ORCID Profile
0000-0003-4210-9613
Current Organisation
Garvan Institute of Medical Research
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Medical Biotechnology | Medical Molecular Engineering of Nucleic Acids and Proteins
Publisher: Frontiers Media SA
Date: 02-02-2018
Publisher: Elsevier
Date: 2011
Publisher: Proceedings of the National Academy of Sciences
Date: 12-05-2014
Abstract: Antibodies are selected to bind microbial but not self-antigens, because binding to self would compete with binding microbes, shorten antibody half-life, and cause autoimmunity. Self-tolerance is actively acquired in part by discarding self-binding antibodies before the body is exposed to a microbe or vaccine. The experiments here provide evidence of an opposite mechanism, allowing antibodies that initially bind both foreign and self-antigens to acquire self/non-self discrimination during the course of an immune response through somatic hypermutation away from self-reactivity. In addition to selection for lower-affinity binding to self, antibody variants were selected with fewer binding sites available to bind self-antigen because most were occupied by N-linked carbohydrate, possibly explaining the frequent occurrence of N-linked glycosylation of antibody variable domains.
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-974-7_10
Abstract: Human antibody single domains are a promising new class of antibody fragments. Here we describe methods for the cloning of human V(H) and V(L) genes into phage and phagemid vectors. Furthermore, we provide detailed protocols for the generation of single domain antibody libraries by Kunkel mutagenesis and the analysis of ersity by DNA sequencing and superantigen binding.
Publisher: Cold Spring Harbor Laboratory
Date: 20-10-2022
DOI: 10.1101/2022.10.19.512954
Abstract: Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS-CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the ergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14) or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.
Publisher: Springer Science and Business Media LLC
Date: 08-12-2017
DOI: 10.1038/S41467-017-01836-2
Abstract: Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus . We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species.
Publisher: Springer Science and Business Media LLC
Date: 02-2012
Abstract: Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.
Publisher: American Chemical Society (ACS)
Date: 19-02-2019
DOI: 10.1021/ACSCHEMBIO.9B00116
Abstract: We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the ribonucleoprotein into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 endosomolytic peptide and outline the development of novel Cas9 or Cas12a ribonucleoprotein complexes with increased editing efficiency.
Publisher: Elsevier BV
Date: 03-2017
DOI: 10.1016/J.JMB.2017.02.003
Abstract: Merozoite surface protein 2 (MSP2) is an intrinsically disordered antigen that is abundant on the surface of the malaria parasite Plasmodium falciparum. The two allelic families of MSP2, 3D7 and FC27, differ in their central variable regions, which are flanked by highly conserved C-terminal and N-terminal regions. In a vaccine trial, full-length 3D7 MSP2 induced a strain-specific protective immune response despite the detectable presence of conserved region antibodies. This work focuses on the conserved C-terminal region of MSP2, which includes the only disulphide bond in the protein and encompasses key epitopes recognised by the mouse monoclonal antibodies 4D11 and 9H4. Although the 4D11 and 9H4 epitopes are overlapping, immunofluorescence assays have shown that the mouse monoclonal antibody 4D11 binds to MSP2 on the merozoite surface with a much stronger signal than 9H4. Understanding the structural basis for this antigenic difference between these antibodies will help direct the design of a broad-spectrum and MSP2-based malaria vaccine. 4D11 and 9H4 were reengineered into antibody fragments [variable region fragment (Fv) and single-chain Fv (scFv)] and were validated as suitable models for their full-sized IgG counterparts by surface plasmon resonance and isothermal titration calorimetry. An alanine scan of the 13-residue epitope 3D7-MSP2
Publisher: Oxford University Press (OUP)
Date: 2022
Abstract: The CRISPR genome editing technology holds great clinical potential for the treatment of monogenetic disorders such as sickle cell disease. The therapeutic in vivo application of the technology relies on targeted delivery methods of the Cas9 and gRNA complex to specific cells or tissues. However, such methods are currently limited to direct organ delivery, preventing clinical application. Here, we show that monoclonal antibodies can be employed to deliver the Cas9/gRNA complex directly into human cells via cell-surface receptors. Using the SpyCatcher/SpyTag system, we conjugated the Fab fragment of the therapeutic antibodies Trastuzumab and Pertuzumab directly to the Cas9 enzyme and observed HER2-specific uptake of the ribonucleoprotein in a human HER2 expressing cell line. Following cellular uptake in the presence of an endosomolytic peptide, modest gene editing was also observed. This finding provides a blueprint for the targeted delivery of the CRISPR technology into specific cells using monoclonal antibodies.
