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T cell and NK cell-redirecting antibodies are emerging as promising modalities in targeted immunotherapy. New technologies are overcoming the limitations of 1st generation immune cell-redirecting bispecifics, especially extending half-life, reducing immunogenicity and improving safety. For example, in the field of T cell engagers (TCEs), a plethora of molecular architectures has entered the clinic, the vast majority generated by genetic engineering of an IgG to bind both a tumor-associated antigen (TAA) and CD3 or 4-1BB. Similarly, recombinant DNA technology enables the C-terminal fusion of a cytokine (e.g. IL-2 or IL-15) to an IgG to obtain NK cell engagers (NKEs). We have shown earlier that the native glycan of a monoclonal antibody provides a privileged site for attachment of cytotoxic payloads,1 providing antibody-drug conjugates (ADCs) with significantly enhanced therapeutic index. This technology, known as GlycoConnect™, has been adapted by various drug developers (e.g. ADC Therapeutics, Mersana Therapeutics and others) and is now applied in two (2) clinical programs as well as multiple late-stage preclinical programs. Here we demonstrate how GlycoConnect™ technology can be applied to generate TCEs or NKEs by selective attachment of scFv (anti-CD3, anti-CD28) or cytokines (IL-15), with concomitant Fc-silencing. Moreover, an existing bispecific TCE (TAA/CD3) can be further boosted by attachment of anti-CD28 scFv for further T cell activation. Molecular architectures can be readily tailored to 2:2 or 2:1 ratio (TAA:scFv or cytokine) to mitigate adverse events like cytokine release syndrome, and even 2:1:1 formats. Structural and functional studies will be presented to corroborate the architecture and biological activity of these GlycoConnect™ bispecifics and trispecifics, in terms of binding, immune cell engagement and cell-killing potential. 1van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. Citation Format: Remon van Geel, Floris van Delft. Generation of bispecific and trispecific T cell engagers (TCEs) and NK cell engagers (NKE)s without prior IgG reengineering [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1872.

Since the early approval of catumaxomab and blinatumomab, T cell-redirecting antibodies have emerged as a promising biologics in cancer treatment. New technologies are overcoming the limitations of the 1st generation T-cell redirecting bispecifics, especially extending half-life to allow intermittent dosing, reducing immunogenicity and improving the safety profile. Today, T cell-engagers are generated as a wide variety of molecular architectures, typically IgG-type. However, nearly without exception, these bispecifics are generated by genetic fusion of a cancer-binding and an anti-CD3 antibody, which inevitably requires genetic reengineering of an existing targeting antibody and/or extensive optimization of antibody format. We have shown earlier that the native glycan of a monoclonal antibody provides a privileged site for controlled attachment of small-molecule cytotoxic drugs.1 The resulting antibody-drug conjugates (ADCs) were found to display significantly improved therapeutic index versus conventional technologies, while improving manufacturability. This proprietary technology, known as GlycoConnect™, has been adapted by various ADC developers, and entered clinical phase early 2019. Here we present the adaption of GlycoConnect™ technology for the generation of immune cell-redirecting antibodies, without requiring prior protein reengineering. Instead, a two-stage chemoenzymatic process is applied that allows the conversion of any IgG isotype into a T cell-engager by selective attachment of anti-CD3 scFv to the antibody glycan. Studies are presented to precisely control conjugation stoichiometry, providing molecular architectures with defined 2:1 or 2:2 ratio (target-binding:CD3-binding), as well as functional studies to corroborate the biological activity of these GlycoConnect™ bispecifics. Finally, early results will be presented regarding extension of the concept to bispecifics designed to redirect natural killer (NK) cells, or to engage T-cells by activation of costimulatory molecules, such as CD137 (4-1BB). 1 van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved NGlycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. Citation Format: Floris L. van Delft, Remon van Geel. Non-genetic generation of IgG-like immune cell-redirecting antibodies lacking effector function [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4524.

We have found that conjugation of toxic payloads to the native glycan of a monoclonal antibody by chemoenzymatic remodeling (GlycoConnect™ technology) consistently provides antibody-drug conjugates (ADCs) with enhanced therapeutic index (TI) increases versus those of ADCs prepared by mainstream clinical technologies. For example, head-to-head comparison of GlycoConnect™ ADCs -based on the same antibody and payload components- with the marketed drugs Adcetris and Kadcyla revealed an improvement in both efficacy and safety, which is further enhanced by use of a polar HydraSpace™ technology. Importantly, a similar improvement in TI was noted for comparison of a GlycoConnect™ ADC versus or a site-specific ADC derived from a cysteine-engineered antibody, the most important emerging technology in the clinic. With the aim to better understand the superiority of ADCs based on combined GlycoConnect™ and HydraSpace™ technologies, we have performed in-depth in vitro and in vivo investigation into factors contributing to the overall performance of ADCs prepared by glycan conjugation with a polar spacer. For example, it is found that stability is significantly improved versus mainstream technologies in terms of aggregation and linker stability. Rapid aggregation was observed for both Kadcyla and Adcetris, as well as ADC decomposition due to retro-Michael reaction resulting in free payload (for Adcetris) or albumin-conjugated DM1 (for Kadcyla), as indicated by LC-MS and immunoassay analysis. The latter image is confirmed by in vivo pharmacokinetic analysis, showing a dramatic difference in total antibody versus total conjugated antibody by premature release of payload. Finally, analysis with a solid 3D tumoroid model indicates enhanced penetration of site-specific versus randomly conjugated ADC, which may also contribute to better efficacy in solid tumors. The data generated further underline the importance of conjugation and linker technology as critical quality attributes for next-generation ADCs with higher therapeutic index. Citation Format: Floris van Delft, Brian Janssen, Remon van Geel, Marloes Wijdeven, Jorge Verkade, Sander van Berkel. Decomposition of parameters contributing to the improved therapeutic index of ADCs obtained by GlycoConnect™ and HydraSpace™ Technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3815.

