An Investigation into the In Vitro Targeted Killing of CD44-Expressing Triple-Negative Breast Cancer Cells Using Recombinant Photoimmunotherapeutics Compared to Auristatin-F-Based Antibody-Drug Conjugates.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer with limited treatment options. The persistence of highly tumorigenic CD44-expressing subpopulation referred to as cancer stem cells (CSCs), endowed with the self-renewal capacity, has been associated with therapeutic resistance, hence clinical relapses. To mitigate these undesired events, targeted immunotherapies using antibody-photoconjugate (APC) or antibody-drug conjugate (ADC), were developed to specifically release cytotoxic payloads within targeted cells overexpressing cognate antigen receptors.

CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate.

Purpose: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products.

Click Chemistry-Generated Auristatin F-Linker-Benzylguanine for a SNAP-Tag-Based Recombinant Antibody-Drug Conjugate Demonstrating Selective Cytotoxicity toward EGFR-Overexpressing Tumor Cells.

Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products.

Non-human primates in the PKPD evaluation of biologics: Needs and options to reduce, refine, and replace. A BioSafe White Paper.

Monoclonal antibodies (mAbs) deliver great benefits to patients with chronic and/or severe diseases thanks to their strong specificity to the therapeutic target. As a result of this specificity, non-human primates (NHP) are often the only preclinical species in which therapeutic antibodies cross-react with the target. Here, we highlight the value and limitations that NHP studies bring to the design of safe and efficient early clinical trials.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging for the Prediction of Monoclonal Antibody Tumor Disposition.

The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts.

Effect of variable domain charge on in vitro and in vivo disposition of monoclonal antibodies.

A growing body of evidence supports the important role of molecular charge on antibody pharmacokinetics (PK), yet a quantitative description of the effect of charge on systemic and tissue disposition of antibodies is still lacking. Consequently, we have systematically engineered complementarity-determining regions (CDRs) of trastuzumab to create a series of variants with an isoelectric point (pI) range of 6.3-8.

Protein engineering of a stable and potent anti-inflammatory IL-37-Fc fusion with enhanced therapeutic potential.

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways.

Challenges of non-clinical safety testing for biologics: A Report of the 9th BioSafe European Annual General Membership Meeting.

New challenges and other topics in non-clinical safety testing of biotherapeutics were presented and discussed at the nineth European BioSafe Annual General Membership meeting in November 2019. The session topics were selected by European BioSafe organization committee members based on recent company achievements, agency interactions and new data obtained in the non-clinical safety testing of biotherapeutics, for which data sharing would be of interest and considered as valuable information. The presented session topics ranged from strategies of testing, immunogenicity prediction, bioimaging, and developmental and reproductive toxicology (DART) assessments to first-in-human (FIH) dose prediction and bioanalytical challenges, reflecting the entire space of different areas of expertise and different molecular modalities.

Nonclinical Development of Biologics: Integrating Safety, Pharmacokinetics, and Pharmacodynamics to Create Smarter and More Flexible Nonclinical Safety Programs Optimizing Animal Use.

Safety assessment of biological drugs has its challenges due to the multiple new different modalities, for example, antibody-drug conjugates, bispecifics, nanobodies, fusion proteins and advanced therapy medicinal products (ATMPs), their different pharmacokinetic and pharmacodynamic properties, and their ability to trigger immunogenicity and toxicity. In the public and in the pharmaceutical industry, there is a strong and general desire to reduce the number of animals used in research and development of drugs and in particular reducing the use of nonhuman primates. Important discussions and activities are ongoing investigating the smarter designs of early research and dose range finding studies, reuse of animals, and replacing animal experiments with in vitro studies.

Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody.

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker.

Subcutaneous Site-of-Absorption Study with the Monoclonal Antibody Tocilizumab in Minipigs: Administration Behind Ear Translates Best to Humans.

Minipigs have been proposed as animal model to study the subcutaneous (SC) absorption of monoclonal antibodies (mAb), because they are more translatable to humans than other species. However, the minipig SC tissue structure differs markedly depending on its location. This study explored different SC administration sites for mAb SC administration, to explore which site translates best to humans.

OMTX705, a Novel FAP-Targeting ADC Demonstrates Activity in Chemotherapy and Pembrolizumab-Resistant Solid Tumor Models.

Purpose: The tumor microenvironment plays a key role in cancer development and progression and is involved in resistance to chemo- and immunotherapy. Cancer-associated fibroblast expressing fibroblast-activating protein α (FAPα) is one of the predominant stroma cell types and is involved in resistance to immunotherapy.

Experimental Design: We generated OMTX705, a novel antibody-drug conjugate from a humanized anti-FAP antibody linked to a new cytolysin.

Heparin chromatography as an predictor for antibody clearance rate through pinocytosis.

The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition.

Are Biotransformation Studies of Therapeutic Proteins Needed? Scientific Considerations and Technical Challenges.

For therapeutic proteins, the currently established standard development path generally does not foresee biotransformation studies by default because it is well known that the clearance of therapeutic proteins proceeds via degradation to small peptides and individual amino acids. In contrast to small molecules, there is no general need to identify enzymes involved in biotransformation because this information is not relevant for drug-drug interaction assessment and for understanding the clearance of a therapeutic protein. Nevertheless, there are good reasons to embark on biotransformation studies, especially for complex therapeutic proteins.

PKPD Assessment of the Anti-CD20 Antibody Obinutuzumab in Cynomolgus Monkey is Feasible Despite Marked Anti-Drug Antibody Response in This Species.

The pharmacokinetics (PK) of the anti-CD20 monoclonal antibody obinutuzumab was assessed after single intravenous dosing to cynomolgus monkeys. In addition, the pharmacokinetic-pharmacodynamic (PKPD) relationship for B-cell depletion was characterized. The PKPD model was used to estimate the B-cell repopulation during the recovery phase of chronic toxicology studies, thereby supporting the study design, in particular planning the recovery phase duration.

Prediction of Human Pharmacokinetics of Fomepizole from Preclinical Species Pharmacokinetics Based on Normalizing Time Course Profiles.

Fomepizole is used as an antidote to treat methanol poisoning due to its selectivity towards alcohol dehydrogenase. In the present study, the goal is to develop a method to predict the fomepizole human plasma concentration versus time profile based on the preclinical pharmacokinetics using the assumption of superimposability on simulated time course profiles of animals and humans. Standard allometric equations with/without correction factors were also assimilated in the prediction.

Simultaneous determination of bendamustine and γ-hydroxybendamustine in mice dried blood spots and its application in a mice pharmacokinetic study.

A selective, sensitive and rapid mice dried blood spot (DBS) method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxy-bendamustine (HBM) as per regulatory guidelines using an LC-MS/MS. Quality control, calibration curve and study sample DBS cards were sonicated with 5% formic acid in water before extraction with ethyl acetate enriched with internal standard (I.S.

Experimental investigations of a manually versus an electrically driven skull drill for bedside usage.

Background: Manual skull drilling is an old but in modern neurosurgery still established procedure which can be applied quickly and universally in emergency situations. Electrical drilling requires more complex equipment and is usually reserved to the Operating Room (OR). It also seems desirable to apply an electrical drill for bedside usage but a suitable product does not exist so far.

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths.

Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths.

Synthesis of a tubugi-1-toxin conjugate by a modulizable disulfide linker system with a neuropeptide Y analogue showing selectivity for hY1R-overexpressing tumor cells.

Tubugi-1 is a small cytotoxic peptide with picomolar cytotoxicity. To improve its cancer cell targeting, it was conjugated using a universal, modular disulfide derivative. This allowed conjugation to a neuropeptide-Y (NPY)-inspired peptide [K(C-βA-),F,L,P]-hNPY, acting as NPY Y1 receptor (hY1R)-targeting peptide, to form a tubugi-1-SS-NPY disulfide-linked conjugate.

Subcutaneous Administration of Biotherapeutics: An Overview of Current Challenges and Opportunities.

Subcutaneous delivery of biotherapeutics has become a valuable alternative to intravenous administration across many disease areas. Although the pharmacokinetic profiles of subcutaneous and intravenous formulations differ, subcutaneous administration has proven effective, safe, well-tolerated, generally preferred by patients and healthcare providers and to result in reduced drug delivery-related healthcare costs and resource use. The aim of this article is to discuss the differences between subcutaneous and intravenous dosing from both health-economic and scientific perspectives.

Biomechanics of common fixation devices for first tarsometatarsal joint fusion-a comparative study with synthetic bones.

Background: Hallux valgus disease is a common deformity of the forefoot. There are currently more than 100 surgical approaches for operative treatment. Because hypermobility of the first tarsometatarsal joint is considered to be causal for hallux valgus disease, fusion of the tarsometatarsal joint is an upcoming surgical procedure.

Prediction of Human Pharmacokinetics of Bendamustine from Preclinical Species Pharmacokinetics Based on Normalizing Time Course Profiles.

Bendamustine, an alkylating anticancer agent, is used to treat chronic lymphocytic leukemia by intravenous infusion alone or in combination. The work aimed to develop a method to predict time vs. concentration profile for humans based on preclinical pharmacokinetics using the assumption of superimposability of normalized time course profiles of animals and humans.

CD20-TCB with Obinutuzumab Pretreatment as Next-Generation Treatment of Hematologic Malignancies.

Despite promising clinical activity, T-cell-engaging therapies including T-cell bispecific antibodies (TCB) are associated with severe side effects requiring the use of step-up-dosing (SUD) regimens to mitigate safety. Here, we present a next-generation CD20-targeting TCB (CD20-TCB) with significantly higher potency and a novel approach enabling safer administration of such potent drug. We developed CD20-TCB based on the 2:1 TCB molecular format and characterized its activity preclinically.