Interchain Disulfide Bridges Research Articles

Seleno-relaxin analogues: effect of internal and external diselenide bonds on the foldability and a fibrosis-related factor of endometriotic stromal cells.

Human relaxin-2 (H2 relaxin) is a peptide hormone of about 6 kDa, first identified as a reproductive hormone involved in vasoregulation during pregnancy. It has recently attracted strong interest because of its diverse functions, including anti-inflammatory, anti-fibrotic, and vasodilatory, and has been suggested as a potential peptide-based drug candidate for a variety of diseases. Mature H2 relaxin is constituted by the A- and B-chains stabilized by two interchain disulfide (SS) bridges and one intrachain SS linkage. In this study, seleno-relaxins, SeRlx-α and SeRlx-β, which are [C11UA,C11UB] and [C10UA,C15UA] variants of H2 relaxin, respectively, were synthesized via a one-pot oxidative chain assembly (folding) from the component A- and B-chains. The substitution of SS bonds in a protein with their analogue, diselenide (SeSe) bonds, has been shown to alter the physical, chemical, and physiological properties of the protein. The surface SeSe bond (U11A-U11B) enhanced the yield of chain assembly while the internal SeSe bond (U10A-U15A) improved the reaction rate of the folding, indicating that these bridges play a major role in controlling the thermodynamics and kinetics, respectively, of the folding mechanism. Furthermore, SeRlx-α and SeRlx-β effectively reduced the expression of a tissue fibrosis-related factor in human endometriotic stromal cells. Thus, the findings of this study indicate that the S-to-Se substitution strategy not only enhances the foldability of relaxin, but also provides new guidance for the development of novel relaxin formulations for endometriosis treatment.

A biotechnological approach for the production of new protein bioplastics.

The future of biomaterial production will leverage biotechnology based on the domestication of cells as biological factories. Plants, algae, and bacteria can produce low-environmental impact biopolymers. Here, two strategies were developed to produce a biopolymer derived from a bioengineered vacuolar storage protein of the common bean (phaseolin; PHSL). The cys-added PHSL* forms linear-structured biopolymers when expressed in the thylakoids of transplastomic tobacco leaves by exploiting the formation of inter-chain disulfide bridges. The same protein without signal peptide (ΔPHSL*) accumulates in Escherichia coli inclusion bodies as high-molar-mass species polymers that can subsequently be oxidized to form disulfide crosslinking bridges in order to increase the stiffness of the biomaterial, a valid alternative to the use of chemical crosslinkers. The E. coli cells produced 300 times more engineered PHSL, measured as percentage of total soluble proteins, than transplastomic tobacco plants. Moreover, the thiol groups of cysteine allow the site-specific PEGylation of ΔPHSL*, which is a desirable functionality in the design of a protein-based drug carrier. In conclusion, ΔPHSL* expressed in E. coli has the potential to become an innovative biopolymer.

The solution structure of the heavy chain–only C5-Fc nanobody reveals exposed variable regions that are optimal for COVID-19 antigen interactions

Heavy chain-only antibodies can offer advantages of higher binding affinities, reduced sizes, and higher stabilities than conventional antibodies. To address the challenge of SARS-CoV-2 coronavirus, a llama-derived single-domain nanobody C5 was developed previously that has high COVID-19 virus neutralization potency. The fusion protein C5-Fc comprises two C5 domains attached to a glycosylated Fc region of a human IgG1 antibody and shows therapeutic efficacy invivo. Here, we have characterized the solution arrangement of the molecule. Two 1443DaN-linked glycans seen in the mass spectra of C5-Fc were removed and the glycosylated and deglycosylated structures were evaluated. Reduction of C5-Fc with 2-mercaptoethylamine indicated three interchain Cys-Cys disulfide bridges within the hinge. The X-ray and neutron Guinier RG values, which provide information about structural elongation, were similar at 4.1 to 4.2nm for glycosylated and deglycosylated C5-Fc. To explain these RG values, atomistic scattering modeling based on Monte Carlo simulations resulted in 72,737 and 56,749 physically realistic trial X-ray and neutron structures, respectively. From these, the top 100 best-fit X-ray and neutron models were identified as representative asymmetric solution structures, similar to that of human IgG1, with good R-factors below 2.00%. Both C5 domains were solvent exposed, consistent with the functional effectiveness of C5-Fc. Greater disorder occurred in the Fc region after deglycosylation. Our results clarify the importance of variable and exposed C5 conformations in the therapeutic function of C5-Fc, while the glycans in the Fc region are key for conformational stability in C5-Fc.

