Engineered scaffold protein

Editor's Notes

• #3: Antibodies have long been regarded as ‘magic bullets’ in human therapy due to their ability to bind targets with high affinity and specificity , over 62 mAbs have been approved by the FDA as therapeutics and many new candidates are presently undergoing preclinical and clinical evaluations.  large size of antibodies (~150 kDa) may impede their ability to penetrate into tumor tissue   planar binding interface makes it difficult to obtain antibodies that bind to grooves and catalytic sites of enzymes. antibody constant region—Fc—can sometimes give rise to adverse effects, such as antibody-dependent-enhancement (ADE) of infection by some viruses e.g., Dengue virus and Zika virus  most mAbs have to be produced in mammalian cells and often require post-translational modifications, such as specific glycosylation patterns.  Antibodies are also difficult to be manipulated for drug conjugation via the conventional conjugation and linker chemistries, as they are too big to be synthesized chemically and too complex to be produced in microorganisms. constant region, which stabilizes the overall protein folding, and multiple variable regions that mediate its binding to a specific target.
• #4: Some of these have already been approved by the FDA for human use, and many are currently being evaluated pre-clinically or in clinical trials. The absence of an Fc may also prove to be beneficial in some cases to avoid the adverse effect. protein fragments are generally very small in size (<20 kDa) and thus may be better able to penetrate tumors. many protein fragments exhibit high thermostability, allowing storage at room temperature for extended periods of time without significant loss of activity, and can be easily produced in microorganisms or be completely chemically synthesized,  ability of some molecules to resist protease degradation and/or chemical denaturation, makes it possible for some protein fragments to be used in oral applications.
• #5: Phage display -  ligand-binding protein fragment can be isolated from other phage by their ability to bind to the ligand, a process called “panning”. The phage that are thus selected are subsequently amplified in E. coli and undergo additional rounds of panning. workhorse for protein engineering One limitation of phage display is its reliance on the transformation of E. coli with plasmids encoding the protein fragment library. For the display of mammalian proteins that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity, yeast has proved to be an attractive choice.  In yeast surface display, the protein fragment is displayed on the surface of the yeast Saccharomyces cerevisiae  In ribosome display, a ternary complex composed of the translated proteins, the ribosome, and its encoding mRNA is used for selection  Translated protein is covalently attached to the mRNA molecule via the adaptor puromycin molecule. The mRNA-protein adduct is subsequently purified from the ribosome and used for selection.
• #7: The structures are displayed using visual molecular dynamics (VMD) (Humphrey et al., 1996). The loops that recognize the antigen are colored in red and the framework residues are indicated in gray. The disulfide bridges are indicated as sticks in element color and the calcium is represented as blue spheres. 
• #12: Applications of monomeric antibody domains. (A) Antibody Drug Conjugate (ADC) drug delivery [58]. Tumour imaging with monobodies conjugated to (B) radioisotopes [62] and (C) microbubbles [52]. (D) Targeted degradation of endogenous intracellular proteins [63] and (E) targeted intracellular fluorescence reporters for endogenous proteins [64].
• #13:  Adnectins were generated for non-invasive imaging of PD-L1 with radioisotopes. PD-L1 is an immune suppression ligand that is overexpressed on the surface of tumours, making it a selective target for the imaging of tumours before and during treatment. However, as PD-L1 is expressed on most cells as part of normal suppression of auto-immune response, the use of antibodies to deliver radioisotopes for imaging is limited since the long circulation time and off-target binding of antibodies results in considerable background signals and reduced tumour penetration while inducing systemic radiation burden to patients. 18F loaded Adnectin, where imaging could be completed on the same day as injection and antibody-based reagents may take several days of radiation burden to clear non-specific binding. This was further confirmed after loading the Adnectin with 64Cu where the monobody was able to provide same-day PET visualisation of tumour cells, resulting in an increase in the monobody uptake in tumours over 24 h post-injection. This concept has been taken even further with a Centyrin domain evolved to bind EGFR and conjugated with a fluorescent dye, which was then applied to guide surgical removal of tumour cells [70].