Publisher: Elsevier BV
Date: 11-2018
Publisher: Springer New York
Date: 2018
DOI: 10.1007/978-1-4939-8648-4_16
Abstract: The therapeutic development of monoclonal antibodies requires robust and reliable methods for their recombinant expression and characterization. In this context, an increasingly important aspect in the antibody development process is to determine the contribution of Fc-mediated immune effector functions to therapeutic activity. Here we describe steps for the cloning and mammalian expression of mouse and human IgG monoclonals with reduced immune effector functions, based on mutation of Fc-gamma receptor and complement-binding sites. The resulting antibody preparations contain low levels of endotoxin and are suitable for testing in animal models of disease.
Publisher: Springer Science and Business Media LLC
Date: 10-08-2022
DOI: 10.1038/S41586-022-05054-9
Abstract: The notion that mobile units of nucleic acid known as transposable elements can operate as genomic controlling elements was put forward over six decades ago
Publisher: American Association for the Advancement of Science (AAAS)
Date: 17-09-2021
Abstract: Prolactin-humanized mice promote growth and metastatic progression of estrogen-dependent patient-derived breast cancer.
Publisher: Humana Press
Date: 2012
DOI: 10.1007/978-1-61779-968-6_23
Abstract: Human antibody variable heavy (VH) domains tend to display poor biophysical properties when expressed in isolation. Consequently, the domains are often characterized by low expression levels, high levels of aggregation, and increased "stickiness." Here, we describe methods that allow the engineering of human VH domains with improved biophysical properties by phage display. The engineered domains withstand challenging conditions, such as high temperature and acidic pH. Engineered human single domains are a promising new class of antibody fragments and represent robust research tools and building blocks for the generation of antibody therapeutics.
Publisher: Springer Science and Business Media LLC
Date: 23-04-2018
DOI: 10.1038/S41557-018-0046-3
Abstract: Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.
Publisher: Springer Science and Business Media LLC
Date: 12-05-2015
DOI: 10.1038/SREP10103
Abstract: Merozoite surface protein 2 (MSP2) is an intrinsically disordered, membrane-anchored antigen of the malaria parasite Plasmodium falciparum . MSP2 can elicit a protective, albeit strain-specific, antibody response in humans. Antibodies are generated to the conserved N- and C-terminal regions but many of these react poorly with the native antigen on the parasite surface. Here we demonstrate that recognition of a conserved N-terminal epitope by mAb 6D8 is incompatible with the membrane-bound conformation of that region, suggesting a mechanism by which native MSP2 escapes antibody recognition. Furthermore, crystal structures and NMR spectroscopy identify transient, strain-specific interactions between the 6D8 antibody and regions of MSP2 beyond the conserved epitope. These interactions account for the differential affinity of 6D8 for the two allelic families of MSP2, even though 6D8 binds to a fully conserved epitope. These results highlight unappreciated mechanisms that may modulate the specificity and efficacy of immune responses towards disordered antigens.
