Multivalent Functionalized Dextran-Antibody Conjugates for Efficient Tumor Cell Killing

Despite impressive progress in medical care and extensive investigation in the field of human malignancies, cancer still represents a major global health issue, and triggering apoptosis of tumor cells has been in focus of cancer research for decades. In addition to classical attempts like surgery, radiotherapy, and chemotherapy, a number of novel methodologies have recently fallen in the limelight, with monoclonal antibodies being the most prominent actors at the therapeutic scene. With their introduction, the so-called targeted therapy has finally become achievable for the treatment of malignant tumors. Having evolved from Paul Ehrlich’s “Magic Bullet” concept that described directing a toxic compound exclusively to a disease-causing organism, antibody-drug conjugates (ADCs) were developed. This class of compounds is aimed at site-selective delivery of cytotoxic agents to target cells expressing a cancer-related antigen. Combining the targeting properties of an antibody with the killing properties of a potent cytotoxin, these constructs were applied in countless approaches intending to treat tumor patients. To date, six ADCs have been marketed and over 60 ones are currently in clinical trials. However, several issues still require improvement, among them toxicity, efficacy and pharmacokinetics. The first investigation in the context of the present cumulative study was focused on the generation of highly hydrophilic ADCs characterized by a high drug-to-antibody ratio (DAR). However, since only a limited number of the administered ADCs is reported to actually reach their cellular target, either highly potent toxins or a higher number of the less-potent ones are prerequisites of these compounds to reach efficacy. Considering the hydrophobic character of most commonly applied potent cytotoxins, these conjugates often suffer from poor hydrophilicity depending on the addressed conjugation site as well as the number and character of the applied toxin units. Further, hydrophobicity of ADCs was reported to raise problems due to aggregation and recognizing by multidrug resistance (MDR) transporters, thus the number, site, and hydrophobicity of the conjugated toxin strongly influences stability, pharmacokinetic properties and the efficacy of ADCs. In this work we addressed these issues simultaneously by designing a novel class of hybrid ADCs combining ability to balance and even recompense the hydrophobicity of commonly applied highly hydrophobic cytotoxins with an option for the attachment of multiple payloads, which may further enable the application of less potent, thus less harmful for the healthy tissues, cytotoxins. To that end, we applied dextran, an FDA-approved polysaccharide, consisting mainly of α-1,6-linked oligo-D-glucose units as multivalency-generating modular scaffold for payload attachment. This glucan, reported to enhance half-life, to improve thermal stability and pharmacokinetic properties, and to reduce immunogenicity of conjugated proteins, opens certain space for chemical modifications, namely conjugation of a) a desired number of payloads to the repeating glucose units at the respective hydroxy groups, and b) at the reducing end that comprises an orthogonally addressable aldehyde. In this work, a strategy to combine an enzyme-catalyzed site-specific conjugation of the dextran scaffold, equipped with multiple reactive moieties for payload loading, to antibodies was developed, resulting in promising constructs for the generation of high-DAR ADCs. Synthetically, a combination of reductive amination of dextran’s reducing end with a protected diamine followed by site-selective carboxyethylation at the C2-position of the glucose repeating units led to dextran bearing dually addressable reactive moieties. Notably, our synthetic procedure allowed adjustment of the number of addressable sites at the repeating glucose monomers for toxin conjugation to the desired amount of copies. Subsequent conjugation of azide-bearing aliphatic amines at the repeating units upon amidation gave a scaffold comprising multiply addressable “click” sites. Demasking of the protected amine at the reducing end yielded a multivalent scaffold combining multiple and solitaire orthogonal addressable sites. Thus, at its amine site it could be easily conjugated to the protein of interest – in this particular case, to the therapeutic antibody trastuzumab – via enzymatic catalysis by microbial transglutaminase (mTG). For this purpose, trastuzumab was engineered to possess an adequate mTG recognition motif at the C-terminus of the heavy chain. The azides at the sugar monomers remained for the decoration with monomethyl auristatin E (MMAE) – a highly toxic and extraordinary hydrophobic compound. To conclude, for the first-time dextran was site-specifically conjugated to a functional antibody via its reducing end, leaving the polysaccharide backbone intact and subsequently equipped with multiple MMAEs by strain-promoted azide-alkyne cycloaddition (SPAAC) in a desired number of copies without corrupting the polysaccharide backbone. These hybrid constructs, called dextramabs, were found not only readily soluble in aqueous buffers, but at least as hydrophilic as the parental antibody trastuzumab, even when conjugated with eleven highly hydrophobic MMAE counterparts. The binding properties of all generated constructs were not affected, as demonstrated by comparable KD values on HER2-positive SK-BR-3 cells (unmodified trastuzumab: KD = 4.9 nM, dextramab (DAR 8): KD = 5.9 nM). Our synthetic dextramabs showed potent subnanomolar cytotoxicity (IC50 = 100 pM) in cell proliferation assays on HER2-positive SK-BR-3 breast cancer cells and no cytotoxicity on HER2-negative control cells in vitro. These site-specifically assembled ADCs may combine the beneficial pharmacokinetic properties, as their protein counterparts are loaded with dextran, therefore possess higher hydrodynamic radius, with the possibility to attach a tailored number of payloads. Generally, our concept represents a promising approach for the generation of highly hydrophilic site-specific ADCs characterized by a high DAR. Follow-up animal studies will unveil if dextramabs hold promise for the novel class of ADCs with high potency, low immunogenicity and enhanced in vivo half-life. In the second investigation we studied applicability of dextran polysaccharide scaffold as carrier for apoptosis-triggering payloads of diverse nature, which act by addressing distinct intra- or extracellular targets. Hence, additionally to the above-mentioned high-DAR ADCs we were focused at validating dextran as a platform for multimerization of cancer-relevant ligands. First, death receptor 5 (DR5) was chosen as a model target expressed on the cell surface. As it is activated by oligomerization/aggregation, we aimed at constructing a flexible scaffold able to bypass the reported need for spatial ligand orientation for efficient DR5-mediated cellular cytotoxicity. Thus, we designed a molecular architecture comprising a polysaccharide scaffold carrying the desired number of DR5 peptidic binders able to efficiently trigger apoptosis upon DR5 receptor clustering. Herein, apoptosis of cancer cells was mediated by multivalent binding to and clustering of a receptor located on the cell surface, which is in fact contrary to the first approach investigated in this work, which relied on a high number of a very potent cytotoxin that only upon internalization inhibits cell division by blocking the polymerization of tubulin. Thus, dextran was loaded with on average 11 or 13.4 peptidic binders, namely death receptor 5 targeting peptides (DR5TP). The resulting constructs were found potent apoptosis-inducing conjugates possessing double-digit nanomolar half-maximal effective concentration (EC50) values on DR5-positive COLO205 colon cells and Jurkat T lymphocytes in vitro. Moreover, conjugation to glutamine 295 of an aglycosylated fragment crystallizable (Fc) fragment of a monoclonal antibody (mAb) by site-specific mTG-catalyzed conjugation resulted in constructs that showed selective DR5 binding upon flow cytometric analysis and further did not impair the potency of the generated multivalent scaffolds. In contrast, these protein-polysaccharide-peptide hybrids demonstrated higher potency in vitro (EC50 = 1.9 - 6.7 nM). Notably, in this approach binding is not mediated by the protein, but rather by the ligand-bearing dextran counterpart. Thus, addition of a second targeting moiety, e.g. application of a full-length antibody, would be an interesting prospective study that opens the possibility of bispecific targeting, which may result in enhanced safety and efficacy. Furthermore, the generated DR5TP-dextran and the Fc-bound counterparts were able to circumvent the mentioned need for spatial orientation of ligands due to additional flexibility provided by dextran scaffold. Our study further underlines the modularity of dextran as carrier for different payloads addressing various targets. In addition, this approach may help overcoming the reported off-target toxicity for multimeric high-affinity protein-based constructs, e.g. TAS266, by the application of low-affinity peptidic binders. This may lead to better tolerability in vivo, conditioned by lower retention on healthy cells expressing minor levels of DR5, which might be additionally improved by prospective bispecific targeting. The third part of this work was aimed at relieving the current lack of satisfactory overviews on mTG-mediated generation of homogeneous ADCs. Since most reviews dealing with ADCs cover a broad scope of topics, but usually very briefly, an in-depth comparative survey was highly required. However, a detailed overview of the factors influencing the resulting architectures in view of stability, potency, efficiency, etc. was still missing. A comprehensive summary of the reported strategies may enable tailoring of existing methods for mTG-promoted conjugation to the needs of particular research projects. On these grounds, the originated review was intended not only to enumerate the applied approaches for site-specific conjugation with respect to ADC assembly, but to map out the research groups and companies working on mTG-mediated generation of antibody-drug conjugates. Our review, gives a thorough overview of conjugation methodologies, addressed conjugation motifs or sites, applied cellular targets, linkers, and cytotoxic cargoes. Thus, it highlights pioneering routes and techniques, recent progress and remaining limitations of mTG-assisted assembly of ADCs. Furthermore, a study aimed at assessment of possibilities offered by dextran as a multivalency-promoting framework was performed. In a preliminary proof-of-concept study dextran was applied as a vehicle for multiple attachment of metal-chelating agents able to carrier valuable ions for radio-imaging or -therapy. Thus, dextran polymer was equipped with a desired number of a widely applied metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The assembled DOTA-dextran conjugates were able to carry 3.2 or 5,3 metal ions per polysaccharide chain, respectively, as shown by photometric analysis of the formed complexes with Cu2+. In vivo biodistribution studies in mice are currently ongoing. This proof-of-concept study should answer the question, whether these novel molecular hybrids are suitable for in vivo applications and whether conjugation to commonly applied binders (affibodies) that suffer from hydrophobicity and in consequence poor solubility, aggregation and precipitation, results in beneficial properties. This approach further strengthens the presumption that dextran represents a promising modular scaffold for multivalent attachment and tailoring of diverse payloads