FEBS Letters 588 (2014) 269–277
`
`j o u r n a l h o m e p a g e : w w w . F E B S L e t t e r s . o r g
`
`Review
`Stability engineering of the human antibody repertoire
`Romain Rouet a,⇑, David Lowe b, Daniel Christ a,c
`
`a Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia
`b MedImmune, Milstein Building, Granta Park, Cambridge CB21 6GH, United Kingdom
`c The University of New South Wales, Faculty of Medicine, St Vincent’s Clinical School, Darlinghurst, Sydney, NSW 2010, Australia
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Human monoclonal antibodies often display limited thermodynamic and colloidal stabilities. This
`behavior hinders their production, and places limitations on the development of novel formulation
`conditions and therapeutic applications. Antibodies are highly diverse molecules, with much of the
`sequence variation observed within variable domain families and, in particular, their complemen-
`tarity determining regions. This has complicated the development of comprehensive strategies for
`the stability engineering of the human antibody repertoire. Here we provide an overview of the
`field, and discuss recent advances in the development of robust and aggregation resistant antibody
`therapeutics.
`Crown Copyright Ó 2013 Published by Elsevier B.V. on behalf of Federation of European Biochemical
`Society. All rights reserved.
`
`Article history:
`Received 1 November 2013
`Revised 20 November 2013
`Accepted 20 November 2013
`Available online 28 November 2013
`
`Edited by Wilhelm Just
`
`Keywords:
`Protein engineering
`Antibody therapeutics
`Protein aggregation
`Stability
`
`1. Introduction
`
`The number of human monoclonal antibody candidates has in-
`creased rapidly in recent years, and now represent the largest sin-
`gle class of molecules entering clinical studies [1]. Despite the
`rapid growth of the antibody therapeutics market over the past
`twenty to thirty years, hurdles remain that limit their manufac-
`ture. Key factors relate to the variable and often limited stability
`of human antibodies, which negatively impact on many production
`processes including expression [2,3], purification [4] and formula-
`tion [5].
`Antibodies are complex multidomain proteins and mechanisms
`governing their thermodynamic [6] and, in particular, colloidal [7]
`stability are not fully understood. Human antibodies of the com-
`monly used immunoglobulin G (IgG) type consist of a total of
`twelve domains, which can be further divided into two chains
`(heavy and light) and variable (VH, VL) and constant (CH1, CH2,
`CH3, CL) domains (Fig. 1). Only limited diversity is observed among
`constant domains, with a total of four isotype (IgG1–4) and two
`light chain ([j] kappa and [k] lambda) classes expressed in hu-
`mans. Consequently, isotype differences can explain only a limited
`proportion of the observed stability variation in the human anti-
`body repertoire [8]. A higher proportion of sequence diversity is
`observed among antibody variable domains, which are assembled
`through genetic recombination of variable, diverse (VH only) and
`
`⇑ Corresponding author.
`
`E-mail address: r.rouet@garvan.org.au (R. Rouet).
`
`joining segments (VDJ-recombination). Further diversity is then
`introduced into framework, and in particular complementarity
`determining regions (CDR), through somatic hypermutation, fol-
`lowed by clonal selection of the repertoire. As a result of these pro-
`cesses, the overwhelming majority of sequence variation within
`the human antibody repertoire is observed within the CDR regions
`of a limited range of human variable domain families. In this re-
`view, we summarize the influence of the observed antibody diver-
`sity on colloidal and thermodynamic stabilities and discuss recent
`advances to improve these properties through engineering
`approaches.
`
`2. Protein aggregation
`
`Aggregation is a complex process by which proteins can form
`alternative colloidal states, which are different from the native
`state, but otherwise energetically favorable [9,10]. It is generally
`believed that such aggregate species are predominantly formed
`via unfolded or partially unfolded states [11]. Protein aggregation
`is increasingly recognized as a problem affecting the manufactura-
`bility of human therapeutic antibodies, shelf life and efficacy [12].
`Importantly, the presence of aggregates has also been linked to in-
`creased immunogenicity, with effects ranging from mild skin irri-
`tation to anaphylaxis [13].
`Although it can be assumed that stabilization of the native state
`over alternative aggregate states has occurred during evolution
`[14], it is important to note that the production of monoclonal anti-
`bodies exposes these molecules to a wide range of non-physiological
`
`0014-5793/$36.00 Crown Copyright Ó 2013 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.
`http://dx.doi.org/10.1016/j.febslet.2013.11.029
`
`Ex. 2033-0001
`
`

`
`270
`
`R. Rouet et al. / FEBS Letters 588 (2014) 269–277
`
`Fig. 1. Structure of the human IgG molecule. The molecule is formed by two heavy chains (consisting of VH, CH1, CH2 and CH3 domains) and two light chains (consisting of VL
`and CL). Figure was generated using PYMOL.
`
`processes and conditions. These include recombinant expression,
`purification, concentration, viral inactivation, filtration, formulation,
`freeze/drying, transport and long-term storage. Throughout these
`steps the antibody molecule may encounter several stress factors
`that can dramatically increase its propensity to aggregate (including
`variations of temperature, pH, protein concentrations, ionic strength,
`exposure to air–water interfaces and mechanical stress). A further
`driver of protein aggregation has been a growing trend towards for-
`mulations that allow sub-cutaneous administration routes. This re-
`quires formulation at high protein concentrations (at around
`100 mg/ml) in syringes for self-injection, which places increased de-
`mands on colloidal stability [15].
`
`3. Stability of human antibody isotypes and constant domains
`
`The four human IgG isotypes differ in their stabilities and bio-
`logical functions, namely their potential to induce cellular killing
`through antibody dependent cellular cytotoxicity (ADCC) and com-
`plement dependent cytotoxicity (CDC). While the human IgG1 iso-
`type induces powerful ADCC and CDC responses, this is not the
`case for human IgG2, which is particularly suitable for applications
`where cellular killing is not required (such as neutralization of sol-
`uble ligands) [16]. Unlike the other human isotypes, IgG4 also has a
`naturally occurring potential to form a bispecific molecule [17] (it
`has been recently shown that this dual specificity can be grafted
`onto other isotypes [18]). Human IgG3 is not commonly used
`due to its longer hinge region, which renders it susceptible to pro-
`teolysis [19]. The vast majority of human antibody therapeutics
`currently in clinical practice and development are of the IgG1 iso-
`type [16].
`Conflicting evidence exists in respect to the colloidal stability of
`the various human IgG isotypes. A recent study of eleven different
`
`IgG1 and IgG2 antibodies concluded that, after high temperature
`storage, the IgG2 isotype is more prone to aggregation [20]. Similar
`findings were also obtained when subjecting these isotypes to high
`salt conditons [21] and from isotype switching studies [22]. The
`latter study confirmed that the human IgG1 isotype is less prone
`to aggregation when compared to IgG2 or IgG4. However, the
`authors also found that this isotype is more prone to fragmenta-
`tion, especially at low pH, due to a non-enzymatic site in the upper
`hinge region. The hinge regions have also been implicated in the
`observed differences in aggregation propensity as the IgG2 hinge
`contains two additional cysteines in each heavy chain compared
`to IgG1, and is prone to the display of free cysteine residues
`[23,24]. Differences in the thermal stability of human constant do-
`mains have also been reported, with the CH3 domains of human
`IgG1 and IgG2 displaying similar melting temperatures, much
`higher than what was observed for IgG4 [8]. In general, CH3 do-
`mains exhibit more favorable biophysical properties than CH2 do-
`mains [25]. This is reflected by melting temperatures of 8–10 °C
`higher than those observed for CH2 domains [8,26].
`Although human IgG1 therefore appears less aggregation
`prone than other isotypes, this is not universally the case. For
`example, a recent study found that the IgG1 isotype variant of
`an anti-LINGO-1 antibody exhibited a much higher propensity
`to aggregate than an IgG2 variant containing identical variable
`regions [27]. Studies such as these demonstrate that predicting
`the aggregation propensities of IgG molecules based on isotype
`can be difficult at the best of times. Moreover, evidence suggests
`that the aggregation propensity of human IgG molecules is greatly
`influenced by their variable domains [8,27,28]. This has the
`effect
`that antibodies with identical constant domains, but
`different variable domains, can vary widely in their stability pro-
`files [5].
`
`Ex. 2033-0002
`
`

`
`4. Stability of human antibody variable domains
`
`5. Increasing antibody stability through formulation
`
`R. Rouet et al. / FEBS Letters 588 (2014) 269–277
`
`271
`
`Human antibody variable domains can be grouped into several
`homologous families, which differ in their biophysical properties. A
`seminal study of consensus domains of human VH and VL families
`has revealed considerable differences of expression yields and
`thermodynamic stability [6]. In the case of human VH domains, sta-
`bilities ranging from DGN–U 14–53 kJ/mol were observed, with sol-
`uble expression yields ranging from 1–2.4 mg/l
`(Table 1).
