Self-inactivating Lentiviral Vector Research Articles

Protection from cisplatin-induced hearing loss with lentiviral vector-mediated ectopic expression of the anti-apoptotic protein BCL-XL

Cisplatin is a highly effective chemotherapeutic agent, but it can cause sensorineural hearing loss (SNHL) in patients. Cisplatin-induced ototoxicity is closely related to the accumulation of reactive oxygen species (ROS) and subsequent death of hair cells (HCs) and spiral ganglion neurons (SGNs). Despite various strategies to combat ototoxicity, only one therapeutic agent has thus far been clinically approved. Therefore, we have developed a gene therapy concept to protect cochlear cells from cisplatin-induced toxicity. Self-inactivating lentiviral (LV) vectors were used to ectopically express various antioxidant enzymes or anti-apoptotic proteins to enhance the cellular ROS scavenging or prevent apoptosis in affected cell types. In direct comparison, anti-apoptotic proteins mediated a stronger reduction in cytotoxicity than antioxidant enzymes. Importantly, overexpression of the most promising candidate, Bcl-xl, achieved an up to 2.5-fold reduction in cisplatin-induced cytotoxicity in HEI-OC1 cells, phoenix auditory neurons, and primary SGN cultures. BCL-XL protected against cisplatin-mediated tissue destruction in cochlear explants. Strikingly, invivo application of the LV BCL-XL vector improved hearing and increased HC survival in cisplatin-treated mice. In conclusion, we have established a preclinical gene therapy approach to protect mice from cisplatin-induced ototoxicity that has the potential to be translated to clinical use in cancer patients.

Sustained Efficacy, Safety, and Improved Quality of Life in Adult and Pediatric Patients with Transfusion-Dependent β-Thalassemia up to 9 Years Post Treatment with Betibeglogene Autotemcel (Beti-cel)

Introduction: Beti-cel gene therapy addresses the underlying cause of transfusion-dependent β-thalassemia (TDT) by adding functional copies of a modified β-globin gene to autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) via a third-generation, self-inactivating lentiviral vector (LVV), BB305, which produces functional adult hemoglobin (Hb), HbA T87Q, in red blood cells (RBCs). Here, we report efficacy, safety, and quality of life (QOL) data from adult and pediatric patients treated with beti-cel who were followed for up to 9 years after treatment. These outcomes may inform patient selection for real-world treatment with beti-cel. Methods: Patients with TDT who completed either a phase 1/2 (HGB-204 [NCT01745120]; HGB-205 [NCT02151526]) or phase 3 (HGB-207 [NCT02906202]; HGB-212 [NCT03207009]) beti-cel parent study and subsequently participated in the long-term, 13-year follow-up study LTF-303 (NCT02633943) were included in this analysis. Analyses were performed with data collected through January 30, 2023. Efficacy (including transfusion independence [TI], defined as a weighted average Hb ≥9 g/dL without packed RBC transfusions for ≥12 months) and safety are reported through last follow-up. QOL data are reported for studies HGB-204, HGB-207, and HGB-212 through month 36. Statistical analyses were conducted to examine key outcomes in subgroups based on patient age at enrollment (pediatric: <18 years; adult: ≥18 years). Results:As of January 30, 2023, 63 patients (median [range] age: 17 [4-35] years) had received beti-cel in a phase 1/2 or 3 study and enrolled in LTF-303, with a median (range) follow-up of 60.1 (23.8-109.5) months. Phase 3 studies used the commercial drug product manufacturing process. Ninety percent (37/41) of phase 3 patients achieved and maintained TI through last follow-up (up to 6 years; Table 1). TI rates by study, genotype, and age for phase 3 patients are also presented in Table 1. In phase 1/2 studies that used an older drug manufacturing process, 68.2% (15/22) of patients achieved TI; 14 of these patients sustained TI through last follow-up (up to 9 years). One patient no longer meets protocol-defined TI as a result of Hb level <9g/dL at year 6 due to acute health events unrelated to β-thalassemia, which were not attributed to loss of beti-cel treatment effect. Approximately 80% of pediatric and adult patients required only one mobilization cycle to achieve the drug product dose. The median percentage of drug product cells transduced with the BB305 LVV was comparable between adult and pediatric populations (78% and 80%, respectively), as were the month 6 median peripheral blood vector copy number (1.4 c/dg and 1.1 c/dg) and HbA T87Q (9.4 g/dL and 8.3 g/dL). Adult patients reported improvements in QOL up to month 36 as assessed by the Short Form-36 Health Survey Questionnaire mental and physical component summary scores, Functional Assessment of Cancer Therapy, and EuroQol ( Table 2). Pediatric Quality of Life Inventory scores will be reported at the time of presentation. Overall, 19% (12/63) of patients experienced ≥1 beti-cel-related adverse event (AE); the most common beti-cel-related AEs (occurring in ≥3 patients) were abdominal pain (experienced in 5/63 [7.9%] patients) and thrombocytopenia (3/63 [4.8%] patients). Five patients experienced serious veno-occlusive liver disease; all 5 received defibrotide and recovered. No malignancies, insertional oncogenesis, or vector-derived replication-competent lentivirus were reported. Age subgroup safety analysis as of last follow-up will be reported in the final presentation. Conclusion: Beti-cel is a potentially curative gene therapy for patients with TDT across ages and genotypes through achievement of TI and normal or near-normal Hb. These data will inform real-world beti-cel treatment decisions for patients with TDT and providers.

Preclinical Efficacy of a Novel CD8-Targeted Fusosome for In Vivo CAR T Cell Therapy

SG299 is a CD8-targeted fusosome; a self-inactivating (SIN) lentiviral vector (LVV) pseudotyped with a CD8-targeted fusogen, which carries a transgene encoding a CD19-directed chimeric antigen receptor (CAR). We evaluated the preclinical efficacy of a representative CD8/CD19CAR fusosome comparable to SG299 for its ability to control tumor growth in vivo in an NSG mouse model engrafted with human CD19 positive tumor cells and human peripheral blood mononuclear cells (PBMC). We compared direct intravenous (IV) administration and ex vivo exposure of fusosome to resting T cells followed by IV injection of vector-cell mixtures, as has been proposed for extracorporeal delivery (ECD) methods. We have also evaluated the performance of multiple lots of fusosomes to assess consistency in manufacturing and have evaluated donor-to-donor variability in five PBMC donors in this model. To measure anti-tumor efficacy, immunodeficient NSG mice were challenged IV with NALM6 tumors expressing firefly luciferase (5E5 cells/mouse) on day 4. For direct IV, PBMC (1E7 cells/mouse) were injected IV on day -1 followed by CD8/CD19CAR fusosome (1E7 IU/mouse) on day 0. For ECD, PBMC were incubated with CD8/CD19CAR fusosome for 1 hr and the cell and fusosome mix were injected IV on day 1. Tumor growth was monitored by bioluminescence imaging over a 28-day period. In addition, we have investigated both pharmacokinetics of fusosome induced CAR T cells and NALM6 tumors by flow cytometry. CD8/CD19CAR fusosome demonstrated statistically significant control of NALM6 tumors by both direct IV and ECD routes of delivery. Kinetic analysis of peripheral blood demonstrated tumor control and in vivo CART cell generation were dose dependent. The peak CAR T cell response was at D14. CARs detected at all timepoints were CD8+ specific; no CD4 + CAR T cells were observed. In addition, all mice that received fusosome demonstrated protection from morbidity and mortality as measured by reduced weight loss and improved survival compared to control or tumor only mice. Lot-to-lot performance variability of fusosomes was minimal; three lots of fusosome that had similar physical and functional attributes showed efficient and equivalent tumor control with multiple PBMC donors. Finally, an assessment of donor-to-donor variability across five PBMC donors using a single lot of fusosome showed donor-specific variations in tumor control as measured by BLI but demonstrated a consistent protection from morbidity and mortality. Collectively, these studies demonstrate preclinical efficacy of our CD8/CD19CAR candidate, lot consistency and verify our ability to generate CD19-directed CAR T cells in vivo using a specifically targeted platform.

Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome

AbstractWiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein–expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10–producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

Preliminary clinical outcomes of ADP-A2M4CD8, a next-generation autologous T-cell receptor T-cell therapy, in patients with advanced urothelial cancer.

517 Background: ADP-A2M4CD8 is a specific peptide enhanced affinity receptor mixed CD4+ and CD8+ T-cell therapy targeting the cancer testis antigen MAGE-A4 and modified with addition of a CD8α co-receptor designed to provide additional functionality to CD4+ T-cells. ADP-A2M4CD8 has demonstrated an acceptable benefit to risk profile in the Phase 1 SURPASS trial (NCT04044859) in HLA A*02–eligible patients with unresectable or metastatic tumors positive for MAGE-A4.1 Here we report updated clinical outcomes in patients with urothelial cancer (UC). Methods: SURPASS is a first-in-human trial consisting of a modified 3+3 dose-escalation design and an expansion cohort. Autologous T-cells are obtained by leukapheresis, transduced with a self-inactivating lentiviral vector expressing the MAGE-A4-specific T-cell receptor and the CD8α co-receptor, and infused back to the patients as ADP-A2M4CD8 following lymphodepleting chemotherapy. Primary and secondary objectives are safety and anti-tumor activity, respectively. Results: At ESMO 2022, we reported promising results from SURPASS in several tumour types.2 In the 43 evaluable patients, the overall response rate was 28%, including 1 complete response and 11 partial responses (PR), and an additional 2 unconfirmed PRs awaiting confirmatory scans (as of August 1, 2022). Data from the 7 evaluable patients in the UC subset (updated September 6, 2022) showed that 3 (43%) had a best overall response of PR, and 1 (14%) had an unconfirmed PR. Disease control rate was 100% (3 PR + 1 unconfirmed PR + 3 stable disease). Adverse events have been consistent with those typically observed with lymphodepletion chemotherapy or cellular therapy. This trial is ongoing; data from additional patients with UC treated by January 2023 and updated translational data will be presented. Conclusions: ADP-A2M4CD8 continues to show an acceptable benefit to risk profile in multiple MAGE-A4+ unresectable or metastatic tumors, and preliminary encouraging evidence of efficacy in UC. An additional treatment cohort has been included in the updated trial protocol to evaluate ADP-A2M4CD8 combined with nivolumab. 1. Hong DS, et al. E-poster 540P: ESMO 2021; Virtual. 2. Hong DS, et al. Ann Oncol 33(suppl_7); S331-S355, Abstract 735MO. ESMO 2022. Clinical trial information: NCT04044859 .