• #14: A core advantage offered by the monobody domain is the absence of a disulphide bond that antibodies may require for stability; this is an advantage, specifically in the reducing cytosolic environment. current immunohistochemistry methods of detecting proteins inside cells often require a cell to be fixed, permeabilised and then soaked with antibodies, which is highly disruptive to the natural environment within a cell.  genetically tagging target proteins with GFP-based fluorescence alters the expression and location of endogenous proteins.   In developing approaches to monitor endogenous proteins in the cell, the FN3 domain was applied as an internal reporting agent of endogenous proteins in live cells. This application was attempted in neuronal cells, where visualising endogenous proteins provided a means of understanding neuronal structure and function.  These intracellular monobodies were then applied to visualise dynamics in living cells without altering expression or location of the endogenous targets. This work was taken even further with the development of state-selective intracellular binders of Ras, a commonly mutated oncoprotein in tumours with poor prognoses. The anti-RAS monobodies specifically targeted K- and H-Ras, two variants that are the most commonly mutated genes in human tumours, and were demonstrated to be highly specific tools for monitoring the conformational or mutational state of RAS proteins while also potentially modulating their signalling pathways.
• #15: Although complete mutational knockouts are important for understanding the importance of a protein to its network, a single protein may interact with an average of four partners in multiple pathways which are all disrupted in a protein knockout. To overcome this, FN3 domains evolved to bind proteins in the Wnt signalling pathway were expressed endogenously to block intermolecular interactions of individual domains within a quaternary protein assembly.  This approach allowed signalling proteins to be annotated by individual domain [71] and was taken into mouse models to target WDR5, a component of mixed lineage leukemia, to effectively suppress leukemogenesis. Further to blocking activity in endogenous intracellular proteins, the monobody domain has been combined with ubiquitin-tagging domains to selectively induce degradation of those intracellular proteins
• #16: Multimeric applications. (A) FN3 domains on the fibronectin chain exhibit binding to multiple partners. Mimicking this beads-on-a-string approach quickly produces (B) bivalent, (C) tetravalent and (D) bispecific monobody constructs. (E) Furthering this fusion approach, tandem monobodies are fused with domains which confer longer circulating half-life or (F) greater avidity.
• #17: Applying monobody advantages to the antibody scaffold. Monobodies are fused with antibody fragments to extend half-life and generate bivalency. Replacing either (A) Antigen-binding fragments or (B) individual variable domains. (C) mAbtyrins extend this combination through developing by specifics by fusing monobodies to 1 of 4 positions on the C- or N-terminal ends of either chain.
• #18: (A) monobody fragment is generated from fragments involved in binding (orange), where the original monobody undergoes loss-of-function mutations in residues to remove affinity to the target and loss-of-stability mutations to disrupt beta-sheet packing (Red). (B) The presence of ligand and fragment allow a fragment-exchange complex to form that brings a Forster Resonance Energy Transfer (FRET) pair together, providing biosensors for the presence of a ligand.
• #24: Knottins (ICKs), which have a macrocycle formed by disulfide bonds between cysteines I and IV and II and V, through which cysteines III and VI are bonded. Cyclotides (CCKs), which include an N-terminal to Cterminal ‘head-to-tail’ peptide bond. Growth factor cystine knots (GFCKs), which have a macrocycle formed by disulfide bonds between cysteines II and V and III and VI, through which cysteines I and IV are bonded. Representative structures taken from PDB are EETI
• #25: This peptide is a component of the venom used by the animals to immobilize its prey. Ziconotide binds and antagonizes the N-type voltage-sensitive calcium channels (NVSCCs) abundant in nerves involved in pain signaling with low picomolar affinity.  In a rat model of neuropathic pain, Ziconotide was found to be more effective than morphine. Ziconotide is approved for intrathecal administration to patients who experience severe chronic pain and who are refractory to other treatments.
• #26: Linaclotide is a high-affinity agonist of guanylate cyclase-C (GC-C). In rodent models, oral administration of linaclotide resulted in increased gastrointestinal transit and reduced visceral pain.
