Biotechnology Advances 61 (2022) 108032 Contents lists available at ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper Protein scaffolds in human clinics Olivia Cano-Garrido a, 1, Naroa Serna a, b, 1, 2, Ugutz Unzueta b, c, d, e, 1, Eloi Parladé a, b, 1, Ramón Mangues d, e, Antonio Villaverde a, b, c, *, Esther Vázquez a, b, c, * a Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès (Barcelona), Spain b CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Instituto de Salud Carlos III, 08193 Cerdanyola del Vallès (Barcelona), Spain c Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès (Barcelona), Spain d Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025 Barcelona, Spain e Josep Carreras Leukaemia Research Institute, 08916 Badalona (Barcelona), Spain A R T I C L E I N F O A B S T R A C T Keywords: Fundamental clinical areas such as drug delivery and regenerative medicine require biocompatible materials as Protein materials mechanically stable scaffolds or as nanoscale drug carriers. Among the wide set of emerging biomaterials, nanomedicine polypeptides offer enticing properties over alternative polymers, including full biocompatibility, biodegrad­ drug delivery ability, precise interactivity, structural stability and conformational and functional versatility, all of them tunable regenerative medicine, self-assembling human protein by conventional protein engineering. However, proteins from non-human sources elicit immunotoxicities that humanization might bottleneck further development and narrow their clinical applicability. In this context, selecting human proteins or developing humanized protein versions as building blocks is a strict demand to design non- immunogenic protein materials. We review here the expanding catalogue of human or humanized proteins tailored to execute different levels of scaffolding functions and how they can be engineered as self-assembling materials in form of oligomers, polymers or complex networks. In particular, we emphasize those that are under clinical development, revising their fields of applicability and how they have been adapted to offer, apart from mere mechanical support, highly refined functions and precise molecular interactions. 1. Introduction is provided by mineral-based complexes (Boskey, 2013). Mainly formed by calcium and phosphorus, the resulting trabecular structures support Structure supports life. Then, living beings are mainly composed by the biomechanical profiling of the whole body and the complex func­ soft matter (cells and tissues). Being highly hydrated and with a primary tions related to both physical resting and movement (LeVeau and gel-like cell organization, those materials require supporting structures Bernhardt, 1984). to acquire sufficient mechanical stability to endure environmental Organic scaffolds include membranous systems and proteins. Among pressures (Dalby et al., 2014; Guimarães et al., 2020) and to interact them, proteins provide multidimensional structural stability and a with the media through bi-directional mechanical signals (Kumar, notable tensegrity (tensional integrity), required for cell sensing, 2014). From lower to higher organization levels, cytoskeleton, mem­ migration and morphogenesis, and to keep the geometry of mature cells branous systems, cell walls, extracellular matrices, cartilage, bones, against deformation forces (Chen et al., 2010; De Santis et al., 2011; xylem and exoskeletons sustain, at different levels of stiffness, the Volokh, 2011; Volokh et al., 2002). These properties are combined with complex functional dynamism required for life (Brule et al., 2016; Chen an unusually dynamic functional versatility, in part mediated by the and Ingber, 1999; Deville and Cordes, 2019). Some of these scaffolds capability of proteins to respond to external stimuli by conformational recruit inorganic components for tailored functionalities. Combined changes (Boehr et al., 2018; Huck, 2008; Orellana, 2019; Shah et al., with cells and organic protein matrices, the resulting composites are 2018; Ulijn and Lampel, 2020). Such a blend of mechanical stability and particularly robust platforms. In higher animals, bones are representa­ responsiveness is not observed in any other organic or inorganic mate­ tive of such hybrid materials, in which approximately 70 % of the mass rial. Protein scaffolds usually consist of combinations of several * Corresponding authors at: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès (Barcelona), Spain. E-mail addresses: Antoni.villaverde@uab.es (A. Villaverde), Esther.vazquez@uab.es (E. Vázquez). 1 Equally contributed. 