Modular Semisynthetic Approach to Generate T Cell-Dependent Bispecific Constructs from Recombinant IgG1 Antibodies.

Redirecting T cells to tumor cells by bispecific antibodies is an effective approach to treat cancer, and T cell-dependent bispecific antibodies (TDBAs) are an emerging class of potent immunotherapeutic agents. By simultaneously targeting antigens on tumor cells and T cells, T cells are activated to kill tumor cells. Herein, we report a platform to generate a novel class of 2:1 structure of T cell-dependent bispecific antibody with bivalency for HER2 receptors on tumor cells and monovalency for CD3 receptors on T cells. For this, we use a biogenic inverse electron-demand Diels-Alder (IEDDA) click reaction on genetically encoded tyrosine residues to install one TCO handle on therapeutically approved antibody trastuzumab. Subsequent TCO-tetrazine click with a tetrazine-functionalized CD3-binding Fab yields a 2:1 HER2 × CD3 TDBA that exhibits a tumor-killing capability at picomolar concentrations. Monovalency toward the CD3 receptor on T cells can lower the chances of cytokine release syndrome, which is a common side effect of such agents. Our semisynthetic approach can generate highly potent TDBA constructs in a few chemoenzymatic and synthetic steps.

Rapid Access to Potent Bispecific T Cell Engagers Using Biogenic Tyrosine Click Chemistry.

Bispecific antibodies as T cell engagers designed to display binding capabilities to both tumor-associated antigens and antigens on T cells are considered promising agents in the fight against cancer. Even though chemical strategies to develop such constructs have emerged, a method that readily converts a therapeutically applied antibody into a bispecific construct by a fully non-genetic process is not yet available. Herein, we report the application of a biogenic, tyrosine-based click reaction utilizing chemoenzymatic modifications of native IgG1 antibodies to generate a synthetic bispecific antibody construct that exhibits tumor-killing capability at picomolar concentrations. Control experiments revealed that a covalent linkage of the different components is required for the observed biological activities. In view of the highly potent nature of the constructs and the modular approach that relies on convenient synthetic methods utilizing therapeutically approved biomolecules, our method expedites the production of potent bispecific antibody constructs with tunable cell killing efficacy with significant impact on therapeutic properties.

Generation of DAR1 Antibody-Drug Conjugates for Ultrapotent Payloads Using Tailored GlycoConnect Technology.

GlycoConnect technology can be readily adapted to provide different drug-to-antibody ratios (DARs) and is currently also evaluated in various clinical programs, including ADCT-601 (DAR2), MRG004a (DAR4), and XMT-1660 (DAR6). While antibody-drug conjugates (ADCs) typically feature a DAR2-8, it has become clear that ADCs with ultrapotent payloads (e.g., PBD dimers and calicheamicin) can only be administered to patients at low doses (<0.5 mg/kg), which may compromise effective biodistribution and may be insufficient to reach target receptor saturation in the tumor. Here, we show that GlycoConnect technology can be readily extended to DAR1 ADCs without the need of antibody re-engineering. We demonstrate that various ultrapotent, cytotoxic payloads are amenable to this methodology. In a follow-up experiment, HCC-1954 tumor spheroids were treated with either an AlexaFluor647-labeled DAR1 or DAR2 PBD-based ADC to study the effect on tumor penetration. Significant improvement of tumor spheroid penetration was observed for the DAR1 ADC compared to the DAR2 ADC at an equal payload dose, underlining the potential of a lower DAR for ADCs bearing ultrapotent payloads.

Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering.

Antibody-drug conjugates (ADCs) are increasingly powerful medicines for targeted cancer therapy. Inspired by the trend to further improve their therapeutic index by generation of homogenous ADCs, we report here how the clinical-stage GlycoConnect™ technology uses the globally conserved N-glycosylation site to generate stable and site-specific ADCs based on enzymatic remodeling and metal-free click chemistry. We demonstrate how an engineered endoglycosidase and a native glycosyl transferase enable highly efficient, one-pot glycan remodeling, incorporating a novel sugar substrate 6-azidoGalNAc. Metal-free click attachment of an array of cytotoxic payloads was highly optimized, in particular by inclusion of anionic surfactants. The therapeutic potential of GlycoConnect™, in combination with HydraSpace™ polar spacer technology, was compared to that of Kadcyla® (ado-trastuzumab emtansine), showing significantly improved efficacy and tolerability.

Cellular-Resolution Imaging of Bystander Payload Tissue Penetration from Antibody-Drug Conjugates.

After several notable clinical failures in early generations, antibody-drug conjugates (ADC) have made significant gains with seven new FDA approvals within the last 3 years. These successes have been driven by a shift towards mechanistically informed ADC design, where the payload, linker, drug-to-antibody ratio, and conjugation are increasingly tailored to a specific target and clinical indication. However, fundamental aspects needed for design, such as payload distribution, remain incompletely understood. Payloads are often classified as "bystander" or "nonbystander" depending on their ability to diffuse out of targeted cells into adjacent cells that may be antigen-negative or more distant from tumor vessels, helping to overcome heterogeneous distribution. Seven of the 11 FDA-approved ADCs employ these bystander payloads, but the depth of penetration and cytotoxic effects as a function of physicochemical properties and mechanism of action have not been fully characterized. Here, we utilized tumor spheroids and pharmacodynamic marker staining to quantify tissue penetration of the three major classes of agents: microtubule inhibitors, DNA-damaging agents, and topoisomerase inhibitors. PAMPA data and coculture assays were performed to compare with the 3D tissue culture data. The results demonstrate a spectrum in bystander potential and tissue penetration depending on the physicochemical properties and potency of the payload. Generally, directly targeted cells show a greater response even with bystander payloads, consistent with the benefit of deeper ADC tissue penetration. These results are compared with computational simulations to help scale the data from in vitro and preclinical animal models to the clinic.

Enzymatic strategies for (near) clinical development of antibody-drug conjugates.

Target-specific killing of tumor cells with antibody-drug conjugates (ADCs) is an elegant concept in the continued fight against cancer. However, despite more than 20 years of clinical development, only four ADC have reached market approval, while at least 50 clinical programs were terminated early. The high attrition rate of ADCs may, at least in part, be attributed to heterogeneity and instability of conventional technologies. At present, various (chemo)enzymatic approaches for site-specific and stable conjugation of toxic payloads are making their way to the clinic, thereby potentially providing ADCs with increased therapeutic window.

A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates.

Despite tremendous efforts in the field of targeted cancer therapy with antibody-drug conjugates (ADCs), attrition rates have been high. Historically, the priority in ADC development has been the selection of target, antibody, and toxin, with little focus on the nature of the linker. We show here that a short and polar sulfamide spacer (HydraSpace™, Oss, The Netherlands) positively impacts ADC properties in various ways: (a) efficiency of conjugation; (b) stability; and (c) therapeutic index. Different ADC formats are explored in terms of drug-to-antibody ratios (DAR2, DAR4) and we describe the generation of a DAR4 ADC by site-specific attachment of a bivalent linker-payload construct to a single conjugation site in the antibody. A head-to-head comparison of HydraSpace™-containing DAR4 ADCs to marketed drugs, derived from the same antibody and toxic payload components, indicated a significant improvement in both the efficacy and safety of several vivo models, corroborated by in-depth pharmacokinetic analysis. Taken together, HydraSpace™ technology based on a polar sulfamide spacer provides significant improvement in manufacturability, stability, and ADC design, and is a powerful platform to enable next-generation ADCs with enhanced therapeutic index.

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3).

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 µM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50-60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.

Phenylglyoxal-based visualization of citrullinated proteins on Western blots.

Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.

Influence of azide incorporation on binding affinity by small papain inhibitors.

In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by 'click' chemistry, in this particular case by copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.

Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA.

Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formed viral RNA molecules possess a covalently linked VPg peptide. It is known that VPg is enzymatically released from the incoming viral RNA by a host protein, called TDP2, but it is still unclear whether the release of VPg is necessary to initiate RNA translation. To study the possible requirement of VPg release for RNA translation, we developed a novel method to modify the genomic viral RNA with VPg linked via a 'non-cleavable' bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne [bicyclo[6.1.0]nonyne (BCN)] by a copper-free 'click' reaction, leading to a VPg-triazole-RNA construct that was 'non-cleavable' by TDP2. We successfully ligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We show that the lack of VPg unlinkase does not influence RNA translation or replication. Thus, the release of the VPg from the incoming viral RNA is not a prerequisite for RNA translation or replication.

Targeting aberrant sialylation in cancer cells using a fluorinated sialic acid analog impairs adhesion, migration, and in vivo tumor growth.

Cancer cells decorate their surface with a dense layer of sialylated glycans by upregulating the expression of sialyltransferases and other glycogenes. Although sialic acids play a vital role in many biologic processes, hypersialylation in particular has been shown to contribute to cancer cell progression and metastasis. Accordingly, selective strategies to interfere with sialic acid synthesis might offer a powerful approach in cancer therapy. In the present study, we assessed the potential of a recently developed fluorinated sialic acid analogue (P-3F(ax)-Neu5Ac) to block the synthesis of sialoglycans in murine melanoma cells and the consequences on cell adhesion, migration, and in vivo growth. The results showed that P-3F(ax)-Neu5Ac readily caused depletion of α2,3-/α2,6-linked sialic acids in B16F10 cells for several days. Long-term inhibition of sialylation for 28 days was feasible without affecting cell viability or proliferation. Moreover, P-3F(ax)-Neu5Ac proved to be a highly potent inhibitor of sialylation even at high concentrations of competing sialyltransferase substrates. P-3F(ax)-Neu5Ac-treated cancer cells exhibited impaired binding to poly-l-lysine, type I collagen, and fibronectin and diminished migratory capacity. Finally, blocking sialylation of B16F10 tumor cells with this novel sialic acid analogue reduced their growth in vivo. These results indicate that P-3F(ax)-Neu5Ac is a powerful glycomimetic capable of inhibiting aberrant sialylation that can potentially be used for anticancer therapy.

The epithelial calcium channel TRPV5 is regulated differentially by klotho and sialidase.

The transient receptor potential vanilloid type 5 (TRPV5) Ca(2+) channel facilitates transcellular Ca(2+) transport in the distal convoluted tubule (DCT) of the kidney. The channel is glycosylated with a complex type N-glycan and it has been postulated that hydrolysis of the terminal sialic acid(s) stimulate TRPV5 activity. The present study delineates the role of the N-glycan in TRPV5 activity using biochemical assays in Human Embryonic Kidney 293 cells expressing TRPV5, isoelectric focusing and total internal reflection fluorescent microscopy. The anti-aging hormone klotho and other glycosidases stimulate TRPV5-dependent Ca(2+) uptake. Klotho was found to increase the plasma membrane stability of TRPV5, via the TRPV5 N-glycan. Sialidase mimicked this stimulatory action. However, this effect was independent of the N-glycosylation state of TRPV5, since the N-glycosylation mutant (TRPV5(N358Q)) was activated to the same extent. We showed that the increased TRPV5 activity after sialidase treatment is caused by inhibition of lipid raft-mediated internalization. In addition, sialidase modified the N-glycan of transferrin, a model glycoprotein, differently from klotho. Previous studies showed that after klotho treatment, galectin-1 binds the TRPV5 N-glycan and thereby increases TRPV5 activity. However, galectin-3, but not galectin-1, was expressed in the DCT. Furthermore, an increase in TRPV5-mediated Ca(2+) uptake was detected after galectin-3 treatment. In conclusion, two distinct TRPV5 stimulatory mechanisms were demonstrated; a klotho-mediated effect that is dependent on the N-glycan of TRPV5 and a sialidase-mediated stimulation that is lipid raft-dependent and independent of the N-glycan of TRPV5.

Interfering with UDP-GlcNAc metabolism and heparan sulfate expression using a sugar analogue reduces angiogenesis.

Heparan sulfate (HS), a long linear polysaccharide, is implicated in various steps of tumorigenesis, including angiogenesis. We successfully interfered with HS biosynthesis using a peracetylated 4-deoxy analogue of the HS constituent GlcNAc and studied the compound's metabolic fate and its effect on angiogenesis. The 4-deoxy analogue was activated intracellularly into UDP-4-deoxy-GlcNAc, and HS expression was inhibited up to ∼96% (IC50 = 16 µM). HS chain size was reduced, without detectable incorporation of the 4-deoxy analogue, likely due to reduced levels of UDP-GlcNAc and/or inhibition of glycosyltransferase activity. Comprehensive gene expression analysis revealed reduced expression of genes regulated by HS binding growth factors such as FGF-2 and VEGF. Cellular binding and signaling of these angiogenic factors was inhibited. Microinjection in zebrafish embryos strongly reduced HS biosynthesis, and angiogenesis was inhibited in both zebrafish and chicken model systems. All of these data identify 4-deoxy-GlcNAc as a potent inhibitor of HS synthesis, which hampers pro-angiogenic signaling and neo-vessel formation.

Site-specific peptide and protein immobilization on surface plasmon resonance chips via strain-promoted cycloaddition.

Surface plasmon resonance (SPR) is a powerful label-free diagnostic tool to study biomolecular interactions. However, one of the drawbacks of SPR is the lack of controlled immobilization of ligands on the sensor surface. We have developed a modular platform for the fast, reagent-free and site-specific immobilization of azide-containing ligands by strain-promoted cycloaddition onto a cyclooctyne-modified SPR sensor surface. The usefulness of the concept was shown in a study with a papain model system, and up to 150 experiments were performed without loss of surface quality. Furthermore, azide-containing green fluorescent protein (GFP) was also effectively immobilized. Taken together, cyclooctyne-modified SPR chips enable smooth and site-selective immobilization of ligands and prove to be more robust than traditionally functionalized systems.

In vitro and in vivo characterization of three 68Ga- and 111In-labeled peptides for cholecystokinin receptor imaging.

Cholecystokinin (CCK) receptors are overexpressed in several human tumor types, such as medullary thyroid carcinomas and small cell lung cancers. Several ligands for the CCK2 receptor (CCK2R) have been developed for radionuclide targeting of these tumors. In this study, we evaluated whether radiolabeled DOTA-sCCK8 and its stabilized derivative, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], are suitable for imaging of CCK2R-positive tumors, using DOTA-MG0 as a reference. In vivo targeting of CCK2R-positive tumors with DOTA-sCCK8, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], and DOTA-MG0, labeled with (111)In or (68)Ga, was evaluated in BALB/c nude mice with a subcutaneous A431-CCK2R tumor. Biodistribution studies and single-photon emission computed tomography (SPECT) and positron emission tomography (PET) were performed at 1 hour postinjection. All peptides specifically accreted in the CCK2R-expressing tumors. Both (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)] showed good tumor retention (4.65% ID/g and 5.44% ID/g, respectively, at 4 hours postinjection). On PET/computed tomographic (CT) and SPECT/CT scans, subcutaneous A431-CCK2R tumors were clearly visualized with low uptake of sCCK8 peptides in the intestines. Whereas radiolabeled DOTA-MG0 showed high kidney uptake (70% ID/g), the sCCK8 peptides showed low uptake in the kidneys. Sulfated CCK8 analogues combined high tumor uptake with low retention in the kidney and are therefore promising tracers for imaging of CCK2R-positive tumors.

Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition.

