Chimeric Adenoviral Vector Research Articles

Chimeric adenovirus-based herpes zoster vaccine with the tPA signal peptide elicits a robust T-cell immune response

Herpes zoster (HZ), or shingles, is caused by reactivation of the varicella-zoster virus (VZV), which remains latent in the sensory ganglia until immunity wanes with age. The representative HZ vaccine, Shingrix is efficacious but causes side effects due to vaccine adjuvants. Therefore, the development of highly efficacious vaccines with minimal side effects is required. We developed chimeric adenovirus vector (ChimAd)-based HZ vaccine candidates encoding the VZV glycoprotein E (gE). These candidates include ChimAd-tPAgE, in which the signal peptide is replaced with tissue plasminogen activator (tPA), and ChimAd-WTgE, which retains the original signal peptide. C57BL/6 mice were immunized with VZV-vaccine candidates, and cellular and humoral immune responses were evaluated using interferon-γ ELISPOT and ELISA. The ChimAd-based HZ vaccines induced high levels of gE-specific antibodies and cell-mediated immunity. ChimAd-tPAgE (optimal dose: 1 × 107 IFU) elicited a more robust gE-specific T-cell response than Shingrix and Zostavax, showing potential as HZ prophylactic vaccines.

Morphological characteristics of the cerebral cortex of a mini-pig under conditions of gene therapy after experimental stroke

Aim. To study the effectiveness of preventive gene therapy (within 2 days) and gene therapy in the acute phase (after 4 hours) of ischemic stroke in mini-pigs using an autologous leucoconcentrate (AutoLeuc) enriched with recombinant genes of vascular endothelial growth factor (VEGF165), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule 1 (NCAM1), as well as the migration of leukocytes transduced with a chimeric adenoviral vector serotype 5 with fiber 35 serotype (Ad5/F35) and the green fluorescent protein (GFP) genome into immune defense organs.Materials and methods. The experiment was conducted on 8-month-old Vietnamese lop-bellied mini-pigs (n=16). An ischemic stroke was created by occlusion of the distal branches of the left middle cerebral artery and the right common carotid artery. Genetically modified AutoLeuc was administered preventively intravenously 2 days before or in the acute phase 4 hours after stroke modelling; the control group was injected with 30 ml of saline solution. The morphology of the cerebral cortex was assessed using histological methods in the areas bordering the infarction and peri-infarction after 21 days. The migration of genetically modified Ad5/F35-GFP leukocytes into the brain, spleen, and submandibular lymph nodes was studied a week after stroke modelling.Results. In the peri-infarction zone, the content of pyknotic neurons in control animals was higher, while the number of capillaries was lower than in the gene therapy groups. In the latter, neurons had a typical morphology with preserved outgrowths; in the control group, the outgrowths were tortuous and fragmented. Fluorescence microscopy after injection of AutoLeuc with Ad5/F35-GFP revealed GFP-positive cells in the spleen and submandibular lymph nodes.Conclusion. 21 days after modeling a stroke in mini-pigs against the background of preventive gene therapy or gene therapy in the acute phase using VEGF165/GDNF/NCAM1 AutoLeuc, greater preservation of neurons and a higher density of capillaries in the peri-infarction zone of ischemic brain damage were established. Leukocytes with Ad5/ F35-GFP were found in the spleen and submandibular lymph nodes.

Toward the understanding of DSG2 and CD46 interaction with HAdV-11 fiber, a super-complex analysis.

The main limitation of oncolytic vectors is neutralization by blood components, which prevents intratumoral administration to patients. Enadenotucirev, a chimeric HAdV-11p/HAdV-3 adenovirus identified by bio-selection, is a low seroprevalence vector active against a broad range of human carcinoma cell lines. At this stage, there's still some uncertainty about tropism and primary receptor utilization by HAdV-11. However, this information is very important, as it has a direct influence on the effectiveness of HAdV-11-based vectors. The aim of this work is to determine which of the two receptors, DSG2 and CD46, is involved in the attachment of the virus to the host, and what role they play in the early stages of infection.

Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model.

