Periodic Reporting for period 4 - BioNanoPattern (Protein nano-patterning using DNA nanotechnology; control of surface-based immune system activation)
Période du rapport: 2022-07-01 au 2023-12-31
Protein nanopatterning concerns the geometric arrangement of individual proteins with nanometre accuracy. Protein nanopatterns are essential for cellular function, and our research has revealed that our immune system is activated by nanopatterned antibody platforms, which initiate the classical Complement pathway by binding to the first component of Complement, the C1 complex.
Control over these nanopatterns will allow us to understand the geometric parameters of immune system activation, modulation and inhibition. Furthermore, control of the nanopatterns will also enable control over the immune system itself.
To gain control, DNA nanotechnology can be used to form self-assembled nanoscale structures, which are ideal for use as templates to pattern proteins with specific geometries and nanometre accuracy. This project uses DNA to nanopattern antigens and other biomolecular moieties with defined geometry to study and control Complement pathway activation by the C1 complex.
The first aim of this proposal aimed to understand the role of nanopatterns in complement activation. We developed DNA nanostructures able to nanopattern antibodies, to which the C1 complex bound with the specific geometry. This allowed us to explore the relationship between geometry and Complement activation, which manifested as a relationship between antibody valency and C4b deposition. C4b is the product of the C1 enzyme, and we therefore discovered how antibody valency can modulate C1 activity. This relationship extended to membrane lysis – the name number of antigens nanopatterned as a hexagon was most potent, and this decreased from 5, 4, 3, and 2 antigens. Note that the same number of antigens were present, but were nanopatterened using DNA to form defined patterns. This work will have important consequences for antibody engineering and therapeutic modulation, but also highlights the importance of protein geometry in immune system activation.
Additionally to the first aim, which focused on antibody-mediated complement activation, we also explored the ability of pentraxins to bind and activate C1. The short pentraxin C-reactive protein (CRP) is predominantly found as a pentamers. We discovered that in solution, CRP can also form inhibitory dimers of pentamers (decamers), whilst on surfaces CRP forms an activatory array comprising a tetramer of pentamers. This geometric switch will have important consequences for CRP activity, and we are now exploring the consequences of CRP geometry. We also discovered that the long pentraxin PTX3 forms an octamer, again highlighting how geometry is important for immune system activation.
The second aim of this proposal was to exploit our understanding of nanopatterns to develop new and highly efficient ways to activate our immune system in vivo, with potential for targeted anti-tumour immunotherapies. This was achieved by determining both the geometric and molecular requirements of activation, and we could distil these down into discrete constituents to form rationally-designed small-molecule activators of complement. This was performed using small peptides that mimicked antibody complexes. We discovered a small (16 amino acid) macrocyclic peptide that can activate complement in the same way as a 1 MDa antibody immune complex. We have demonstrated the ability of this peptide to recruit C1 from human serum, activate the C1 proteases on surfaces, and even induce membrane lysis. We are now exploring the therapeutic applications of this peptide.
Control over these nanopatterns will allow us to understand the geometric parameters of immune system activation, modulation and inhibition. Furthermore, control of the nanopatterns will also enable control over the immune system itself.
To gain control, DNA nanotechnology can be used to form self-assembled nanoscale structures, which are ideal for use as templates to pattern proteins with specific geometries and nanometre accuracy. This project uses DNA to nanopattern antigens and other biomolecular moieties with defined geometry to study and control Complement pathway activation by the C1 complex.
The first aim of this proposal aimed to understand the role of nanopatterns in complement activation. We developed DNA nanostructures able to nanopattern antibodies, to which the C1 complex bound with the specific geometry. This allowed us to explore the relationship between geometry and Complement activation, which manifested as a relationship between antibody valency and C4b deposition. C4b is the product of the C1 enzyme, and we therefore discovered how antibody valency can modulate C1 activity. This relationship extended to membrane lysis – the name number of antigens nanopatterned as a hexagon was most potent, and this decreased from 5, 4, 3, and 2 antigens. Note that the same number of antigens were present, but were nanopatterened using DNA to form defined patterns. This work will have important consequences for antibody engineering and therapeutic modulation, but also highlights the importance of protein geometry in immune system activation.
