A tetrameric ACE2 protein broadly neutralizes SARS-CoV-2 spike variants of concern with elevated potency.

The SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) was previously engineered into a high affinity tetravalent format (ACE2-Fc-TD) that is a potential decoy protein in SARS-CoV-2 infection.We report that this protein shows greatly enhanced binding to SARS-CoV-2 spike proteins of the SARS-CoV-2 variants of concern B.1.1.7 (alpha variant, originally isolated in the United Kingdom) and B.1.351 (beta variant, originally isolated in South Africa) with picomolar compared with nanomolar Kd values. In addition, ACE2-Fc-TD displays greater neutralization of SARS-CoV-2 pseudotype viruses compared to a dimeric ACE2-Fc, with enhanced activity on variant B.1.351. This tetrameric decoy protein would be a valuable addition to SARS-CoV-2 therapeutic approaches, especially where vaccination cannot be used but also should there be any future coronavirus pandemics.

An antibody-drug conjugate with intracellular drug release properties showing specific cytotoxicity against CD7-positive cells.

Refractory T cell acute leukaemias that no longer respond to treatment would benefit from new modalities that target T cell-specific surface proteins. T cell associated surface proteins (the surfaceome) offer possible therapy targets to reduce tumour burden but also target the leukaemia-initiating cells from which tumours recur. Recent studies of the T cell leukaemia surfaceome confirmed that CD7 is highly expressed in overt disease. We have used an anti-CD7 antibody drug conjugate (ADC) to show that the binding of antibody to surface CD7 protein results in rapid internalization of the antigen together with the ADC. As a consequence, cell killing was observed via induction of apoptosis and was dependent on cell surface CD7. The in vitro cytotoxic activity (EC50) of the anti-CD7 ADC on T cell acute leukaemia (T-ALL) cells Jurkat and KOPT-K1 was found to be in the range of 5-8 ng/mL. In a pre-clinical xenograft model of human tumour growth expressing CD7 antigen, growth was curtailed by a single dose of ADC. The data indicate that CD7 targeting ADCs may be developed into an important second stage therapy for T cell acute leukaemia, for refractory CD7-positive leukaemias and for subsets of acute myeloid leukaemia (AML) expressing CD7.

A super-potent tetramerized ACE2 protein displays enhanced neutralization of SARS-CoV-2 virus infection.

Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.

Implementing a method for engineering multivalency to substantially enhance binding of clinical trial anti-SARS-CoV-2 antibodies to wildtype spike and variants of concern proteins.

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.

A cell-based screening method using an intracellular antibody for discovering small molecules targeting the translocation protein LMO2.

Intracellular antibodies are tools that can be used directly for target validation by interfering with properties like protein-protein interactions. An alternative use of intracellular antibodies in drug discovery is developing small-molecule surrogates using antibody-derived (Abd) technology. We previously used this strategy with an in vitro competitive surface plasmon resonance method that relied on high-affinity antibody fragments to obtain RAS-binding compounds. We now describe a novel implementation of the Abd method with a cell-based intracellular antibody-guided screening method that we have applied to the chromosomal translocation protein LMO2. We have identified a chemical series of anti-LMO2 Abd compounds that bind at the same LMO2 location as the inhibitory anti-LMO2 intracellular antibody combining site. Intracellular antibodies could therefore be used in cell-based screens to identify chemical surrogates of their binding sites and potentially be applied to any challenging proteins, such as transcription factors that have been considered undruggable.

Competitive SPR using an intracellular anti-LMO2 antibody identifies novel LMO2-interacting compounds.

