Thiol-maleimide Conjugation Research Articles

Nanobodies Outperform Antibodies - Rapid Functionalization with Equal In Vivo Targeting Properties.

Highly specific targeting of dendritic cells in vivo is crucial for the development of effective tumor nanovaccines. This group recently presented an antibody-functionalized nanocarrier system able to maintain its targeting properties when transferred from in vitro to in vivo studies. However, producing this system requires long synthesis times and involves high expenses due to the involved site-specific enzymatic multi-step modification procedure of the antibody. Consequently, improving the previously proposed system is necessary in order to accelerate the development. Here, a novel system utilizing nanobodies for the targeting of dendritic cells is presented. A C-terminal cysteine tag facilitates an easy attachment of the nanobody to the nanocarrier via a thiol-maleimide conjugation technique. This reduces the functionalization time from several days to mere hours. Using in vitro and in vivo assays, it is shown that the optimized system possesses equal targeting properties as the antibody-based system. As a result, nanobodies and the coupling chemistry are found to be a superior strategy for the in vivo targeting of dendritic cells when compared to antibodies, due to their rapid attachment to nanocarriers and equal targeting specificity. This would replace antibodies as the current "gold standard" of targeting moieties.

Abstract 740: CNTN4 as a novel target for solid cancer with antibody-drug conjugate

Abstract As a novel immune checkpoint, we previously confirmed that contactin 4 (CNTN4) regulates T cell activity negatively by binding to amyloid precursor protein (APP) on T cells. CNTN4 is highly expressed in various types of tumors, including gallbladder, pancreas, stomach, endometrium, liver, prostate, bladder cancer, melanoma, and other tumors, with positive rates over 70% through immunohistochemistry analysis in contrast to low-level expression in normal tissues. Based on the tumor-specific expression profile of CNTN4, we evaluated the potential of CNTN4 as a novel target for antibody-drug conjugate (ADC). First, the internalization of the anti-CNTN4 antibody in the IND submission stage, GENA-104A16, into CNTN4-positive cancer cells was confirmed. For investigation of the potential of targeting CNTN4 using ADC, clinically validated various linker-payloads (MMAE, MMAF, SN-38, and exatecan derivate) were conjugated to GENA-104A16 using thiol maleimide conjugation with average drug-to-antibody ratio (DAR) in the range of 4.0 to 4.8. After preparing ADCs, we confirmed the binding affinity for CNTN4 was maintained as high even after the payload conjugation through an ELISA. In vitro studies have demonstrated the highly cytotoxic effect of MMAF conjugate among the conjugates of GENA-104A16 on CNTN4-positive cancer cells compared to negative cancer cells. And there was no cytotoxic effect on normal cells. Furthermore, treating MMAF conjugate of GENA-104A16 showed promising efficacy in the Pan02 pancreatic orthotopic mouse model and the patient-derived xenograft model. These results suggest that CNTN4 could be a highly potential ADC target that could expand the options for cancer treatment strategies. Citation Format: Mi Young Cha, Hyunuk Kim, Seungmin Byun, Youngeun Ha, Kitae Park, Hyunkyung Yu, Bu-Nam Jeon, Mira Kim, Soojung Moon, Kyung Mi Park, Hansoo Park. CNTN4 as a novel target for solid cancer with antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 740.

Total Chemical Synthesis of Palmitoyl-Conjugated Insulin.

Commercially available insulins are manufactured by recombinant methods for the treatment of diabetes. Long-acting insulin drugs (e.g., detemir and degludec) are obtained by fatty acid conjugation at LysB29 ε-amine of insulin via acid-amide coupling. There are three amine groups in insulin, and they all react with fatty acids in alkaline conditions. Due to the lack of selectivity, such conjugation reactions produce non-desired byproducts. We designed and chemically synthesized a novel thiol-insulin scaffold (CysB29-insulin II), by replacing the LysB29 residue in insulin with the CysB29 residue. Then, we conjugated a fatty acid moiety (palmitic acid, C16) to CysB29-insulin II by a highly efficient and selective thiol-maleimide conjugation reaction. We obtained the target peptide (palmitoyl-insulin) rapidly within 5 min without significant byproducts. The palmitoyl-insulin is shown to be structurally similar to insulin and biologically active both in vitro and in vivo. Importantly, unlike native insulin, palmitoyl-insulin is slow and long-acting.

Abstract 1760: The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer

Abstract Antibody-drug conjugates (ADCs) are considered as promising cancer treatment modalities that combine the selectivity of antibodies and the cytotoxic properties of payloads using chemical linkers. However, despite their success, ADCs still suffer from drawbacks, e.g., systemic toxicity, which limit their potential clinical applications. The systemic toxicity of an ADC is mainly related to the stability of its linker, and to the selectivity of its antibody towards the targeted antigen expressed on cancer cells. Lu/BCAM (Lutheran/basal cell adhesion molecule) is a member of the immunoglobulin superfamily and is a receptor for laminin, a protein that facilitates cell adhesion, migration, and invasion. A growing number of studies show that BCAM plays an essential role in tumor progression and is overexpressed on epithelial cancers e.g., skin cancer. Here we describe GENA-111, a human monoclonal anti-BCAM IgG4 (S228P) antibody that binds to human BCAM with high affinity, and that is significantly internalized by BCAM-positive tumor cells. We also describe the GENA-111-auristatin F ADC, wherein the GENA-111 antibody has been armed with an auristatin F derivative using a new linker technology and a stabilized thiol maleimide conjugation. This new linker technology comprises a cleavable peptidic sequence that facilitates multidrug attachment and the production of ADCs with tailored drug-to-antibody ratios (DARs). Cytotoxic drugs are rapidly and selectively released from the linker by the carboxypeptidase activity of Cathepsin B. In vitro cytotoxicity examination showed the potent cytotoxic effects of this GENA-111-auristatin F ADC on BCAM-expressing tumor cells, with a positive correlation between cytotoxicity and BCAM expression. Moreover, this ADC was also shown to significantly reduce the growth of tumor cells, including A431, T47D, and Huh7. The GENA-111-auristatin F ADC was evaluated in a xenograft mouse model established by subcutaneous injection of A431 cells, a BCAM positive human skin cancer cell line. The results of this study will be presented and discussed. Taken together, our data suggest that an ADC targeting BCAM e.g., GENA-111-auristatin F, might be a promising treatment strategy for BCAM positive epithelial cancer patients. Citation Format: Hyunkyung Yu, Nathalie Bellocq, Youngeun Ha, Hyunuk Kim, Yunyeon Kim, Bu-Nam Jeon, Léo Marx, Mathilde Pantin, Hyunjin Yoo, Seungmin Byun, Joo-Yeon Chung, Mi Young Cha, Patrick Garrouste, Frédéric Lévy. The antibody-drug conjugate GENA-111 conjugated to auristatin F shows therapeutic potency in BCAM positive epithelial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1760.

Fast-Forming Dissolvable Redox-Responsive Hydrogels: Exploiting the Orthogonality of Thiol–Maleimide and Thiol–Disulfide Exchange Chemistry

Fast-forming yet easily dissolvable hydrogels (HGs) have potential applications in wound healing, burn incidences, and delivery of therapeutic agents. Herein, a combination of a thiol–maleimide conjugation and thiol–disulfide exchange reaction is employed to fabricate fast-forming HGs which rapidly dissolve upon exposure to dithiothreitol (DTT), a nontoxic thiol-containing hydrophilic molecule. In particular, maleimide disulfide-terminated telechelic linear poly(ethylene glycol) (PEG) polymer and PEG-based tetrathiol macromonomers are employed as gel precursors, which upon mixing yield HGs within a minute. The selectivity of the thiol–maleimide conjugation in the presence of a disulfide linkage was established through 1H NMR spectroscopy and Ellman’s test. Rapid degradation of HGs in the presence of thiol-containing solution was evident from the reduction in storage modulus. HGs encapsulated with fluorescent dye-labeled dextran polymers and bovine serum albumin were fabricated, and their cargo release was investigated under passive and active conditions upon exposure to DTT. One can envision that the rapid gelation and fast on-demand dissolution under relatively benign conditions would make these polymeric materials attractive for a range of biomedical applications.

A chitosan-based nanosystem as pneumococcal vaccine delivery platform.

Chitosan-based nanosystems have been described as interesting tools for antigen delivery and for enhancing the immunogenicity of nasally administered vaccines. As a possible vaccine delivery method, the chemical conjugation of chitosan nanocapsules with the Streptococcus pneumoniae cell membrane protein PsaA (pneumococcal surface adhesin A) is suggested here. The antigen PsaA, common to all pneumococcus serotypes, is expected to improve its uptake by immune cells and to activate specific T cells, generating an adaptive immune response against pneumococcus.With this aim, chitosan nanocapsules with thiol-maleimide conjugation between the polymer (chitosan) and the antigen (PsaA) were designed to enable the surface presentation of PsaA for immune cell recognition. Spherical-shaped particles, with a size of 266 ± 32nm, positive charge of +30 ± 1mV, and good stability profiles in simulated nasal fluids (up to 24h) were achieved. PsaA association rates were three times higher compared with nanocapsules without covalent polymer-protein conjugation. Cytotoxicity studies in cell culture media showed non-toxic effect under 150µg/mL concentration of nanocapsules, and subsequent studies on the maturation of immature dendritic cells in the presence of antigen-conjugated nanocapsules displayed peripheral blood mononuclear cell activation and lymphocyte differentiation after their presentation by dendritic cells. Secretion of TNFα following exposure to nanocapsules and the ability of nanocapsules to activate CD4 and CD8 T lymphocytes had also been studied. Antigen loaded nanocarrier uptake and presentation by professional presenting cells.

Fabrication of Patterned Hydrogel Interfaces: Exploiting the Maleimide Group as a Dual Purpose Handle for Cross-Linking and Bioconjugation.

Functional hydrogels that can be obtained through facile fabrication procedures and subsequently modified using straightforward reagent-free methods are indispensable materials for biomedical applications such as sensing and diagnostics. Herein a novel hydrogel platform is obtained using polymeric precursors containing the maleimide functional group as a side chain. The maleimide groups play a dual role in fabrication of functional hydrogels. They enable photochemical cross-linking of the polymers to yield bulk and patterned hydrogels. Moreover, the maleimide group can be used as a handle for efficient functionalization using the thiol-maleimide conjugation and Diels-Alder cycloaddition click reactions. Obtained hydrogels are characterized in terms of their morphology, water uptake capacity, and functionalization. Micropatterned hydrogels are obtained under UV-irradiation using a photomask to obtain reactive micropatterns, which undergo facile functionalization upon treatment with thiol-containing functional molecules such as fluorescent dyes and bioactive ligands. The maleimide group also undergoes conjugation through the Diels-Alder reaction, where the attached molecule can be released through thermal treatment via the retro Diels-Alder reaction. The antibiofouling nature of these hydrogel micropatterns enables efficient ligand-directed biomolecular immobilization, as demonstrated by attachment of streptavidin-coated quantum dots.

Application of Fmoc-SPPS, Thiol-Maleimide Conjugation, and Copper(I)-Catalyzed Alkyne-Azide Cycloaddition "Click" Reaction in the Synthesis of a Complex Peptide-Based Vaccine Candidate Against Group A Streptococcus.

Fmoc solid-phase peptide synthesis (SPPS) is the most common approach used to synthesize natural and unnatural peptides. However, the synthesis of sequences longer than 30-60 amino acids is associated with a drastic reduction in peptide quality. Thus, large and complex peptides are normally synthesized as fragments, which are then conjugated together. Here, we describe the synthesis of a large, branched peptide, a multi-epitope vaccine candidate against Group A Streptococcus, with the help of microwave-assisted Fmoc-SPPS, thiol-maleimide conjugation, and copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) "click" reaction.

Simple and universal signal labeling of cell surface for amplified detection of cancer cells via mild reduction

Membrane protein, a novel surface biomarker, plays an important role in cell recognition and disease diagnosis. Accurate recognition of membrane protein ensure high specificity of cell identification, while introducing signal molecules onto cell membrane is critical to achieve high sensitivity. In this work, we introduced a simple and universal signal labeling approach for cancer cell detection based on mild reduction-mediated cell engineering. This approach included the mild reduction of disulfide bonds within membrane proteins and the introduction of DNA bridge complex-templated silver nanoclusters (DNA bridge-AgNCs) through the thiol-maleimide conjugation. The mild reduction reactions on the cell surface significantly increased the binding sites for signal labeling, and DNA bridge-AgNCs served as a scaffold of signal amplification, resulting in a wide linear range from 50–2 × 106 cells, and a detection limit of 15 cells. In addition, the method also showed good selectivity in complex environment. Therefore, this method may have great application space in the field of cell detection and even disease diagnosis in the near future.

Block Copolymer/Plasmid DNA Micelles Postmodified with Functional Peptides via Thiol-Maleimide Conjugation for Efficient Gene Delivery into Plants.

Introducing exogenous genes into plant cells is essential for a wide range of applications in agriculture and plant biotechnology fields. Cationic peptide carriers with cell-penetrating and DNA-binding domains successfully deliver exogenous genes into plants. However, their cell-penetrating activity may be attenuated by undesired electrostatic interactions between the cell-penetrating peptide (CPP) domain and DNA cargo, resulting in limited gene delivery efficiency. Here, we developed the block copolymer maleimide-conjugated tetra(ethylene glycol) and poly(l-lysine) (MAL-TEG-PLL). Through electrostatic interactions with plasmid DNA (pDNA), MAL-TEG-PLL formed a micelle that presented maleimide groups on its surface. The micelle enabled postmodification with cysteine-containing functional peptides, including a CPP (BP100-Cys) and nuclear localization signal (Cys-NLS) via thiol-maleimide conjugation, thereby avoiding undesired interactions. According to a comparison of gene delivery efficiencies among the peptide-postmodified micelles, the amount of BP100-Cys on the micelle surface was key for efficient gene delivery. The BP100-postmodified micelle showed more efficient delivery compared with that of the BP100-premodified micelle. Thus, postmodification of polymeric micelles with functional peptides opens the door to designing highly efficient plant gene delivery systems.

A Two-Step Immunocapture LC/MS/MS Assay for Plasma Stability and Payload Migration Assessment of Cysteine–Maleimide-Based Antibody Drug Conjugates

Plasma stability assessment under physiological temperature is an essential step for developing and optimizing antibody drug conjugate (ADC) molecules, especially those with cleavable linkers. The assessment of plasma stability often requires monitoring multiple analytes using a combination of bioanalytical assays for free payloads, conjugated payloads (or conjugated antibodies), total antibodies, and payloads that have migrated from antibodies to plasma constituent proteins. Bioanalytical assays are needed in early drug discovery to quickly screen diverse ADC candidates of different antibody constructs, linker variants, and antibody anchor sites. To improve the sensitivity and selectivity of LC/MS/MS-based assays for the assessment, immunocapture has been widely used for extracting ADCs and unconjugated antibodies from plasma samples. In this study, a novel two-step immunocapture LC/MS/MS assay was described to allow the quantification of conjugated payloads, total antibodies, and migrated payloads forming adducts with albumin in the plasma samples for stability assessment. A target antigen immobilized on magnetic beads was used to exhaustively extract the ADC and antibody-associated species. The remaining supernatant was then extracted further with anti-albumin beads for recovering the albumin-associated adducts for quantification. The method was optimized for higher efficiency and cost-effectiveness using microwave enhanced papain-based enzymatic cleavage for measuring conjugated payloads of ADCs and lysyl endopeptidase cleavage in the total antibody assay. A maleimide linker-based ADC with a proprietary payload, TAK-001, was used to demonstrate the streamlined workflow of the ADC stability assessment. The method could provide valuable evaluation of the stability of the ADC as well as the quantitative assessment of the albumin adducts formed from the linker-payload migration in mouse and human plasma. Furthermore, the method should be readily adaptable for other ADCs using thiol-maleimide conjugation chemistry.

Surface-Anchored Thiol-Reactive Soft Interfaces: Engineering Effective Platforms for Biomolecular Immobilization and Sensing.

Fabrication of antibiofouling, specifically reactive polymeric coatings that undergo facile functionalization with thiol-bearing small molecules and ligands, yields effective platforms for biomolecular immobilization and sensing. Poly(ethylene glycol) (PEG)-based copolymers containing alkoxysilyl groups to enable surface-anchoring and furan-protected maleimide groups as latent thiol-reactive moieties as side-chains were synthesized. Reactive interfaces were obtained by coating these copolymers onto Si/SiO2 or glass surfaces and activating the maleimide groups to their thiol-reactive forms via thermal treatment. A series of surfaces modified with copolymers containing varying amounts of maleimide groups were synthesized. Effectiveness of surface modification was probed using Fourier transform infrared spectroscopy, contact angle goniometry, ellipsometry and X-ray photoelectron spectroscopy. Facile surface modification through thiol-maleimide conjugation was established by attachment of a thiol-containing fluorescent dye, namely BODIPY-SH. It was demonstrated that these surfaces allow spatially localized modification through microcontact printing. Importantly, the extent of surface modification could be tuned by varying the initial composition of the copolymer used for coating. Using fluorescence microscopy, it was observed that increasing amount of fluorescent dye was attached onto surfaces fabricated with copolymers with increasing amount of masked maleimide groups. Thereafter, the thiol-maleimide conjugation was utilized to decorate these surfaces with biotin, a protein-binding ligand. It was observed that though these biotinylated surfaces were able to bind Streptavidin effectively, some nonspecific binding was observed on places that were not in conformal contact with the stamp during microcontact printing. This nonspecific binding was eliminated upon neutralizing the residual maleimide units on the printed surface using thiol-containing PEG. Notably, fluorescence analysis of Streptavidin immobilized onto biotinylated surfaces fabricated using varying amounts of maleimide demonstrated that the amount of immobilized protein could be tuned by varying surface composition. It can be envisioned that facile fabrication of these maleimide-containing polymeric surfaces, their effective functionalization in a tunable manner to engineer interfaces for effective immobilization or sensing of biomolecules in a spatially controlled manner would make them attractive candidates for various biotechnological applications.

"Clickable" Nanogels via Thermally Driven Self-Assembly of Polymers: Facile Access to Targeted Imaging Platforms using Thiol-Maleimide Conjugation.

Multifunctionalizable nanogels are fabricated using thermally driven self-assembly and cross-linking of reactive thermoresponsive copolymers. Nanogels thus fabricated can be easily conjugated with various appropriately functionalized small molecules and/or ligands to tailor them for various applications in delivery and imaging. In this study, a poly(ethylene glycol)-methacrylate-based maleimide-bearing copolymer was cross-linked with a dithiol-based cross-linker to synthesize nanogels. Because of lower critical solution temperature (LCST) around 55 °C in aqueous media, these copolymers assemble into nanosized aggregates when heated to this temperature, and they are cross-linked using the thiol-maleimide conjugation. Nanogels thus fabricated contain both thiol and maleimide groups in the same cross-linked nanogels. Postgelation functionalization of the residual maleimide and thiol groups is demonstrated through conjugation of a thiol-bearing hydrophobic dye (BODIPY-SH) and N-(fluoresceinyl) maleimide, respectively. In addition, to demonstrate the utility of multifunctionality of these nanogels, a thiol-bearing cyclic-peptide-based targeting group, cRGDfC, and N-(fluoresceinyl)-maleimide-based fluorescent tag was conjugated to nanogels in aqueous media. Upon treatment with breast cancer cell lines, MDA-MB-231, it was deduced from cellular internalization studies using fluorescence microscopy and flow cytometry that the peptide carrying constructs were preferentially internalized. Overall, a facile synthesis of multifunctionalizable nanogels that can be tailored using effective conjugation chemistry under mild conditions can serve as promising candidates for various applications.

'Dual' peptidyl-oligonucleotide conjugates: Role of conformational flexibility in catalytic cleavage of RNA.

Traditional therapeutic interventions against abnormal gene expression in disease states at the level of expressed proteins are becoming increasingly difficult due to poor selectivity, off-target effects and associated toxicity. Upstream catalytic targeting of specific RNA sequences offers an alternative platform for drug discovery to achieve more potent and selective treatment through antisense interference with disease-relevant RNAs. We report a novel class of catalytic biomaterials, comprising amphipathic RNA-cleaving peptides placed between two RNA recognition motifs, here demonstrated to target the TΨC loop and 3'- acceptor stem of tRNAPhe. These unique peptidyl-oligonucleotide 'dual' conjugates (DCs) were created by phosphoramidate or thiol-maleimide conjugation chemistry of a TΨC-targeting oligonucleotide to the N-terminus of the amphipathic peptide sequence, followed by amide coupling of a 3'-acceptor stem-targeting oligonucleotide to the free C-terminal carboxylic acid functionality of the same peptide. Hybridization of the DCs bearing two spatially-separated recognition motifs with the target tRNAPhe placed the peptide adjacent to a single-stranded RNA region and promoted cleavage within the 'action radius' of the catalytic peptide. Up to 100% cleavage of the target tRNAPhe was achieved by the best candidate (i.e. DC6) within 4h, when conformational flexibility was introduced into the linker regions between the peptide and oligonucleotide components. This study provides the strong position for future development of highly selective RNA-targeting agents that can potentially be used for disease-selective treatment at the level of messenger, micro, and genomic viral RNA.

Bactericidal Hydrogels via Surface Functionalization with Cecropin A.

The immobilization of antimicrobial peptides (AMPs) to surfaces, enabling their utilization in biosensor and antibacterial/antifouling coating applications, is typically performed using rigid, solid support materials such as glass or gold and may require lengthy, temperamental protocols. Here, we employ a hydrogel immobilization platform to afford facile fabrication and surface functionalization while offering improved biocompatibility for evaluating the influence of linker length, surface density, and AMP conjugation site on retained peptide activity. Rapid, interfacial photo-polymerization using the radical-mediated thiol-ene addition mechanism was used to generate cross-linked, polymeric coatings bearing residual thiol moieties on prefabricated poly(ethylene glycol) (PEG)-based hydrogel supports. The photo-polymerized coatings were 60 μm thick and contained 0.55 nmol of unreacted free thiols, corresponding to a concentration of 410 μM, for use as cecropin A (CPA) immobilization handles via thiol-maleimide conjugation, where the CPA-bound maleimide moiety was localized at either the carboxyl terminus or midsequence between Ala22 and Gly23. Surface presentation of the thiol handles was controlled by varying the thiolated PEG monomer (PEGSH) used in the photo-polymerizable formulation. Bactericidal activity of CPA functionalized hydrogels against E. coli K235 indicated that CPA immobilized at the carboxyl terminus killed 94 ± 6% of the inoculated pathogens when coatings were prepared with high molecular weight PEGSH and 99 ± 1% when prepared with low molecular weight PEGSH. E. coli cell death demonstrated a stronger dependence on peptide concentration than PEG linker length or degree of thiol functionalization, with activity ranging from 34 ± 13% to 99 ± 1% bacterial cells killed as the prefunctionalization thiol concentration in the coatings was increased from 90 to 990 μM. Finally, the immobilization site on the surface-bound CPA strongly affected antibacterial activity; when midsequence modified CPA was bound to a hydrogel coating bearing 990 μM thiol, only 20 ± 4% of the E. coli population was killed.

Multireactive Poly(2-oxazoline) Nanofibers through Electrospinning with Crosslinking on the Fly.

Crosslinked hydrophilic poly(2-oxazoline)-based nanofibers amenable to facile multifunctionalization are fabricated using alkene-containing poly(2-alkyl-2-oxazoline)s (PAOx) via in situ photoinitiated radical thiol-ene crosslinking during electrospinning. The resulting crosslinked nanofibers are demonstrated to be multifunctionalizable using different chemistries as they contain two functional handles, being the alkene moieties from the parent copolymer and the residual thiol groups from the tetra-thiol-based crosslinker. While the thiol groups in these nanofibers could be passivated or conjugated to install functional molecules through thiol-maleimide conjugation, the alkene groups could sequentially be modified with thiol-containing molecules using photoinitiated radical thiol-ene reactions. Utilization of the photochemically induced conjugation of thiol-bearing molecules to the alkene groups on the nanofibers is used to obtain functionalization in a spatially controlled manner.

Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation.

For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

Orthogonal functionalization of a fullerene building block through copper-catalyzed alkyne–azide and thiol–maleimide click reactions

In this study, the synthesis of an orthogonally clickable fullerene building block mono-adduct bearing on one side an alkyne unit and a maleimide moiety on the other side is presented. This derivative has been involved in CuAAC and thiol–maleimide click reactions using stepwise or one-pot processes with benzyl azide and 1-octanethiol. The one-pot process involving the CuAAC reaction followed by the thiol–maleimide conjugation gives the highest yield. This new platform could pave the way for the synthesis of a wide range of fullerene derivatives exploiting this set of orthogonal reactions.

