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Bispecific antibodies have become important formats for therapeutic discovery. They allow for potential synergy by simultaneously engaging two separate targets and enable new functions that are not possible to achieve by using a combination of two monospecific antibodies. Antagonistic antibodies dominate drug discovery today, but only a limited number of agonistic antibodies (i.e. those that activate receptor signaling) have been described. For receptors formed by two components, engaging both of these components simultaneously may be required for agonistic signaling. As such, bispecific antibodies may be particularly useful in activating multicomponent receptor complexes. Here, we describe a biparatopic (i.e. targeting two different epitopes on the same target) format that can activate the endocrine fibroblast growth factor (FGF) 21 receptor (FGFR) complex containing β-Klotho and FGFR1c. This format was constructed by grafting two different antigen-specific VH domains onto the VH and VL positions of an IgG, yielding a tetravalent binder with two potential geometries, a close and a distant, between the two paratopes. Our results revealed that the biparatopic molecule provides activities that are not observed with each paratope alone. Our approach could help address the challenges with heterogeneity inherent in other bispecific formats and could provide the means to adjust intramolecular distances of the antibody domains to drive optimal activity in a bispecific format. In conclusion, this format is versatile, is easy to construct and produce, and opens a new avenue for agonistic antibody discovery and development.

A dearth of biologics have been developed and used as therapeutics for numerous disease indications, including IgG monoclonal antibodies, non-IgG recombinant proteins, bi- and multi-specific antibodies, and antibody drug conjugates. A remarkable portion of these biologic constructs exhibits a short plasma half-life that results in a significant reduction in therapeutic efficacy. Frequently, biologic drugs need to be designed to maintain the effective concentration range during the therapeutic window with an extended serum half-life. Provided here is a comprehensive overview of various half-life extension methods involving FcRn engagement, chemical and genetic fusion, post-translational modifications and formulation.

AMG 386 is an investigational first-in-class peptide-Fc fusion protein (peptibody) that inhibits angiogenesis by preventing the interaction of angiopoietin-1 (Ang1) and Ang2 with their receptor, Tie2. Although the therapeutic value of blocking Ang2 has been shown in several models of tumorigenesis and angiogenesis, the potential benefit of Ang1 antagonism is less clear. To investigate the consequences of Ang1 neutralization, we have developed potent and selective peptibodies that inhibit the interaction between Ang1 and its receptor, Tie2. Although selective Ang1 antagonism has no independent effect in models of angiogenesis-associated diseases (cancer and diabetic retinopathy), it induces ovarian atrophy in normal juvenile rats and inhibits ovarian follicular angiogenesis in a hormone-induced ovulation model. Surprisingly, the activity of Ang1 inhibitors seems to be unmasked in some disease models when combined with Ang2 inhibitors, even in the context of concurrent vascular endothelial growth factor inhibition. Dual inhibition of Ang1 and Ang2 using AMG 386 or a combination of Ang1- and Ang2-selective peptibodies cooperatively suppresses tumor xenograft growth and ovarian follicular angiogenesis; however, Ang1 inhibition fails to augment the suppressive effect of Ang2 inhibition on tumor endothelial cell proliferation, corneal angiogenesis, and oxygen-induced retinal angiogenesis. In no case was Ang1 inhibition shown to (a) confer superior activity to Ang2 inhibition or dual Ang1/2 inhibition or (b) antagonize the efficacy of Ang2 inhibition. These results imply that Ang1 plays a context-dependent role in promoting postnatal angiogenesis and that dual Ang1/2 inhibition is superior to selective Ang2 inhibition for suppression of angiogenesis in some postnatal settings. Mol Cancer Ther; 9(10); 2641–51. ©2010 AACR.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:low-density lipoprotein receptor complex formation reduce serum low-density lipoprotein-cholesterol (LDL-C) in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect. Administration of high-affinity mAb1:PCSK9 complex (1:2) to mice resulted in significantly lower mAb1 exposure compared with mAb1 dosed alone in normal mice or in PCSK9 knockout mice lacking antigen. To identify mAb-binding characteristics that minimize lysosomal disposition, the pharmacokinetic behavior of four mAbs representing a diverse range of PCSK9-binding affinities at neutral (serum) and acidic (endosomal) pH was evaluated in cynomolgus monkeys. Results revealed an inverse correlation between affinity and both mAb exposure and duration of LDL-C lowering. High-affinity mAb1 exhibited the lowest exposure and shortest duration of action (6 days), whereas mAb2 displayed prolonged exposure and LDL-C reduction (51 days) as a consequence of lower affinity and pH-sensitive PCSK9 binding. mAbs with shorter endosomal PCSK9:mAb complex dissociation half-lives (

Mutations in the lacZ gene were constructed that allowed the detection of mutators that caused specific transversion mutations. This allowed the detection of two new mutator genes, mutt and mutM, which are involved in the repair of the 8-oxodguanine (8oxodG) lesion.

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.

High levels of translational errors, both truncation and misincorporation in an Fc-fusion protein were observed. Here, we demonstrate the impact of several commercially available codon optimization services, and compare to a targeted strategy. Using the targeted strategy, only codons known to have translational errors are modified. For an Fc-fusion protein expressed in Escherichia coli, the targeted strategy, in combination with appropriate fermentation conditions, virtually eliminated misincorporation (proteins produced with a wrong amino acid sequence), and reduced the level of truncation. The use of full optimization using commercially available strategies reduced the initial errors, but introduced different misincorporations. However, truncation was higher using the targeted strategy than for most of the full optimization strategies. This targeted approach, along with monitoring of translation fidelity and careful attention to fermentation conditions is key to minimizing translational error and ensuring high-quality expression. These findings should be useful for other biopharmaceutical products, as well as any other transgenic constructs where protein quality is important.

Active oxygen species can damage DNA and may play a role in aging and carcinogenesis. We have tested MutY glycosylase for activity on undamaged mispairs as well as mispairs formed with the oxidatively damaged substrates, 8-oxo-7,8-dihydrodeoxyguanine (GO) or 8-oxo-7,8-dihydrodeoxyadenine (AO). MutY acts as a glycosylase on four of the heteroduplexes tested, A/G, A/GO, A/C, and A/AO, removing the undamaged adenine from each substrate. Genetic data suggest that the primary substrate for MutY glycosylase in vivo is the A/GO mispair. We present biochemical evidence demonstrating that MutY glycosylase is an important part of a repair system that includes the MutM and MutT proteins. The GO repair system is dedicated to the repair of the oxidatively damaged guanine and the mutations it can induce.

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a beta-strand. We find that residues surrounding a beta-bulge structure within the beta-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested.

It has been previously shown both in vivo and in vitro that DNA synthesis past an oxidatively damaged form of guanine, 7,8-dihydro-8-oxoguanine (8-oxoG), can result in the misincorporation of adenine (A) opposite the 8-oxodG. In this study we show that MutY glycosylase is active on a site-specific, oxidatively damaged A/8-oxoG mispair and that it removes the undamaged adenine from this mispair. Strains that lack active MutY protein have elevated rates of G.C----T.A transversions. We find that the mutator phenotype of a mutY strain can be fully complemented by overexpressing MutM protein (Fpg protein) from a plasmid clone. The MutM protein removes 8-oxoG lesions from DNA. In addition, we have isolated a strain with a chromosomal mutation that suppresses the mutY phenotype and found that this suppressor also overexpresses MutM. Finally, a mutY mutM double mutant has a 25- to 75-fold higher mutation rate than either mutator alone. The data strongly suggest that MutY is part of an intricate repair system directed against 8-oxoG lesions in nucleic acids and that the primary function of MutY in vivo is the removal of adenines that are misincorporated opposite 8-oxoG lesions during DNA synthesis.

We have measured the accuracy of in vitro synthesis by DNA polymerase I (large fragment) during translesion synthesis past an aminofluorene (AF) adduct. These studies were carried out using a site-specifically modified template which contained a single AF adduct. The template was prepared by first modifying the lone guanine in a 17 base long oligonucleotide and extensively purifying and characterizing this product. The modified 17mer was then ligated to a synthetic duplex to produce a 31 nucleotide long template strand containing the AF adduct annealed to a 14mer, such that the 3'-hydroxyl primer terminus was four nucleotides before the modified guanine. Synthesis on this template by DNA polymerase I efficiently bypassed the AF adduct and produced full-length duplex 31mers. T7 DNA polymerase, on the other hand, was unable to utilize the AF-modified template though it was active on an identical unmodified one. The strand synthesized by DNA polymerase I was then separated from the modified strand, annealed to a complementary oligonucleotide, and the resulting heteroduplex cloned into M13. Each of the 49 clones isolated had sequences which indicated that cytidine had been incorporated opposite the AF-modified guanine.

We have cloned chromosomal DNA bordering an insert that inactivates mutM. Sequencing of this clone has revealed that the insertion element is located between the promoter and structural gene for formamidopyrimidine-DNA glycosylase (Fapy-DNA glycosylase). An overproducing clone of Fapy-DNA glycosylase complements the original mutM strain that had been isolated after EMS mutagenesis. Thus, we conclude that MutM is actually Fapy-DNA glycosylase. mutM has previously been characterized as a mutator strain that leads specifically to G.C----T.A transversions. This in vivo characterization correlates well with the mutagenic potential of one of the lesions Fapy-DNA glycosylase removes, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-OxodG).