SARS-CoV-2 ORF8 dimerization and binding mode analysis with class I MHC: computational approaches to identify COVID-19 inhibitors.

SARS-CoV-2 encodes eight accessory proteins, one of which, ORF8, has a poorly conserved sequence with SARS-CoV and its role in viral pathogenicity has recently been identified. ORF8 in SARS-CoV-2 has a unique functional feature that allows it to form a dimer structure linked by a disulfide bridge between Cys20 and Cys20 (S-S). This study provides structural characterization of natural mutant variants as well as the identification of potential drug candidates capable of binding directly to the interchain disulfide bridge. The lead compounds reported in this work have a tendency to settle in the dimeric interfaces by direct interaction with the disulfide bridge. These molecules may disturb the dimer formation and may have an inhibition impact on its potential functional role in host immune evasion and virulence pathogenicity. This work provides detailed insights on the sequence and structural variability through computational mutational studies, as well as potent drug candidates with the ability to interrupt the intermolecular disulfide bridge formed between Cys20 and Cys20. Furthermore, the interactions of ORF8 peptides complexed with MHC-1 is studied, and the binding mode reveals that certain ORF8 peptides bind to MHC-1 in a manner similar to other viral peptides. Overall, this study is a narrative of various computational approaches used to provide detailed structural insights into SARS-CoV-2 ORF8 interchain disulfide bond disruptors.

Extending Native Top-Down Electron Capture Dissociation to MDa Immunoglobulin Complexes Provides Useful Sequence Tags Covering Their Critical Variable Complementarity-Determining Regions.

Native top-down mass spectrometry (MS) is gaining traction for the analysis and sequencing of intact proteins and protein assemblies, giving access to their mass and composition, as well as sequence information useful for identification. Herein, we extend and apply native top-down MS, using electron capture dissociation, to two submillion Da IgM- and IgG-based oligomeric immunoglobulins. Despite structural similarities, these two systems are quite different. The ∼895 kDa noncovalent IgG hexamer consists of six IgG subunits hexamerizing in solution due to three specifically engineered mutations in the Fc region, whereas the ∼935 kDa IgM oligomer results from the covalent assembly of one joining (J) chain and 5 IgM subunits into an asymmetric “pentamer” stabilized by interchain disulfide bridges. Notwithstanding their size, structural differences, and complexity, we observe that their top-down electron capture dissociation spectra are quite similar and straightforward to interpret, specifically providing informative sequence tags covering the highly variable CDR3s and FR4s of the Ig subunits they contain. Moreover, we show that the electron capture dissociation fragmentation spectra of immunoglobulin oligomers are essentially identical to those obtained for their respective monomers. Demonstrated for recombinantly produced systems, the approach described here opens up new prospects for the characterization and identification of IgMs circulating in plasma, which is important since IgMs play a critical role in the early immune response to pathogens such as viruses and bacteria.

Enhancement of Pharmaceutical Urate Oxidase Thermostability by Rational Design of De Novo Disulfide Bridge.

Background and Purpose: As a therapeutic enzyme, urate oxidase is utilized in the reduction of uric acid in various conditions such as gout or tumor syndrome lysis. However, even bearing kinetical advantage over other counterparts, it suffers from structural instability most likely due to its subcellular and fungal origin.Objectives: In this research, by using rational design and introduction of de novo disulfide bridge in urate oxidase structure, we designed and created a thermostable urate oxidase for the first time.Materials and Methods:Utilizing site-directed mutagenesis and only with one point mutation we constructed two separate mutants: Ala6Cys and Ser282Cys which covalently linked subunits of enzyme each other. Single mutation to cysteine created three inter-chain disulfide bridges and one hydrogen bond in Ala6Cys and two disulfide bridges in Ser282Cys.Results: Both mutants showed 10 °C increase in optimum activity compared to wild-type enzyme while the Km values for both increased by 50% and their specific activity compromised. The thermal stability of Ser282Cys increased remarkably by comparing Ala6Cys and wild-type enzymes. Estimation of half life for wild-type enzyme demonstrated 38.5 min, while for Ala6Cys and Ser282Cys were 138 and 115 min, respectively. Interestingly, the optimal pH of both mutants was broaden from 7 to 10, which could make them candidates for industrial applications.Conclusion: It seemed that introducing disulfide bridges resulted in local and overall rigidity by bringing two adjacent sites of enzyme together and decreasing the conformational entropy of unfolding state is responsible for the enhancement of thermostability.

Development of a Versatile and Modular Linker for Antibody-Drug Conjugates Based on Oligonucleotide Strand Pairing.

Linker design is crucial to the success of antibody-drug conjugates (ADCs). In this work, we developed a modular linker format for attaching molecular cargos to antibodies based on strand pairing between complementary oligonucleotides. We prepared antibody-oligonucleotide conjugates (AOCs) by attaching 18-mer oligonucleotides to an anti-HER2 antibody through thiol-maleimide chemistry, a method generally applicable to any immunoglobulin with interchain disulfide bridges. The hybridization of drug-bearing complementary oligonucleotides to our AOCs was rapid, stoichiometric, and sequence-specific. AOCs loaded with cytotoxic payloads were able to selectively target HER2-overexpressing cell lines such as SK-BR-3 and N87, with in vitro potencies similar to that of the marketed ADC Kadcyla (T-DM1). Our results demonstrated the potential of utilizing AOCs as a highly versatile and modular platform, where a panel of well-characterized AOCs bearing DNA, RNA, or various nucleic acid analogs, such as peptide nucleic acids, could be easily paired with any cargo of choice for a wide range of diagnostic or therapeutic applications.

Middle Level IM-MS and CIU Experiments for Improved Therapeutic Immunoglobulin Subclass Fingerprinting.

Most of the current FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are based on humanized or human IgG1, 2, or 4 subclasses and engineered variants. On the structural side, these subclasses are characterized by specific interchain disulfide bridge connections. Different analytical techniques have been reported to assess intact IgGs subclasses, with recently special interest in native ion mobility (IM) and collision induced unfolding (CIU) mass spectrometry (MS). However, these two techniques exhibit significant limitations to differentiate mAb subclasses at the intact level. In the present work, we aimed at developing a unique IM-MS-based approach for the characterization of mAb subclasses at the middle level. Upon IdeS-digestion, the unfolding patterns of the F(ab')2 and Fc domains were simultaneously analyzed in a single run to provide deeper structural insights of the mAb scaffold. The unfolding patterns associated with the F(ab')2 domains are completely different in terms of unfolding energies and number of transitions. Thereby, F(ab')2 regions are the diagnostic domain to provide specific unfolding signatures to differentiate IgG subclasses and provide more confident subclass categorization than CIU on intact mAbs. In addition, the potential of middle-level CIU was evaluated through the characterization of eculizumab, a hybrid therapeutic IgG2/4 mAb. The unfolding signatures of both domains were allowed to corroborate, within a single run, the hybrid nature of eculizumab as well as specific subclass domain assignments to the F(ab')2 and Fc regions. Altogether, our results confirm the suitability of middle-level CIU of F(ab')2 domains for subclass categorization of canonical and more complex new generation engineered antibodies and related products.

The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs.

A large number of bacterial toxins consist of active and cell binding protomers linked by an interchain disulfide bridge. The largest family of such disulfide‐bridged exotoxins is that of the clostridial neurotoxins that consist of two chains and comprise the tetanus neurotoxins causing tetanus and the botulinum neurotoxins causing botulism. Reduction of the interchain disulfide abolishes toxicity, and we discuss the experiments that revealed the role of this structural element in neuronal intoxication. The redox couple thioredoxin reductase–thioredoxin (TrxR‐Trx) was identified as the responsible for reduction of this disulfide occurring on the cytosolic surface of synaptic vesicles. We then discuss the very relevant finding that drugs that inhibit TrxR‐Trx also prevent botulism. On this basis, we propose that ebselen and PX‐12, two TrxR‐Trx specific drugs previously used in clinical trials in humans, satisfy all the requirements for clinical tests aiming at evaluating their capacity to effectively counteract human and animal botulism arising from intestinal toxaemias such as infant botulism.

Ligand binding and retention in snake gourd seed lectin (SGSL). A crystallographic, thermodynamic and molecular dynamics study.

Snake gourd seed lectin (SGSL) is a non-toxic homolog of type II ribosome-inactivating proteins (RIPs) which contain a catalytic domain and a lectin domain. Isothermal titration calorimetry (ITC) measurements of the interactions of the protein with LacNAc, Lac, Gal, Me-α-Gal were carried out and the crystal structures of the native protein and its complex with Lac were determined. The crystal structure of the Me-α-Gal complex has already been determined. While the crystal structure showed the presence of two-sugar-binding sites, one on each of the two domains of the lectin chain, ITC measurements indicated the presence of only one binding site. In order to resolve this anomaly, molecular dynamics (MD) simulations were carried out on the native protein and on its complexes with Me-α-Gal and Lac. Simulations were also performed on the protein after reducing the inter-chain disulfide bridge between the two chains. The crystal structures and the simulations confirmed the robustness of the protein structure, irrespective of the presence or absence of the disulfide bridge. The simulations indicated that although two sites can bind sugar, only the ligand at one site is retained in a dynamic situation. The studies thus bring out the subtle relationship between binding and retention of the ligand.

Similar Albeit Not the Same: In-Depth Analysis of Proteoforms of Human Serum, Bovine Serum, and Recombinant Human Fetuin.

Fetuin, also known as alpha-2-Heremans Schmid glycoprotein (AHSG), belongs to some of the most abundant glycoproteins secreted into the bloodstream. In blood, fetuins exhibit functions as carriers of metals and small molecules. Bovine fetuin, which harbors 3 N-glycosylation sites and a suggested half dozen O-glycosylation sites, has been used often as a model glycoprotein to test novel analytical workflows in glycoproteomics. Here we characterize and compare fetuin in depth, using protein from three different biological sources: human serum, bovine serum, and recombinant human fetuin expressed in HEK-293 cells, with the aim to elucidate similarities and differences between these proteins and the post-translational modifications they harbor. Combining data from high-resolution native mass spectrometry and glycopeptide centric LC-MS analysis, we qualitatively and quantitatively gather information on fetuin protein maturation, N-glycosylation, O-glycosylation, and phosphorylation. We provide direct experimental evidence that both the human serum and part of the recombinant proteins are processed into two chains (A and B) connected by a single interchain disulfide bridge, whereas bovine fetuin remains a single-chain protein. Although two N-glycosylation sites, one O-glycosylation site, and a phosphorylation site are conserved from bovine to human, the stoichiometry of the modifications and the specific glycoforms they harbor are quite distinct. Comparing serum and recombinant human fetuin, we observe that the serum protein harbors a much simpler proteoform profile, indicating that the recombinant protein is not ideally engineered to mimic human serum fetuin. Comparing the proteoform profile and post-translational modifications of human and bovine serum fetuin, we observe that, although the gene structures of these two proteins are alike, they represent quite distinct proteins when their glycoproteoform profile is also taken into consideration.

PSMA-targeted bispecific Fab conjugates that engage T cells

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity.

Cross-Linked Collagen Triple Helices by Oxime Ligation.

Covalent cross-links are crucial for the folding and stability of triple-helical collagen, the most abundant protein in nature. Cross-linking is also an attractive strategy for the development of synthetic collagen-based biocompatible materials. Nature uses interchain disulfide bridges to stabilize collagen trimers. However, their implementation into synthetic collagen is difficult and requires the replacement of the canonical amino acids (4R)-hydroxyproline and proline by cysteine or homocysteine, which reduces the preorganization and thereby stability of collagen triple helices. We therefore explored alternative covalent cross-links that allow for connecting triple-helical collagen via proline residues. Here, we present collagen model peptides that are cross-linked by oxime bonds between 4-aminooxyproline (Aop) and 4-oxoacetamidoproline placed in coplanar Xaa and Yaa positions of neighboring strands. The covalently connected strands folded into hyperstable collagen triple helices (Tm ≈ 80 °C). The design of the cross-links was guided by an analysis of the conformational properties of Aop, studies on the stability and functionalization of Aop-containing collagen triple helices, and molecular dynamics simulations. The studies also show that the aminooxy group exerts a stereoelectronic effect comparable to fluorine and introduce oxime ligation as a tool for the functionalization of synthetic collagen.

