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Fig S1. ELISA formats for detection of various analytes. Fig S2. Biophysical characterization of CAT-02-106. Fig S3. Competitive ELISA data showing CAT-02-106 binding to CD22. Fig S4. CAT-02-106 internalization on Ramos, Granta-519, and WSU-DLCL2 cells. Fig S5. CAT-02-106 in vitro cytotoxicity test on CD22-negative cells. Fig S6. The CAT-02-106-related ADC, anti-HER2 RED-106, does not induce bystander killing. Fig S7. Comparison of cell surface CD22 expression levels on Ramos and WSU-DLCL2 cells. Fig S8. Ramos and WSU-DLCL2 xenograft study data plotted as individual animals. Fig S9. Mouse body weights from Ramos and WSU-DLCL2 xenograft studies. Fig S10. CAT-02-106 binding to cynomolgus monkey B cells as assessed by flow cytometry. Fig S11. CAT-02-106 cross-reactivity to human and cynomolgus tissues. Table S1. Summary of pharmacokinetic findings in rats dosed at 3 mg/kg with CAT-02-106.

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. CD22 is a clinically validated target for the treatment of NHL, but no anti-CD22 agents have yet been approved for this indication. Recent approval of an anti-CD22 antibody–drug conjugate (ADC) for the treatment of relapsed/refractory ALL supports the rationale for targeting this protein. An opportunity exists for a next-generation anti-CD22 antibody–drug conjugate (ADC) to address unmet medical needs in the relapsed/refractory NHL population. We describe a site-specifically conjugated antibody–drug conjugate, made using aldehyde tag technology, targeted against CD22 and bearing a noncleavable maytansine payload that is resistant to MDR1-mediated efflux. The construct was efficacious against CD22+ NHL xenografts and could be repeatedly dosed in cynomolgus monkeys at 60 mg/kg with no observed significantly adverse effects. Exposure to total ADC at these doses (as assessed by AUC0-inf) indicated that the exposure needed to achieve efficacy was below tolerable limits. Together, the data suggest that this drug has the potential to be used effectively in patients with CD22+ tumors that have developed MDR1-related resistance to prior therapies. Mol Cancer Ther; 17(1); 161–8. ©2017 AACR.

Contains 3 tables and 8 figures with legends.

Supplementary Figure Legends

Supplementary Methods and Synthesis of RED-106

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. Here we report the observation of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell culture, the first example of Acr-induced degradation of CRISPR-Cas nucleases in human cells. Optimized expression of AcrIIA1 in human cells provided robust inhibition of SpyCas9 editing but had no impact on Type V CRIPSR-Cas12a, consistent with its binding site on the HNH domain. Targeted Cas9 protein degradation by AcrIIA1 could modulate Cas9 nuclease activity in human therapies. The small size and specificity of AcrIIA1 could be used in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), providing a tool for developing safe and precise gene editing applications.

Small CRISPR-Cas effectors are key to developing gene editing therapies due to the packaging constraints in 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. Here we describe μCas, a novel class of miniature (

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, leading 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. Only a few reports describe methods for optimizing peptide linkers to maintain efficient and potent tumor payload delivery while enhancing circulating stability. Herein, we address these critical limitations through the development of a tandem-cleavage linker strategy, where two sequential enzymatic cleavage events mediate payload release. We prepared dipeptides that are protected from degradation in the circulation by a sterically-encumbering glucuronide moiety. Upon ADC internalization and lysosomal degradation, the monosaccharide is removed and the exposed dipeptide is degraded, liberating the attached payload inside the target cell. We used CD79b-targeted monomethyl auristatin E (MMAE) conjugates as our model system, and compared the stability, efficacy, and tolerability of ADCs made with tandem-cleavage linkers to ADCs made using standard technology with the vedotin linker. The results—where rat studies showed dramatically improved tolerability in the hematopoietic compartment—highlight the role that linker stability plays in efficacy and tolerability, and offer a means of improving an ADC’s therapeutic index for improved patient outcomes.

