ABSTRACT
Bovine colostral antibodies, purified from cow's milk produced immediately after calving, have enhanced resistance to degradation by intestinal proteases relative to antibodies from human or bovine serum, making them of particular interest as orally administered therapeutic agents. However, the basis of this resistance is not well defined. We evaluated the stability of AVX-470, a bovine colostral anti-tumor necrosis factor (TNF) polyclonal antibody used in early clinical studies for treatment of ulcerative colitis, using conditions that mimic the human small intestine. AVX-470 was degraded â¼3 times more slowly than human IgG antibodies or infliximab (a monoclonal mouse-human chimeric IgG). Bovine IgG1 antibodies, the primary component of AVX-470, were slowly cleaved to F(ab')2 fragments. In contrast, bovine IgG2 and human IgG1 antibodies were cleaved rapidly into Fab and smaller fragments, pointing to specific regions where additional stability might be gained. Infliximab was modified to incorporate the sequences from these regions, including the bovine IgG1 hinge region and a predicted disulfide bonding motif linking the upper hinge region, the CH1 domain, and the light chain. This infliximab-bovine IgG1 chimera (bovinized infliximab) retained the antigen binding and neutralization activity of the WT sequence but was degraded 9-fold more slowly than the unmodified infliximab. This remarkable increase in stability with as few as 18 amino acid substitutions suggests that this bovinization process is a means to enable oral delivery of proven therapeutic antibodies as well as novel antibodies to targets that have been previously inaccessible to therapies delivered by injection.
Subject(s)
Colostrum/chemistry , Immunoglobulin G/chemistry , Intestines/chemistry , Proteolysis , Animals , Cattle , Female , Humans , Protein StabilityABSTRACT
The use of hyper-immune bovine colostrum as a human therapeutic platform is an emerging technology with potential to deliver the efficacy of antibody therapeutics with the convenience and safety of oral or topical application. It is necessary to understand how the bovine immune system responds to immunization with foreign proteins, both in terms of the serum antibody response and the transfer of antigen-specific antibodies into the colostrum to enable efficient large-scale production of therapeutic antibodies. We have immunized 25 cows with recombinant human tumor necrosis factor (rhTNF) and measured the levels of rhTNF-specific antibodies in the serum and colostrum of these animals. We observed a decline of 84±9% in serum IgG1 concentrations in the final weeks of pregnancy that presumably reflects rapid transport of IgG1 into colostrum. The serum IgG2 levels remained constant, such that the serum IgG1 to IgG2 ratio was 1:20 at parturition. We observed substantial animal-to-animal variability in the levels of anti-rhTNF antibodies in both serum and colostrum samples. In particular, a subset of 4 cows had extraordinarily high colostral anti-rhTNF antibody production. Only a weak correlation was found between the peak serum anti-rhTNF activity and the colostral anti-rhTNF activity in these animals. The 4 cows with high colostral anti-rhTNF activities trended toward higher serum IgG1 loss relative to average colostral anti-rhTNF producers, but this difference was not statistically significant in this small sample. The high-anti-rhTNF-producing cows also exhibited a greater proportion of rhTNF-specific antibodies that bound to bovine IgG1- and IgG2-specific detection antibodies relative to the total anti-rhTNF immunoglobulin population. This finding suggests that the isotype distribution of the anti-rhTNF response is varied between individuals and genetic or environmental factors may increase the yield of antigen-specific colostral antibodies.
Subject(s)
Colostrum/immunology , Immunoglobulin G/blood , Tumor Necrosis Factor-alpha/immunology , Animals , Cattle , Colostrum/chemistry , Female , Humans , Hydrogen-Ion Concentration , Immunization/veterinary , Immunoglobulin G/biosynthesis , Linear Models , Parturition/immunology , Pregnancy , Recombinant Proteins/administration & dosage , Recombinant Proteins/immunology , Tumor Necrosis Factor-alpha/administration & dosageABSTRACT
BACKGROUND AND AIMS: AVX-470 is an orally administered, bovine-derived, anti-tumour necrosis factor (TNF) antibody with local activity in the gastrointestinal tract. In the first-in-human clinical trial of AVX-470 in active ulcerative colitis, we evaluated inflammatory biomarkers in colon tissue as measures of disease activity and early response to treatment. METHODS: Thirty-six patients received active drug (AVX-470 at 0.2, 1.6 or 3.5g/day) or placebo over 4 weeks. Colon biopsy samples were collected from 5 regions of colon at baseline and week 4. Tissue inflammatory biomarkers were evaluated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), epithelial cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and bovine immunoglobulin by immunohistochemistry and mass spectrometry. Endoscopic activity (Ulcerative Colitis Endoscopic Index of Severity [UCEIS]) at colonoscopy was assessed in each colonic region by a central reader. RESULTS: Bovine immunoglobulin was observed in mucosal tissue before and after dosing in lamina propria and submucosal layers of biopsy tissue. Baseline levels of TNF, myeloperoxidase (MPO), CD68 and interleukin (IL)-1ß and, to a lesser extent, IL-6 mRNA were 2- to 3-fold higher in distal vs proximal colon tissue, corresponding to the 2- to 3-fold differences in baseline severities of endoscopic scores. Reductions of >10-fold in TNF and, to lesser extents, in MPO and epithelial cell apoptosis were observed in proximal and distal colon biopsies after 4 weeks of AVX-470 3.5g/day treatment. Reductions in TNF scores were correlated with changes in MPO and CD3 immunohistochemistry scores. CONCLUSIONS: These results are consistent with anti-TNF activity of orally administered AVX-470 in colon mucosal tissue in ulcerative colitis patients and demonstrate the utility of tissue biomarkers in assessing disease and treatment response in early clinical studies. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered with Clinicaltrials.gov as study NCT01759056 and with EudraCT as study 2012-004859-27.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antibodies/therapeutic use , Biomarkers/metabolism , Colitis, Ulcerative/drug therapy , Colon/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Administration, Oral , Adolescent , Adult , Aged , Colitis, Ulcerative/diagnostic imaging , Colitis, Ulcerative/metabolism , Colonoscopy , Double-Blind Method , Drug Administration Schedule , Drug Monitoring , Female , Humans , Immunohistochemistry , In Situ Nick-End Labeling , Male , Mass Spectrometry , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Severity of Illness Index , Treatment Outcome , Tumor Necrosis Factor-alpha/metabolism , Young AdultABSTRACT
Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called "acidic species" that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity.
Subject(s)
Antibodies, Monoclonal/chemistry , Citric Acid/chemistry , Amines/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Mass Spectrometry , Peptide Mapping , Peptides/analysis , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-Ig™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the flexibility of parental mAbs is important for designing and retaining functionality of DVD-Ig™ molecules. The architecture and dynamics of a DVD-Ig™ molecule and its parental mAbs was examined using single particle electron microscopy. Hinge angles measured for the DVD-Ig™ molecule were similar to the inner antigen parental mAb. The outer binding domain of the DVD-Ig™ molecule was highly mobile and three-dimensional (3D) analysis showed binding of inner antigen caused the outer domain to fold out of the plane with a major morphological change. Docking high-resolution X-ray structures into the 3D electron microscopy map supports the extraordinary domain flexibility observed in the DVD-Ig™ molecule allowing antigen binding with minimal steric hindrance.
Subject(s)
Antibodies, Bispecific/chemistry , Antibodies, Monoclonal/chemistry , Immunoglobulin G/chemistry , Immunoglobulin Variable Region/chemistry , Immunotherapy , Antibodies, Monoclonal/therapeutic use , Antigens/immunology , Crystallography, X-Ray , Humans , Interleukin-12/chemistry , Interleukin-12/immunology , Interleukin-18/chemistry , Interleukin-18/immunology , Microscopy, Electron, Transmission , Protein Binding , Protein Engineering/methods , Protein Structure, TertiaryABSTRACT
Many applications in pharmaceutical development, clinical diagnostics, and biological research demand rapid detection of multiple analytes (multiplexed detection) in a minimal volume. This need has led to the development of several novel array-based sensors. The most successful of these so far have been suspension arrays based on polystyrene beads. However, the 5 microm beads used for these assays are incompatible with most microfluidic chip technologies, mostly due to clogging problems. The challenge, then, is to design a detection particle that has high information content (for multiplexed detection), is compatible with miniaturization, and can be manufactured easily at low cost. DNA is a solid molecular wire that is easily produced and manipulated, which makes it a useful material for nanoparticles. DNA molecules are very information-rich, readily deformable, and easily propagated. We exploit these attributes in a suspension array sensor built from specialized recombinant DNA, Digital DNA, that carries both specific analyte-recognition units, and a geometrically encoded identification pattern. Here we show that this sensor combines high multiplexing with high sensitivity, is biocompatible, and has sufficiently small particle size to be used within microfluidic chips that are only 1 microm deep. We expect this technology will be the foundation of a broadly applicable technique to identify and quantitate proteins, nucleic acids, viruses, and toxins simultaneously in a minimal volume.
Subject(s)
DNA/analysis , Microfluidics/methods , Proteins/analysis , ImmunoassayABSTRACT
ATP hydrolysis by AAA+ ClpX hexamers powers protein unfolding and translocation during ClpXP degradation. Although ClpX is a homohexamer, positive and negative allosteric interactions partition six potential nucleotide binding sites into three classes with asymmetric properties. Some sites release ATP rapidly, others release ATP slowly, and at least two sites remain nucleotide free. Recognition of the degradation tag of protein substrates requires ATP binding to one set of sites and ATP or ADP binding to a second set of sites, suggesting a mechanism that allows repeated unfolding attempts without substrate release over multiple ATPase cycles. Our results rule out concerted hydrolysis models involving ClpX(6)*ATP(6) or ClpX(6)*ADP(6) and highlight structures of hexameric AAA+ machines with three or four nucleotides as likely functional states. These studies further emphasize commonalities between distant AAA+ family members, including protein and DNA translocases, helicases, motor proteins, clamp loaders, and other ATP-dependent enzymes.
Subject(s)
Adenosine Triphosphate/metabolism , Bacterial Proteins/metabolism , Bacterial Proton-Translocating ATPases/metabolism , Endopeptidase Clp/metabolism , Escherichia coli Proteins/metabolism , Adenosine Diphosphate/metabolism , Allosteric Regulation/physiology , Allosteric Site/physiology , Binding Sites/physiology , Binding, Competitive/physiology , Endopeptidase Clp/chemistry , Energy Metabolism/physiology , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Hydrolysis , Models, Molecular , Nucleotides/metabolism , Protein Folding , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
ATP-dependent protein degradation is controlled principally by substrate recognition. The AAA+ HslU ATPase is thought to bind protein substrates, denature them, and translocate the unfolded polypeptide into the HslV peptidase. The lack of well-behaved high-affinity substrates for HslUV (ClpYQ) has hampered understanding of the rules and mechanism of substrate engagement. We show that HslUV efficiently degrades Arc repressor, especially at heat-shock temperatures. Degradation depends on sequences near the N terminus of Arc. Fusion protein and peptide-binding experiments demonstrate that this sequence is a degradation tag that binds directly to HslU. Strong binding of this tag to the enzyme requires ATP and Mg(2+). Furthermore, fusion of this sequence to a protein with marked mechanical stability leads to complete degradation. Thus, these experiments demonstrate that HslUV is a powerful protein unfoldase and that initial substrate engagement by the HslU ATPase must occur after ATP binding.
Subject(s)
Adenosine Triphosphate/metabolism , Endopeptidase Clp/metabolism , Escherichia coli Proteins/metabolism , Magnesium/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/physiology , Binding Sites , Heat-Shock Proteins/metabolism , Heat-Shock Proteins/physiology , Protein Denaturation/physiology , Protein Folding , Protein Interaction Mapping , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Substrate Specificity/physiology , Viral Proteins/metabolism , Viral Regulatory and Accessory ProteinsABSTRACT
Machines of protein destruction-including energy-dependent proteases and disassembly chaperones of the AAA(+) ATPase family-function in all kingdoms of life to sculpt the cellular proteome, ensuring that unnecessary and dangerous proteins are eliminated and biological responses to environmental change are rapidly and properly regulated. Exciting progress has been made in understanding how AAA(+) machines recognize specific proteins as targets and then carry out ATP-dependent dismantling of the tertiary and/or quaternary structure of these molecules during the processes of protein degradation and the disassembly of macromolecular complexes.
Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Peptide Hydrolases/metabolism , Proteome/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Adenosine Triphosphatases/genetics , Animals , Binding Sites/physiology , Humans , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Conformation , Peptide Hydrolases/genetics , Proteome/geneticsABSTRACT
ClpX and related AAA+ ATPases of the Clp/Hsp100 family are able to denature native proteins. Here, we explore the role of protein stability in ClpX denaturation and subsequent ClpP degradation of model substrates bearing ssrA degradation tags at different positions. ClpXP degraded T. thermophilus RNase-H* with a C-terminal ssrA tag very efficiently, despite the very high global stability of this thermophilic protein. In fact, global thermodynamic stability appears to play little role in susceptibility to degradation, as a far less stable RNase-H*-ssrA mutant was degraded more slowly than wild type by ClpXP and a completely unfolded mutant variant was degraded less than twice as fast as the wild-type parent. When ssrA peptide tags were covalently linked to surface cysteines at positions 114 or 140 of RNase-H*, the conjugates were proteolyzed very slowly. This resistance to degradation was not caused by inaccessibility of the ssrA tag or an inability of ClpXP to degrade proteins with side-chain linked ssrA tags. Our results support a model in which ClpX denatures proteins by initially unfolding structural elements attached to the degradation tag, suggest an important role for the position of the degradation tag and direction of force application, and correlate well with the mapping of local protein stability within RNase-H* by native-state hydrogen exchange.
Subject(s)
Ribonuclease H/metabolism , Adenosine Triphosphatases/metabolism , Endopeptidase Clp , Enzyme Stability , Escherichia coli/genetics , Escherichia coli Proteins/metabolism , Kinetics , Molecular Probes , Mutagenesis, Site-Directed , Protein Denaturation , Ribonuclease H/genetics , Thermus thermophilus/enzymologyABSTRACT
ClpX requires ATP to unfold protein substrates and translocate them into the proteolytic chamber of ClpP for degradation. The steady-state parameters for hydrolysis of ATP and ATPgammaS by ClpX were measured with different protein partners and the kinetics of degradation of ssrA-tagged substrates were determined with both nucleotides. ClpX hydrolyzed ATPgammaS to ADP and thiophosphate at a rate (6/min) significantly slower than ATP hydrolysis (140/min), but the hydrolysis of both nucleotides was increased by ssrA-tagged substrates and decreased by ClpP. K(M) and k(cat) for hydrolysis of ATP and ATPgammaS were linearly correlated over a 200-fold range, suggesting that protein partners largely affect k(cat) rather than nucleotide binding, indicating that most bound ATP leaves the enzyme by hydrolysis rather than dissociation, and placing an upper limit of approximately 15 micro M on K(D) for both nucleotides. Competition studies with ClpX and fluorescently labeled ADP gave inhibition constants for ATPgammaS ( approximately 2 micro M) and ADP ( approximately 3 micro M) under the reaction conditions used for steady-state kinetics. In the absence of Mg(2+), where hydrolysis does not occur, the inhibition constant for ATP ( approximately 55 micro M) was weaker but very similar to the value for ATPgammaS ( approximately 45 micro M). Compared with ATP, ATPgammaS supported slow but roughly comparable rates of ClpXP degradation for two Arc-ssrA substrates and denatured GFP-ssrA, but not of native GFP-ssrA. These results show that the processing of protein substrates by ClpX is closely coupled to the maximum rate of nucleotide hydrolysis.