Biology subjects

Cragg, M. S.

Publications and source records attributed to Cragg, M. S..

11 recordsLinked to original sources

bioRxiv2026

Duality Vaccination through Nanodesign of Click-Ligated B-Cell Inhibitory Receptor Bridging

Synthesis of precise and robust molecular architectures is critical to harness the power of the immune system. The dimensions, rigidity and multivalency of nanoparticles contribute to eliciting high levels of antibodies from vaccination. However, antibodies vary hugely in quality: some antibodies are highly effective at neutralizing pathogens, while others provide little or no benefit. Here we establish switchable and modular platforms for protein and nanoparticle assembly that program the target of antibody responses. We introduce Duality vaccination: the simultaneous delivery of activating and inhibitory stimuli for focusing immune responses. To construct activatory immunogens, we employ click biology with spontaneous amidation to functionalize plug-and-display self-assembling porous dodecahedral nanocages. We construct inhibitory immunogens using SpyMask protease-gated bispecific assembly, ligating the antigen to binders that bridge the B cell receptor to immune checkpoint receptors. This dual immunogen architecture tailors the elicited antibodies to down-modulate undesired B cell responses. We establish the effect of temporal and spatial separation of activatory and inhibitory immunogen delivery. We further explore sensitivity to valency, linker flexibility and adjuvant delivery, employing model targets and coronavirus antigens. Dual programmable functionalization identifies key nanodesign features to control in vivo outputs, providing logic-gating towards urgent challenges in the creation of protective vaccines.

synthetic biology↗

bioRxiv2026

Bispecific Antibody Architecture and TNFRSF Target Selection Determine CD8+ T Cell Differentiation and Anti-tumour Immunity

Tumour necrosis factor receptor superfamily (TNFRSF)-targeting bispecific antibodies (bsAb) enable tumour-localised T cell co-stimulation, yet how antibody architecture and receptor choice govern activity remains unclear. Here we define how bsAb format and TNFRSF target selection shape CD8+ T cell responses and anti-tumour immunity. Using B7-H3 as a tumour-associated antigen, we show that dual-bivalent 2x2 bsAb elicit maximal agonistic activity, whereas the corresponding monovalent 1x1 format is least active. A 2x1 format retaining TNFRSF bivalency but monovalent B7-H3 binding preserves substantial activity, identifying co-stimulatory receptor bivalency as a key determinant of efficacy. Across TNFRSF targets, 4-1BB drives the strongest cytotoxic CD8+ T cell differentiation and anti-tumour response. This superiority is conserved in human T cells and reproduced by cognate ligands, indicating that the observed functional hierarchy reflects receptor-intrinsic biology. Mechanistically, efficacy requires tumour-associated B7-H3 and T cell-dependent 4-1BB signalling. Together, these findings establish general principles linking antibody architecture and receptor biology to co-stimulatory bsAb efficacy and provide a framework for rational design leading to optimal therapeutics.

immunology↗

bioRxiv2025

Energy-guided combinatorial co-optimization of antibody affinity and stability

Affinity maturation is an essential process in antibody engineering. Although powerful, it is iterative, time-consuming, and can result in trade-offs, where affinity is gained at the cost of important properties, such as specificity and stability. We present a scalable strategy, called LAffAb, that starts from a crystallographic structure of the antibody-antigen complex and introduces combinations of mutations to optimize its energy. A combinatorial library comprising 7,000 variants with up to nine mutations results in gains of up to 30-fold in affinity while co-optimizing stability. Surprisingly, the library does not converge on a single solution, instead favoring diverse variants with a high mutational load. Small-scale screening of 10 designs against a potential drug target results in an order-of-magnitude improvement in affinity while maintaining high developability. We also apply LAffAb to improve the developability of a therapeutic antibody without degrading affinity. We envision that LAffAb can be used to design stable, specific, and high-affinity binders and to improve our understanding of sequence, structure, and function relationships in antibodies.

synthetic biology↗

bioRxiv2025

A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

The DNA damage response (DDR) is a central regulator of cancer cell fate, coordinating both pro-death and pro-survival pathways in response to genotoxic stress. Here, we reveal an unexpected role for the DDR at the cell surface, in mediating immune evasion from complement-dependent cytotoxicity (CDC), an innate immune mechanism exploited by therapeutic monoclonal antibodies (mAbs). In the context of diffuse large B-cell lymphoma (DLBCL), where the anti-CD20 mAb rituximab utilizes CDC, we show that genotoxic chemotherapy induces expression of membrane-bound complement regulatory proteins (mCRPs) CD46, CD55, and CD59, thereby reducing CDC sensitivity and compromising rituximab activity. In this setting, CD59 emerged as the dominant DDR-induced inhibitor of complement-mediated killing. A high-throughput kinase inhibitor screen identified checkpoint kinase 1 (Chk1) as a critical mediator of this response. Mechanistically, DNA damage activates Chk1, enhancing CD59 transcription via an Sp1-bound promoter. Co-immunoprecipitation mass spectrometry revealed a Chk1 dependent remodelling of Sp1-associated complexes to a transcriptionally active state with recruitment of the histone acetyltransferase KAT2A. These findings expand the role of the DDR in immune resistance at the tumor cell surface, and highlight a negative interaction between chemotherapy and monoclonal antibodies that may require sequential administration or targeting of the Chk1- Sp1-CD59 axis. SignificanceThe DNA Damage Response upregulates complement-protective proteins, extending its role in modulating immune evasion at the cell surface, with direct implications for combinations of chemotherapy and monoclonal antibodies widely used in cancer.