Double modular modification of thiolactone-containing polymers: towards polythiols and derived structures

A conceptual proof for the double modification (aminolysis and subsequent thiol-click modification) of thiolactone units, incorporated in linear polymer scaffolds, was elaborated. These polymers were prepared by either reversible addition–fragmentation chain transfer (RAFT) or nitroxide mediated radical polymerization (NMP) starting from a stable, readily available styrenic thiolactone monomer (St-TLa). Successful copolymerization of the latter with styrene (St) or methyl methacrylate (MMA) yielded linear polymers with varying thiolactone content (4–25%). Upon amine treatment, the ring-opening of the pendent thiolactones resulted in the formation of linear polythiols. Reaction conditions were optimized to avoid cross-linking via disulfide formation, thus preserving the linear nature of the polymer. Different primary amines (propylamine, benzylamine, ethanolamine and Jeffamine M-1000) were attached to the polymer backbone, while the PDIs remained low. The resulting polythiols are versatile scaffolds for further modification by various thiol-click reactions. In this respect, thiol–maleimide conjugation was used as a model reaction. NMR- and SEC-analyses revealed a near-quantitative double modification of thiolactone containing polystyrene (PS) and poly(methylmethacrylate) (PMMA) by subsequent treatment with propylamine and N-benzylmaleimide.

Abstract 3619: Tolerability in mice, monkeys, and rabbits of new antibody (MAb)-drug (SN-38) immunoconjugates

Abstract Antibody drug conjugates (ADCs) of the topoisomerase I inhibitor, SN-38, that allowed for a slow release of SN-38 from the ADC over time, were prepared using anti-CEACAM5 MAb, labetuzumab (hMN-14), anti-TROP-2 MAb, hRS7, and other MAbs by thiol-maleimide conjugation chemistry. These were shown previously to have significant anti-tumor activity in human cancer-xenograft models at cumulative doses frequently ≤ 100 mg/kg (≤ 8 mg/kg human equivalent dose; HED) without toxicity. To determine tolerability and dose-limiting toxicity, hRS7-SN-38 was examined in Swiss-Webster (S-W) mice and Cynomolgus monkeys (CMs), and labetuzumab-SN-38 was evaluated in New Zealand White rabbits. CMs do not express CEACAM5, but similarity in tissue cross-reactivity of hRS7 renders toxicity evaluation of hRS7-SN-38 conjugates in CM feasible. Male S-W mice (n = 14; ∼ 40 g) were administered i.p with 0 (control), 250, 500, or 750 mg/kg (0, 4, 8, and 12 mg/kg SN-38 equivalents) of hRS7-SN-38 on days 1 and 4. Half of the animals were necropsied 7 d, and the remainder 15 d, after the last dose. Blood counts, serum chemistry, and histopathology were performed. This ADC was also administered to CMs (3 M + 3 F/group), via i.v. infusion, at 0, 60, or 120 mg/kg (0, 0.96 and 1.92 mg/kg SN-38 equivalents, respectively) on days 1 and 4. Two males and 2 females from each group were euthanized and necropsied day 11, while the remaining animals were terminated on day 32. Blood counts and chemistries were collected pre-study and periodically through termination. The labetuzumab-SN-38 conjugate was examined in rabbits (n = 6-10) at 0, 30, 60, and 90 mg/kg, administered on days 1 and 4. Mice experienced no hematopoietic toxicity, and only transient weight loss of <10%, but there was a transient elevation in ALT and AST 7 days after treatment, which resolved in most animals one week later. There was no evidence of organ damage. Thus, mice tolerated a cumulative dose of 1500 mg/kg (HED ∼120 mg/kg) of the ADC. CMs receiving 2×60 mg/kg (HED = 2×20 mg/kg) had <7.3% weight loss, showed no abnormal serum chemistries, and while blood counts decreased, they remained within normal limits. Histopathological changes in the terminal group ranged from mild to moderate, but trended to normalcy in the recovery group. The 2×120 mg/kg (HED = 2×40 mg/kg) dose resulted in severe GI and hematologic toxicity. Rabbits tolerated labetuzumab-SN-38 conjugate at all the doses (HED up to 2×30 mg/kg), with uneventful histopathology findings. Pharmacokinetic analysis of rabbit serum showed the IgG cleared with a half-life of ∼ 2 d, and SN-38 clearance was consistent with in vitro stability of the ADC in rabbit serum. ADC and free SN-38 were found to have similar mechanisms of action in vitro. These findings, coupled with previous efficacy studies in xenograft models, indicate a favorable therapeutic window for these ADCs and support the clinical evaluation of these agents in their appropriate indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3619. doi:10.1158/1538-7445.AM2011-3619