RESUMO
Clinical applications of CAR-T cells are limited by the scarcity of tumor-specific targets and are often afflicted with the same on-target/off-tumor toxicities that plague other cancer treatments. A new promising strategy to enforce tumor selectivity is the use of logic-gated, two-receptor systems. One well-described application is termed Tmod™, which originally utilized a blocking inhibitory receptor directed towards HLA-I target antigens to create a protective NOT gate. Here we show that the function of Tmod blockers targeting non-HLA-I antigens is dependent on the height of the blocker antigen and is generally compatible with small, membrane-proximal targets. We compensate for this apparent limitation by incorporating modular hinge units to artificially extend or retract the ligand-binding domains relative to the effector cell surface, thereby modulating Tmod activator and blocker function. By accounting for structural differences between activator and blocker targets, we developed a set of simple geometric parameters for Tmod receptor design that enables targeting of blocker antigens beyond HLA-I, thereby broadening the applications of logic-gated cell therapies.
Assuntos
Neoplasias , Linfócitos T , Humanos , Antígenos/metabolismoRESUMO
BACKGROUND: Mesothelin (MSLN) is a classic tumor-associated antigen that is expressed in lung cancer and many other solid tumors. However, MSLN is also expressed in normal mesothelium which creates a significant risk of serious inflammation for MSLN-directed therapeutics. We have developed a dual-receptor (Tmod™) system that exploits the difference between tumor and normal tissue in a subset of patients with defined heterozygous gene loss (LOH) in their tumors. METHODS: T cells engineered with the MSLN CAR Tmod construct described here contain (1) a novel MSLN-activated CAR and (2) an HLA-A*02-gated inhibitory receptor (blocker). A*02 binding is intended to override T-cell cytotoxicity, even in the presence of MSLN. The Tmod system is designed to treat heterozygous HLA class I patients, selected for HLA LOH. When A*02 is absent from tumors selected for LOH, the MSLN Tmod cells are predicted to mediate potent killing of the MSLN(+)A*02(-) malignant cells. RESULTS: The sensitivity of the MSLN Tmod cells is comparable with a benchmark MSLN CAR-T that was active but toxic in the clinic. Unlike MSLN CAR-T cells, the Tmod system robustly protects surrogate "normal" cells even in mixed-cell populations in vitro and in a xenograft model. The MSLN CAR can also be paired with other HLA class I blockers, supporting extension of the approach to patients beyond A*02 heterozygotes. CONCLUSIONS: The Tmod mechanism exemplified by the MSLN CAR Tmod construct provides an alternative route to leverage solid-tumor antigens such as MSLN in safer, more effective ways than previously possible.
Assuntos
Antígeno HLA-A2/genética , Imunoterapia Adotiva/métodos , Mesotelina/imunologia , Neoplasias/terapia , Receptores de Antígenos Quiméricos/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Antígeno HLA-A2/imunologia , Humanos , Perda de Heterozigosidade , Camundongos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neoantigens are among the most intriguing potential immuno-oncology targets because, unlike many cancer targets that are expressed on normal tissues, they are by definition restricted to cancer cells. Medicines directed at common neoantigens such as mutant KRAS are especially interesting because they may offer the convenience and cost of an off-the-shelf therapy. However, all common KRAS mutations produce proteins that differ from the wild type at a single amino acid, creating challenges for molecular discrimination. We have undertaken an effort to optimize single-chain variable fragments (scFv) against peptide/major histocompatibility antigen complexes composed of HLA-A*11 and either G12V- or G12D-mutant KRAS peptides. These scFvs could in principle be used in chimeric antigen receptor (CAR) T-cell therapies for selected patients whose tumors bear either of these mutations. Here we show that optimization of such CARs involves a trade-off between potency and selectivity. We further show that targeting this family without high selectivity engenders risks of cross-reactivity against other members of the G-protein family to which KRAS belongs. Significance: We report an effort to generate high potency, selective CARs directed at mutant KRAS peptides. Although the heavily optimized CARs maintain high selectivity against wild-type KRAS, they lose selectivity against other KRAS-related peptides derived from human proteins. To our knowledge, this work is the first to examine the trade-off between potency and selectivity with regard to KRAS pMHC-directed CARs, illustrating the challenge to achieve both sufficient potency and high selectivity.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Anticorpos de Cadeia Única , Humanos , Receptores de Antígenos Quiméricos/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Imunoterapia Adotiva , Anticorpos de Cadeia Única/genéticaRESUMO
Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients' T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629-38 complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.