2 Present address: Nanoligent SL. Edifici Eureka, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain. https://doi.org/10.1016/j.biotechadv.2022.108032 Received 30 April 2022; Received in revised form 30 July 2022; Accepted 3 September 2022 Available online 9 September 2022 0734-9750/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). O. Cano-Garrido et al. Biotechnology Advances 61 (2022) 108032 polypeptides or multiple copies of a single protein species, that form combine mechanical properties and refined functionalities. The spec­ supramolecular structures supported by self-assembling domains. Found trum of those protein-based platforms (Fig. 1) spans from single poly­ in many polypeptides, cross-interacting protein motives allow gener­ peptidic chains as mere drug carriers or stabilizers (such as human ating a wide spectrum of protein materials, in form of fibers, particles, serum albumin, HSA, in the paclitaxel nanoscale formulation called cages, matrices or layers (Corchero et al., 2014). These elements sustain Abraxane (Ma and Mumper, 2013), Table 1) to complex macromolecular subcellular structures such as amyloids, viral capsids and vaults, but also entities such as protein-based matrices, hydrogels or related architec­ whole cells, tissues and organs. Furthermore, protein materials show tures. Mimicking the extracellular matrix (Bhattacharjee et al., 2017; supportive roles out of the body. Tools involved in biofilm formation in Wu et al., 2018), these later offer mechanical support in regenerative single-cell organisms (pili and related bacterial structures (Epler Bar­ medicine (Fig. 1, Table 2). In an intermediate concept, relatively simple bercheck et al., 2018)), adhesive pads in climbing plants (Burris et al., nanoscale oligomers (Cespedes et al., 2018a; Molino and Wang, 2014) 2018) and animals (Hallahan et al., 2009), viscoelastic gels in marine (including virus-like particles (Hill et al., 2018)) are highly convenient invertebrates (Smith, 2002), adhesive worm secretions (Corrales-Ureña as selective carriers for targeted drug delivery and theragnosis (Fig. 1, et al., 2017), silks in spiders (Yarger et al., 2018) and underwater ad­ Table 1). Most of these materials result from the biological fabrication of hesive fibers of mussels (Park et al., 2019) among others, are based on recombinant building blocks in cell factories followed by spontaneous or particular protein species often found as nanostructured entities. induced assembly. The recombinant production approach makes thus A significant part of industries relies on the fabrication of materials possible a genetic tuning of the amino acid sequence of such poly­ with supportive roles. When looking for application in biological in­ peptides (Corchero et al., 2013). Aiming at enhancing biocompatibility, terfaces, apart from the needed geometry and mechanical properties, humanizing the resulting materials, mostly based on non-human pro­ biocompatibility and biodegradability are required to avoid the often teins, is a particularly challenging task. In the next sections, the observed toxicity linked to xenobiotic substances (Raftis and Miller, complexity of such clinically-oriented protein materials is exemplified 2019; Yuan et al., 2019). Because of the above-mentioned properties of by three main categories, namely plain monomeric polypeptides, natural proteins and their intrinsic biocompatibility, they emerge as nanoscale oligomers and complex protein networks. ideal biomaterials in clinics. Apart from the mimicry of natural protein functions in synthetic constructs (Hansen and Khare, 2020; Wang et al., 2020; Xu et al., 2020) the capability of de novo designing polypeptides expand the spectrum of application. This would cover functionally inert entities useful as plain scaffolds but also bioactive materials that Fig. 1. Engineering and application of protein scaffolds in biomedicine. In the vertical axis, different protein oligomerization levels are presented, spanning from plain monomeric forms to progressively complex structures. Two main sets of applications are indicated; while monomeric or nanoscale protein complexes have been mainly used as carriers for drug delivery (upper section), more complex polymers or networks (usually within the microscale) are essentially tailored as scaffolds in the context of tissue engineering (bottom section). On the other hand, the horizontal axis indicates the extent of protein engineering. It starts from plain recombinant versions of natural proteins and moves towards conferring advanced functions, including oligomerization (by engineering protein-protein contacts) or the incor­ poration of functional domains such as toxins, growth factors or hormones, by their genetic fusion to the building blocks. The abolition of undesired interactivity of these proteins with cell or body components and their de-immunization or humanization is in general required when intended for systemic administration. 2 O. Cano-Garrido et al. Biotechnology Advances 61 (2022) 108032 Table 1 Table 2 Representative examples of polypeptides used as scaffolds in drug delivery. Representative examples of polypeptides used as scaffolds in regenerative Protein Origin Form Application or Reference medicine. target tissue Protein Origin Form Application Reference or target (Klem et al., tissue Ferritin Human Nanocages MRI contrast 2008) (Zhen et al., Self-assembling (Achilli et al., Collagen Human Vascular Cancer therapy 2013) lattices 2012) (Alamo et al., Cross-linked (Merrett et al., Nidogen G2 Human Nanoparticles Cancer therapy 2021a) neoglycopolymer Cornea 2009) (Woodman Hemostat and (Spotnitz, Fibrin Human Sealant Stefin A Human Unassembled Peptide display et al., 2005) wound repair 2014) (Serna