An Innovative PTD-IVT-mRNA Delivery Platform for CAR Immunotherapy of ErbB(+) Solid Tumor Neoplastic Cells (original) (raw)
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Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells
Cancer Gene Therapy, 2001
Targeted gene transfer by nonviral vectors can be achieved through incorporation of specific ligand(s) into the vectors. In this study, the effects of incorporation of an anti-ErbB2 single-chain antibody fragment (ScFv) into nonviral vectors for targeted gene delivery were investigated. The ML39 ScFv, selected from a human ScFv phage display library and affinity matured in vitro (K d =1Â10 À 9 M), was used as ligand specific for the extracellular domain of the tumor surface protein, ErbB2. Two approaches were taken: (a) development of a vector that is composed of a bifunctional fusion protein capable of binding DNA with the ErbB2-specific ML39 ScFv at its N-terminus and a truncated form of human protamine at its C-terminus, and (b) formulation and evaluation of delivery vectors consisting of three independent components including ML39 ScFv, protamine, and cationic lipids. We demonstrate that fusion proteins comprised of the ML39 ScFv and a truncated form of protamine, denoted as ScFv-P-S, can selectively deliver exogenous DNA into ErbB2(+) cells, with an 8-to 10-fold increase in expression levels of the luciferase reporter gene in ErbB2(+) cells as compared to ErbB2(À) cells. In addition, vectors formulated by appropriately mixing DNA, ScFv, protamine, and lipids in vitro could even more efficiently deliver the reporter gene into ErbB2(+) cells with approximately 5-fold increase in gene expression in ErbB2(+) cell as compared to ErbB2(À) cells. Expression and refolding of the ScFv fusion proteins, in addition to determination of optimal conditions for vector development using these approaches, are discussed.
Chimeric Receptor mRNA Transfection as a Tool to Generate Antineoplastic Lymphocytes
Human Gene Therapy, 2008
mRNA transfection is a useful approach for temporal cell reprogramming with minimal risk of transgenemediated mutagenesis. We applied this to redirect lymphocyte cytotoxicity toward malignant cells. Using the chimeric immune receptor (CIR) constructs anti-CD19 CIR and 8H9 CIR, we achieved uniform expression of CIRs on virtually the entire population of lymphocytes. We reprogrammed CD3 + CD8 + , CD3 + CD4 + , and natural killer (NK) cells toward autologous and allogeneic targets such as B cells, Daudi lymphoma, primary melanoma, breast ductal carcinoma, breast adenocarcinoma, and rhabdomyosarcoma. The reprogramming procedure is fast. Although most of the experiments were performed on lymphocytes obtained after 7-day activation, only 1-day activation of T cells with anti-CD3, anti-CD28 antibodies, and interleukin-2 is sufficient to develop both lymphocyte cytotoxicity and competence for mRNA transfer. The entire procedure, which includes lymphocyte activation and reprogramming, can be completed in 2 days. The efficiency of mRNA-modified human T cells was tested in a murine xenograft model. Human CD3 + CD8 + lymphocytes expressing anti-CD19 CIR mRNA inhibited Daudi lymphoma growth in NOD=SCID mice. These results demonstrate that a mixed population of cytotoxic lymphocytes, including T cells together with NK cells, can be quickly and simultaneously reprogrammed by mRNA against autologous malignancies. With relatively minor modifications the described method of lymphocyte reprogramming can be scaled up for cancer therapy.
OncoImmunology, 2020
Despite the recent success of CAR T cells targeting CD19 and CD22 in hematological malignancies, the production of CAR T cells still requires an extensive manufacturing process. The well-established NK-92 cell line provides a promising alternative to produce CAR-modified effector cells in a GMP-compliant, costeffective way. NK-92 can be redirected against a variety of surface antigens by our adapter CAR (AdCAR) system utilizing biotinylated antibodies (bAb) as adapter molecules. Selected bAb were capable of inducing significant AdCAR NK-92-mediated lysis of non-Hodgkin lymphoma (NHL) and mantle-cell lymphoma (MCL) cell lines as well as primary MCL and chronic lymphocytic leukemia (CLL) cells. AdCAR specificity was proven using a JeKo-1 CD19/CD20 knockout antigen-loss model. Moreover, through combinations of bAb, AdCAR NK-92 cells are capable of combatting tumor antigen evasion mechanisms. In conclusion, we successfully generated the AdCAR NK-92 cell line which can be manufactured as an "offthe-shelf, on-demand" product allowing universal and tunable tumor targeting.