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes, which affect the potency of the functional recovery after spinal cord injury (SCI). Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue. In our previous studies for delivering the therapeutic genes at the site of spinal cord injury, we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses (Ad5/35) carrying recombinant cDNA. In the present study, the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF), and neural cell adhesion molecule (NCAM) was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury. Experimental animals were randomly divided into two groups of 4 pigs each: the therapeutic (infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35-VEGF165, Ad5/35-GDNF, and Ad5/35-NCAM1) and control groups (infused with intact leucoconcentrate). The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed: (1) higher sparing of the grey matter and increased survivability of the spinal cord cells (lower number of Caspase-3-positive cells and decreased expression of Hsp27); (2) recovery of synaptophysin expression; (3) prevention of astrogliosis (lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells); (4) higher growth rates of regenerating βIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region. These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF, GDNF, and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury. Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

Chimeric adenoviral (Ad5.F35) and listeria vector prime-boost immunization is safe and effective for cancer immunotherapy

Strategies to augment immunity to self/neoantigens expressed by cancers are urgently needed to expand the proportion of patients benefiting from immunotherapy, particularly for GI cancers where only a fraction of patients respond to immunotherapies. However, current vaccine strategies are limited by poor immunogenicity, pre-existing vector-specific immunity, and vaccine-induced vector-specific immunity. Here, we examined a prime-boost strategy using a chimeric adenoviral vector (Ad5.F35) that resists pre-existing immunity followed by recombinant Listeria monocytogenes (Lm) to amplify immunity to the GI cancer antigen GUCY2C. This previously unexplored combination enhanced the quantity, avidity, polyfunctionality, and antitumor efficacy of GUCY2C-specific effector CD8+ T cells, without toxicity in any tissue, including GUCY2C-expressing intestines and brain. Importantly, this combination was partially resistant to pre-existing immunity to Ad5 which is endemic in human populations and vector-specific immunity did not limit the ability of multiple Lm administrations to repeatedly enhance GUCY2C-specific responses. Broadly, these findings suggest that cancer patient immunizations targeting self/neoantigens, as well as immunizations for difficult infectious diseases (HIV, malaria, etc), may be most successful using a combination of Ad5.F35-based priming, followed by Lm-based boosting. More specifically, Lm-GUCY2C may be utilized to amplify GUCY2C-specific immunity in patients receiving adenovirus-based GUCY2C vaccines currently in clinical trials to prevent or treat recurrent GI cancer.

Abstract 582: Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy

Abstract Introduction: Engineered cell therapies have demonstrated significant clinical activity against hematologic malignancies, but responses have been rare in solid tumors. Our previously developed human chimeric antigen receptor macrophage (CAR-M) platform has shown potent anti-tumor activity in pre-clinical solid tumor models1, and the anti-HER2 CAR-M CT-0508 is currently being evaluated in a Phase I trial. The use of myeloid cells as a platform for cell therapy provides the tools to overcome critical solid tumor challenges such as infiltration, immunosuppression within the tumor microenvironment, lymphocyte exclusion, and target antigen heterogeneity. Currently, CAR-M are generated in a week-long ex-vivo process in which peripheral blood monocytes are differentiated into macrophages prior to genetic manipulation. Here, we demonstrate the production feasibility, phenotype, pharmacokinetics, cellular fate, specificity, and anti-tumor activity of human CD14+ CAR monocytes. Experimental: Using the chimeric adenoviral vector Ad5f35, we engineered primary human CD14+ monocytes to express a CAR (CAR-mono) targeted against HER2. We established a process that allowed for same day manufacturing (from Leukopak to cryopreserved CAR-mono cell product). Results: CAR-mono showed high CAR expression and viability (>90%), and efficiently differentiated into CAR-expressing macrophages. Adenoviral transduction led to pre-conditioning of CAR-mono, resulting in a strong M1 phenotype upon differentiation into CAR-M. CAR-mono derived macrophages demonstrated potent anti-tumor activity regardless of exposure to GM-CSF or M-CSF, and were protected against M2 switching by immunosuppressive factors. Treating CAR-mono with GM-CSF and IL-4 resulted in their differentiation to monocyte-derived CAR-DCs with an activated phenotype, indicating that these cells retained their myeloid differentiation potential. In vivo, CAR-mono induced anti-tumor activity in various HER2+ solid tumor xenograft models. Following IV administration, CAR-mono demonstrated the ability to traffic to both GM-CSFhigh and GM-CSFlow expressing tumors. Notably, CAR-mono showed long-term CAR expression and persistence (>180 days) in both NSG and NSG-S mouse models, demonstrating lasting persistence irrespective of human cytokine support. Conclusions: The CAR-mono platform enables an automated, same-day manufacturing process while maintaining the key characteristics of CAR-M therapy. The use of Ad5f35 for human monocyte transduction primes the cells toward M1 macrophage differentiation and produces a cell population phenotypically and functionally similar to our established CAR-M platform. These data provide strong pre-clinical support to advance the CAR-mono platform into clinical testing.1Klichinsky M, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nature Biotechnology. March 2020. Citation Format: Daniel Blumenthal, Linara Gabitova, Brett Menchel, Patricia Reyes-Uribe, Sabrina Ceeraz DeLong, Sascha Abramson, Michael Klichinsky. Pre-clinical development of CAR Monocytes (CAR Mono) for solid tumor immunotherapy [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 582.

Abstract 2112: Chimeric antigen receptor macrophages (CAR-M) sensitize solid tumors to anti-PD1 immunotherapy

Abstract Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, achieving efficacy against solid tumors has been challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Given that CAR-M are M1-polarized macrophages with the potential to remodel the tumor microenvironment (TME) and act as professional antigen presenting cells, we developed an immunocompetent animal model to evaluate the interaction of CAR-M with the endogenous immune system and to study the combinatorial approach of CAR-M with blockade of the PD1/PDL1 T cell checkpoint axis. Murine bone marrow-derived macrophages were engineered to express an anti-HER2 CAR using the chimeric adenoviral vector Ad5f35. In addition to efficient gene delivery, Ad5f35 transduction promoted a pro-inflammatory (M1) phenotype in murine macrophages. Anti-HER2 CAR-M, but not control macrophages, phagocytosed and killed HER2-overexpressing tumor cell lines. CAR-M induced MHC-I expression on tumor cells and enhanced the cytotoxicity of CD8+ T cells. To evaluate the safety and efficacy of CAR-M therapy, immunocompetent mice were engrafted with HER2+ tumors and treated with syngeneic HER2-CAR or untransduced (UTD) macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cell responsiveness to tumor-associated antigens, indicating enhanced epitope spreading. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with anti-PD1 in tumors resistant to anti-PD1 monotherapy and found that the combination further reprogrammed the TME, significantly enhanced tumor control, and improved overall survival compared to monotherapy with either agent. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2-negative rechallenge model, indicating long-term anti-tumor immunity. Finally, the combination of CAR-M with anti-PD1 did not trigger sustained elevations of serum analytes associated with cytokine release syndrome (CRS) and was well tolerated across numerous safety assessments. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Alison Worth, Ilyssa Ramos, Daniel Blumenthal, Yumi Ohtani, Linara Gabitova, Michael Ball, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) sensitize solid tumors to anti-PD1 immunotherapy [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 2112.

Cancer Cell-specific Transfection of hCas9 Gene Using Ad5F35 Vector.

The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) is thought to have promising clinical potential. However, the off-target effects of Cas9 are a major concern for its application. Therefore, we hypothesized that the adverse effects of off-target gene editing might be minimized if the human codon-optimized Streptococcus pyogenes Cas9 (hCas9) could be specifically expressed in cancer cells. We constructed a chimeric adenoviral vector, Ad5F35-MKp-hCas9, and infected human bladder cancer cell lines with this vector. The confirmation of hCas9 gene expression was performed in 3-4 days after from infection. hCas9 gene expression was observed in Ad5F35-MKp-hCas9 infected bladder cancer cells but not in non-malignant cells. Our study showed that the Ad5F35-MKp-hCas9 vector is capable of expressing the hCas9 gene with high specificity in bladder cancer cells. These findings may help in minimizing the risk of off-target effects of gene editing.

Abstract 1530: Anti-HER2 CAR monocytes demonstrate targeted anti-tumor activity and enable a single day cell manufacturing process

