Immunotherapy has become a powerful clinical strategy for treating cancer. The number of immunotherapy drug approvals has been increasing, with numerous treatments in clinical and preclinical development. However, a key challenge in the broad implementation of immunotherapies for cancer remains the controlled modulation of the immune system, as these therapeutics have serious adverse effects including autoimmunity and nonspecific inflammation. Understanding how to increase the response rates to various classes of immunotherapy is key to improving efficacy and controlling these adverse effects. Advanced biomaterials and drug delivery systems, such as nanoparticles and the use of T cells to deliver therapies, could effectively harness immunotherapies and improve their potency while reducing toxic side effects. Here, we discuss these research advances, as well as the opportunities and challenges for integrating delivery technologies into cancer immunotherapy, and we critically analyse the outlook for these emerging areas.

Delivery technologies for cancer immunotherapy

Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

/pdf/applications-of-genome-editing-technology-in-the-targeted-2giausc34s.pdf

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

The immune system evolved to distinguish non-self from self to protect the organism. As cancer is derived from our own cells, immune responses to dysregulated cell growth present a unique challenge. This is compounded by mechanisms of immune evasion and immunosuppression that develop in the tumour microenvironment. The modern genetic toolbox enables the adoptive transfer of engineered T cells to create enhanced anticancer immune functions where natural cancer-specific immune responses have failed. Genetically engineered T cells, so-called 'living drugs', represent a new paradigm in anticancer therapy. Recent clinical trials using T cells engineered to express chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs) have produced stunning results in patients with relapsed or refractory haematological malignancies. In this Review we describe some of the most recent and promising advances in engineered T cell therapy with a particular emphasis on what the next generation of T cell therapy is likely to entail.

Engineered T cells: the promise and challenges of cancer immunotherapy

Recent advances in the development of genome editing technologies based on programmable nucleases have substantially improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress toward developing programmable nuclease–based therapies as well as future prospects and challenges.

Therapeutic genome editing: prospects and challenges

The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR-Cas-based tools towards clinical use in gene and cell therapies.

https://par.nsf.gov/servlets/purl/10132911

The next generation of CRISPR-Cas technologies and applications.

Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases.Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.Results: The CRISPR gene-edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene-edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells.Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 23(9); 2255-66. ©2016 AACR.

https://clincancerres.aacrjournals.org/content/clincanres/23/9/2255.full.pdf

Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition

Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude. High-affinity cells recognized target expressed at any level, including at levels in normal cells that were undetectable by flow cytometry. Affinity-tuned cells exhibited robust antitumor efficacy similar to high-affinity cells, but spared normal cells expressing physiologic target levels. The use of affinity-tuned scFvs offers a strategy to empower wider use of CAR T cells against validated targets widely overexpressed on solid tumors, including those considered undruggable by this approach.

https://cancerres.aacrjournals.org/content/canres/75/17/3596.full.pdf

Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice

Chimeric antigen receptor (CAR)-modified adoptive T-cell therapy has been successfully applied to the treatment of hematologic malignancies, but faces many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced in both naturally occurring and genetically modified tumor-infiltrating lymphocytes (TIL) by inhibitory receptors (IR), namely PD1. We hypothesized that interfering with PD1 signaling would augment CAR T-cell activity against solid tumors. To address this possibility, we introduced a genetically engineered switch receptor construct, comprising the truncated extracellular domain of PD1 and the transmembrane and cytoplasmic signaling domains of CD28, into CAR T cells. We tested the effect of this supplement, "PD1CD28," on human CAR T cells targeting aggressive models of human solid tumors expressing relevant tumor antigens. Treatment of mice bearing large, established solid tumors with PD1CD28 CAR T cells led to significant regression in tumor volume due to enhanced CAR TIL infiltrate, decreased susceptibility to tumor-induced hypofunction, and attenuation of IR expression compared with treatments with CAR T cells alone or PD1 antibodies. Taken together, our findings suggest that the application of PD1CD28 to boost CAR T-cell activity is efficacious against solid tumors via a variety of mechanisms, prompting clinical investigation of this potentially promising treatment modality.

https://cancerres.aacrjournals.org/content/canres/76/6/1578.full.pdf

A Chimeric Switch-Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors

// Jiangtao Ren 1 , Xuhua Zhang 1 , Xiaojun Liu 1 , Chongyun Fang 1 , Shuguang Jiang 1 , Carl H. June 1, 2 , Yangbing Zhao 1, 2 1 Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 2 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA Correspondence to: Yangbing Zhao, email: yangbing@upenn.edu Keywords: CRISPR/CAS9, T lymphocytes, chimeric antigen receptors, PD-1, CD95 Received: November 18, 2016      Accepted: January 27, 2017      Published: February 09, 2017 ABSTRACT The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector. High efficient double knockout of endogenous TCR and HLA class I could be easily achieved to generate allogeneic universal CAR T cells. We also generated Fas-resistant universal CAR T cells by triple gene disruption. Simultaneous gene editing of four gene loci using the one-shot CRISPR protocol to generate allogeneic universal T cells deficient of both PD1 and CTLA-4 was also attempted.

/pdf/a-versatile-system-for-rapid-multiplex-genome-edited-car-t-i8dmn8spxk.pdf

A versatile system for rapid multiplex genome-edited CAR T cell generation

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB) The expanded T cells were phenotypically and functionally similar to T cells expanded by OKT3/IL-2 Moreover, co-electroporation of CD86 and 4-1BBL could further change the phenotype and enhance the in vivo anti-tumor activity Although T cells expanded by the co-electroporation of OKT3-28BB with CD86 and 4-1BBL showed an increased central memory phenotype, the T cells still maintained tumor lytic activities as potent as those of OKT3/IL-2 or OKT3-28BB-stimulated T cells In different tumor mouse models, T cells expanded by OKT3-28BB RNA electroporation showed anti-tumor activities superior to those of OKT3/IL-2 T cells Hence, T cells with both a less differentiated phenotype and potent tumor killing ability can be generated by RNA electroporation, and this T cell manufacturing procedure can be further optimized by simply co-delivering other splices of RNA, thus providing a simple and cost-effective method for generating high-quality T cells for adoptive immunotherapy

/pdf/novel-t-cells-with-improved-in-vivo-anti-tumor-activity-kkgm5jx23f.pdf

Chongyun Fang

Papers

Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition

Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice

A Chimeric Switch-Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors

A versatile system for rapid multiplex genome-edited CAR T cell generation

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation