Random Conjugation Research Articles

Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase.

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.

Labeling of Anti-HER2 Nanobodies with Astatine-211: Optimization and the Effect of Different Coupling Reagents on Their in Vivo Behavior.

The use of nanobodies (Nbs) as vehicles in targeted alpha therapy (TAT) has gained great interest because of their excellent properties. They combine high in vivo affinity and specificity of binding with fast kinetics. This research investigates a novel targeted therapy that combines the α-particle emitter astatine-211 (211At) and the anti-HER2 Nb 2Rs15d to selectively target HER2+ cancer cells. Two distinctive radiochemical methodologies are investigated using three different coupling reagents. The first method uses the coupling reagents, N-succinimidyl 4-(1,2-bis-tert-butoxycarbonyl)guanidinomethyl-3-(trimethylstannyl)benzoate (Boc2-SGMTB) and N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE), which are both directed to amino groups on the Nb, resulting in random conjugation. The second method aims at obtaining a homogeneous tracer population, via a site-specific conjugation of the N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide (MSB) reagent onto the carboxyl-terminal cysteine of the Nb. The resulting radioconjugates are evaluated in vitro and in vivo. 2Rs15d is labeled with 211At using Boc2-SGMTB, m-MeATE, and MSB. After astatination and purification, the binding specificity of the radioconjugates is validated on HER2+ cells, followed by an in vivo biodistribution assessment in SKOV-3 xenografted mice. α-camera imaging is performed to determine uptake and activity distribution in kidneys/tumors. 2Rs15d astatination resulted in a high radiochemical purity >95% for all radioconjugates. The biodistribution studies of all radioconjugates revealed comparable tumor uptake (higher than 8% ID/g at 1 h). [211At]SAGMB-2Rs15d showed minor uptake in normal tissues. Only in the kidneys, a higher uptake was measured after 1 h, but decreased rapidly after 3 h. Astatinated Nbs consisting of m-MeATE or MSB reagents revealed elevated uptake in lungs and stomach, indicating the presence of released 211At. α-Camera imaging of tumors revealed a homogeneous activity distribution. The radioactivity in the kidneys was initially concentrated in the renal cortex, while after 3 h most radioactivity was measured in the medulla, confirming the fast washout into urine. Changing the reagents for Nb astatination resulted in different in vivo biodistribution profiles, while keeping the targeting moiety identical. Boc2-SGMTB is the preferred reagent for Nb astatination because of its high tumor uptake, its low background signals, and its fast renal excretion. We envision [211At]SAGMB-2Rs15d to be a promising therapeutic agent for TAT and aim toward efficacy evaluation.

Site-Specific Immuno-PET Tracer to Image PD-L1.

The rapid ascension of immune checkpoint blockade treatments has placed an emphasis on the need for viable, robust, and noninvasive imaging methods for immune checkpoint proteins, which could be of diagnostic value. Immunoconjugate-based positron emission tomography (immuno-PET) allows for sensitive and quantitative imaging of target levels and has promising potential for the noninvasive evaluation of immune checkpoint proteins. However, the advancement of immuno-PET is currently limited by available imaging tools, which heavily rely on full-length IgGs with Fc-mediated effects and are heterogeneous mixtures upon random conjugation with chelators for imaging. Herein, we have developed a site-specific αPD-L1 Fab conjugate with the chelator 1,4,7-triazacyclononane- N, N', N″-triacetic acid (NOTA), enabling radiolabeling for PET imaging, using the amber suppression-mediated genetic incorporation of unnatural amino acid (UAA), p-azidophenylalanine. This Fab conjugate is homogeneous and demonstrated tight binding toward the PD-L1 antigen in vitro. The radiolabeled version, 64Cu-NOTA-αPD-L1, has been employed in PET imaging to allow for effective visualization and mapping of the biodistribution of PD-L1 in two normal mouse models, including the capturing of different PD-L1 expression levels in the spleens of the different mouse types. Follow-up in vivo blocking studies and ex vivo fluorescent staining further validated specific tissue uptakes of the imaging agent. This approach illustrates the utility of UAA-based site-specific Fab conjugation as a general strategy for making sensitive PET imaging probes, which could facilitate the elucidation of the roles of a wide variety of immune checkpoint proteins in immunotherapy.

'Clickable lectins': bioorthogonal reactive handles facilitate the directed conjugation of lectins in a modular fashion.

Lectins are carbohydrate-binding proteins with specificity for their target ligands. They play diverse roles in cellular recognition and signalling processes, as well as in infections and cancer metastasis. Owing to their specificity, lectins find application in biotechnology and medicine, e.g. for blood group typing, purification of glycoproteins or lipids and as markers that target cancer cells. For some applications, lectins are immobilized on a solid support, or they are conjugated with other molecules. Classical protein conjugation reactions at nucleophilic amino acids such as cysteine or lysine are often non-selective, and the site of conjugation is difficult to pre-define. Random conjugation, however, can interfere with protein function. Therefore, we sought to equip lectins with a unique reactive handle, which can be conjugated with other molecules in a pre-defined manner. We site-specifically introduced non-canonical amino acids carrying bioorthogonal reactive groups into several lectins. As a proof of principle, we conjugated these 'clickable lectins' with small molecules. Furthermore, we conjugated lectins with different ligand specificities with one another to produce superlectins. The 'clickable lectins' might be useful for any process where lectins shall be conjugated with another module in a selective, pre-defined and site-specific manner.

Chemical structure analysis of diamond-like carbon by Raman spectroscopy

The chemical structure of diamond-like carbon (DLC) films was analyzed by Raman spectroscopy. The samples were DLC films synthesized by photoemission-assisted Townsend discharge (PATD). Group theory of the fundamental molecular structures suggested that the Raman spectrum consists of five bands with specific vibration modes, and the lineshape was represented by a modified Voigt-type formula. Analysis of the areas and positions of the bands resulted in the chemical structure of the DLC films with the sp2 cluster model. The model comprises conjugated and conductive clusters of sp2 carbons (sp2 clusters) floating in a non-conjugated and dielectric matrix of sp2 carbon, sp3 carbon, and hydrogen. The sp2 clusters were rather aliphatic for DLC films formed in low concentration of methane. The clusters grew to become aromatic with increasing methane concentration. The number of defects or dangling bonds increased similarly but were terminated with hydrogen for the films formed in a high methane concentration. The essential structure of DLC is the result of the development of random conjugation represented by the sp2 cluster model. We consider that DLC is a carbonaceous material in which conjugation increases slowly with time during the deposition process and which exhibits dielectric characteristics.

Applications of SNAP-tag technology in skin cancer therapy.

BackgroundCancer treatment in the 21st century has seen immense advances in optical imaging and immunotherapy. Significant progress has been made in the bioengineering and production of immunoconjugates to achieve the goal of specifically targeting tumors.DiscussionIn the 21st century, antibody drug conjugates (ADCs) have been the focus of immunotherapeutic strategies in cancer. ADCs combine the unique targeting of monoclonal antibodies (mAbs) with the cancer killing ability of cytotoxic drugs. However, due to random conjugation methods of drug to antibody, ADCs are associated with poor antigen specificity and low cytotoxicity, resulting in a drug to antibody ratio (DAR) >1. This means that the cytotoxic drugs in ADCs are conjugated randomly to antibodies, by cysteine or lysine residues. This generates heterogeneous ADC populations with 0 to 8 drugs per an antibody, each with distinct pharmacokinetic, efficacy, and toxicity properties. Additionally, heterogeneity is created not only by different antibody to ligand ratios but also by different sites of conjugation. Hence, much effort has been made to find and establish antibody conjugation strategies that enable us to better control stoichiometry and site‐specificity. This includes utilizing protein self‐labeling tags as fusion partners to the original protein. Site‐specific conjugation is a significant characteristic of these engineered proteins. SNAP‐tag is one such engineered self‐labeling protein tag shown to have promising potential in cancer treatment. The SNAP‐tag is fused to an antibody of choice and covalently reacts specifically in a 1:1 ratio with benzylguanine (BG) substrates, eg, fluorophores or photosensitizers, to target skin cancer. This makes SNAP‐tag a versatile technique in optical imaging and photoimmunotherapy of skin cancer.ConclusionSNAP‐tag technology has the potential to contribute greatly to a broad range of molecular oncological applications because it combines efficacious tumor targeting, minimized local and systemic toxicity, and noninvasive assessment of diagnostic/prognostic molecular biomarkers of cancer.

Efficient Production of Homogeneous Lysine-Based Antibody Conjugates Using Microbial Transglutaminase.

Random conjugation of chemical linkers to endogenous lysines or cysteines within an antibody yields a heterogeneous mixture of conjugates with various drug-to-antibody ratios. One approach for generating homogeneous antibody conjugates utilizes enzymatic transfer of payloads onto a specific glycan or amino acid residue. Microbial transglutaminase (MTG) is an enzyme that catalyzes the formation of a stable isopeptide bond between a glutamine and a lysine. We have previously identified and reported several sites throughout the antibody structure where an engineered lysine is sufficient for transfer of a glutamine-based substrate onto the antibody. Whereas other enzymatic transfer strategies typically require significant antibody engineering to either modify the N-glycans or introduce a multi-amino acid enzyme recognition site, the lower contextual specificity of MTG for lysines allows just a single lysine point mutation in an antibody to be efficiently transamidated. Here we describe the molecular positioning of these single engineered lysine residues and the conjugation conditions for producing homogeneous antibody conjugates exemplified using azido- and auristatin F-based acyl donor substrates.

Site-specific PEGylation of Human Growth Hormone by Mutated Sortase A

Human growth hormone(hGH), a classic therapeutic protein, which promotes growth and wound healing, is released from the pituitary gland. As a protein drug, its short half-life is its main barrier to therapeutic efficacy. Various strategies have been designed to prolong its serum half-life, the most common of which is the conjugation with polyethylene glycol(PEG), as this has been shown to significantly extend protein’s serum half-life. However, PEGylation often results in random conjugation, which can lead to impaired protein function and hinder purification, characterization and evaluation of the PEGylated protein. Therefore, site specific PEGylation is a promising direction for PEG-protein conjugation. Here we took advantages of the mutated sortase A(7M) enzyme, which can enzymatically ligate the universal α-amino acids to a C-terminal tagged protein. This then allows specific modification of the C-terminal of hGH with PEG. This site-specific bound PEG-hGH has similar efficacy, receptor binding and cell proliferation as wild-type hGH; however, pharmacokinetic analysis demonstrates that its serum half-life is almost 24 times that of wild-type hGH. Herein, we provided a promising advancement in the development of site specific PEGylated therapeutic proteins.

Site-specific and hydrophilic ADCs through disulfide-bridged linker and branched PEG

Kadcyla® (T-DM1), an antibody–drug conjugates (ADCs) for HER2+ breast cancer treatment, has been approved by the Food and Drug Administration (FDA) in 2013. An ADC of random lysine conjugation, it has difficulties in DAR control and unsatisfactory PK due to uneven DAR distribution. It also gives rise to aggregation during conjugation because of the hydrophobicity nature of the cytotoxin, DM1. The linker-drug in T-DM1, SMCC-DM1 is hydrophobic and requires certain percentage of organic solvent such as DMA in the conjugation solution, limiting the manufacturing process in an organic-solvent-compatible device and adding extra costs. To address these problems, a site-specific conjugation method was developed involving full reduction of antibody and full conjugation with the bridge-like conjugator-drug, based on the work of Caddick and co-workers, to obtain a site-directed antibody-drug conjugate with DAR 4. The bridge-like conjugator was assembled with SMCC-DM1 and different lengths of hydrophilic polyethylene glycol (PEG) moiety. By applying PEG moiety in the side chain of the linker-drug, the organic solvent used in the conjugation can be reduced. When the PEG length is about 26 units, organic solvent is no longer needed in the conjugation. Reducing the amount of organic solvent in conjugation could also diminish the aggregation occurrence during the conjugation. Moreover, the conjugation configuration with the designed conjugator was also discussed in the article. The binding affinity of the resulting ADCs did not show significant decrease and the cell based assay and animal study have shown the comparable results with T-DM1.

A Switchable Site-Specific Antibody Conjugate.

Genetic incorporation of unnatural amino acids (UAAs) provides a unique approach to the synthesis of site-specific antibody conjugates that are homogeneous and better defined constructs than random conjugates. Yet, the yield varies for every antibody, and the process is costly and time-consuming. We have developed a switchable αGCN4-Fab conjugate that incorporates UAA p-acetylphenylalanine. The GCN4 peptide is used as a switch, and antibodies fused by GCN4 can direct the αGCN4-Fab conjugate to target different cancer cells for diagnosis, imaging, or therapeutic treatment. More importantly, this switchable conjugate demonstrated an impressive potential for pretargeted imaging in vivo. This approach illustrates the utility of an orthogonal switch as a general strategy to endow versatility to a single antibody conjugate, which should facilitate the application of UAA-based site-specific conjugates for a host of biomedical uses in the future.

Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification.

Glycoconjugate vaccines obtained by chemical linkage of a carbohydrate antigen to a protein are part of routine vaccinations in many countries. Licensed antimicrobial glycan-protein conjugate vaccines are obtained by random conjugation of native or sized polysaccharides to lysine, aspartic or glutamic amino acid residues that are generally abundantly exposed on the protein surface. In the last few years, the structural approaches for the definition of the polysaccharide portion (epitope) responsible for the immunological activity has shown potential to aid a deeper understanding of the mode of action of glycoconjugates and to lead to the rational design of more efficacious and safer vaccines. The combination of technologies to obtain more defined carbohydrate antigens of higher purity and novel approaches for protein modification has a fundamental role. In particular, methods for site selective glycoconjugation like chemical or enzymatic modification of specific amino acid residues, incorporation of unnatural amino acids and glycoengineering, are rapidly evolving. Here we discuss the state of the art of protein engineering with carbohydrates to obtain glycococonjugates vaccines and future perspectives.

Abstract 65: RESPECT (REsidue-SPEcific Conjugation Technology): A platform technology utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates

Abstract The random conjugation of toxins, dyes, peptides, or other payloads to monoclonal antibodies often targets free thiol groups generated by partial reduction methods or lysine residues using succinimide- or isothiocyanate-based chemistry. There remains a need for conjugation technologies targeting specific amino acid residues as a way to produce a homogeneous antibody-drug conjugate (ADC) product with a defined drug-to-antibody ratio (DAR). Our REsidue-SPEcific Conjugation Technology (RESPECT) utilizes two methods by which payloads can be conjugated to specific residues in an antibody. Our cysteine-specific conjugation method exploits a unique intrachain disulfide bond in the light chain of rabbit antibodies between residues 80 and 171 of the variable and constant domains, respectively. Our humanization strategy allows retention of the cysteine at position 80 with a free thiol group that is both amenable for residue-specific conjugation and compatible with optimal antibody biophysical properties including antigen binding and structural stability. This platform has been optimized via antibody engineering strategy stems from in silico modeling, extensive mutagenesis, and crystallographic studies, which have allowed defining the contribution of neighboring residues to the retention of a reactive thiol group as well as the desired humanized antibody’s properties. Our C-terminal lysine-specific linkage method employs the transglutaminase enzyme that catalyzes the formation of a stable isopeptide bond between the γ-carboxyamide group (acyl donor) of a glutamine and the ϵ-amino group (acyl acceptor) of a lysine. While we found no acyl acceptor sites in recombinant wild-type IgG, all antibodies investigated lacked the C-terminal Lys447 due to cleavage by carboxypeptidase B in the antibody production cell line. Blocking the cleavage of Lys447 by addition of a C-terminal amino acid resulted in transamidation of Lys447 by a variety of acyl donor substrates in the presence of any non-acidic, non-proline amino acid residue at position 448. Antibody-drug conjugates (ADCs) prepared using our RESPECT technology targeting the tumor associated-mesothelin protein produced uniform drug-to-antibody ratios (DAR) and were shown to be highly potent and specific in vitro and effective in vivo in reduction of tumor growth in a highly aggressive mesothelin-expressing xenograft tumor model. Citation Format: Earl Albone, Jared Spidel, Xin Cheng, Young Chul Park, Sara Jacob, Arielle Verdi, Andrew Milinichik, Ben Vaessen, J. Bradford Kline, Luigi Grasso. RESPECT (REsidue-SPEcific Conjugation Technology): A platform technology utilizing native cysteine and lysine residues for the generation of homogeneous antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 65. doi:10.1158/1538-7445.AM2017-65

Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity

Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity.

Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules.

Quantification of PEGylated proteases with varying degree of conjugation in mixtures: An analytical protocol combining protein precipitation and capillary gel electrophoresis

PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times.In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase® which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis.

Abstract 1341: PRINT: A protein bioconjugation method with exquisite N-terminal specificity

Abstract The use of proteins and peptides for therapeutic applications are often compromised by low biological stability, high renal clearance, and non-optimal biodistribution. Chemical attachment of poly-(ethylene glycol) (PEGylation) is often considered the most effective way to improve these pharmacologic properties by increasing circulation half-life as well as reduced renal clearance, immunogenicity and protease mediated degradation. However, random conjugation results in heterogeneous derivatives with undefined composition and can substantially lower the bioactivity of the modified protein, leading to unpredictable in vivo behavior. Site-specific modification of proteins is therefore an attractive approach to circumvent the non-specificity resulting from random conjugation. We have developed a novel technique named PRINT (PRotect, INcise Tag) for N-terminal specific bioconjugation of proteins and peptides. Conceptually, PRINT can be performed on any protein that has any N-terminal tag for purification and a protease cleavage site following the tag. The recombinant protein is first treated with an excess of citraconic anhydride to reversibly block all reactive primary amine sites. Proteolytic cleavage then exposes only a single amine (the primary amine at the N-terminus) for desired bioconjugation by amine-reactive NHS ester chemistry. Lowering of reaction pH results in removal of the citraconates, leaving N-terminal specific mono PEGylated protein molecules. We used Tumor Necrosis Factor-α (TNF-α) as a model protein as it suffers from inherent instability and short biological half-life, and exhibits toxic side effects at therapeutic concentrations in both small animals and human patients. We demonstrate that PRINT results in a single product with exquisite selectivity and specificity in contrast to conventional reaction using the same NHS reagent, which was further confirmed by mass spectrometric analyses. Subsequent de-blocking generated an N-terminal protected TNF-α molecule with enhanced serum stability, superior pharmacokinetic properties, and reduced systemic toxicity. Importantly, N-terminal protection by PRINT did not affect the bioactivity of TNF-α. Existing site-selective bioconjugation approaches are either specific to amino acid tags or involve substantial non-trivial chemical or biotechnological manipulations to synthesize a desired bioconjugate. In contrast, PRINT employs ubiquitously used recombinant DNA techniques and easily acquired commercial reagents to generate exquisite N-terminal selective protection. We show that PRINT is a robust, reproducible and mild strategy which is able to target the α -amine and provide N-terminal specific protection to proteins or peptides that suffer from similar issues. We believe that this approach is strongly orthogonal to current methods and will be applicable to many biotherapeutics and bioprobes that are currently being designed to treat cancer. Citation Format: Surojit Sur, Yuan Qiao, Anja C. Fries, Robert N. O’Meally, Robert N. Cole, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. PRINT: A protein bioconjugation method with exquisite N-terminal specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1341.

Enhanced Biosensor Platforms for Detecting the Atherosclerotic Biomarker VCAM1 Based on Bioconjugation with Uniformly Oriented VCAM1-Targeting Nanobodies.

Surface bioconjugation of biomolecules has gained enormous attention for developing advanced biomaterials including biosensors. While conventional immobilization (by physisorption or covalent couplings using the functional groups of the endogenous amino acids) usually results in surfaces with low activity, reproducibility and reusability, the application of methods that allow for a covalent and uniformly oriented coupling can circumvent these limitations. In this study, the nanobody targeting Vascular Cell Adhesion Molecule-1 (NbVCAM1), an atherosclerotic biomarker, is engineered with a C-terminal alkyne function via Expressed Protein Ligation (EPL). Conjugation of this nanobody to azidified silicon wafers and Biacore™ C1 sensor chips is achieved via Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) “click” chemistry to detect VCAM1 binding via ellipsometry and surface plasmon resonance (SPR), respectively. The resulting surfaces, covered with uniformly oriented nanobodies, clearly show an increased antigen binding affinity, sensitivity, detection limit, quantitation limit and reusability as compared to surfaces prepared by random conjugation. These findings demonstrate the added value of a combined EPL and CuAAC approach as it results in strong control over the surface orientation of the nanobodies and an improved detecting power of their targets—a must for the development of advanced miniaturized, multi-biomarker biosensor platforms.

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

BackgroundThe 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.ResultsThe 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 %.ConclusionsWe 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 materialThe online version of this article (doi:10.1186/s12896-016-0254-0) contains supplementary material, which is available to authorized users.

Site-Specifically Labeled Immunoconjugates for Molecular Imaging--Part 1: Cysteine Residues and Glycans.

Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created via the random conjugation of bifunctional probes (e.g., DOTA-NCS) to amino acids within the antibody (e.g., lysines). Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates—both antibody-drug conjugates as well as constructs for in vivo imaging—has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities.

Hydrolytically Stable Site-Specific Conjugation at the N-Terminus of an Engineered Antibody.

Antibody-drug conjugates (ADCs) have emerged as an important class of therapeutics for cancer treatment that combine the target specificity of antibodies with the killing activity of anticancer chemotherapeutics. Early conjugation technologies relied upon random conjugation to either lysine or cysteine residues, resulting in heterogeneous ADCs. Recent technology advancements have resulted in the preparation of homogeneous ADCs through the site-specific conjugation at engineered cysteines, glycosylated amino acids, and bioorthogonal unnatural amino acids. Here we describe for the first time the conjugation of an anti-mitotic drug to an antibody following the mild and selective oxidation of a serine residue engineered at the N-terminus of the light chain. Using an alkoxyamine-derivatized monomethyl auristatine E payload, we have prepared a hydrolytically stable ADC that retains binding to its antigen and displays potent in vitro cytotoxicity and in vivo tumor growth inhibition.