Team:Bielefeld-CeBiTec/Nanobodies Project

Bielefeld-Cebitec-2020-Entrepeneurship

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Nanobodies & scFv's

Antibodies

Antibodies are a class of glycoproteins from the immunoglobulin superfamily (IgSF). They are used in the mammalian immune system to detect and neutralize pathogens. Produced by B-cell derived plasma cells, antibodies recognize specific antigens [1], [2]. Antibody monomers consists of two heavy chains (H) and two light chains (L), linked together via disulfide bonds. The shape of a complete antibody monomer resembles the letter "Y". Each chain contains an N-terminal variable (V) domain (referred to as “VH” and “VL”) and one or more C-terminal constant (C) domains: Whereas light chains contain only one C-domain (C¬L), heavy chains contain between three and four C-domains (CH) [3]. The lower part of the Y shaped structure is formed by the CH and defines the isotype of the antibody: There are five isotypes of mammalian immunoglobulins - IgA, IgD, IgE, IgG and IgM. This determining region is termed Fc (crystallizable fragment). An antibody binds its specific antigen via the V-domains at the tips of the Y shaped structure. This region is called Fab (fragment, antigen binding) region. The Fab-region consists of the complementary V-domains of L- and H-chains. Each V-domain consists of three hypervariable regions, termed complementarity determining regions (CDRs) 1 to 3, and four regions with relatively constant sequence making up the framework of the V-domain [3].

Figure 1: Schematic overview of a typical mammalian Ig antibody.

Because antibodies bind specific antigens, they are not only a very important part of our immune system but also a highly valuable tool for scientific use and healthcare. In research, typical applications of antibodies are the detection and localization of specific intra- and extracellular proteins, flow cytometry, immunoprecipitation and enzyme-linked immunosorbent assays (ELISAs). Downsides of antibodies are their large size (on average ~150 kDa) as well as the crucial need of inter- and intrachain disulfide bridges and glycosylations for formation of their correct structure and function. Therefore, smaller antibody variants with the same strong binding functionality and a fold independent of disulfide bonds are desirable. Additionally, antibodies that are not human antibodies or at least humanized antibodies poses the risk of evoking an immune response in the patient making the use of some antibodies problematic in medicine. Therefore, smaller antibody variants present a considerably reduced risk [4].

A group of antibody variants fulfilling these requirements are Nanobodies. Nanobodies are antibodies consisting of only the heavy chain, they are naturally found in the sera of camelids (so called “single variable domain on a heavy chain or “VHH”) and of cartilaginous fish (so called “variable new antigen receptor (VNAR) single domain antibodies). VHH antibodies are only around 15 kDa and thus better suited for scientific and medical application than normal Ig antibodies [4]–[6]. Another group of antibody-variants are single chain variable fragments (scFv). In this approach the VH and the VL domains are linked by a short peptide linker, thus creating a significantly smaller binding fragment which does not need glycosylation. Furthermore scFv´s are a single polypeptide and not several chains held together via disulfide bonds, an advantage they have in common with Nanobodies [7].

Nanobodies

First described in 1993 [6], nanobodies are an important research field for multiple pharmaceutical applications today. Mainly, nanobodies are divided in two types of different structures. The most common nanobodies are the VHH fragments, which consist only of a variable domain of a heavy chain and are found in Camelidae species [8]. Another type of nanobodies are the VNAR fragments based on the IgNAR (“Immunoglobin new antigen receptor”) from cartilaginous fish [9]. Having a molecular mass of ∼12 kDa, the VNAR domain is the smallest antibody-like antigen binding domain known so far in animals [10]. VNAR domains have only two complementarity determining regions CDR1 and CDR3, in contrast to variable mammalian domains [11]. Nanobodies are the smallest known antigen-binding proteins with a length of 4 nm and a diameter of 2.5 nm [12]. However, like a whole antibody, they are still able to recognize and bind specifically their target molecules using the single variable domain. Their compact structure and light molecular weight of only 12-15 kDa (compared to conventional antibodies with 150-160 kDa) make nanobodies the smallest active antigen-binding fragments [13]. Due to their versatile binding properties, high stability, manipulable characteristics as well as improved tissue penetration ability nanobodies are crucial in the field of biotechnology or medicine today [14], [15].

Figure 2: Schematic overview of a conventional Ig-antibody (left side). Enlarged overview over the characteristic features of a VHH nanobody on the right side.

Antibodies consist of two glycosylated heavy chains (H) with a molecular weight of 50 kDA each and two light chains (L) of 25 kDA each, which are covalently linked via disulfide bridges. [16]. At the one end, two N-terminal variable domains (V) of the heavy (VH) and light chain (VL) form the antigen-binding domain, also called paratope. At the other end, the C-terminal regions form the three constant domains CH1, CH2 and CH3. Compared to common antibodies, the nanobody family consist of conventional heterotetramic antibodies with low-affinity binders and as well as unique functional heavy(H)-chain antibodies (HCAbs) with high affinity binders [13]. The H chain of these homodimeric antibodies consist of one antigen-binding domain, the VHH, and two constant domains. The HCAbs fail to incorporate light (L)-chains owing to the deletion of the first constant domain and a reshaped surface of the VHH side, which normally associates with the L-chain in common antibodies [17]. This structural change leads to an enlargement, allowing the nanobodies to bind their antigen with high affinity. Furthermore, the hydrophobic amino acids within the conserved region (frame region; FR), which ensure the interaction of VH and VL, are replaced by hydrophilic amino acids [12], [18].

Nanobodies are usable in very diverse field of applications. Their use in diagnostic test systems and therapeutic options is increasing. In contrast to conventional antibodies, nanobodies merge as a new technical achievement due to their low molecular weight (half the size of single-chain variable elements and ten times lighter than conventional antibodies) and small size (2-3 nm) [17]. Moreover, a high expression rate of nanobodies was shown in E. coli with high functionality and stability even in absence of conserved disulfide bonds [19]. The loss of the L-chain leads to missing disulfide modifications in nanobodies, which makes them suitable for production in different bacteria and yeast [20], [21]. Additionally, their high tolerance against acid conditions and temperature compared to mouse monoclonal antibodies [8], high solubility due to a tetrahedron of highly conserved hydrophilic substitutions [22] and very few cleavage sites for enzymes [13] make nanobodies very promising and an interesting research subject for new applications in biotechnology. Furthermore, the recombinant nature and single domain of the nanobodies allows easy generation, production and molecular biological manipulation. These include sequential modifications, the generation of fusion proteins with other substances (e.g. pharmaceuticals), the transfer of antigen specificities as well as the transfer of affinity from one nanobody to another [23].

Due to their low mass, the nanobodies are able to easily cross the blood-brain barrier and make them able to quickly leave the human body's circulation via urine after resolving their function inside the cell. Furthermore, nanobodies show a higher availability to tissue penetration and due to their smaller size, they trigger less immunological reaction, which leads to better pharmacokinetics [24]. All stated points are an important property for therapeutic options (e.g. cancer therapies) and diagnostic processes (e.g. molecular imaging) [23].

Nanobody Grafting

If you want to produce specific nanobodies by yourself, without ordering them, you would typically need a camelid immune library for this task and for this library one would need to keep animals and take care of them. This may raise ethical concerns. One can also generate nanobodies from synthetic libraries, but this requires large libraries and sophisticated selection mechanisms. An alternative is the so-called grafting of nanobodies. In this method, the CDRs of any antibody are transferred to an existing nanobody to create a mixed nanobody [4]. However, not only the three CDR regions 1 to 3 are transferred, but also amino acids important for the structure and function are transferred from the donor antibody to the nanobody scaffold [25].

Figure 3: Schematic representation of the in silico grafting process. To start grafting, an acceptor and a suitable CDR donor must be selected. The CDRs 1 to 3 are transferred from the donor (any antibody) to the acceptor (a nanobody suitable for grafting). The resulting initial graft is usually a weak binder. The grafted sequence must therefore be subjected to a suitable affinity maturation method, for example a phage display. After affinity maturation a new synthetic nanobody with high affinity is obtained. Abbreviations: CDR, complementarity-determining region; VH, variable domain of the heavy chain; VHH, single variable domain on a heavy chain antibody; VL, variable domain of the light chain. Created with BioRender.