• #28:  these molecules non-immunogenic in humans. 
• #29: The Fynomer 2C1 was engineered via phage display to bind the proinflammatory cytokine interleukin-17A (IL-17A)   2C1 was subsequently fused to the Fc domain of a human antibody to prolong its circulation half-life Fynomer-Fc fusion proteins represent new drug candidates for the treatment of IL-17A mediated inflammatory conditions such as psoriasis, psoriatic arthritis, or rheumatoid arthritis.
• #30: Inspired by the success of the 2C1-Fc fusion, the same group subsequently engineered FynomAb COVA322 Bispecific FynomAb COVA322 inhibited IL-17A and TNF with in vitro 
• #31: Anticalin proteins are an emerging class of clinical-stage biopharmaceuticals with high potential as an alternative to antibodies.
• #32:  retinol-binding protein (RBP) transports the poorly soluble and chemically sensitive vitamin A from its storage site in the liver through the bloodstream to various tissues.  apolipoprotein D—in plasma associated with high-density lipoprotein (HDL) particles—is involved in the transport of progesterone and arachidonic acid.  Other lipocalins have scavenger functions, such as the neutrophil gelatinase-associated lipocalin [NGAL], which tightly binds certain bacterial siderophores and thus restricts iron supply to invading microbes 
• #33: Natural lipocalins are abundant plasma proteins in humans, and compact fold dominated by a central β-barrel, supporting four structurally variable loops that form a binding site. Reshaping and reconstruction of different loops can lead to development of antcalin protein that can recognize wide range of medically relevant targets, from small molecules to peptides and proteins.
• #34: five Anticalin therapeutic candidates have reached the clinical development stage.  first Tlc-derived Anticalin protein was selected against human vascular endothelial growth factor (VEGF)-A, a key factor of tumor angiogenesis and ocular diseases.  Intravitreal administration of the Anticalin protein in a rabbit model suppressed VEGF-induced blood–retinal barrier breakdown. PEGylated PRS-050 (“Angiocal”) was subjected to a first-in-human, dose-escalation phase I study in patients with advanced solid tumors. significant reductions in circulating matrix metalloproteinase 2 (MMP-2) levels indicated an anti-angiogenic effect. An Anticalin protein that targets proprotein convertase subtilisin/kexin type 9 (PCSK9) showed convincing preclinical activity in the treatment of dyslipidemia. PCSK9 binds to the low-density lipoprotein receptor (LDL-R), inducing its internalization and degradation. An anti-PCSK9 antibody blocking the binding of PCSK9 to LDL-R was demonstrated to lower plasma levels of LDL cholesterol (LDL-C) and, consequently, led to fewer cardiovascular events in patients with atherosclerotic cardiovascular disease receiving standard statin therapy. PRS-080 is another Anticalin protein with prolonged circulation, this time via site-specific PEGylation, which targets hepcidin (hepatic bactericidal protein) alias liver-expressed antimicrobial peptide 1 (LEAP1). Hepcidin plays a major role in the regulation of iron metabolism [36], particularly in patients with functional iron deficiency (FID) anemia [37]. FID is a major cause of anemia of chronic disease (ACD), which particularly develops in patients with infections, inflammatory diseases, cancer, or chronic kidney disease (CKD). Routine use of erythropoietin-stimulating agents (ESAs) and high-dose intravenous iron supplementation corrects the condition in most patients; however, ESAs may result in a number of adverse clinical outcomes, and concerns about the long-term safety of intravenous iron administration have been raised. Hepcidin binds to ferroportin, the only iron transporter on the surface of absorptive enterocytes, macrophages, hepatocytes, and placental cells, and causes its internalization and degradation [40], thereby blocking iron export from the body’s depositories and reducing its availability for erythropoiesis [37]. Consequently, antagonizing hepcidin has the potential to improve iron availability and erythropoiesis while avoiding overload with exogenous iron and, thus, may allow lower dosing of ESAs