The 1,3-dipolar cycloaddition of azides with ring-strained alkynes is one of the few bioorthogonal reactions suitable for specific biomolecule labeling in complex biological systems. Nevertheless, azide-independent labeling of proteins by strained alkynes can occur to a varying extent, thereby limiting the sensitivity of assays based on strain-promoted azide-alkyne cycloaddition (SPAAC). In this study, a subset of three cyclooctynes, dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]nonyne (BCN), was used to evaluate the azide-independent labeling of proteins in vitro. For all three cyclooctynes, we show that thiol-yne addition with reduced peptidylcysteines is responsible for most of the azide-independent polypeptide labeling. The identity of the reaction product was confirmed by LC-MS and NMR analysis. Moreover, we show that undesired thiol-yne reactions can be prevented by alkylating peptidylcysteine thiols with iodoacetamide (IAM). Since IAM is compatible with SPAAC, a more specific azide-dependent labeling is achieved by preincubating proteins containing reduced cysteines with IAM.

Fluorogenic peptide-based substrates for monitoring thrombin activity.

The synthesis of a series of peptides containing C-terminal 7-amino-4-methylcoumarin (AMC) for use in the thrombin generation test (TGT) is described. The lead structure in this project was H-Gly-Gly-Arg-AMC, of which the water solubility and kinetic parameters (K(M) and k(cat)) are greatly improved over those of the substrate in current use in the TGT: Cbz-Gly-Gly-Arg-AMC. A series of N-terminally substituted Gly-Gly-Arg-AMC derivatives were synthesized, as well as implementation of structural changes at either the P(2) or P(3) position of the peptide backbone. Furthermore, two substrates were synthesized that have structural similarities to the chromogenic thrombin substrate SQ68 or that contain a 1,2,3-triazole moiety in the peptide chain, mimicking an amide bond. To determine the applicability of newly synthesized fluorogenic substrates for monitoring continuous thrombin generation, the K(M) and k(cat) values of the conversion of these fluorogenic substrates by thrombin (FIIa) and factor Xa (FXa) were quantified. An initial selection was made on basis of these data, and suitable substrates were further evaluated as substrates in the thrombin generation assay. Assessment of the acquired data showed that several substrates, including the SQ68 derivative Et-malonate-Gly-Arg-AMC and N-functionalized Gly-Gly-Arg-AMC derivatives, are suitable candidates for replacement of the substrate currently in use.

Clickable enzyme-linked immunosorbent assay.

Click chemistry is explored as a potential cost-effective and selective immobilization method for the production of an enzyme-linked immunosorbent assay (ELISA). Coatings were formulated containing either a terminal alkyne or a bicyclo[6.1.0]non-4-yne (BCN) chemical handle, and a diagnostic peptide was subsequently immobilized onto these coatings by the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) or copper-free strain-promoted azide-alkyne 1,3-dipolar cycloaddition (SPAAC), respectively. The terminal alkyne-containing coating showed high background levels in subsequent ELISA's due to the copper catalyst used in the immobilization step. The BCN-containing coating, however, was successfully employed and presents a cost-effective alternative to existing (strept)avidin-biotin immobilization methods. This technology was illustrated with an ELISA used for the diagnosis of rheumatoid arthritis (RA) but could be easily applied to a wide range of diagnostic tests.

Papain-catalyzed peptide bond formation: enzyme-specific activation with guanidinophenyl esters.

The substrate mimetics approach is a versatile method for small-scale enzymatic peptide-bond synthesis in aqueous systems. The protease-recognized amino acid side chain is incorporated in an ester leaving group, the substrate mimetic. This shift of the specific moiety enables the acceptance of amino acids and peptide sequences that are normally not recognized by the enzyme. The guanidinophenyl group (OGp), a known substrate mimetic for the serine proteases trypsin and chymotrypsin, has now been applied for the first time in combination with papain, a cheap and commercially available cysteine protease. To provide insight in the binding mode of various Z-X(AA)-OGp esters, computational docking studies were performed. The results strongly point at enzyme-specific activation of the OGp esters in papain through a novel mode of action, rather than their functioning as mimetics. Furthermore, the scope of a model dipeptide synthesis was investigated with respect to both the amino acid donor and the nucleophile. Molecular dynamics simulations were carried out to prioritize 22 natural and unnatural amino acid donors for synthesis. Experimental results correlate well with the predicted ranking and show that nearly all amino acids are accepted by papain.