Abstract Recent advances in cell therapy have led to significant efficacy in hematologic malignancies, but solid tumors remain an intractable challenge. We have previously developed a CAR macrophage (CAR-M) adoptive cell therapy platform and demonstrated potent anti-tumor activity in pre-clinical models. CAR-M overcome several of the barriers to efficacy in the solid tumor setting - trafficking, immunosuppression, lymphocyte exclusion, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. In order to streamline the manufacturing process, improve cell yields, and potentially improve tumor infiltration, we sought to evaluate the feasibility of directly engineering CD14+ monocytes to express CAR. Using the chimeric adenoviral vector Ad5f35, we engineered primary human CAR-monocytes to target tumors overexpressing HER2. CAR expression and viability both exceeded 90%. Anti-HER2 CAR-monocytes produced pro-inflammatory cytokines in response to antigen, specifically phagocytosed HER2 overexpressing target cells, and eradicated HER2-overexpressing tumor cells in a time and dose-dependent manner. CAR-monocytes efficiently differentiated into CAR-expressing macrophages in response to various maturation factors and cytokines within 3-5 days. Adenoviral based gene modification resulted in the upregulation of several pro-inflammatory markers on CAR-monocytes and pre-conditioned these cells to differentiate into M1 macrophages in the absence of exogenous M1 polarization factors. The M1 phenotype was maintained when CAR-monocytes were challenged with immunosuppressive cytokines in vitro. These cells demonstrated potent effector function after differentiation into macrophages, regardless of exposure to GM-CSF, M-CSF, or immunosuppressive factors. Anti-HER2 CAR monocytes carried broadly expressed myeloid chemokine receptors and responded to a panel of chemotactic factors. In order to optimize the cell manufacturing process associated with CAR-M, we established a closed-system, ultra-rapid CAR monocyte process. This process enabled same day manufacturing of the final CAR monocyte cell product, significantly reducing the cost of goods associated with autologous cell therapy as well as “vein to vein” time. This study demonstrated the successful development of CAR-monocytes with direct anti-tumor activity and capacity to differentiate into M1 polarized CAR-M. In addition, we established an ultra-rapid same-day CAR monocyte manufacturing process. The CAR-monocyte platform represents an advance in the field of adoptive cell therapy. Citation Format: Linara Gabitova, Brett Menchel, Rashid Gabbasov, Stefano Pierini, Andrew Best, Kayleigh Ross, Yumi Ohtani, Daniel Blumenthal, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Anti-HER2 CAR monocytes demonstrate targeted anti-tumor activity and enable a single day cell manufacturing process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1530.

Abstract 63: Chimeric antigen receptor macrophages (CAR-M) induce anti-tumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid tumor models

Abstract Despite the remarkable efficacy achieved by CAR-T cell therapy in hematologic malignancies, translating these results in solid tumors remains challenging. We previously developed human CAR-M and demonstrated that adoptive cell transfer of CAR-M into xenograft models of human cancer controls tumor progression and improves overall survival1. Herein, we established a fully immunocompetent syngeneic mouse model and evaluated the interaction of CAR-M with the tumor microenvironment (TME) and the endogenous adaptive immune system. Murine bone marrow-derived CAR-expressing macrophages (muCAR-M) were efficiently engineered to express an anti-huHER2 CAR using the chimeric adenoviral vector Ad5f35. MuCAR-M, but not control untransduced (UTD) macrophages, specifically phagocytosed HER2+ target cancer cell lines and killed HER2-expressing murine CT26 colorectal and human AU-565 (HER2+) breast cancer cells in a dose-dependent manner. Moreover, CAR-M induced MHC-I and MHC-II expression on tumor cells and cross-presented tumor-associated antigens (TAA) resulting in CD8 T cell activation. To evaluate muCAR-M in an immunocompetent in vivo setting, BALB/c mice were engrafted with subcutaneous CT26-HER2+ tumors and treated with HER2-CAR or UTD macrophages. CAR-M treated mice showed significant tumor control and improved survival compared to control groups. Analysis of the TME showed increased intratumoral immune infiltration - as well as an increase in T cells reactive to the CT26 MHC-I antigen gp70, indicating enhanced epitope spreading. Mice that achieved complete responses (CRs) after CAR-M therapy were protected against antigen-negative relapse in a HER2- CT26 rechallenge model, indicating the induction of long-term T cell memory against TAA. To evaluate the systemic anti-tumor immune response, we simultaneously engrafted BALB/c mice with CT26-HER2+ and CT26-Wt tumors on opposite flanks and treated mice with local administration of CAR-M restricted to the HER2+ tumors. After CAR-M treatment, 75% of mice cleared their CT26-HER2+ tumors and the growth rate of the contralateral CT26-Wt tumors was significantly reduced, demonstrating an abscopal effect. Given the impact of CAR-M on the endogenous adaptive immune system, we evaluated the combination of CAR-M with PD1 checkpoint inhibitor therapy in the CT26-HER2 model, which is resistant to anti-PD1 monotherapy, and found that the combination further improved tumor control and overall survival. These results demonstrate that CAR-M reprogram the TME, induce epitope spreading, and orchestrate a systemic immune response against solid tumors. Moreover, our findings provide rationale for the combination of CAR-M with immune checkpoint inhibitors. The anti-HER2 CAR-M, CT-0508, is under evaluation in a phase I clinical trial for patients with HER2 overexpressing solid tumors. Citation Format: Stefano Pierini, Rashid Gabbasov, Linara Gabitova, Yumi Ohtani, Olga Shestova, Saar Gill, Sascha Abramson, Thomas Condamine, Michael Klichinsky. Chimeric antigen receptor macrophages (CAR-M) induce anti-tumor immunity and synergize with T cell checkpoint inhibitors in pre-clinical solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 63.