• #35: The concept of costimulatory T cell engagement. Following binding of the Anticalin–antibody fusion protein to the tumor cell and interaction with a T cell in its vicinity, the clustering of the costimulatory tumor necrosis factor receptor (TNFR) 4-1BB provides a local co-activation signal to the latter, thus further enhancing its T cell receptor-mediated activity and leading to tumor destruction. As T cells in the periphery should not get activated, toxic side effects are expected to be manageable. HER2 human epidermal growth factor receptor 2
• #36: Formatting opportunities for Anticalin proteins. Anticalin therapeutics can be developed as stand-alone small biologics but can also be used as building blocks to generate multispecific fusion proteins. Multispecific Anticalin-based biologics are accessible by mutually fusing several Anticalin proteins or by fusing Anticalin proteins to antibodies or an Ig Fc part. mAb monoclonal antibody
• #37: Kunitz-type protease inhibitors belong to the family of serine protease inhibitors that are found in almost all organisms.  Kunitz proteins have been involved in various physiological processes such as blood coagulation, fibrinolysis, inflammation, and ion channel blocking 
• #38: hereditary angioedema (HAE), a rare, autosomal dominantly inherited blood disorder that manifests as an episodic swelling of the body 
• #39: DX890 was shown to reduce neutrophil trans-epithelia migration and inflammation ex vivo
• #42: A – the structure of the consensus ankyrin repeat; the constant part is shown in gray; the variable regions are shown in red.  B – the structure of a DARPin molecule. Two or three binding motifs form the binding surface through variable amino acids (shown in red); the hydrophobic regions are shielded by the C-cap and N-cap.  C – 3D structure of a DARPin molecule, the variable amino acids are shown in red
• #43:  a Ribbon representation of an ankyrin repeat domain (PDB ID: 1MJ0) consisting of five repeats, with randomized interaction colored in orange and a transparent surface superimposed.  b Molecular model of MP0250, a DARPin® drug candidate consisting of four DARPin® domains (molecular weight approximately 62 kDa). The scaffold is dark blue, and the potential target interaction residues of the individual domains are orange (human serum albumin [HSA]), cyan (hepatocyte growth factor [HGF]), and red (vascular endothelial growth factor [VEGF])
• #44: MP0112 was tested in a series of clinical trials for treating age-related macular degeneration (AMD) and diabetic macular edema (DME), both of which are eye conditions that can cause significant vision impairment. Although the pathogenesis of these diseases is not completely understood, VEGF antagonists have been shown to retard the disease progression  MP0112 demonstrated encouraging results in phase I/II studies. In the DME trial, MP0112 was well-tolerated in patients and exhibited an ocular half-life of more than 13 days  Inflammation was reported for some patients, ostensibly due to impurities present in the protein preparation purified from the E. coli culture. Similarly, in the AMD trial, MP0112 was effective for up to 8 weeks following a single intraocular dosage with inflammation reported in some patients. Subsequently, the protein purification process was improved, and MP0112 was reformulated to contain a PEG molecule and renamed as Abicipar Pegol.  Phase I/II trials of Abicipar Pegol showed lower incidence of inflammation when compared to the trials using MP0112. Abicipar clinical phase III data indicate that the drug candidate has the potential to reduce the need for frequent injections in ophthalmic diseases.
• #45: The molecule is also able to bind human serum albumin (HSA), conferring it with an increased serum half-life and potentially enhanced tumor penetration.
• #47: DARPins can inhibit cell signaling molecules, thus suppressing cell proliferation, or serve as targeting modules for the delivery of various agents: radionuclides, nanoparticles or liposomes, photosensitizers, protein toxins, oncolytic viruses, and lymphocytes with chimeric antigenic receptors. HER2 – human epidermal growth factor receptor 2; NP – nanoparticle; ROS – reactive oxygen species; PI3K – phosphoinositide-3-kinase; Ras – small GTPase Ras; CAR – chimeric antigen receptor; CAR-T – T-lymphocyte expressing the chimeric antigen receptor; FAS – death receptor (CD95, APO-1), an inducer of extrinsic apoptosis pathway; FASL – ligand of the FAS receptor (CD95L, CD178); ETA – truncated Pseudomonas aeruginosa exotoxin A
• #51: affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. more than 400 studies have been published in which affibody molecules have been selected against more than 40 different targets
• #52: Large ‘naïve’ libraries of affibody molecules are constructed via combinatorial protein engineering of typically 13 positions (green) in helices one and two (H1/H2) of the 58-residue cysteine-free three-helix bundle Z-domain scaffold (brown). Selection of target-binding variants, including optional affinity maturations, can be performed using any suitable technology, including phage, cell, or ribosome/mRNA display. Selected variants can be recombinantly produced in several different hosts (e.g., bacteria, yeast, mammalian, or insect cells), either as single-affinity domains or as fusion partners (N- or C-terminal) in a variety of recombinant contexts (e.g., for circulation half-life extension, bivalency, bi/multispecificity, or toxin fusions). Addition of a unique cysteine (e.g., in the N or C terminus) allows for site-specific labeling and/or conjugation to functionalizing groups (e.g., fluorophores, biotin, metal chelators, or cytotoxic drugs) using thiol-directed chemistry. Peptide-based chelators for technetium (Tc), for example, can be fused N or C terminally. Production using solid-phase peptide chemistry provides further options for site-specific modifications, also at internal positions (including click chemistries for crosslinking). Using the second-generation scaffold provides improved properties for synthetic production. Structure of the complex between a human epidermal growth factor receptor 2 (HER2)-binding affibody (brown with the 13 engineered positions in green) and its HER2 target (gray). For size comparison, one Fab fragment of the HER2-binding antibody trastuzumab (red), binding to a different epitope, is shown. The composite structure was generated in Pymol using the Protein Data Bank (PDB) entries 3MZW.pdb and 1N8Z.pdb.
• #53: Affibodies are protein fragments derived from the Z-domain of the Ig-binding region of Staphylococcus aureus protein A which adopt a three-helix bundle motif and contain no cysteines. The small size (58 amino acids, 7 kDa) of affibodies allow them to be produced by chemical synthesis.
• #54: (A) Affibody molecules have been shown to exert biological activity by blocking various disease-associated protein–protein interactions. Examples include: blocking of receptor signaling by either ligand binding or receptor binding, as well as by binding to aggregation-prone parts of amyloid-forming peptides to inhibit aggregation
• #55: (B) Another well-investigated mode of action is affibody-targeted delivery of various payloads. Examples include fusions or conjugations to toxins and cytotoxic drugs, as well as targeted nanoparticles for drug delivery. Arming affibody molecules with a cytotoxic payload appears to be a seemingly straightforward and efficient way to direct the action of the payload to desired cell populations. The concept has been evaluated in different preclinical studies and, so far, the payload has been a cytotoxic substance (biologically derived toxin, cytotoxic drug, or therapeutic radionuclide) intended for cancer therapy.