The use of intracellular antibodies as templates to derive surrogate compounds is an important objective because intracellular antibodies can be employed initially for target validation in pre-clinical assays and subsequently employed in compound library screens. LMO2 is a T cell oncogenic protein activated in the majority of T cell acute leukaemias. We have used an inhibitory intracellular antibody fragment as a competitor in a small molecule library screen using competitive surface plasmon resonance (cSPR) to identify compounds that bind to LMO2. We selected four compounds that bind to LMO2 but not when the anti-LMO2 intracellular antibody fragment is bound to it. These findings further illustrate the value of intracellular antibodies in the initial stages of drug discovery campaigns and more generally antibodies, or antibody fragments, can be the starting point for chemical compound development as surrogates of the antibody combining site.

Pan RAS-binding compounds selected from a chemical library by inhibiting interaction between RAS and a reduced affinity intracellular antibody.

Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.

A potent KRAS macromolecule degrader specifically targeting tumours with mutant KRAS.

Tumour-associated KRAS mutations are the most prevalent in the three RAS-family isoforms and involve many different amino-acids. Therefore, molecules able to interfere with mutant KRAS protein are potentially important for wide-ranging tumour therapy. We describe the engineering of two RAS degraders based on protein macromolecules (macrodrugs) fused to specific E3 ligases. A KRAS-specific DARPin fused to the VHL E3 ligase is compared to a pan-RAS intracellular single domain antibody (iDAb) fused to the UBOX domain of the CHIP E3 ligase. We demonstrate that while the KRAS-specific DARPin degrader induces specific proteolysis of both mutant and wild type KRAS, it only inhibits proliferation of cancer cells expressing mutant KRAS in vitro and in vivo. Pan-RAS protein degradation, however, affects proliferation irrespective of the RAS mutation. These data show that specific KRAS degradation is an important therapeutic strategy to affect tumours expressing any of the range of KRAS mutations.

Multimeric antibodies with increased valency surpassing functional affinity and potency thresholds using novel formats.

The success of therapeutic antibodies is largely attributed for their exquisite specificity, homogeneity, and functionality. There is, however, a need to engineer antibodies to extend and enhance their potency. One parameter is functional affinity augmentation, since antibodies matured in vivo have a natural affinity threshold. Generation of multivalent antibodies is one option capable of surpassing this affinity threshold through increased avidity. In this study, we present a novel platform consisting of an array of multivalent antibody formats, termed Quads, generated using the self-assembling tetramerization domain from p53. We demonstrate the versatility of this tetramerization domain by engineering anti-tumor necrosis factor (TNF) Quads that exhibit major increases in binding potency and in neutralizing TNF-mediated cytotoxicity compared to parental anti-TNF molecules. Further, Quads are amenable to fusion with different binding domains, allowing generation of novel multivalent monospecific and bispecific formats. Quads are thus a novel group of molecules that can be engineered to yield potential therapeutics with novel modalities and potencies.

Immunopolymer Lipid Nanoparticles for Delivery of Macromolecules to Antigen-Expressing Cells.

Macromolecules such as antibodies and antibody fragments have been reported to interfere with intracellular targets, but their use is limited to delivery systems where expression is achieved from vectors such as plasmids or viruses. We have developed PEGylated nanoparticles of poly-lactic acid (PLA), including the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), which are functionalized with monoclonal anti-CD7, anti-CD53, or anti-GPR56 antibodies for receptor-mediated endocytic delivery into T-cell leukemia cell lines. Incorporation of DOTAP as the lipid component of the PLA nanoparticles enhanced the release of the immuno-nanoparticles from the endosomes into the cytosol compared to nanoparticles made from PLA alone. Systemic delivery of these anti-CD7 immuno-nanoparticles into humanized CD7 transgenic mice resulted in localization in the spleen, enhanced uptake into CD7-expressing splenocytes, and release of low amounts of reporter mRNA for translation. These functionalized polymer lipid nanoparticles are the basis for elaboration and optimization for realizing their potential in therapeutic applications to carry specific macromolecules such as mRNAs for translation into therapeutic proteins that can target intracellular proteins which mediate disease.

KRAS-specific inhibition using a DARPin binding to a site in the allosteric lobe.

