Prodrug-Activating Chain Exchange (PACE) converts targeted prodrug derivatives to functional bi- or multispecific antibodies.

Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive chain-exchange reactions between them. Importantly, Prodrug-Activating Chain Exchange (PACE) could be an attractive option to conditionally activate therapeutics at the target site. Several examples are provided that demonstrate the efficacy of PACE as a new principle of cancer immunotherapy in vitro and in a human xenograft model.

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder.

PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.

Antibody-targeted chromatin enables effective intracellular delivery and functionality of CRISPR/Cas9 expression plasmids.

We report a novel system for efficient and specific targeted delivery of large nucleic acids to and into cells. Plasmid DNA and core histones were assembled to chromatin by salt gradient dialysis and subsequently connected to bispecific antibody derivatives (bsAbs) via a nucleic acid binding peptide bridge. The resulting reconstituted vehicles termed 'plasmid-chromatin' deliver packaged nucleic acids to and into cells expressing antigens that are recognized by the bsAb, enabling intracellular functionality without detectable cytotoxicity. High efficiency of intracellular nucleic acid delivery is revealed by intracellular expression of plasmid encoded genes in most (∼90%) target cells to which the vehicles were applied under normal growth/medium conditions in nanomolar concentrations. Specific targeting, uptake and transgene expression depends on antibody-mediated cell surface binding: plasmid chromatin of identical composition but with non-targeting bsAbs or without bsAbs is ineffective. Examples that demonstrate applicability, specificity and efficacy of antibody-targeted plasmid chromatin include reporter gene constructs as well as plasmids that enable CRISPR/Cas9 mediated genome editing of target cells.

Quantitative fluorescence imaging determines the absolute number of locked nucleic acid oligonucleotides needed for suppression of target gene expression.

Locked nucleic acid based antisense oligonucleotides (LNA-ASOs) can reach their intracellular RNA targets without delivery modules. Functional cellular uptake involves vesicular accumulation followed by translocation to the cytosol and nucleus. However, it is yet unknown how many LNA-ASO molecules need to be delivered to achieve target knock down. Here we show by quantitative fluorescence imaging combined with LNA-ASO microinjection into the cytosol or unassisted uptake that ∼105 molecules produce >50% knock down of their targets, indicating that a substantial amount of LNA-ASO escapes from endosomes. Microinjected LNA-ASOs redistributed within minutes from the cytosol to the nucleus and remained bound to nuclear components. Together with the fact that RNA levels for a given target are several orders of magnitude lower than the amounts of LNA-ASO, our data indicate that only a minor fraction is available for RNase H1 mediated reduction of target RNA. When non-specific binding sites were blocked by co-administration of non-related LNA-ASOs, the amount of target LNA-ASO required was reduced by an order of magnitude. Therefore, dynamic processes within the nucleus appear to influence the distribution and activity of LNA-ASOs and may represent important parameters for improving their efficacy and potency.

Engineering therapeutic bispecific antibodies using CrossMab technology.

Bispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct association of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various approaches have been described during recent years to tackle this challenge. We have developed the so-called CrossMab technology that enforces correct light chain association based on the domain crossover of immunoglobulin domains in the Fab region of the bispecific antibody. This versatile technology allows the generation of different bispecific antibody formats including asymmetric heterodimeric monovalent 1 + 1 bispecific antibodies and asymmetric heterodimeric bispecific antibodies with 2 + 1 valency in combination with approaches enabling Fc-hetermodimerization like knob-into-hole technology as well as the generation of tetravalent symmetric bispecific antibodies with 2 + 2 valency, also known as Tandem-Fab based IgG antibodies, using processes suitable for the large scale production of therapeutic bispecific antibodies. Notably, as of now, at least eight different bispecific antibodies using CrossMab technology entered clinical development, and additional CrossMabs are in late preclinical development. This review provides a summary of the status and progress with the engineering and generation of CrossMab technology based bispecific antibodies as well as their therapeutic application.

Engineered hapten-binding antibody derivatives for modulation of pharmacokinetic properties of small molecules and targeted payload delivery.

Hapten-binding antibodies have for more than 50 years played a pivotal role in immunology, paving the way to antibody generation (as haptens are very important and robust immunogens), to antibody characterization (as the first structures generated more than 40 years ago were those of hapten binders), and enabled and expanded antibody engineering technologies. The latter field of engineered antibodies evolved over many years and many steps resulting in recombinant humanized or human-derived antibody derivatives in multiple formats. Today, hapten-binding antibodies are applied not only as reagents and tools (where they still play an important part) but evolved also to engineered targeting and pretargeting vehicles for disease diagnosis and therapy. Here we describe recent applications of hapten-binding antibodies and of engineered mono- and bispecific hapten-binding antibody derivatives. We have designed and applied these molecules for the modulation of the pharmacokinetic properties of small compounds or peptides. They are also integrated as additional binding entities into bispecific antibody formats. Here they serve as non-covalent or covalent coupling modules to haptenylated compounds, to enable targeted payload delivery to disease tissues or cells.