Assuntos
Imunoterapia Adotiva , Neoplasias/terapia , Infecções por Papillomavirus/terapia , Receptores de Antígenos Quiméricos/imunologia , Linhagem Celular , Proteínas de Fluorescência Verde , Antígeno HLA-A2/imunologia , Humanos , Interferon gama/imunologia , Luciferases de Vaga-Lume , Neoplasias/imunologia , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/imunologia , Peptídeos/imunologia , Proteínas Repressoras/imunologia , Anticorpos de Cadeia Única/imunologiaRESUMO
We designed variant human TCRs composed of the full-length TCRα/ß or extracellular and transmembrane domains of the associated CD3 subunits fused to polypeptides derived from proteins thought to either enhance or inhibit normal T cell function. First, we showed that the C termini of both the TCR α- and ß-chains can accommodate specific additional sequences, without abrogating complex formation or acute sensitivity of the receptor. Replacement of ITAMs with ITIM-containing intracellular domains inverted the TCR signal (i.e., created a ligand-dependent inhibitory receptor). The normal signaling function of the CD3 complex was transferable to the TCR by eliminating all CD3 ITAMs and grafting three to six ITAMs onto the C termini of the α/ß-chains, with no effect on acute sensitivity. The observation that TCR variants of such diverse C-terminal composition can fold and function as signaling receptors demonstrates substantial structural and functional malleability of TCRs. These results add to knowledge about TCR structure-function with regard to acute signaling and may provide a route to use TCRs in different ways for T cell therapy.
Assuntos
Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
In 2013, an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity. The safety problems were hypothesized to originate from off-target T-cell receptor activity against a closely related MAGE-A12 peptide. A combination of published and new data led us to test this hypothesis with current technology. Our results call into question MAGE-A12 as the source of the neurotoxicity. Rather, the data imply that an alternative related peptide from EPS8L2 may be responsible. Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, these findings suggest that MAGE-A3 may deserve further consideration as a cancer target. As a step in this direction, the authors isolated 2 MAGE-A3 peptide-major histocompatibility complex-directed chimeric antigen receptors, 1 targeting the same peptide as the clinical T-cell receptor. Both chimeric antigen receptors have improved selectivity over the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics, showing that there may be other options for MAGE-A3 cell therapy.
Assuntos
Antígenos de Neoplasias/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Células HCT116 , Células HEK293 , Humanos , Células Jurkat , Leucócitos Mononucleares/imunologia , Células MCF-7 , Complexo Principal de Histocompatibilidade/imunologia , Neoplasias/imunologia , Células PC-3 , Receptores de Antígenos Quiméricos/imunologiaRESUMO
We describe an approach to cancer therapy based on exploitation of common losses of genetic material in tumor cells (loss of heterozygosity) (Basilion et al., 1999; Beroukhim et al., 2010). This therapeutic concept addresses the fundamental problem of discrimination between tumor and normal cells and can be applied in principle to the large majority of tumors. It utilizes modular activator/blocker elements that integrate signals related to the presence and absence of ligands displayed on the cell surface (Fedorov et al., 2013). We show that the targeting system works robustly in vitro and in a mouse cancer model where absence of the HLA-A*02 allele releases a brake on engineered T cells activated by the CD19 surface antigen. This therapeutic approach potentially opens a route toward a large, new source of cancer targets.
Assuntos
Perda de Heterozigosidade/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Alelos , Animais , Antígenos CD19/imunologia , Linhagem Celular Tumoral , Feminino , Antígenos HLA-A/imunologia , Humanos , Células Jurkat , Ligantes , Camundongos , Camundongos Endogâmicos NODRESUMO
Chimeric antigen receptors (CARs) and their parent signaling molecule, the T cell receptor (TCR), are fascinating proteins of increasing relevance to disease therapy. Here we use a collection of 1221 pMHC-directed CAR constructs representing 10 pMHC targets to study aspects of CAR structure-activity relationships (SAR), with particular focus on the extracellular and transmembrane structural components. These experiments that involve pMHC targets whose number/cell can be manipulated by peptide dosing in vitro enable systematic analysis of the SAR of CARs in carefully controlled experimental situations (Harris and Kranz, 2016). We find that CARs tolerate a wide range of structural variation, with the ligand-binding domains (LBDs) dominating the SAR of CAR antigen sensitivity. Notwithstanding the critical role of the LBD, CAR antigen-binding on the cell surface, measured by pMHC tetramer staining, is not an effective predictor of functional sensitivity. These results have important implications for the design and testing of CARs aimed toward the clinic.
Assuntos
Antígenos HLA-A/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Sítios de Ligação/imunologia , Antígenos HLA-A/metabolismo , Humanos , Células Jurkat , Ligantes , Células MCF-7 , Domínios Proteicos/imunologia , Multimerização Proteica/imunologia , Receptores de Antígenos Quiméricos/imunologia , Relação Estrutura-Atividade , Linfócitos T/metabolismoRESUMO
Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, but vector manufacturing remains a challenge. New methods are needed to increase rAAV yields and reduce costs. Past efforts to improve rAAV production have focused on optimizing a single variable at a time, but this approach does not account for the interactions of multiple factors that contribute to vector generation. Here, we utilized a design-of-experiment (DOE) methodology to optimize rAAV production in a HEK293T suspension cell system. We simultaneously varied the transgene, packaging, and helper plasmid ratios, the total DNA concentration, and the cell density to systematically evaluate the impact of each variable across 52 conditions. The results revealed a unique set of parameters with a lower concentration of transgene plasmid, a higher concentration of packaging plasmid, and a higher cell density than previously described protocols. Using this DOE-optimized protocol, we achieved unpurified yields approaching 3 × 1014 viral genomes (VGs)/L of cell culture. Additionally, we incorporated polyethylene glycol (PEG)-based virus precipitation, pH-mediated protein removal, and affinity chromatography to our downstream processing, enabling average purified yields of >1 × 1014 VGs/L for rAAV-EGFPs across 13 serotypes and capsid variants.
RESUMO
Inhibitors that block the programmed cell death-1 (PD-1) pathway can potentiate endogenous antitumor immunity and have markedly improved cancer survival rates across a broad range of indications. However, these treatments work for only a minority of patients. The efficacy of anti-PD-1 inhibitors may be extended by cytokines, however, the incorporation of cytokines into therapeutic regimens has significant challenges. In their natural form when administered as recombinant proteins, cytokine treatments are often associated with low response rates. Most cytokines have a short half-life which limits their exposure and efficacy. In addition, cytokines can activate counterregulatory pathways, in the case of immune-potentiating cytokines this can lead to immune suppression and thereby diminish their potential efficacy. Improving the drug-like properties of natural cytokines using protein engineering can yield synthetic cytokines with improved bioavailability and tissue targeting, allowing for enhanced efficacy and reduced off-target effects. Using structure guided engineering we have designed a novel class of antibody-cytokine fusion proteins consisting of a PD-1 targeting antibody fused together with an interleukin-21 (IL-21) cytokine mutein. Our bifunctional fusion proteins can block PD-1/programmed death-ligand 1 (PD-L1) interaction whilst simultaneously delivering IL-21 cytokine to PD-1 expressing T cells. Targeted delivery of IL-21 can improve T cell function in a manner that is superior to anti-PD-1 monotherapy. Fusion of engineered IL-21 variants to anti-PD1 antibodies can improve the drug-like properties of IL-21 cytokine leading to improved cytokine serum half-life allowing for less frequent dosing. In addition, we show that targeted delivery of IL-21 can minimize any potential detrimental effect on local antigen-presenting cells. A highly attenuated IL-21 mutein variant (R9E:R76A) fused to a PD-1 antibody provides protection in a humanized mouse model of cancer that is refractory to anti-PD-1 monotherapy. Collectively, our preclinical data demonstrate that this approach may improve upon and extend the utility of anti-PD-1 therapeutics currently in the clinic.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Imunoterapia , Interleucinas/uso terapêutico , Neoplasias/terapia , Animais , Anticorpos Monoclonais/imunologia , Antígeno B7-H1/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias/imunologia , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.
Assuntos
Envelhecimento/metabolismo , Osso e Ossos/lesões , Osso e Ossos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Osteogênese/fisiologia , Envelhecimento/efeitos dos fármacos , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/farmacologia , Densidade Óssea/efeitos dos fármacos , Doenças Ósseas Metabólicas/sangue , Doenças Ósseas Metabólicas/fisiopatologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Linhagem Celular , Estrogênios/deficiência , Feminino , Fêmur/diagnóstico por imagem , Fêmur/efeitos dos fármacos , Fêmur/patologia , Consolidação da Fratura/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Vértebras Lombares/efeitos dos fármacos , Vértebras Lombares/patologia , Masculino , Camundongos , Osteogênese/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos , Via de Sinalização Wnt/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Osteoporosis is a chronic condition that is typically treated by the long-term repeated administration of antiresorptive agents. Gene therapy has the potential to deliver protein-based antiresorptive agents without the need for repeated administration. Osteoprotegerin (OPG) is a naturally occuring protein that prevents bone resorption by inhibiting osteoclast formation, function and survival. We tested whether adeno-associated virus (AAV) could deliver OPG at levels that are sufficient to reverse established osteopenia in ovariectomized (OVX) mice without causing liver toxicity. Tibial bone mineral density (BMD) was measured by peripheral quantitative computed tomography (pQCT) in 12-week-old CDF1 mice prior to OVX or sham surgery. Six weeks later, BMD was significantly reduced in OVX mice compared to sham controls or pre-surgery values. Sham and OVX mice were then injected once IV with an AAV vector carrying cDNA for recombinant hOPG (AAV-OPG) or beta-galactosidase (AAV-betaGal). BMD and bone histomorphometry were assessed 10 weeks after treatment. A single injection of AAV-OPG led to the appearance of human OPG (hOPG) in the serum of mice within 7 days, and high serum levels of hOPG were maintained for the duration of the 10-week study. At the end of the study, OVX mice given AAV-OPG had significantly greater tibial BMD compared to age-matched OVX animals given AAV-betaGal. In sham-operated mice, AAV-OPG also significantly increased tibial BMD compared to AAV-betaGal. The increased BMD in AAV-OPG animals was accompanied by significantly increased bone volume and significantly reduced osteoclast surfaces in the proximal tibial metaphysis. Liver histology was normal, and circulating activities of hepatocyte cytosolic enzymes were unaffected by AAV exposure. In an accompanying experiment, young (3-4 weeks) C57BL/6 mice treated once IV with AAV-OPG maintained pharmacologically active levels of OPG in serum for at least 16 months. In summary, a single AAV-OPG treatment reversed established osteopenia in OVX mice without evidence of liver toxicity. AAV delivery appears to be a safe and effective method for producing sustained systemic exposure to OPG.