Diversity of insulin-like peptide signaling system proteins in Calanus finmarchicus (Crustacea; Copepoda) – Possible contributors to seasonal pre-adult diapause

Calanus finmarchicus, an abundant calanoid copepod in the North Atlantic Ocean, is both a major grazer on phytoplankton and an important forage species for invertebrate and vertebrate predators. One component of the life history of C. finmarchicus is the overwintering dormancy of sub-adults, a feature key for the annual recruitment of this species in early spring. While little is known about the control of dormancy in C. finmarchicus, one hypothesis is that it is an insect-like diapause, where the endocrine system is a key regulator. One group of hormones implicated in the control of insect diapause is the insulin-like peptides (ILPs). Here, C. finmarchicus transcriptomic data were used to predict ILP signaling pathway proteins. Four ILP precursors were identified, each possessing a distinct A- and B-chain peptide; these peptides are predicted to form bioactive heterodimers via inter-chain disulfide bridging. Two ILP receptors, which likely represent splice variants of a common gene, were identified. Three insulin-degrading enzymes were also discovered, as were proteins encoding the transcription factor FOXO, a downstream target of ILP that has been implicated in the regulation of insect diapause, and insulin receptor substrate, a protein putatively linking the ILP receptor and FOXO. RNA-Seq data suggest that some C. finmarchicus insulin pathway transcripts are differentially expressed across development. As in insects, the ILP signaling system may be involved in controlling C. finmarchicus’ organism-environment interactions (e.g., regulation of seasonal sub-adult diapause), a hypothesis that can now be investigated using these data.

Site-selective in situ growth of fluorescent polymer–antibody conjugates with enhanced antigen detection by signal amplification

This paper reports a new and general in situ methodology to grow fluorescent polymer conjugates from the interchain disulfide bridging sites of a monoclonal antibody. Atom transfer radical polymerization (ATRP) initiators were attached to a monoclonal antibody at its interchain disulfide bridging sites by disulfide re-bridging to yield a macroinitiator. Subsequent in situ ATRP of PEG-like monomers with dye-functionalized monomers from the macroinitiator formed antibody-polymer-dye conjugates with site-selectivity and tunable dye-to-antibody ratios. Notably, these conjugates can amplify antigen detection signal by reducing label-density dependent fluorescence quenching and by increasing dye-to-antibody ratios. The method developed may be applicable to a variety of antibodies, dyes and drugs to create a number of antibody-polymer-dye/drug conjugates for advanced diagnosis and therapy of diseases.

A Heterologous Reporter Defines the Role of the Tetanus Toxin Interchain Disulfide in Light-Chain Translocation.

Botulinum neurotoxins (BoNTs) and tetanus toxin (TeNT) are the most potent toxins for humans and elicit unique pathologies due to their ability to traffic within motor neurons. BoNTs act locally within motor neurons to elicit flaccid paralysis, while retrograde TeNT traffics to inhibitory neurons within the central nervous system (CNS) to elicit spastic paralysis. BoNT and TeNT are dichain proteins linked by an interchain disulfide bond comprised of an N-terminal catalytic light chain (LC) and a C-terminal heavy chain (HC) that encodes an LC translocation domain (HCT) and a receptor-binding domain (HCR). LC translocation is the least understood property of toxin action, but it involves low pH, proteolysis, and an intact interchain disulfide bridge. Recently, Pirazzini et al. (FEBS Lett 587:150-155, 2013, http://dx.doi.org/10.1016/j.febslet.2012.11.007) observed that inhibitors of thioredoxin reductase (TrxR) blocked TeNT and BoNT action in cerebellar granular neurons. In the current study, an atoxic TeNT LC translocation reporter was engineered by fusing β-lactamase to the N terminus of TeNT [βlac-TeNT(RY)] to investigate LC translocation in primary cortical neurons and Neuro-2a cells. βlac-TeNT(RY) retained the interchain disulfide bond, showed ganglioside-dependent binding to neurons, required acidification to promote βlac translocation, and was sensitive to auranofin, an inhibitor of thioredoxin reductase. Mutation of βlac-TeNT(RY) at C439S and C467S eliminated the interchain disulfide bond and inhibited βlac translocation. These data support the requirement of an intact interchain disulfide for LC translocation and imply that disulfide reduction is a prerequisite for LC delivery into the host cytosol. The data also support a model that LC translocation proceeds from the C to the N terminus. βlac-TeNT(RY) is the first reporter system to measure translocation by an AB single-chain toxin in intact cells.

Cryostructuring of polymer systems. Proteinaceous wide-pore cryogels generated by the action of denaturant/reductant mixtures on bovine serum albumin in moderately frozen aqueous media.