Trastuzumab and the related antibody-drug conjugate (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. Interestingly, in the clinic, T-DM1 cannot always replace the use of trastuzumab plus chemotherapy administered together as single agents. We hypothesize that this failure may be due in part to the limited systemic exposure achieved by T-DM1 relative to trastuzumab because of toxicity-related dosing constraints on the ADC. We have developed a trastuzumab-based ADC site-specifically conjugated to maytansine through a noncleavable linker. This construct, termed CAT-01-106, has a drug-to-antibody ratio (DAR) of 1.8, approximately half the average DAR of T-DM1, which comprises a mixture of antibodies variously conjugated with DARs ranging from 0-8. The high DAR species present in T-DM1 contribute to its toxicity and limit its clinical dose. CAT-01-106 showed superior in vivo efficacy compared to T-DM1 at equal payload dosing and was equally or better tolerated compared to T-DM1 at equal payload dosing up to 120 mg/kg in Sprague-Dawley rats and 60 mg/kg in cynomolgus monkeys. CAT-01-106 also showed improved pharmacokinetics in rats relative to T-DM1, with 40% higher ADC exposure levels. Together, the data suggest that CAT-01-106 may be sufficiently tolerable to enable clinical dosing at trastuzumab-equivalent exposure levels, combining the functions of both the antibody and the payload in one drug and potentially improving patient outcomes.

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. In this work, we investigate the effect of a new parameter, cross-linker sequence. A support-free synthesis of PEGylated, sequence-defined cross-linkers was developed and applied to the synthesis of three constitutionally isomeric ADCs containing PEG side chains and a monomethyl auristatin E payload. Placement of PEG side chains distally from the payload was found to yield an ADC with altered hydrophilicity, antigen binding, and in vitro potency. This work establishes a versatile method for synthesizing multifunctional cross-linkers and identifies cross-linker sequence as a new handle for modulating the performance of ADCs.

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. Scanning insertion of the human formylglycine generating enzyme (FGE) recognition sequence, LCTPSR, at each of the 436 positions in the light and heavy chain antibody constant regions followed by co-expression with FGE yielded a library of antibodies bearing an aldehyde functional group ready for conjugation. Each of the variants was expressed, purified, and conjugated to a cytotoxic payload using the Hydrazinyl Iso-Pictet-Spengler ligation to generate an antibody-drug conjugate (ADC), which was analyzed in terms of conjugatability (assessed by drug-to-antibody ratio, DAR) and percent aggregate. We searched for insertion sites that could generate manufacturable ADCs, defined as those variants yielding reasonable antibody titers, DARs of ≥ 1.3, and ≥ 95% monomeric species. Through this process, we discovered 58 tag insertion sites that met these metrics, including 14 sites in the light chain, a location that had proved refractory to the placement of manufacturable tag sites using in silico modeling/rational approaches.

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. Knowing where the drug goes and how it is processed allows mechanistic connections to be drawn with commonly observed clinical toxicities. The field is also starting to consider ADC interactions with the immune system and potential synergistic therapeutic opportunities therein. In an indication of future directions for the field, antibody conjugates bearing non-cytotoxic small-molecule payloads are being developed to reduce side effects associated with treatment of chronic diseases. ADCs are not a magic bullet to cure disease. However, they will increasingly become valuable therapeutic tools to improve patient outcomes across a variety of indications.

International audience; 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. We report here the first multi-level characterization of a site-specific ADC by state-of-the-art mass spectrometry (MS) methods, including native MS and its hyphenation to ion mobility (IM-MS). We demonstrate the versatility of native MS methodologies for site-specific ADC analysis, with the unique ability to provide several critical quality attributes within one single run, along with a direct snapshot of ADC homogeneity/heterogeneity without extensive data interpretation. The capabilities of native IM-MS to directly access site-specific ADC conformational information are also highlighted. Finally, the potential of these techniques for assessing an ADC's heterogeneity/homogeneity is illustrated by comparing the analytical characterization of a site-specific DAR4 ADC to that of first-generation ADCs. Altogether, our results highlight the compatibility, versatility, and benefits of native MS approaches for the analytical characterization of all types of ADCs, including site-specific conjugates. Thus, we envision integrating native MS and IM-MS approaches, even in their latest state-of-the-art forms, into workflows that benchmark bioconjugation strategies.

