Substituted diaryl ether compounds as retinoic acid-related orphan Receptor-γt (RORγt) agonists.

The discovery of a series of substituted diarylether compounds as retinoic acid related orphan receptor γt (RORγt) agonists is described. Compound 1 was identified from deck mining as a RORγt agonist. Hit-to-lead optimization led to the identification of lead compound 5, which possesses improved potency (10x). Extensive SAR exploration led to the identification of a potent and selective compound 22, that demonstrated an improved pharmacokinetic profile and a dose-dependent pharmacodynamic response. However, when dosed in a MC38 syngeneic tumor model, no evidence of efficacy was observed. ©2020 Elsevier Science Ltd. All rights reserved.

Discovery of BMS-986251: A Clinically Viable, Potent, and Selective RORγt Inverse Agonist.

Novel tricyclic analogues were designed, synthesized, and evaluated as RORγt inverse agonists. Several of these compounds were potent in an IL-17 human whole blood assay and exhibited excellent oral bioavailability in mouse pharmacokinetic studies. This led to the identification of compound 5, which displayed dose-dependent inhibition of IL-17F production in a mouse IL-2/IL-23 stimulated pharmacodynamic model. In addition, compound 5 was studied in mouse acanthosis and imiquimod-induced models of skin inflammation, where it demonstrated robust efficacy comparable to a positive control. As a result of this excellent overall profile, compound 5 (BMS-986251) was selected as a clinically viable developmental candidate.

Substituted benzyloxytricyclic compounds as retinoic acid-related orphan receptor gamma t (RORγt) agonists.

Substituted benzyloxy aryl compound 2 was identified as an RORγt agonist. Structure based drug design efforts resulted in a potent and selective tricyclic compound 19 which, when administered orally in an MC38 mouse tumor model, demonstrated a desired pharmacokinetic profile as well as a dose-dependent pharmacodynamic response. However, no perceptible efficacy was observed in this tumor model at the doses investigated.

Rationally Designed, Conformationally Constrained Inverse Agonists of RORγt-Identification of a Potent, Selective Series with Biologic-Like in Vivo Efficacy.

RORγt is an important nuclear receptor that regulates the production of several pro-inflammatory cytokines such as IL-17 and IL-22. As a result, RORγt has been identified as a potential target for the treatment of various immunological disorders such as psoriasis, psoriatic arthritis, and inflammatory bowel diseases. Structure and computer-assisted drug design led to the identification of a novel series of tricyclic RORγt inverse agonists with significantly improved in vitro activity in the reporter (Gal4) and human whole blood assays compared to our previous chemotype. Through careful structure activity relationship, several potent and selective RORγt inverse agonists have been identified. Pharmacokinetic studies allowed the identification of the lead molecule 32 with a low peak-to-trough ratio. This molecule showed excellent activity in an IL-2/IL-23-induced mouse pharmacodynamic study and demonstrated biologic-like efficacy in an IL-23-induced preclinical model of psoriasis.

Structure-based Discovery of Phenyl (3-Phenylpyrrolidin-3-yl)sulfones as Selective, Orally Active RORγt Inverse Agonists.

A new phenyl (3-phenylpyrrolidin-3-yl)sulfone series of RORγt inverse agonists was discovered utilizing the binding conformation of previously reported bicyclic sulfonamide 1. Through a combination of structure-based design and structure-activity relationship studies, a polar set of amides at N1-position of the pyrrolidine ring and perfluoroisopropyl group at para-position of the 3-phenyl group were identified as critical structural elements to achieve high selectivity against PXR, LXRα, and LXRß. Further optimization led to the discovery of (1R,4r)-4-((R)-3-((4-fluorophenyl)sulfonyl)-3-(4-(perfluoropropan-2-yl)phenyl)pyrrolidine-1-carbonyl)cyclohexane-1-carboxylic acid (26), which displayed excellent selectivity, desirable liability and pharmacokinetic properties in vitro, and a good pharmacokinetic profile in mouse. Oral administration of 26 demonstrated dose-dependent inhibition of IL-17 production in a mouse IL-2/IL-23-induced pharmacodynamic model and biologic-like efficacy in an IL-23-induced mouse acanthosis model.

Identification and Preclinical Pharmacology of ((1 R,3 S)-1-Amino-3-(( S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986166): A Differentiated Sphingosine-1-phosphate Receptor 1 (S1P1) Modulator Advanced into Clinical Trials.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life ( T1/2) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P1. Herein, we describe our efforts to discover highly potent, partial agonists of S1P1 with a shorter T1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments are discussed. Furthermore, efficacy of 14a in multiple preclinical models of autoimmune diseases are presented.

Identification of bicyclic hexafluoroisopropyl alcohol sulfonamides as retinoic acid receptor-related orphan receptor gamma (RORγ/RORc) inverse agonists. Employing structure-based drug design to improve pregnane X receptor (PXR) selectivity.

We disclose the optimization of a high throughput screening hit to yield benzothiazine and tetrahydroquinoline sulfonamides as potent RORγt inverse agonists. However, a majority of these compounds showed potent activity against pregnane X receptor (PXR) and modest activity against liver X receptor α (LXRα). Structure-based drug design (SBDD) led to the identification of benzothiazine and tetrahydroquinoline sulfonamide analogs which completely dialed out LXRα activity and were less potent at PXR. Pharmacodynamic (PD) data for compound 35 in an IL-23 induced IL-17 mouse model is discussed along with the implications of a high Ymax in the PXR assay for long term preclinical pharmacokinetic (PK) studies.

Asymmetric Hydroboration Approach to the Scalable Synthesis of ((1R,3S)-1-Amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986104) as a Potent S1P1 Receptor Modulator.

We describe a highly efficient route for the synthesis of 4a (BMS-986104). A key step in the synthesis is the asymmetric hydroboration of trisubstituted alkene 6. Particularly given the known difficulties involved in this type of transformation (6 → 7), the current methodology provides an efficient approach to prepare this class of compounds. In addition, we disclose the efficacy of 4a in a mouse EAE model, which is comparable to 4c (FTY720). Mechanistically, 4a exhibited excellent remyelinating effects on lysophosphatidylcholine (LPC) induced demyelination in a three-dimensional brain cell culture assay.

Discovery and Structure-Activity Relationship (SAR) of a Series of Ethanolamine-Based Direct-Acting Agonists of Sphingosine-1-phosphate (S1P1).

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates a multitude of physiological processes such as lymphocyte trafficking, cardiac function, vascular development, and inflammation. Because of the ability of S1P1 receptor agonists to suppress lymphocyte egress, they have great potential as therapeutic agents in a variety of autoimmune diseases. In this article, the discovery of selective, direct acting S1P1 agonists utilizing an ethanolamine scaffold containing a terminal carboxylic acid is described. Potent S1P1 agonists such as compounds 18a and 19a which have greater than 1000-fold selectivity over S1P3 are described. These compounds efficiently reduce blood lymphocyte counts in rats through 24 h after single doses of 1 and 0.3 mpk, respectively. Pharmacodynamic properties of both compounds are discussed. Compound 19a was further studied in two preclinical models of disease, exhibiting good efficacy in both the rat adjuvant arthritis model (AA) and the mouse experimental autoimmune encephalomyelitis model (EAE).

Dual Inhibition of Interleukin-23 and Interleukin-17 Offers Superior Efficacy in Mouse Models of Autoimmunity.

Therapies targeting either interleukin (IL)-23 or IL-17 have shown promise in treating T helper 17 (Th17)-driven autoimmune diseases. Although IL-23 is a critical driver of IL-17, recognition of nonredundant and independent functions of IL-23 and IL-17 has prompted the notion that dual inhibition of both IL-23 and IL-17 could offer even greater efficacy for treating autoimmune diseases relative to targeting either cytokine alone. To test this hypothesis, we generated selective inhibitors of IL-23 and IL-17 and tested the effect of either treatment alone compared with their combination in vitro and in vivo. In vitro, using a novel culture system of murine Th17 cells and NIH/3T3 fibroblasts, we showed that inhibition of both IL-23 and IL-17 completely suppressed IL-23-dependent IL-22 production from Th17 cells and cooperatively blocked IL-17-dependent IL-6 secretion from the NIH/3T3 cells to levels below either inhibitor alone. In vivo, in the imiquimod induced skin inflammation model, and in the myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis model, we demonstrated that dual inhibition of IL-17 and IL-23 was more efficacious in reducing disease than targeting either cytokine alone. Together, these data support the hypothesis that neutralization of both IL-23 and IL-17 may provide enhanced benefit against Th17 mediated autoimmunity and provide a basis for a therapeutic strategy aimed at dual targeting IL-23 and IL-17.