Freeze-thaw processing of bovine serum albumin (BSA) aqueous solutions, which contain also the additives of denaturants (urea in this case) and thiol-bearing reductants [cysteine (Cys) in this case] leads to the formation of wide-pore cryogels. The properties and porous morphology of these spongy gel matrices were demonstrated to depend on the initial concentration of all precursors and on the freezing/frozen storage temperature. The optimum conditions for preparing such BSA-based cryogels were found to be as follows: [BSA] = 3-5 g dL(-1), [urea] = 0.5-2.0 mol L(-1), [Cys] = 0.01 mol L(-1), and freezing temperatures in the range of -15 to -20 °C. The size of gross pores in thus prepared cryogels is ∼50-150 μm. The spatial network of BSA-cryogels was shown to be cross-linked chemically via interchain disulfide bridges. The significant role of hydrophobic interactions in the stabilization of 3D networks of these cryogels is inferred, as well as the supposition about the relay-race sequence mechanism of the intermolecular disulfide cross-link formation is made.

Recombinant p35 from bacteria can form Interleukin (IL-)12, but Not IL-35.

The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.

The Cysteine-rich Region of Type VII Collagen Is a Cystine Knot with a New Topology

Collagens are a group of extracellular matrix proteins with essential functions for skin integrity. Anchoring fibrils are made of type VII collagen (Col7) and link different skin layers together: the basal lamina and the underlying connective tissue. Col7 has a central collagenous domain and two noncollagenous domains located at the N and C terminus (NC1 and NC2), respectively. A cysteine-rich region of hitherto unknown function is located at the transition of the NC1 domain to the collagenous domain. A synthetic model peptide of this region was investigated by CD and NMR spectroscopy. The peptide folds into a collagen triple helix, and the cysteine residues form disulfide bridges between the different strands. The eight cystine knot topologies that are characterized by exclusively intermolecular disulfide bridges have been analyzed by molecular modeling. Two cystine knots are energetically preferred; however, all eight disulfide bridge arrangements are essentially possible. This novel cystine knot is present in type IX collagen, too. The conserved motif of the cystine knot is CX3CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus.

Abstract C238: Polyacetal polymer-based anti-HER2 antibody-drug conjugate employing cysteine bioconjugation through thioether linkage allows a high drug loading of dolastatin-derived payload with excellent pharmacokinetics and potent anti-tumor activity.

Abstract The application of biodegradable, polyacetal polymers to antibody-drug conjugate (ADC) design can provide numerous advantages, including significantly higher capacity for drug payload (∼20 drugs per antibody), the use of payloads with low potency that are not suitable for direct conjugation, the improvement of physicochemical properties for ADC, especially with highly hydrophobic payloads and the use of protein recognition scaffolds beyond the commonly used IgGs. The basis of this novel polyacetal polymer-based conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal, or Fleximer®) modified with chemically orthogonal linkers. One linker is used to covalently attach a targeting moiety (mAb or alternative), while a second, chemically distinct linker is used to attach a drug payload and to control the mechanism and rate of drug release. Previously, we have reported potent anti-tumor activity with trastuzumab-s-Dolaflexin™, an anti-HER2 ADC composed of trastuzumab and a proprietary dolastatin derivative coupled to a Fleximer scaffold (Dolaflexin™). In that example, Dolaflexin was conjugated to the antibody through interchain cysteine residues via a hindered disulfide linkage (AACR Annual Meeting 2013 Abstract #4331). Unlike direct drug-cysteine linked ADCs that can result in destabilization of antibody by disruption of interchain disulfide bridges, we have shown that Dolaflexin conjugation via cysteines in the antibody hinge region stabilizes the resulting ADCs through the formation of inter-chain bridge structures. Trastuzumab-s-Dolaflexin ADC exhibited a prolonged plasma half-life, tumor-specific accumulation, and potent anti-tumor activity in vivo. Here, we report a novel trastuzumab-m-Dolaflexin ADC using a maleimide linker that further enhances the pharmacokinetics of the ADC and demonstrates complete regressions of established HER2+ BT-474 xenograft tumors in SCID mice even at a single dose of 2.5 mg/kg. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C238. Citation Format: Joshua Thomas, Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Patrick Conlon, Cheri Stevenson, Alex Uttard, Liu Qin, Dmitry Gumerov, Elena Ter-Ovaneysan, Michael DeVit, Laura L. Poling, Peter U. Park, Timothy B. Lowinger. Polyacetal polymer-based anti-HER2 antibody-drug conjugate employing cysteine bioconjugation through thioether linkage allows a high drug loading of dolastatin-derived payload with excellent pharmacokinetics and potent anti-tumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C238.