The grafting itself is carried out in silico, therefore the amino acid residues from the VH domain from the donor antibody and from the accepting nanobody have to be numbered according to a numbering scheme like Kabat or AHo [26] and CDR1 to 3 have to be defined accordingly [25]. The correct structure of the graft can be checked with the help of visualization programs such as ChimeraX [27]. Here you can see the results of our grafting.

Error Prone PCR

For means of affinity maturation we needed a library of our grafted sequence. In nature, antibodies with initial binding properties are further refined in a process called “affinity maturation”. This process combines random mutagenesis of the binding amino acids and continuous selection of antibody specificity as well as binding strength to finally obtain mature antibodies with extremely high binding specificity and strength. For improving our grafted nanobody by affinity maturation we needed a diverse library of the DNA of our grafted nanobody. To introduce the required diversity into the grafted sequence, we chose a random mutagenesis approach by polymerase chain reaction (PCR), first described by Cadwell and Joyce in 1992 [28]. This variant of the PCR-technique is termed error prone PCR (epPCR). In this technique, the natural error rate of the Taq polymerase is increased by unfavorable reaction conditions. This is accomplished by: 1) Increased concentration of Taq DNA polymerase; 2) extended polymerase extension time; 3) increased concentration of MgCl2 ions; 4) unbalanced rate of dNTPs; and 5) addition of MnCl2 ions [29].

Phage Display

Over the last years, the phage display has proven to be a very powerful biotechnological approach to display and select recombinant libraries of many different peptides and proteins. These libraries are displayed on the surface of bacteriophages found on Escherichia coli. In the last decades, this library method based on the use of filamentous phage has been established and developed [30]. Initially, George Smith's Phage Display was limited to the selection of peptides [30]. In 1990, scientists succeeded in displaying the first antibodies on the surface of a phage [31]. This was achieved by fusing the coding sequence of the antibody's variable region (V) to the amino terminus of the phage coat protein pIII. The antibody was generated using a phage vector based on the genome of a filamentous phage and its geneIII as fusion partner [32].

Figure 4: Schematic illustration of a phagemid display. Involving the steps of library preparation, cloning into the phagemid vector, transforming into a suitable E. coli strain. Production of the phagemid with the gene of interest as fusion protein to geneIII surface protein by helper phage M13. Subsequently panning on target antigen, washing steps and elution are conducted. Eluted phages can be analysed by means of e.g. phage ELISA. Then, the next round of panning in the phagemid display can be started using the eluted phages. Created with BioRender.

In the used vector, the coding sequence for the antibody scFv fragment was cloned between the signal and coding sequence of phage geneIII. In this fusion construct the VH and VL domains fold correctly and are connected and stabilized by an intramolecular disulfide bridge, thus a functional scFv is formed [33], [34]. Initially, vectors were used that carry all the genetic information necessary for the life cycle of the phage [31]. Today, small minimal plasmids, called phagemids [35] –[37] are used. These carry geneIII, suitable cloning sites to fuse the individual sequences from the library to geneIII and a phage packaging signal. In addition to the phagemid an additional helper phage is required: Since the phagemid is a minimal plasmid without the necessary proteins or assembly machinery, no phage can be formed with the information on the phagemid alone. Therefore, an additional phage, called helper phage is needed. This phage carries the entire M13 genome and confers the information regarding capsid production, phage assembly, chromosome replication and budding [38].

One of the most widespread phage display formats is the described genetic fusion of variable chains to the coat protein pIII. This fusion can be achieved by direct engineering of the phage genome [30], [31], [39] or by a geneIII expression plasmid in combination with a helper phage [35], [37], [40]. The first approach is called phage display format. In this format, all expressed geneIII molecules are fused to the antibody. The 3-5 copies of modified gene III per phage are displayed on the tip of the bacteriophage. In contrast, the number of copies of geneIII fused together with the to be presented antibody in the phagemid format is largely dependent on the helper phage and leads to a mostly monovalent display [36], [41]. Monovalent in this case means that the resulting antibodies can only bind one specific antigen. Due to the fact, that phagemids have a high transformation efficiency, they are perfectly suited for using large libraries [37], [42]. Suitable antibody formats for the phage display based on a phagemid are scFvs [31], Fab fragments [36], [37], scFv’s [43] as well as nanobodies [44].

