Contextual Reprogramming of CAR-T Cells for Treatment of HER2+ Cancers

Yang, Zhifen; Li, Lingyu; Turkoz, Ahu; Chen, Pohan; Harari-Steinfeld, Rona; Kearney, Alper Y.; Patel, Dharmesh; Balan, Vitaly; Bobbin, Maggie; Choi, Heechul; Stefanson, Ofir; Inel, Damla; Vinod, Veena; Cesano, Alessandra; Wang, Bing; Roh, Kyung-Ho; Qi, Lei; Marincola, Francesco M.

doi:10.2139/ssrn.3656606

Cited by 3 publications

(10 citation statements)

References 13 publications

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“…Effective cell therapy of cancer should challenge the disease simultaneously through multiple modalities to address cell fitness [4,28,29], and resilience in a metabolically and immunologically hostile TME [3,30,31]. Recent advances in synthetic biology allow improvements of CAR architecture to prolong CAR-T cell persistence in vivo and enhance efficacy as previously shown by reversible prevention of PD-1 expression with CRISPRi in response to antigen encounter through [13].…”

Section: Discussionmentioning

confidence: 99%

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Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

et al. 2023

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Background Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection. Methods We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR). Results The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)-γ production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2+ FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects. Conclusions RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.

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Section: Discussionmentioning

confidence: 99%

“…We designed such construct to be inducible upon T cell activation to successfully downregulate PD-1 gene expression, a system referred to as CRISPR interference (CRISPRi). The engineered CAR-T cell product demonstrated enhanced cellular persistence and tumor eradication [13].…”

Section: Introductionmentioning

confidence: 99%

Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

et al. 2023

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“…Introduction of premature STOP codons into the CD7 and CD3 loci of anti-CD7 and anti-CD3 CAR T-cells prevents the induction of CAR-mediated fratricide during the manufacturing and infusion process [ 106 ]. dCas9 tethered to transcriptional repressor KRAB targeting the PDCD1 gene is effective in generating anti-HER2 CAR T-cells with low PD-1 expression to confer checkpoint inhibition resistance [ 107 ]. Prime editors, while still in their infancy, can efficiently edit the genomes of human cell lines and human primary T-cells [ 108 ].…”

Section: Main Textmentioning

confidence: 99%

Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing

2022

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Chimeric Antigen Receptor (CAR) T-cells represent a breakthrough in personalized cancer therapy. In this strategy, synthetic receptors comprised of antigen recognition, signaling, and costimulatory domains are used to reprogram T-cells to target tumor cells for destruction. Despite the success of this approach in refractory B-cell malignancies, optimal potency of CAR T-cell therapy for many other cancers, particularly solid tumors, has not been achieved. Factors such as T-cell exhaustion, lack of CAR T-cell persistence, cytokine-related toxicities, and bottlenecks in the manufacturing of autologous products have hampered the safety, effectiveness, and availability of this approach. With the ease and accessibility of CRISPR-Cas9-based gene editing, it is possible to address many of these limitations. Accordingly, current research efforts focus on precision engineering of CAR T-cells with conventional CRISPR-Cas9 systems or novel editors that can install desired genetic changes with or without introduction of a double-stranded break (DSB) into the genome. These tools and strategies can be directly applied to targeting negative regulators of T-cell function, directing therapeutic transgenes to specific genomic loci, and generating reproducibly safe and potent allogeneic universal CAR T-cell products for on-demand cancer immunotherapy. This review evaluates several of the ongoing and future directions of combining next-generation CRISPR-Cas9 gene editing with synthetic biology to optimize CAR T-cell therapy for future clinical trials toward the establishment of a new cancer treatment paradigm.

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“…For example, Schmidt et al [46] developed a CRISPRa and CRISPRi platform to perform genomewide screens for functional regulators of cytokine production in response to T cell stimulation [46]. Yang et al [47] developed a CAR-T cell product called RB-340-1, which was engineered through a CRISPRi circuit to prevent Programmed cell Death protein 1 (PD-1) expression upon antigen-encounter [47]. RB-340-1 is the first application of CRISPRi toward a clinically relevant product and allows the conditional and reversible suppression of PD-1.…”

Section: Talens Crispr/casmentioning

confidence: 99%

“…The reversible nature of this editing also allows fine tuning of the degree of PD-1 expression. RB-340-1 demonstrated resilience to checkpoint inhibition and increased persistence and effectiveness against HER2expressing cancer xenografts [47].…”

Section: Talens Crispr/casmentioning

confidence: 99%

Current strategies employed in the manipulation of gene expression for clinical purposes

et al. 2022

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Abnormal gene expression level or expression of genes containing deleterious mutations are two of the main determinants which lead to genetic disease. To obtain a therapeutic effect and thus to cure genetic diseases, it is crucial to regulate the host’s gene expression and restore it to physiological conditions. With this purpose, several molecular tools have been developed and are currently tested in clinical trials. Genome editing nucleases are a class of molecular tools routinely used in laboratories to rewire host’s gene expression. Genome editing nucleases include different categories of enzymes: meganucleses (MNs), zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR associated protein (Cas) and transcription activator-like effector nuclease (TALENs). Transposable elements are also a category of molecular tools which includes different members, for example Sleeping Beauty (SB), PiggyBac (PB), Tol2 and TcBuster. Transposons have been used for genetic studies and can serve as gene delivery tools. Molecular tools to rewire host’s gene expression also include episomes, which are divided into different categories depending on their molecular structure. Finally, RNA interference is commonly used to regulate gene expression through the administration of small interfering RNA (siRNA), short hairpin RNA (shRNA) and bi-functional shRNA molecules. In this review, we will describe the different molecular tools that can be used to regulate gene expression and discuss their potential for clinical applications. These molecular tools are delivered into the host's cells in the form of DNA, RNA or protein using vectors that can be grouped into physical or biochemical categories. In this review we will also illustrate the different types of payloads that can be used, and we will discuss recent developments in viral and non-viral vector technology.

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Contextual Reprogramming of CAR-T Cells for Treatment of HER2+ Cancers

Cited by 3 publications

References 13 publications

Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment

Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing

Current strategies employed in the manipulation of gene expression for clinical purposes

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