IgG Domain Research Articles

Treatment of Parkinson's disease with biologics that penetrate the blood-brain barrier via receptor-mediated transport.

Parkinson's disease (PD) is characterized by neurodegeneration of nigral-striatal neurons in parallel with the formation of intra-neuronal α-synuclein aggregates, and these processes are exacerbated by neuro-inflammation. All 3 components of PD pathology are potentially treatable with biologics. Neurotrophins, such as glial derived neurotrophic factor or erythropoietin, can promote neural repair. Therapeutic antibodies can lead to disaggregation of α-synuclein neuronal inclusions. Decoy receptors can block the activity of pro-inflammatory cytokines in brain. However, these biologic drugs do not cross the blood-brain barrier (BBB). Biologics can be made transportable through the BBB following the re-engineering of the biologic as an IgG fusion protein, where the IgG domain targets an endogenous receptor-mediated transcytosis (RMT) system within the BBB, such as the insulin receptor or transferrin receptor. The receptor-specific antibody domain of the fusion protein acts as a molecular Trojan horse to ferry the biologic into brain via the BBB RMT pathway. This review describes the re-engineering of all 3 classes of biologics (neurotrophins, decoy receptor, therapeutic antibodies) for BBB delivery and treatment of PD. Targeting the RMT pathway at the BBB also enables non-viral gene therapy of PD using lipid nanoparticles (LNP) encapsulated with plasmid DNA encoding therapeutic genes. The surface of the lipid nanoparticle is conjugated with a receptor-specific IgG that triggers RMT of the LNP across the BBB in vivo.

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite β-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

Neutralizing antibody activity against the B.1.617.2 (delta) variant 8 months after two-dose vaccination with BNT162b2 in health care workers

ObjectivesHumoral immunity wanes over time after two-dose BNT162b2 vaccination. Emerging variants of concern, such as the B.1.617.2 (delta) variant, are increasingly responsible for breakthrough infections owing to their higher transmissibility and partial immune escape. Longitudinal data on neutralization against the B.1.617.2 (delta) variant are urgently needed to guide vaccination strategies. MethodsIn this prospective longitudinal observational study, anti-S1 IgG and surrogate neutralizing antibodies were measured in 234 collected samples from 60 health care workers after two-dose vaccination with BNT162b2 at five different time points over an 8-month period. In addition, antibodies against various severe acute respiratory syndrome coronavirus 2 epitopes, neutralization against wild-type, and cross-neutralization against the B.1.617.2 (delta) variant using a live virus assay were measured 6 weeks (second time point) and 8 months (last time point) after first vaccine dose. ResultsMedian (interquartile range) anti-S1 IgG, surrogate neutralizing, and receptor-binding domain antibodies decreased significantly from a maximum level of 147 (102–298), 97 (96–98), and 20 159 (19 023–21 628) to 8 (4–13), 92 (80–96), and 15 324 (13 055–17 288) at the 8-month follow-up, respectively (p < 0.001 for all). Neutralization against the B.1.617.2 (delta) variant was detectable in all 36 (100%) participants at 6 weeks and in 50 of 53 (94%) participants 8 months after first vaccine dose. Median (interquartile) ID50 as determined by a live virus assay decreased from 160 (80–320) to 40 (20–40) (p < 0.001). DiscussionAlthough humoral immunity wanes over time after two-dose BNT162b2 vaccination in healthy individuals, most individuals still had detectable neutralizing activity against the B.1.617.2 (delta) variant after 8 months.

Kinetics of Blood-Brain Barrier Transport of Monoclonal Antibodies Targeting the Insulin Receptor and the Transferrin Receptor.

Biologic drugs are large molecule pharmaceuticals that do not cross the blood–brain barrier (BBB), which is formed by the brain capillary endothelium. Biologics can be re-engineered for BBB transport as IgG fusion proteins, where the IgG domain is a monoclonal antibody (MAb) that targets an endogenous BBB transporter, such as the insulin receptor (IR) or transferrin receptor (TfR). The IR and TfR at the BBB transport the receptor-specific MAb in parallel with the transport of the endogenous ligand, insulin or transferrin. The kinetics of BBB transport of insulin or transferrin, or an IRMAb or TfRMAb, can be quantified with separate mathematical models. Mathematical models to estimate the half-time of receptor endocytosis, MAb or ligand exocytosis into brain extracellular space, or receptor recycling back to the endothelial luminal membrane were fit to the brain uptake of a TfRMAb or a IRMAb fusion protein in the Rhesus monkey. Model fits to the data also allow for estimates of the rates of association of the MAb in plasma with the IR or TfR that is embedded within the endothelial luminal membrane in vivo. The parameters generated from the model fits can be used to estimate the brain concentration profile of the MAb over time, and this brain exposure is shown to be a function of the rate of clearance of the antibody fusion protein from the plasma compartment.

\u201cNon-criteria\u201d antiphospholipid antibodies add value to antiphospholipid syndrome diagnoses in a large Chinese cohort

BackgroundDespite expansion in the 2006 Sydney antiphospholipid syndrome (APS) classification criteria to include IgG/IgM anti-β2-glycoprotein (aβ2GPI) antibodies in addition to IgG/IgM anti-cardiolipin antibodies (aCL) and lupus anticoagulant (LAC), some individuals with clinical features of APS remain seronegative (seronegative APS or SNAPS) and are at risk of recurrent thrombosis and pregnancy morbidities. Our aim was to assess the value of “non-criteria” aPL antibodies to detect these SNAPS patients.MethodsOne hundred ninety-two APS patients, 90 SNAPS patients, 193 autoimmune disease controls, and 120 healthy controls were evaluated. Ten antiphospholipid antibodies (aPLs) were tested using commercial kits, including 5 non-criteria aPLs: anti-phosphatidylserine/prothrombin antibodies (aPS/PT) IgG/IgM, aCL IgA, aβ2GPI IgA, and anti-β2GPI Domain 1 (aβ2GPI-D1) IgG.ResultsUp to 60.9% of the SNAPS and 93.5% of APS patients were detected by at least one non-criteria aPL. aPS/PT IgG had the highest Youden index in classifying APS and SNAPS from controls. aPS/PT IgG and aβ2GPI Domain 1 IgG seem to be the most significant risk factors for thrombotic events and pregnancy morbidity, respectively. aPS/PT IgG/IgM and aβ2GPI-D1 IgG were detected in some SNAPS patients, while IgA isotypes of aCL/aβ2GPI tended to appear together with other biomarkers. The combined analysis showed enhanced diagnostic performance with the inclusion of non-criteria aPLs.ConclusionsRecognition of SNAPS patients is critical for clinical management and prevention of potential thrombotic and obstetric adverse events. The non-criteria antiphospholipid antibodies help to identify a considerable portion (60.9%) of these patients who otherwise may remain untreated and at clinical risk.

Bi-functional IgG-lysosomal enzyme fusion proteins for brain drug delivery

Most lysosomal storage disorders affect the central nervous system. However, lysosomal enzymes do not cross the blood-brain barrier (BBB), and intravenous enzyme infusion is not effective for the brain. Lysosomal enzymes can be re-engineered for BBB transport as IgG-enzyme fusion proteins, where the IgG domain is a monoclonal antibody (MAb) against an endogenous BBB receptor/transporter, and which acts as a molecular Trojan horse to deliver the enzyme to brain. However, the problem is retention of high enzyme activity following enzyme fusion to the IgG. The present investigation shows this is possible with a versatile approach that employs fusion of the enzyme to either the IgG heavy chain or light chain using a long flexible linker. The model IgG is a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR). The enzyme activity of the HIRMAb-enzyme fusion protein is preserved for hexosaminidase A, which is mutated in Tay Sachs disease, for protein palmitoylthioesterase-1, which is mutated in Batten disease type 1, acid sphingomyelinase, which is mutated in Niemann Pick disease type A, and beta galactosidase-1, which is mutated in GM1 gangliosidosis.

Domain unfolding of monoclonal antibody fragments revealed by non-reducing SDS-PAGE

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)2, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications.

Neurocognitive and somatic stabilization in pediatric patients with severe Mucopolysaccharidosis Type I after 52 weeks of intravenous brain-penetrating insulin receptor antibody-iduronidase fusion protein (valanafusp alpha): an open label phase 1-2 trial

BackgroundMucopolysaccharidosis (MPS) Type I (MPSI) is caused by mutations in the gene encoding the lysosomal enzyme, α-L-iduronidase (IDUA), and a majority of patients present with severe neurodegeneration and cognitive impairment. Recombinant IDUA does not cross the blood-brain barrier (BBB). To enable BBB transport, IDUA was re-engineered as an IgG-IDUA fusion protein, valanafusp alpha, where the IgG domain targets the BBB human insulin receptor to enable transport of the enzyme into the brain. We report the results of a 52-week clinical trial on the safety and efficacy of valanafusp alpha in pediatric MPSI patients with cognitive impairment. In the phase I trial, 6 adults with attenuated MPSI were administered 0.3, 1, and 3 mg/kg doses of valanafusp alpha by intravenous (IV) infusion. In the phase II trial, 11 pediatric subjects, 2-15 years of age, were treated for 52 weeks with weekly IV infusions of valanafusp alpha at 1, 3, or 6 mg/kg. Assessments of adverse events, cognitive stabilization, and somatic stabilization were made. Outcomes at 52 weeks were compared to baseline.ResultsDrug related adverse events included infusion related reactions, with an incidence of 1.7%, and transient hypoglycemia, with an incidence of 6.4%. The pediatric subjects had CNS involvement with a mean enrollment Development Quotient (DQ) of 36.1±7.1. The DQ, and the cortical grey matter volume of brain, were stabilized by valanafusp alpha treatment. Somatic manifestations were stabilized, or improved, based on urinary glycosaminoglycan levels, hepatic and spleen volumes, and shoulder range of motion.ConclusionClinical evidence of the cognitive and somatic stabilization indicates that valanafusp alpha is transported into both the CNS and into peripheral organs due to its dual targeting mechanism via the insulin receptor and the mannose 6-phosphate receptor. This novel fusion protein offers a pharmacologic approach to the stabilization of cognitive function in MPSI.Trial registrationClinical Trials.Gov, NCT03053089. Retrospectively registered 9 February, 2017; Clinical Trials.Gov, NCT03071341. Registered 6 March, 2017.

Reduction in Brain Heparan Sulfate with Systemic Administration of an IgG Trojan Horse-Sulfamidase Fusion Protein in the Mucopolysaccharidosis Type IIIA Mouse.

Mucopolysaccharidosis Type IIIA (MPSIIIA), also known as Sanfilippo A syndrome, is an inherited neurodegenerative disease caused by mutations in the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH), also known as sulfamidase. Mutations in the SGSH enzyme, the only mammalian heparan N-sulfatase, cause accumulation of lysosomal inclusion bodies in brain cells comprising heparan sulfate (HS) glycosaminoglycans (GAGs). Treatment of MPSIIIA with intravenous recombinant SGSH is not possible because this large molecule does not cross the blood-brain barrier (BBB). BBB penetration by SGSH was enabled in the present study by re-engineering this enzyme as an IgG-SGSH fusion protein, where the IgG domain is a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), designated the cTfRMAb. The IgG domain of the fusion protein acts as a molecular Trojan horse to deliver the enzyme into brain via transport on the endogenous BBB TfR. The cTfRMAb-SGSH fusion protein bound to the mouse TfR with high affinity, ED50 = 0.74 ± 0.07 nM, and retained high SGSH enzyme activity, 10 043 ± 1003 units/mg protein, which is comparable to recombinant human SGSH. Male and female MPSIIIA mice, null for the SGSH enzyme, were treated for 6 weeks with thrice-weekly intraperitoneal injections of vehicle, 5 mg/kg of the cTfRMAb alone, or 5 mg/kg of the cTfRMAb-SGSH fusion protein, starting at the age of 2 weeks, and were euthanized 1 week after the last injection. Brain and liver HS, as determined by liquid chromatography-mass spectrometry, were elevated 30-fold and 36-fold, respectively, in the MPSIIIA mouse. Treatment of the mice with the cTfRMAb-SGSH fusion protein caused a 70% and 85% reduction in brain and liver HS, respectively. The reduction in brain HS was associated with a 28% increase in latency on the rotarod test of motor activity in male mice. The mice exhibited no injection related reactions, and only a low titer end of study antidrug antibody response was observed. In conclusion, substantial reductions in brain pathologic GAGs in a murine model of MPSIIIA are produced by chronic systemic administration of an IgG-SGSH fusion protein engineered to penetrate the BBB via receptor-mediated transport.

Peptides of the variable IgG domain as potential biomarker candidates in primary open-angle glaucoma (POAG).

Autoantibody profiling has gained increasing interest in the research field of glaucoma promising the detection of highly specific and sensitive marker candidates for future diagnostic purposes. Recent studies demonstrated that immune responses are characterized by the expression of congruent or similar complementarity determining regions (CDR) in different individuals and could be used as molecular targets in biomarker discovery. Main objective of this study was to characterize glaucoma-specific peptides from the variable region of sera-derived immunoglobulins using liquid chromatography--mass spectrometry (LC-MS)-based quantitative proteomics. IgG was purified from sera of 13 primary open-angle glaucoma patients (POAG) and 15 controls (CTRL) and subsequently digested into Fab and Fc by papain. Fab was further purified, tryptic digested and measured by LC-MS/MS. Discovery proteomics revealed in total 75 peptides of the variable IgG domain showing significant glaucoma-related level changes (P < 0.05; log2 fold change ≥ 0.5): 6 peptides were high abundant in POAG sera, whereas 69 peptides were low abundant in comparison to CTRL group. Via accurate inclusion mass screening strategy 28 IgG V domain peptides were further validated showing significantly decreased expression levels in POAG sera. Amongst others 5 CDR1, 2 CDR2 and 1 CDR3 sequences. In addition, we observed significant shifts in the variable heavy chain family distribution and disturbed κ/λ ratios in POAG patients in contrast to CTRL. These findings strongly indicate that glaucoma is accompanied by systemic effects on antibody production and B cell maturation possibly offering new prospects for future diagnostic or therapy purposes.

Expression of cancer cell-derived IgG and extra domain A-containing fibronectin in salivary adenoid cystic carcinoma

ObjectiveCancer-IgG is a newly-discovered molecule, mainly derived from epithelial carcinoma cells and is significantly correlated with differentiation, metastasis, local invasion, and poor prognosis of many cancers. In our previous study we detected IgG expression in oral epithelial carcinoma, including salivary adenoid cystic carcinoma (SACC), using an IgG-specific commercial antibody. Here, we explored the correlation between cancer-IgG and clinicopathological features of SACC. DesignA total of 68 human SACC tissue specimens and 2 siRNAs were used to analyze the correlation between cancer-IgG and extra domain A (EDA+)-containing fibronectin using the cancer-IgG-specific monoclonal antibody, RP215. ResultsWe found an unexpected correlation between cancer-IgG and EDA+ fibronectin, both of which showed aberrant expression in SACC tissue samples. Both were highly expressed in SACC with nerve invasion. In our previous study, EDA+ fibronectin overexpression in SACC cells decreased N-cadherin expression. In the present study, we used SACC-83 cells, wherein EDA+ fibronectin is overexpressed and cancer-IgG is knocked down. EDA+ fibronectin expression was reduced with cancer-IgG knockdown, while cancer-IgG expression did not affect EDA+ fibronectin overexpression. Furthermore, knockdown of non-B cell-derived IgG in SACC cells decreased cellular motility (P<0.05) as well as increased E-cadherin and alpha-smooth muscle actin levels. ConclusionThe results suggest that cancer IgG potentially regulates EDA+ fibronectin expression, thereby suggesting possible new therapeutic approaches for SACC.

Very High Plasma Concentrations of a Monoclonal Antibody against the Human Insulin Receptor Are Produced by Subcutaneous Injection in the Rhesus Monkey.

Brain penetration of recombinant protein drugs is possible following the re-engineering of the drug as an IgG fusion protein. The IgG domain is a monoclonal antibody (mAb) against an endogenous blood-brain barrier (BBB) receptor transporter, such as the insulin receptor. One such mAb targets the human insulin receptor (HIR) and is active in Rhesus monkeys. Prior work has measured the plasma pharmacokinetics of HIRMAb-derived fusion proteins following intravenous (IV) infusion. However, an alternative method of administration for chronic treatment of brain disease is the subcutaneous (SQ) route. The extent to which an antibody against the insulin receptor undergoes systemic distribution and clearance is unknown. Therefore, in the present study, the rate of plasma clearance of the HIRMAb is measured in Rhesus monkeys following IV or SQ administration of 3, 10, and 30 mg/kg doses of the antibody. The HIRMAb is readily absorbed into the systemic circulation following SQ injection with a 42% plasma bioavailability. The rate of plasma clearance of the antibody, 0.04-0.06 mL/min/kg, is the same following either IV or SQ administration. Owing to the slow rate of plasma clearance of the antibody, high concentrations of the HIRMAb are sustained in plasma for days after the SQ injection. The plasma concentration of the HIRMAb exceeds 0.8 mg/mL, which is 9% of the entire plasma IgG pool in the primate, after the SQ injection of the high dose, 30 mg/kg, of the antibody. In summary, the pharmacokinetics of plasma clearance of the HIRMAb are such that HIRMAb-derived fusion proteins can be developed as protein therapeutics for the brain with chronic SQ administration on a weekly or twice-weekly regimen.

Insulin Receptor Antibody-α-N-Acetylglucosaminidase Fusion Protein Penetrates the Primate Blood-Brain Barrier and Reduces Glycosoaminoglycans in Sanfilippo Type B Fibroblasts.

Mucopolysaccharidosis Type IIIB (MPSIIIB) is caused by mutations in the gene encoding the lysosomal enzyme, α-N-acetylglucosaminidase (NAGLU). MPSIIIB presents with severe disease of the central nervous system, but intravenous NAGLU enzyme replacement therapy has not been developed because the NAGLU enzyme does not cross the blood-brain barrier (BBB). A BBB-penetrating form of the enzyme was produced by re-engineering NAGLU as an IgG-enzyme fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb traverses the BBB via transport on the endogenous insulin receptor and acts as a molecular Trojan horse to ferry the fused NAGLU across the BBB from blood. The NAGLU was fused to the carboxyl terminus of each heavy chain of the HIRMAb via an extended 31-amino acid linker, and the fusion protein is designated HIRMAb-LL-NAGLU. The fusion protein retains high affinity binding to the HIR, and on a molar basis has an enzyme activity equal to that of recombinant human NAGLU. Treatment of MPSIIIB fibroblasts with the fusion protein normalizes intracellular NAGLU enzyme activity and reduces sulfate incorporation into intracellular glycosoaminoglycan. The fusion protein is targeted to the lysosomal compartment of the cells as shown by confocal microscopy. The fusion protein was radiolabeled with the [(125)I]-Bolton-Hunter reagent and injected intravenously in the adult Rhesus monkey. The fusion protein was rapidly cleared from plasma by all major peripheral organs. The high brain uptake of the fusion protein, 1% injected dose/brain, enables normalization of brain NAGLU enzyme activity with a therapeutic dose of 1 mg/kg. The HIRMAb-LL-NAGLU fusion protein is a new treatment of the brain in MPSIIIB, which can be administered by noninvasive intravenous infusion.

Insulin receptor antibody‐iduronate 2‐sulfatase fusion protein: Pharmacokinetics, anti‐drug antibody, and safety pharmacology in Rhesus monkeys

Mucopolysaccharidosis (MPS) Type II is caused by mutations in the gene encoding the lysosomal enzyme, iduronate 2-sulfatase (IDS). The majority of MPSII cases affect the brain. However, enzyme replacement therapy with recombinant IDS does not treat the brain, because IDS is a large molecule drug that does not cross the blood-brain barrier (BBB). To enable BBB penetration, IDS has been re-engineered as an IgG-IDS fusion protein, where the IgG domain is a monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and the HIRMAb domain of the fusion protein acts as a molecular Trojan horse to ferry the fused IDS into brain from blood. The present study reports on the first safety pharmacology and pharmacokinetics study of the HIRMAb-IDS fusion protein. Juvenile male Rhesus monkeys were infused intravenously (IV) weekly for 26 weeks with 0, 3, 10, or 30 mg/kg of the HIRMAb-IDS fusion protein. The plasma clearance of the fusion protein followed a linear pharmacokinetics profile, which was equivalent either with measurements of the plasma concentration of immunoreactive HIRMAb-IDS fusion protein, or with assays of plasma IDS enzyme activity. Anti-drug antibody (ADA) titers were monitored monthly, and the ADA response was primarily directed against the variable region of the HIRMAb domain of the fusion protein. No infusion related reactions or clinical signs of immune response were observed during the course of the study. A battery of safety pharmacology, clinical chemistry, and tissue histopathology showed no signs of adverse events, and demonstrate the safety profile of chronic treatment of primates with 3-30 mg/kg weekly IV infusion doses of the HIRMAb-IDS fusion protein.

Insulin receptor antibody-sulfamidase fusion protein penetrates the primate blood-brain barrier and reduces glycosoaminoglycans in Sanfilippo type A cells.

Mutations in the lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (SGSH), also called sulfamidase, cause accumulation of lysosomal inclusion bodies in the brain of children born with mucopolysaccharidosis type IIIA, also called Sanfilippo type A syndrome. Enzyme replacement therapy with recombinant SGSH does not treat the brain because the enzyme is a large molecule drug that does not cross the blood–brain barrier (BBB). A BBB-penetrating form of SGSH was produced by re-engineering the enzyme as an IgG fusion protein, where the IgG domain is a monoclonal antibody (mAb) against the human insulin receptor (HIR). The HIRMAb domain of the HIRMAb–SGSH fusion protein acts as a molecular Trojan horse to ferry the fused enzyme across the BBB. The HIRMAb–SGSH was produced in stably transfected host cells and purified to homogeneity by protein A chromatography. The fusion protein reacted with antibodies against either human IgG or SGSH on Western blotting. High affinity binding to the HIR was retained following SGSH fusion to the HIRMAb, with an EC50 of 0.33 ± 0.05 nM in an HIR binding ELISA. The SGSH enzyme activity of the HIRMAb–SGSH fusion protein was 4712 ± 388 units/mg protein based on a two-step fluorometric enzyme assay. The HIRMAb–SGSH was taken up by lysosomes in MPSIIIA fibroblasts, and treatment of these cells with the fusion protein caused an 83% reduction in sulfate incorporation into lysosomal glycosoaminoglycans. The HIRMAb–SGSH fusion protein was radiolabeled with the [125I]-Bolton–Hunter reagent and injected intravenously in the Rhesus monkey. The brain uptake of the fusion protein was high, ∼1% injected dose/brain. Calculations, based on this level of brain uptake, suggest normalization of brain SGSH enzyme activity is possible following administration of therapeutic doses of the fusion protein. These studies describe a novel IgG–SGSH fusion protein that is a new noninvasive treatment of the brain in MPS type IIIA.

Blood-Brain Barrier Molecular Trojan Horse Enables Imaging of Brain Uptake of Radioiodinated Recombinant Protein in the Rhesus Monkey

Recombinant proteins are large molecule drugs that do not cross the blood-brain barrier (BBB). However, BBB-penetration of protein therapeutics is enabled by re-engineering the recombinant protein as IgG fusion proteins. The IgG domain is a monoclonal antibody (mAb) against an endogenous BBB receptor-mediated transport system, such as the human insulin receptor (HIR), and acts as a molecular Trojan horse to ferry the fused protein across the BBB. In the present study, a recombinant lysosomal enzyme, iduronate 2-sulfatase (IDS), is fused to the HIRMAb, and BBB penetration of the IDS alone vs the HIRMAb-IDS fusion protein is compared in the Rhesus monkey. Recombinant IDS and the HIRMAb-IDS fusion protein were radiolabeled with indirect iodination with the [(125)I]-Bolton-Hunter reagent and with direct iodination with Iodogen/[(125)I]-idodine. IDS and the HIRMAb-IDS fusion protein have comparable plasma pharmacokinetics and uptake by peripheral organs. IDS does not cross the BBB. The HIRMAb-IDS fusion protein crosses the BBB and the brain uptake is 1% of injected dose/brain. Brain imaging shows HIRMAb-IDS penetration to all parts of brain, and immunoprecipitation of brain radioactivity shows intact fusion protein in brain. The use of BBB molecular Trojan horses enables brain imaging of recombinant proteins that are re-engineered for BBB transport.

IgG-Enzyme Fusion Protein: Pharmacokinetics and Anti-Drug Antibody Response in Rhesus Monkeys

The chronic administration of recombinant fusion proteins in preclinical animal models may generate an immune response and the formation of antidrug antibodies (ADA). Such ADAs could alter the plasma pharmacokinetics of the fusion protein, and mask any underlying toxicity of the recombinant fusion protein. In the present study, a model IgG-enzyme fusion protein was evaluated with chronic dosing of rhesus monkeys. The IgG domain of the fusion protein is a genetically engineered monoclonal antibody (mAb) against the human insulin receptor (HIR), which is shown to cross-react with the primate insulin receptor. The enzyme domain of the fusion protein is human iduronidase (IDUA), the lysosomal enzyme mutated in Mucopolysaccharidosis Type I (MPSI). MPSI affects the brain, but enzyme replacement therapy is not effective for the brain, because IDUA does not cross the blood-brain barrier (BBB). The HIRMAb domain of the fusion protein acts as a molecular Trojan horse to deliver the IDUA across the BBB. The HIRMAb-IDUA fusion protein was administered to rhesus monkeys with weekly intravenous infusions of 3-30 mg/kg for 6 months, and the pharmacokinetics, immune response, and tissue toxicology were assessed. The pharmacokinetics of plasma clearance of the fusion protein was determined with measurements of plasma IDUA enzyme activity. ADAs formed during the course of the 6 months of treatment, as determined by a sandwich ELISA. However, the plasma clearance of the fusion protein at the start and end of the 6-month study was comparable at all drug doses. Fusion protein administration for 6 months showed no evidence of chronic tissue toxicity. These studies demonstrate that the immune response produced with chronic treatment of primates with an IgG-enzyme fusion protein has no effect on the pharmacokinetics of plasma clearance of the fusion protein.

Selective plasma pharmacokinetics and brain uptake in the mouse of enzyme fusion proteins derived from species-specific receptor-targeted antibodies

Enzymes may be re-engineered for brain drug targeting as an IgG-enzyme fusion protein, where the IgG is a monoclonal antibody (MAb) against an endogenous blood–brain barrier (BBB) receptor transporter, such as the insulin receptor or transferrin receptor (TfR). Iduronate 2-sulfatase (IDS) is fused to the heavy chain of a genetically engineered MAb against the human insulin receptor (HIR). Neither the HIRMAb alone, nor the HIRMAb–IDS fusion protein, is delivered across the BBB in the mouse, owing to lack of cross-reactivity of the HIRMAb with the insulin receptor in the mouse. The uptake of the HIRMAb–IDS fusion protein in peripheral organs exceeds that of the HIRMAb, which is attributed to uptake mediated via the mannose-6 phosphate receptor in non-brain organs. In contrast to the lack of BBB transport of the HIRMAb–IDS fusion protein, there is high BBB penetration in the mouse of an IDS fusion protein and a chimeric MAb against the mouse TfR. The comparison of the brain distribution of two different IgG-IDS fusion proteins, with different reactivity for an endogenous BBB receptor, illustrates the difference in brain targeting of a biopharmaceutical caused by the targeting properties of the IgG domain of the fusion protein.

Glycemic Control and Chronic Dosing of Rhesus Monkeys with a Fusion Protein of Iduronidase and a Monoclonal Antibody Against the Human Insulin Receptor

Hurler's syndrome, or mucopolysaccharidosis type I, is a lysosomal storage disorder caused by mutations in the gene encoding the lysosomal enzyme iduronidase (IDUA). The disease affects both peripheral tissues and the central nervous system (CNS). Recombinant IDUA treatment does not affect the CNS, because IDUA does not cross the blood-brain barrier (BBB). To enable BBB penetration, human IDUA was re-engineered as an IgG-IDUA fusion protein, where the IgG domain is a genetically engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb penetrates the brain from the blood via transport on the endogenous BBB insulin receptor and acts as a molecular Trojan horse to deliver the fused IDUA to the brain. Before human testing, the HIRMAb-IDUA fusion protein was evaluated in a 6-month weekly dosing toxicology study at doses of 0, 3, 9, and 30 mg/kg/week of the fusion protein administered to 40 rhesus monkeys. The focus of the present study is the effect of chronic high dose administration of this fusion protein on plasma glucose and long-term glycemic control. The results show that the HIRMAb has weak insulin agonist activity and causes hypoglycemia at the high dose, 30 mg/kg, after intravenous infusion in normal saline. When dextrose is added to the saline infusion solution, no hypoglycemia is observed at any dose. An intravenous glucose tolerance test performed at the end of the 6 months of chronic treatment showed no change in glucose tolerance at any dose of the HIRMAb-IDUA fusion protein.

Exogenous Recombinant Dimeric Neuropilin-1 Is Sufficient to Drive Angiogenesis

Neuropilin-1 (NRP-1) is present on the cell surface of endothelial cells, or as a soluble truncated variant. Membrane NRP-1 is proposed to enhance angiogenesis by promoting the formation of a signaling complex between vascular endothelial growth factor-A(165) (VEGF-A(165)), VEGF receptor-2 (VEGFR-2) and heparan sulfate, whereas the soluble NRP-1 is thought to act as an antagonist of signaling complex formation. We have analyzed the angiogenic potential of a chimera comprising the entire extracellular NRP-1 region dimerized through an Fc IgG domain and a monomeric truncated NRP-1 variant. Both NRP-1 proteins stimulated tubular morphogenesis and cell migration in HDMECs and HUVECs. Fc rNRP-1 was able to induce VEGFR-2 phosphorylation and expression of the VEGFR-2 specific target, regulator of calcineurin-1 (RCAN1.4). siRNA mediated gene silencing of VEGFR-2 revealed that VEGFR-2 was required for Fc rNRP-1 mediated activation of the intracellular signaling proteins PLC-γ, AKT, and MAPK and tubular morphogenesis. The stimulatory activity was independent of VEGF-A(165). This was evidenced by depleting the cell culture of exogenous VEGF-A(165), and using instead for routine culture VEGF-A(121), which does not interact with NRP-1, and by the inability of VEGF-A sequestering antibodies to inhibit the angiogenic activity of the NRP proteins. Analysis of angiogenesis over a period of 6 days in an in vitro fibroblast/endothelial co-culture model revealed that Fc rNRP-1 could induce endothelial cell tubular morphogenesis. Thus, we conclude that soluble Fc rNRP-1 is a VEGF-A(165)-independent agonist of VEGFR-2 and stimulates angiogenesis in endothelial cells.