BOXR1030, an anti-GPC3 CAR with exogenous GOT2 expression, shows enhanced T cell metabolism and improved anti-cell line derived tumor xenograft activity.

PURPOSE: The solid tumor microenvironment (TME) drives T cell dysfunction and inhibits the effectiveness of immunotherapies such as chimeric antigen receptor-based T cell (CAR T) cells. Early data has shown that modulation of T cell metabolism can improve intratumoral T cell function in preclinical models. EXPERIMENTAL DESIGN: We evaluated GPC3 expression in human normal and tumor tissue specimens. We developed and evaluated BOXR1030, a novel CAR T therapeutic co-expressing glypican-3 (GPC3)-targeted CAR and exogenous glutamic-oxaloacetic transaminase 2 (GOT2) in terms of CAR T cell function both in vitro and in vivo. RESULTS: Cell surface expression of tumor antigen GPC3 was observed by immunohistochemical staining in tumor biopsies from hepatocellular carcinoma, liposarcoma, squamous lung cancer, and Merkel cell carcinoma patients. Compared to control GPC3 CAR alone, BOXR1030 (GPC3-targeted CAR T cell that co-expressed GOT2) demonstrated superior in vivo efficacy in aggressive solid tumor xenograft models, and showed favorable attributes in vitro including an enhanced cytokine production profile, a less-differentiated T cell phenotype with lower expression of stress and exhaustion markers, an enhanced metabolic profile and increased proliferation in TME-like conditions. CONCLUSIONS: Together, these results demonstrated that co-expression of GOT2 can substantially improve the overall antitumor activity of CAR T cells by inducing broad changes in cellular function and phenotype. These data show that BOXR1030 is an attractive approach to targeting select solid tumors. To this end, BOXR1030 will be explored in the clinic to assess safety, dose-finding, and preliminary efficacy (NCT05120271).

Drug-drug interaction of the anti-TFPI aptamer BAX499 and factor VIII: studies of spatial dynamics of fibrin clot formation in hemophilia A.

BACKGROUND: In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. OBJECTIVE: To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro. METHODS: Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6pmole/m(2) by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C<5%, but the effect was only 25% if FVIII:C>30%). CONCLUSIONS: The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.

ARC15105 is a potent antagonist of von Willebrand factor mediated platelet activation and adhesion.

OBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 µmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. CONCLUSIONS: The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor.

Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

Multiple sequence signals direct recognition and degradation of protein substrates by the AAA+ protease HslUV.

Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V(max). By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease.

Altered tethering of the SspB adaptor to the ClpXP protease causes changes in substrate delivery.

SspB is a dimeric adaptor protein that increases the rate at which ssrA-tagged substrates are degraded by tethering them to the ClpXP protease. Each SspB subunit consists of a folded domain that forms the dimer interface and a flexible C-terminal tail. Ternary delivery complexes are stabilized by three sets of tethering interactions. The C-terminal XB peptide of each SspB subunit binds ClpX, the body of SspB binds one part of the ssrA-tag sequence, and ClpX binds another part of the tag. To test the functional importance of these tethering interactions, we engineered monomeric SspB variants and dimeric variants with different length linkers between the SspB body and the XB peptide and employed substrates with degradation tags that bind ClpX weakly and/or contain extensions between the binding sites for SspB and ClpX. We find that monomeric SspB variants can enhance ClpXP degradation of a subset of substrates, that doubling the number of tethering interactions stimulates degradation via changes in Km and Vmax, and that major alterations in the length of the 48-residue SspB linker cause only small changes in the efficiency of substrate delivery. These results indicate that the properties of the degradation tag and the number of SspB.ClpX tethering interactions are the major factors that determine the extent to which the substrate and ClpX are engaged in ternary delivery complexes.

Engineering controllable protein degradation.

Complex biological networks are regulated via alterations in protein expression, degradation, and function. Synthetic control of these processes allows dissection of natural systems and the design of new networks. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. To engineer controlled degradation, we have designed a series of modified ssrA tags that have weakened interactions with ClpXP. When SspB is present, ClpXP degrades purified substrates bearing these engineered peptide tags 100-fold more efficiently. Importantly, substrates bearing these tags are stable in the absence of SspB in vivo but are rapidly degraded upon SspB induction. Our studies supply a conceptual foundation and working components for controllable degradation, improve mechanistic understanding of adaptor-mediated proteolysis, and demonstrate that the relative importance of adaptor proteins in degradation is correlated with the strength of protease-substrate contacts.

Ribosomal protein S1 binds mRNA and tmRNA similarly but plays distinct roles in translation of these molecules.

Ribosomes stalled during protein synthesis can be rescued by tmRNA, which acts first as a tRNA and then as an mRNA to direct addition of a C-terminal degradation tag to the nascent polypeptide. Ribosomal protein S1 binds tmRNA, but its functional role in tmRNA-mediated tagging is uncertain. To probe interactions between S1 and tmRNA, truncated variants missing one or more of the six contiguous S1 domains were studied. The third S1 domain (R1) plays a critical role in binding tmRNA and mRNA but requires additional N- or C-terminal S1 domains. The binding of S1 and its fragments to tmRNA and mRNA is positively cooperative, and the essential role of the R1 domain may be to mediate protein-protein interactions. Overproduction of N-terminal fragments of S1 in Escherichia coli displaces endogenous S1 from ribosomes, inhibits general protein synthesis, and slows growth but causes little if any disruption of tmRNA-mediated tagging. Moreover, tagging of proteins translated from model mRNAs with either no or an increased requirement for S1 is indistinguishable. These results raise the possibility that S1 plays little or no role in tmRNA-mediated tagging.

In search of an RNA replicase ribozyme.

The theory that an RNA world played a pivotal role in life's evolutionary past has prompted investigations into the scope of RNA catalysis. These efforts have attempted to demonstrate the plausibility of an RNA-based genetic system, which would require RNA molecules that catalyze their own replication. The mechanistic features of modern protein polymerases have been used to guide the laboratory evolution of catalytic RNAs (ribozymes) that exhibit polymerase-like activity. Ribozymes have been developed that recognize a primer-template complex in a general way and catalyze the template-directed polymerization of mononucleotides. These experiments demonstrate that RNA replicase behavior is likely within the catalytic repertoire of RNA, although many obstacles remain to be overcome in order to demonstrate that RNA can catalyze its own replication in a manner that could have sustained a genetic system on the early Earth.

Continuous in vitro evolution of a ribozyme that catalyzes three successive nucleotidyl addition reactions.

Variants of the class I ligase ribozyme, which catalyzes joining of the 3' end of a template bound oligonucleotide to its own 5' end, have been made to evolve in a continuous manner by a simple serial transfer procedure that can be carried out indefinitely. This process was expanded to allow the evolution of ribozymes that catalyze three successive nucleotidyl addition reactions, two template-directed mononucleotide additions followed by RNA ligation. During the development of this behavior, a population of ribozymes was maintained against an overall dilution of more than 10(406). The resulting ribozymes were capable of catalyzing the three-step reaction pathway, with nucleotide addition occurring in either a 5'-->3' or a 3'-->5' direction. This purely chemical system provides a functional model of a multi-step reaction pathway that is undergoing Darwinian evolution.

RNA-catalyzed RNA ligation on an external RNA template.

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside 5'-triphosphates onto the 3' end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.

Substitution of ribonucleotides in the T7 RNA polymerase promoter element.

A systematic analysis was carried out to examine the effects of ribonucleotide substitution at various locations within the promoter element for T7 RNA polymerase. Ribonucleotides could be introduced at most positions without significantly decreasing transcription efficiency. A critical window of residues that were intolerant of RNA substitution was defined for both the nontemplate and template strands of the promoter. These residues are involved in important contacts with the AT-rich recognition loop, specificity loop, and beta-intercalating hairpin of the polymerase. These results highlight the malleability of T7 RNA polymerase in recognizing its promoter element and suggest that promoters with altered backbone conformations may be used in molecular biology applications that use T7 RNA polymerase for in vitro transcription.