Publisher: Elsevier BV
Date: 05-2015
Publisher: Oxford University Press (OUP)
Date: 22-07-2014
Abstract: Protein aggregation hinders the development of biologics and underpins the molecular basis of many human diseases. Considerable variation of aggregation propensity exists not only between different proteins, but also within a single homologous family, which complicates analyses. A classic ex le is observed among human antibody light chains, which aggregate in a clonally specific manner, driven by sequence ersity within their variable domains. Here, we utilise a library versus library strategy, based on phage display and a chemical library of FDA approved drugs, to overcome this limitation. Our approach allowed the identification of small molecule drugs that inhibit the aggregation of the human light chain repertoire. It also provides a general template for the small molecule targeting of erse protein families.
Publisher: Proceedings of the National Academy of Sciences
Date: 27-06-2012
Abstract: The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V H and V L ) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence ersity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30–33, 35 in V H and 24, 49–53, 56 in V L ). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V H 3 and V κ 1). The effects of the mutations were highly positional and independent of sequence ersity at other positions. Moreover, crystal structures of mutant V H and V L domains revealed a surprising degree of structural conservation, indicating compatibility with V H /V L pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.
Publisher: Springer Science and Business Media LLC
Date: 12-08-2019
DOI: 10.1038/S41467-019-10968-6
Abstract: Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.
Publisher: American Chemical Society (ACS)
Date: 22-03-2019
DOI: 10.1021/ACS.BIOCHEM.9B00037
Abstract: We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.
Publisher: Oxford University Press (OUP)
Date: 19-05-2013
Abstract: Aggregation limits the recombinant production of many commercially important proteins. We have recently identified mutations that control the aggregation behavior of human antibody variable domains (Dudgeon K., Rouet R., Kokmeijer I., Schofield P., Stolp J., Langley D., Stock D. and Christ D. (2012) Proc Natl Acad Sci USA, 109, 10879-10884. This has allowed the generation of a panel of human antibody variable heavy domains with a defined range of aggregation propensities. Here we utilize this unique resource to validate a previously reported heat-denaturation method on phage (Jespers L., Schon O., Famm K. and Winter G. (2004) Nat Biotechnol, 22, 1161-1165. Our experiments revealed that the method is not only robust in respect to denaturation conditions on phage, but also highly indicative of solution behavior. In particular, it is an excellent predictor of expression and refolding yields.
Publisher: Wiley
Date: 28-11-2013
DOI: 10.1016/J.FEBSLET.2013.11.029
Abstract: Human monoclonal antibodies often display limited thermodynamic and colloidal stabilities. This behavior hinders their production, and places limitations on the development of novel formulation conditions and therapeutic applications. Antibodies are highly erse molecules, with much of the sequence variation observed within variable domain families and, in particular, their complementarity determining regions. This has complicated the development of comprehensive strategies for the stability engineering of the human antibody repertoire. Here we provide an overview of the field, and discuss recent advances in the development of robust and aggregation resistant antibody therapeutics.
Publisher: Springer Science and Business Media LLC
Date: 02-2014
DOI: 10.1038/NBT.2812
Publisher: Proceedings of the National Academy of Sciences
Date: 25-09-2019
Abstract: The adaptive immune system forms our primary defense against bacteria and viruses. Key players of this system are antigen receptors, dimeric molecules formed by two different types of immunoglobulin domains. It is generally believed that these receptors evolved from an ancestral dimer formed by only a single type of immunoglobulin. Using laboratory evolution, we have recreated such homodimeric receptors and characterized their interactions by X-ray crystallography. Our findings provide molecular insights and support of long-held theories concerning the evolution of the adaptive immune system. They also provide a blueprint for the experimental reconstruction of ancestral proteins in the large number of cases in which evolution has obscured sequence similarities beyond recognition, and which cannot be analyzed using current sequence-based approaches.
Publisher: American Chemical Society (ACS)
Date: 18-04-2018
DOI: 10.1021/JACS.8B01551
Start Date: 04-2019
End Date: 01-2022
Amount: $409,574.00
Funder: Australian Research Council
View Funded Activity