cancer stem cell/ niche-specific signature. When stimulated with CD44v6+ autologous monocytes, CD44v6-CAR28z+ T cells expanded significantly, but also displayed background cytoxicity. To increase the safety of our strategy, we implemented the thymidine kinase (tk) suicide gene. Co-transfer of tk enabled the elimination of CD44v6-CAR28z+ T cells upon administration of the prodrug ganciclovir, providing a safety switch in case of unexpected toxicity. These results warrant the clinical implementation of CD44v6-redirected suicide gene-modified T cells for the safe and effective eradication of LSC.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
784. Pre-Clinical Safety and Toxicity DoseFinding of a Novel Bi-Cistronic High-Capacity Adenoviral Vector Encoding Flt3L and TK: Identification of a Maximally Tolerated Dose in the Brain with Restricted Biodistribution of Vector Genomes
Mariana Puntel,1 A. K. M. G. Muhammad,1 Weidong Xiong,1 Kyle Kelson,1 Alireza Salem,1 Kurt M. Kroeger,1 Chunyan Liu,1 Catherine Farrokhi,1 Liliana Lacayo,1 Robert N. Pechnick,1 Donna Palmer,2 Philip Ng,2 Maria G. Castro,1 Pedro R. Lowenstein.1 1 Cedars-Sinai Medical Center/UCLA, Los Angeles, CA; 2Baylor College of Medicine, Houston, TX. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, is highly invasive, and invariably recurs killing the patient. Using a combined immunotherapy/conditional cytotoxic approach, we have previously demonstrated that intratumoral delivery of first generation adenoviral vectors (Ad) encoding the conditional cytotoxic gene, herpes simplex virus type 1-thimidine kinase (TK) and Fms-like Tyrosine Kinase 3 ligand (Flt3L) generates a tumor antigen specific anti-GBM immune response resulting in s long-term survival and immunological memory in over eight orthotopic rat and mouse models of GBM. The large cloning capacity of HC-Ads has allowed us to develop a single novel, bi-cistronic HC-Ad vector engineered for inducible expression of Flt3L using the TetOn regulatory system and constitutive expression of HSV1-TK; this simplifies downstream process development, improves the immunological safety profile, and facilitates clinical implementation. We assessed the safety profile of this novel dual HC-Ad vector in naive Lewis rats at three doses of HCAd vector, at short-term (5 days), medium-term (1 month) and longterm (12 months) time points. To identify the maximally tolerated dose in the brain, we assessed safety and toxicity as determined by biodistribution of vector genomes using real-time quantitative PCR (qPCR), levels of Flt3L in the serum, Ad-specific neutralizing antibodies, TK-specific serum antibodies, serum biochemistry, and S300
a comprehensive panel of behavioral tests. HC-Ad vector genomes were below detectable limits in all peripheral organs tested and restricted to the tumor bearing brain hemisphere. At the highest dose tested (1x10^10 vp), neuropathological analysis of brain sections reveled considerable disruption of the neuronal architecture and transient short-term inflammation. Levels of Flt3L in serum were only detectable at 5 days, but not at 1 month and 12 months post treatment. Biochemical laboratory parameters indicated normal liver and renal function for all doses. Analysis of amphetamine-induced rotational behavior, total locomotor activity and rearing activity did not reveal any behavioral abnormalities compared to age matched control animals, even at the high dose that caused neuropathology. These data reveal that 1x10^9 vp is the MTD in the naïve Lewis rat brain, thus further demonstrating the high safety profile of the dual HC-Ad vector administration, and supports further downstream process development for eventual implementation in human clinical trials.
785. Targeting of Myeloid Leukemia Stem Cells through Genetic Redirection of T Cells Against CD44 Variant 6
Monica Casucci,1 Laura Falcone,1 Barbara Camisa,1 Zulma Magnani,1 Claudio Bordignon,2 Barbara Savoldo,3 Gianpietro Dotti,3 Chiara Bonini,1 Attilio Bondanza.1 1 Division of Regenerative Medicine, Stem Cells, and Gene Therapy, Experimental Hematology Unit, San Raffaele Scientific Institute, Milano, Italy; 2MolMed S.p.A, Milano, Italy; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX. Acute myeloid leukemia (AML) is organized as a hierarchy dominated by leukemia stem cells (LSC). LSC are resistant to radiochemotherapy and ultimately responsible for disease relapse. The results of allogeneic hematopoietic stem cell transplantation (allo-SCT) suggest that LSC are sensitive to adoptive T-cell therapy. Nevertheless, the frequent occurrence of HLA-loss variants after transplantation recommends the targeting of antigens crucial for LSC. LSC self-renewal depends on signals provided within the SC-niche, among which those mediated by CD44 and its variant isoforms. Accordingly, CD44-specific mAb have been shown to specifically eradicate LSC in immunodeficient mouse models. With the aim of targeting LSC, we constructed a second-generation chimeric antigen receptor (CAR) specific for CD44 variant 6 (CD44v6-CAR.28z). Retroviral-mediated gene transfer after stimulation with cell-sized beads coupled to anti-CD3 and anti-CD28 antibodies allowed robust transgene expression. Culture with IL-7 and IL-15 efficiently expanded CD44v6-CAR28z+ T cells, while preserving a central memory T-cell phenotype. In co-culture experiments, CD44v6-CAR28z+ T cells specifically cleared AML cell lines expressing CD44v6. CD44v6CAR28z+ T cells were also effective against a panel of primary AML cells isolated from patients and expressing CD44v6, but not against healthy CD34+ stem cells. Moreover, CD44v6 expression on primary AML and recognition by CD44v6-CAR28z+ T cells was enhanced upon culture on bone marrow-derived stromal cells. Interestingly, similar results were observed on multiple myeloma (MM) cell lines and primary MM cells, suggesting a common cancer stem cell/ niche-specific signature. When stimulated with CD44v6+ autologous monocytes, CD44v6-CAR28z+ T cells expanded significantly, but also displayed background cytoxicity. To increase the safety of our strategy, we implemented the thymidine kinase (tk) suicide gene. Co-transfer of tk enabled the elimination of CD44v6-CAR28z+ T cells upon administration of the prodrug ganciclovir, providing a safety switch in case of unexpected toxicity. These results warrant the clinical implementation of CD44v6-redirected suicide gene-modified T cells for the safe and effective eradication of LSC.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy