A long-acting C-natriuretic peptide for achondroplasia.

The C-natriuretic peptide (CNP) analog vosoritide has recently been approved for treatment of achondroplasia in children. However, the regimen requires daily subcutaneous injections in pediatric patients over multiple years. The present work sought to develop a long-acting CNP that would provide efficacy equal to or greater than that of vosoritide but require less frequent injections. We used a technology for half-life extension, whereby a drug is attached to tetra-polyethylene glycol hydrogels (tetra-PEG) by ß-eliminative linkers that cleave at predetermined rates. These hydrogels-fabricated as uniform ∼60-µm microspheres-are injected subcutaneously, where they serve as a stationary depot to slowly release the drug into the systemic circulation. We prepared a highly active, stable CNP analog-[Gln6,14]CNP-38-composed of the 38 C-terminal amino acids of human CNP-53 containing Asn to Gln substitutions to preclude degradative deamidation. Two microsphere [Gln6,14]CNP-38 conjugates were prepared, with release rates designed to allow once-weekly and once-monthly administration. After subcutaneous injection of the conjugates in mice, [Gln6,14]CNP-38 was slowly released into the systemic circulation and showed biphasic elimination pharmacokinetics with terminal half-lives of ∼200 and ∼600 h. Both preparations increased growth of mice comparable to or exceeding that produced by daily vosoritide. Simulations of the pharmacokinetics in humans indicated that plasma [Gln6,14]CNP-38 levels should be maintained within a therapeutic window over weekly, biweekly, and likely, monthly dosing intervals. Compared with vosoritide, which requires ∼30 injections per month, microsphere [Gln6,14]CNP-38 conjugates-especially the biweekly and monthly dosing-could provide an alternative that would be well accepted by physicians, patients, and patient caregivers.

Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids.

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

A few good reasons to use nanobodies for cancer treatment.

mAbs have been instrumental for targeted cancer therapies. However, their relatively large size and physicochemical properties result in a heterogenous distribution in the tumor microenvironment, usually restricted to the first cell layers surrounding blood vessels, and a limited ability to penetrate the brain. Nanobodies are tenfold smaller, resulting in a deeper tumor penetration and the ability to reach cells in poorly perfused tumor areas. Nanobodies are rapidly cleared from the circulation, which generates a fast target-to-background contrast that is ideally suited for molecular imaging purposes but may be less optimal for therapy. To circumvent this problem, nanobodies have been formatted to noncovalently bind albumin, increasing their serum half-life without majorly increasing their size. Finally, nanobodies have shown superior qualities to infiltrate brain tumors as compared to mAbs. In this review, we discuss why these features make nanobodies prime candidates for targeted therapy of cancer.

Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates.

Thromboprophylaxis is indicated in patients at an elevated risk of developing thrombotic disorders, typically using direct oral anticoagulants or low-molecular-weight heparins. We postulated that transient thromboprophylaxis (days-weeks) could be provided by a single dose of an anticoagulant engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) was used as a model anticoagulant to test the hypothesis that conjugation of thrombin inhibitors to the surface of albumin would provide durable protection against thrombotic insults. Covalent conjugates were formed between albumin and PPACK using click chemistry, and they were tested in vitro using a thrombin activity assay and a clot formation assay. Thromboprophylactic efficacy was tested in mouse models of arterial thrombosis, both chemically induced (FeCl3) and following ischemia-reperfusion (transient middle cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown to have nanomolar potency in both in vitro assays, and following intravenous injection had prolonged circulation. Conjugates did not impact hemostasis (tail clipping) or systemic coagulation parameters in normal mice. Intravenous injection of conjugates prior to FeCl3-induced thrombosis provided significant protection against occlusion of the middle cerebral and common carotid arteries, and injection immediately following ischemia-reperfusion reduced stroke volume measured 3 days after injury by ∼40% in the tMCAO model. The data presented here provide support for the use of albumin-linked anticoagulants as an injectable, long-circulating, safe thromboprophylactic agent. In particular, albumin-PPACK provides significant protection against thrombosis induced by multiple mechanisms, without adversely affecting hemostasis.

A Charge Variant of Bevacizumab Offers Enhanced FcRn-Dependent Pharmacokinetic Half-Life and Efficacy.

BACKGROUND: Lysine variants of monoclonal antibodies (mAbs) result from incomplete clipping of the C-terminal lysine residues of the heavy chain. Although the structure of the lysine variants has been determined for several mAb products, a detailed study that investigates the impact of lysine charge variants on PK/PD and preclinical safety is yet to be published. OBJECTIVE: An in-depth investigation of the impact of C- terminal lysine clipping of mAbs on safety and efficacy for bevacizumab charge variants. METHOD: Charge variant isolation using semi-preparative chromatography is followed by a comparative analysis of FcRn binding, pharmacokinetics, and pharmacodynamics in relevant animal models. RESULTS: K1 variant exhibited improved FcRn binding affinity (4-fold), half-life (1.3-fold), and anti-tumor activity (1.3-fold) as compared to the K0 (main) product. However, the K2 variant, even though exhibited higher FcRn affinity (2-fold), displayed lower half-life (1.6-fold) and anti-tumor activity at medium and low doses. Differential proteomic analysis revealed that seven pathways (such as glycolysis, gluconeogenesis, carbon metabolism, synthesis of amino acids) were significantly enriched. Higher efficacy of the K1 variant is likely due to higher bioavailability of the drug, leading to complete downregulation of the pathways that facilitate catering of the energy requirements of the proliferating tumor cells. On the contrary, the K2 variant exhibits a shorter half-life, resulting only in partial reduction in the metabolic/energy requirements of the growing tumor cells. CONCLUSION: Overall, we conclude that the mAb half-life, dosage, and efficacy of a biotherapeutic product are significantly impacted by the charge variant profile of a biotherapeutic product.

Half-Life Extension and Biodistribution Modulation of Biotherapeutics via Red Blood Cell Hitch-Hiking with Novel Anti-Band 3 Single-Domain Antibodies.

Small therapeutic proteins are receiving increased interest as therapeutic drugs; however, their clinical success has been limited due to their rapid elimination. Here, we report a half-life extension strategy via strategy via red blood cell red blood cell (RBC) hitch-hiking. This manuscript details the development and characterization of novel anti-RBC single-domain antibodies (sdAbs), their genetic fusion to therapeutic antibody fragments (TAF) as bispecific fusion constructs, and their influence on TAF pharmacokinetics and biodistribution. Several sdAbs specific to the band 3 antigen were generated via phage-display technology. Binding affinity to RBCs was assessed via flow cytometry. Affinity maturation via random mutagenesis was carried out to improve the binding affinity of the sdAbs. Bi-specific constructs were generated by fusing the anti-RBC sdAbs with anti-tissue necrosis factor alpha (TNF-α) TAF via the use of a glycine-serine flexible linker, and assessments for binding were performed via enzyme-linked immunosorbent assay and flow cytometry. Pharmacokinetics of anti-RBC sdAbs and fusion constructs were evaluated following intravenous bolus dosing in mice at a 1 mg/kg dose. Two RBC-binding sdAbs, RB12 and RE8, were developed. These two clones showed high binding affinity to human RBC with an estimated KD of 17.7 nM and 23.6 nM and low binding affinity to mouse RBC with an estimated KD of 335 nM and 528 nM for RB12 and RE8, respectively. Two derivative sdAbs, RMA1, and RMC1, with higher affinities against mouse RBC, were generated via affinity maturation (KD of 66.9 nM and 30.3 nM, respectively). Pharmacokinetic investigations in mice demonstrated prolonged circulation half-life of an anti-RBC-TNF-α bispecific construct (75 h) compared to a non-RBC binding control (1.3 h). In summary, the developed anti-RBC sdAbs and fusion constructs have demonstrated high affinity in vitro, and sufficient half-life extension in vivo.

GlycoTAIL and FlexiTAIL as Half-Life Extension Modules for Recombinant Antibody Fragments.

Many therapeutic proteins are small in size and are rapidly cleared from circulation. Consequently, half-life extension strategies have emerged to improve pharmacokinetic properties, including fusion or binding to long-lasting serum proteins, chemical modifications with hydrophilic polymers such as PEGylation, or, more recently, fusion to PEG mimetic polypeptides. In the present study, two different PEG mimetic approaches, the GlycoTAIL and the FlexiTAIL, were applied to increase the hydrodynamic radius of antibody fragments of different sizes and valencies, including scFv, diabody, and scFv-EHD2 fusion proteins. The GlycoTAIL and FlexiTAIL sequences of varying lengths are composed of aliphatic and hydrophilic residues, with the GlycoTAIL furthermore comprising N-glycosylation sites. All modified proteins could be produced in a mammalian expression system without reducing stability and antigen binding, and all modified proteins exhibited a prolonged half-life and increased drug disposition in mice. The strongest effects were observed for proteins comprising a FlexiTAIL of 248 residues. Thus, the GlycoTAIL and FlexiTAIL sequences represent a flexible and modular system to improve the pharmacokinetic properties of proteins.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

Ocular and systemic pharmacokinetics of BI-X, a nanobody targeting VEGF and Ang-2, after intravitreal dosing in cynomolgus monkeys - Evidence for half-life extension by albumin.

Half-life extension strategies to reduce the intravitreal dosing frequency of biomolecules for the treatment of retinal neovascular diseases are attracting increasing interest. This study investigated ocular and systemic pharmacokinetics of the trivalent nanobody BI-X (with affinity to VEGF, Ang-2 and human albumin) in cynomolgus monkeys after intravitreal injection. BI-X concentrations were measured in serial samples of plasma, vitreous humor, aqueous humor and retina. Ocular pharmacokinetics of BI-X exhibited two phases. Initially up to 2-4 weeks after dosing, BI-X concentrations in vitreal, aqueous humor and retina declined with half-lives of around 3 days, which is comparable to macromolecules with a similar molecular weight. Thereafter, only vitreal concentrations were measurable, with a terminal half-life of 13.2 days, which is considerably longer than expected based on the BI-X molecular weight or hydrodynamic radius. It is hypothesized that binding of BI-X to low levels of intraocular albumin resulted in this half-life extension. BI-X was detectable in plasma up to 10 weeks post-dosing. Plasma pharmacokinetics of BI-X exhibited a similar biphasic disposition profile to the vitreous body, with a terminal half-life of 11.8 days, thus reflecting input kinetics from the eye. In conclusion, an important half-life extension principle based on vitreal albumin binding could be confirmed in a primate model, and the data obtained can potentially be translated to humans taking into account the differing vitreal albumin concentrations.

Small Bispecific Affinity Proteins for Simultaneous Target Binding and Albumin-Associated Half-Life Extension.

Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. Albumin-derived affinity proteins (ADAPTs) are a new type of scaffold proteins based on one of the albumin-binding domains of streptococcal protein G, with engineered binding specificities against numerous targets. Here, we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to possess affinity toward both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin-binding fusion partner. To accomplish this, we designed a phage display library and used it to successfully select for single-domain bispecific binders toward a panel of targets: TNFα, prostate-specific antigen (PSA), C-reactive protein (CRP), renin, angiogenin, myeloid-derived growth factor (MYDGF), and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex consisting of the ADAPT together with albumin and each of the five targets, TNFα, PSA, angiogenin, MYDGF, and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and noninvasive administration routes while still maintaining a sufficient in vivo half-life.

Multivalent Albumin-Neonatal Fc Receptor Interactions Mediate a Prominent Extension of the Serum Half-Life of a Therapeutic Protein.

Human serum albumin (HSA) has been used to extend the serum half-life of therapeutic proteins owing to its exceptionally long serum half-life via the neonatal Fc receptor (FcRn)-mediated recycling mechanism. In most cases, only one HSA molecule was conjugated to a therapeutic protein, leading to a limited extension of the serum half-life. In this study, we hypothesized that conjugation of multiple HSA molecules to a therapeutic protein significantly further extends the serum half-life via multivalent HSA-FcRn interactions. We chose urate oxidase (Uox), a tetrameric therapeutic protein used for the treatment of gout, as a model. In previous studies, only one HSA molecule was site-specifically conjugated to one Uox because of poor conjugation yield of the relatively slow bio-orthogonal chemistry, strain-promoted azide-alkyne cycloaddition (SPAAC). To increase the number of HSA molecules conjugated to one Uox, we employed the faster bio-orthogonal chemistry, inverse electron demand Diels-Alder reaction (IEDDA). We site-specifically introduced the phenylalanine analog with a fast-reacting tetrazine group (frTet) into position 174 of each subunit of Uox. We then achieved site-specific HSA conjugation to each subunit of Uox via IEDDA, generating Uox conjugated to four HSA molecules (Uox-HSA4), with a small portion of Uox conjugated to three HSA molecules (Uox-HSA3). We characterized Uox-HSA4 as well as Uox variants conjugated to one or two HSA molecules prepared via SPAAC (Uox-HSA1 or Uox-HSA2). The enzyme activity of all three Uox-HSA conjugates was comparable to that of unmodified Uox. We found out that an increase in HSA molecules conjugated to Uox (multiple albumin-conjugated therapeutic protein) enhanced FcRn binding and consequently prolonged the serum half-life in vivo. In particular, the conjugation of four HSA molecules to Uox led to a prominent extension of the serum half-life (over 21 h), which is about 16-fold longer than that of Uox-WT.

Challenges and advancements in the pharmacokinetic enhancement of therapeutic proteins.

Nowadays, proteins are frequently administered as therapeutic agents in human diseases. However, the main challenge regarding the clinical application of therapeutic proteins is short circulating plasma half-life that leads to more frequent injections for maintaining therapeutic plasma levels, increased therapy costs, immunogenic reactions, and low patient compliance. So, the development of novel strategies to enhance the pharmacokinetic profile of therapeutic proteins has attracted great attention in pharmaceuticals. So far, several techniques, each with their pros and cons, have been developed including chemical bonding to polymers, hyper glycosylation, Fc fusion, human serum albumin fusion, and recombinant PEG mimetics. These techniques mainly classify into three strategies; (i) the endosomal recycling of neonatal Fc receptor which is observed for immunoglobulins and albumin, (ii) decrease in receptor-mediated clearance, and (iii) increase in hydrodynamic radius through chemical and genetic modifications. Recently, novel PEG mimetic peptides like proline/alanine/serine repeat sequences are designed to overcome pitfalls associated with the previous technologies. Biodegradability, lack of or low immunogenicity, product homogeneity, and a simple production process, currently make these polypeptides as the preferred technology for plasma half-life extension of therapeutic proteins. In this review, challenges and pitfalls in the pharmacokinetic enhancement of therapeutic proteins using PEG-mimetic peptides will be discussed in detail.

PASylated Thymosin α1: A Long-Acting Immunostimulatory Peptide for Applications in Oncology and Virology.

Thymosin α1 (Tα1) is an immunostimulatory peptide for the treatment of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections and used as an immune enhancer, which also offers prospects in the context of COVID-19 infections and cancer. Manufacturing of this N-terminally acetylated 28-residue peptide is demanding, and its short plasma half-life limits in vivo efficacy and requires frequent dosing. Here, we combined the PASylation technology with enzymatic in situ N-acetylation by RimJ to produce a long-acting version of Tα1 in Escherichia coli at high yield. ESI-MS analysis of the purified fusion protein indicated the expected composition without any signs of proteolysis. SEC analysis revealed a 10-fold expanded hydrodynamic volume resulting from the fusion with a conformationally disordered Pro/Ala/Ser (PAS) polypeptide of 600 residues. This size effect led to a plasma half-life in rats extended by more than a factor 8 compared to the original synthetic peptide due to retarded kidney filtration. Our study provides the basis for therapeutic development of a next generation thymosin α1 with prolonged circulation. Generally, the strategy of producing an N-terminally protected PASylated peptide solves three major problems of peptide drugs: (i) instability in the expression host, (ii) rapid degradation by serum exopeptidases, and (iii) low bioactivity because of fast renal clearance.

Hydrophilic Small Molecules That Harness Transthyretin To Enhance the Safety and Efficacy of Targeted Chemotherapeutic Agents.

The hydrophobicity of many chemotherapeutic agents usually results in their nonselective passive distribution into healthy cells and organs causing collateral toxicity. Ligand-targeted drugs (LTDs) are a promising class of targeted anticancer agents. The hydrophilicity of the targeting ligands in LTDs limits its nonselective passive tissue distribution and toxicity to healthy cells. In addition, the small size of LTDs allows for better tumor penetration, especially in the case of solid tumors. However, the short circulation half-life of LTDs, due to their hydrophilicity and small size, remains a significant challenge for achieving their full therapeutic potential. Therefore, extending the circulation half-life of targeted chemotherapeutic agents while maintaining their hydrophilicity and small size will represent a significant advance toward effective and safe cancer treatment. Here, we present a new approach for enhancing the safety and efficacy of targeted chemotherapeutic agents. By endowing hydrophobic chemotherapeutic agents with a targeting moiety and a hydrophilic small molecule that binds reversibly to the serum protein transthyretin, we generated small hydrophilic drug conjugates that displayed enhanced circulation half-life in rodents and selectivity to cancer cells. To the best of our knowledge, this is the first demonstration of a successful approach that maintains the small size and hydrophilicity of targeted anticancer agents containing hydrophobic payloads while at the same time extending their circulation half-life. This was demonstrated by the superior in vivo efficacy and lower toxicity of our conjugates in xenograft mouse models of metastatic prostate cancer.

Establishment of an In Vitro-In Vivo Correlation for Melanin Binding and the Extension of the Ocular Half-Life of Small-Molecule Drugs.

A large variety of drugs bind effectively to melanin, and this binding influences their ocular pharmacokinetic and distribution profiles. We aimed to establish a correlation between in vitro melanin binding and in vivo ocular pharmacokinetics (PK). The extent of melanin binding in vitro was determined for a set of model drugs; binding kinetics and binding isotherms were generated and fitted to a mechanistic model to derive the drug-melanin binding parameters (Bmax, KD, kon, and koff). In addition, in vitro ADME properties such as cellular permeability, P-glycoprotein-mediated efflux, plasma protein binding, and octanol partition coefficients were determined. Moreover, cellular uptake was measured in the nonpigmented ARPE-19 cells and in lightly pigmented human epidermal melanocytes. Finally, in vivo ocular PK studies were performed in albino and pigmented rats using intravenous injections. Substantial drug enrichment accompanied by a very long residence time was observed in pigmented ocular tissues, which could be linked to the melanin binding determined in vitro and to the intracellular drug uptake into the pigmented cells. The resulting ocular PK profile is shown to be a consequence of the interplay of melanin binding with concurrent processes such as systemic clearance, plasma protein binding, cellular permeation, P-glycoprotein efflux, pH partitioning, and tissue binding. Understanding this interplay at a mechanistic level could help in the rational design and development of new small-molecule drug candidates with the desired PK/pharmacodynamic profile to target the back of the eye.

Half-life extension of porcine interferon-α by fusion to the IgG-binding domain of streptococcal G protein.

Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability.

Fusion with an albumin-binding domain improves pharmacokinetics of an αvß3-integrin binding fibronectin scaffold protein.

Fusion with an albumin-binding domain (ABD) of streptococcal protein G represents a popular approach for half-life extension of small protein therapeutics in the organism. To increase the circulation time of engineered αvß3-integrin-binding protein (JCL) based on the 10th human fibronectin type III domain (10 Fn3), we have constructed several fusions with ABD with different orientations of the partner proteins and linker length. The recombinant proteins were expressed in Escherichia coli cells and purified by nickel-affinity chromatography. All fusion proteins bound human serum albumin (HSA) in ELISA assay; however, fusions with longer linkers demonstrated better performance. Interaction of ABD-L15 -JCL and JCL-L14 -ABD with HSA was confirmed by analytical size exclusion chromatography and pull-down assays. Surprisingly, the thermal stability of ABD-L15 -JCL was dramatically decreased in comparison with JCL and JCL-L14 -ABD proteins. Pharmacokinetic studies revealed that JCL-L14 -ABD circulated in murine blood about 10 times longer than ABD-L15 -JCL and 960 times longer than JCL. Biodistribution studies of JCL-L14 -ABD in mice revealed its increased level in blood and a decreased accumulation in liver and kidneys in comparison with JCL. Obtained results demonstrate the utility of the fusion with ABD for half-life extension of the binding proteins based on 10 Fn3.

