Effects of Conformational Changes in Peptide-CRM197 Conjugate Vaccines.

Conjugate vaccines prepared with the cross-reactive material 197 (CRM197) carrier protein have been successful in the clinic and are of great interest in the field of immunotherapy. One route to preparing peptide-CRM197 conjugate vaccines involves an activation-conjugation strategy, effectively coupling lysine residues on the protein to cysteine thiolate groups on the peptide of interest using a heterobifunctional linker as an activation agent. This method has been found to result in two distinct populations of conjugates, believed to be the result of a conformational change of CRM197 during preparation. This report explores the factors that lead to this conformational change, pointing to a model in which the unintentional alkylation of histidine-21 by the activating agent promotes the "opening" of the monomeric protein. This exposes a new set of lysine residues that are modified by additional activation agents. Subsequent peptide ligation to these sites results in the two conformers. This is the first time that a specific chemical modification is demonstrated to induce a defined conformational change for this carrier protein. Importantly, alternative conditions and reagents have been found to minimize this effect, improving the conformational homogeneity of peptide-CRM197 conjugates.

Process development of a FGF21 protein-antibody conjugate.

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

The Effect of Physicochemical Modification on the Function of Antibodies Induced by Anti-Nicotine Vaccine in Mice.

Smoking remains one of the major causes of morbidity and mortality worldwide. One approach to assisting smoking cessation is via anti-nicotine vaccines, composed of nicotine-like haptens conjugated to a carrier protein plus adjuvant(s). We have previously shown that the carrier, hapten, linker, hapten load, degree of conjugate aggregation, and presence of adducts can each influence the function (nicotine-binding capacity) of the antibody (Ab) induced. Herein, we extend those findings and show that tertiary structure is also critical to the induction of functional immune responses and that this can be influenced by conjugation conditions. We evaluated immunogenicity in mice using six lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7), and a single point (glycine 52 to glutamic acid) mutant nontoxic form of diphtheria toxin, cross-reactive material 197 (CRM197), which were synthesized under different reaction conditions resulting in conjugates with equivalent molecular characteristics (hapten load, aggregates, adducts), but a different tertiary structure. When tested in mice, better functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with conjugates with a more closed structure than those with an open conformation. These studies highlight the need for a better understanding of the physicochemical properties of small molecule conjugate vaccines.

Systematic Investigation of EDC/sNHS-Mediated Bioconjugation Reactions for Carboxylated Peptide Substrates.

1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet-to-be-reported side products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show that the trends established with model peptides hold for more complex systems.

Anti-nicotine vaccines: Comparison of adjuvanted CRM197 and Qb-VLP conjugate formulations for immunogenicity and function in non-human primates.

Anti-nicotine vaccines comprise nicotine-like haptens conjugated to a carrier protein plus adjuvant(s). Unfortunately, those tested clinically have failed to improve overall long term quit rates. We had shown in mice that carrier, hapten, linker, hapten load (number of haptens per carrier molecule), aggregation and adducts, as well as adjuvants influence the function of antibodies (Ab) induced. Herein, we tested an optimized antigen, NIC7-CRM, comprised of 5-aminoethoxy-nicotine (NIC7) conjugated to genetically detoxified diphtheria toxin (CRM197), with hapten load of ~16, no aggregation (~100% monomer) and minimal adducts. NIC7-CRM was tested in non-human primates (NHP) and compared to NIC-VLP, which has the same hapten and carrier as the clinical-stage CYT002-NicQb but a slightly different linker and lower hapten load. With alum as sole adjuvant, NIC7-CRM was superior to NIC-VLP for Ab titer, avidity and ex vivo function (83% and 27% nicotine binding at 40ng/mL respectively), but equivalent for in vivo function after intravenous [IV] nicotine challenge (brain levels reduced ~10%). CpG adjuvant added to NIC7-CRM/alum further enhanced the Ab responses and both ex vivo function (100% bound) and in vivo function (~80% reduction in brain). Thus, both optimal antigen design and CpG adjuvant were required to achieve a highly functional vaccine. The compelling NHP data with NIC7-CRM with alum/CpG supported human testing, currently underway.

Molecular attributes of conjugate antigen influence function of antibodies induced by anti-nicotine vaccine in mice and non-human primates.

Anti-nicotine vaccines aim to prevent nicotine entering the brain, and thus reduce or eliminate the reward that drives nicotine addiction. Those tested in humans to date have failed to improve quit rates over placebo, possibly because antibody (Ab) responses were insufficient to sequester enough nicotine in the blood in the majority of subjects. We have previously shown in mice that the carrier, hapten and linker used in the nicotine conjugate antigen each influence the function (nicotine-binding capacity) of the Ab induced. Herein we have evaluated immunogenicity in mice of 27 lots of NIC7-CRM, a conjugate of 5-aminoethoxy-nicotine (Hapten 7) and a mutant nontoxic form of diphtheria toxin (CRM197), that differed in three antigen attributes, namely hapten load (number of haptens conjugated to each molecule of CRM197), degree of conjugate aggregation and presence of adducts (small molecules attached to CRM197 via a covalent bond during the conjugation process). A range of functional responses (reduced nicotine in the brain of immunized animals relative to non-immunized controls) were obtained with the different conjugates, which were adjuvanted with aluminum hydroxide and CpG TLR9 agonist. Trends for better functional responses in mice were obtained with conjugates having a hapten load of 11 to 18, a low level of high molecular mass species (HMMS) (i.e., not aggregated) and a low level of adducts and a more limited testing in cynomolgus monkeys confirmed these results. Thus hapten load, conjugate aggregation and presence of adducts are key antigen attributes that can influence Ab function induced by NIC7-CRM.

Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain.

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

Structure-guided inhibitor design for human FAAH by interspecies active site conversion.

The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-A crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.

Direct interaction of soluble human recombinant tau protein with Abeta 1-42 results in tau aggregation and hyperphosphorylation by tau protein kinase II.

We report here that aggregated beta-amyloid (Abeta) 1-42 promotes tau aggregation in vitro in a dose-dependent manner. When Abeta-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and Abeta 1-42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer's disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of Abeta 1-42 from these cells was impaired. Taken together, these in vitro results suggest that Abeta 1-42 promotes both tau aggregation and hyperphosphorylation.