cancer biology↗

bioRxiv2024

ACE2 decoy Fc-fusions and bi-specific killer engager (BiKEs) require Fc engagement for in vivo efficacy against SARS-CoV-2

SARS-CoV-2 virus has continued to evolve over time necessitating the adaptation of vaccines to maintain efficacy. Monoclonal antibodies (mAbs) against SARS-CoV-2 were a key line of defense for unvaccinated or immunocompromised individuals. However, these mAbs are now ineffective against current SARS-CoV-2 variants. Here, we tested three aspects of SARS-CoV-2 therapeutics. First, we tested whether Fc engagement is necessary for in vivo clearance of SARS-CoV-2. Secondly, we tested bi-specific killer engagers (BiKEs) that simultaneously engage SARS-CoV-2 and a specific Fc receptor. Benefits of these engagers include the ease of manufacturing, stability, more cell-specific targeting, and high affinity binding to Fc receptors. Using both mAbs and BiKEs, we found that both neutralization and Fc receptor engagement were necessary for effective SARS-CoV-2 clearance. Thirdly, due to ACE2 being necessary for viral entry, ACE2 will maintain binding to SARS-CoV-2 despite viral evolution. Therefore, we used an ACE2 decoy Fc-fusion or BiKE, instead of an anti-SARS-CoV-2 antibody sequence, as a potential therapeutic that would withstand viral evolution. We found that the ACE2 decoy approach also required Fc receptor engagement and, unlike traditional neutralizing antibodies against specific variants, enabled the clearance of two distinct SARS-CoV-2 variants. These data show the importance of Fc engagement for mAbs, the utility of BiKEs as therapies for infectious disease, and the in vivo effectiveness of the ACE2 decoy approach. With further studies, we predict combining neutralization, the cellular response, and this ACE2 decoy approach will benefit individuals with ineffective antibody levels. AbbreviationsACE2, scFv, mAb, BiKE, COVID-19, Fc, CD16, CD32b, CD64, d.p.i Key pointsO_LIWith equal dosing, both neutralization and Fc engagement are necessary for the optimal efficacy of in vivo antibodies and bi-specific killer engagers (BiKEs) against SARS-CoV-2. C_LIO_LIBiKEs can clear SARS-CoV-2 virus and protect against severe infection in the hACE2-K18 mouse model. C_LIO_LIACE2 decoys as part of Fc-fusions or BiKEs provide in vivo clearance of two disparate SARS-CoV-2 variants. C_LI

immunology↗

bioRxiv2023

Soluble CTLA-4 raises the threshold for T-cell activation and modulates anti-tumour immunity

CTLA-4 is a crucial immune checkpoint receptor involved in the maintenance of immune homeostasis, tolerance, and tumour control. Antibodies targeting CTLA-4 have been promising treatment for numerous cancers, but the mechanistic basis of their anti-tumoral immune boosting effects are poorly understood. Although the ctla4 gene also encodes an alternatively-spliced soluble variant (sCTLA-4), preclinical/clinical evaluation of anti-CTLA-4-based immunotherapies have not considered the contribution of this isoform. Here, we explore the functional properties of sCTLA-4 and evaluate the efficacy of isoform-specific anti-sCTLA-4 antibody targeting in murine cancer model. We show that expression of sCTLA-4 in tumour cells suppresses CD8+ T-cells in vitro, and accelerates growth and experimental metastasis of murine tumours in vivo. These effects were accompanied by modification of the immune infiltrate, notably restraining CD8+ T-cells in a non-effector state. sCTLA-4 blockade with isoform-specific antibody reversed this restraint, enhancing intratumoural CD8+ T-cell activation and cytolytic potential, correlating with therapeutic efficacy and tumour control. This previously unappreciated role of sCTLA-4 suggests that better understanding of the biology and function of multi-gene products of immune checkpoint receptors needs to be fully elucidated for improved cancer immunotherapy.

cancer biology↗

bioRxiv2023

Synthetic STING agonists elicit powerful vaccine adjuvancy providing robust central memory and anti-tumour effects