Engineering of a novel anti-CD40L domain antibody for treatment of autoimmune diseases.

CD40-CD40L interactions play a critical role in regulating immune responses. Blockade of CD40L by Abs, such as the anti-CD40L Ab 5c8, demonstrated positive clinical effects in patients with autoimmune diseases; however, incidents of thromboembolism (TE) precluded further development of these molecules. In this study, we examined the role of the Fc domain interaction with FcγRs in modulating platelet activation and potential for TE. Our results show that the interaction of the 5c8 wild-type IgG1 Fc domain with FcγRs is responsible for platelet activation, as measured by induction of PAC-1 and CD62P. A version of 5c8 with a mutated IgG1 tail was identified that showed minimal FcγR binding and platelet activation while maintaining full binding to CD40L. To address whether Fc effector function is required for immunosuppression, a potent Ab fragment, termed a "domain Ab" (dAb), against murine CD40L was identified and fused to a murine IgG1 Fc domain containing a D265A mutation that lacks Fc effector function. In vitro, this dAb-Fc demonstrated comparable potency to the benchmark mAb MR-1 in inhibiting B cell and dendritic cell activation. Furthermore, the anti-CD40L dAb-Fc exhibited a notable efficacy comparable to MR-1 in various preclinical models, such as keyhole limpet hemocyanin-induced Ab responses, alloantigen-induced T cell proliferation, "heart-to-ear" transplantation, and NZB × NZW F1 spontaneous lupus. Thus, our data show that immunosuppression and TE can be uncoupled and that a CD40L dAb with an inert Fc tail is expected to be efficacious for treating autoimmune diseases, with reduced risk for TE.

A highly selective, orally bioavailable, vascular endothelial growth factor receptor-2 tyrosine kinase inhibitor has potent activity in vitro and in vivo.

Angiogenesis is a complex process that relies on a variety of growth factors and signaling pathways to stimulate endothelial cell responses and establish functional blood vessels. Signaling through the vascular endothelial growth factor (VEGF) receptors is an important mediator of angiogenesis, a hallmark of tumor growth and metastasis. Inhibition of signaling through VEGF has been clinically validated with FDA-approvals of bevacizumab, sorafenib, and suntinib. Our goal was to discover an orally available, selective VEGFR-2 inhibitor. A novel oxime, 1-{4-[6-amino-5-(methoxyimino-methyl)-pyrimidin-4-yloxy]-2-chloro-phenyl}-3-ethyl-urea (JNJ-38158471), was identified as a potent and selective inhibitor of VEGFR-2. While JNJ-38158471 shares some structure features with sorafenib, unlike sorafenib, it lacks Raf kinase activity. JNJ-38158471 inhibits VEGFR-2 (IC50 = 40 nM) and closely related tyrosine kinases, Ret (180 nM) and Kit (500 nM); it has no significant activity (>1 microM) against VEGFR-1 and VEGFR-3. At nanomolar levels, it inhibits VEGF-stimulated autophosphorylation of VEGFR-2 in a whole cell assay and inhibits VEGF-dependent endothelial migration. Once-daily oral dosing of JNJ-3815871 to nude mice bearing human A431, HCT116, and A375 tumors resulted in up to 90% tumor growth inhibition. Strikingly, after termination of JNJ-38158471 monotherapy-treatment of A375 xenografts, tumor growth delay was significantly prolonged up to 4 weeks. Anti-tumor efficacy correlated well with the observed dose concentrations (on a mg/kg basis) necessary to inhibit VEGF-induced corneal angiogenesis in C57BL/6J mice. In addition, the compound inhibited spontaneous polyp formation in the APC min-mouse model. These data demonstrate that JNJ-38158471 is a well tolerated, orally available, highly selective VEGFR-2 inhibitor that may have therapeutic benefit in human malignancies.

Cellular and in vivo activity of JNJ-28871063, a nonquinazoline pan-ErbB kinase inhibitor that crosses the blood-brain barrier and displays efficacy against intracranial tumors.