`Intriguingly, Ewert et al. also reported that members of the (odd
`numbered) VH 1, 3 and 5 families generally displayed superior bio-
`physical properties in comparison with the (even numbered) 2, 4
`and 6 families. These differences were observed for both thermo-
`dynamic stabilities and expression yield. In particular, the human
`VH3 family was identified as uniquely stable and well expressed.
`This family is also among the most abundant in the human reper-
`toire [29]. The overall stability trends were confirmed in a more re-
`cent study, which analyzed the effect of pairing of different human
`variable domain families on expression, thermal and colloidal sta-
`bilities of Fab fragments and IgG monoclonals [30].
`More limited differences in biophysical properties were ob-
`served among human VL families, with thermodynamic stabilities
`ranging from DGN–U 15–24 kJ/mol for lambda and DGN–U 29–
`35 kJ/mol for kappa [6]. Similarly, higher expression yields were
`observed for kappa variable light domains (5–17 mg/l) than for
`lambda domains (0.3–2 mg/l). The observed differences between
`antibody variable light domains were much less pronounced when
`pairing these with human VH in an scFv format, while considerable
`differences between variable heavy domain families remained.
`Overall, the authors found the VH/VL combinations H3j3, H1bj3,
`H5j3 and H3j1 to be superior in respect to expression yield and
`thermodynamic stability [6]. However, despite the clear influence
`of variable domain family (and framework regions), the study also
`observed a strong influence of CDR sequence on biophysical prop-
`erties. Indeed, initial attempts of the authors to express human VH
`domain in isolation failed to yield protein (with the exception of
`VH3). Only the grafting of a solubilizing CDR3 region allowed the
`soluble expression of human VH1, and of VH2, VH4, VH5 and VH6
`through refolding from inclusion bodies.
`Although several of the human variable domain families are
`thermodynamically stable, they nevertheless often display poor
`colloidal stability and readily aggregate when heated above their
`melting temperatures. This behavior is exemplified by the VH3
`model domain DP47 [31] and by studies of synthetic antibody rep-
`ertoires on phage, which have demonstrated that as few as one in a
`thousand VH domains resists heat induced aggregation [32].
`
`Table 1
`Biophysical properties of human antibody variable domains. Adapted from Ref. [6].
`
`Human
`family
`
`Solubilizing
`CDR3
`required
`
`Expression
`yield
`
`DGN–U
`
`VH
`
`VL
`
`1a
`1b
`2
`3
`4
`5
`6
`
`j1
`j2
`j3
`j4
`k1
`k2
`k3
`
`Y
`Y
`Y
`N
`Y
`Y
`Y
`
`N
`N
`N
`N
`N
`N
`N
`
`+
`+
`Refold
`+
`Refold
`Refold
`Refold
`
`+
`+++
`+++
`++
`+
`+
`+
`
`+
`++
`nd
`+++
`nd
`+
`nd
`
`++
`++
`+++
`-
`++
`+
`+
`
`The most common method for controlling the biophysical prop-
`erties of monoclonal antibodies and other biologics is through
`changes of buffer conditions and excipients. Formulation allows
`for the control of ionic strength, pH and the addition of excipient
`agents such as sugars or surfactants, which have been shown to
`stabilize monoclonal antibody preparations
`[33]. Although
`formulation can shield proteins from some chemical and physical
`challenges (including deamidation, oxidation, hydrolysis/fragmen-
`tation and isomerization), aggregation remains difficult to control
`[12]. Consequently, finding appropriate formulation conditions
`for an individual product is often an arduous process. This is
`exemplified by the therapeutic monoclonal antibody cetuximab
`(Erbitux). Its initial formulation conditions (2 mg/ml in phosphate
`buffer pH 6.0) generated visible aggregates under mechanical
`stress
`[34]. To address
`this
`issue, conditions had to be
`empirically optimized and were changed to 5 mg/ml in citrate
`buffer pH 5.5 with the addition of glycine and polysorbate 80 as
`stabilizers.
`In recent years, more high-throughput techniques for formula-
`tion optimization have been developed [35], and stabilizers and
`excipients with more general applicability have been identified.
`Among these, arginine is commonly used to improve refolding
`yields [36,37], and more recently has been used in formulation to
`reduce heat-induced aggregation [38]. Another commonly used
`class of excipient molecules are polysorbates, which accumulate
`at the air–solvent interface and offer protection against shaking
`and other mechanical stresses [39]. Despite recent advances, the
`identification of suitable formulation conditions for a specific
`monoclonal antibody remains challenging and cannot be deter-
`mined from its amino acid sequence. Indeed, a considerable pro-
`portion of human monoclonal
`antibody
`candidates
`fail
`formulation studies, often at relatively late pre-clinical stage, plac-
`ing heavy burdens onto drug development pipelines [5].
`
`6. Stability engineering of human antibody constant domains
`
`Many efforts have been made to increase the stability of the dif-
`ferent human antibody isotypes. Early work in this field focused on
`sequences analyses to predict stabilizing mutations. This approach
`allowed the identification of a set of substitutions (371K, 376G and
`392L), which considerably increased thermal stability (from 76 °C
`to 86 °C) of the CH3 domain [40]. The study was based on frequency
`analyses within 19 mammalian species to generate a consensus se-
`quence. Structural approaches have also been utilized to increase
`stability of the CH1/CL interface [41]. More recently, it was shown
`that the introduction of additional intra-domain disulfide bridges
`in the human CH3 domain resulted in the thermal stabilization of
`an isolated IgG1 Fc fragment [42] (Fig. 2). In particular, the intro-
`duction of a link between the N-terminal A strand (position 343)
`and the F strand (position 431) caused a 8 °C increase in the melt-
`ing temperature, while a linkage between positions 375 and 396
`resulted in a 4 °C increase (combined 13 °C). These findings were
`successfully applied to a HER2 specific Fcab molecule (Fc fragment
`with engineered binding loops) resulting in an increased in the
`thermal stability of 19 °C. A similar finding was observed for a hu-
`man IgG1 CH3 single domain, which could be stabilized by intro-
`duction of an intra-domain disulfide bridge [43]. The authors
`used a two-step approach, by initially introducing mutations that
`rendered the CH3 domain monomeric, resulting in a overall low
`thermal stability (41 °C compared to 82 °C for the native dimeric
`form). Using structure-based design, two additional cysteines (at
`positions 343 and 431) were then introduced to generate a disul-
`fide bridge, which increased the thermal stability to 76 °C (a
`
`Ex. 2033-0003
`
`

`
`272
`
`R. Rouet et al. / FEBS Letters 588 (2014) 269–277
`
`Fig. 2. Stability engineering of human constant domains.
`
`35 °C increase) [43]. Similar strategies have been also reported for
`CH2 domains [26]. Here the authors reported the engineering of an
`isolated human CH2 domain, resulting in an increase in thermal
`stability of 20 °C (from 54 °C to 74 °C) [26]. More recently, the
`same group reported the stabilization of a CH2 domain through re-
`moval of an unstructured region at the N-terminus. Combined with
`the intra-domain disulfide bond, this resulted in a further increase
`of melting temperature of 10 °C [44]. For all of the above studies,
`stability of constant domains was evaluated in the context of frag-
`ments or isolated domains.
`Other studies have investigated the effects of such mutations on
`the stability of full-length human IgG. In the case of human IgG4,
`alteration of the disulfide bond network in the Fab region was
`shown to lead to an increase of thermal stability of between 3
`and 7 °C [45]. This effect was achieved through removal of a cys-
`teine at position 127 in the CH1 domain, while an additional in-
`ter-domain disulfide bond between position 229 and CL was
`introduced (similar to what is observed in human IgG1). Impor-
`tantly, the authors reported that the introduced mutations did
`not affect antigen binding [45]. More recently, molecular dynamic
`simulations have been used to predict the spatial aggregation pro-
`pensity (SAP) of human IgG molecules [46]. The authors later
`showed that by mutating identified surface-exposed hydrophobic
`patches within the hinge and Fc regions of an IgG, aggregation
`resistance and, to a more limited extent, thermodynamic stability
`could be improved [47].
`
`7. Increased stability through charged fusion tags
`
`In addition to the modification of constant domains, it was re-
`cently shown that the addition of charged fusion tags to the N-ter-
`minus of human variable domains could considerably improve
`aggregation propensity [48]. The authors noted that batches of
`the same IgG monoclonal expressed in yeast were less prone to
`aggregation than those expressed in mammalian cells. Mass spec-
`trometry and sequencing analyses revealed the presence of a non-
`processed ‘‘EAEA’’ leader sequence within both heavy and light
`chains. In contrast to colloidal stability, the authors did not observe
`improved thermodynamic stability, and the effect seemed to be
`particularly pronounced for antibodies with a low global net
`charge. In addition to terminal fusion tags, a different study ex-
`plored the internal use of a ‘‘DED’’ motif [49]. It was shown that
`the insertion of the tag into the CDR3 region of aggregation-prone
`VH domain antibodies reduced their aggregation propensity, while
`reducing retention on gel filtration matrices.
`
`8. Stability engineering of human antibody variable domains
`
`8.1. Engineering of human VH domains through mutation of the light
`chain interface
`
`The poor biophysical properties of isolated human antibody
`variable domains are in marked contrast to those of the variable
`domains of camels and llamas [50]. Although these immunoglobu-
`lin domains (VHH) display a relatively high level of sequence iden-
`tity with human VH domains, they are generally well expressed
`and soluble [51] and reversibly unfold after incubation at temper-
`atures as high as 90 °C [52]. In addition to the observed differences
`in biophysical nature, camelid domains also display an important
`difference in chain structure, as they are naturally devoid of light
`chain partners [50]. Their single domain nature results in unique
`structural features, compared to human antibody VH domains.
`These include CDR length and conformation, as well as framework
`residues. A particular feature of VHH domains is their long flexible
`CDR3 loop capable of folding back on itself, thereby protecting an
`area forming the VH/VL interface in human antibodies. In addition,
`these naturally occurring single domains display a marked reduc-
`tion in hydrophobicity of the former light chain interface, due to
`a limited number of changes in their respective germlines
`(Fig. 3). The most common and significant changes compared to
`humans, coined the ‘‘VHH tetrad’’, are located at positions: G44Q,
`L45R/C, W47G/I and V37F/Y (human/camelid). The latter residue
`is nucleating a small core involving Y91, W103, R45 and other
`hydrophobic residues within the CDR3 loop [53]. Structural inves-
`tigation of VHH has also shown the CDR3 loop covering residue 37,
`thereby shielding hydrophobicity in an area corresponding to
`the light chain interface in human VH [54]. In addition, camelid
`VHH often contain an additional disulfide bridge between CDR1
`and CDR3, which increases their thermal stability [55,56].
`In order to improve the biophysical properties of human VH do-
`mains, elements of the VHH tetrad have been incorporated into hu-
`man frameworks (‘‘camelization’’) [57]. Although the introduced
`framework changes improved the solubility of human VH, they also
`reduced expression levels, induced conformational changes, de-
`creased thermodynamic stability and did not considerably improve
`the colloidal stability of the domains [58,59]. In contrast, combining
`the approach with the introduction of an additional disulfide bond
`between CDR1 and CDR3, yielded domains with high colloidal sta-
`bility, although at the expense of antigen binding affinity [60].
`As an alternative to camelid domains, shark single domain anti-
`bodies (VNAR) could also potentially serve as a blueprint for improving
`
`Ex. 2033-0004
`
`

`
`R. Rouet et al. / FEBS Letters 588 (2014) 269–277
`
`273
`
`Fig. 3. Engineering of human VH domains through mutation of the light chain interface. Shown are surface representations of the light chain interface in human VH (PDB
`2VXS) and of the equivalent surfaces in camelized VH (PDB 1VHP), camelid VHH (PDB 1ZVH) and shark VNAR domains (PDB 1T6V). Residues forming the camelid ‘tetrad’ at
`positions 37, 44, 45 and 47 are highlighted in green. Camelized VH, VHH and VNAR domains display a considerable reduction in surface hydrophobicity in comparison to human
`VH domains. Figure was generated using PYMOL.
`
`the stability of human antibody VH domains (although they are more
`distantly related to human domains [61]). As observed for camelid
`domains, shark VNAR domains are characterized by a long protruding
`CDR3 [62]. The CDR loop is constrained by additional disulfide bonds,
`thereby folding over an area equivalent to the former VH/VL interface
`of human antibodies [61]. A recent study revealed that VNAR domains
`are resistant to thermal denaturation, acidic denaturation, and lyo-
`philisation denaturation and are capable of returning to their native
`state after thermal unfolding [63].
`Rather than focusing on direct grafting of camelid residues,
`more recent approaches for the generation of autonomous single
`domains have focused on the identification of novel mutations.
`This has been possible due to the use of phage display based selec-
`tion strategies and the use of protein A to select for folded domains
`(pA superantigen binds to folded human VH3 domains, but not to
`unfolded or aggregated domains). Using this approach, an exten-
`sive mutagenesis study has identified hydrophilic substitutions
`within the VH3/VL interface [53]. The authors were able to demon-
`strate that their mutations (including H35G, Q39R, L45E, R50S) im-
`proved solubility, thermal refolding and melting temperature.
`Importantly, some of the mutations improved protein A binding
`(and presumably stability), independently of CDR3 sequence diver-
`sity. As CDR3 of VH provides the majority of binding energy in anti-
`body–antigen interactions, this indicated broad compatibility with
`antigen binding and suggested that the engineered frameworks
`could be used for the construction of stable repertoires of autono-
`mous VH domains.
`
`8.2. Engineering of human VH domains through CDR grafting and
`framework mutations
`
`strategies relates to the use of CDR grafting for the improvement
`of biophysical properties. Developed by Jones and Winter in the
`mid-1980s, CDR grafting was initially used to reduce the immuno-
`genicity of mouse monoclonals, by transplantation of murine CDR
`regions onto a human framework [64]. However, it was later
`shown that by grafting onto a stable and well-behaved framework
`(such as the Herceptin 4D5 [VH3] framework) considerably im-
`proved expression levels and thermodynamic stability, while
`maintaining binding affinity of the parental antibody fragment
`[65]. A second approach is based on the use of consensus se-
`quences to improve stability [66]. This approach has been applied
`to a range of protein scaffolds [67], and utilizes amino acid ob-
`served with high frequency within the repertoire [68]. This ap-
`proach was also used in the construction of the HuCAL human
`antibody library, for which the authors have reported good expres-
`sion yields [69]. More recent studies from the same group have
`identified additional framework mutations that transfer biophysi-
`cal properties between human VH3 and VH6 families [70,71].
`As an alternative to homology-based approaches, other studies
`have focused on the computational prediction of aggregation hot-
`spots in antibody sequences. Early studies on peptides and proteins
`involved in neurodegenerative diseases have highlighted physico-
`chemical properties that correlate with an increase in aggregate
`formation, namely hydrophobicity, b-sheet propensity and re-
`duced net charge [72]. Algorithms have been developed to detect
`such aggregation-prone regions [73–76]. Two of these algorithms,
`TANGO and PAGE, have been used to identify potential aggrega-
`tion-prone regions in commercial therapeutic antibodies, which
`are predominately located in variable domains, and particularly
`within CDR and adjacent framework residues [28].
`
`In addition to their use in the engineering of autonomous anti-
`body single domains, homology-based approaches have also been
`used for the identification of mutations outside the interface re-
`gions, thereby retaining the ability of human VH to interact with
`variable light domains. One of the earliest examples of such
`
`8.3. Engineering of human variable domains through CDR mutations
`
`The role of CDR residues in determining thermodynamic and in
`particular colloidal stability of human antibody variable domains is
`further highlighted by studies on the biophysical properties of the
`
`Ex. 2033-0005
`
`

`
`274
`
`R. Rouet et al. / FEBS Letters 588 (2014) 269–277
`
`HEL4 model domain [31]. HEL4 was originally isolated through
`phage display selection from a synthetic CDR-only repertoire based
`on the human VH3-23 DP47 germline [77]. As phage is remarkably
`resistant to chemical, proteolytic and thermal denaturation [78–
`80], the authors were able to heat the phage displayed antibody
`fragments at high temperature (80 °C), followed by cooling and
`binding to antigen (hen egg white lysozyme). The method thereby
`selected for domains that unfolded reversibly, and could be cap-
`tured by binding to antigen or superantigen. Unlike other human
`VH3 domains, HEL4 displayed highly favorable biophysical proper-
`ties, including heat-refoldability, high expression levels in bacteria,
`and the absence of ‘stickiness’ on gel-filtration [31]. Indeed, it was
`later shown that aggregation resistance on phage is a general indi-
`cator of the solution behavior of human VH domains [81]. In addi-
`tion to HEL4, several other VH domains, that were isolated using
`this method, showed a high level of resistance to aggregation.