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. Aldehyde tag technology was employed for linking, and either one or two tags were inserted into the antibody heavy chain, to produce site-specifically conjugated ADCs with drug-to-antibody ratios of either two or four. Both cleavable and non-cleavable linkers were combined with two different antimitotic toxins—MMAE (monomethylauristatin E) and maytansine. Nine different 2G10 ADCs were produced and tested for their ability to target uPAR in cell-based assays and a mouse model. The anti-uPAR ADC that resulted in tumor regression comprised an MMAE payload with a cathepsin B cleavable linker, 2G10-RED-244-MMAE. This work demonstrates in vitro activity of the 2G10-RED-244-MMAE in TNBC cell lines and validates uPAR as a therapeutic target for TNBC.

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. Here we describe a detailed method for generating formylglycine-containing proteins in a mammalian system developed specifically for the production of antibody-drug conjugates (ADCs) but applicable to a wide range of proteins.

International audience; 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. However, peptide mapping strategies can be challenging when dealing with more complex and heterogeneous mAb formats, like antibody-drug conjugates (ADCs). We report here, for the first time, MD MS analysis of a third-generation site-specific DAR4 ADC using different fragmentation techniques, including higher-energy collisional- (HCD), electron-transfer (ETD) dissociation and 213 nm ultraviolet photodissociation (UVPD). UVPD used as a standalone technique for ADC subunit analysis afforded, within the same liquid chromatography-MS/MS run, enhanced performance in terms of primary sequence coverage compared to HCD- or ETD-based MD approaches, and generated substantially more MS/MS fragments containing either drug conjugation or glycosylation site information, leading to confident drug/glycosylation site identification. In addition, our results highlight the complementarity of ETD and UVPD for both primary sequence validation and drug conjugation/glycosylation site assessment. Altogether, our results highlight the potential of UVPD for ADC MD MS analysis for drug conjugation/glycosylation site assessment, and indicate that MD MS strategies can improve structural characterization of empowered next-generation mAb-based formats, especially for PTMs and drug conjugation sites validation.

Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years. However, the molecular mechanisms by which ADCs are internalized and activated within cells remain unclear. Here we use CRISPR-Cas9 screens to identify genes that control the toxicity of ADCs. Our results demonstrate critical roles for a range of known and novel endolysosomal trafficking regulators in ADC toxicity. We identify and characterize C18orf8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of CRISPR screens with ADCs bearing a noncleavable linker versus a cleavable valine-citrulline (VC) linker, we show that a subset of late endosomal and lysosomal regulators are selectively essential for toxicity of noncleavable linker ADCs. We further show that cleavable VC linkers are rapidly processed upon internalization and therefore surprisingly appear to bypass the requirement of lysosomal delivery. Lastly, we show that inhibition of sialic acid biosynthesis sensitizes cells to ADC treatment by increasing the rate of ADC internalization. This sensitization was observed using several ADCs targeting different antigens in diverse cancer cell types, including the FDA-approved ADC trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal novel regulators of endolysosomal trafficking, provide important insights to guide future ADC design, and identify candidate combination therapy targets as well as potential mechanisms of ADC resistance.

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. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine-citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.

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. Here we describe a detailed method for generating formylglycine-containing proteins in a mammalian system developed specifically for the production of antibody-drug conjugates (ADCs) but applicable to a wide range of proteins.

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). This enzymatic modification is carried out by the formylglycine-generating enzyme (FGE). The broad recognition of this short sequence by FGE within the context of heterologous proteins allows for the introduction of fGly residues at chosen sites in proteins expressed in prokaryotic and eukaryotic systems. The protocol presented here describes the methods for expressing fGly-containing antibodies in eukaryotic cells and subsequent site-specific conjugation with a payload-linker using aldehyde-specific Hydrazino-Iso-Pictet-Spengler (HIPS) chemistry.

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 pyrazolone reacts with an aldehyde to generate an enone, which undergoes subsequent attack by a second pyrazolone to generate a bis-pyrazolone species. This rapid and facile ligation technique is performed under mild conditions in the absence of catalyst to generate new architectures that were previously inaccessible via conventional ligation reactions. Using this unique ligation, we generated three site-specifically labeled antibody-drug conjugates (ADCs) with an average of four drugs to one antibody. The in vitro and in vivo efficacies along with pharmacokinetic data of the site-specific ADCs are reported.

We hypothesized that systematic liquid chromatography-tandem mass spectrometry investigations of an antibody&ndash, 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. Nearly quantitative bioconversion from cysteine to formylglycine was observed in the monoclonal antibody, and bioorthogonal conjugation was detected only on the formylglycine residues in the ADC. Using our method, both conjugatable and nonconjugatable modifications were discovered in the linker/payload, however, only conjugatable modifications were observed on the ADC. Based on these results, we anticipate that our approach to systematic mass spectrometric investigations can be successfully applied to other ADCs and therapeutic bioconjugates for investigational new drug (IND)-enabling extended characterization.

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. However, these agents are systemically delivered and indiscriminately cytotoxic to proliferating cells. By contrast, antibody-drug conjugates can selectively deliver a cytotoxic payload to a tumor, sparing most healthy cells. The ability of antibody-drug conjugates to induce immunogenic cell death in target cells has not yet been determined, although preclinical in vivo studies suggest this possibility. Here, we describe for the first time production of the in vitro hallmarks of immunogenic cell death – ecto-calreticulin and secreted ATP and HMGB1 protein – by cells in response to treatment with antibody-drug conjugates bearing a maytansine payload.

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). fGly-containing proteins contain a bioorthogonal aldehyde on their surface that can be modified selectively in the presence of the 20 canonical amino acids. Here, we describe protocols for the generation of a site-specifically modified protein, an antibody-drug conjugate (ADC), using aldehyde-tagging protocols and aldehyde-reactive conjugation chemistry.

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). fGly-containing proteins contain a bioorthogonal aldehyde on their surface that can be modified selectively in the presence of the 20 canonical amino acids. Here, we describe protocols for the generation of a site-specifically modified protein, an antibody-drug conjugate (ADC), using aldehyde-tagging protocols and aldehyde-reactive conjugation chemistry.

Background: Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo. Results: Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro. Conclusion: FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags. Significance: Understanding FGE biochemistry informs research on sulfatases and enables expanded biotechnology applications of the aldehyde tag., To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.

SummaryThere is a need for facile chemistries that allow for chemo- and regioselectivity in bioconjugation reactions. To address this need, we are pioneering site-specific bioconjugation methods that use formylglycine as a bioorthogonal handle on a protein surface. Here we introduce aldehyde-specific bioconjugation chemistry, the trapped-Knoevenagel ligation. The speed and stability of the trapped-Knoevenagel ligation further advances the repertoire of aldehyde-based bioconjugations and expands the toolbox for site-specific protein modifications. The trapped-Knoevenagel ligation reaction can be run at near neutral pH in the absence of catalysts to produce conjugates that are stable under physiological conditions. Using this new ligation, we generated an antibody-drug conjugate that demonstrates excellent efficacy in vitro and in vivo.

Genetic fusions of either full enzymes or peptide tags with a protein of interest have enabled the synthesis of protein conjugates with precise control over the site of attachment and number of payloads incorporated. Engineering of protein glycans, depending on the application, can lead to similar control for nonengineered glycoproteins. Recent advances in the field of chemoenzymatic modifications of proteins for site-specific protein conjugation will be reviewed. These techniques have been used in an array of fields for the execution of innovative and valuable experiments. Specific industrial applications of these technologies will be highlighted.

In the context of antibody–drug conjugates (ADCs), noncleavable linkers provide a means to deliver cytotoxic small molecules to cell targets while reducing systemic toxicity caused by nontargeted release of the free drug. Additionally, noncleavable linkers afford an opportunity to change the chemical properties of the small molecule to improve potency or diminish affinity for multidrug transporters, thereby improving efficacy. We employed the aldehyde tag coupled with the hydrazino- iso -Pictet-Spengler (HIPS) ligation to generate a panel of site-specifically conjugated ADCs that varied only in the noncleavable linker portion. The ADC panel comprised antibodies carrying a maytansine payload ligated through one of five different linkers. Both the linker-maytansine constructs alone and the resulting ADC panel were characterized in a variety of in vitro and in vivo assays measuring biophysical and functional properties. We observed that slight differences in linker design affected these parameters in disparate ways, and noted that efficacy could be improved by selecting for particular attributes. These studies serve as a starting point for the exploration of more potent noncleavable linker systems.

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. Additionally, nonspecific surface adhesion hinders studies of α–helical proteins, which unfold at low forces and low extensions. Here, we overcame these limitations by merging two developments: (i) a polyprotein with versatile, genetically encoded short peptide tags functionalized via a mechanically robust Hydrazino-Pictet Spengler ligation and (ii) the efficient site-specific conjugation of biomolecules to PEG-coated surfaces. Heterobifunctional anchoring of this polyprotein construct and DNA via copper-free click chemistry to PEG-coated substrates and a strong but reversible streptavidin-biotin linkage to PEG-coated AFM tips enhanced data quality and throughput. For example, we achieved a 75-fold increase in the yield of high-quality data and repeatedly probed the same individual polyprotein to deduce its dynamic force spectrum in just 2 h. The broader utility of this polyprotein was demonstrated by measuring three diverse target proteins: an α-helical protein (calmodulin), a protein with internal cysteines (rubredoxin), and a computationally designed three-helix bundle (α3D). Indeed, at low loading rates, α3D represents the most mechanically labile protein yet characterized by AFM. Such efficient SMFS studies on a commercial AFM enable the rapid characterization of macromolecular folding over a broader range of proteins and a wider array of experimental conditions (pH, temperature, denaturants). Further, by integrating these enhancements with optical traps, we demonstrate how efficient bioconjugation to otherwise nonstick surfaces can benefit diverse single-molecule studies.

Expansion of antibody scaffold diversity has the potential to expand the neutralizing capacity of the immune system and to generate enhanced therapeutics and probes. Systematic exploration of scaffold diversity could be facilitated with a modular and chemical scaffold for assembling proteins, such as DNA. However, such efforts require simple, modular, and site-specific methods for coupling antibody fragments or bioactive proteins to nucleic acids. To address this need, we report a modular approach for conjugating synthetic oligonucleotides to proteins with aldehyde tags at either terminus or internal loops. The resulting conjugates are assembled onto DNA-based scaffolds with low nanometer spatial resolution and can bind to live cells. Thus, this modular and site-specific conjugation strategy provides a new tool for exploring the potential of expanded scaffold diversity in immunoglobulin-based probes and therapeutics.

It is becoming increasingly clear that site-specific conjugation offers significant advantages over conventional conjugation chemistries used to make antibody–drug conjugates (ADCs). Site-specific payload placement allows for control over both the drug-to-antibody ratio (DAR) and the conjugation site, both of which play an important role in governing the pharmacokinetics (PK), disposition, and efficacy of the ADC. In addition to the DAR and site of conjugation, linker composition also plays an important role in the properties of an ADC. We have previously reported a novel site-specific conjugation platform comprising linker payloads designed to selectively react with site-specifically engineered aldehyde tags on an antibody backbone. This chemistry results in a stable C–C bond between the antibody and the cytotoxin payload, providing a uniquely stable connection with respect to the other linker chemistries used to generate ADCs. The flexibility and versatility of the aldehyde tag conjugation platform has enabled us to undertake a systematic evaluation of the impact of conjugation site and linker composition on ADC properties. Here, we describe the production and characterization of a panel of ADCs bearing the aldehyde tag at different locations on an IgG1 backbone conjugated using Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. We demonstrate that in a panel of ADCs with aldehyde tags at different locations, the site of conjugation has a dramatic impact on in vivo efficacy and pharmacokinetic behavior in rodents; this advantage translates to an improved safety profile in rats as compared to a conventional lysine conjugate.

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. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10nM with 370pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step.

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. For example, antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology, where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression, the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE), which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation. Results The yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen, driving enzymatic turnover. Building upon that work, here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE, which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture, as demonstrated in fed-batch cell cultures with antibody titers of 5 g · L−1, specific cellular production rates of 75 pg · cell−1 · d−1, and fGly conversion yields of 95–98 %. Conclusions We describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE, which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0254-0) contains supplementary material, which is available to authorized users.

We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with α-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

Atomic force microscopy (AFM) is a powerful tool for single molecule force spectroscopy, allowing researchers to directly probe the energy landscape of protein folding and unfolding. Despite the power of this technique, it has been hindered by non-specific attachment of protein to the AFM surface and tip. Non-specific attachment allows for simple experimental set up, but results in low attachment efficiency, multiple attachments, incomplete unfolding, and adhesion to the surface which can mask low force unfolding events. Not only does this make data collection incredibly time consuming, it also makes it difficult to test mechanically labile proteins. Despite recent advances in tip and surface functionalization, protein labeling continues to be a major hurdle for many labs. Furthermore, most current labeling protocols require cysteine tag modification which is unsuitable for cysteine containing proteins. To address this issue, we developed a simple and versatile protein cassette utilizing aldehyde tags to facilitate the specific attachment of proteins to both the AFM surface and tip. Combining our labeled protein cassette with newly developed techniques to functionalize the surface and tip resulted in a 75-fold increase in attachment efficiency while greatly reducing the number of incomplete unfolding events and surface adhesion. We have shown the ability to quickly insert new proteins into our cassette and rapidly characterize their response to force. Moreover, we were able to rapidly collect a dynamic force spectrum for α3D, an α-helical protein which unfolds at very low force. Our protein cassette is a powerful tool that not only increases the efficiency of AFM based single molecule force spectroscopy, but also enhances the ability to test mechanically labile proteins.

In the past decade, numerous chemical technologies have been developed to allow the site-specific post-translational modification of proteins. Traditionally covalent chemical protein modification has been accomplished by the attachment of synthetic groups to nucleophilic amino acids on protein surfaces. These chemistries, however, are rarely sufficiently selective to distinguish one residue within a literal sea of chemical functionality. One solution to this problem is to introduce a unique chemical handle into the target protein that is orthogonal to the remainder of the proteome. In practice, this handle can be a novel peptide sequence, which forms a “tag” that is selectively and irreversibly modified by enzymes. Furthermore, if the enzymes can tolerate substrate analogs, it becomes possible to engineer chemically modified proteins in a site-specific fashion. This review details the significant progress in creating techniques for the chemoenzymatic generation of protein-small molecule constructs and provides examples of novel applications of these methodologies.

Quantifying and controlling the orientation of surface-bound macromolecules is crucial to a wide range of processes in areas as diverse as biology, materials science, and nanotechnology. Methods capable of directing orientation, as well as an understanding of the underlying physical mechanisms are, however, lacking. In this paper, we describe experiments in which the conformations of structurally well-defined polymers anchored to fluid lipid membranes were probed using Fluorescence Interference Contrast Microscopy (FLIC), an optical technique that provides topographic information with few-nanometer precision. The novel rodlike polymers mimic the architecture of mucin glycoproteins and feature a phospholipid tail for membrane incorporation and a fluorescent optical probe for FLIC imaging situated at the opposite termini of the densely glycosylated polymeric backbones. We find that the orientation of the rigid, approximately 30 nm long glycopolymers depends profoundly on the properties of the optical reporter. Molecules terminated with Alexa Fluor 488 projected away from the lipid bilayer by 11 +/- 1 nm, consistent with entropy-dominated sampling of the membrane-proximal space. Molecules terminated with Texas Red lie flat at the membrane (height, 0 +/- 2 nm), implying that interactions between Texas Red and the bilayer dominate the polymers' free energy. These results demonstrate the design of macromolecules with specific orientational preferences, as well as nanometer-scale measurement of their orientation. Importantly, they reveal that seemingly minute changes in molecular structure, in this case fluorophores that comprise only 2% of the total molecular weight, can significantly alter the molecule's presentation to the surrounding environment.

Lipid-derived desiccation resistance in membranes is a rare, unique ability previously observed only with trehalose dimycolate (TDM), an abundant mycobacterial glycolipid. Here we present the first synthetic trehalose glycolipids capable of providing desiccation protection to membranes of which they are constituents. The synthetic glycolipids consist of a simple trehalose disaccharide headgroup, similar to TDM, with hydrophobic tail groups of two 15- or 18-carbon chains. The synthetic trehalose glycolipids protected supported monolayers of phospholipids against dehydration even as minority components of the overall membrane, down to as little as 20 mol % trehalose glycolipid as assessed by assays of membrane fluidity. The dependence of the desiccation protection on the synthetic trehalose glycolipid fraction is nearly identical to that of TDM. The striking similarity of the desiccation resistance observed with TDM and the synthetic trehalose glycolipids, despite the variety of hydrophobic tail structures employed, suggests that interactions between the trehalose headgroup and surrounding molecules are the determining factor in dehydration protection.

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. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

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. In some respects, the ADC space has become divided into the clinical realm-where the proven technologies continue to represent the bulk of clinical candidates with a few exceptions-and the research realm-where innovations in conjugation chemistry and linker technologies have suggested that there is much room for improvement in the conventional methods. Now, two and four years after the approvals of Kadcyla™ and Adcetris™, respectively, consensus may at last be building that these two drugs rely on rather unique target antigens that enable their success. It is becoming increasingly clear that future target antigens will require additional innovative approaches. Next-generation ADCs have begun to move out of the lab and into the clinic, where there is a pressing need for continued innovation to overcome the twin challenges of safety and efficacy.

A library of 4,5- and 4,6-linked bivalent aminoglycoside (AMG) antibiotics consisting of neamine and nebramine pharmacophores have been synthesized. We probed the effect of the linker on antibiotic activity with a series of selected synthetic analogues with varied length and substituents. A number of compounds demonstrated in vitro activity against several bacterial strains and showed activity against drug resistant strains of Pseudomonas aeruginosa. Among the compounds prepared, analogues 12a–d were novel 4,6-linked AMGs containing the nebramine pharmacophore. In addition the lead compound OPT-11 possessed an ED50 of ⩽5 mg/kg in a Staphylococcus aureus ATCC 29213 mouse protection model.

A new and efficient route to a ketolide aglycon served as a basis for the unprecedented 5-O-glyco-modification of ketolide antibiotics. Combined with an effective copper-catalyzed triazole-forming reaction a series of novel and potent ketolide antibiotics were synthesized.

Glyco-optimization (OPopS TM ) of aminoglycosides has been performed by replacing the existing sugar moiety with a variety of sugar derivatives. Glycosylation of the 6-position of nebramine provided a library of novel 4,6-linked aminoglycosides (AMGs). Among them, compounds 8b , g , i , l , and 8u with 2″-amino, 2″,3″-diamino, 2″,4″-diamino, 3″,4″-diamino, 3″-amino groups, respectively, showed significant antimicrobial activity against Gram-(+) and -(−) bacteria. Several were particularly potent against Pseudomonus aeruginosa with MICs in the 1–2 μg/mL range.