JNJ-28871063 is a potent and highly selective pan-ErbB kinase inhibitor from a novel aminopyrimidine oxime structural class that blocks the proliferation of epidermal growth factor receptor (EGFR; ErbB1)- and ErbB2-overexpressing cells but does not affect the growth of non-ErbB-overexpressing cells. Treatment of human cancer cells with JNJ-28871063 inhibited phosphorylation of functionally important tyrosine residues in both EGFR and ErbB2 and blocked downstream signal transduction pathways responsible for proliferation and survival. A single dose of compound reduced phosphorylation of ErbB2 receptors in tumor-bearing mice, demonstrating target suppression in vivo. Tissue distribution studies show that JNJ-28871063 crosses the blood-brain barrier and penetrates into tumors, where it is able to accumulate to higher levels than those found in the plasma. JNJ-28871063 showed oral antitumor activity in human tumor xenograft models that overexpress EGFR and ErbB2. In an intracranial ErbB2-overexpressing tumor model, JNJ-28871063 extended survival relative to untreated animals. The brain is a primary site of metastasis for EGFR-overexpressing lung cancers and ErbB2-overexpressing breast cancers. Therefore, the ability to penetrate into the brain could be an advantage over existing therapies such as trastuzumab (Herceptin) and cetuximab (Erbitux), which are antibodies and do not cross the blood-brain barrier. These results show that JNJ-28871063 is orally bioavailable, has activity against EGFR and ErbB2-dependent tumor xenografts, and can penetrate into the brain and inhibit ErbB2-overexpressing tumor growth.

Delivery of G3139 using releasable PEG-linkers: impact on pharmacokinetic profile and anti-tumor efficacy.

In order to overcome the problems of enzymatic degradation and short plasma half life, which can limit the delivery of antisense oligonucleotides, and the potential immuno-stimulatory effects of CpG motifs, we utilized a polyethylene glycol (PEG) technology that employed various releasable linkers (rPEG). 5'-20 kDa-PEGylation of an anti-Bcl-2 5'-aminoalkyl-oligonucleotide with the same sequence as G3139 (Compound 1) did not alter its binding to the heparin-binding protein bFGF, nor the release of cytochrome c from isolated mitochondria treated with the conjugates. However, in 518A2 melanoma cells in vitro, PEGylation resulted in greatly diminished cellular uptake. In striking contrast, PEGylation of 1 resulted in dramatically improved pharmacokinetic profiles in vivo, with a prolonged half-life (t1/2), increased plasma concentration, and increased area under the plasma concentration-time curve (AUC). In an in vivo melanoma 518A2 xenograft mouse model, treatment with either 5'-20 kDa-PEG-1 or 1 demonstrated similar tumor growth inhibition. Furthermore, in an in vitro mouse splenocyte culture system, attachment of a PEG moiety to 1 through releasable linkers abolished the immunostimulatory response that was observed for G3139. Our results demonstrate the potential of the in vivo use of PEGylated oligonucleotides, and point out the profound differences between in vitro and in vivo models of oligonucleotide activity.

Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.

Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics. Specifically, ADI with either 12 kDa or 20 kDa PEG attachments on 33% of the primary amines retained about 60% or 48% of enzyme activity, respectively; the Km and pH profiles were nearly unchanged; IC50 values were diminished by less than 30%; while stability studies demonstrated full retention of activity at 4 degrees C for 5 months. A comparison of the enzymatic properties of a second ADI from Pseudomonas putida illustrated the superior characteristics of the M. arthritidis ADI enzyme.

Role of the ABCG2 drug transporter in the resistance and oral bioavailability of a potent cyclin-dependent kinase/Aurora kinase inhibitor.

Cell cycle kinase inhibitors have advanced into clinical trials in oncology. One such molecule, JNJ-7706621, is a broad-spectrum inhibitor of the cyclin-dependent kinases and Aurora kinases that mediate G(2)-M arrest and inhibits tumor growth in xenograft models. To determine the putative mechanisms of resistance to JNJ-7706621 that might be encountered in the clinic, the human epithelial cervical carcinoma cell line (HeLa) was exposed to incrementally increasing concentrations of JNJ-7706621. The resulting resistant cell population, designated HeLa-6621, was 16-fold resistant to JNJ-7706621, cross-resistant to mitoxantrone (15-fold) and topotecan (6-fold), and exhibited reduced intracellular drug accumulation of JNJ-7706621. ABCG2 was highly overexpressed at both the mRNA ( approximately 163-fold) and protein levels. The functional role of ABCG2 in mediating resistance to JNJ-7706621 was consistent with the following findings: (a) an ABCG2 inhibitor, fumitremorgin C, restored the sensitivity of HeLa-6621 cells to JNJ-7706621 and to mitoxantrone; (b) human embryonic kidney-293 cells transfected with ABCG2 were resistant to both JNJ-7706621 and mitoxantrone; and (c) resistant cells that were removed from the drug for 12 weeks and reverted to susceptibility to JNJ-7706621 showed near-normal ABCG2 RNA levels. ABCG2 is likely to limit the bioavailability of JNJ-7706621 because oral administration of JNJ-7706621 to Bcrp (the murine homologue of ABCG2) knockout mice resulted in an increase in the plasma concentration of JNJ-7706621 compared with wild-type mice. These findings indicate that ABCG2 mediates the resistance to JNJ-7706621 and alters the absorption of the compound following administration.

