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1.
Bioconjug Chem ; 2024 May 29.
Article in English | MEDLINE | ID: mdl-38809610

ABSTRACT

Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.

2.
ACS Chem Biol ; 2024 May 11.
Article in English | MEDLINE | ID: mdl-38733564

ABSTRACT

The intracellular delivery of cargos via cell penetrating peptides (CPPs) holds significant promise as a drug delivery vehicle, but a major issue is their lack of cell type specificity, which can lead to detrimental off-target effects. We use an ADEPT-like concept to introduce conditional and selective activation of cellular uptake by using the lysine-rich, cationic, and amphiphilic L17E peptide as a model CPP. By masking the lysine residues of the L17E peptide with enzyme-cleavable acetyl protecting groups, the delivery of the covalently conjugated fluorophore TAMRA to HeLa cells was diminished. Recovery of cellular uptake could be achieved by deacetylation of the masked acetylated L17E peptide using the NAD-dependent sirtuin 2 (SirT2) deacetylase in vitro. Finally, trastuzumab-SirT2 and anti-B7H3-SirT2 antibody-enzyme conjugates were generated for the conditional and selective delivery of a cryptophycin cytotoxin by the L17E peptide. While the masked peptide still demonstrated some cytotoxicity, selective cell killing mediated by the antibody-enzyme conjugates was observed.

3.
Antibodies (Basel) ; 13(2)2024 May 02.
Article in English | MEDLINE | ID: mdl-38804305

ABSTRACT

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a VL domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells.

4.
Antibodies (Basel) ; 13(2)2024 May 02.
Article in English | MEDLINE | ID: mdl-38804304

ABSTRACT

The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.

5.
Molecules ; 29(5)2024 Mar 04.
Article in English | MEDLINE | ID: mdl-38474651

ABSTRACT

Valued for their ability to rapidly kill multiple tumor cells in succession as well as their favorable safety profile, NK cells are of increasing interest in the field of immunotherapy. As their cytotoxic activity is controlled by a complex network of activating and inhibiting receptors, they offer a wide range of possible antigens to modulate their function by antibodies. In this work, we utilized our established common light chain (cLC)-based yeast surface display (YSD) screening procedure to isolate novel B7-H3 and TIGIT binding monoclonal antibodies. The chicken-derived antibodies showed single- to low-double-digit nanomolar affinities and were combined with a previously published CD16-binding Fab in a 2+1 format to generate a potent NK engaging molecule. In a straightforward, easily adjustable apoptosis assay, the construct B7-H3xCD16xTIGIT showed potent apoptosis induction in cancer cells. These results showcase the potential of the TIGIT NK checkpoint in combination with activating receptors to achieve increased cytotoxic activity.


Subject(s)
Immunotherapy , Killer Cells, Natural , Antibodies, Monoclonal , Receptors, Immunologic , Apoptosis
6.
Front Immunol ; 15: 1323049, 2024.
Article in English | MEDLINE | ID: mdl-38455046

ABSTRACT

T cell engaging bispecific antibodies have shown clinical proof of concept for hematologic malignancies. Still, cytokine release syndrome, neurotoxicity, and on-target-off-tumor toxicity, especially in the solid tumor setting, represent major obstacles. Second generation TCEs have been described that decouple cytotoxicity from cytokine release by reducing the apparent binding affinity for CD3 and/or the TAA but the results of such engineering have generally led only to reduced maximum induction of cytokine release and often at the expense of maximum cytotoxicity. Using ROR1 as our model TAA and highly modular camelid nanobodies, we describe the engineering of a next generation decoupled TCE that incorporates a "cytokine window" defined as a dose range in which maximal killing is reached but cytokine release may be modulated from very low for safety to nearly that induced by first generation TCEs. This latter attribute supports pro-inflammatory anti-tumor activity including bystander killing and can potentially be used by clinicians to safely titrate patient dose to that which mediates maximum efficacy that is postulated as greater than that possible using standard second generation approaches. We used a combined method of optimizing TCE mediated synaptic distance and apparent affinity tuning of the TAA binding arms to generate a relatively long but persistent synapse that supports a wide cytokine window, potent killing and a reduced propensity towards immune exhaustion. Importantly, this next generation TCE induced significant tumor growth inhibition in vivo but unlike a first-generation non-decoupled benchmark TCE that induced lethal CRS, no signs of adverse events were observed.


Subject(s)
Antibodies, Bispecific , Neoplasms , Humans , T-Lymphocytes , Cytokines/metabolism , Receptor Tyrosine Kinase-like Orphan Receptors
7.
Biol Chem ; 2024 Feb 01.
Article in English | MEDLINE | ID: mdl-38297991

ABSTRACT

Antibody-based therapies are revolutionizing cancer treatment and experience a steady increase from preclinical and clinical pipelines to market share. While the clinical success of monoclonal antibodies is frequently limited by low response rates, treatment resistance and various other factors, multispecific antibodies open up new prospects by addressing tumor complexity as well as immune response actuation potently improving safety and efficacy. Novel antibody approaches involve simultaneous binding of two antigens on one cell implying increased specificity and reduced tumor escape for dual tumor-associated antigen targeting and enhanced and durable cytotoxic effects for dual immune cell-related antigen targeting. This article reviews antibody and cell-based therapeutics for oncology with intrinsic dual targeting of either tumor cells or immune cells. As revealed in various preclinical studies and clinical trials, dual targeting molecules are promising candidates constituting the next generation of antibody drugs for fighting cancer.

8.
Biol Chem ; 2024 Feb 20.
Article in English | MEDLINE | ID: mdl-38373142

ABSTRACT

In this work we have generated cattle-derived chimeric ultralong CDR-H3 antibodies targeting tumor necrosis factor α (TNF-α) via immunization and yeast surface display. We identified one particular ultralong CDR-H3 paratope that potently neutralized TNF-α. Interestingly, grafting of the knob architecture onto a peripheral loop of the CH3 domain of the Fc part of an IgG1 resulted in the generation of a TNF-α neutralizing Fc (Fcknob) that did not show any potency loss compared with the parental chimeric IgG format. Eventually, grafting this knob onto the CH3 region of adalimumab enabled the engineering of a novel TNF-α targeting antibody architecture displaying augmented TNF-α inhibition.

9.
Mater Today Bio ; 24: 100897, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38169974

ABSTRACT

In vitro cellular models denote a crucial part of drug discovery programs as they aid in identifying successful drug candidates based on their initial efficacy and potency. While tremendous headway has been achieved in improving 2D and 3D culture techniques, there is still a need for physiologically relevant systems that can mimic or alter cellular responses without the addition of external biochemical stimuli. A way forward to alter cellular responses is using physical cues, like 3D topographical inorganic substrates, to differentiate macrophage-like cells. Herein, protein secretion and gene expression markers for various macrophage subsets cultivated on a 3D topographical substrate are investigated. The results show that macrophages differentiate into anti-inflammatory M2-type macrophages, secreting increased IL-10 levels compared to the controls. Remarkably, these macrophage cells are differentiated into the M2d subset, making up the main component of tumour-associated macrophages (TAMs), as measured by upregulated Il-10 and Vegf mRNA. M2d subset differentiation is attributed to the topographical substrates with 3D fractal-like geometries arrayed over the surface, else primarily achieved by tumour-associated factors in vivo. From a broad perspective, this work paves the way for implementing 3D topographical inorganic surfaces for drug discovery programs, harnessing the advantages of in vitro assays without external stimulation and allowing the rapid characterisation of therapeutic modalities in physiologically relevant environments.

10.
Nat Chem ; 16(4): 564-574, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38049652

ABSTRACT

Artificial cells are biomimetic microstructures that mimic functions of natural cells, can be applied as building blocks for molecular systems engineering, and host synthetic biology pathways. Here we report enzymatically synthesized polymer-based artificial cells with the ability to express proteins. Artificial cells were synthesized using biocatalytic atom transfer radical polymerization-induced self-assembly, in which myoglobin synthesizes amphiphilic block co-polymers that self-assemble into structures such as micelles, worm-like micelles, polymersomes and giant unilamellar vesicles (GUVs). The GUVs encapsulate cargo during the polymerization, including enzymes, nanoparticles, microparticles, plasmids and cell lysate. The resulting artificial cells act as microreactors for enzymatic reactions and for osteoblast-inspired biomineralization. Moreover, they can express proteins such as a fluorescent protein and actin when fed with amino acids. Actin polymerizes in the vesicles and alters the artificial cells' internal structure by creating internal compartments. Thus, biocatalytic atom transfer radical polymerization-induced self-assembly-derived GUVs can mimic bacteria as they are composed of a microscopic reaction compartment that contains genetic information for protein expression upon induction.


Subject(s)
Artificial Cells , Polymerization , Micelles , Actins , Polymers/chemistry , Unilamellar Liposomes/chemistry
11.
Nanoscale ; 15(48): 19486-19492, 2023 Dec 14.
Article in English | MEDLINE | ID: mdl-38051112

ABSTRACT

Through the innovative use of surface-displayed horseradish peroxidase, this work explores the enzymatic catalysis of both bioRAFT polymerization and bioATRP to prompt polymer synthesis on the surface of Saccharomyces cerevisiae cells, with bioATRP outperforming bioRAFT polymerization. The resulting surface modification of living yeast cells with synthetic polymers allows for a significant change in yeast phenotype, including growth profile, aggregation characteristics, and conjugation of non-native enzymes to the clickable polymers on the cell surface, opening new avenues in bioorthogonal cell-surface engineering.


Subject(s)
Polymers , Saccharomyces cerevisiae , Polymerization , Saccharomyces cerevisiae/metabolism , Catalysis , Horseradish Peroxidase/metabolism
12.
Front Immunol ; 14: 1238313, 2023.
Article in English | MEDLINE | ID: mdl-37942319

ABSTRACT

In this work we present a novel symmetric bispecific antibody format based on engraftments of cattle-derived knob paratopes onto peripheral loops of the IgG1 Fc region. For this, knob architectures obtained from bovine ultralong CDR-H3 antibodies were inserted into the AB loop or EF loop of the CH3 domain, enabling the introduction of an artificial binding specificity into an IgG molecule. We demonstrate that inserted knob domains largely retain their binding affinities, resulting into bispecific antibody derivatives versatile for effector cell redirection. Essentially, generated bispecifics demonstrated adequate biophysical properties and were not compromised in their Fc mediated functionalities such as FcRn or FcγRIIIa binding.


Subject(s)
Antibodies, Bispecific , Immunoglobulin G , Cattle , Animals , Binding Sites, Antibody
13.
Protein Eng Des Sel ; 362023 Jan 21.
Article in English | MEDLINE | ID: mdl-37903068

ABSTRACT

Most proteins are flexible molecules that coexist in an ensemble of several conformations. Point mutations in the amino acid sequence of a protein can trigger structural changes that drive the protein population to a conformation distinct from the native state. Here, we report a protein engineering approach to better understand protein dynamics and ligand binding of the FK506-binding protein 51 (FKBP51), a prospective target for stress-related diseases, metabolic disorders, some types of cancers and chronic pain. By randomizing selected regions of its ligand-binding domain and sorting yeast display libraries expressing these variants, mutants with high affinity to conformation-specific FKBP51 selective ligands were identified. These improved mutants are valuable tools for the discovery of novel selective ligands that preferentially and specifically bind the FKBP51 active site in its open conformation state. Moreover, they will help us understand the conformational dynamics and ligand binding mechanics of the FKBP51 binding pocket.


Subject(s)
Protein Engineering , Tacrolimus Binding Proteins , Tacrolimus Binding Proteins/chemistry , Ligands , Amino Acid Sequence , Catalytic Domain , Protein Conformation , Protein Binding
14.
Front Immunol ; 14: 1258700, 2023.
Article in English | MEDLINE | ID: mdl-37841262

ABSTRACT

Cancerous B cells are almost indistinguishable from their non-malignant counterparts regarding their surface antigen expression. Accordingly, the challenge to be faced consists in elimination of the malignant B cell population while maintaining a functional adaptive immune system. Here, we present an IgM-specific antibody-drug conjugate masked by fusion of the epitope-bearing IgM constant domain. Antibody masking impaired interaction with soluble pentameric as well as cell surface-expressed IgM molecules rendering the antibody cytotoxically inactive. Binding capacity of the anti-IgM antibody drug conjugate was restored upon conditional protease-mediated demasking which consequently enabled target-dependent antibody internalization and subsequent induction of apoptosis in malignant B cells. This easily adaptable approach potentially provides a novel mechanism of clonal B cell lymphoma eradication to the arsenal available for non-Hodgkin's lymphoma treatment.


Subject(s)
Immunoconjugates , Lymphoma, B-Cell , Lymphoma, Non-Hodgkin , Humans , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Lymphoma, B-Cell/drug therapy , Immunoglobulin M
15.
Pharmaceutics ; 15(10)2023 Sep 22.
Article in English | MEDLINE | ID: mdl-37896133

ABSTRACT

Here we report the generation of nanobody dextran polymer conjugates (dextraknobs) that are loaded with small molecules, i.e., fluorophores or photosensitizers, for potential applications in cancer diagnostics and therapy. To this end, the molecules are conjugated to the dextran polymer which is coupled to the C-terminus of an EGFR-specific nanobody using chemoenzymatic approaches. A monovalent EGFR-targeted nanobody and biparatopic version modified with different dextran average molecular weights (1000, 5000, and 10,000) were probed for their ability to penetrate tumor spheroids. For monovalent Cy5-labeled dextraknobs, the utilization of smaller sized dextran (MW 5000 vs. 10,000) was found to be beneficial for more homogeneous penetration into A431 tumor spheroids over time. For the biparatopic dual nanobody comprising MW 1000, 5000, and 10,000 dextran labeled with photosensitizer IRDye700DX, penetration behavior was comparable to that of a direct nanobody-photosensitizer conjugate lacking a dextran scaffold. Additionally, dextraknobs labeled with IRDye700DX incubated with cells in 2D and 3D showed potent cell killing upon illumination, thus inducing photodynamic therapy (PDT). In line with previous results, monovalent nanobody conjugates displayed deeper and more homogenous penetration through spheroids than the bivalent conjugates. Importantly, the smaller size dextrans did not affect the distribution of the conjugates, thus encouraging further development of dextraknobs.

16.
Methods Mol Biol ; 2702: 227-243, 2023.
Article in English | MEDLINE | ID: mdl-37679622

ABSTRACT

The adaptive immune system of sharks comprises a unique heavy chain-only antibody isotype, termed immunoglobulin new antigen receptor (IgNAR), in which antigen binding is mediated by a single variable domain, referred to as vNAR. In recent years, efforts were made to harness these domains for biomedical and biotechnological applications particularly due to their high affinity and specificity combined with a small size and high stability. Herein, we describe protocols for the construction of semisynthetic, CDR3-randomized vNAR libraries for the isolation of target-specific paratopes by yeast surface display. Additionally, we provide guidance for affinity maturation of a panel of antigen-enriched vNAR domains through CDR1 diversification of the FACS-selected, antigen-enriched population and sublibrary establishment.


Subject(s)
Antibodies , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Receptors, Antigen, B-Cell , Antibodies, Fungal , Immunoglobulin Isotypes , Immunoglobulin Heavy Chains
17.
J Biol Chem ; 299(11): 105270, 2023 11.
Article in English | MEDLINE | ID: mdl-37734558

ABSTRACT

Synthetic cytokine receptors can modulate cellular functions based on an artificial ligand to avoid off-target and/or unspecific effects. However, ligands that can modulate receptor activity so far have not been used clinically because of unknown toxicity and immunity against the ligands. Here, we developed a fully synthetic cytokine/cytokine receptor pair based on the antigen-binding domain of the respiratory syncytial virus-approved mAb Palivizumab as a synthetic cytokine and a set of anti-idiotype nanobodies (AIPVHH) as synthetic receptors. Importantly, Palivizumab is neither cross-reactive with human proteins nor immunogenic. For the synthetic receptors, AIPVHH were fused to the activating interleukin-6 cytokine receptor gp130 and the apoptosis-inducing receptor Fas. We found that the synthetic cytokine receptor AIPVHHgp130 was efficiently activated by dimeric Palivizumab single-chain variable fragments. In summary, we created an in vitro nonimmunogenic full-synthetic cytokine/cytokine receptor pair as a proof of concept for future in vivo therapeutic strategies utilizing nonphysiological targets during immunotherapy.