Advancements in mRNA Encoded Antibodies for Passive Immunotherapy
Vaccines, 2021
Monoclonal antibodies are the fastest growing therapeutic class in medicine today. They hold great promise for a myriad of indications, including cancer, allergy, autoimmune and infectious diseases. However, the wide accessibility of these therapeutics is hindered by manufacturing and purification challenges that result in high costs and long lead times. Efforts are being made to find alternative ways to produce and deliver antibodies in more expedient and cost-effective platforms. The field of mRNA has made significant progress in the last ten years and has emerged as a highly attractive means of encoding and producing any protein of interest in vivo. Through the natural role of mRNA as a transient carrier of genetic information for translation into proteins, in vivo expression of mRNA-encoded antibodies offer many advantages over recombinantly produced antibodies. In this review, we examine both preclinical and clinical studies that demonstrate the feasibility of mRNA-encoded anti...
Molecular Therapy - Oncolytics, 2020
Vg9Vd2 T cell-based anticancer immunotherapy has shown some promise in early-phase clinical trials but there is still large room for improvement. Using the extracellular domain of the human NKG2D, a stimulatory receptor expressed by Vg9Vd2 T cells, we constructed NKG2D ligand-specific chimeric antigen receptors (CARs). We adopted a non-viral CAR approach via mRNA electroporation to modify Vg9Vd2 T cells and demonstrated that, upon interaction with the NKG2D ligand-positive cancer cells, the CARs substantially enhanced the cytotoxic activity of the modified cells toward multiple cultured solid tumor cell lines, including those resistant to Zometa treatment. Repeated doses of the CAR-expressing cells resulted in tumor regression in mice with established tumors, extending median survival time by up to 132% as compared to the PBS control group. The findings suggest clinical potential for RNA CAR-modified Vg9Vd2 T cells to treat a wide variety of NKG2D ligand-expressing cancers.
The Journal of Gene Medicine, 2012
Direct vaccination with mRNA encoding tumor antigens is a novel and promising approach in cancer immunotherapy. CureVac's mRNA vaccines contain free and protamine-complexed mRNA. Such two-component mRNA vaccines support both antigen expression and immune stimulation. These self-adjuvanting RNA vaccines, administered intradermally without any additional adjuvant, induce a comprehensive balanced immune response, comprising antigen specific CD4+ T cells, CD8+ T cells and B cells. The balanced immune response results in a strong anti-tumor effect and complete protection against antigen positive tumor cells. This tumor inhibition elicited by mRNA vaccines is a result of the concerted action of different players. After just two intradermal vaccinations, we observe multiple changes at the tumor site, including the up-regulation of many genes connected to T and natural killer cell activation, as well as genes responsible for improved infiltration of immune cells into the tumor via chemotaxis. The two-component mRNA vaccines induce a very fast and boostable immune response. Therefore, the vaccination schedules can be adjusted to suit the clinical situation. Moreover, by combining the mRNA vaccines with therapies in clinical use (chemotherapy or anti-CTLA-4 antibody therapy), an even more effective anti-tumor response can be elicited. The first clinical data obtained from two separate Phase I/IIa trials conducted in PCA (prostate cancer) and NSCLC (non-small cell lung carcinoma) patients have shown that the twocomponent mRNA vaccines are safe, well tolerated and highly immunogenic in humans.
