Engineering substrates of transglutaminase using the Glutamine-Walk Strategy for specific modification of IgG1 antibodies

Please click Additional Files below to see the full abstract

Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B- cell lymphomas, the tumor cells express a tumor-speci fi c and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-speci fi c binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identi fi ed, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fl uorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed speci fi c binding to the parental SUP-B8 cell line con fi rming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a signi fi cant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable speci fi c killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy

Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy

Arming Antibodies for Cancer Therapy: Transglutaminase-Mediated Toxin Conjugation

The growing number of malignant cancer cases worldwide is an enormous threat for modern society. Therefore, the efforts of scientists from various research fields are focused on the development of efficient and viable therapeutic approaches. Recently, site-specific conjugation of highly potent organic compounds to tumour-binding immunoglobulins towards antibody-drug conjugates (ADCs) emerged as a promising strategy for the treatment of cancer. To allow for the precise modification of antibodies, multiple chemical and enzymatic methodologies have been developed within the last decades. Among them, application of microbial transglutaminase (mTG) from Streptomyces mobaraensis represents a promising approach. This enzyme that natively catalyses formation of isopeptide bonds between glutamine and lysine side chains, has been recently used for the assembly of ADCs. Although native residues within human antibodies cannot be employed in mTG catalysis, incorporation of reactive peptidyl linker sequences or truncation of the CH2 glycan to expose Gln295 were shown to overcome this limitation. In the present work, we focused on improving the performance of mTG catalysis towards efficient and rapid generation of ADCs by engineering both the enzyme and the targeted immunoglobulin. To that end, three independent studies were conducted: (1) Microbial transglutaminase is a useful tool for the crosslinking of diverse molecules due to its ability to form stable isopeptide bonds between proteinaceous glutamine residues and primary amines. However, tailoring of transglutaminase is desired because the substrate indiscrimination of mTG can lead to heterogeneous product formation. In this part of my thesis, we aimed at the development of a molecular platform for the engineering of transglutaminase towards altered activity and selectivity by yeast surface display (YSD) in combination with fluorescence-activated cell sorting (FACS). Due to its intracellular cytotoxicity, mTG was displayed on the surface of Saccharomyces cerevisiae cells as an inactive zymogen. The precursor was converted into the mature enzyme by the addition of different proteases and a biotinylated glutamine-donor peptide LLQG was added. The surface-anchored enzyme catalyses the respective transamidation of the supplemented substrate at available lysine residues, thus enabling the identification of active variants. We demonstrated that genotype-phenotype correlation is provided and constructed a randomised library comprising 3×108 different mutants. After five rounds of FACS-screening against biotin-GSGLLQG, 150 individual clones were analysed on the surface of yeast cells and compared to the wildtype enzyme. Upon recombinant expression, six mutants revealed improved consumption of the corresponding peptide in an HPLC-controlled assay and a triple mutant R5.2 (S2G/R15C/M234L) labelled LLQG-tagged therapeutic antibody trastuzumab more efficiently compared to the wildtype counterpart. Site-specific conjugation mediated by mutant transglutaminases with elevated catalytic performance might improve coupling efficiency and increase yields upon the construction of antibody-drug conjugates. (2) In addition to enzyme optimization, improvement of catalytic efficiency can be achieved upon engineering of the genetically incorporated mTG recognition sequence. The reactivity of the enzyme is influenced by charge and polarity of the amino acids surrounding the addressed glutamine as well as by the spatial arrangement of the targeted protein. In previous work, Siegmund et al. demonstrated that mimicking glutamine-donor sites of natural mTG substrates is a fruitful approach to yield peptidyl-linker sequences, which mediate proper modification by the enzyme. We aimed at the identification of novel recognition sequences derived from intrinsic mTG substrates dispase-autolysis inducing protein (DAIP) and Streptomyces papain inhibitor (SPIP) towards efficient modification of therapeutic antibodies. To that end, sequences derived from Gln6 of SPIP as well as Gln39 and Gln298 of DAIP were synthesized as oligopeptides by microwave-assisted Fmoc solid-phase peptide synthesis (SPPS) and their reactivity in mTG-mediated conjugation of different linker substrates was determined. Upon C-terminal fusion of the respective sequences to the heavy chain of HER2-targeting antibody trastuzumab, mTG-promoted modification of the resulting constructs with model substrate N-(biotinyl)cadaverine (MBC) was investigated. Having demonstrated high reactivity in preliminary experiments, SPIP-derived sequence DIPIGQGMTG (SPI7G) was chosen for the assembly of an ADC in a twostep chemo-enzymatic approach. Engineered antibodies were labelled with N-[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl]-1,8-diamino-3,6-dioxaoctane (NH2-PEG2-BCN) by mTG within a drastically shortened reaction time. Subsequently, BCN-modified monoclonal antibodies (mAb) were armed with the cytotoxic agent monomethyl auristatin E (MMAE) for selective growth inhibition of HER2-overexpressing breast cancer cells by strain-promoted alkyne-azide cycloaddition (SPAAC). Due to the superior labelling efficiency, an ADC assembled from the newly identified sequence SPI7G was significantly more potent on HER2-positive SKBR3-cells compared to a conventional LLQG-motif. We were able to show that intrinsic mTG substrates can be rapidly harnessed as a source of potent bioconjugation tags. Within a short reaction time of 1 h, a novel sequence SPI7G outperformed the conventional tag LLQG when located at the C-terminus of the heavy chain of trastuzumab. Such a short reaction time minimizes the risk of yield-limiting protein denaturation that often occurs during commonly applied overnight reactions. (3) Incorporation of specific mTG recognition tags into the framework of antibodies proved to be a powerful approach for their labelling. However, longer sequences may interfere with the antibody’s intrinsic stability and trigger the patient’s immune response during treatment. We investigated different positions of a human IgG Fc (fragment crystallisable) to identify sites that allow for mTG-mediated labelling upon single residue substitution to reduce undesired immunogenicity and instability risks. In a first step, a straightforward strategy for the recombinant expression of the Fc in E. coli cells was established. The fragment was purified by Ni2+-affinity chromatography in sufficient yields and analysed by non-reducing SDS-PAGE, thermal shift assays and liquid chromatography-mass spectrometry (LC-MS) regarding dimeric assembly and native disulfide formation. Site-directed mutagenesis was used to specifically introduce glutamine substitutions within the Fc and the mutants were screened for labelling by mTG in a rapid fluorescent assay. Out of 30 analysed positions, two mutations, namely I253Q and Y296Q were efficiently labelled with a fluorescent amine substrate by mTG. These promising results need to be further confirmed in the context of a full-length antibody to study their usefulness for the mTG-mediated construction of ADCs

