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

Abstract

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.

Tyrosine kinase signaling-independent MET-targeting with CAR-T cells

“…Another checkpoint inhibitor (tremelimumab) developed for targeting cytotoxic T-lymphocyte associated protein 4 (CTLA-4) also showed promising clinical results for treating HCC patients [ 52 ]. Of note, genetic editing of CAR vectors to transcriptionally suppress PD-1 expression or adding PD-1 blockage as a combination strategy was able to improve CAR-T cell persistence and anti-tumor efficacy in vivo [ 26 , 53 ]. Thus, future optimization to overcome MET-CAR-T cell exhaustion via PD-L1/PD-1 and other essential inhibitory signaling pathways such as CTLA-4 [ 52 ] and LAG3 [ 54 ] could improve MET-CAR-T cell therapeutic efficacy.…”

Shifting the paradigm: engaging multicellular networks for cancer therapy

“…1 B). The vicarious modulation of components of the multi-cellular network (MCN) in the TME can be achieved by small molecules that activate multiple anti-tumoral mechanisms [ 3 ] that we refer to as ‘smart’ small molecules, pathway inhibitors [ 4 ], biologics such as ADCs [ 5 , 6 ], chimeric antigen receptors (CAR) T cells or tumor infiltrating lymphocytes (TIL) [ 7 ] and genetic engineering modalities such as polycistronic mRNA or DNA constructs that can simultaneously deliver pleiotropic payloads [ 8 ]. Such modalities can result in amplification of anti-cancer responses through the secondary engagement of endogenous immune effector functions.…”

“…Production of pro-inflammatory cytokines such as IL-12 [ 71 ] or the IL-1 family members such as IL-18 [ 72 ], together with IFN- can redirect the immune suppressive milieu toward an immune effector polarization. This is exemplified by the M2 to M1 transition of macrophages that elicit macrophage-mediated extracellular killing and potentiate IL-12-mediated Th1 responses [ 8 , 12 , 63 – 65 , 71 ] or by the Th1 polarization of otherwise immune suppressive tumor associated stromal cells [ 73 ].…”

“…Indeed, the ability of ACT products to survive and proliferate upon reaching the TME by maintaining a stem cell like phenotype is critical and it has been clearly demonstrated in the context of solid [ 51 , 75 , 76 ] and hematological malignancies [ 77 ]. Moreover, overcoming CIR remains the ultimate frontier for all immunotherapies including ACT and this can be attained by building smart ACT products [ 47 ] that can safely redirect the multicellular network of cancer toward an ICR-like phenotype through the contextual delivery, limited for safety reasons to the TME, of powerful immunostimulatory factors such as IL-1 family members [ 78 ] or IL-12 [ 8 , 61 ].…”