2015
For many years, the instability of RNA had raised doubts as to whether it was possible to effectively use mRNA for gene therapy. However, rapid advances in messenger RNA-based technologies in the last decade have transformed mRNA into an increasingly popular therapeutic modality, especially in the field of vaccination against cancer and viral infections. Today, mRNA is considered a safer alternative to pDNA-based therapeutics, as it does not pose the risk of genomic integration, unlike DNA. Furthermore, mRNA-based approaches offer immediate expression of a protein of interest even in non-dividing cells. In Chapter 2 of the dissertation we reviewed the general properties and advantages of RNA as a therapeutic modality. Moreover, we discussed specific attributes, limitations and benefits of unmodified, modified and self-replicating mRNA platforms. Additionally, we also provide insights into the instability of the mRNA molecule and strategies to improve the efficiency of the transfecti...
Cancer Immunology, Immunotherapy, 2004
Over the past years, monoclonal antibodies have attracted enormous interest as targeted therapeutics, and a number of such reagents are in clinical use. However, responses could not be achieved in all patients with tumors expressing high levels of the respective target antigens, suggesting that other factors such as limited recruitment of endogenous immune effector mechanisms can also influence treatment outcome. This justifies the search for alternative, potentially more effective reagents. Antibody-toxins and cytolytic effector cells genetically modified to carry antibody-based receptors on the surface, represent such tailor-made targeting vehicles with the potential of improved tumor localization and enhanced efficacy. In this way, advances in recombinant antibody technology have made it possible to circumvent problems inherent in chemical coupling of antibodies and toxins, and have allowed construction via gene fusion of recombinant molecules which combine antibody-mediated recognition of tumor cells with specific delivery of potent protein toxins of bacterial or plant origin. Likewise, recombinant antibody fragments provide the basis for the construction of chimeric antigen receptors that, upon expression in cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells, link antibody-mediated recognition of tumor antigens with these effector cellsÕ potent cytolytic activities, thereby making them promising cellular therapeutics for adoptive cancer therapy. Here, general principles for the derivation of cytotoxic proteins and effector cells with antibody-dependent tumor specificity are summarized, and current strategies to employ these molecules and cells for directed cancer therapy are discussed, focusing mainly on the tumor-associated antigens epidermal growth factor receptor (EGFR) and the closely related ErbB2 (HER2) as targets.
Frontiers in Immunology, 2021
The progress in the isolation and characterization of tumor antigen (TA)-specific T lymphocytes and in the genetic modification of immune cells allowed the clinical development of adoptive cell therapy (ACT). Several clinical studies highlighted the striking clinical activity of T cells engineered to express either Chimeric Antigen (CAR) or T Cell (TCR) Receptors to target molecularly defined antigens expressed on tumor cells. The breakthrough of immunotherapy is represented by the approval of CAR-T cells specific for advanced or refractory CD19+ B cell malignancies by both the Food and Drug Administration (FDA) and the European Medicinal Agency (EMA). Moreover, advances in the manufacturing and gene editing of engineered immune cells contributed to the selection of drug products with desired phenotype, refined specificity and decreased toxicity. An important step toward the optimization of CAR-T cell therapy is the development of “off-the shelf” T cell products that allow to reduce...
2000
NK cells hold great potential for improving the immunotherapy of cancer. Nevertheless, tumor cells can effectively escape NK cell-mediated apoptosis through interaction of MHC molecules with NK cell inhibitory receptors. Thus, to harness NK cell effector function against tumors, we used Amaxa gene transfer technology to gene-modify primary mouse NK cells with a chimeric single-chain variable fragment (scFv) receptor specific for the human erbB2 tumor-associated Ag. The chimeric receptor was composed of the extracellular scFv anti-erbB2 Ab linked to the transmembrane and cytoplasmic CD28 and TCR-signaling domains (scFv-CD28-). In this study we demonstrated that mouse NK cells gene-modified with this chimera could specifically mediate enhanced killing of an erbB2 ؉ MHC class I ؉ lymphoma in a perforin-dependent manner. Expression of the chimera did not interfere with NK cell-mediated cytotoxicity mediated by endogenous NK receptors. Furthermore, adoptive transfer of genemodified NK cells significantly enhanced the survival of RAG mice bearing established i.p. RMA-erbB2 ؉ lymphoma. In summary, these data suggest that use of genetically modified NK cells could broaden the scope of cancer immunotherapy for patients.