title: Arming Antibodies for Cancer Therapy: Transglutaminase-Mediated Toxin Conjugation
Creators: name: Deweid, Ludwig Lukas
Publisher: name: UNSPECIFIED
Year: 2020

description: 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.