A Simple, Safe and Effective Approach for Personalized Precision Ex Vivo Gene Therapy Based on Autoinfusion of Gene-Modified Leucoconcentrate (GML) Prepared from Routine Unit of Patient's Peripheral Blood

Nowadays gene and cell therapy become the basic methods in regenerative medicine. However only few gene and cell products are currently approved for clinical usage. Biosafety problems, complexity of cell and gene technologies and high cost of manufacturing are the main reasons for the slow introduction of such approaches in practical medicine. Treatment of hereditary diseases of the immune system based on the correction of the mutant gene by delivering functional recombinant gene into WBC is the first successfully employed in the clinical practice approach of cell-mediated or ex vivo gene therapy. Earlier we have reported the strategy of the cell-mediated gene therapy based on umbilical cord blood mononuclear cells transduced with adenoviral vectors carrying recombinant genes encoding neurotrophic factors for treatment neurodegenerative diseases, neurotrauma and stroke. Significant disadvantage of this method is the usage of the umbilical cord blood mononuclear cells as a cell carrier for the therapeutic genes. Considering immunodeficiency treatment and our own data we developed a new approach of recombinant gene delivery for personalized ex vivo gene therapy. The method is based on autoinfusion of patient's WBC transduced with recombinant therapeutic genes for correction of certain pathological conditions. In the present study for the first time the human gene-modified leucoconcentrate (GML) producing recombinant reporter gene encoding green fluorescent protein (GFP) was obtained without culturing WBC in vitro. The routine unit of peripheral blood (450 ml) was collected into the plastic blood bag and the leucocyte- and platelet-rich concentrates (50 ml) were obtained by standard method using Macopress Smart (Macopharma, France). Afterwards the equal volume of hydroxyethyl starch 6% was added into the plastic blood bag which was centrifuged (DP-2065 R PLUS, Centrifugal Presvac RV; Presvac, Buenos Aires, Argentina) at 350 rpm for 10 min at 10°C. The obtained supernatant was transferred into the new plastic blood bag using manual plasma extractor FK-01 (Leadcore, Russia) and 200 ml of saline was added into the bag which was centrifuged at 1300 rpm for 10 min at 10°C and the supernatant was expressed out of the bag so that the remaining solution in the bag (30 ml) contained leucoconcentrate (WBC - 45.56 ± 23.93 × 106/ml and RBC - 1.76 ± 3.33 × 109/ml). Transduction of WBC with chimeric adenoviral vector (Ad5/35) carrying GFP gene was performed in the plastic bag with MOI 5 according to the count of WBC in the leucoconcentrate. After transduction for 12 hours, 200 ml of saline was added to the bag with leucoconcentrate, the mixture was centrifuged at 1000 rpm for 10 min at 10°C and the supernatant was squeezed out of the bag. The remained in the bag solution (30 ml) was considered as gene-modified leucoconcentrate carrying GFP gen (WBC - 22.63 ± 8.90 × 106/ml and RBC - 1.77 ± 1.21 × 109/ml). For in vitro study of GFP gene expression the samples of GML-GFP were cultivated for 60 hours after GML-GFP preparation. Fluorescent microscopy in the cytoplasm of the transduced WBC showed specific intensive green fluorescence. Flow cytometry analysis demonstrated that 2.5% of WBC from the GML-GFP efficiently expressed GFP. Thus leucoconcentrate after 72 h of transduction with Ad5/35-GFP with MOI 5 resulted in 2.5% of the GFP-positive cells. Thus the results of this study represent a simple, safe and effective approach for preparation of GML for personalized ex vivo gene therapy aimed at temporary production of the specific recombinant biologically active molecules for pathogenetic therapy of the varied nosological form, such as trauma, ischemic, degenerative, autoimmune, infection and other diseases. This study was supported by the grant of Russian Science Foundation 19-75-10030. Disclosures No relevant conflicts of interest to declare.

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury.

We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases, neurotrauma, and stroke. In this study, using a mini pig model of spinal cord injury, we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy. Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector (Ad5/35F) that carried an enhanced green fluorescent protein (EGFP) reporter gene in the plastic blood bag. The day after blood donation, the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate. A week after gene-modified leucoconcentrate therapy, fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury. In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed. In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes. Thus, the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions. This paper presents a proof-of-concept of simple, safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 5) on May 27, 2014.

