CDR3 Variants of the TXB2 Shuttle with Increased TfR1 Association Rate and Enhanced Brain Penetration.

Since the delivery of biologic drugs to the brain is greatly hampered by the existence of the blood-brain barrier (BBB), brain shuttles are being developed to enhance therapeutic efficacy. As we have previously shown, efficient and selective brain delivery was achieved with TXB2, a cross-species reactive, anti-TfR1 VNAR antibody. To further explore the limits of brain penetration, we conducted restricted randomization of the CDR3 loop, followed by phage display to identify improved TXB2 variants. The variants were screened for brain penetration in mice using a 25 nmol/kg (1.875 mg/kg) dose and a single 18 h timepoint. A higher kinetic association rate to TfR1 correlated with improved brain penetration in vivo. The most potent variant, TXB4, showed a 3.6-fold improvement over TXB2, which had on average 14-fold higher brain levels when compared to an isotype control. Like TXB2, TXB4 retained brain specificity with parenchymal penetration and no accumulation in other organs. When fused with a neurotensin (NT) payload, it led to a rapid drop in body temperature upon transport across the BBB. We also showed that fusion of TXB4 to four therapeutic antibodies (anti-CD20, anti-EGFRvIII, anti-PD-L1 and anti-BACE1) improved their brain exposure between 14- to 30-fold. In summary, we enhanced the potency of parental TXB2 brain shuttle and gained a critical mechanistic understanding of brain delivery mediated by the VNAR anti-TfR1 antibody.

Single domain shark VNAR antibodies neutralize SARS-CoV-2 infection in vitro.

Single domain shark variable domain of new antigen receptor (VNAR) antibodies can offer a viable alternative to conventional Ig-based monoclonal antibodies in treating COVID-19 disease during the current pandemic. Here we report the identification of neutralizing single domain VNAR antibodies selected against the severe acute respiratory syndrome coronavirus 2 spike protein derived from the Wuhan variant using phage display. We identified 56 unique binding clones that exhibited high affinity and specificity to the spike protein. Of those, 10 showed an ability to block both the spike protein receptor binding domain from the Wuhan variant and the N501Y mutant from interacting with recombinant angiotensin-converting enzyme 2 (ACE2) receptor in vitro. In addition, three antibody clones retained in vitro blocking activity when the E484K spike protein mutant was used. The inhibitory property of the VNAR antibodies was further confirmed for all 10 antibody clones using ACE2 expressing cells with spike protein from the Wuhan variant. The viral neutralizing potential of the VNAR clones was also confirmed for the 10 antibodies tested using live Wuhan variant virus in in vitro cell infectivity assays. Single domain VNAR antibodies, due to their low complexity, small size, unique epitope recognition, and formatting flexibility, should be a useful adjunct to existing antibody approaches to treat COVID-19.

Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1.

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

Brain delivery of biologics using a cross-species reactive transferrin receptor 1 VNAR shuttle.

Transferrin receptor 1 (TfR1) mediated transcytosis is an attractive strategy to enhance brain uptake of protein drugs, but translation remains a challenge. Here, a single domain shark antibody VNAR fragment (TXB2) with similar affinity to murine and human TfR1 was used to shuttle protein cargo into the brain. TXB2 was fused to a human IgG1 Fc domain (hFc) or to the amyloid-ß (Aß) antibody bapineuzumab (Bapi). TXB2-hFc displayed 20-fold higher brain concentrations compared with a control VNAR-hFc at 18 hours post-injection in wt mice. At the same time point, brain concentrations of Bapi-TXB2 was threefold higher than Bapi. In transgenic mice overexpressing human Aß, the brain-to-blood concentration ratio increased with time due to interaction with intracerebral Aß deposits. The relatively stable threefold difference between Bapi-TXB2 and Bapi was observed for up to 6 days after injection. PET imaging and ex vivo autoradiography revealed more parenchymal distribution of Bapi-TXB2 compared with Bapi. In conclusion, the TXB2 VNAR shuttle markedly increased brain uptake of protein cargo and increased brain concentrations of the Aß binding antibody Bapi.

VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry.

B cell-activating factor (BAFF) plays a dominant role in the B cell homeostasis. However, excessive BAFF promotes the development of autoreactive B-cells and several antibodies have been developed to block its activity. Bispecific antibodies with added functionality represent the next wave of biologics that may be more effective in the treatment of complex autoimmune disease. The single variable domain from the immunoglobulin new antigen receptor (VNAR) is one of the smallest antibody recognition units that could be combined with monospecific antibodies to develop bispecific agents. We isolated a panel of BAFF-binding VNARs with low nM potency from a semi-synthetic phage display library and examined their functional activity. The anti-BAFF VNARs blocked the binding of BAFF to all three of its receptors (BR3, TACI and BCMA) and the presence of the conserved DXL receptor motif found in the CDR3 regions suggests molecular mimicry as the mechanism of antagonism. One clone was formatted as an Fc fusion for functional testing and it was found to inhibit both mouse and human BAFF with equal potency ex vivo in a splenocyte proliferation assay. In mice, subchronic administration reduced the number of immature and transitional intermediates B cells and mature B cell subsets. These results indicate that VNAR single domain antibodies function as selective B-cell inhibitors and offer an alternative molecular format for targeting B-cell disorders.

Pharmacogenomic, physiological, and biochemical investigations on safety and efficacy biomarkers associated with the peroxisome proliferator-activated receptor-gamma activator rosiglitazone in rodents: a translational medicine investigation.

Peroxisome proliferator-activated receptor (PPAR)-gamma modulators, a class of antidiabetic drugs, have been associated with cardiovascular risks in type 2 diabetes in humans. The objective of this study was to explore possible cardiovascular risk biomarkers associated with PPAR-gamma in rodents that could provide an alert for risk to humans. Normal, myocardial infarction-induced heart failure (HF) or Zucker diabetic fatty (ZDF) rats were used. Rats (n = 5-6) were treated with either vehicle or rosiglitazone (RGZ; 3 or 45 mg/kg/day p.o.) for 4 weeks. Biomarkers for potential cardiovascular risks were assessed, including 1) ultrasound for cardiac structure and function; 2) neuroendocrine and hormonal plasma biomarkers of cardiovascular risk; 3) pharmacogenomic profiling of cardiac and renal tissue by targeted tissue low-density gene array representing ion channels and transporters, and components of the renin-angiotensin-aldosterone system; and 4) immunohistochemistry for cardiac fibrosis, hypertrophy, and inflammation (macrophages and tumor necrosis factor-alpha). HF was confirmed by increase in cardiac brain natriuretic peptide expression (p < 0.01) and echocardiography. Adequate exposure of RGZ was confirmed by pharmacokinetics (plasma drug levels) and the pharmacodynamic biomarker adiponectin. In normal or HF rats, RGZ had no negative effects on any of the biomarkers investigated. Similarly, RGZ had no significant effects on gene expression except for the increase in interleukin-6 mRNA expression in the heart and decrease in epithelial sodium channel beta in the kidney. In contrast, echocardiography showed improved cardiac structure and function after RGZ in ZDF rats. Taken together, this study suggests a limited predictive power of these preclinical models in respect to observed clinical adverse effects associated with RGZ.

Correlation between 2- and 3-dimensional assessment of tumor volume and vascular density by ultrasonography in a transgenic mouse model of mammary carcinoma.

OBJECTIVE: Visualization and quantification of angiogenesis are instrumental in development of antiangiogenic therapy. Although both 2-dimensional (2D) and 3-dimensional (3D) ultrasonography have been used to monitor tumor growth and vasculature development, the correlation between them has not been sufficiently investigated. We hereby investigated the 2D and 3D sonographic correlation for tumor volume and vascular density confirmed by histologic assessment in the polyoma virus middle T antigen (PyMT) mouse model of mammary carcinoma. METHODS: Female PyMT mouse tumors were evaluated by ultrasonography in the 2D region of interest (ROI), 3D tumor volume, and 2D and 3D microvascular density after a bolus infusion of a nontargeted contrast-enhanced microbubble agent. Texas Red-dextran was used for quantitative histologic assessment of the tumor microvascular density. RESULTS: The individual 2D tumor ROI area correlated with the 3D tumor volume throughout the 2-week period. However, the extent of the increase in the 3D volume (380%; P < .01; n = 10) was higher than that of the 2D ROI area (72%; P < .01; n = 8-11). A significant and comparable increase in vascular density accessed by both 2D (87%; P < .05; n = 8) and 3D (64%; P < .05; n = 8) imaging was documented. Vascular density obtained through 3D imaging correlated significantly with 2D measurement. These data were confirmed by Texas Red-dextran quantification of vascular density. Conclusions. This study showed a valid application of sonographically based imaging technology in tumor volume and vascular density assessment as well as their 2D and 3D correlation, of which tumor vascular density measured by 2D ultrasonography appeared to be better correlated with the 3D data. Our data indicate that ultrasonography can be applied for real-time, accurate, noninvasive imaging of the tumor volume and vascular density in preclinical models.

Translational medicine--a paradigm shift in modern drug discovery and development: the role of biomarkers.

The success rate of novel medical entities that are submitted for registration by the regulatory agencies and followed successful marketing has been stagnating for the past decade. Failure in efficacy and safety continue to be the prime hurdles and causes of failure. Translational medicine is a new function within the pharmaceutical industry R&D organization aimed to improve the predictability and success of drug discovery and development. Biomarkers are the essence of the translational medicine strategy focus on disease biomarker, patient selection, pharmacodynamic responses (efficacy and safety) target validation, compound-target interaction). Successful deployment of biomarkers research, validation and implementation is adopted and embraced as key strategy to improved the drug discovery and development towards new medical entities.

Cognitive endpoints as disease biomarkers: optimizing the congruency of preclinical models to the clinic.

Cognition is a complex set of processes, including attention, learning and memory, that refers to the capacity to encode, consolidate, store and retrieve recent and remotely stored fact (semantic) and experience-based (episodic) memory. The development of cognitive enhancers is of particular importance to society and the pharmaceutical industry, as cognitive dysfunctions are observed across a wide range of neuropsychiatric and neurodegenerative disorders; however, developing such therapeutics has proven difficult. There is poor congruency between the abundance of positive results observed in animal studies compared with clinical outcome. For example, from 1982 to 2002 there was a 6000% increase in studies on cognitive processing in rodents that had little or no impact on the outcome of phase II and III clinical trials. The effects of therapeutics on models of cognition that demonstrate the potential to improve preclinical-to-clinical congruency, focusing on attention, impulsivity and episodic memory, are summarized in this review. Changes in attention, impulsivity and episodic memory are tractable 'disease biomarkers' that correlate with the disease phenotypes that are potential therapeutic targets. In the context of the development of cognitive enhancing drugs, one of the major goals of translational medicine is to improve the congruency between preclinical models and clinical results. Improved translatability could improve discovery, validation and implementation of biomarkers to inform clinical outcome studies and decision making, and to establish proof-of-concept for efficacy and safety based on targeted mechanisms of action.