Subject(s)
Receptors, Artificial , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Humans , Palivizumab/pharmacology , Palivizumab/therapeutic use , Receptors, Artificial/metabolism , Receptors, Artificial/therapeutic use , Receptors, Cytokine , Cytokines , Respiratory Syncytial Virus Infections/drug therapy , Ligands , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use
18.
MAbs ; 15(1): 2236265, 2023.
Article in English | MEDLINE | ID: mdl-37469014

ABSTRACT

Here, we generated bispecific antibody (bsAb) derivatives that mimic the function of interleukin (IL)-18 based on single domain antibodies (sdAbs) specific to IL-18 Rα and IL-18 Rß. For this, camelids were immunized, followed by yeast surface display (YSD)-enabled discovery of VHHs targeting the individual receptor subunits. Upon reformatting into a strictly monovalent (1 + 1) bispecific sdAb architecture, several bsAbs triggered dose-dependent IL-18 R downstream signaling on IL-18 reporter cells, as well as IFN-γ release by peripheral blood mononuclear cells in the presence of low-dose IL-12. However, compared with IL-18, potencies and efficacies were considerably attenuated. By engineering paratope valencies and the spatial orientation of individual paratopes within the overall design architecture, we were able to generate IL-18 mimetics displaying significantly augmented functionalities, resulting in bispecific cytokine mimetics that were more potent than IL-18 in triggering proinflammatory cytokine release. Furthermore, generated IL-18 mimetics were unaffected from inhibition by IL-18 binding protein decoy receptor. Essentially, we demonstrate that this strategy enables the generation of IL-18 mimetics with tailor-made cytokine functionalities.


Subject(s)
Antibodies, Bispecific , Single-Domain Antibodies , Interleukin-18 , Leukocytes, Mononuclear , Binding Sites, Antibody
19.
Methods Mol Biol ; 2681: 113-129, 2023.
Article in English | MEDLINE | ID: mdl-37405646

ABSTRACT

Cattle are known for their repertoire of antibodies harboring extremely long CDR3H regions that form extensive "knob on stalk" cysteine-rich structures. The compact knob domain allows for the recognition of epitopes potentially not accessible to classical antibodies. To effectively access the potential of bovine-derived antigen-specific ultra-long CDR3 antibodies, a straightforward and effective high-throughput method based on yeast surface display and fluorescence-activated cell sorting is described.


Subject(s)
Antibodies , Saccharomyces cerevisiae , Cattle , Animals , Saccharomyces cerevisiae/genetics , Antigens , Immunization , Vaccination
20.
Methods Mol Biol ; 2681: 213-229, 2023.
Article in English | MEDLINE | ID: mdl-37405650

ABSTRACT

Classical yeast surface display (YSD) antibody immune libraries are generated by a separate amplification of heavy- and light-chain antibody variable regions (VH and VL, respectively) and subsequent random recombination during the molecular cloning procedure. However, each B cell receptor comprises a unique VH-VL combination, which has been selected and affinity matured in vivo for optimal stability and antigen binding. Thus, the native variable chain pairing is important for the functioning and biophysical properties of the respective antibody. Herein, we present a method for the amplification of cognate VH-VL sequences, compatible with both next-generation sequencing (NGS) and YSD library cloning. We employ a single B cell encapsulation in water-in-oil droplets, followed by a one-pot reverse transcription overlap extension PCR (RT-OE-PCR), resulting in a paired VH-VL repertoire from more than a million B cells in a single day.


Subject(s)
Antibodies , Reverse Transcription , Polymerase Chain Reaction , Gene Library , Cloning, Molecular
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