et al., (Kneser et al., Bone Nanoparticles Cancer therapy 2022) Hydrogel 2005; Peretti regeneration (Serna et al., et al., 2006) CTP Human Nanoparticles Cancer therapy 2022) Non- (Arkudas et al., Unassembled, (Gradishar, Silk human Hydrogel Angiogenesis 2007) Albumin Human clustered Cancer therapy 2006) (Ding et al., (Chen et al., Fibroin Bone 2021) Nanoparticles Bioimaging 2016) Non- Wound (Suzuki et al., Ocular drug (Tiwari et al., Gelatin human Gel dressing 2013) Nanoparticles delivery 2021) Nerve (Sierpinski Elastin Human Nanoparticles Cancer therapy (Hu et al., 2015) Keratin Human Hydrogel regeneration et al., 2008) (Taylor and (Aboushwareb Nerve Sakiyama- Hydrogel Hemostat et al., 2009) Fibrin Human Matrix regeneration Elbert, 2006) Cation formed Wound (Fujii and Ide, Multivalent film dressing 2004) Chaperonin peptide display (Alsultan et al., Proof-of- 10 Human Nanoparticle and targeting. 2016) Protein cross- concept (Bloom and Albumin Human linking (bone) (Li et al., 2014) Calabro, 2009; Non- Proof-of- (Chien et al., Peptide display Getmanova Soy protein human Freeze-dried concept 2013) FN3 Human Unassembled and targeting et al., 2006) Proof-of- (Ramji and (Goldberg et al., Nanofiber concept Shah, 2014) Targeted drug 2016; Klein Proof-of- Chien et al., Centyrin Human Unassembled delivery et al., 2021) 3D bioprinting concept 2013 Targeted (Klein et al., (Seras-Franzoso Unassembled imaging 2021) FGF-2 Human Inclusion bodies Wound repair et al., 2014) (Shen et al., Artificial (Serna et al., mCH3 Human Unassembled Cancer therapy 2019) inclusion bodies Wound repair 2020) Non- (Kundu et al., Non- (Stamm et al., Silk human Hydrogel Drug delivery 2012) Lipoxygenase Human Inclusion bodies Wound repair 2018) Non- (Rouse and Van (Fernández- Keratin human Sponge Drug delivery Dyke, 2010) Tissue Colino et al., Non- (Cespedes et al., Elastin Human Hydrogel engineering 2018) GFP human Nanoparticles Cancer therapy 2018b) genetically fused, to be internalized triggering cytotoxic activity in 2. Results and discussion cancer cells. Both types of antibody-based materials must be adminis­ tered at their maximal tolerated dose to be effective, a procedure that 2.1. Single polypeptides as plain drug carriers often associates with severe side effects. Therefore, their clinical use is usually limited to a single cancer type that lacks an effective therapy Some proteins themselves are drugs, which do not need additional (Jen et al., 2020; Serna et al., 2018). The side effects induced by the structural support apart from the appropriate formulation. Enzymes ADCs and ITs could be due to the induction of cytotoxicity through the used in enzyme-replacement therapies, (Li, 2018; Marchetti et al., 2022) release of the transported drug on normal cells that express the targeted antibodies or antibody fragments in oncotherapy (Duan and Luo, 2021) receptor (on-target effect). Moreover, the adverse effects could also have an intrinsic dual role as drug and structural agent. For instance, derive form the lack of biological neutrality of the antibody scaffold that antibody-only drugs can suppress tumor growth, either by inhibiting interacts with Fc receptors expressed on normal cells (off-target effect) signaling from specific receptors in cancer cells to induce their death (Criscitiello et al., 2021; Wilkinson et al., 2021). In addition, both (Trastuzumab, Cetuximab), by blocking endothelial receptors to inhibit particular nanomedical approaches have been extensively reviewed, tumor angiogenesis (Bevacizumab), or instead by blocking immune also by us (Sanchez-Garcia et al., 2016; Serna et al., 2018), and are out of checkpoints to activate the immune system against the tumor (Pem­ the scope of the present review that focuses on the plain scaffolding uses brolizumab, Nivolumab, Ipilimumab) (Wong et al., 2021; Zahavi and of proteins in clinics. Weiner, 2020). Antibody-drug nanoconjugates (ADCs), such as Gemtu­ On the other hand, different categories of recombinant, single-chain zumab Ozogamicin (Jen et al., 2018), Trastuzumab Deruxtecan (Keam, polypeptides have been exploited, as natural or engineered forms, to be 2020), Brentuximab Vedotin (Richardson et al., 2019) and others used as convenient partners for drug delivery. They are intended for (Kadkhoda et al., 2021; Lu et al., 2021; Serna et al., 2018) use the stabilizing the drug or for increasing the size of the whole pharmaco­ antibody scaffold to achieve targeting to specific cancer cells to subse­ logical complex, thus preventing renal clearance. In advanced con­ quently trigger the release of conjugated conventional drugs in their structs, the carrier might also confer cell type selectivity in the delivery cytosol. Similarly, immunotoxins (ITs), such as Denileukin difitox, process. The human serum albumin (HSA) was the first FDA-approved Tagraxofusp-erzs or Moxetumomab pasudotox (Khirehgesh et al., 2021; human protein scaffold for drug delivery (in form of Abraxane (Gra­ Kreitman and Pastan, 2021) also use antibody scaffolds as targeted dishar, 2006)). Its structure is well known, stable, and non- carriers to transport a bacterial toxin, either covalently linked or immunogenic, exhibiting high biocompatibility and long plasma 3 O. Cano-Garrido et al. Biotechnology Advances 61 (2022) 108032 circulation time. In contrast, HSA presents complex post-translational extracellular matrix components and thus, to generate a GFP-like non- modifications and multiple interactions with receptors in several body fluorescent human scaffold, called HSNBT (Alamo et al., 2021a). This tissues (Merlot et al., 2014), what impedes biological neutrality and was achieved by applying in silico-predicted mutations of four residues aborts any potential for receptor-targeted delivery. In this sense, a plain implicated in the interaction with collagen IV and perlecan, that resulted HSA has been widely used as human scaffold for several nanomedical into a biologically neutral, novel protein scaffold successfully validated applications (An and Zhang, 2017) in absence of precise engineering in drug delivery (Alamo et al., 2021a). addressed to improve its performance. Interestingly, alternative human The human chaperonin 10 (hCpn10) is an intracellular homo- scaffolds have also attracted attention as tools for drug delivery. Still oligomeric protein composed by 7 subunits that assist protein folding under development, they exhibit some superior properties to those or re-folding. It is a stable beta-barrel core that does not undergo post- shown by HSA, including high manipulability and less molecular translational modifications, shows low immunogenicity and it has interactivity. In contrast to HSA, these alternative scaffolds have been been safely tested in clinical trials (Alsultan et al., 2016; Broadley et al., engineered at different levels of complexity, paying special attention to 2009). The loop responsible for the natural interactivity of Cpn60 has strategies to oligomerize them in form of multimeric materials, by a been successfully substituted for different target-specific ligands, thus proteomic control of cross-molecular contacts and self-assembling. The preventing the native binding and conferring multivalent ligand display views and concepts underlying these strategies are illustrated in the next with different clinical applications. Molecular dynamic modelling was section through representative examples. used to design and insert linkers at the junctures of the mobile loop to prevent interferences of inserted ligands with the subunit interface and 2.2. Nanoscale protein oligomers in drug delivery to maintain the heptameric structure that allows multivalent peptide presentation (Alsultan et al., 2016). In the context of drug delivery, Stefin A-derived proteins represent a The human 10th fibronectin type III domain (FN3) is a structurally paradigmatic example of rational design of human protein scaffolds for stable small protein with a beta-sandwich fold similar to the variable nanomedical applications (Woodman et al., 2005). Stefin A is an intra­ domain of antibodies. It shows no post-translational modifications and cellular small single-chain, single-domain protease inhibitor with high consequently it has been successfully produced in a variety of eukaryotic structural stability and no post-translational modifications. Its structure and prokaryotic expression systems. Moreover, its high levels in extra­ has been also completely solved and solvent-exposed regions that are cellular body fluids permit to envisage minimal immunogenicity for any suitable for drug conjugation or peptide presentation have been iden­ FN3-based scaffold (Chandler and Buckle, 2020; Koide et al., 1998). FN3 tified (Jenko et al., 2003). On this basis, a structurally stable variant naturally binds multiple targets involved in the formation of the extra­ called STM (Stefin A triple mutant) was created so that it abolished the cellular matrix, and natural ligand-binding loops have been successfully natural interactivity of the protein. This version is based on two selected engineered to confer new affinities for alternative therapeutic targets. amino acid substitutions involved in protein-protein interactions plus an For that, the human FN3 has been used as a scaffold to display peptide additional modification in a solvent exposed loop to allow peptide libraries in randomized loops in directed protein evolution studies and insertion for surface presentation (Woodman et al., 2005). Through this to generate new target-specific proteins, called monobodies (Bloom and engineered accommodation site, STM has been successfully used as a Calabro, 2009; Getmanova et al., 2006). A derived scaffold based on two scaffold to display peptide libraries (Woodman et al., 2005) and to different proteins has been generated by a consensus sequence approach perform specific target protein interactions (Evans et al., 2008). Also, of different FN3 domains within human Fibronectin and Tenascin-C and by engineering its self-assembling through short end-terminal ar­ (Centyrins). Additional rational design was then applied to still in­ chitectonic peptides, STM-based oligomeric nanoparticles have been crease the structural stability of the consensus FN3 domain. This com­ used to selectively deliver the ultra-potent anti-mitotic drug mono­ plex approach resulted in a very stable scaffold that was robustly methyl Auristatin E in acute myeloid leukemia models (Serna et al., expressed in prokaryotic systems such as E. coli and was also able to 2022). Later, the versatility of the scaffold has been expanded by display functional peptides accommodated in its exposed loops (Jacobs introducing additional multiple peptide insertion sites (SQM, Stefin A et al., 2012). Later, an EGFR-targeted FN3 consensus scaffold has been quadruple mutant) (Hoffmann et al., 2010). A trivalent SQM version has further engineered to introduce site-specific Cys residues to be used for proved to be successful in the detection of target proteins (Song et al., drug conjugation, devoted to generate a receptor-targeted drug delivery 2011) and in simultaneous peptide presentation (Hoffmann et al., 2010). platform. In this sense, centyrins have been then successfully conjugated Starting from such trivalent SQM version, a new variant has been with MMAF (Goldberg et al., 2016) or siRNA (Klein et al., 2021) mol­ generated called SQT (stefin A quadruple mutant-Tracy), with signifi­ ecules to be specifically delivered into EGFR+ cells. The same EGFR- cantly improved structural stability and higher tolerance to multiple targeted FN3 scaffold has been also used for fluorescent dye conjuga­ peptide presentation (Stadler et al., 2011). SQT has been used for the tion and in vivo fluorescence-guided surgery applications (Mahalingam presentation of BH3 domains for interaction with pro-apoptotic effectors et al., 2017). In a similar approach, the antimicrotubule agent DM1 has (Stadler et al., 2014). Recently, it has been shown that SQT with AU1 been conjugated in a Cys-functionalized consensus FN3 scaffold (Shi and Myc peptide insertions (called SQT-1C) spontaneously oligomerized et al., 2018). into dimers and tetramers by a loop-mediated domain swapping process Finally, the human Ig-G constant domain (Fc) is a homodimer pro­ (Zalar et al., 2019). The slightly minimized stability of this mutant was tein composed by one glycosylated CH2 and non-glycosylated CH3 do­ recovered by introducing a disulfide bond that locked the monomeric mains that show long half-live in plasma but no interaction with the state (Zalar and Golovanov, 2019). target antigen due to the missing variable region. However, the Fc As a further example, the human nidogen is a multi-domain struc­ domain still naturally binds different effectors such as Fcɣ receptors, the tural protein from the basement membranes that naturally binds FcRn receptor and the complement factor C1q. In this sense, several collagen IV, perlecan and laminin (Takagi et al., 2003). Interestingly, the mutations that inhibit the interaction with most of those receptors have G2 domain of nidogen shows a stable β-barrel structure identical to already been described (Davis et al., 2007; Glaesner et al., 2010). Other Aequorea Victoria Green fluorescent protein (Hopf et al., 2001), which mutations have also been reported to minimize the interaction with the has already proved to be structurally robust and stable in blood circu­ FcRn receptor (Jain et al., 2007). To generate a less complex scaffold, lation when used in oligomeric vehicles for drug delivery (Cespedes several mutations have been introduced that generate highly stable et al., 2018b; Pallares et al., 2020). Moreover, since this protein natu­ monomeric Fc versions, that can be efficiently produced as non- rally lacks post-translational modifications, its easy production in bac­ glycosylated forms in E. coli (Ying et al., 2012). Also, an unglycosy­ terial cell factories is feasible. Then, the G2 domain of the human lated CH2 domain has been further isolated and produced in bacteria nidogen has been recently engineered to abolish its natural binding to resulting in a smaller protein scaffold with conserved structure 4 O. Cano-Garrido et al. Biotechnology Advances 61 (2022) 108032 (Prabakaran et al., 2008). Its lower stability and aggregation tendency (Hussey et al., 2018), and it is expected to offer sufficient plasticity, has been further corrected by the deletion of several critical residues biocompatibility and biodegradability in a range appropriate for given (Gong et al., 2013). Similar strategies have been used to develop an applications (Boekhoven and Stupp, 2014). In this context, protein isolated monomeric CH3 domain scaffold by introducing four punctual polymers are exploited as scaffolds in cartilage regeneration, wound mutations. Very interestingly, the new mCH3 scaffold shows weaker healing, vascular grafts and tracheal splints. Synthetic polymers such as FcRn receptor binding than Fc and a complete loss of Fcɣ and C1q re­ poly-glycolic acid (PLG) and poly-lactic acid (PLA) show relatively high ceptor binding. Moreover, the incorporation of an additional disulfide tunability (through the modification of the molecular weight and bond within the protein structure has considerably improved its stability crosslinking process) but relatively low capacity to induce appropriate (Ying et al., 2013; Ying et al., 2014). In this sense, the mutated mCH3 cell responses through mechanical and biological stimuli (Elmowafy scaffold has been successfully used to improve the pharmacokinetics of a et al., 2019). In addition, their capacity to affect the performance and genetically fused immunomodulatory Tα1 peptide by significantly activities of the immune system (through a recognized immunomodu­ increasing its circulation half-life (up to 47h) and its therapeutic activity lation potential) (Dobrovolskaia et al., 2016; Khademi et al., 2018) in tumor xenograft models (Shen et al., 2019). Other representative represents a matter of concern when used for non-vaccine purposes. examples are summarized in Table 1. Natural human and non human protein polymers such as collagen, Of course, the engineering of human proteins for functional gains or elastin and silk offer optimal biochemical signals for communication to confer new interactions or higher stability, even limited to specific with cells (Costa et al., 2018). In this sense, generating protein-only and usually short regions, might generate new non-natural epitopes with scaffolds exclusively made of human proteins is feasible, and a design potential to trigger an inappropriate immune response upon adminis­ based on a modular repetitive pattern would allow the ordered con­ tration in humans. Even plausible, such effects have not been generically struction of complex, humanized supramolecular structures (Corchero described (Fleishman and Mariuzza, 2022; Narayanan et al., 2021) even et al., 2014). This type of design should be based on repeating structural using non site-directed approaches, such as molecular evolution (Liu units of the natural protein components of human extracellular matrix et al., 2019), and protein engineering in the context of bio­ that include collagen, elastin and fibrillin, among others. These building pharmaceuticals has proved to be successful and safe (Lagasse et al., blocks self-assemble into hierarchical structures such as peptide fibrils 2017; Narayanan et al., 2021; Radziwon and Weeks, 2021). Importantly, that form the structural backbone of the biomaterial. The predominant the analyses of remote effects of mutations on protein structure and format to formulate these proteins is the hydrogel, which permits to stability offer a limited but important catalogue of alternative mutation incorporate cells in an aqueous media suitable for proliferation and targets in a protein engineering process (Wilding et al., 2019). Finally, it differentiation. Those hydrogels also provide the possibility of biological must be noted that the risk for immunotoxicities of modified human interactions for cell adhesion and degradability (Lee and Kim, 2018), proteins should globally remain much lower than when straightforward and they are used as a glue in many surgical uses (Ahmed et al., 2008; using non-human proteins. Apel et al., 2008). In addition, further protein engineering allows the incorporation of a 2.3. Protein scaffolds for complex materials in tissue engineering and set of biologically active proteins for enhanced functionalities (Bichara regeneration et al., 2012; Lorentz et al., 2011) (Fig. 1). The integrin RGD sequence either naturally present in keratin or added in elastin by protein engi­ Tissue engineering recruits a set of strategies aimed to regenerate neering increases cell adhesion and growth on scaffold surfaces (Daa­ damaged tissues assisted by biomimetic platforms, which combining men et al., 2007; Mogosanu et al., 2014). The use of the heparin-binding cells, scaffolds and bioactive factors should bring an appropriate envi­ site A from fibrin permits capturing growth factors for their slow release ronment for regeneration or improvement of tissue function (Fig. 2). The (Noh et al., 2015; Yang et al., 2010; Yang et al., 2011). Cross-linking scaffold moiety plays a critical role in presenting biochemical, structural scaffolds are also under development towards more stable sponge-like and mechanical properties similar to those of the extracellular matrix surface in which cells could adhere but at the same time allowing free Fig. 2. Paradigm of human protein-based materials for tissue engineering. In tissue engineering, plain scaffolds (left) can be functionalized or combined with functional agents (right) for multifaceted interactions with cells (center). FGF2: Fibroblast growth factor 2; VEGF: Vascular endothelial growth factor; BMPs: Bone morphogenetic proteins; NCAM: Neural cell adhesion molecule; tPA: tissue plasminogen activator; uPA: urokinase-type plasminogen activator; MMP: Matrix met­ allopeptidases; RGD: arginylglycylaspartic acid motif is the integrin recognition domain found in fibronectin; PHSRN: Fibronectin motif; CS5: residues 90-109 of the type III connecting segment region of fibronectin. IKVAV and YIGSR: Laminin motifs. 5 O. Cano-Garrido et al. Biotechnology Advances 61 (2022) 108032 movement of water and nutrients. These structures can be polymerized be used directly, or upon mutagenesis through directed evolution. The by protein–carbohydrate interactions (Merrett et al., 2009) and covalent incorporation of mutations looks for robustness and lack of interactivity proteins bonds (Li et al., 2014), among others. of the building block by introducing energetically-favorable residues From a different point of view, bacterial inclusion bodies are self- toward a proper folding, removing the affinity towards other molecules assembling secretory amyloids in between the nano- and micro-scales or tightening the tertiary structure by means of disulfide bridges, that act (de Marco et al., 2019), that spontaneously formed in recombinant as molecular-level staples (Bhardwaj et al., 2016; Silva et al., 2018). In bacteria provide mechanical and biological stimuli when decorating this regard, aside from the finite catalogue of available proteins, there is surfaces for cell growth, motility and differentiation (Martinez-Miguel a limitation in the number of mutations that can be introduced to a et al., 2020; Seras-Franzoso et al., 2015; Tatkiewicz et al., 2018). In­ protein without making it lose its native structure. Proteins have evolved clusion bodies formed by growth factors such as the human FGF-2, the to operate efficiently in specific cellular environments and temperature Mexican axolotl lipoxygenase and others offer a combination of me­ ranges, sometimes at the expense of unfavorable backbone angles chanical and biological stimuli favoring among other effects, wound (Herzberg and Moult, 1991) and often trading off stability for specific healing (Seras-Franzoso et al., 2014; Stamm et al., 2018). This is because functionality (Beadle and Shoichet, 2002; Schreiber et al., 1994). the forming protein is slowly released under physiological conditions Because of this compromise, an excessive number of stabilizing muta­ through a slow self-disintegrating process (Cespedes et al., 2020; tions leaves the protein at the risk of exhibiting a folded state that di­ Sánchez et al., 2022), and therefore, the functional building blocks are verges too much from the native conformation. This could make, even a available for biological activities. Synthetic versions of inclusion bodies, human protein, susceptible to activate an immunogenic response to­ fabricated in vitro from pure protein (Alamo et al., 2021b; López-Laguna wards unfamiliar epitopes. In this same context, aggregation-prone et al., 2021; Sanchez et al., 2020), offer regulatory friendly versions of proteins are known to easily trigger undesired immune responses such functional topographies (Serna et al., 2020). These and other ex­ (Sauerborn et al., 2010; van Beers et al., 2010). Then, a high solubility of amples are summarized in Table 2. a target protein is vital to secure its success as a scaffold or scaffold component. 2.4. De novo design of protein scaffolds On the other hand, the emergence of computational de novo protein design has revolutionized the protein engineering field, making it Apart from exploiting well-known proteins from nature, constructing possible to design a virtually infinite amount of sequences with unique de novo protein scaffolds for clinical applications is perfectly plausible structures not found in nature (Huang et al., 2016). The process starts (Fig. 3). Generically, the main traits of an ideal protein building block with the backbone design. Fragment assembly is generally the most intended for supportive roles should include those generically contrib­ successful method. It embraces the use of well-known structural motifs uting to protein stability and solubility, apart from a lack of undesired (5–50 residues) that can be chained in iteration to construct new pro­ interactivity. A robust candidate should contain properly arranged teins with either globular or non-globular architectures, depending on segments of secondary structure connected by short loops. Also, the the type and number of repeating units acting as components (Brunette hydrophobic residues should be tightly packed at its core and most of the et al., 2015; Parmeggiani and Huang, 2017). Alternative and analogous polar residues being surface-exposed or having satisfied its hydrogen approaches employ curated secondary structures of known proteins to bonding potential (Worth and Blundell, 2009). While listing the desired create new tertiary structures (Jacobs et al., 2016), or fully mathemat­ traits of a protein might be trivial, coming across a sequence that folds ical approaches (i.e. parametric design) to build arrangements of sec­ appropriately to satisfy such needs is not straightforward. ondary structures (Schafmeister et al., 1997). Several methods for In this regard, two different approaches can be pursued to design backbone design often require a final generation of loops to connect the new building blocks for protein scaffolds. On the one hand, conventional projected secondary structures (Canutescu and Dunbrack Jr., 2003; protein engineering has been the source of new constructs from already Stein and Kortemme, 2013). Next, the precise amino acid sequence is to existing proteins in nature, selecting stable human globular domains to be optimized based on the side chain interactions that best minimize the Fig. 3. Simplified overview of the main strategies to generate novel protein scaffolds. The traditional approach (top) involves screening known human proteins for stable and soluble globular domains, assuming the convenience to introduce stabilizing mutations. Alternatively, de novo design (bottom) involves three main steps; defining the protein backbone, optimizing the amino acid sequence to promote favorable side chain interactions, and testing the folding compatibility of the resulting candidate. Immunogenicity prediction can be observed as a generic common and convenient step. 6 O. Cano-Garrido et al. Biotechnology Advances 61 (2022) 108032 energy function of the chosen backbone. This process is often inter­ useful to combine several desired functions in a single polypeptide chain spersed with the steps of backbone perturbation and minimization, in a by domain recruiting, conferring additional biological activities beyond way that more diverse lower energy structures can ultimately be ach­ a plain structural role. In addition, protein materials used in human ieved compared to fixed-backbone approaches (Murphy et al., 2012). clinics should be non-immunogenic. In this regard, immunoreactivities, Finally, the compatibility between sequence and structure must be mainly associated to non-human proteins, can be minimized by site- evaluated to ensure that no other folding state is favored over the pre­ directed mutagenesis allowing foreign proteins to be ‘humanized’ and dicted design. Working towards the stabilization of one sequence is then usable in human medicine. On the other hand, advanced compu­ considered positive design, while intentionally building the protein in a tational methods permit to design, fully de novo, non-natural poly­ way that alternative states are destabilized is known as negative design. peptides that fulfill precise structural requirements. Ensuring high Ideally, both strategies should be simultaneously applied although the solubility and structural stability of these constructs, that do not have a latter is not always implementable since it is computationally more natural origin, will in turn results in non-immunogenic materials. Even intense. in early stages, this approach complements and is expected to largely Pressed by the need of highly stable and soluble proteins to avoid expand, in a close future, the catalogue of protein-based materials with unwanted immune responses, many de novo design approaches often medical usability. resulted in proteins with highly regular secondary structure patterns with compact cores (Bhardwaj et al., 2016; Kuhlman et al., 2003). This is Acknowledgements setting the best possible scenario even for non-human protein constructs or for fully de novo designed materials with no specific source. Recent The authors are indebted to Agencia Española de Investigación for studies with small hyper stable peptides in murine models showed close granting projects on the construction of protein materials of clinical to no-immune response in intranasal, intraperitoneal and intravenous interest (PID2019-105416RB-I00/AEI/10.13039/501100011033 to EV administration (Chevalier et al., 2017; Silva et al., 2019). Despite a and PID2020-116174RB-I00 to AV), to AGAUR for projects 2017 SGR- handful of examples cannot be extrapolated to a general trend, these 229 to AV and 2017 SGR-865 to RM, and to ISCIII for projects P21/ promising results set the stage for the evaluation of upcoming a la carte 00150 to RM and PI20/00400 to UU co-funded by European Regional design of protein scaffolds or scaffold components. Development Fund (ERDF, “a way to make Europe”). We also appreciate Regardless the selected approach to design a novel scaffold protein, the funding from the CIBER -Consorcio Centro de Investigación Bio­ current in silico methods allow a very efficient search for antigenic epi­ médica en Red- (CB06/01/0014), Instituto de Salud Carlos III, Minis­ topes. Such tools can serve as a last quality check before proteins are terio de Ciencia e Innovación, through projects (Nanoremote, Nanopills, tested experimentally and can then provide a temporary assumption of Venom4Cancer, Nanoprother II, Nanoscape and Nanolink). U.U. was safety. Predictors of either lineal (continuous) or conformational supported by a Miguel Servet contract (CP19/00028) from ISCIII co- (discontinuous) epitopes have achieved significant progresses in the last funded by European Social Fund (ESF investing in your future). A.V. decade (Sanchez-Trincado et al., 2017). This is even despite the intrinsic received an ICREA ACADEMIA award. Molecular graphics were gener­ difficulties particularly inherent to conformational antigenic de­ ated with UCSF ChimeraX, developed by the Resource for Biocomputing, terminants, which require molecular dynamics simulations limited to Visualization, and Informatics at the University of California, San only infer the tested interactivity. Francisco, with support from National Institutes of Health R01- To assess whether a lineal antigen will be able to trigger an immune GM129325 and the Office of Cyber Infrastructure and Computational response, its likelihood to be presented by the major histocompatibility Biology, National Institute of Allergy and Infectious Diseases. complex, and hence, potentially recognizable by T-lymphocytes, is typically evaluated. 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