Highly flexible, IgG-shaped, trivalent antibodies effectively target tumor cells and induce T cell-mediated killing.

A novel bispecific antibody format was applied to generate T cell-engaging antibodies. The TriFab format is a trivalent IgG-shaped entity composed of two Fab arms that bind to antigens on the surface of tumor cells, which are linked via flexible peptides to a CD3 binding moiety that replaces the CH2 domains of conventional IgGs. The distinctive feature of these T cell recruiting bispecifics is that their CD3 variable regions are incorporated between domains, rather than N- or C-terminally fused to an Fc or antibody fragments. T cell recruiting TriFabs resemble in size and shape, are expressed and show biophysical properties similar to regular IgGs. Transmission electron microscopy (TEM) demonstrates high flexibility of the cell surface binding arms as well as target antigen accessibility of the interspersed CD3 binding domain. Functional co-culturing assays of peripheral blood mononuclear cells (PBMCs) and different tumor cell lines (MCF7 and A431) revealed a dose-dependent T cell-mediated cytotoxicity that was induced by the TriFabs targeting either LeY or EGFR cell surface antigens.

Interplay between reversible phosphorylation and irreversible ADP-ribosylation of eukaryotic translation elongation factor 2.

The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 'modifier region' triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.

Transcytosis of payloads that are non-covalently complexed to bispecific antibodies across the hCMEC/D3 blood-brain barrier model.

A transcellular shuttle system was generated for the delivery of non-covalently linked payloads across blood-brain barrier (BBB) endothelial cells. Transcytosis-enabling shuttles are composed of bispecific antibodies (bsAbs) that simultaneously bind transferrin receptor (TfR) and haptens such as digoxigenin or biocytinamide. Haptenylated payloads are attached to these vehicles via non-covalent hapten-antibody complexation. This enables targeting to and internalization into human BBB-derived microvascular endothelial hCMEC/D3 cells. In contrast to other shuttles, this system does not require special affinities or formats of their TfR-binding moieties for transcytosis and subsequent release. Non-covalent payload complexation to bsAb is flexible and robust, works for a multitude of payloads and enables separation of payloads from shuttles during transcytosis. Released payloads can enter the brain without connected bsAb entities, minimizing potential interference with distribution or functionality. Intracellular separation of shuttle and payload and recycling to cell surfaces may also enable recharging of the cell-bound BBB shuttle with payload for subsequent (merry-go-round) transport cycles.

Loss of diphthamide pre-activates NF-κB and death receptor pathways and renders MCF7 cells hypersensitive to tumor necrosis factor.

The diphthamide on human eukaryotic translation elongation factor 2 (eEF2) is the target of ADP ribosylating diphtheria toxin (DT) and Pseudomonas exotoxin A (PE). This modification is synthesized by seven dipthamide biosynthesis proteins (DPH1-DPH7) and is conserved among eukaryotes and archaea. We generated MCF7 breast cancer cell line-derived DPH gene knockout (ko) cells to assess the impact of complete or partial inactivation on diphthamide synthesis and toxin sensitivity, and to address the biological consequence of diphthamide deficiency. Cells with heterozygous gene inactivation still contained predominantly diphthamide-modified eEF2 and were as sensitive to PE and DT as parent cells. Thus, DPH gene copy number reduction does not affect overall diphthamide synthesis and toxin sensitivity. Complete inactivation of DPH1, DPH2, DPH4, and DPH5 generated viable cells without diphthamide. DPH1ko, DPH2ko, and DPH4ko harbored unmodified eEF2 and DPH5ko ACP- (diphthine-precursor) modified eEF2. Loss of diphthamide prevented ADP ribosylation of eEF2, rendered cells resistant to PE and DT, but does not affect sensitivity toward other protein synthesis inhibitors, such as saporin or cycloheximide. Surprisingly, cells without diphthamide (independent of which the DPH gene compromised) were presensitized toward nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) and death-receptor pathways without crossing lethal thresholds. In consequence, loss of diphthamide rendered cells hypersensitive toward TNF-mediated apoptosis. This finding suggests a role of diphthamide in modulating NF-κB, death receptor, or apoptosis pathways.

Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies.

Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a "universal" coupling position (52(+2)) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95-100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten-mediated positioning is necessary as hapten-thiol-payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide-linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharmacokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction-releasable manner to tumor- or tissue-targeting delivery vehicles.

A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties.

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the "knob-into-hole" technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.

TriFabs--Trivalent IgG-Shaped Bispecific Antibody Derivatives: Design, Generation, Characterization and Application for Targeted Payload Delivery.

TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with "knob"-mutations, and the variable region of the light chain (VL) fused to CH3 with matching "holes". The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent "in between" these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads.

Bispecific digoxigenin-binding antibodies for targeted payload delivery.

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.

Diphthamide - a conserved modification of eEF2 with clinical relevance.

Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases.

Functional Integrity of Radical SAM Enzyme Dph1•Dph2 Requires Non-Canonical Cofactor Motifs with Tandem Cysteines.

The Dph1•Dph2 heterodimer from yeast is a radical SAM (RS) enzyme that generates the 3-amino-3-carboxy-propyl (ACP) precursor for diphthamide, a clinically relevant modification on eukaryotic elongation factor 2 (eEF2). ACP formation requires SAM cleavage and atypical Cys-bound Fe-S clusters in each Dph1 and Dph2 subunit. Intriguingly, the first Cys residue in each motif is found next to another ill-defined cysteine that we show is conserved across eukaryotes. As judged from structural modeling, the orientation of these tandem cysteine motifs (TCMs) suggests a candidate Fe-S cluster ligand role. Hence, we generated, by site-directed DPH1 and DPH2 mutagenesis, Dph1•Dph2 variants with cysteines from each TCM replaced individually or in combination by serines. Assays diagnostic for diphthamide formation in vivo reveal that while single substitutions in the TCM of Dph2 cause mild defects, double mutations almost entirely inactivate the RS enzyme. Based on enhanced Dph1 and Dph2 subunit instability in response to cycloheximide chases, the variants with Cys substitutions in their cofactor motifs are particularly prone to protein degradation. In sum, we identify a fourth functionally cooperative Cys residue within the Fe-S motif of Dph2 and show that the Cys-based cofactor binding motifs in Dph1 and Dph2 are critical for the structural integrity of the dimeric RS enzyme in vivo.

Novel assay format for total anti-adeno-associated virus antibody detection with low capsid consumption and built-in specificity control.

Aim: To develop an assay format for detection of total anti-adeno-associated virus 2 (AAV2) antibodies with low capsid material consumption. Methods: An immune complex (IC) assay format was developed. The format is based on the formation of ICs in solution and their subsequent detection using an anti-AAV2 antibody for capture and an antibody against the study species IgG for detection. Results: The feasibility of the IC assay for detection of preexisting and treatment-emergent anti-AAV2 antibodies was demonstrated in cynomolgus monkey and human serum samples, including samples from a preclinical study with AAV2-based therapies. Conclusion: The presented IC assay is an easy-to-perform total anti-AAV2 antibody assay that requires a small amount of unlabeled capsid material and provides an intrinsic specificity control.

[Box: see text].

The present and future of bispecific antibodies for cancer therapy.

Bispecific antibodies (bsAbs) enable novel mechanisms of action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. Consequently, development of these molecules has garnered substantial interest in the past decade and, as of the end of 2023, 14 bsAbs have been approved: 11 for the treatment of cancer and 3 for non-oncology indications. bsAbs are available in different formats, address different targets and mediate anticancer function via different molecular mechanisms. Here, we provide an overview of recent developments in the field of bsAbs for cancer therapy. We focus on bsAbs that are approved or in clinical development, including bsAb-mediated dual modulators of signalling pathways, tumour-targeted receptor agonists, bsAb-drug conjugates, bispecific T cell, natural killer cell and innate immune cell engagers, and bispecific checkpoint inhibitors and co-stimulators. Finally, we provide an outlook into next-generation bsAbs in earlier stages of development, including trispecifics, bsAb prodrugs, bsAbs that induce degradation of tumour targets and bsAbs acting as cytokine mimetics.

Human-protein-derived peptides for intracellular delivery of biomolecules.

Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve CPPs (cell-penetrating peptides) or protein transduction domains. Most of these require rather high concentrations to elicit cell-penetrating functionality, are non-human, pathogen-derived or synthetic entities, and may therefore not be tolerated or even immunogenic. We identified novel human-protein-derived CPPs by a combination of in silico and experimental analyses: polycationic CPP candidates were identified in an in silico library of all 30-mer peptides of the human proteome. Of these peptides, 60 derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA (small interfering RNA) transfection assays revealed that 20 out of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human NRTN (neurturin), which contains an α-helix, performed the best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.