Abstract 3242: CT-0508 is an anti-HER2 chimeric antigen receptor (CAR) macrophage with targeted anti-tumor activity that promotes a pro-inflammatory solid tumor microenvironment

Abstract Despite recent advances in T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are often the most abundant immune cell in the tumor microenvironment (TME) where, as immunosuppressive tumor associated macrophages (TAMs), they participate in disease progression. Currently, most macrophage based immunotherapeutic approaches are focused on the depletion, repolarization, or phagocytic disinhibition of TAMs. We have developed a new paradigm based on the adoptive transfer of genetically engineered CAR macrophages (CAR-M) for the treatment of human cancer. CAR-M can be efficiently produced using the chimeric adenoviral vector Ad5f35. We have previously shown that the primary mechanism of action of CAR-M is antigen dependent phagocytosis, and that a single dose of primary human anti-HER2 CAR-M leads to significantly improved overall survival in multiple solid tumor xenograft models. Given that Ad5f35-transduced anti-HER2 CAR-M (CT-0508) adopt a unique pro-inflammatory M1-like phenotype, we hypothesized that CT-0508 may have the capacity to reprogram the TME toward an activated state. Functional evaluation and transcriptome-wide characterization revealed that CT-0508 maintain a pro-inflammatory phenotype despite challenge with immunosuppressive environments in vitro. By engrafting immunodeficient mice with human hematopoietic cells and human cancer cells we established a novel xenografted human TME model. We demonstrate with single cell resolution that CT-0508 maintain their M1 phenotype within the human TME. Additionally, CT-0508 augmented the human TME by inducing a pro-inflammatory signature in surrounding immune cells, characterized by induction of MHC-II and TNFα. To further investigate the potential of CT-0508 for TME activation, we modeled the interaction of CT-0508 with primary human M2 macrophages, dendritic cells, and T cells in vitro. CT-0508 repolarized bystander M2 macrophages toward a pro-inflammatory phenotype, induced activation and maturation markers on immature dendritic cells, and recruited resting as well as activated T cells in chemotaxis assays. CT-0508 demonstrated enhanced antigen presentation when compared to control human macrophages and cross-presented tumor derived intracellular antigens to CD8 T cells after tumor phagocytosis. Our results show that in addition to direct anti-tumor activity, the anti-HER2 CAR macrophage cell product CT-0508 is capable of promoting a pro-inflammatory tumor microenvironment and has the potential to induce epitope spreading via T cell recruitment and antigen presentation. Citation Format: Michael Klichinsky, Konrad Gabrusiewicz, Nicholas Anderson, Maggie Schmierer, Andrew Best, Martha Zeeman, Sotheavy Chhum, Yumi Ohtani, Olga Shestova, Xueqing Lu, Nicholas Petty, Xinhe Shan, Feng Shen, Saar Gill. CT-0508 is an anti-HER2 chimeric antigen receptor (CAR) macrophage with targeted anti-tumor activity that promotes a pro-inflammatory solid tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3242.

Abstract 2180: Genetically engineered chimeric antigen receptor (CAR) monocytes demonstrate targeted anti-tumor activity and differentiate into M1-polarized CAR macrophages

Abstract Despite rapid advances in cancer immunotherapy, clinical responses in metastatic solid tumors have been limited. Macrophages are the most abundant immune cell in the solid tumor microenvironment (TME) and are primarily recruited as monocytes by TME-derived chemokines. When not under the control of the TME, macrophages are potent immune effector cells capable of phagocytosis, T cell recruitment, and antigen presentation. We have previously demonstrated that CAR macrophages (CAR-M) have potent anti-tumor activity and overcome several of the barriers to success in solid tumor immunotherapy - trafficking, immunosuppression, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. To more closely recapitulate normal biologic behavior, we attempted to create CAR monocytes that could traffic and differentiate into CAR macrophages upon tumor penetration. Toward that goal, we genetically engineered CD14+ human monocytes without ex vivo differentiation and with minimal cell culture. Using the chimeric adenoviral vector Ad5f35, we engineered human CAR-monocytes targeted against HER2. CAR expression and viability both exceeded 90%. Ad5f35 transduced CAR monocytes survived and maintained CAR expression ex vivo for at least 21 days. CAR monocytes efficiently differentiated into CAR-expressing macrophages when treated with GM-CSF as determined by FACS-based phenotypic characterization and Wright-Giemsa staining. Anti-HER2 CAR monocytes eradicated HER2 expressing tumor cells in a time and dose-dependent manner, and had comparable potency to anti-HER2 CAR-M. Additionally, the CAR monocyte manufacturing process offered the logistical advantage of a short manufacturing process (approximately two days). We have previously demonstrated that CAR-M are polarized toward a pro-inflammatory M1 phenotype after transduction with Ad5f35. Similarly, CAR monocytes demonstrated elevated expression of M1 markers, and intriguingly after differentiation into CAR-expressing macrophages, HLA-DR, CD80, CD86, and other M1 markers remained elevated - suggesting that transduction prior to differentiation does not impact the pro-inflammatory impact of adenoviral vectors on myeloid cells. Taken together, this abstract describes the successful development of CAR-monocytes with the potential for a rapid manufacturing process. In addition to direct anti-tumor activity while in the monocyte phase, CAR monocytes have the capacity to differentiate into CAR macrophages in situ, which are in turn capable of phagocytosis, T cell recruitment, TME activation, and antigen presentation. Given the previously demonstrated pre-clinical efficacy of CT-0508 (an anti-HER2 CAR macrophage), the CAR monocyte platform described herein offers a shortened manufacturing process and a potential advantage in tumor penetration, which will be directly evaluated in upcoming studies. Citation Format: Konrad Gabrusiewicz, Maggie Schmierer, Andrew Best, Martha Zeeman, Yumi Ohtani, Linara Gabitova, Daniel Cushing, Saar Gill, Michael Klichinsky. Genetically engineered chimeric antigen receptor (CAR) monocytes demonstrate targeted anti-tumor activity and differentiate into M1-polarized CAR macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2180.

Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity.

BackgroundAdenovirus serotype 5 (Ad5) is a commonly used viral vector for transient delivery of transgenes, primarily for vaccination against pathogen and tumor antigens. However, endemic infections with Ad5 produce virus-specific neutralizing antibodies (NAbs) that limit transgene delivery and constrain target-directed immunity following exposure to Ad5-based vaccines. Indeed, clinical trials have revealed the limitations that virus-specific NAbs impose on the efficacy of Ad5-based vaccines. In that context, the emerging focus on immunological approaches targeting cancer self-antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectors.MethodsHere, we evaluated the ability of a chimeric adenoviral vector (Ad5.F35) derived from the capsid of Ad5 and fiber of the rare adenovirus serotype 35 (Ad35) to induce immune responses to the tumor-associated antigen guanylyl cyclase C (GUCY2C).ResultsIn the absence of pre-existing immunity to Ad5, GUCY2C-specific T-cell responses and antitumor efficacy induced by Ad5.F35 were comparable to Ad5 in a mouse model of metastatic colorectal cancer. Furthermore, like Ad5, Ad5.F35 vector expressing GUCY2C was safe and produced no toxicity in tissues with, or without, GUCY2C expression. Importantly, this chimeric vector resisted neutralization in Ad5-immunized mice and by sera collected from patients with colorectal cancer naturally exposed to Ad5.ConclusionsThese data suggest that Ad5.F35-based vaccines targeting GUCY2C, or other tumor or pathogen antigens, may produce clinically relevant immune responses in more (≥90%) patients compared with Ad5-based vaccines (~50%).

Abstract PR07: Human chimeric antigen receptor (CAR) macrophages for cancer immunotherapy