For the selection of a phagmid or phage library, various methods can be used. In general, the aim of the selection process is the separation of binding clones from non-binding clones. One well known method is the selection by biopanning [45]-[47]. Therefore, the antigen is either immobilized on surfaces or biotinylated [48]. For example, the antigen can be immobilized on the surface of an Immunotube and a phage solution can be added. After several washing steps, the bound phages can be eluted and, after a round of amplification in e.g. E. coli, used for the next round of panning. In general, the phage display allows the selection of antibodies against an unlimited array of biological [31] and non-biological [49] targets. In addition, screening assays are important to find the most suitable antibody according to the requirements. For this purpose, numerous variants of an antibody with few different properties are subjected to screening. In the first rounds of panning can be washed with mild conditions to achieve a sufficient size of the library. As the number of panning rounds increases, the conditions become more and more severe, so that in the end only the strongest binders with the highest affinity remain.[47], [50].

Phage ELISA

The ELISA (enzyme-linked immunosorbent assay) is a technique which allows the detection and quantification of proteins and antigens from several samples using enzyme-linked antibodies. Phage ELISA is commonly used to determine whether there is an accumulation of phage binders to the specific antigen within the pool. To perform ELISA, the antigen must first be immobilized on microtiter plates, followed by the addition of different dilutions of the phage pool from each round. Subsequently, the binding of phages can then be detected using an anti-M13 phage antibody [51].

Figure 5: Schematic overview of the phage ELISA. If nanobody-fusion protein expressing phages bind the via streptavidin-biotin immobilized antigen, the phages can be detected after washing by the enzymatic reaction catalyzed by the horseradish peroxidase (HRP). HRP is conjugated to an antibody targeting protein VIII (pVIII) from phage M13. The strength of the signal correlates to the number of binding phages: The more phages bind, the stronger the signal.

This antibody recognizes a phage protein (e.g. pVIII). Furthermore, the used antibody for detection is coupled with an enzyme, which reaction can be measured by spectrophotometric methods, like the horseradish peroxidase (HRP). HRP oxidizes various organic substrates in the presence of hydrogen peroxide [52]. Some of its substrates are chromogenic or chemiluminescent substrates: The most common used ones are luminol, TMB (3,3',5,5'-Tetramethylbenzidine), DAB (3,3'-Diaminobenzidine) or ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) [53] - [56].

Single Chain Fragment Variable

A single chain fragment variable (scFv) represents one of the smallest functional structures of an antibody. It consists of a VL and a complementary VH domain, which are synthesized as a single polypeptide chain and are required for antigen binding. . The linker connects the carboxyl terminus of one domain with the amino terminus of the other variable domain - the order of whether VH is attached to VL or VL is attached to VH is up to the user [58].

Figure 6: Schematic overview of a conventional Ig-antibody on the left side. Enlarged overview over the characteristic features of a single chain fragment variable (scFv) on the right side.

A recombinant scFv can be produced in a variety of different systems ranging from bacteria to mammalian cells [59]. The idea for the design of scFvs emerged to overcome problems of whole antibody expression in E. coli. Due to their small size and homogeneity, scFvs offer significant advantages over polyclonal and monoclonal antibodies and can be easily expressed as unique units or as fusion proteins in bacteria. ScFvs are easier and cheaper to produce in bacterial systems compared to conventional antibodies due to the lack of glycosylation, disulfide bridges and their small size [7]. However, some scFvs have a toxic effect on E. coli, which impairs their expression and production in E. coli. The toxic effect is due to the composition of amino acids of the V-domains [60]. An scFv can be designed in silico, no immunization process of animals is required. Therefore, to obtain new antibodies, no animals need to be kept, only the sequences of the VH and VL domains are needed. Furthermore, the immobilization of the scFv fragments on surfaces in a high density is possible due to their small size. This is often done by introducing certain amino acids into the linker [58].