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 robust, generally applicable, nongenetic technology is presented to convert monoclonal antibodies into stable and homogeneous ADCs. Starting from a native (nonengineered) mAb, a chemoenzymatic protocol allows for the highly controlled attachment of any given payload to the N-glycan residing at asparagine-297, based on a two-stage process: first, enzymatic remodeling (trimming and tagging with azide), followed by ligation of the payload based on copper-free click chemistry. The technology, termed GlycoConnect, is applicable to any IgG isotype irrespective of glycosylation profile. Application to trastuzumab and maytansine, both components of the marketed ADC Kadcyla, demonstrate a favorable in vitro and in vivo efficacy for GlycoConnect ADC. Moreover, the superiority of the native glycan as attachment site was demonstrated by in vivo comparison to a range of trastuzumab-based glycosylation mutants. A side-by-side comparison of the copper-free click probes bicyclononyne (BCN) and a dibenzoannulated cyclooctyne (DBCO) showed a surprising difference in conjugation efficiency in favor of BCN, which could be even further enhanced by introduction of electron-withdrawing fluoride substitutions onto the azide. The resulting mAb-conjugates were in all cases found to be highly stable, which in combination with the demonstrated efficacy warrants ADCs with a superior therapeutic index.

Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme able to catalyze the formation of e(γ-glutamyl)-lysine crosslinks between polypeptides, resulting in high molecular mass multimers. We have developed a bioorthogonal chemical method for the labeling of TG2 glutamine-donor proteins. As amine-donor substrates we used a set of azide- and alkyne-containing primary alkylamines that allow, after being crosslinked to glutamine-donor proteins, specific labeling of these proteins via the azide-alkyne cycloaddition. We demonstrate that these azide- and alkyne-functionalized TG2 substrates are cell permeable and suitable for specific labeling of TG2 glutamine-donor substrates in HeLa and Movas cells. Both the Cu(I)-catalyzed and strain promoted azide-alkyne cycloaddition proved applicable for subsequent derivatization of the TG2 substrate proteins with the desired probe. This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins. Electronic supplementary material The online version of this article (doi:10.1007/s00726-011-1198-2) contains supplementary material, which is available to authorized users.

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.

While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall.

BACKGROUND: The aging related decline of heat shock factor-1 (HSF1) signaling may be causally related to protein aggregation diseases. To model such disease, we tried to cripple HSF1 signaling in the Xenopus tadpole. RESULTS: Over-expression of heat shock factor binding protein-1 did not inhibit the heat shock response in Xenopus. RNAi against HSF1 mRNA inhibited the heat shock response by 70% in Xenopus A6 cells, but failed in transgenic tadpoles. Expression of XHSF380, a dominant-negative HSF1 mutant, was embryonic lethal, which could be circumvented by delaying expression via a tetracycline inducible promoter. HSF1 signaling is thus essential for embryonic Xenopus development. Surprisingly, transgenic expression of the XHSF380 or of full length HSF1, whether driven by a ubiquitous or a neural specific promoter, was not detectable in the larval brain. CONCLUSIONS: Our finding that the majority of neurons, which have little endogenous HSF1, refused to accept transgene-driven expression of HSF1 or its mutant suggests that HSF1 levels are strictly controlled in neuronal tissue.

In this paper, a straightforward and generic protocol is presented to label the C-terminus of a peptide with any desired moiety that is functionalized with a primary amine. Amine-functional molecules included are polymers (useful for hybrid polymers), long alkyl chains (used in peptide amphiphiles and stabilization of peptides), propargyl amine and azido propyl-amine (desirable for 'click' chemistry), dansyl amine (fluorescent labeling of peptides) and crown ethers (peptide switches/hybrids). In the first part of the procedure, the primary amine is attached to an aldehyde-functional resin via reductive amination. To the secondary amine that is produced, an amino acid sequence is coupled via a standard solid-phase peptide synthesis protocol. Since one procedure can be applied for any given amine-functional moiety, a robust method for C-terminal peptide labeling is obtained. Copyright  2006 European Peptide Society and John Wiley & Sons, Ltd.