Modern Biopharmaceuticals (Recent Success Stories) || Massive Mutagenesis®: The Path to Smarter Genetic Libraries

6Massive Mutagenesis®: The Path to Smarter Genetic LibrariesJulien Sylvestre, St�ephane Blesa, Ingrid Marchal, Philippe Thullier,Olivier Dubreuil, and Marc Delcourt

6.1Introduction

6.1.1Directed Evolution and Biopharmaceuticals

Directed evolution is inspired by Darwinian evolution and mimics its basic princi-ples at the molecular level [1–4]. Directed evolution can be regarded as an ensembleof stochastic molecular algorithms that aim at improving virtually any protein ornucleic acid. Thousands of successful results are now published in the fields ofenzymes, antibodies, hormones, cytokines, peptides, RNAs, and vaccines. Directedevolution can also be seen as a form of combinatorial chemistry and in fact these twofields are reciprocally linked [5].The target for evolutionary improvement itself varies, from activity, specificity,

affinity, thermostability, and solubility to stability in vivo, or toxicity. Most of theseproperties are difficult to predict and an approach that gives room for chance is thusappropriate. One can envision that just as Darwinian evolution has given rise to acentral theory on the formidable diversity of life and has revolutionized ourunderstanding of biology, similarly directed evolution will reveal completely newperspectives for the creation of new biomolecules and, in particular, newbiopharmaceuticals.Directed evolution, which creates and sorts in an almost “blind” manner large

genetic libraries, is often opposed to rational design, which uses structural andfunctional information to precisely engineer proteins, according to molecularmodels. It remains clear that we cannot modify proteins at will in a purely rationalway, because much of the protein work still escapes our understanding. Never-theless, recent progress enables a semirational approach. We describe here howMassive Mutagenesis1, Biomethodes’ unique high-throughput combinatorial site-directed mutagenesis technology, bridges the gap between directed evolution andrational design, gathering the best properties from both worlds.

Modern Biopharmaceuticals: Recent Success Stories, First Edition. Edited by Jörg Knäblein.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

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6.1.2Directed Evolution: The Process

Directed evolution mimics natural evolution in the test tube. It begins with a geneticdiversity – a collection of genotypes – then sorts it on the basis of correspondingphenotypes, either by assaying them one by one (screening) or by devising a way toisolate en masse the best ones (selection). Although directed evolution happens to bea powerful tool in the evolution of functional nucleic acids (in which case thegenotype and the phenotype are either identical or complementary), we restrict thischapter to the directed evolution of proteins, because they are the major biophar-maceuticals. In the case of proteins, directed evolution creates a collection ofdiversified nucleic acids (in general, DNAs) and sorts them based on the activityof corresponding proteins. Several rounds of diversification and sorting are used toprogressively improve a given target. In vitro recombination (a molecular equivalentof sexual reproduction) can also be integrated in the process to shuffle functionalelements and further increase the diversity.Fundamental to the directed evolution scheme is the library creation step. One can

start with an existing group of natural genes from different organisms thenrecombine it, using techniques such as DNA-shuffling and variants or the staggeredextension process (StEP) [6–9]. One can also directly harvest natural nucleic aciddiversity, without cultivating corresponding organisms (classic laboratory cultivationbeing a step thought to lead to a 100-fold reduction of diversity), using techniquescalled metagenomics [10,11]. Both approaches, although undoubtedly useful, arerelatively labor-intensive and can be troublesome; moreover, they strictly rely onpreexisting natural diversity.By contrast, most directed evolution experiments start with a single gene from

which one generates a variety of mutants. After each round, obtained “hits” are oftendiversified again, within the new local sequence space, in order to gain furtherimprovements. Mutagenesis, as a primary library creation or as a secondary librarydiversification method, hence appears as a central feature of directed evolution[3,12,4].