Abstract Despite recent landmark advances in T-cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are often the most abundant immune cell in the tumor microenvironment (TME), where they may convert into immunosuppressive (M2) tumor-associated macrophages (TAMs) and participate in disease progression. Currently, macrophage-orientated immunotherapeutic approaches under clinical development in oncology seek to reduce TAM infiltration (CSF-1 antagonists) or enhance TAM phagocytosis (CD47 antagonists). Transfer of autologous, activated, but nontargeted macrophages failed to demonstrate antitumor efficacy in past clinical trials. We hypothesized that genetically engineering human macrophages with CARs against tumor-associated antigens could redirect their phagocytic activity and lead to therapeutic efficacy with the potential for the induction of an antitumor T-cell response. We first demonstrate that CD3-zeta-based CARs are capable of inducing phagocytosis in human THP-1 macrophages, while truncated intracellular-domain deficient CARs were not. Targeted phagocytosis and clearance of CD19+, mesothelin +, and HER2+ cells by CARs targeted against each respective antigen was significantly superior to that by control untransduced (UTD) macrophages. We demonstrate that primary human macrophages, which are resistant to most viral vectors, are efficiently transduced by the chimeric fiber adenoviral vector Ad5f35 (~70% in 10 normal donors). Using Ad5f35, we engineered primary human macrophages with a CD3-zeta-based CAR against HER2. Anti-HER2 primary human CAR macrophages demonstrated targeted phagocytosis against HER2+ but not HER2- cell lines, with phagocytic activity dependent on both the CAR and antigen densities. Furthermore, CAR, but not UTD, macrophages led to potent dose-dependent killing of three distinct HER2-high cell lines in vitro. We sought to test the efficacy of anti-HER2 primary human macrophages in xenograft models of human HER2+ ovarian cancer. A single dose of CAR, but not UTD macrophages, led to tumor regression and improved overall survival in both intraperitoneal and disseminated models of disease. We show that macrophage transduction with Ad5f35, a double-stranded DNA virus, leads to a broad gene expression change, an interferon signaling signature, and phenotypic clustering toward classically activated M1 macrophages. CAR macrophages upregulated co-stimulatory ligand and antigen processing/presentation genes and led to enhanced T-cell stimulation in vitro and in vivo. Lastly, CAR, but not UTD, macrophages showed a broad resistance for M2 conversion in response to immunosuppressive cytokines. In conclusion, we show that primary human CAR macrophages are capable of targeted tumor phagocytosis, lead to improved overall survival in xenograft models, and demonstrate enhanced T-cell stimulation. CAR macrophages are a novel cell therapy platform for the treatment of human cancer. This abstract is also being presented as Poster B29. Citation Format: Michael Klichinsky, Marco Ruella, Olga Shestova, Andrew Best, Kristin Blouch, Xueqing M. Lu, Saad S. Kenderian, Miriam Y. Kim, Roddy O'Connor, Stephen Wallace, Miroslaw Kozlowski, Dylan M. Marchione, Maksim Shestov, Benjamin A. Garcia, Carl June, Saar Gill. Human chimeric antigen receptor (CAR) macrophages for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR07.

Human chimeric antigen receptor macrophages for cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1-4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity.

A novel fiber chimeric conditionally replicative adenovirus-Ad5/F35 for tumor therapy

ABSTRACTSignificant progress has been made in the diagnosis and treatment of cancer; however, significant challenges remain. Conditionally replicating adenoviruses (CRAds), which not only kill cancer cells, but also serve as vectors to express therapeutic genes, are a novel and effective method to treat cancer. However, most adenoviruses are Ad5, which infect cells through the coxsackie and adenovirus receptor (CAR). The transduction efficacy of Ad5 is restricted because of the absent or low expression of CAR on several cancer cells. Ad serotype 35 has a different tropism pattern to Ad5. Ad35 attaches to cells via a non-CAR receptor, CD46, which is expressed widely on most tumor cells. Thus, chimeric adenoviral vectors consisting of the knob and shaft of Ad35 combined with Ad5 have been constructed. The chimeric fiber adenoviral vectors can transduce CAR-positive and CAR-negative cell lines. In this review, we explore the application of the novel fiber chimeric conditionally replicative adenovirus-Ad5/F35 in tumor therapy in terms of safety, mechanism, transduction efficacy, and antitumor effect.

Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator.

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

Angiogenesis-related polymorphisms in thalidomide survivors

Apurinic/apyrimidinic endonuclease (APE1), a bifunctional AP endonuclease/redox factor, is important in DNA repair and redox signaling, may be associated with chemoresistance. In this study, we first investigated APE1 expression and its correlation with cisplatin resistance and prognosis in non-small cell lung cancer (NSCLC) patients. Then, we investigated the effect of chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA (Ad5/F35-APE1 siRNA) on the sensitivity of cisplatin in A549 human lung adenocarcinoma cells.Tumor specimens from 103 patients with operable NSCLC were obtained from 1999 to 2001. Among these patients, 72 patients have been treated with at least three cycles of cisplatin-based chemotherapy. APE1 protein expression was examined by immunohistochemistry and Western blot on the tumor samples and a cultured A549 cell line, respectively. Cell survival and apoptosis were determined by MTT and TUNEL, respectively.83.3% (20/24) cisplatin-resistant tumors showed high APE1 expression levels, while 8.3% (4/48) cisplatin-sensitive tumors showed high APE1 expression levels (p < 0.01). Univariate analysis indicated that overall survival and disease-free survival were significantly better in NSCLC patients with low vs those with high APE1 expression levels (p < 0.01). Treatment with cisplatin resulted in a dose-dependent increase in APE1 protein expression in A549 cells, and Ad5/F35-APE1 siRNA effectively inhibited APE1 expression. Ad5/F35-APE1 siRNA significantly enhanced sensitivity of A549 cells to cisplatin, associated with increased cell apoptosis.Our results indicate that APE1 is a new promising target for the combination of cisplatin-based chemotherapy in NCSLC patients.