Safety and efficacy from the phase 1 SURPASS trial of ADP-A2M4CD8, a next-generation T-cell receptor T-cell therapy, in patients with advanced esophageal, esophagogastric junction, or gastric cancer.

349 Background: ADP-A2M4CD8 is a specific peptide enhanced affinity receptor mixed CD4+ and CD8+ T-cell therapy targeting the cancer testis antigen MAGE-A4 and modified with addition of a CD8α co-receptor designed to provide additional functionality to CD4+ T-cells. ADP-A2M4CD8 has demonstrated a favorable benefit to risk profile in the Phase 1 SURPASS trial (NCT04044859) in HLA A*02–eligible patients (pts) with unresectable or metastatic tumors positive for MAGE-A4 (Hong DS, et al. E-poster 540P: ESMO 2021; Virtual). Here we report updated clinical outcomes in pts with esophageal, esophagogastric junction (EGJ), or gastric cancer. Methods: SURPASS is a first-in-human trial consisting of a modified 3+3 dose-escalation design and an expansion cohort. Autologous T-cells are obtained by leukapheresis, transduced with a self-inactivating lentiviral vector expressing the MAGE-A4-specific T-cell receptor and the CD8α co-receptor, and infused back to the pts as ADP-A2M4CD8 following lymphodepleting chemotherapy. Primary and secondary objectives are safety and anti-tumor activity, respectively. Results: As September 6, 2022, 13 pts (11 male, 2 female) with esophageal (3), EGJ (8), and gastric cancer (2) received ADP-A2M4CD8 (range: 1.02–9.9x109 transduced T-cells). All pts had adenocarcinoma, median age was 55 years (range: 31–71), median MAGE-A4 expression H-score was 245 (range: 160–300), and pts received median of 2 lines of prior therapy (range 1–5). Adverse events (AEs) were consistent with those typically associated with lymphodepleting chemotherapy, cellular therapy, and/or disease. One pt had a Grade 5 (fatal) AE of pancytopenia. Overall response rate per RECIST v1.1 by investigator review was 15% (2 partial response [PR]). Disease control rate was 77% (2 PR+8 stable disease). This trial is ongoing, and additional data will be presented. Conclusions: Results indicate an acceptable benefit to risk profile and encouraging anti-tumor activity of ADP-A2M4CD8. An additional treatment cohort has been included in the updated SURPASS trial protocol to evaluate ADP-A2M4CD8 combined with nivolumab. A Phase 2, open-label trial in advanced esophageal and EGJ cancers has been initiated (SURPASS-2; NCT04752358). Clinical trial information: NCT04044859 .

Abstract 560: Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs

Abstract Background: We have previously established the ability of CD3-directed lentiviral vectors encoding for CD19 or CD22 CARs to mediate robust anti-tumor immunity in humanized lymphoreplete mouse models. We now present additional mechanistic data for this novel subcutaneous (SC) CAR-T approach. Methods: Human PBMCs loaded with a self-inactivating lentiviral vector (LV) encoding CD19 CAR with a synthetic driver element were injected SC into autologous PBMC humanized NSG MHC I/II double knock out (DKO) mice. The LV was packaged with a modified envelope with the ability to target and activate CD3+ T cells. To first track the site of CAR formation in vivo following SC injection, PBMCs were loaded with an LV encoding a CD19 CAR and luciferase, and bioluminescence imaging (BLI) was performed. Additionally, histopathology of the site of injection and distal organs were examined. Evaluation for CAR+ cells was performed through immunohistochemistry and PCR detection. To examine the tropism of the CD3-directed CD19 CAR LV and characterize CAR+ cells, the phenotype of in vivo expanded CAR+ cells was evaluated. Further characterization of CAR+ cells was performed with in vitro studies. Results: Following SC injection of LV-loaded PBMCs, the first evidence of transgene expression utilizing BLI for luciferase was detected four to five days following SC injection. At the same time point, histologic examination of the SC site of injection revealed the formation of tertiary lymphoid structures (TLS) consisting of human CD8+ and CD4+ T cells, CD68+ macrophages, CD68+ dendritic cells, and a few CD20+ B cells. On day 13 post-SC injection, BLI detected the presence of CAR+ cells systemically beyond the site of injection and within subcutaneous Raji tumors implanted on the contralateral side. On day 14 post-SC injection, histologic examination showed sustained TLS within the SC tissue without signs of dermal acute inflammation or ulceration and evidence of CAR+ cells appearing in the spleen. CAR+ cells exhibited robust anti-tumor immunity with expansion into peripheral blood. CAR+ cells consisted of a distinct population of CD8+ T cells with NK-like features (TaNKs) and a CD3+ CD8+ CD56+ NKG2D+ cell phenotype. In vitro transduction of CD56 NK cell-depleted PBMC with the CD3-directed LV also led to CAR-TaNK formation. Conclusion: The subcutaneous injection of CD3-directed LV-loaded PBMCs leads to the formation of tertiary lymphoid structures at the site of injection and the development of distinct CD3+ CD8+ CD56+ NKG2D+ CAR-TaNK cells. These cells possess enhanced systemic proliferative capacity compared to traditional ex vivo manufactured 41BB CAR-T cells in a lymphoreplete mouse model and the ability to eliminate target cells in vivo with low numbers of starting cells (10,000 cells). Citation Format: Ani Kundu, Dongming Zhang, Frederic Vigant, Junyi Zhang, Greg Schreiber, Ewa Jaruga-Killeen, Alissa Kerner, Michelle Andraza, Wei Zhang, John Henkelman, Renata Soares, Gregory I. Frost, Sid P. Kerkar. Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 560.

Abstract 3294: In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion

Abstract Introduction: Chimeric antigen receptor therapies (CAR-T) are highly effective against hematologic malignancies but require a lymphodepleting chemotherapy regimen and are faced with many challenges including manufacturing time, scalability, and cost of production due to the need for ex vivo culturing of cells and complex chain of custody requirements. In vivo delivery of self-inactivating lentiviral vectors (LV) encoding for CAR-T transgenes represents a promising strategy to improve the time to treatment, scalability, and cost of current CAR-T therapies. Methods: Lentiviral vectors encoding a CD19 CAR with a synthetic driver element were manufactured in a chemically defined cell substrate that incorporates a modified envelope designed to target and activate CD3+ T cells. Two different humanized mouse models were utilized. In a PBMC humanized mouse model, NSG MHC Class I/II knock-out mice (DKO) were injected with 1E7 human PBMC and followed one day later with 2E7 TU of LV encoding for CD19 CAR. In parallel, CD34+ humanized NSG SGM3 mice were also administered 2E7 TU LV encoding CD19 CAR. Flow cytometry of peripheral blood samples was evaluated at various time points for the presence of CAR+ cells and CD20+ B cells. Additionally, tissue samples were examined by histopathology and PCR for vector copy numbers. Results: The NSG SGM3 humanized CD34+ mouse model exhibited efficient chimerism of human CD45+ hematopoietic cells (>50% of live cells in peripheral blood). Apart from CD15+ neutrophils, all major human immune cell components were well represented in peripheral blood including CD14+ monocytes, CD20+ B cells, and CD3+ lymphocytes. At study initiation, the T cell compartment in the SGM3 mice exhibited skewing towards CD4+ T cells. Injection of CD19 CAR-LV resulted in a significant reduction of CD20+ B cells as early as 5 days post-injection. CD3+ CAR+ cells were detected in peripheral blood by day 5 with evidence of CAR+ cell expansion at subsequent time points. Loss of CD20+ B cells was stable throughout the observation period with some mice exhibiting a complete elimination of B cells. Similarly, the PBMC humanized NSG DKO mice injected with CD19 CAR-LV also showed evidence of CAR+ cells in peripheral blood. Conclusions: CD3-directed self-inactivating lentiviral vectors can efficiently deliver an integrating CAR gene into T lymphocytes following in vivo administration. These in vivo generated CD19 CAR T cells expand systemically and effectively eliminate pre-existing B cells. These data show that the targeting of CD3 through in vivo delivery can produce functional CAR T cells and represents an innovative therapeutic opportunity to potentially overcome current manufacturing times, scalability, and cost challenges facing cell therapies. Citation Format: Frederic Vigant, Ani Kundu, Dongming Zhang, Wei Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Gregory Schreiber, Alissa Kerner, Junyi Zhang, John Henkelman, Renata Soares, Ramya Yarlagadda, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3294.

Intranasal Lentiviral Vector-Mediated Antibody Delivery Confers Reduction of SARS-CoV-2 Infection in Elderly and Immunocompromised Mice.

Vaccines for COVID-19 are now a crucial public health need, but the degree of protection provided by conventional vaccinations for individuals with compromised immune systems is unclear. The use of viral vectors to express neutralizing monoclonal antibodies (mAbs) in the lung is an alternative approach that does not wholly depend on individuals having intact immune systems and responses. Here, we identified an anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibody, NC0321, which can efficiently neutralize a range of SARS-CoV-2 variants, including alpha, beta, delta, and eta. Both prophylactic and therapeutic NC0321 treatments effectively protected mice from SARS-CoV-2 infection. Notably, we adopted viral vector-mediated delivery of NC0321 IgG1 as an attractive approach to prevent SARS-CoV-2 infection. The NC0321 IgG1 expression in the proximal airway, expressed by a single direct in-vivo intranasal (I.N.) administration of a self-inactivating and recombinant lentiviral vector (rSIV.F/HN-NC0321), can protect young, elderly, and immunocompromised mice against mouse-adapted SARS-CoV-2 surrogate challenge. Long-term monitoring indicated that rSIV.F/HN-NC0321 mediated robust IgG expression throughout the airway of young and SCID mice, importantly, no statistical difference in the NC0321 expression between young and SCID mice was observed. A single I.N. dose of rSIV.F/HN-NC0321 30 or 180 days prior to SARS-CoV-2 challenge significantly reduced lung SARS-CoV-2 titers in an Ad5-hACE2-transduced mouse model, reconfirming that this vectored immunoprophylaxis strategy could be useful, especially for those individuals who cannot gain effective immunity from existing vaccines, and could potentially prevent clinical sequelae.

Hurdles to the Adoption of Gene Therapy as a Curative Option for Transfusion-Dependent Thalassemia.

Beta-thalassemia is one of the most common monogenic disorders. Standard treatment of the most severe forms, i.e., transfusion-dependent thalassemia (TDT) with long-term transfusion and iron chelation, represents a considerable medical, psychological, and economic burden. Allogeneic hematopoietic stem cell transplantation from an HLA-identical donor is a curative treatment with excellent results in children. Recently, several gene therapy approaches were evaluated in academia or industry-sponsored clinical trials as alternative curative options for children and young adults without an HLA-identical donor. Gene therapy by addition of a functional beta-globin gene using self-inactivating lentiviral vectors in autologous stem cells resulted in transfusion independence for a majority of TDT patients across different age groups and genotypes, with a current follow-up of multiple years. More recently, promising results were reported in TDT patients treated with autologous hematopoietic stem cells edited with the clustered regularly interspaced short palindromic repeats-Cas9 technology targeting erythroid BCL11A expression, a key regulator of the normal switch from fetal to adult globin production. Patients achieved high levels of fetal hemoglobin allowing for discontinuation of transfusions. Despite remarkable clinical efficacy, 2 major hurdles to gene therapy access for TDT patients materialized in 2021: (1) a risk of secondary hematological malignancies that is complex and multifactorial in origin and not limited to the risk of insertional mutagenesis, (2) the cost—even in high-income countries—is leading to the arrest of commercialization in Europe of the first gene therapy medicinal product indicated for TDT despite conditional approval by the European Medicines Agency.

SURPASS-2 trial design: A phase 2, open-label study of ADP-A2M4CD8 SPEAR T cells in advanced esophageal or esophagogastric junction cancers.

TPS363 Background: ADP-A2M4CD8 is an autologous specific peptide enhanced affinity receptor (SPEAR) mixed CD4 and CD8 T-cell product that expresses an engineered T-cell receptor (TCR) designed to target the MAGE-A4 antigen in HLA-A*02-positive patients. These SPEAR T-cells also express wild-type CD8α co-receptors, designed to provide additional functionality to CD4 T-cells. MAGE-A4 expression has been described in several solid tumors, including esophageal and esophagogastric junction (EGJ) cancers[1]. In an ongoing phase 1 study (NCT04044859) of ADP-A2M4CD8 in patients with different tumor types, most adverse events have been consistent with those typically experienced by patients undergoing chemotherapy and/or adoptive T-cell therapies, and as of 2 August 2021, among evaluable patients with esophageal and EGJ cancers, best overall responses were 1 partial response (EGJ), 4 stable disease (2 EGJ, 2 esophageal), and 1 progressive disease (EGJ)[2]. Methods: SURPASS-2 (NCT04752358) is a phase 2, open-label, single-arm trial to assess safety and efficacy of ADP-A2M4CD8 SPEAR T-cells in HLA-A*02–positive patients with advanced esophageal or EGJ cancers that express MAGE-A4 antigen. A total of 45 patients between the ages of 18 and 75 years old will be treated across sites in North America and Europe. Key eligibility criteria include measurable disease per RECIST v1.1; ECOG performance status of 0 or 1; no active autoimmune or immune-mediated disease; no leptomeningeal disease, carcinomatous meningitis, or symptomatic CNS metastases; and ≤2 prior lines of combination or single agent systemic treatment for advanced or metastatic disease before treatment with ADP-A2M4CD8 as the next therapy. Leukapheresis can be performed before initiating or at the end of second-line treatment. Collected T-cells will be transduced with a self-inactivating lentiviral vector expressing the high affinity MAGE-A4-specific TCR and the CD8α co-receptor. Lymphodepleting chemotherapy, consisting of intravenous (IV) cyclophosphamide 600 mg/m2/day for 3 days and IV fludarabine 30 mg/m2/day for 4 days, will be given approximately 1 week prior to treatment with ADP-A2M4CD8. ADP-A2M4CD8 dose will range between 1 x 109 to 10 x 109 transduced cells administered by a single IV infusion. The primary endpoint is overall response rate per RECIST v1.1 by an independent radiological assessment committee. Safety evaluations will include adverse events (AEs); serious AEs; incidence, severity, and duration of the AEs of special interest; replication competent lentivirus; and T-cell clonality and insertional oncogenesis. All patients receiving ADP-A2M4CD8 cellular therapy will be followed for 15 years from time of last T-cell infusion for observation of delayed AEs. To date, one site is activated. [1] Ishihara et al. BMC Cancer 20, 606 (2020). [2] Hong et al. E-poster 540P: ESMO (2021). Clinical trial information: NCT04752358.