Drugs that target the innate immune sensor STING are known to be effective in modulating the immune infiltrate of the tumour microenvironment. STING agonists have potential to enhance responses to checkpoint inhibitor therapy, however, their ability to influence and shape adaptive immune responses is poorly understood. Here, we investigated the impact of a range of synthetic STING agonists on antigen specific CD8+ T-cell responses to soluble antigen using the murine OT-1 adoptive transfer model with Ovalbumin as the antigen to monitor T cell responses. Our data demonstrate that synthetic STING agonists are able to stimulate antigen specific T-cell expansion in response to challenge in mice. This effect required expression of STING, an intact myeloid compartment and Type-I IFN and TNF signalling. Expanded T-cells post treatment differed from those induced by the established immune adjuvant, anti-CD40 antibody through lower induction of the immune checkpoint receptor PD-1. Furthermore, our data revealed a marked increase in the induction and persistence of CD8+ central memory cells after STING agonist and antigen challenge. Finally, we demonstrate that following rechallenge, STING agonism produced larger secondary responses that could be translated into enhanced tumour protection and survival. Therefore, synthetic STING agonists are capable of acting as potent immune adjuvants and can induce robust memory formation leading to better recall and tumour control. Critically, these benefits along with the lower expression of PD-1, have implications for their use as adjuvants for multiple immunotherapy and vaccine applications.

immunology↗

bioRxiv2022

Role of the circadian clock "Death-Loop" in the DNA damage response underpinning cancer treatment resistance

The Circadian Clock (CC) drives the normal cell cycle and reciprocally regulates telomere elongation. However, it can be deregulated in cancer, embryonic stem cells (ESC), and the early embryo. Here, its role in the resistance of cancer cells to genotoxic treatments was assessed in relation to whole-genome duplication (WGD) and telomere regulation. We first evaluated the DNA damage response of polyploid cancer cells and observed a similar impact on the cell cycle to that seen in ESC - overcoming G1/S, adapting DNA damage checkpoints, tolerating DNA damage, and coupling telomere erosion to accelerated cell senescence, favouring transition by mitotic slippage into the ploidy cycle (reversible polyploidy). Next, we revealed a positive correlation between cancer WGD and deregulation of CC assessed by bioinformatics on 11 primary cancer datasets (rho=0.83; p<0.01). As previously shown, the cancer cells undergoing mitotic slippage cast off telomere fragments with TERT, restore the telomeres by recombination and return their depolyploidised mitotic offspring to TERT-dependent telomere regulation. Through depolyploidisation and the CC "death loop" the telomeres and Hayflick limit count are thus again renewed. This mechanism along with similar inactivity of the CC in early embryos support a life-cycle (embryonic) concept of cancer.

cancer biology↗

bioRxiv2021

Replicative senescence dictates the emergence of disease-associated microglia and contributes to Abeta pathology

The sustained proliferation of microglia is a key hallmark of Alzheimers disease (AD), accelerating its progression. Here, we sought to understand the long-term impact of the early and prolonged microglial proliferation observed in AD, hypothesising that extensive and repeated cycling would engender a distinct transcriptional and phenotypic trajectory. We found that the early and sustained microglial proliferation seen in an AD-like model promotes replicative senescence, characterised by increased {beta}gal activity, a senescence-associated transcriptional signature and telomere shortening, correlating with the appearance of disease-associated microglia (DAM) and senescent microglial profiles in human post-mortem AD cases. Prevention of early microglial proliferation hindered the development of senescence and DAM, impairing the accumulation of A{beta} and associated neuritic damage. Overall, our results support that excessive microglial proliferation leads to the generation of senescent DAM, which contribute to early A{beta} pathology in AD.

neuroscience↗

bioRxiv2020

Domain Binding and Isotype Dictate the Activity of Anti-human OX40 Antibodies

Previous data suggests that anti-OX40 mAb can elicit anti-tumour effects in mice through deletion of Tregs. However, OX40 also has powerful costimulatory effects on T cells which could evoke therapeutic responses. The contributions of these different effector mechanisms has not previously been systematically evaluated, particularly for mAb directed to human OX40. Therefore, we generated a novel human OX40 knock-in (KI) mouse to evaluate a panel of anti-hOX40 mAb and show that their activities relate directly to two key properties: 1) isotype – with mIgG1 mAb evoking receptor agonism and CD8+ T cell expansion and mIgG2a mAb evoking deletion of Treg and; 2) epitope - with membrane-proximal mAb delivering more powerful agonism. Intriguingly, both isotypes acted therapeutically in tumour models by engaging these different mechanisms. These findings highlight the significant impact of isotype and epitope on the modulation of anti-hOX40 mAb therapy, and indicate that CD8+ T cell expansion or Treg depletion might be preferable according to the composition of different tumours.Summary Human trials with anti-OX40 antibodies have been disappointing indicating that optimal reagents have not yet been developed. Here, using a new panel of antibodies, we show that isotype and epitope combine to determine agonistic and therapeutic activity.Competing Interest StatementMSC is a retained consultant for BioInvent International and has performed educational and advisory roles for Baxalta and Boehringer Ingleheim. He has received research funding from Roche, Gilead, Bioinvent International and GSK. BF, IT, LM and MS are employees of Bioinvent International.View Full Text

immunology↗

bioRxiv2020

Activation of the myeloid checkpoint inhibitor LILRB3 (ILT5) elicits profound immununomodulation

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin (Ig)-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress for this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAb) was generated. LILBR3-specific mAb bound to discrete epitopes in either Ig-like domain two or four. LILRB3 ligation on primary human monocytes by agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppresive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.

immunology↗