scFv Grafting

Given their small size, scFvs can easily be expressed as unique units or as fusion proteins in bacteria. By selecting suitable donor antibodies, more precisely their variable domains, it is theoretically possible to produce scFv against all kinds of antigens. For the in silico grafting process, the two variable domains are combined and connected by a flexible peptide linker, usually 15 amino acids long. The sequence of the linker can be modified, according to how you want to use the grafted scFv [58]. The resulting scFv can be ordered as a gene synthesis product and is ready for cloning and further experiments.

Cloning

We wanted to purify scFv against estradiol and progesterone. These scFv´s were grafted by ourselfs. In order to clone [61] the scFv´s into the expression vector pTXB1, we applied different cloning strategies . On the one hand we used Gibson Assembly. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes. In this strategy, vector and insert contain complementary overhangs and are assembled in a single enzymatic reaction. [62]. Furthermore, we applied the seamless cloning of thermo fisher, which is also based on an enzymatic reaction. The Invitrogen™ GeneArt™ Seamless Cloning and Assembly Kit allows in vitro cloning in almost any linearized vector, in around 30 minutes, without additional DNA sequences, restriction endonucleases or ligation [63]. As a final strategy we used cloning with restriction enzymes. Restriction enzymes are nucleases that cut the DNA at specific sites. To clone a DNA insert into a vector, both have to be treated with two restriction enzymes that produce compatible ends. Depending on the enzyme used, digestion produces so-called sticky or blunt ends. The complementary ends are connected by a ligase [64]. Have a look at our results to see exactly how we proceeded.

Purification of Nanobodies and scFvs

The process of the scFv's or nanobody creation can be summarized in three different reaction steps: the cloning of the scFv's or nanobody sequences into a vector, its expression and the purification. For the expression and following purification of the scFv's and nanobody sequences, the expression vector pTXB1 was used. By integration of the scFv's or nanobody sequence into the vector upstream of the Mxe intein/Chitin-binding domain, a fusion protein is created. For gene expression, the induction with IPTG is required. Due to the chitin-binding domain, the fusion proteins can be purified by affinity chromatography, where the fusion protein is bound to chitin residues and can be released by a thiol-induced cleavage reaction of the intein/chitin binding domain from the protein of interest. For the purification process, usually a purchasable kit is used. In order to make conclusions about the specificity and quality of the scFv's and nanobodies, the optimal expression conditions must be tested afterwards. [65]–[67].

Immobilization of Nanobodies and scFvs

The process of the scFv's or nanobody production can be summarized in three different steps: the cloning of the scFv's or nanobody sequences into a vector, its expression and the purification. For the expression and following purification of the scFv's and nanobody sequences, the expression vector pTXB1 was used. By cloning of the scFv's or nanobody sequence into the vector upstream of the Mxe intein/Chitin-binding domain, the coding sequence a fusion protein is created. Due to the chitin-binding domain, the fusion proteins can be purified by affinity chromatography on chitin residues. The fusion protein is bound to the chitin column and can be cleaved in a thiol-induced cleavage reaction. In this reaction the intein/chitin binding domain cleaves itself from the protein of interest. Afterwards the protein of interest, now without the chitin binding domain, elutes from the columns and is thus purified with only minimal additional amino acids (in contrast to e.g. His-tag mediated purification). In order to make conclusions about the specificity and quality of the scFv's and nanobodies, the optimal expression conditions must be tested afterwards. [65] – [67].

Figure 7: Schematic representation of the immobilization of a nanobody.(1) and (2) illustrate an immobilization variant which is likely to impair the activity. Figure 3 displays an immobilization where the functionality is not inhibited.

Figure 8: Schematic representation of the immobilization of a single chain fragment variable (scFv).(1) and (2) illustrate an immobilization variant which is likely to impair the activity.(3) displays an immobilization where the functionality is not inhibited.

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