Structure-function engineering of interferon-beta-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation.

Recombinant interferon-beta-1b (IFN-beta-1b) is used clinically in the treatment of multiple sclerosis. In common with many biological ligands, IFN-beta-1b exhibits a relatively short serum half-life, and bioavailability may be further diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increase the blood residency time of protein therapeutics, there is a further requisite for molecular engineering approaches to also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins. We investigated these five parameters of recombinant human IFN-beta-1b in over 20 site-selective mono-PEGylated or multi-PEGylated IFN-beta-1b bioconjugates. Primary amines were modified by single or multiple attachments of poly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternate approaches, site-directed mutagenesis was independently employed in the construction of designed IFN-beta-1b variants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugate preparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and a comparison of the structural and functional properties of the IFN-beta-1b proteins and their PEGylated counterparts was conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sites of the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferation bioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusion HPLC, and immunoassays demonstrated that the functional activities of the designed IFN-beta-1b conjugates were maintained, while the formation of soluble or insoluble aggregates of IFN-beta-1b was ameliorated. Immunogenicity and pharmacokinetic studies of selected PEGylated IFN-beta-1b compounds in mice and rats demonstrated both diminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. The results demonstrate the capacity of this macromolecular engineering strategy to address both pharmacological and formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN-beta-1b, were further investigated in formulation optimization and biological studies.

Antitumor activity of poly(ethylene glycol)-camptothecin conjugate: the inhibition of tumor growth in vivo.

Antitumor effect of poly(ethylene glycol)-camptothecin conjugate (PEG-CPT) was studied in the nude mouse model of human colon cancer xenografts. The animals were treated intravenously with 15 mg/kg of camptothecin (CPT) or PEG-CPT conjugate at equivalent CPT dose. Antitumor activity, apoptosis induction and caspase-dependent signaling pathways were studied 12, 24, 48 and 96 h after single injection. In addition, pharmacokinetics, tumor distribution and accumulation of PEG polymer labeled with green fluorescence protein (GFP) were studied. The data obtained showed that the conjugation of low molecular weight anticancer drug CPT with low solubility to high molecular weight water-soluble PEG polymer provides several advantages over the native drug. First, the conjugation improves drug pharmacokinetics in the blood and tumor. Second, such conjugation provides passive tumor targeting by the Enhanced Permeability and Retention (EPR) effect, increasing drug concentration in the tumor. Third, the coupling increases the bioavailability of CPT, induces apoptosis in tumor and, therefore, enhances anticancer activity of PEG-CPT. Thus, the use of macromolecular conjugate provided passive tumor targeting of the drug, improved pharmacokinetics and increased the stability of the drug during circulation. It offered better uptake by the targeted tumor cells and substantially enhanced apoptosis and antitumor activity of the conjugated drug in the tumor and decreased apoptosis in liver and kidney as compared with the native drug. All these characteristics make PEG-CPT conjugate an attractive anticancer drug for the effective chemotherapy of solid tumors.

Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation.

The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V(L)-linker-V(H) and V(H)-linker-V(L). High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Independent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.

Anticancer drug delivery systems: multi-loaded N4-acyl poly(ethylene glycol) prodrugs of ara-C. II. Efficacy in ascites and solid tumors.

The synthesis of branched PEG (40,000) acids has been achieved using aspartic acid (Asp) and AspAsp dendrons. Complete conjugation of these dendritic acids with cytosine arabinoside (ara-C) was achieved by the use of spacers that allowed a greater separation of the branches to accommodate several large ara-C molecules in proximity to each other. The tetrameric and octameric PEG-ara-C amide prodrugs were much more effective in the treatment of solid and ascites tumors compared to the native drug. The greater loading of the PEG backbone appears to have achieved a minimum threshold concentration for the therapeutic delivery of ara-C.