Unraveling the Interaction between Carboxylesterase 1c and the Antibody-Drug Conjugate SYD985: Improved Translational PK/PD by Using Ces1c Knockout Mice.

Carboxylesterase 1c (CES1c) is responsible for linker-drug instability and poor pharmacokinetics (PK) of several antibody-drug conjugates (ADC) in mice, but not in monkeys or humans. Preclinical development of these ADCs could be improved if the PK in mice would more closely resemble that of humans and is not affected by an enzyme that is irrelevant for humans. SYD985, a HER2-targeting ADC based on trastuzumab and linker-drug vc-seco-DUBA, is also sensitive to CES1c. In the present studies, we first focused on the interaction between CES1c and SYD985 by size- exclusion chromatography, Western blotting, and LC/MS-MS analysis, using recombinant CES1c and plasma samples. Intriguingly, CES1c activity not only results in release of the active toxin DUBA but also in formation of a covalent bond between CES1c and the linker of vc-seco-DUBA. Mass spectrometric studies enabled identification of the CES1c cleavage site on the linker-drug and the structure of the CES1c adduct. To assess the in vivo impact, CES1c-/- SCID mice were generated that showed stable PK for SYD985, comparable to that in monkeys and humans. Patient-derived xenograft (PDX) studies in these mice showed enhanced efficacy compared with PDX studies in CES1c+/+ mice and provided a more accurate prediction of clinical efficacy of SYD985, hence delivering better quality data. It seems reasonable to assume that CES1c-/- SCID mice can increase quality in ADC development much broader for all ADCs that carry linker-drugs susceptible to CES1c, without the need of chemically modifying the linker-drug to specifically increase PK in mice. Mol Cancer Ther; 17(11); 2389-98. ©2018 AACR.

Synthesis and Evaluation of a Series of Long-Acting Glucagon-Like Peptide-1 (GLP-1) Pentasaccharide Conjugates for the Treatment of Type†2 Diabetes.

The present study details the development of a family of novel D-Ala(8) glucagon-like peptide-1 (GLP-1) peptide conjugates by site specific conjugation to an antithrombin III (ATIII) binding carrier pentasaccharide through tetraethylene glycol linkers. All conjugates were found to possess potent insulin-releasing activity. Peptides with short linkers (<25 atoms) conjugated at Lys(34) and Lys(37) displayed strong GLP-1 receptor (GLP-1-R) binding affinity. All D-Ala(8) GLP-1 conjugates exhibited prominent glucose-lowering action. Biological activity of the Lys(37) short-linker peptide was evident up to 72 h post-injection. In agreement, the pharmacokinetic profile of this conjugate (t1/2 , 11 h) was superior to that of the GLP-1-R agonist, exenatide. Once-daily injection of the Lys(37) short-linker peptide in ob/ob mice for 21 days significantly decreased food intake and improved HbA1c and glucose tolerance. Islet size was decreased, with no discernible change in islet number. The beneficial effects of the Lys(37) short-linker peptide were similar to or better than either exenatide or liraglutide, another GLP-1-R agonist. In conclusion, GLP-1 peptides conjugated to an ATIII binding carrier pentasaccharide have a substantially prolonged bioactive profile compatible for possible once-weekly treatment of type 2 diabetes in humans.

Identification of low abundant secreted proteins and peptides from primary culture supernatants of human T-cells.

Live cells continually communicate with their surroundings by the secretion of biomolecules, among which proteins and/or peptides are an important class. As such, these protein/peptide signals which end up in the extracellular medium, reflect the state of a cell in a certain condition, and as by definition are potential biomarkers indicative for specific physiological/pathological processes. We here report on a mass spectrometry based method for the detection and analysis of peptides and proteins secreted in a highly complex background, such as cell culture supernatant. Our method, which combines chromatography, high duty cycle tandem mass spectrometry and bio-informatics, enables the detection of interleukin-2 (IL-2), a cytokine secreted by activated T-cells, present in cell supernatant while representing only 0.006‰ of the total protein content. Moreover, the method allows the mass spectrometric analysis of signaling proteins in a non-targeted way and without any prior immunodepletion of the highest abundant cell culture medium proteins. In this study this is exemplified by the detection of yet two other secretory peptides, i.e., the granulins A and B, in the primary culture supernatant of non-activated T-cells.

Therapeutic antibody targeting of CD97 in experimental arthritis: the role of antigen expression, shedding, and internalization on the pharmacokinetics of anti-CD97 monoclonal antibody 1B2.

CD97 is a member of the EGF-TM7 family of adhesion class receptors, with a proposed role in inflammatory cell recruitment. Neutralization of murine CD97 with the anti-mCD97 mAb 1B2 was efficacious in prevention of murine collagen-induced arthritis, a model with features resembling rheumatoid arthritis. Here, the therapeutic potential of neutralizing CD97 in arthritis was studied with emphasis on the 1B2 pharmacokinetics. Mice with established arthritis were treated with anti-mCD97 or anti-TNF-alpha serum. Ab pharmacokinetics and biodistribution were studied in diseased and nondiseased mice using labeled 1B2. The impact of CD97 expression on Ab pharmacokinetics was studied using CD97 knockout mice. Treatment with 1B2 showed an efficacy comparable to anti-TNF-alpha treatment. Pharmacokinetic analysis of 1B2 in wild-type and CD97 knockout mice indicated a dose-dependent Ab clearance, due to specific interaction with CD97. Biodistribution studies showed accumulation of 1B2 in spleen and lung. In vitro studies using murine splenocytes revealed that CD97 when bound to Ab was internalized. Moreover, soluble CD97 was detected in the supernatant, suggesting Ag shedding. Finally, in arthritic mice, higher levels of soluble CD97 were found and 1B2 treatment led to specific targeting of inflamed paws, resulting in a higher clearance rate of 1B2 in arthritic mice than in wild-type mice. In conclusion, our data support a therapeutic value of CD97 neutralization in experimental arthritis. The pharmacokinetic profile of the 1B2 Ab illustrates the complexity of Ab elimination from an organism and stresses the importance of understanding Ag-Ab interactions when developing therapeutic mAbs.

Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263-275) epitope: an MHC anchor variant peptide for immune modulation.

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263-275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263-275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.

Antigens up the nose: identification of putative biomarkers for nasal tolerance induction functional studies combined with proteomics.

Intranasal autoantigen delivery is the most effective means of inducing mucosal tolerance and suppression of autoimmune disease. In an effort to identify markers of the "tolerant state", we employed proteomics technology at the level of the cervical lymph node. The analysis revealed that nasal antigen administration (without adiuvant) led to modulation of various proteins among which the most prominent were haptoglobin, nonintegrin 67 kDa laminin receptor, and MRP8. The immunoregulatory haptoglobin may qualify as (bio)marker for effective immunotherapy.

Compositional analyses of a human menopausal gonadotrophin preparation extracted from urine (menotropin). Identification of some of its major impurities.

Recently, a highly purified human menopausal gonadotrophin preparation (HMG) was launched. The composition and purity of this HMG (Menopur); Ferring Pharmaceuticals) with a claimed 1:1 ratio of FSH and LH was determined. Three gonadotrophins were observed: FSH, LH and human chorionic gonadotrophin (HCG). The immunoactivity for HCG was three-fold higher than the immunoactivity for LH. Because of the longer half-life of HCG as compared with LH, about 95% of the in-vivo LH-receptor-mediated bioactivity is attributable to the presence of HCG. This is substantiated by biochemical analyses. To the best of the authors' knowledge, this relatively high amount of HCG can only be explained by assuming the addition of HCG from external sources, which is a well established practice for standardization purposes. In addition to gonadotrophins, a number of other proteins were detected. The amount of these impurities, as determined by reversed-phase high-performance liquid chromatography on a peak-area basis, is at least 30%. Therefore, it is concluded that this HMG preparation contains at most 70% gonadotrophins. Via a proteomics approach three major impurities were identified: leukocyte elastase inhibitor, protein C inhibitor, and zinc-alpha(2)-glycoprotein. On the basis of the results obtained in this study, a comparison is made with recombinant FSH.

Crystal structure and carbohydrate-binding properties of the human cartilage glycoprotein-39.

The human cartilage glycoprotein-39 (HCgp-39 or YKL40) is expressed by synovial cells and macrophages during inflammation. Its precise physiological role is unknown. However, it has been proposed that HCgp-39 acts as an autoantigen in rheumatoid arthritis, and high expression levels have been associated with cancer development. HCgp-39 shares high sequence homology with family 18 chitinases, and although it binds to chitin it lacks enzymatic activity. The crystal structure of HCgp-39 shows that the protein displays a (beta/alpha)8-barrel fold with an insertion of an alpha + beta domain. A 43-A long carbohydrate-binding cleft is present at the C-terminal side of the beta-strands in the (beta/alpha)8 barrel. Binding of chitin fragments of different lengths identified nine sugar-binding subsites in the groove. Protein-carbohydrate interactions are mainly mediated by stacking of side chains of aromatic amino acid residues. Surprisingly, the specificity of chitin binding to HCgp-39 depends on the length of the oligosaccharide. Although chitin disaccharides tend to occupy the distal subsites, longer chains bind preferably to the central subsites in the groove. Despite the absence of enzymatic activity, long chitin fragments are distorted upon binding, with the GlcNAc at subsite -1 in a boat conformation, similar to what has been observed in chitinases. The presence of chitin in the human body has never been documented so far. However, the binding features observed in the complex structures suggest that either chitin or a closely related oligosaccharide could act as the physiological ligand for HCgp-39.