6.1.3Aiming for Bigger and Smarter Libraries

Existing mutagenesis technologies are usually divided into site-directed mutagene-sis and random mutagenesis (for a review of existing library creation protocols, seeRef. [13]).In 1993, Michael Smith shared the Nobel Prize in chemistry with Kary B. Mullis,

the inventor of PCR, “for his fundamental contributions to the establishment ofoligonucleotide-based, site-directed mutagenesis and its development for proteinstudies.” He had performed the first site-directed mutagenesis experiments in thelate 1970s [14], and since then, site-directedmutagenesis has been central inmodern

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biology as it has provided ameans to demonstrate hypotheses on structure–functionrelationships in genes and proteins.Site-directed mutagenesis targets one or a handful of residues in a gene and

modifies them in a specific manner, producing one mutant gene or a smallcollection of mutant genes (e.g., 10–100 distinct mutants). A particular aspect ofsite-directed mutagenesis that has gained much interest in recent years issaturation mutagenesis. Saturation mutagenesis – a term coined in the 1980s –

aims at substituting a particular residue in a protein with all other possible aminoacids, thus exhausting the diversity available at this specific position [15,16].In 1995, Olins and colleagues [17] performed the saturation of 105 residues inthe IL-3 gene. Saturation mutagenesis has since then been further improved andgeneralized [18] and methods allowing saturation mutagenesis of one or at best ahandful of residues are now becoming part of the standard mutagenesis toolkit.Site-directed and saturation mutagenesis often gains from the knowledge ofstructure–function relationships of the protein of interest: residues that arebelieved to be crucial for activity can be specifically targeted and in return, site-directed and saturation mutagenesis results are usually informative about theprotein evolved.On the other hand, random mutagenesis [19,20] is a technique that modifies a

gene in a completely blind manner, by way of uncontrolled substitutions occurringat uncontrolled positions. The only two parameters that are controllable are theaverage number of mutations per molecule, and with significantly more effort, thecontiguous region(s) on whichmutations are allowed to happen or which residues toexclude [21]. Contrary to site-directed mutagenesis, current random mutagenesistechnologies, which rely on error-prone PCR, facilitate the rapid generation of largegenetic diversities and neither require nor incorporate any structural or functionalinformation.Despite this lack of control and quality, the large library sizes readily achieved by

random mutagenesis are clearly suitable for efficiently sampling relatively “vast”regions of the sequence space. The advent of more and more selection techniquesand high throughput or ultrahigh throughput screening techniques [22,23], whichare capable of sorting larger and larger libraries, make such large-scale approachesrealistic. Obviously, compared to the total theoretical sequence diversity, the sam-pling remains very sparse and the quality of the sample is thus of prime importance(up to 107 variants are typically screened and up to 1013 selected, whereas there are20n different proteins of n residues,�1011 yeast cells in a 1 l culture and . . . “only”�1080 atoms in the universe . . . ) [4].Ideally, one would therefore require a simple technique that allows the rapid

generation of genetic libraries, which are both bigger and “smarter.” This iswhat Massive Mutagenesis is about. Figure 6.1 summarizes the positioning ofMassive Mutagenesis among library creation protocols. As we clearly show,Massive Mutagenesis ushers in both important qualitative improvementsfor random mutagenesis techniques and huge quantitative improvements forsite-directed mutagenesis.

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6.2Massive Mutagenesis

6.2.1Principle

MassiveMutagenesis was first developed a few years ago and is described in detail inUS Patent 7,202,086 and European Patent EP1311670. It is based on a single-strandcircular amplification reaction that uses oligonucleotides, which incorporate one ora small number of mismatches, as well as a thermostable polymerase and athermostable ligase.Figure 6.2 illustrates the principles underlying Massive Mutagenesis. One starts

with a plasmid containing the gene of interest and a collection of phosphorylatedoligonucleotides. The oligonucleotides are all complementary to the same strand ofthe plasmid. The oligonucleotides incorporate 1–10 base mismatches, typically intheir center that correspond with the substitutions to be introduced. The oligonu-cleotides can be phosphorylated either chemically, during their synthesis, orenzymatically afterward. To this mixture of plasmid and oligonucleotides, a ther-mostable DNA ligase, a thermostable DNA polymerase, dNTPs, and a compatible

Figure 6.1 Library creation protocols. Site-directed mutagenesis or gene synthesis createssmall custom diversities, whereas randommutagenesis by error-prone PCR or in vitrorecombination by DNA shuffling generates

large diversities but without any control over thenature and position of the mutationsintroduced. Massive Mutagenesis achievesboth high-throughput and controlleddiversification.