Inhibiting the RAS oncogenic protein has largely been through targeting the switch regions that interact with signalling effector proteins. Here, we report designed ankyrin repeat proteins (DARPins) macromolecules that specifically inhibit the KRAS isoform by binding to an allosteric site encompassing the region around KRAS-specific residue histidine 95 at the helix α3/loop 7/helix α4 interface. We show that these DARPins specifically inhibit KRAS/effector interactions and the dependent downstream signalling pathways in cancer cells. Binding by the DARPins at that region influences KRAS/effector interactions in different ways, including KRAS nucleotide exchange and inhibiting KRAS dimerization at the plasma membrane. These results highlight the importance of targeting the α3/loop 7/α4 interface, a previously untargeted site in RAS, for specifically inhibiting KRAS function.

Surfaceome interrogation using an RNA-seq approach highlights leukemia initiating cell biomarkers in an LMO2 T cell transgenic model.

The surfaceome is critical because surface proteins provide a gateway for internal signals and transfer of molecules into cells, and surfaceome differences can influence therapy response. We have used a surfaceome analysis method, based on comparing RNA-seq data between normal and abnormal cells (Surfaceome DataBase Mining or Surfaceome DBM), to identify sets of upregulated cell surface protein mRNAs in an LMO2-mediated T-ALL mouse model and corroborated by protein detection using antibodies. In this model the leukemia initiating cells (LICs) comprise pre-leukaemic, differentiation inhibited thymocytes allowing us to provide a profile of the LIC surfaceome in which GPR56, CD53 and CD59a are co-expressed with CD25. Implementation of cell surface interaction assays demonstrates fluid interaction of surface proteins and CD25 is only internalized when co-localized with other proteins. The Surfaceome DBM approach to analyse cancer cell surfaceomes is a way to find targetable surface biomarkers for clinical conditions where RNA-seq data from normal and abnormal cell are available.

Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds.

The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein-protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein-protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein-protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein-protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.

Two distinct conformations of factor H regulate discrete complement-binding functions in the fluid phase and at cell surfaces.

Factor H (FH) is the major regulator of C3b in the alternative pathway of the complement system in immunity. FH comprises 20 short complement regulator (SCR) domains, including eight glycans, and its Y402H polymorphism predisposes those who carry it to age-related macular degeneration. To better understand FH complement binding and self-association, we have studied the solution structures of both the His-402 and Tyr-402 FH allotypes. Analytical ultracentrifugation revealed that up to 12% of both FH allotypes self-associate, and this was confirmed by small-angle X-ray scattering (SAXS), MS, and surface plasmon resonance analyses. SAXS showed that monomeric FH has a radius of gyration (Rg ) of 7.2-7.8 nm and a length of 25 nm. Starting from known structures for the SCR domains and glycans, the SAXS data were fitted using Monte Carlo methods to determine atomistic structures of monomeric FH. The analysis of 29,715 physically realistic but randomized FH conformations resulted in 100 similar best-fit FH structures for each allotype. Two distinct molecular structures resulted that showed either an extended N-terminal domain arrangement with a folded-back C terminus or an extended C terminus and a folded-back N terminus. These two structures are the most accurate to date for glycosylated full-length FH. To clarify FH functional roles in host protection, crystal structures for the FH complexes with C3b and C3dg revealed that the extended N-terminal conformation accounted for C3b fluid-phase regulation, the extended C-terminal conformation accounted for C3d binding, and both conformations accounted for bivalent FH binding to glycosaminoglycans on the target cell surface.

Small molecule inhibitors of RAS-effector protein interactions derived using an intracellular antibody fragment.

