Viral Delivery of Compact CRISPR-Cas12f for Gene Editing Applications.

Treating human genetic conditions requires efficient delivery of the CRISPR gene editing machinery to the affected cells and organs. The gene editing field has seen clinical advances with therapies and with delivery to the liver using lipid nanoparticle technology. Adeno-associated virus (AAV) serotypes have been discovered and engineered to deliver genetic material to nearly every organ in the body.

Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications.

The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here, we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a).

Cas9 degradation in human cells using phage anti-CRISPR proteins.

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9.

Discovery and Characterization of Novel Type V Cas12f Nucleases with Diverse Protospacer Adjacent Motif Preferences.

Small Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) effectors are key to developing gene editing therapies due to the packaging constraints of viral vectors. While Cas9 and Cas12a CRISPR-Cas effectors have advanced into select clinical applications, their size is prohibitive for efficient delivery of both nuclease and guide RNA in a single viral vector. Type V Cas12f effectors present a solution given their small size.

Tandem-Cleavage Linkers Improve the In Vivo Stability and Tolerability of Antibody-Drug Conjugates.

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, which leads to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia.

A Novel HER2-targeted Antibody-drug Conjugate Offers the Possibility of Clinical Dosing at Trastuzumab-equivalent Exposure Levels.

Trastuzumab and the related ADC, ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in both early-stage and metastatic settings for HER2 breast cancer. T-DM1 retains the antibody functionalities of trastuzumab and adds the potency of a cytotoxic maytansine payload.

Antibody-Drug Conjugates Targeting the Urokinase Receptor (uPAR) as a Possible Treatment of Aggressive Breast Cancer.

A promising molecular target for aggressive cancers is the urokinase receptor (uPAR). A fully human, recombinant antibody that binds uPAR to form a stable complex that blocks uPA-uPAR interactions (2G10) and is internalized primarily through endocytosis showed efficacy in a mouse xenograft model of highly aggressive, triple negative breast cancer (TNBC). Antibody-drug conjugates (ADCs) of 2G10 were designed and produced bearing tubulin inhibitor payloads ligated through seven different linkers.

Hydrophilic Sequence-Defined Cross-Linkers for Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are an established modality for the tissue-specific delivery of chemotherapeutics. However, due to the hydrophobic nature of many cytotoxic payloads, challenges remain in developing chemically stable ADCs with high drug loading. In previous studies, payload structure, unique stimuli-responsive chemistries, and PEGylated cross-linkers have been used to decrease ADC hydrophobicity.

Systematic LC/MS/MS Investigations for the IND-Enabling Extended Characterization of Antibody-Drug Conjugate Modifications.

We hypothesized that systematic liquid chromatography-tandem mass spectrometry investigations of an antibody-drug conjugate (ADC), its small and large molecular components, and surrogate small-molecule conjugates might comprise a simple and efficient approach for the extended characterization of ADCs. Furthermore, we envisioned that results from this work might allow us to assign specific composition changes in the ADC based on monoisotopic mass shifts of conjugatable modifications as detected in the surrogate small-molecule conjugates. We tested our hypothesis with a case study using an aldehyde-tag-based ADC conjugated to a noncleavable linker bearing a maytansine payload.

CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity.

Antibody-drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity.

A Case Study to Identify the Drug Conjugation Site of a Site-Specific Antibody-Drug-Conjugate Using Middle-Down Mass Spectrometry.

Middle-down mass spectrometry (MD MS) has emerged as a promising alternative to classical bottom-up approaches for protein characterization. Middle-level experiments after enzymatic digestion are routinely used for subunit analysis of monoclonal antibody (mAb)-related compounds, providing information on drug load distribution and average drug-to-antibody ratio (DAR). However, peptide mapping is still the gold standard for primary amino acid sequence assessment, post-translational modifications (PTM), and drug conjugation identification and localization.

Site-Specific Bioconjugation Using SMARTag Technology: A Practical and Effective Chemoenzymatic Approach to Generate Antibody-Drug Conjugates.

As a critical feature of the next generation of antibody-drug conjugates (ADCs), site-specific bioconjugation approaches can help to optimize stability, pharmacokinetics, efficacy, and safety as well as improve manufacturing consistency. The SMARTag technology platform offers a practical and efficient chemoenzymatic solution for site-specific protein modifications. A bioorthogonal aldehyde handle is introduced through the oxidation of a cysteine residue, embedded in a specific peptide sequence (CxPxR), to the aldehyde-bearing formylglycine (fGly).

Site-Specific Labeling of Proteins Using the Formylglycine-Generating Enzyme (FGE).

Use of the formylglycine generating enzyme (FGE)-a copper-dependent posttranslational protein modifier-represents a particularly elegant method taken directly from nature of introducing a unique amino acid into the larger context of a protein. Formylglycine (fGly) is a crucial component of the active site of sulfatases, where it directly participates in the breakdown of sulfate ester substrates. In the context of bioconjugation this aldehyde containing amino acid can be an invaluable reactive handle for the chemical conjugation of molecules.

Maytansine-bearing antibody-drug conjugates induce in vitro hallmarks of immunogenic cell death selectively in antigen-positive target cells.

Oncology treatment has been revolutionized by the introduction of immune checkpoint inhibitor drugs, which enable 20-40% of patients to generate anti-tumor immune responses. Combination treatment approaches with chemotherapeutic drugs may enable responses in the remaining patient cohorts. In this regard, a handful of drugs are promising due to their ability to induce immunogenic cell death in target cells.

Antibody-drug conjugate library prepared by scanning insertion of the aldehyde tag into IgG1 constant regions.

The advantages of site-specific over stochastic bioconjugation technologies include homogeneity of product, minimal perturbation of protein structure/function, and - increasingly - the ability to perform structure activity relationship studies at the conjugate level. When selecting the optimal location for site-specific payload placement, many researchers turn to in silico modeling of protein structure to identify regions predicted to offer solvent-exposed conjugatable sites while conserving protein function. Here, using the aldehyde tag as our site-specific technology platform and human IgG1 antibody as our target protein, we demonstrate the power of taking an unbiased scanning approach instead.

Leveraging Formylglycine-Generating Enzyme for Production of Site-Specifically Modified Bioconjugates.

Enzymatic modification of proteins can generate uniquely reactive chemical functionality, enabling site-specific reactions on the protein surface. Formylglycine-generating enzyme (FGE) is one enzyme that can be exploited in this fashion. FGE binds its consensus sequence (CXPXR, known as the "aldehyde-tag") and converts the cysteine to a formylglycine (fGly).

CAT-02-106, a Site-Specifically Conjugated Anti-CD22 Antibody Bearing an MDR1-Resistant Maytansine Payload Yields Excellent Efficacy and Safety in Preclinical Models.

Hematologically derived tumors make up ∼10% of all newly diagnosed cancer cases in the United States. Of these, the non-Hodgkin lymphoma (NHL) designation describes a diverse group of cancers that collectively rank among the top 10 most commonly diagnosed cancers worldwide. Although long-term survival trends are improving, there remains a significant unmet clinical need for treatments to help patients with relapsed or refractory disease, one cause of which is drug efflux through upregulation of xenobiotic pumps, such as MDR1.

Recent Developments in ADC Technology: Preclinical Studies Signal Future Clinical Trends.

The antibody-drug conjugate (ADC) field is in a transitional period. Older approaches to conjugate composition and dosing regimens still dominate the ADC clinical pipeline, but preclinical work is driving a rapid evolution in how we strategize to improve efficacy and reduce toxicity towards better therapeutic outcomes. These advances are largely based upon a body of investigational studies that together offer a deeper understanding of the absorption, distribution, metabolism, and excretion (ADME) and drug metabolism and pharmacokinetics (DMPK) fates of both the intact conjugate and its small-molecule component.

Rapid Characterization of a Mechanically Labile α-Helical Protein Enabled by Efficient Site-Specific Bioconjugation.

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize mechanical properties of biomolecules. Historically, accessibility relies upon the nonspecific adhesion of biomolecules to a surface and a cantilever and, for proteins, the integration of the target protein into a polyprotein. However, this assay results in a low yield of high-quality data, defined as the complete unfolding of the polyprotein.

Insights from native mass spectrometry approaches for top- and middle- level characterization of site-specific antibody-drug conjugates.

Antibody-drug conjugates (ADCs) have emerged as a family of compounds with promise as efficient immunotherapies. First-generation ADCs were generated mostly via reactions on either lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds, resulting in heterogeneous populations with a variable number of drug loads per antibody. To control the position and the number of drug loads, new conjugation strategies aiming at the generation of more homogeneous site-specific conjugates have been developed.

Site-Specific Tandem Knoevenagel Condensation-Michael Addition To Generate Antibody-Drug Conjugates.

Expanded ligation techniques are sorely needed to generate unique linkages for the growing field of functionally enhanced proteins. To address this need, we present a unique chemical ligation that involves the double addition of a pyrazolone moiety with an aldehyde-labeled protein. This ligation occurs via a tandem Knoevenagel condensation-Michael addition.

A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations.

Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity.

Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II).

Background: The ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development.

An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro.

An emerging playbook for antibody-drug conjugates: lessons from the laboratory and clinic suggest a strategy for improving efficacy and safety.

Antibody-drug conjugates (ADCs) have become de rigueur for pharmaceutical oncology drug development pipelines. There are more than 40 ADCs undergoing clinical trials and many more in preclinical development. The field has rushed to follow the initial successes of Kadcyla™ and Adcetris™, and moved forward with new targets without much pause for optimization.