Figure 6.2 Massive Mutagenesis. Starting withthe gene of interest cloned in a standardplasmid and using biochemical data andsequence alignments to design a mutagenesisstrategy, Massive Mutagenesis makes use ofhundreds of oligonucleotides to introducemutations of controlled nature at controlledposition. After mixing the oligonucleotides, athermostable DNA polymerase and athermostable DNA ligase, as well as dNTPs inan appropriate buffer, the mixture isthermocycled – typically: 12 cycles of (94 �C,

1min; 50 �C, 1min; 68 �C, 10min) – to generatesmall fragments complementary of the sameplasmid strand that are subsequently ligatedtogether. Selection against parental plasmid canbe achieved using the methylation-sensitiveenzyme DpnI. The gene library obtained is thenelectroporated and individual clones can bescreened individually or the library can besubject to en masse selection. Positive clone(s)can be sequenced and subject to furthermutagenesis round(s).

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buffer are added. Amixture of several polymerases (for instance, 1 : 10 Taq/Pfu, u/u)can be used. The resulting mix is then subjected to thermocycling for a few hours. Atypical thermocycling protocol is 12 cycles of (94 �C, 1min; 50 �C, 1min; 68 �C,10min). The exact duration of the extension steps at 68 �C depends on the length ofthe plasmid to be amplified and the processivity of the polymerase(s) used.After the last cycle, the product is recovered and digested at 37 �C by the DpnI

restriction endonuclease [24].DpnI acts as a selection force against parental plasmidsand strands. Alternatively, other classical methods to select mutant molecules can beused. In one particular embodiment of Massive Mutagenesis, for instance, Kunkel’ssystem using Ung- bacterial strains is used (see Ref. [25] for details).After 30–60min, the product of the digestion is desalted by simple membrane

dialysis and used to electrotransform competent bacteria, which are plated andgrown overnight. At this stage, up to 50% of the bacterial clones that are harvestedcontain plasmids, which incorporate one or a combination of several targetedsubstitutions. The clones can then be screened or selected. Alternatively, to increasethe number of mutations per molecule, the whole plate can be scrapped, itsplasmidic DNA content prepared, and the resulting DNA subjected to anotherround of Massive Mutagenesis, which increases the number of mutant clones aswell as the number of mutations per molecule.

6.2.2Properties

Massive Mutagenesis harbors a number of interesting properties that we shall nowdescribe and are listed in Table 6.1.First, large library sizes (up to 109 different variants) have been successfully

generated by Massive Mutagenesis. Moreover, any mutation can be introduced –

one just has to synthesize the corresponding oligonucleotide. The simultaneoussubstitution of two or three adjacent codons can also be targeted using slightly longeroligonucleotides; this can prove useful if these codons all happen to be important inthe protein activity. In some cases, deletions or insertions can be more efficient thansubstitutions and this triggered the development of relatively complicated specificmutagenesis tools [26]. In fact,MassiveMutagenesis is readily amenable to generatingtargeted codon deletions as well as codon insertions after obviousmodifications of the

Table 6.1 Main properties of Massive Mutagenesis.

Library sizes Up to 109

Position of mutations AnyNature (insertion, deletion, substitution) AnyCloning NoAmplification bias NoneSaturation Standard, degenerated, or chip-eluted oligos

List of the main properties of Massive Mutagenesis. See text for details, discussion, and comparisonwith other mutagenesis procedures.


oligonucleotide design. No special reagents are used and manipulations are fairlysimple so the total cost is close to that of the oligonucleotides used. Importantly, nocloning is necessary since everything occurs within the same plasmid. Cloning isrelatively work-intensive, can sometimes be troublesome, and most often involves adouble-strand ligation step. This step reduces the library diversity and frequentlynonrecombinant plasmids are observed, so avoiding any cloning steps is a significantadvantage. Plasmids up to 12 kb have been successfully mutated using MassiveMutagenesis, which makes the technique appropriate for long genes. The amplifica-tion process copies only one strand and therefore the number of copies producedincreases linearly andnot exponentiallywith the number of cycles. This eliminates theso-called exponential amplification biases, where mutations occurring during firststeps tend to be overrepresented in thefinal library.MassiveMutagenesis initially usedoligonucleotides complementary to both strands of the plasmid but this lead topreferential amplification of short mutated fragments. To overcome this problem,we copied only one strand using oligonucleotides all oriented in the same direction –

this in fact prevents the formation of short fragments. Less than 10% unwantedmutations are routinely observed in the final library.

6.2.3Chip-Eluted Oligonucleotide Libraries for Mutagenesis

When a full randomization is sought, Massive Mutagenesis uses only one oligo-nucleotide per mutation to be introduced. This way of conducting saturationmutagenesis, while circumventing biases and constraints associated with the useof degenerated oligonucleotides, is both simple and flexible. However, in large-scaleprojects, the price of oligonucleotides becomes a major part of the total cost. Forinstance, if oligonucleotides are synthesized one by one at an individual cost of D5,complete saturation mutagenesis of a typical 300 codon gene would cost around300� 19� 5 � D30 k. Nevertheless, these oligonucleotides are not used singly, butare actually pooled before adding them to themixture that contains the plasmid to bemutated. Handling thousands of separate oligonucleotides and pooling them can betedious, time-consuming, and a source of errors. We hence investigated the use ofDNA chips as a tool to produce much cheaper oligonucleotide pools [27,28].Figure 6.3 highlights the Chip/Massive Mutagenesis (ChipMM) approach, from in

situ parallel oligonucleotide synthesis to oligonucleotide pool elution and purificationand subsequent use in Massive Mutagenesis. The chips used were built by Rosatech(Grenoble, France) and include porous silicon surfaces to increase the yield.Our initial results, based on a model experiment in which 300 oligonucleotides

were used, showed that sufficient amounts of oligonucleotides could be eluted fromchips and used successfully to perform Massive Mutagenesis [29]. The low biasesobserved and the absence of untargeted mutations moreover demonstrated thequality of the oligonucleotides when synthesized on a chip and subsequently elutedand purified. As chip-eluted oligonucleotide pools become commercially available,this ChipMM approach lowers the overall cost of Massive Mutagenesis by at least anorder of magnitude.


6.2.4Comparison with Existing Mutagenesis Procedures

Whenever an efficient screening or selection procedure has been developed andwhen a high library quality can be guaranteed, generating large genetic libraries ispreferable. Actually, the precise relation between the library size and the probabilityof obtaining one evolved protein that is improved according to a particular criterionnaturally depends on the protein and the criterion. It has been shown that forantibodies the relation is strongly nonlinear implying that below a certain librarysize, the probability of the library containing a “hit” remains almost null [30]. Thispoint eliminates existing site-directed mutagenesis technologies, which can onlygenerate a handful of different sequences, as well as gene synthesis.It has been proposed, in recent years, to use degenerated oligonucleotides in a

gene assembly reaction to synthesize a diversified gene from scratch. The degener-ated oligonucleotides can for instance be synthesized using trinucleotide phosphor-amidites or dinucleotide phosphoramidites (described in Maxygen’s PatentUS6436675). The process is similar, conceptually, to DNA shuffling and has beentermed synthetic shuffling. Owing to synthesis errors, oligonucleotides are producedwith 2–5 deletions or insertions per 1000 bases. When incorporated into a gene,these deletions or insertions result in a frame shift and, almost always, an inactiveprotein. The absence of template hence results in an unacceptable proportion ofincorrect products. For instance, for a 1000 bp gene, if as few as 0.2% of the bases inthe oligonucleotides are inserted or deleted, this results in 1� (0.998)1000¼ 86%frame shifted proteins (the same calculation for a 2000 bp gene gives 98% frameshifted proteins). This problem is central to gene synthesis and whole-genomereassembly strategies, and the process remains quite tedious in spite of some recentimprovements [31,32]. By comparison, Massive Mutagenesis is template-based, andthese oligonucleotide-based errors occur much less frequently since the number ofnucleotides used to create a mutant gene is much lower. For a gene in which twomutations are introduced, only two oligonucleotides are incorporated, whatever thelength of the gene. If these oligonucleotides are 30-mer and have 0.2% of their basesdeleted or inserted, only 11% (1� (0.998)60) of the library will be frameshifted.Additional errors in Massive Mutagenesis can be due to polymerase, but the errorrates of the polymerases used are in the range (10�5–10�6) and therefore such errorsremain negligible since only one strand is copied.Asmentioned previously (also in Figure 6.1), two techniques that accommodate the

generation of large synthetic libraries are in vitro recombination and random muta-genesis. In vitro recombination obviously requires some genes to recombine, whichcan be difficult to obtain if the protein of interest is not found in many organisms.In vitro recombination is alsounable to generate anything that is not present in startingsequences, which can constitute an important limitation if the goal is to create a newproperty. Overall, recombination, just as in Darwinian evolution, is very useful andoften complementary to mutation, but can never be a substitute for it.Random mutagenesis, on the other hand, has become the method of choice for

the rapid creation of large repertoires of single, or multiple, mutant genes. The use


ofmutator bacteria (e.g., Stratagene’sXL1-Red strain), aswell as of various chemical(e.g., alkylating agents) or physical (UVs) procedures, has been described, yetrandom mutagenesis is currently almost always being carried out using error-pronePCR. Error-pronePCR is simply a reactionmodified in such away that a smallnumber of misincorporations occur. This can be done for instance using non-proofreading polymerases, using a high Mg2þ concentration, including someMn2þ, choosing a low annealing temperature, using low or unequal dNTP concen-trations, increasing the number of cycles, or by combining two or more of thesestrategies.Although recent results appear to challenge this idea [33], it has long beenobserved that the fraction of proteins remaining functional after mutation declinessharply as the average number ofmutations per gene increases.Hence, the numberofmutations permolecule is usually kept low (1–5). In any case, the probability thatmore than one nucleotide is modified in any given codon is close to 0. As shown inFigure 6.3, thismeans that randommutagenesis, although it is clearly uncontrolled,is actually not random at all, when one looks at its products at the relevant level – thepolypeptide level. Starting from a given codon and modifying only one nucleotide,one has access to only 9 (3� 3) new codons out of 63 (43� 1) possible differentcodons.Moreover, due to the genetic code redundancy, these nine codons only codean average of about six new amino acids, some more frequently than others and inmore than one-third of the cases, one stop codon. For example, starting from a AAAcodon, which encodes lysine (when using the standard genetic code), error-pronePCR can produce AAG (lysine also), AAT, AAC (both asparagine), CAA (glycine),GAA (glutamate), TAA (a terminator), ACA (threonine), AGA (arginine), and ATA(isoleucine). To further deteriorate this situation, depending on the error-pronePCRmethodology adopted, all misincorporations do not have the same probabilityof occurrence, further affecting the randomness of the diversity produced.Exponential amplification bias, which was mentioned previously is yet anothersignificant problem in error-prone PCR. It is also almost impossible to mutagenizeefficiently small contiguous regions (30–100 bp) or several separate segments (e.g.,5 separate 30 bp gene regions) using error-prone PCR.Hence (as boldly highlighted in Figure 6.3), if the mere prospect of a mutagenesis

strategy is to randomize a gene, Massive Mutagenesis does the job a lot better thanso-called randommutagenesis by error-prone PCR. Arguably, other techniques havebeen described that partially address this issue, yet all fall far from offering thesimplicity and flexibility inherent to Massive Mutagenesis.Moreover, MassiveMutagenesis is notmerely a nonbiased, truly randommutagen-

esis technique but is a combinatorial form of site-directed mutagenesis, extended tolarge libraries. Due to quick progress in genomics, structural genomics, and bio-informatics, more and more information becomes available that can help choosing amutagenesis strategy. For instance, when the target is an enzyme andwhen structuralinformation is available, one can choose to preferentially substitute specific residuesthat are known to be in direct contact with the substrate or that are in or near the activesite. When the target is an antibody, a kind of molecule whose structure–functionrelationships have been studied in depth for decades, there are similar regions andtypes of substitutions that can be purported as “smarter” than others [34,35].


Due to the progress in sequencing, homologous sequences are growingly availa-ble, and they can also help to define sequence regions to target for mutagenesis. Forinstance, based on sequence alignments, NMR, or X-ray crystallographic data, onecan decide to mutate only residues that are located on a precise region of a protein,which is key to its function, or decide to avoid a number of residues whosesubstitution is known to destabilize the protein structure, or decide to keep theamino acid class of the targets that are substituted (substituting codons encodingleucine only by codons encoding other hydrophobic amino acids for instance) [36].In fact, all available information regarding protein structure and function can be

incorporated in the mutagenesis strategy since Massive Mutagenesis appears as thefirst library creation technique that allows the generation of combinatorial librariesthat incorporate any custom-designed diversity – it allows to target any number ofmutations simultaneously. This is in contrast to all other available methods, forexample, Quick Change Multi Site1 (Agilent/Stratagene), which, as specified in themanufacturer booklet, is strictly limited to targeting five residues at most and thusnot adapted to sizeable library creation (amaximum of 400 doublemutants and 8000triple mutants only).The next part illustrates, in the context of industrial problems that have been

solved using Massive Mutagenesis, the broad applicability of the method, itsefficiency, as well as the kind of relatively sophisticated mutagenesis strategies itallows for.

6.3Sample Applications of Massive Mutagenesis

6.3.1Fine-Tuning of the Specificity of an Antibody to be Used in Diagnostics

The ability of antibodies to selectively bind a wide range of molecules makes them ofgreat interest for diagnostic and therapeutic applications, especially since thedevelopment of the monoclonal antibody technology [34,35].However, despite their high sensitivity, antibodies often present some cross-

reactivity with molecules whose structure is close to that of their main target (a lackin selectivity, or specificity). Directed evolution can thus aim at decreasing the cross-reactivity of an antibody (i.e., increasing its selectivity), while maintaining a highsensitivity. This signifies less side effects for therapeutic antibodies and less falsepositives in diagnostics.Massive Mutagenesis has been used, in collaboration with Dr Ducancel’s group, to

improve an antibody scFv fragment binding the progesterone hormone. The initialantibody presented a high (subnanomolar) affinity for progesterone, but displayedcross-reactionsof20%and35%, for twohepaticmetabolites,5a-dihydroxyprogesteroneand 5b-dihydroxyprogesterone (5a-DHP and 5b-DHP), respectively. The aim was toobtain a scFv displaying cross-reactions for 5a-DHP and 5b-DHP decreased to 5% orless while maintaining an affinity inferior to 0.1 nM.

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Based on crystallographic data (Figure 6.4), targets were located in the region ofinteraction with the antigen and its close environment, as well as at the interfacebetween VH and VL. Targets at position VH57 and VH58 in the bindingsite were substituted to saturation; others were substituted to amino acidsfrom the same subclass or to the following three hydrophobic amino acids(I, L, V). A single residue insertion in the binding site was also designed. Table 6.2lists 13 residues targeted and illustrates the custom-designed diversity that wasintroduced. Although the importance of particular residues could be guessed,knowing which combinations were to be most efficient was not possible. In thiscontext of semirational design, Massive Mutagenesis appeared as an appropriatetechnology to generate all these combinations in the form of a library.The library was produced using appropriate oligonucleotides and screened by

competitive ELISA. The originality of the system resided in the fact that the scFvvariants were directly and functionally expressed in Escherichia coli fused to a dopedalkaline phosphatase double mutant (CEA patent). Thus, and contrary to classicalELISA protocols that use a secondary antibody coupled to peroxidase for revelation,the extracted hybrid molecules were used directly and this without any purificationstep in competitive ELISAperformed in the presence of progesterone, or competitor:5a-DHP and 5b-DHP. This allowed determination of IC50 values of the differentvariants in comparison with the wild-type scFv, leading to an easy and fast analysis ofthe cross-reactivity evolution upon Massive Mutagenesis strategy.Mutants showing only 5% cross-reactivity with both metabolites were obtained.

Among all the mutations designed, it appeared that those involved in the improve-ment of the selectivity of the scFv were located in a small region formed by threeconsecutive VL residues and two consecutive VH residues. These mutants did notsignificantly lose their sensitivity to progesterone, but from the total library, as muchas 4% ofmutants displayed improved characteristics. These impressive results showthe value of the described approach to successfully incorporate structural data into amutagenesis strategy to create a biopharmaceutical with the desired features!