Additionally to the first aim, which focused on antibody-mediated complement activation, we also explored the ability of pentraxins to bind and activate C1. The short pentraxin C-reactive protein (CRP) is predominantly found as a pentamers. We discovered that in solution, CRP can also form inhibitory dimers of pentamers (decamers), whilst on surfaces CRP forms an activatory array comprising a tetramer of pentamers. This geometric switch will have important consequences for CRP activity, and we are now exploring the consequences of CRP geometry. We also discovered that the long pentraxin PTX3 forms an octamer, again highlighting how geometry is important for immune system activation.
The second aim of this proposal was to exploit our understanding of nanopatterns to develop new and highly efficient ways to activate our immune system in vivo, with potential for targeted anti-tumour immunotherapies. This was achieved by determining both the geometric and molecular requirements of activation, and we could distil these down into discrete constituents to form rationally-designed small-molecule activators of complement. This was performed using small peptides that mimicked antibody complexes. We discovered a small (16 amino acid) macrocyclic peptide that can activate complement in the same way as a 1 MDa antibody immune complex. We have demonstrated the ability of this peptide to recruit C1 from human serum, activate the C1 proteases on surfaces, and even induce membrane lysis. We are now exploring the therapeutic applications of this peptide.
We initially consolidated a team including me, 2 PhD students and 1 Postdoc. Together, we determined which DNA nanotemplate is optimal for complement activation and successfully demonstrated the first instance of DNA-mediated immune system activation. We then determined how different antibody geometries affect complement activation by exploring multiple nanopatterns, including the valency, height and geometry of the activating molecules. We performed high-resolution cryo-electron tomography of DNA-mediated complement activation and can clearly see stepwise binding, initiation, activation and termination of the entire immune pathway.
As a separate project, we determined the structures of other immune-system activators, and have discovered that these are stimulated by pH changes and oxidative stresses induced in wound- and infection environments. These complexes are involved in immune defense and autoimmune disease progression, but also found in neurological disorders such as Alzheimers. As such, the structure-function relationship of these molecules will have far-reaching consequences for structural biologists, immunologists and neurologists.
Developing small-molecule complement activators has also progressed, but not in the direction initially envisaged. We were unable to derive agonistic aptamers based on nucleic acids. However, we were successful at deriving peptide-based activators. We are currently pursuing these are potential immunotheraputics.
Developing DNA nanotechnology as a nanopatterning platform has also enabled us to form collaborations with various groups in immunology, organic chemistry, biotechnology and microscopy groups.
As a separate project, we determined the structures of other immune-system activators, and have discovered that these are stimulated by pH changes and oxidative stresses induced in wound- and infection environments. These complexes are involved in immune defense and autoimmune disease progression, but also found in neurological disorders such as Alzheimers. As such, the structure-function relationship of these molecules will have far-reaching consequences for structural biologists, immunologists and neurologists.
Developing small-molecule complement activators has also progressed, but not in the direction initially envisaged. We were unable to derive agonistic aptamers based on nucleic acids. However, we were successful at deriving peptide-based activators. We are currently pursuing these are potential immunotheraputics.
Developing DNA nanotechnology as a nanopatterning platform has also enabled us to form collaborations with various groups in immunology, organic chemistry, biotechnology and microscopy groups.
Using DNA to regulate complement activation is a novel method to gain control over a complex biological system. Our data show that we can now modify DNA to display biomolecules that are able to activate complement. This is a novel mechanism to control the spatial organization of antibody-binding sites to enable us to understand complement activation at both a structural and functional level. We have now applied this to other molecules of the immune system, including tumour necrosis factor receptors (TNFRs) and human leukocyte antigens (HLA) complexes, to explore how these function on, and induce signalling within, whole cells.
We have identified small-molecule activators of complement with cell-killing properties. We are now developing these as novel immunotherapeutics for the treatment of diseases such as cancer, autoimmune diseases and antimicrobials.
We have identified small-molecule activators of complement with cell-killing properties. We are now developing these as novel immunotherapeutics for the treatment of diseases such as cancer, autoimmune diseases and antimicrobials.