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.

Sensitivity and drug tolerance of antidrug antibody assays in relation to positive control characteristics.

AIM: Detection and characterization of antidrug antibodies (ADA) play a key role in understanding the relation of ADA with safety and efficacy. Positive controls (PCs) are used to estimate the sensitivity and assay sensitivity in the presence of drug (drug tolerance) of assays to detect ADA. We investigated a number of factors that may drive sensitivity and drug tolerance. RESULTS: We found no correlation between affinity and sensitivity and sensitivity in the presence of drug in multiple assays with two antibody-drug conjugates. Multiple factors that influenced sensitivity and drug tolerance were observed, yet these had limited overall predictive value for all investigated assay formats, PCs and compounds. CONCLUSION: Assay sensitivity and drug tolerance as studied here, are likely driven by multiple factors such as cut point confidence level, stoichiometry of PC-drug complexes and presentation of epitopes.

Feedback from the European Bioanalysis Forum: focus workshop on current analysis of immunogenicity: best practices and regulatory hurdles.

European Bioanalysis Forum Workshop, Lisbon, Portugal, September 2016: At the recent European Bioanalysis Forum Focus Workshop, 'current analysis of immunogenicity: best practices and regulatory hurdles', several important challenges facing the bioanalytical community in relation to immunogenicity assays were discussed through a mixture of presentations and panel sessions. The main areas of focus were the evolving regulatory landscape, challenges of assay interferences from either drug or target, cut-point setting and whether alternative assays can be used to replace neutralizing antibody assays. This workshop report captures discussions and potential solutions and/or recommendations made by the speakers and delegates.

Rapid screening of IgG quality attributes - effects on Fc receptor binding.

The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High-throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress-induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D-N) reduced the binding of IgG to the low-affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short-term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in-process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.

Rapid Buffer and Ligand Screening for Affinity Chromatography by Multiplexed Surface Plasmon Resonance Imaging.

Protein purifications are often based on the principle of affinity chromatography, where the protein of interest selectively binds to an immobilized ligand. The development of affinity purification requires selecting proper wash and elution conditions. In recent years, miniaturization of the purification process is applied to speed up the development (e.g., microtiterplates, robocolumns). The application of surface plasmon resonance imaging (SPRi) as a tool to simultaneously screen many buffer conditions for wash and elution steps in an affinity-based purification process is studied. Additionally, the protein A ligand stability after exposure to harsh cleaning conditions often limits the reuse of resins and is determined at lab scale. The SPRi technology to screen ligand life-time with respect to alkali stability is used. It is also demonstrated that SPRi can successfully be applied in screening experiments for process developments in a miniaturized approach. The amount of resin, protein and buffer in these studies is reduced 30-300-fold compared to 1 mL column scale, and approximately 10-1000-fold compared to filter plate experiments. The overall development time can be decreased from several months towards days. The multiplexed SPRi can be applied in screening affinity chromatography conditions in early stage development for ligand development and recombinant protein production.

Switching from branded to generic glatiramer acetate: 15-month GATE trial extension results.

BACKGROUND: Open-label 15-month follow-up of the double-blind, placebo-controlled Glatiramer Acetate clinical Trial to assess Equivalence with Copaxone® (GATE) trial. OBJECTIVE: To evaluate efficacy, safety, and tolerability of prolonged generic glatiramer acetate (GTR) treatment and to evaluate efficacy, safety, and tolerability of switching from brand glatiramer acetate (GA) to GTR treatment. METHODS: A total of 729 patients received GTR 20 mg/mL daily. Safety was assessed at months 12, 15, 18, 21, and 24 and Expanded Disability Status Scale and magnetic resonance imaging (MRI) scans at months 12, 18, and 24. The presence of glatiramer anti-drug antibodies (ADAs) was tested at baseline and months 1, 3, 6, 9, 12, 18, and 24. RESULTS: The mean number of gadolinium-enhancing lesions in the GTR/GTR and GA/GTR groups was similar at months 12, 18, and 24. The change in other MRI parameters was also similar in the GTR/GTR and GA/GTR groups. The annualized relapse rate (ARR) did not differ between the GTR/GTR and GA/GTR groups, 0.21 and 0.24, respectively. The incidence, spectrum, and severity of reported adverse events did not differ between the GTR/GTR and GA/GTR groups. Glatiramer ADA titers were similar in the GTR/GTR and GA/GTR groups. CONCLUSION: Efficacy and safety of GTR is maintained over 2 years. Additionally, switching from GA to GTR is safe and well tolerated.

Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis.

Chemical protein biotinylation and streptavidin or anti-biotin-based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine-tuning of critical parameters is important to reduce biotinylation in a protein binding site.

A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA.

Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece - coinciding with the loss of high-mannose type N-glycan species - as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated anti-proliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.

Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers.

SYD985 is a HER2-targeting antibody-drug conjugate (ADC) based on trastuzumab and vc-seco-DUBA, a cleavable linker-duocarmycin payload. To evaluate the therapeutic potential of this new ADC, mechanistic in vitro studies and in vivo patient-derived xenograft (PDX) studies were conducted to compare SYD985 head-to-head with T-DM1 (Kadcyla), another trastuzumab-based ADC. SYD985 and T-DM1 had similar binding affinities to HER2 and showed similar internalization. In vitro cytotoxicity assays showed similar potencies and efficacies in HER2 3+ cell lines, but in cell lines with low HER2 expression, SYD985 was 3- to 50-fold more potent than T-DM1. In contrast with T-DM1, SYD985 efficiently induced bystander killing in vitro in HER2-negative (HER2 0) cells mixed with HER2 3+, 2+, or 1+ cell lines. At pH conditions relevant for tumors, cathepsin-B cleavage studies showed efficient release of the active toxin by SYD985 but not by T-DM1. These in vitro data suggest that SYD985 might be a more potent ADC in HER2-expressing tumors in vivo, especially in low HER2-expressing and/or in heterogeneous tumors. In line with this, in vivo antitumor studies in breast cancer PDX models showed that SYD985 is very active in HER2 3+, 2+, and 1+ models, whereas T-DM1 only showed significant antitumor activity in HER2 3+ breast cancer PDX models. These properties of SYD985 may enable expansion of the target population to patients who have low HER2-expressing breast cancer, a patient population with still unmet high medical need.

Preclinical profile of the HER2-targeting ADC SYD983/SYD985: introduction of a new duocarmycin-based linker-drug platform.

A linker-drug platform was built on the basis of a cleavable linker-duocarmycin payload for the development of new-generation antibody-drug conjugates (ADC). A leading ADC originating from that platform is SYD983, a HER2-targeting ADC based on trastuzumab. HER2-binding, antibody-dependent cell-mediated cytotoxicity and HER2-mediated internalization are similar for SYD983 as compared with trastuzumab. HER2-expressing cells in vitro are very potently killed by SYD983, but SYD983 is inactive in cells that do not express HER2. SYD983 dose dependently reduces tumor growth in a BT-474 mouse xenograft in vivo. The ADC is stable in human and cynomolgus monkey plasma in vitro but shows relatively poor stability in mouse plasma due to mouse-specific carboxylesterase. SYD983 could be dosed up to 30 mg/kg in cynomolgus monkeys with high exposure, excellent stability in blood, and without severe toxic effects. The monkey safety study showed no SYD983-induced thrombocytopenia and no induction of peripheral sensory neuropathy, both commonly observed in trials and studies with ADCs based on tubulin inhibitors. Finally, to improve homogeneity, SYD983 was further purified by hydrophobic interaction chromatography resulting in an ADC (designated SYD985) predominantly containing DAR2 and DAR4 species. SYD985 showed high antitumor activity in two patient-derived xenograft models of HER2-positive metastatic breast cancers. In conclusion, the data obtained indicate great potential for this new HER2-targeting ADC to become an effective drug for patients with HER2-positive cancers with a favorable safety profile. More generally, this new-generation duocarmycin-based linker-drug technology could be used with other mAbs to serve more indications in oncology.

Analysis of obstetric complications and uterine connective tissue in tenascin-X-deficient humans and mice.

Tenascin-X (TNX) is a large, multi-domain, extracellular matrix glycoprotein. Complete deficiency of TNX in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), and TNX haploinsufficiency is a cause of hypermobility type EDS. EDS patients appear to have a higher risk of several complications during pregnancy, such as pelvic instability, premature rupture of membranes, and postpartum hemorrhage. Here, we present a study of genitourinary and obstetric complications in TNX-deficient women of reproductive age. We have found complications, such as uterus prolapses, that are in agreement with previous findings in other EDS types. In TNX knockout (KO) mice, we have observed mild pregnancy-related abnormalities. Morphological and immunohistological analysis of uterine tissues has not revealed obvious quantitative or spatial differences between TNX KO and wildtype mice with respect to collagen types I, III, V, and XII or elastic fibers. We conclude that TNX-deficient women are at risk of obstetric complications, but that TNX KO mice show only a mild phenotype. Furthermore, we show that TNX is involved in the stability of elastic fibers rather than in their initial deposition.

Wound healing in tenascin-X deficient mice suggests that tenascin-X is involved in matrix maturation rather than matrix deposition.

Tenascin-X (TNX) is an extracellular matrix glycoprotein whose absence in humans leads to a recessive form of Ehlers-Danlos Syndrome (EDS). TNX deficient patients have hypermobile joints and fragile skin, but unlike the classical type of EDS, no atrophic scars were observed. Anecdotal evidence suggested that wound healing in TNX deficient patients is abnormal, but no detailed study has been performed so far. To address the role of TNX in wound healing, we analyzed skin wound morphology and mechanical properties of scars in TNX knockout (KO) mice. Breaking strength of unwounded skin of KO mice is significantly lower (<50%) than that of wild-type (WT) mice. In the early stage of wound healing when TNX is hardly expressed in WT wounds (day 7), WT and KO skin are of similar strength. After 14 days, when TNX starts to be expressed at moderate levels in wounds of WT mice, the WT scars gain a further increase in breaking strength, whereas KO scars do not progress beyond the mechanical strength of uninjured KO skin. No obvious differences between KO and WT mice were noted in the rate of wound closure, or in expression of fibrillar collagens during wound healing. We conclude that TNX is unlikely to be involved in matrix deposition in the early phase of wound healing, but it is required in the later phase when remodeling and maturation of the matrix establishes and improves its biomechanical properties.

Interactions of human tenascin-X domains with dermal extracellular matrix molecules.

Tenascin-X (TNX) is a large 450 kDa extracellular matrix protein expressed in a variety of tissues including skin, joints and blood vessels. Deficiency of TNX causes a recessive form of Ehlers-Danlos syndrome characterized by joint hypermobility, skin fragility and hyperextensible skin. Skin of TNX deficient patients shows abnormal elastic fibers and reduced collagen deposition. The mechanism by which TNX deficiency leads to connective tissue alterations is unknown. Here we report that C-terminal domains of human TNX bind to major dermal fibrillar collagens and tropoelastin. We have mapped these interactions to the fibronectin type III repeat 29 (FNIII29) and the C-terminal fibrinogen domain (FbgX) of TNX. In addition we found that FNIII29 of TNX accelerates collagen fibrillogenesis in vitro. We hypothesize that TNX contributes to matrix stability and is possibly involved in collagen fibril formation.

Tenascin-X, collagen, elastin, and the Ehlers-Danlos syndrome.

Tenascin-X is an extracellular matrix protein initially identified because the gene encoding it overlaps with the human CYP21B gene. Because studies of gene and protein function of other tenascins had been poorly predictive of essential functions in vivo, we used a genetic approach that critically relied on an understanding of the genomic locus to uncover an association between inactivating tenascin-X mutations and novel recessive and dominant forms of Ehlers-Danlos syndrome (EDS). Tenascin-X provides the first example of a gene outside of the fibrillar collagens and their processing enzymes that causes EDS. Tenascin-X null mice recapitulate the skin findings of the human disease, confirming a causative role for this gene in EDS. Further evaluation of these mice showed that tenascin-X is an important regulator of collagen deposition in vivo, suggesting a novel mechanism of disease in this form of EDS. Further studies suggest that tenascin-X may do this through both direct and indirect interactions with the collagen fibril. Recent studies show that TNX effects on matrix extend beyond the collagen to the elastogenic pathway and matrix remodeling enzymes. Tenascin-X serves as a compelling example of how human "experiments of nature" can guide us to an understanding of genes whose function may not be evident from their sequence or in vitro studies of their encoded proteins.