Figure 6.4 The antibody ScFv binding progesterone. Residue numbering as in Table 6.2.Representation of the antibody-binding site with the two antigens and the mutated residues inimproved mutants.


6.3.2Biocatalysis of APIs

Besides the use of proteins and enzymes as biopharmaceuticals, many pharmaceu-tical manufacturers and CRAMs are now turning to biocatalysis instead of chemicalroutes as it can help reduce cost, water, and energy use and yield purer products withcontrolled chirality. Massive Mutagenesis has been used to improve an enzymeinvolved in the synthesis of a precursor for a major small molecule antibiotic. A firstround of Massive Mutagenesis using oligonucleotides with mismatches encodingan alanine codon was used to perform a so-called alanine scan of the whole geneencoding the enzyme of interest. This actually constituted the first exhaustivealanine scan of a protein and led to a functional cartography of the enzyme, whichwas in accordance with previous characterization studies, but also revealed newtargets important for the activity, which were never described before. Then, a newlibrary was constructed in which these “hot spots” were substituted specifically.Some were substituted toward all the amino acids of the same subclass; othersubstitutions were designed based on structural data; yet others were based on theresidues observed at the same position in homologous proteins (a kind of artificialshuffling). The library obtained, containing amajority of single and double mutants,was then screened for clones showing an improved activity. In the end, after sixrounds of Massive Mutagenesis and screening, a clone containing 10 mutationscompared to the initial enzyme showed an important improvement in activity, with a40-fold Vm/Km increase. Further biochemical analysis determined that in fact bothVm and Km were increased. This mutant enzyme obviously yields in much fasterbioconversion into the desired pharmaceutical compound and at the same timemuch smaller amounts of the enzyme are required for this reaction – and above all,the synthesized product can be obtained at much higher purity!

6.3.3Improvement of an Antibody Neutralizing the Anthrax Toxin

Anthrax pathogenicity depends on the lethal toxin (LT).MassiveMutagenesiswasusedto improve the affinity of an antibody against anthrax toxin in amuch focusedway so asto avoid modifying framework regions, which are important for tolerance [34,35].Starting from a Fab (35PA83) with a 3.4 nMaffinity, a library of 5� 108 variants was

constructed that targeted simultaneously to saturation all 6CDRs (73 positions on sixdifferent regions of the molecule). The library, with 3.5 mutations per molecule onaverage, was phage-displayed and panned with adsorbed antigen. It was eluted withincreasingly stringent washing then submitted to an additional selection procedureto identify themost improved variants. Two selectionmethods were used: one with alow concentration (1 pM) of soluble antigen and a newly adapted method usingadsorbed antigens (0.6mM) and longer incubation times. The former methodproved more efficient. The best variant obtained had three mutations and showeda 19-fold affinity improvement (180 pM – 40% lower IC50), which is, to ourknowledge, the best result obtained so far in a single round of affinity maturation.


LT neutralization correlates with affinity for PA and the affinity of an antibody (orantibody fragment) interacting with PA has to be lower than the affinity of PA for itsreceptor (1 nM) for optimal LT neutralization. In cases where mutations arenonadditive, which has sometimes been observed even for antibodies, startingwith a larger library is more efficient than doing several rounds of improvements.This result is representative of what large, custom libraries combined with anefficient selection scheme can bring in a single selection step. It is described infurther details in Ref. [37]. Figure 6.5 showsmutated residues in all variants that hada subnanomolar affinity.

Figure 6.5 Anti-anthrax Fab antibody. Mutatedresidues in a three-dimensional model ofantibody 35PA83. VL and VH variable domainsare colored in light and medium gray,respectively. CDR loops of VL and VH arecolored in light blue and green, respectively.Residues mutated in variants with KD< 1 nM

are colored in red. From left to right: His24,Ser58, and Gln68-Ser69 in VL; Ser74, His55, andSer117 in VH. In orange: residues that weremutated in antibodies with decreased but >1nM KD. In yellow: the only residue that wasmutated in a mutant antibody that had anincreased KD.