• #56: (C) Various strategies have been reported for increasing selectivity to reduce adverse effects, thus widening the therapeutic window. Examples include affibody-based prodrugs, where masked (inactive) affibody molecules are proteolytically activated at the site of disease, and peptide nucleic acid (PNA)-based pretargeting strategies, in which the payload is decoupled from the affibody molecule and the two components are administered consecutively, followed by formation of the complex at the site of disease, to reduce uptake in nontargeted organs. The biodistribution properties and time points of administration of the components are critical parameters for the success of the pretargeting strategy One way to potentially reduce the adverse effects is to use a pretargeting strategy, where a tagged but unarmed targeting affibody is first injected and allowed to accumulate at the surface of the receptor-bearing tumor cells, while simultaneously being cleared from nontumor organs. This is followed by injection of the cytotoxic drug, which is modified to bind specifically to the tag of the targeting affibody and designed to be otherwise cleared rapidly without any extensive uptake in nontumor organs. I
• #63: Representative images of knottins used for in vivo imaging. BLZ-100 viewed with SIRIS (an optimized in vivo imaging system) illuminates injected LL229 human glioblastoma cells. BLZ-100 viewed with an Odyssey CLx Infrared Imaging System illuminates spontaneous medulloblastomas and metastases highlighted in pink. MicroPET imaging shows high signal-to-background with an engineered 64Cu-labeled knottin and minimal organ uptake in mice bearing U87MG human glioblastoma xenografts. Coronal SPECT/CT imaging shows moderate signal-to-background with an engineered 99mTc-labeled knottin in mice bearing HCC4006 human lung adenocarcinoma xenografts
• #64: Schematic illustration of duration of action engineering of abicipar. Drug concentration of abicipar (black line) and ranibizumab (gray line) are shown over time. Assuming reference ranibizumab is administered 50 μl/0.5 mg intravitreally, and the drug has a half-life in the 1.5 ml rabbit eye of 3 days and an inhibition constant IC50 of about 100 pM, then the drug would stop being efficacious when reaching a concentration equivalent to the IC50, i.e. duration of action of about 48 days. Three aspects help increase the duration of action of abicipar: (I) improving the potency to lower values in combination with high drug stability ensure that the drug is also active at very low amounts; (II) engineering the half-life as such, requiring high drug stability; (III) applying higher doses. Assuming abicipar exhibits an IC50 of about 25 pM [26], has an ocular half-life of 6 days in the rabbit eye, and that it can be applied at about 5.5 times the molar amounts of ranibizumab, then it would take 124 days (4 months) for the vitreal concentration of abicipar reaches the IC50 concentration of 25 pM
• #65: Schematic representation of the differentiation of localized immune cell modulator MP0310 from classical immune cell modulators. Antibodies activating immune cells via 4-1BB typically activate immune cells in the tumor as well as in healthy tissue, leading to promising efficacy linked to a problematic toxicology profile. MP0310 binds to fibroblast-activating protein (blue dots) on tumor-associated fibroblast and clusters, providing an opportunity for immune cell activation. In healthy tissue, with a lack of opportunity to cluster, immune cells are not modulated by MP0310. mAb monoclonal antibody
• #66: A schematic overview of all development-stage DARPin® drug candidates depicted on the two dimensions “degree of novel biology” vs. “degree of novel drug concept” on the drug development programs in the context of the three-horizon strategy of Molecular Partners. A first-horizon program such as abicipar is built on known biology and takes advantage of the favorable DARPin® drug properties for the generation of a “biobetter compound.” Second-horizon programs such as MP0250 and MP0274 are based on biology that has been validated in third-party phase II clinical trials; at the same time, they are multidomain drug candidates, a key strength of the DARPin® platform. In third-horizon programs, biology may be less validated and novel drug concepts may be applied, as illustrated by MP0310 (see Fig. 5) and MP0317
• #67: (A) The Type 3 Fibronectin domains 7 to 10 from human fibronectin with the original RGD binding sequence highlighted in red. 
• #69: (D) amino acid sequence, sharing only the F-Strand sequence across the domains, which leads to (E) a large variation in overall sequence identity between derivatives. Colouring: (D) Sequence alignment: */green—identical amino-acid,:/purple—strongly similar,/blue—weakly similar.; (E) Sequence pairwise identity matrix: green—highly identical sequences, pale green—strongly identical sequences, pink—weakly identical, red—low identical amino-acid matches.
• #71: Affibody dimers are shown in gold. Please note that these peptide-specific binders have an unusual and relatively complex interaction mechanism that is distinct from that of typical affibody molecules, involving structural rearrangements of both the affibody molecules and the target peptide upon binding. The first part of each affibody domain that is typically folded into an a helix is instead forming a b strand (highlighted in red) and the aggregation prone peptide is folded into a b hairpin conformation; together, they comprise a four-stranded b sheet. Affibody dimer in complex with a-synuclein peptide (in orange) Affibody dimer in complex with amyloid b peptide in blue. Affibody dimer in complex with human islet amyloid polypeptide