Dextramabs: A Novel Format of Antibody‐Drug Conjugates Featuring a Multivalent Polysaccharide Scaffold

Abstract Antibody‐drug conjugates (ADCs) are multicomponent biomolecules that have emerged as a powerful tool for targeted tumor therapy. Combining specific binding of an immunoglobulin with toxic properties of a payload, they however often suffer from poor hydrophilicity when loaded with a high amount of toxins. To address these issues simultaneously, we developed dextramabs, a novel class of hybrid antibody‐drug conjugates. In these architectures, the therapeutic antibody trastuzumab is equipped with a multivalent dextran polysaccharide that enables efficient loading with a potent toxin in a controllable fashion. Our modular chemoenzymatic approach provides an access to synthetic dextramabs bearing monomethyl auristatin as releasable cytotoxic cargo. They possess high drug‐to‐antibody ratios, remarkable hydrophilicity, and high toxicity in vitro

Streamlining the Transition From Yeast Surface Display of Antibody Fragment Immune Libraries to the Production as IgG Format in Mammalian Cells

Yeast-surface display (YSD) is commonly applied to screen Fab immune or naïve libraries for binders of predefined target molecules. However, reformatting of isolated variants represents a time-intensive bottleneck. Herein, we present a novel approach to facilitate a lean transition from antibody screening using YSD Fab libraries to the production of full-length IgG antibodies in Expi293-F cells. In this study, utilizing Golden Gate Cloning (GGC) and a bidirectional promoter system, an exemplary Fab-displaying YSD library was generated based on immunised transgene rats. After subsequent screening for antigen-specific antibody candidates by fluorescence-activated cell sorting (FACS), the Fab-encoding genes were subcloned into a bidirectional mammalian expression vector, exhibiting CH2-CH3 encoding genes, in a GGC-mediated, PCR-free manner. This novel, straightforward and time-saving workflow allows the VH/VL pairing to be preserved. This study resulted in antibody variants exhibiting suitable biophysical properties and covered a broad VH diversity after two rounds of FACS screening, as revealed by NGS analysis. Ultimately, we demonstrate that the implication of such a gene transfer system streamlines antibody hit discovery efforts, allowing the faster characterisation of antibodies against a plethora of targets that may lead to new therapeutic agents

Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.ISSN:2399-364

A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein-Protein Conjugates

A highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels-Alder cycloaddition with inverse electron demand (DAinv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines