scholarly journals Novel Fluorinated Spermine and Small Molecule PEI to Deliver Anti-PD-L1 and Anti-VEGF siRNA for Highly Efficient Tumor Therapy

Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2058
Author(s):  
Yihui Zhang ◽  
Zihan Yuan ◽  
Yi Jin ◽  
Wenkai Zhang ◽  
Wei-En Yuan
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Small Molecule ◽  
Tumor Therapy ◽  
Sirna Delivery ◽  
Death Receptor ◽  
Target Cells ◽  
Anti Vegf ◽  
Tumor Killing ◽  
Death Receptor Ligand

Small interfering RNA (siRNA) can specifically silence disease gene expression. This project investigated the overexpression of programmed death receptor ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF) on the surface of tumor cells. However, the main obstacle to the development of gene therapy drugs is the lack of an efficient delivery vector, which should be able to overcome multiple delivery barriers and protect siRNA to enter the target cells. Therefore, a novel fluorine-modified endogenous molecular carrier TFSPEI was constructed by linking fluorinated groups with hydrophobic and hydrophilic characteristics on the surface of PEI and spermine. The results showed that lower toxicity, higher endocytosis, and silencing efficiency were achieved. We found that the inhibition of VEGF targets can indirectly activate the immune response to promote the tumor-killing and invasion effects of T cells. The combined delivery of anti-VEGF siRNA and anti-PD-L1 siRNA could inhibit the expression of corresponding proteins, restore the anti-tumor function of T cells and inhibit the growth of neovascularization, and obtained significant anti-tumor effects. Therefore, this safe and efficient fluorinated spermine and small molecule PEI-based anti-PD-L1 and anti-VEGF siRNA delivery system is expected to provide a new strategy for gene therapy of tumors.

AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 455-455 ◽  
Author(s):  
Federico Mingozzi ◽  
Marcela V. Maus ◽  
Denise E. Sabatino ◽  
Daniel J. Hui ◽  
John E.J. Rasko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Cd8 T Cell ◽  
Target Cells

Abstract Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes. It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

scholarly journals Prevention of Autoimmunity and Control of Recall Response to Exogenous Antigen by Fas Death Receptor Ligand Expression on T Cells

Immunity ◽  
2008 ◽  
Vol 29 (6) ◽  
pp. 922-933 ◽  
Author(s):  
Imed Mabrouk ◽  
Stéphanie Buart ◽  
Meriem Hasmim ◽  
Christelle Michiels ◽  
Elizabeth Connault ◽  
...  
Keyword(s):  
T Cells ◽  
Death Receptor ◽  
Receptor Ligand ◽  
Recall Response ◽  
Death Receptor Ligand ◽  
Ligand Expression ◽  
Exogenous Antigen ◽  
And Control

IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2167-2174 ◽  
Author(s):  
Els Verhoeyen ◽  
Valerie Dardalhon ◽  
Odile Ducrey-Rundquist ◽  
Didier Trono ◽  
Naomi Taylor ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Lentiviral Vectors ◽  
Target Cells ◽  
Interleukin 7 ◽  
Efficient Gene ◽  
Cognate Antigen ◽  
Therapy Target

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell–based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3–displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)–displaying HIV-1–derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7–displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7–displaying vectors could constitute interesting tools for T-cell–targeted gene therapy.

scholarly journals Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

2015 ◽  
Vol 89 (13) ◽  
pp. 6761-6772 ◽  
Author(s):  
Renier Myburgh ◽  
Sandra Ivic ◽  
Michael S. Pepper ◽  
Gustavo Gers-Huber ◽  
Duo Li ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Immune System ◽  
Humanized Mice ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Hiv 1 ◽  
Selection Of

ABSTRACTGene-engineered CD34+hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIVin vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric micein vivoare anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+cells that produce a constant supply of HIV-resistant progeny.IMPORTANCEMajor issues in experimental long-termin vivoHIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a selective enrichment of genetically targeted cells because positive selection of modified cells is likely to be insufficient below this threshold. This selection approach may be beneficial not only for HIV patients but also for other patients requiring transplantation of genetically modified cells.

scholarly journals Optimizing CD4+ T-Cells Transduction Protocol for Gene Therapy of HIV-1 Infected Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4429-4429
Author(s):  
Amani Ouedrani ◽  
Lounes Djerroudi ◽  
Isabelle Hmitou ◽  
Marina Cavazzana ◽  
Fabien Touzot
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
Cd4 T Cells ◽  
Conflicts Of Interest ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Effector Memory ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Hiv 1

Abstract Gene therapy represents an alternative and promising strategy that could provide a path to a curative therapy for HIV-1 infection. One approach involves the introduction of protective gene into a cell, thereby conferring protection against HIV. We plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma. The patients will received autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells. CD34+ and CD4+ will be ex vivo transduced by the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor. We developed a transduction process for CD4+ T-cells using the TransAct™ reagent (Miltenyi Biotec, Bergisch Gladbach , Germany) for CD4+ T-cells activation. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an equivalent efficacy of transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, CD4+ T-cells ex vivo transduced after activation with the TransAct™ reagent display very few changes in their surface marker with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 15-25% of cells suggesting a more "gentle " activation of the transduced CD4+ T-cells. Our transduction process had no significant impact in TCRβ repertoire diversity as evaluated by high-throughput sequencing and analyzis of diversity through the Gini-Simpson index or the Shannon index. Finally, transduced CD4 + T-cells retained the ability to to be primed towards the TH1, TH2 and TH17 pathways suggesting that the transduction protocol used did not alter the functional properties of the target cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals 258 AB308 is an anti-TIGIT antibody that enhances immune activation and anti-tumor immunity alone and in combination with other I-O therapeutic agents