We have developed a system for rapidly cloning chromosomal DNA that flanks the site of insertion of Tn10 derivatives. A central portion of the tetracycline-resistance gene (tet) from Tn10 was cloned into a recombinant M13mp vector that carries a cat marker. Infection of a strain that contains a Tn10 derivative (mini-tet) leads to homologous recombination between the chromosomal tet gene and the cloned segment on the bacteriophage. Correct M13 lysogens can be identified by the inactivation of the tet gene and the gain of the cat gene. Digestion, ligation and transformation of chromosomal DNA from an M13 lysogen produces phage which carry a portion of the Tn10 as well as adjoining chromosomal DNA. The phage can be sequenced directly and are very useful for probing libraries for the wild type gene. Recombinant M13 clones have also been developed for the cloning of sequences adjacent to a Tn10 derivative which confers kanamycin resistance (mini-kan).

The mutY gene of Escherichia coli, which codes for an adenine glycosylase that excises the adenine of a G-A mispair, has been cloned and sequenced. The mutY gene codes for a protein of 350 amino acids (Mr = 39,123) and the clone genetically complements the mutY strain. The protein shows significant sequence homology to E. coli endonuclease III, an enzyme that has previously been shown to have glycosylase activity on damaged base pairs. Sequence analysis suggests that, like endonuclease III, MutY is an iron-sulfur protein with a [4Fe-4S]2+ cluster.

Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.

We have cloned and sequenced the mutR gene from Escherichia coli, which results in an increased frequency of spontaneous deletions, by using a strain carrying a Tn10 derivative inserted into mutR. The analysis of 1,286 bp of mutR sequence shows that this gene is identical to the topB gene, which encodes topoisomerase III. The increased deletion formation is the first reported phenotype for cells lacking topoisomerase III, and this suggests that topoisomerase III is involved in reactions that normally reduce the levels of spontaneous deletions.

Two transversion-specific mutator loci, mutA and mutC, were identified in Escherichia coli. Mutators with high rates of A.T----T.A transversions were identified using a screening technique that relied upon the reversion of an altered lacZ gene back to wild-type via a specific A.T----T.A transversion. Among the mutators collected, one class mapped to a previously unidentified locus that we designate mutA. Analysis of reverse mutations in lacZ and forward nonsense mutations in lacI showed that the mutA strain has higher levels of A.T----T.A and G.C----T.A transversions, and to a lesser degree A.T----C.G transversions. The mutA locus maps very near to, but is separable from, mutL, at about 95 min on the E. coli chromosome. Both its mutagenic specificity and complementation experiments confirmed that mutA is distinct from mutL and from a nearby mutator locus, miaA. The phenotype of a mutA mutL double mutator strain suggests that the mutA gene product prevents some replication errors. Another mutator, designated mutC, maps very near uvrC, at 42 min, but is distinguishable from uvrC, which has no mutator effects. The specificity of reversion of lacZ mutations in a mutC strain is identical to that in a mutA strain. Also, the behavior of a mutC mutS double mutant is identical to that of a mutA mutL double mutant. It is likely that mutA and mutC are components of the same error-avoidance system.

By using site-directed oligonucleotide mutagenesis, amber nonsense stop codons (5'-TAG-3') have been introduced at 20 sites in the Escherichia coli thymidylate synthase gene. By transforming the thyA mutant plasmids into 13 strains, each of which harbor different amber suppressor tRNAs, we were able to generate over 245 amino acid substitutions in E. coli thymidylate synthase (EC 2.1.1.45). Growth characteristics of these mutants have been studied, yielding a body of information that includes some surprising results in light of the recently published crystal structure of the enzyme.

Fibroblast growth factor-21 (FGF21) signaling requires the presence of β-Klotho, a co-receptor with a very short cytoplasmic domain. Here we show that FGF21 binds directly to β-Klotho through its C-terminus. Serial C-terminal truncations of FGF21 weakened or even abrogated its interaction with β-Klotho in a Biacore assay, and led to gradual loss of potency in a luciferase reporter assay but with little effect on maximal response. In contrast, serial N-terminal truncations of FGF21 had no impact on β-Klotho binding. Interestingly, several of them exhibited characteristics of partial agonists with minimal effects on potency. These data demonstrate that the C-terminus of FGF21 is critical for binding to β-Klotho and the N-terminus is critical for fibroblast growth factor receptor (FGFR) activation.Structured summaryMINT-6799939: FGFR1c (uniprotkb:P11362) binds (MI:0407) to β-Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)MINT-6799907, MINT-6799922: FGF21 (uniprotkb: Q9NSA1) binds (MI:0407) to β-Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)