Targeting transgenic proteins to alpha granules for platelet-directed gene therapy

Platelets are anucleate blood cells that are shed from megakaryocytes (MKs) into the bloodstream to maintain hemostasis and promote wound healing after vascular injury. To carry out their functions, platelets become activated and release bioactive substances from their secretory granules. As alpha granules (αGs) in resting platelets store proteins and release them only after activation, the packaging of proteins into αGs is an attractive strategy to deliver therapeutic proteins. Here, we propose an adjustable model for targeting transgenic proteins to platelet αGs using third-generation self-inactivating lentiviral vectors. The vectors express from the murine platelet factor 4 promoter (mPf4P), restricting transgene expression to the MK lineage. For the delivery and retention of expressed proteins in αGs, proteins are fused to short peptide sorting signals derived from the human cytokine RANTES or from the transmembrane protein P-selectin. We demonstrate effective targeting of GFP to αGs of murine and human in vitro-differentiated MKs and murine platelets in vivo. Furthermore, interferon-α (IFNα), as a potentially therapeutic cytokine, was successfully delivered to and stored in murine platelets in vivo, was released after activation, and inhibited virus replication in vitro. Our vectors create possibilities for numerous applications in cell therapy utilizing platelets as carriers of therapeutic proteins.

Cell membrane-anchored anti-HIV single-chain antibodies and bifunctional inhibitors targeting the gp41 fusion protein: new strategies for HIV gene therapy

ABSTRACT Emerging studies indicate that infusion of HIV-resistant cells could be an effective strategy to achieve a sterilizing or functional cure. We recently reported that glycosylphosphatidylinositol (GPI)-anchored nanobody or a fusion inhibitory peptide can render modified cells resistant to HIV-1 infection. In this study, we comprehensively characterized a panel of newly isolated HIV-1-neutralizing antibodies as GPI-anchored inhibitors. Fusion genes encoding the single-chain variable fragment (scFv) of 3BNC117, N6, PGT126, PGT128, 10E8, or 35O22 were constructed with a self-inactivating lentiviral vector, and they were efficiently expressed in the lipid raft sites of target cell membrane without affecting the expression of HIV-1 receptors (CD4, CCR5 and CXCR4). Significantly, transduced cells exhibited various degrees of resistance to cell-free HIV-1 infection and cell-associated HIV-1 transmission, as well as viral Env-mediated cell–cell fusion, with the cells modified by GPI-10E8 showing the most potent and broad anti-HIV activity. In mechanism, GPI-10E8 also interfered with the processing of viral Env in transduced cells and attenuated the infectivity of progeny viruses. By genetically linking 10E8 with a fusion inhibitor peptide, we subsequently designed a group of eight bifunctional constructs as cell membrane-based inhibitors, designated CMI01∼CMI08, which rendered cells completely resistant to HIV-1, HIV-2, and simian immunodeficiency virus (SIV). In human CD4+ T cells, GPI-10E8 and its bifunctional derivatives blocked both CCR5- and CXCR4-tropic HIV-1 isolates efficiently, and the modified cells displayed robust survival selection under HIV-1 infection. Therefore, our studies provide new strategies for generating HIV-resistant cells, which can be used alone or with other gene therapy approaches.

Hematopoietic Stem Cell Gene Therapy for Cystinosis: From Bench-to-Bedside.

Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The gene involved is the CTNS gene that encodes cystinosin, a seven-transmembrane domain lysosomal protein, which is a proton-driven cystine transporter. Cystinosis is characterized by the lysosomal accumulation of cystine, a dimer of cysteine, in all the cells of the body leading to multi-organ failure, including the failure of the kidney, eye, thyroid, muscle, and pancreas, and eventually causing premature death in early adulthood. The current treatment is the drug cysteamine, which is onerous and expensive, and only delays the progression of the disease. Employing the mouse model of cystinosis, using Ctns−/− mice, we first showed that the transplantation of syngeneic wild-type murine hematopoietic stem and progenitor cells (HSPCs) led to abundant tissue integration of bone marrow-derived cells, a significant decrease in tissue cystine accumulation, and long-term kidney, eye and thyroid preservation. To translate this result to a potential human therapeutic treatment, given the risks of mortality and morbidity associated with allogeneic HSPC transplantation, we developed an autologous transplantation approach of HSPCs modified ex vivo using a self-inactivated lentiviral vector to introduce a functional version of the CTNS cDNA, pCCL-CTNS, and showed its efficacy in Ctns−/− mice. Based on these promising results, we held a pre-IND meeting with the Food and Drug Administration (FDA) to carry out the FDA agreed-upon pharmacological and toxicological studies for our therapeutic candidate, manufacturing development, production of the GMP lentiviral vector, design Phase 1/2 of the clinical trial, and filing of an IND application. Our IND was cleared by the FDA on 19 December 2018, to proceed to the clinical trial using CD34+ HSPCs from the G-CSF/plerixafor-mobilized peripheral blood stem cells of patients with cystinosis, modified by ex vivo transduction using the pCCL-CTNS vector (investigational product name: CTNS-RD-04). The clinical trial evaluated the safety and efficacy of CTNS-RD-04 and takes place at the University of California, San Diego (UCSD) and will include up to six patients affected with cystinosis. Following leukapheresis and cell manufacturing, the subjects undergo myeloablation before HSPC infusion. Patients also undergo comprehensive assessments before and after treatment to evaluate the impact of CTNS-RD-04 on the clinical outcomes and cystine and cystine crystal levels in the blood and tissues for 2 years. If successful, this treatment could be a one-time therapy that may eliminate or reduce renal deterioration as well as the long-term complications associated with cystinosis. In this review, we will describe the long path from bench-to-bedside for autologous HSPC gene therapy used to treat cystinosis.

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Gene Therapy for Beta Thalassemia: Preliminary Results from the PHASE I/II Tiget-Bthal Trial of Autologous Hematopoietic Stem Cells Genetically Modified with GLOBE Lentiviral Vector

Engineering Precision Medicine to Enhance Graft-Versus-Lymphoma Activity: Hematopoietic Stem Cells Modified with Chimeric Antigen Receptors

Background: Gene-modified human hematopoietic stem cells (HSC) have been used to introduce genes to correct, prevent or treat diseases. Clinical trials using infusion of autologous T cells engineered with anti-CD19 chimeric antigen receptors (CAR) have demonstrated complete remissions even in chemotherapy-resistant malignancies, but the persistence of the effector cells is transient, limiting clinical efficacy and allowing the cancer to recur. Current trials of CAR T cells have recommended patients in remission post-T cell therapy to undergo hematopoietic stem cell transplantation (HSCT) due to the limited persistence of the CAR+ T cells. Seeking to foster persistence of the CAR-modified immune cells, we propose using gene modification of HSC to create persistent generation of multilineage immune effectors to directly target cancer cells. Concerns regarding malignant transformation, abnormal hematopoiesis and autoimmunity exist, making the co-delivery of a suicide gene a necessary safety measure. We evaluated a second-generation anti-CD19 chimeric antigen receptor (CAR) to human HSC in a humanized mouse model, co-delivered with a truncated epidermal growth factor receptor (EGFRt) as a suicide gene system.Significance: CAR+ HSC can be infused in the context of HSCT, when the chemotherapy-conditioning regimen favors engraftment of gene-modified cells, decreases residual tumor burden and decreases the chance of CAR immunogenicity. The prospect of modifying autologous cells to enhance GVL bears the possibility of decreased morbidity and mortality, a concern for specifically vulnerable populations such as children and elderly patients. In addition, this approach will offer alternative therapy for those without cell sources available for allogeneic HSCT, benefiting ethnic minorities.Methods: Third-generation self-inactivating lentiviral vectors were used to co-deliver an anti-CD19 CAR and EGFRt. In vitro, gene-modified HSC were differentiated into myeloid cells to allow transgene expression. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used incubating target cells with leukocytes and monoclonal antibody cetuximab to determine percentage of surviving cells. In vivo, gene-modified HSC were engrafted into immunodeficient NSG mice treated or not with intraperitoneal cetuximab 1mg/mouse/day for 12 days. Persistence of gene-modified cells was assessed by flow cytometry, ddPCR, and PET imaging using 89Zr-Cetuximab.Results: Gene modification of HSC did not affect engraftment, proliferation or differentiation, at efficiency of 20-40%. Antigen specificity of myeloid, NK and T cells were successfully redirected against CD19. Ablation of gene-modified cells was significantly increased (p=0.01) in target cells expressing EGFRt after incubation with leukocytes and cetuximab 1µg/mL, compared to EGFRt+ cells without cetuximab, and non-transduced cells with or without cetuximab. This was seen at all effector-to-target ratios. Mice engrafted with CAR-modified HSC presented prolonged persistence of gene-modified cells in bone marrow, spleen, thymus and peripheral blood, and CAR+ cells elicited protection against CD19+ tumors, with inhibition or elimination of tumor development and survival advantage. Mice humanized with gene-modified HSC for the expression of the suicide gene had significant ablation of gene-modified cells after treatment with cetuximab (p=0.002). Remaining gene-modified cells were close to background on flow cytometry and within two logs of decrease of vector copy numbers by ddPCR in mouse tissues. PET imaging of humanized mice documented engraftment of EGFRt-expressing cells in bone marrow, spleen and liver, and confirmed ablation with an average decrease of 82.5% after cetuximab treatment.Conclusions: These results give proof of principle for persistence and anti-tumor activity of CAR-modified HSC regulated by suicide gene, and further studies are needed to enable clinical translation. Cetuximab ADCC of EGFRt-modified cells caused effective killing. Different ablation approaches, such as inducible caspase 9 or co-delivery of other inert cell markers should be evaluated. DisclosuresNo relevant conflicts of interest to declare.

Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency

BackgroundSevere combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency.MethodsWe treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up.ResultsOverall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade.ConclusionsTreatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.)

Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1αshort promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19- deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor

Self-inactivating lentiviral vectors (LVVs) are used regularly for genetic modification of cells, including T cells and hematopoietic stem cells for cellular gene therapy. As vector demand grows, scalable and controllable methods are needed for production. LVVs are typically produced in HEK293T cells in suspension bioreactors using serum-free media or adherent cultures with serum. The iCELLis® is a packed-bed bioreactor for adherent or entrained cells with surface areas from 0.53 to 500 m2. Media are pumped through the fixed bed and overflows, creating a thin film that is replenished with oxygen and depleted of CO2 as media return to the reservoir. We describe the optimization and scale-up of the production of GPRTG-EF1α-hγc-OPT LVV using a stable packaging cell line in the iCELLis Nano 2-cm to the 10-cm bed height low compaction bioreactors (0.53 and 2.6 m2 surface area) and compare to the productivity and efficacy of GPRTG-EF1α-hγc-OPT LVV manufactured under current Good Manufacturing Practice (cGMP) using 10-layer cell factories for the treatment of X-linked severe combined immunodeficiency. By optimizing fetal bovine serum (FBS) concentration, pH post-induction, and day of induction, we attain viral yields of more than 2 × 107 transducing units/mL. We compared transduction efficiency between LVVs produced from the iCELLis Nano and cell factories on healthy, purified CD34+ cells and found similar results.

Open-label pilot study of genetically engineered NY-ESO-1–specific t cells (GSK3377794) alone or in combination with pembrolizumab in relapsed/refractory multiple myeloma.

TPS8555 Background: Adoptive cellular therapy may be practice-changing in relapsed/refractory multiple myeloma (MM). NY-ESO-1 TCR T (GSK3377794) are autologous polyclonal T cells transduced by a self-inactivating lentiviral vector to express an affinity-enhanced TCR capable of recognizing NY-ESO-1 or LAGE-1a antigenic peptides in complex with HLA-A*02. GSK3377794 has shown clinical activity in synovial sarcoma, melanoma, myxoid/round cell liposarcoma, and MM after autologous stem cell transplant. NY-ESO-1 and LAGE-1a are cancer/testis antigens frequently overexpressed in MM and linked to poor clinical outcome. PD-1 expression on CD8 T cells, which has been observed in MM patients previously treated with GSK3377794 as well as with CD19 CAR T-cell therapy, can limit adaptive immune response. We hypothesize that GSK3377794 alone, or in combination with the anti-PD-1 inhibitor pembrolizumab, may result in an antitumor effect in MM. Methods: This is an open-label, pilot study (NCT03168438) of GSK3377794 in patients with relapsed/refractory MM positive for HLA-A*02:01, HLA-A*02:05, ± HLA-A*02:06 and NY-ESO-1/LAGE-1a. Patients (n = 20) who have received ≥3 prior therapies containing ≥1 immunomodulatory imide, proteasome inhibitor, alkylator, CD38 monoclonal antibody, or glucocorticoid will be assigned to either single-infusion GSK3377794 (Arm 1, n = 10) or single-infusion GSK3377794 + pembrolizumab 200 mg IV every 3 weeks (Arm 2, n = 10). Arm 1 enrollment will be completed first. In Arm 2, pembrolizumab will begin in Week 3 (Week 6 if precluded by toxicity). Patients in both arms will provide cells via leukapheresis to manufacture autologous NY-ESO-1–specific T cells, undergo lymphodepletion (fludarabine + cyclophosphamide), and then receive GSK3377794 infusion (1−8x109 transduced T cells). Primary and secondary objectives are to assess safety/tolerability and antitumor activity, respectively, of GSK3377794 (± pembrolizumab). Arm 2 enrollment will pause for a 3-week safety review after 3 patients have received a first dose of pembrolizumab. Efficacy, safety, and biomarkers will be assessed every visit. The treatment phase will last 108 weeks, or until disease progression; follow-up will last ≤15 years. As of January 2020, 3 patients have been treated. Funding: GlaxoSmithKline (208470) Clinical trial information: NCT03168438 .