6.3.4Thermostable Vaccines

Vaccines’ thermostability and shelf life can be an important issue for countries withdeficient cold chain. It is believed that half the vaccines end up being thrown away asthey experienced too extreme temperatures. Some vaccines support neither freezing(which can happen accidentally during transportation in ice-cube chilled packs) norprolonged heating at temperatures (40–45 �C), which are not uncommon in theSouth. TheWorld Health Organization estimates that more than $200million couldbe saved annually by cold chain and logistics savings. Introducing thermostablevaccines would also increase vaccine availability in remote areas. Progress informulation using sugar glass to encapsulate vaccines has led to a renewed interestin thermostable vaccines. Massive Mutagenesis offers an alternative solution toimprove the thermostability of monovalent recombinant protein vaccines. HepatitisB is a vaccine of particular interest because of the high prevalence of the virus (1/3 ofworld population has been exposed, 3–6% is infected).To maximize the chance of obtaining thermostabilized variants, large libraries

created by Massive Mutagenesis are selected using a unique, dedicated,

Figure 6.6 Direct thermostability selectionusing THRTM technology. A large library ofgenes, synthesized by Massive Mutagenesis orother techniques is expressed in T. thermophilusin fusion with kanamycin-nucleotidyltransferase, a gene conferring resistance to theantibiotic kanamycin. When grown on agar

plate containing kanamycin, only clonesexpressing a soluble, well-folded, fusion proteinmake colonies. These clones contain athermostabilized variant of the protein ofinterest and they are further assayed for activityindividually.


thermostability selection scheme called THRTM that is based on a specific life-or-death reporter expressed in the thermophilic organism Thermus thermophilus. Thisdirect selection technique alleviates the need for the fastidious screening of mutantsone by one. THRTM has not yet been applied for vaccines yet it has been successfullyused to increase the thermostability of several interferons as well as enzymes. It wasdeveloped in collaboration with Jos�e Berenguer, a Thermus expert at MadridUniversity, and is described in details in Ref. [38] and schematized in Figure 6.6.Briefly, a library of �5 million variants of IFNc with an average of 1 mutation permolecule was built. The library consisted of IFNc variants (N-terminal) fused with areporter protein, kanamycin nucleotidyl transferase (C-terminal). An improperlyfolded N-terminus of the protein of interest has a deleterious effect on the reporterprotein. The library was transformed into T. thermophilus and the cells were platedand grown at 70 �C on kanamycin. Non-thermostable interferon variants lead to animproperly folded fusion protein and kanamycin-sensitive clones. By contrast, mostclones that grew contained thermostabilized variants of IFNc (some contained“accidents” in the form of truncated IFNc protein). As shown in Figure 6.7, variantF159C displays an improved resistance to incubation at 60 �C (50% activity after30min against <5% for the wild-type IFNc). THRTM has been successfully used tostabilize other interferons and is currently applied to other proteins. Interestingly,

Figure 6.7 THRTM isolates thermoresistant IFNc variants. Activity assay of wild-type (blue) andmutant (red) interferon isolated using the THRTM technique after incubation for various times at60 �C.


it was shown that disulfide bond formation could occur correctly in T. thermophiluscytoplasm. The same approach to thermostabilization (which is directly linked toshelf-life) is currently being considered for application to thermostable vaccines andother biopharmaceuticals.

6.4Conclusion and Perspectives

Massive Mutagenesis is a unique and flexible technology that is used to create verylarge (108–1010) yet focused libraries that are suitable for HTS and en masse selectionapproaches. Structure and homology data can be easily included in the strategy. Inlarge-scale projects, Massive Mutagenesis makes use of numerous oligonucleotidesand the feasibility of using chip-eluted oligonucleotides pools to lower the work andtime has now been established. Alongside applications in API biocatalysis andbiofuels/biorefinery and in conjunction with powerful activity and stability selectiontechniques, Massive Mutagenesis is currently being used at Biom�ethodes to tailornext generation biopharmaceuticals, for example, in the area of hemophilia.

Acknowledgments

The authors thank Fr�ed�eric Ducancel, Michel Jolivet, and Andr�e Menez for criticalreading of the manuscript and Anne-Laure Swiercz for her helpful contributionto Part 3.

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