Engineering a long-acting, potent GLP-1 analog for microstructure-based transdermal delivery.

Antidiabetic treatments aiming to reduce body weight are currently gaining increased interest. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist administered twice daily via s.c. injection, improves glycemic control, often with associated weight reduction. To further improve the therapeutic efficacy of exendin-4, we have developed a novel peptide engineering strategy that incorporates a serum protein binding motif onto a covalent side-chain staple and applied to the peptide to enhance its helicity and, as a consequence, its potency and serum half-life. We demonstrated that one of the resulting peptides, E6, has significantly improved half-life and glucose tolerance in an oral glucose tolerance test in rodents. Chronic treatment of E6 significantly decreased body weight and fasting blood glucose, improved lipid metabolism, and also reduced hepatic steatosis in diet-induced obese mice. Moreover, the high potency of E6 allowed us to administer this peptide using a dissolvable microstructure-based transdermal delivery system. Pharmacokinetic and pharmacodynamic studies in guinea pigs showed that a single 5-min application of a microstructure system containing E6 significantly improved glucose tolerance for 96 h. This delivery strategy may offer an effective and patient-friendly alternative to currently marketed GLP-1 injectables and can likely be extended to other peptide hormones.

Process development of a FGF21 protein-antibody conjugate.

A scalable, viable process was developed for the Fibroblast Growth Factor 21 (FGF21) protein-antibody conjugate, CVX-343, an extended half-life therapeutic for the treatment of metabolic disease. CVX-343 utilizes the CovX antibody scaffold technology platform that was specifically developed for peptide and protein half-life extension. CVX-343 is representative of a growing number of complex novel peptide- and protein-based bioconjugate molecules currently being explored as therapeutic candidates. The complexity of these bioconjugates, assembled using well-established chemistries, can lead to very difficult production schemes requiring multiple starting materials and a combination of diverse technologies. Key improvements had to be made to the original CVX-343 Phase 1 manufacturing process in preparation for Phase 3 and commercial manufacturing. A strategy of minimizing FGF21A129C dimerization and stabilizing the FGF21A129C Drug Substance Intermediate (DSI), linker, and activated FGF21 intermediate was pursued. The use of tris(2-carboxyethyl)phosphine (TCEP) to prevent FGF21A129C dimerization through disulfide formation was eliminated. FGF21A129C dimerization and linker hydrolysis were minimized by formulating and activating FGF21A129C at acidic instead of neutral pH. An activation use test was utilized to guide FGF21A129C pooling in order to minimize misfolds, dimers, and misfolded dimers in the FGF21A129C DSI. After final optimization of reaction conditions, a process was established that reduced the consumption of FGF21A129C by 36% (from 4.7 to 3.0 equivalents) and the consumption of linker by 55% (from 1.4 to 0.95 equivalents for a smaller required amount of FGF21A129C ). The overall process time was reduced from ∼5 to ∼3 days. The product distribution improved from containing ∼60% to ∼75% desired bifunctionalized (+2 FGF21) FGF21-antibody conjugate in the crude conjugation mixture and from ∼80% to ∼85% in the final CVX-343 Drug Substance (DS), while maintaining the same overall process yield based on antibody scaffold input.

Neoglycolipids for Prolonging the Effects of Peptides: Self-Assembling Glucagon-like Peptide 1 Analogues with Albumin Binding Properties and Potent in Vivo Efficacy.

Novel principles for optimizing the properties of peptide-based drugs are needed in order to leverage their full pharmacological potential. We present the design, synthesis, and evaluation of a library of neoglycolipidated glucagon-like peptide 1 (GLP-1) analogues, which are valuable drug candidates for treatment of type 2 diabetes and obesity. Neoglycolipidation of GLP-1 balanced the lipophilicity, directed formation of soluble oligomers, and mediated albumin binding. Moreover, neoglycolipidation did not compromise bioactivity, as in vitro potency of neoglycolipidated GLP-1 analogues was maintained or even improved compared to native GLP-1. This translated into pronounced in vivo efficacy in terms of both decreased acute food intake and improved glucose homeostasis in mice. Thus, we propose neoglycolipidation as a novel, general method for modulating the properties of therapeutic peptides.