Synthesis of Erythropoietins Site-Specifically Conjugated with Complex-Type N-Glycans.

The biological activity of the glycoprotein hormone erythropoietin (EPO) is dependent mainly on the structure of its N-linked glycans. We aimed to readily attach defined N-glycans to EPO through copper-catalyzed azide alkyne cycloaddition. EPO variants with an alkyne-bearing non-natural amino acid (Plk) at the N-glycosylation sites 24, 38, and 83 were obtained by amber suppression followed by protein purification and refolding. Click conjugation of the alkynyl EPOs with biantennary N-glycan azides provided biologically active site-specifically modified EPO glycoconjugates.

Generation, Characterization, and Quantitative Bioanalysis of Drug/Anti-drug Antibody Immune Complexes to Facilitate Dedicated In Vivo Studies.

PURPOSE: Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies. METHODS: Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum. RESULTS: Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum. CONCLUSIONS: The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.

Intelligence as the efficiency of cue-driven retrieval from secondary memory.

Complex-span (working-memory-capacity) tasks are among the most successful predictors of intelligence. One important contributor to this relationship is the ability to efficiently employ cues for the retrieval from secondary memory. Presumably, intelligent individuals can considerably restrict their memory search sets by using such cues and can thereby improve recall performance. We here test this assumption by experimentally manipulating the validity of retrieval cues. When memoranda are drawn from the same semantic category on two successive trials of a verbal complex-span task, the category is a very strong retrieval cue on its first occurrence (strong-cue trial) but loses some of its validity on its second occurrence (weak-cue trial). If intelligent individuals make better use of semantic categories as retrieval cues, their recall accuracy suffers more from this loss of cue validity. Accordingly, our results show that less variance in intelligence is explained by recall accuracy on weak-cue compared with strong-cue trials.

Biodistribution of Drug/ADA Complexes: The Impact of Immune Complex Formation on Antibody Distribution.

The clinical use of therapeutic monoclonal antibodies (mAbs) for the treatment of cancer, inflammation, and other indications has been successfully established. A critical aspect of drug-antibody pharmacokinetics is immunogenicity, which triggers an immune response via an anti-drug antibody (ADA) and forms drug/ADA immune complexes (ICs). As a consequence, there may be a reduced efficacy upon neutralization by ADA or an accelerated drug clearance. It is therefore important to understand immunogenicity in biological therapies. A drug-like immunoglobulin G (IgG) was radiolabeled with tritium, and ICs were formed using polyclonal ADA, directed against the complementary-determining region of the drug-IgG, to investigate in vivo biodistribution in rodents. It was demonstrated that 65% of the radioactive IC dose was excreted within the first 24 h, compared with only 6% in the control group who received non-complexed 3H-drug. Autoradiographic imaging at the early time point indicated a deposition of immune complexes in the liver, lung, and spleen indicated by an increased radioactivity signal. A biodistribution study confirmed the results and revealed further insights regarding excretion and plasma profiles. It is assumed that the immune complexes are readily taken up by the reticuloendothelial system. The ICs are degraded proteolytically, and the released radioactively labeled amino acids are redistributed throughout the body. These are mainly renally excreted as indicated by urine measurements or incorporated into protein synthesis. These biodistribution studies using tritium-labeled immune complexes described in this article underline the importance of understanding the immunogenicity induced by therapeutic proteins and the resulting influence on biological behavior.

Phase 1, first-in-human study of TYRP1-TCB (RO7293583), a novel TYRP1-targeting CD3 T-cell engager, in metastatic melanoma: active drug monitoring to assess the impact of immune response on drug exposure.

Introduction: Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy. Methods: TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352). Results: Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1-2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro. Discussion: This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed.

High ionic strength dissociation assay reduces dimeric target interference in immunogenicity testing.

Aim: The presence of di-/multi-meric forms of soluble target in biological samples can interfere in anti-drug antibody (ADA) assays, leading to increased background values and potentially false positivity. The authors investigated the use of the high ionic strength dissociation assay (HISDA) to reduce target interference in two different ADA assays. Results: Interference caused by homodimeric FAP was successfully eliminated to enable cut point determination after applying HISDA. Biochemical experiments confirmed the dissociation of homodimeric FAP after treatment with high ionic strength conditions. Conclusion: HISDA is a promising approach to simultaneously achieve high drug tolerance and reduced interference by noncovalently bound dimeric target molecules in ADA assays without extensive optimization, which is particularly advantageous in routine use.

Enhanced immunogenic potential of cancer immunotherapy antibodies in human IgG1 transgenic mice.

ABBREVIATIONS: ADA Anti-Drug Antibodies; BCR B Cell Receptor; BId Idiotype-specific B Cell; BiTE Bispecific T cell Engager; BMC Bone Marrow Chimeric Mice; BSA Bovine Serum Albumin; CDR Complementary Determining Region; CEA Carcinoembryonic Antigen; CIT Cancer Immunotherapy; CitAbs Cancer Immunotherapy Antibodies; DC Dendritic Cell; ELISA Enzyme-Linked Immunosorbent Assay; FcRn Neonatal Fc Receptor; FcyR Fc gamma Receptor; GM-CSF Granulocyte-Macrophage Colony Stimulating Factor; gMFI Geometric Mean Fluorescence Intensity; H Heavy Chain; IC Immune Complex; Id Idiotype; IgA Immunoglobulin alpha; IgG1 Immunoglobulin gamma 1; IL-2 Interleukin 2; IL-2R Interleukin 2 Receptor; IL2v Interleukin 2 Variant; IVIG1 Intravenous Immunoglobulin 1; KLH Keyhole Limpet Hemocyanin; L Light Chain; MAPPs MHC-associated Peptide Proteomics; MHC Major Histocompatibility Complex; PBMC Peripheral Blood Mononuclear Cells; PBS Phosphate Buffered Saline; SHM Somatic Hypermutation; scFv Single-chain Variable Fragment; TCR T cell Receptor; TFc Fc-specific T cell; TId Id-specific T cell; UV Ultraviolet; V Variable.

The impact of immunogenicity on therapeutic antibody pharmacokinetics: A preclinical evaluation of the effect of immune complex formation and antibody effector function on clearance.

The occurrence of an immune response against therapeutic proteins poses a major risk for the development of biologics and for successful treatment of patients. Generation of anti-drug antibodies (ADAs) can lead to formation of immune complexes (ICs), consisting of drug and ADAs, with potential impact on safety, efficacy and exposure. Here, we focus on the effects of IC formation, i.e., specific IC sizes, ADA and drug properties, on drug pharmacokinetics. Pre-formed IC preparations of an IgG1 drug (with wild type or with an ablated effector function at the Fc domain) and different ADA surrogates (directed against the complementarity-determining regions or Fc domain of the drug) were administered to rats and collected serum was analyzed to determine the total drug concentration. A combination of size-exclusion chromatography and ELISA enabled a size-specific evaluation of IC profiles in serum and their changes over time. Within five minutes, total drug concentration decreased by ~20-60% when the drug was complexed. Independent of the ADA surrogate and drug variant used, increasing IC size led to increased clearance. Comparing ICs formed with the same ADA surrogate but different IgG1 variants, we observed that complexed drug with a wildtype Fc domain showed faster clearance compared to immune effector function modified drug. Data generated in this study indicated that clearance of drug due to ADA generation is driven by size and structure of the formed ICs, but also by the immune effector functions of the Fc domains of IgGs.Abbreviations Ab: antibody, ADA: anti-drug antibody, AUC: area under the curve, Bi: biotin, CDR: complementary-determining region, cmax: maximal concentration, Dig: digoxigenin, ELISA: enzyme-linked immunosorbent assay, Fc: fragment crystallizable, FcRn: neonatal Fc receptor, HMW: high molecular weight, IC: immune complex, IC-QC: immune complex quality control, IgG: immunoglobulin G, mAb: monoclonal antibody, mADA: monoclonal ADA, pAb: polyclonal antibody, pADA: polyclonal ADA, PD: pharmacodynamics; PK: pharmacokinetic, QC: quality control, SEC: size-exclusion chromatography, WT: wildtype.