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A280-A280
Author(s):  
Dana Piovesan ◽  
Alejandra Lopez ◽  
Patrick Schweickert ◽  
Ferdie Soriano ◽  
Soonweng Cho ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Immunity ◽  
Small Molecule ◽  
Immune Activation ◽  
Human Tumor ◽  
Immune Checkpoints ◽  
Tumor Growth Inhibition ◽  
Target Cells ◽  
Mouse Tumor

BackgroundTIGIT (T-cell immunoreceptor with Ig and ITIM domains) is an inhibitory receptor expressed on natural killer (NK) cells, CD8< sup >+</sup > T cells, CD4< sup >+</sup > T cells and regulatory T cells (T < sub >regs</sub >). On the surface of these cells, TIGIT competes with another receptor, CD226, for shared receptor ligands (mainly CD155) that are expressed by cancer and antigen-presenting cells. Binding of CD155 to TIGIT results in immune suppression through multiple mechanisms. When TIGIT is blocked, binding of CD155 to CD226 promotes immune activation and anti-tumor immunity. We describe the preclinical characterization of AB308, a humanized wild-type IgG1 anti-TIGIT antibody that is currently undergoing clinical evaluation.MethodsBinding of AB308 to TIGIT and inhibition of the TIGIT/CD155 interaction were evaluated < i >in vitro</i >. Functional assays were used to evaluate the immunomodulatory activity of AB308 alone or in combination with zimberelimab (anti-PD-1) or etrumadenant (a small molecule A< sub >2a</sub >A< sub >2b</sub > adenosine receptor antagonist). Surrogate Fc-silent and Fc-enabled antibodies that recognize mouse TIGIT or PD-1 were leveraged to interrogate TIGIT biology in syngeneic mouse tumor models.ResultsHuman tumor-infiltrating lymphocytes from a variety of cancer types expressed appreciable levels of TIGIT on relevant immune populations, including tumor reactive CD39< sup >+</sup >CD103< sup >+</sup > CD8< sup >+</sup > T cells and T< sub >regs</sub >. AB308 has a high binding affinity for human TIGIT, potently blocks the TIGIT-CD155 interaction, and induces Fcγ receptor (FcγR)-mediated signaling. In line with FcγRIII binding, AB308 also demonstrated the ability to induce NK cell-driven antibody-dependent cell-mediated cytotoxicity against TIGIT-expressing target cells. AB308 significantly increased IL-2 secretion by peripheral blood mononuclear cells activated with superantigen A, an activity that was further enhanced with zimberelimab. Blocking TIGIT with AB308 potently activated CD226 signaling in Jurkat T cells co-cultured with CD155-expressing cells, and combination of AB308 with etrumadenant in this system abrogated adenosine-mediated T cell suppression that occurred even in the presence of checkpoint inhibition. In mice, while combining Fc-silent or Fc-enabled anti-mouse TIGIT antibody with anti-PD-1 resulted in greater tumor growth inhibition than with anti-PD-1 alone, the activity of Fc-enabled anti-TIGIT was associated with intratumoral T< sub >regs</sub >depletion.ConclusionsAB308 is a potent and highly effective anti-TIGIT antibody. Concurrent blockade of multiple immune checkpoints has the potential to confer effective and durable responses in the treatment of cancer. The data presented here support the clinical use of AB308 and provides a rationale for combination with zimberelimab and adenosine pathway blocking agents such as etrumadenant and CD73 small molecule inhibitor, AB680.Ethics ApprovalAnimal experiments were performed at Arcus Biosciences, Inc. in accordance with federal, state and Institutional guidelines and were approved by Arcus’ Institutional Animal Care and Use Committee.

scholarly journals Design and Characterization of an “All-in-One” Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines

Cancers ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 375 ◽  
Author(s):  
Katharina Zimmermann ◽  
Johannes Kuehle ◽  
Anna Christina Dragon ◽  
Melanie Galla ◽  
Christina Kloth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell Activation ◽  
Transgene Expression ◽  
Cell Activation ◽  
Lentiviral Vector ◽  
Tumor Therapy ◽  
Vector System ◽  
Target Cells ◽  
Efficient Production ◽  
Car T

Genetically modified T cells expressing chimeric antigen receptors (CARs) so far have mostly failed in the treatment of solid tumors owing to a number of limitations, including an immunosuppressive tumor microenvironment and insufficient CAR T cell activation and persistence. Next-generation approaches using CAR T cells that secrete transgenic immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“T cells redirected for universal cytokine-mediated killing”), are currently being explored. As TRUCKs were engineered by the transduction of T cells with two separate vectors, we developed a lentiviral modular “all-in-one” vector system that combines constitutive CAR expression and inducible nuclear factor of activated T cells (NFAT)-driven transgene expression for more efficient production of TRUCKs. Activation of the GD2-specific CAR via GD2+ target cells induced NFAT promoter-driven cytokine release in primary human T cells, and indicated a tight linkage of CAR-specific activation and transgene expression that was further improved by a modified NFATsyn promoter. As proof-of-concept, we showed that T cells containing the “all-in-one” vector system secrete the immunomodulatory cytokines interleukin (IL)12 or IL18 upon co-cultivation with primary human GD2+ tumor cells, resulting in enhanced effector cell properties and increased monocyte recruitment. This highlights the potential of our system to simplify application of TRUCK-modified T cells in solid tumor therapy.

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