`Intriguingly, despite their high level of colloidal stability, all of
`the domains actually had lower thermodynamic stabilities than
`the DP47 domain from which they had been derived from
`(DGN–U = 15–20 kJ/mol vs 35 kJ/mol). In contrast, DP47 readily
`aggregated upon heating, further highlighting differences between
`
`thermodynamic and colloidal stability. As an alternative to heat-
`based selection on phage, a more recent study explored the use
`of pH stress (pH 3.2) for the selection of aggregation-resistant VH
`domains [82].
`In contrast to domains isolated after thermal
`the selected VH domains displayed increased
`denaturation,
`thermodynamic stabilities and increased colloidal stability at low
`pH, reflecting the different denaturation conditions experienced
`during the selection process.
`Although the denaturation method on phage was capable of
`selecting human VH domains with CDR sequences that promoted
`a high level of aggregation resistance, it was also evident that such
`domains were rare within the repertoire. To increase the frequency
`of such clones, a human VH library was constructed through com-
`binatorial assembly of CDR regions that had been pre-selected for
`aggregation resistance in phage. Binders were successfully selected
`from this repertoire, which exhibited good antigen-binding prop-
`erties together with favorable biophysical properties [32].
`Despite clear improvements over other human VH domains, de-
`tailed determinants of the observed aggregation resistance initially
`had remained unclear. As HEL4 and other clones had been selected
`from synthetic CDR-only repertoires, it was apparent that the
`
`Fig. 4. Effect of mutations in human antibody VH and VL domains on aggregation resistance. Surface residues in variable heavy and light domains were targeted for
`substitution with aspartate. Aggregation resistance of the domains was determined by measuring binding to protein A and protein L superantigen after heating to 80 °C on
`phage. Complementarily determining regions are indicated (A) Mutations in human VH. (B) Mutations in human VL. Introduction of aspartate into CDR H1 and CDR L2
`increases aggregation resistance of human variable domains up to 40-fold for VH and up to 80-fold for VL. (C) Mutations mapped onto the human VH surface. (D) Mutations
`mapped onto the human VL surface (blue: 100% retained superantigen binding, white: 0%). (E) Stability engineering of human VH repertoire. (F) Stability engineering of
`human VL repertoire. The mutant repertoires were generated through introduction of aspartate mutations in CDR H1 and CDR L2 (at positions 32/33 and 52/53, respectively).
`Introduction of the mutations significantly increases the aggregation resistance of human antibody repertoires, independent of sequence diversity at other CDR positions.
`Adapted from Ref. [7].
`
`Ex. 2033-0006
`
`

`
`R. Rouet et al. / FEBS Letters 588 (2014) 269–277
`
`275
`
`exhibited biophysical properties were a result of changes in the
`complementarity determining regions, without any further influ-
`ence of framework residues [31]. Crystal structure of the HEL4
`model domain had indicated a potential effect of a serine to glycine
`mutation at position 35 [77]. Indeed, introduction of this mutation
`into DP47, resulted in increased solubility and reduced ‘‘stickiness’’
`on gel-filtration. However, the mutant domain continued to readily
`aggregate when heated above its melting temperature. The authors
`also reported that many of the selected domains had a low isoelec-
`tric point (pI). Similar findings were also obtained in a second
`study, which reported that a low pI, together with the non-canon-
`ical inter-CDR disulfide linkages, contribute to improved biophysi-
`cal properties human VH domains [83]. Using transient heating to
`pre-select for proteins reversible thermal unfolding properties,
`the authors found that many of the isolated domains had pI values
`of less than six, although they also observed domains with values
`higher than eight. The authors concluded that the improved bio-
`physical characteristics of these VH domains were caused by a
`combination of global pI effects, together with an increase of sta-
`bility through inter-CDR disulfide linkage (other studies by the
`same group demonstrated that, although the additional disulfide
`bridge significantly increased thermal stability and protease resis-
`tance [84], it also induced structural changes in human VH as indi-
`cated by a marked reduction of protein A superantigen binding
`[85]). A global increase in charge was also observed in a different
`study, analyzing larger sets of VH domain sequences after heat
`selection on phage, together with an overall reduction of hydro-
`phobicity and a decrease in beta-sheet propensity [86].
`More recently, an extensive mutagenesis study on phage has
`provided detailed insights into mutations that increase the colloi-
`dal stability of human VH and VL antibody domains [7]. Using
`superantigen binding after heat denaturation on phage as a mean
`to