Abstract LB-390: Intratumoral electroporation of plasmid-encoded IL-12 and membrane-bound anti-CD3 increases tumor immunogenicity and augments the function of T cell subsets
Mia HanAnandaroop MukhopadhyayBianca NguyenJack Y. LeeErica BrowningJon SalazarReneta HermizLauren SvensonChris A. R. BakerDaniel O’ConnorKellie MalloyDavid A. CantonChristopher G. Twitty
0
Citation
0
Reference
10
Related Paper
Abstract:
Abstract Intratumoral (IT) delivery of plasmid IL-12 (tavokinogene telseplasmid; tavo) via electroporation (EP), collectively referred as IT-tavo-EP, generates immunologically-relevant levels of localized IL-12, triggering regression of both treated and distant tumors with minimal toxicity in preclinical and clinical studies. Our previous clinical trial data from melanoma patients treated with IT-tavo-EP identified a treatment-related increase of infiltrating T cells and transcripts related to immune activation, as well as a significant increase in the IFN-γ score of patients with a clinical benefit, suggesting that CD3+ tumor-infiltrating lymphocytes (TIL) may be critical in maximizing the anti-tumor effects of IT-tavo-EP. Furthermore, in-house biomarker data have identified an abundance of non-tumor reactive TIL that, if mobilized could additionally contribute to a clinical response. Accordingly, a plasmid-encoded membrane-bound polyclonal T cell-stimulating anti-CD3 (αCD3) hybrid antibody (scFv) was developed and used in combination with tavo (IT-tavo-αCD3-EP) to broaden the scope and depth of the T cell response. We previously demonstrated that membrane expression of αCD3 on neoplastic and stromal cells could activate CD3+ TIL, driving enhanced proliferation and cytotoxicity in a B16-OVA murine model. Here, using immune profiling of the tumor microenvironment (TME), we have demonstrated that this membrane-bound αCD3 therapeutic can significantly upregulate frequencies of CXCR3+CD8+ T cells and short-lived effector T cells, while reducing PD-1 expression on CD8+ T cells in vivo. Critically, naïve T cells, Treg cells, and exhausted T cells (subsets not typically associated with strong anti-tumor responses) displayed enhanced effector function (IFN-γ and granzyme B release) with engagement of membrane-bound αCD3 and IL-12. Furthermore, we found that this therapeutic approach could equally enhance proliferation of T cells regardless of the affinity for their cognate peptide:MHC, suggesting a TCR independent mechanism. Collectively, these observations demonstrate that IT-tavo-αCD3-EP can mobilize broad subsets of T cells beyond dominant anti-tumor effectors demonstrated. Thus, while enhanced cytolytic function is associated with this therapy, inclusion of additional atypical anti-tumor T cell subsets may also promote reshaping of the TME by production of effector cytokines upon engagement of surface-bound αCD3. Moreover, functional restoration of TIL isolated from a melanoma patient with active clinical progression on anti-PD-1 therapy, was possible with engagement of membrane-bound αCD3 in the presence of IL-12. Collectively, these data continue to support the utility of IT-tavo-αCD3-EP as a promising therapeutic approach for patients with melanoma and other accessible solid tumors. Citation Format: Mia Han, Anandaroop Mukhopadhyay, Bianca Nguyen, Jack Y. Lee, Erica Browning, Jon Salazar, Reneta Hermiz, Lauren Svenson, Chris Baker, Daniel O'Connor, Kellie Malloy, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of plasmid-encoded IL-12 and membrane-bound anti-CD3 increases tumor immunogenicity and augments the function of T cell subsets [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 LB-390.Keywords:
Tumor-infiltrating lymphocytes
The mechanism of electroporation has been successfully exploited to deliver gene and drugs into tissue. Electroporation has become an important role to enhance the delivery of genes into tissue. This technique can also be effectively utilized with marginal effect on viability. Electroporation technique can also provide high transfection efficiency. Therefore, there is an increasing interest in electroporation method for delivering gene into tissue. One important area is the delivery of genes into tissues as a therapy. This paper presents the overview of optimal parameters of electroporation for gene and tissue in vivo. This article reviews the obtained results, including the background of electroporation and essential parameters for transfection. This study discusses the evidence based on the success of delivering gene in vivo and effective delivery of drugs in electroporation.
Cite
Citations (5)
It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.
Cuvette
Primary cell
Cite
Citations (1)
瞄准：在老鼠在调停 electroporation 的基因转移期间在 transgene 表示上调查 Ca2+ 的特定的效果。方法：骨胳的肌肉和皮肤与一个酶记者原生质标志受到在活体内 electroporation，与或没有 Ca2+ 和许多其他的离子。结果：为在活体内 electroporation，在 DNA 解决方案的 10 就 mmol/L Ca2+ 的存在急速地减少了产生 transgene 表达式，到不到 5% 控制值。仅仅 Ca2+ ，不是另外的离子，引起的抑制，和效果不是织物 specific。更令人惊讶地，甚至当 Ca2+ 离子被 electroporation 在 DNA 管理前后交付时，类似的效果仍然被观察。结论：由 electroporation 的在活体内基因转移上的 Ca2+ 的禁止的效果是特定的，即，禁止的效果可能与在 electroporation 和随后的重新封上的事件以后的房间膜性质有关。
Gene transfer
Cite
Citations (0)
Electroporation is a process in which a controlled electrical pulse is applied to cells, inducing a transient destabilization of the cell membrane. During this time, the cells are highly permeable to exogenous substances in the surrounding media. DNA, proteins, and small molecules are all taken up by cells during electroporation; introduction of DNA into cells is the most common application. Gene transfer by electroporation offers many advantages for analysis of gene expression. The technique is simple, rapid, and reproducible. It is especially suited to suspension cultures and certain cell types that are poorly trans-fected by other means. Because all cells are transfected instantaneously, and essentially simultaneously, it is particularly suited to quantitation of gene transfer. Finally, single-copy, stable transfectants can often be isolated (1). Whereas the basic mechanisms of electroporation are still largely unknown, optimizing the conditions for electroporation of any particular cell type is primarily empirical.
Exogenous DNA
Gene transfer
Cite
Citations (33)
Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternative techniques. This unit describes electroporation of mammalian cells, including ES cells, for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer, as well as for DNA vaccination. © 2017 by John Wiley & Sons, Inc.
Cite
Citations (14)
The present study on tumor infiltrating lymphocytes (TILs) from tumors of cattle has revealed 13% MHC-II+ve cells in TILs from horn cancer while those from nasal granuloma had 18% cells expressing MHC-II. In case of horn cancer, 54% TlLs were CD4+ve and 18% were CD8+ve whereas in case of nasal granuloma, 43% of TlLs were CD4+ve and 19% were CD8+ve. This indicates that TILs comprise mostly of T-cells with a predominance of helper cells in these tumors of cattle. The substantial percentage of MHC-ll+ve cells in TILs may indicate the presence of activated lymphocytes.
Tumor-infiltrating lymphocytes
Cite
Citations (0)
It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.
Cuvette
Primary cell
Cite
Citations (11)
Abstract Electroporation—the use of high‐voltage electric shocks to introduce DNA into cells—can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knock‐out, knock‐in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination. Also described are modifications for preparation and transfection of plant protoplasts. Curr. Protoc. Neurosci . 57:A.1E.1‐A.1E.11. © 2011 by John Wiley & Sons, Inc.
Exogenous DNA
Cite
Citations (34)
Cuvette
Cite
Citations (2)
Abstract Adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TILs) can induce durable complete tumor regression in patients with advanced melanoma. Efforts are currently underway to expand this treatment modality to other cancer types. In the microenvironment of ovarian cancer, the engagement of co-inhibitory immune checkpoint molecules such as CTLA-4 can lead to the inactivation of TILs. Thus, approaches that directly manipulate co-inhibitory pathways within the tumor microenvironment might improve the expansion of tumor-reactive TILs. The initial expansion of TILs for ACT from tumor fragments provides a window of opportunity to manipulate an intact tumor microenvironment and improve CD8 + T-cell output and TIL tumor reactivity. To exploit this, we used a CTLA-4-blocking antibody, added during the initial TIL culture, and found that the blockade of CTLA-4 favored the propagation of CD8 + TILs from ovarian tumor fragments. Interestingly, adding the CTLA-4 blocking antibody in the initial phase of the TIL culture resulted in more potent anti-tumor TILs in comparison to standard TIL cultures. This phenotype was preserved during the rapid expansion phase. Thus, targeting CTLA-4 within the intact tumor microenvironment of tumor fragments enriches tumor-reactive TILs and may improve clinical outcome of TIL-based ACT in ovarian cancer.
Tumor-infiltrating lymphocytes
Immune checkpoint
CTLA-4
Cancer Immunotherapy
Blocking antibody
Cite
Citations (54)