Targeting specific protein-protein interactions (PPIs) is an attractive concept for drug development, but hard to implement since intracellular antibodies do not penetrate cells and most small-molecule drugs are considered unsuitable for PPI inhibition. A potential solution to these problems is to select intracellular antibody fragments to block PPIs, use these antibody fragments for target validation in disease models and finally derive small molecules overlapping the antibody-binding site. Here, we explore this strategy using an anti-mutant RAS antibody fragment as a competitor in a small-molecule library screen for identifying RAS-binding compounds. The initial hits are optimized by structure-based design, resulting in potent RAS-binding compounds that interact with RAS inside the cells, prevent RAS-effector interactions and inhibit endogenous RAS-dependent signalling. Our results may aid RAS-dependent cancer drug development and demonstrate a general concept for developing small compounds to replace intracellular antibody fragments, enabling rational drug development to target validated PPIs.

BRET-based RAS biosensors that show a novel small molecule is an inhibitor of RAS-effector protein-protein interactions.

The RAS family of proteins is amongst the most highly mutated in human cancers and has so far eluded drug therapy. Currently, much effort is being made to discover mutant RAS inhibitors and in vitro screening for RAS-binding drugs must be followed by cell-based assays. Here, we have developed a robust set of bioluminescence resonance energy transfer (BRET)-based RAS biosensors that enable monitoring of RAS-effector interaction inhibition in living cells. These include KRAS, HRAS and NRAS and a variety of different mutations that mirror those found in human cancers with the major RAS effectors such as CRAF, PI3K and RALGDS. We highlighted the utility of these RAS biosensors by showing a RAS-binding compound is a potent pan-RAS-effector interactions inhibitor in cells. The RAS biosensors represent a useful tool to investigate and characterize the potency of anti-RAS inhibitors in cells and more generally any RAS protein-protein interaction (PPI) in cells.

Cloning, purification and structure determination of the HIV integrase-binding domain of lens epithelium-derived growth factor.

Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å. The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixture of monomers and dimers in concentrated solutions, but the dimers are unlikely to be biologically relevant.

Intracellular immunization against HIV infection with an intracellular antibody that mimics HIV integrase binding to the cellular LEDGF protein.

Preventing the protein-protein interaction of the cellular chromatin binding protein Lens Epithelium-Derived Growth Factor (LEDGF) and human immunodeficiency virus (HIV) integrase is an important possible strategy for anti-viral treatment for AIDS. We have used Intracellular Antibody Capture technology to isolate a single VH antibody domain that binds to LEDGF. The crystal structure of the LEDGF-VH complex reveals that the single domain antibody mimics the effect of binding of HIV integrase to LEDGF which is crucial for HIV propagation. CD4-expressing T cell lines were constructed to constitutively express the LEDGF-binding VH and these cells showed interference with HIV viral replication, assayed by virus capsid protein p24 production. Therefore, pre-conditioning cells to express antibody fragments confers effective intracellular immunization for preventing chronic viral replication and can be a way to prevent HIV spread in infected patients. This raises the prospect that intracellular immunization strategies that focus on cellular components of viral integrase protein interactions can be used to combat the problems associated with latent HIV virus re-emergence in patients. New genome editing development, such as using CRISPR/cas9, offer the prospect intracellularly immunized T cells in HIV+ patients.

Structure-based Discovery of Novel Small Molecule Wnt Signaling Inhibitors by Targeting the Cysteine-rich Domain of Frizzled.

Frizzled is the earliest discovered glycosylated Wnt protein receptor and is critical for the initiation of Wnt signaling. Antagonizing Frizzled is effective in inhibiting the growth of multiple tumor types. The extracellular N terminus of Frizzled contains a conserved cysteine-rich domain that directly interacts with Wnt ligands. Structure-based virtual screening and cell-based assays were used to identify five small molecules that can inhibit canonical Wnt signaling and have low IC50 values in the micromolar range. NMR experiments confirmed that these compounds specifically bind to the Wnt binding site on the Frizzled8 cysteine-rich domain with submicromolar dissociation constants. Our study confirms the feasibility of targeting the Frizzled cysteine-rich domain as an effective way of regulating canonical Wnt signaling. These small molecules can be further optimized into more potent therapeutic agents for regulating abnormal Wnt signaling by targeting Frizzled.