Design and Application of Antibody Cysteine Variants - Bioconjugate

Bioconjugate Chem. 2010, 21, 385–392

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Design and Application of Antibody Cysteine Variants Vladimir Voynov,† Naresh Chennamsetty,† Veysel Kayser,† Hans-Joachim Wallny,‡ Bernhard Helk,‡ and Bernhardt L. Trout*,† Massachusetts Institute of Technology, Chemical Engineering, Cambridge, Massachusetts, and Novartis Pharma AG, CH-4057, Basel, Switzerland. Received November 19, 2009; Revised Manuscript Received December 30, 2009

Antibodies are multidomain proteins that are extensively used as a research tool in molecular biology and as therapeutics in medicine. In many cases, antibodies are engineered to contain surface cysteines for the sitespecific conjugation of payloads. These antibodies can serve as payload vehicles in targeting a diseased cell to which the conjugated molecules exercise their activity. Here, we design and analyze a set of fourteen new IgG1 cysteine variants, with at least one variant per immunoglobulin fold domain. The cross-linking propensity of these mutants correlates very well with a tool we have developed for measuring aggregation propensity in silico, called spatial aggregation propensity (SAP). Our results indicate the utility of the SAP technology in selecting antibody cysteine variants with desired properties. Moreover, the different oligomerization propensity of the variants suggests a variety of applications in molecular biology and medicine, such as payload delivery, structural analysis, electrophoresis, and chromatography.

INTRODUCTION Monoclonal antibodies are of significant laboratory and therapeutic use. Antibody derivatives with engineered sitespecific fluorescence or binding properties have been developed and used for many years. Antibodies have also been developed as therapeutic agents, currently the fastest growing class of pharmaceuticals (1). Antibodies are multidomain proteins of two light and two heavy chains held together by disulfide bonds. The variable regions specify binding to a particular antigen, and part of the constant region is responsible for effector functions via binding to Fc receptors on the surface of immune cells. Engineering of antibody conjugates has further increased the versatility of antibody applications. In many laboratory techniques, enzymes or fluorescent probes are conjugated to antibodies to carry out an assay function, for example, quantification of antigen abundance. In targeted therapy, conjugation is used to generate new therapeutics with highly specific functions (2). For example, toxic small molecules are attached to antibodies that specifically bind biomarkers on diseased cells, and the targeted cells are destroyed by the toxic molecules (3–6). Various approaches to antibody conjugation have been pursued, for example, attachment to surface lysines (7), to Fc carbohydrates (8), or to partially reduced interchain disulfides (9). Antibody conjugation to engineered surface cysteines remains a very attractive option because most antibodies do not have cysteines other than the ones involved in intra- and interchain disulfide bonds. Small molecules can be attached at the specific site of cysteine substitution via thiol-reactive chemistry with molecules such as maleimides (10–16). Engineering the CH1 and CH3 domains has been favored to avoid interference with antigen binding of the variable regions and effector function of CH2. Different criteria for successful antibody conjugation via engineered cysteines have been considered. For example, the antibody domain in which to carry out mutations, the exposure of the mutated site, and the amino acid to be substituted are * To whom correspondence should be addressed. E-mail: trout@ mit.edu. † Massachusetts Institute of Technology. ‡ Novartis Pharma AG.

several of the aspects to take into account. A high-throughput screening approach to identifying sites suitable for cysteine engineering and conjugation has been developed (17). Yet, there is no universal tool for predicting whether an antibody cysteine variant will be stable and efficiently conjugated, and cysteine variants currently exist only for the CL, CH1, and CH3 domains (10, 11, 13, 14, 17). In this report, we present a new approach to design cysteine variants and the application of that approach to develop a new set of 14 human IgG1 cysteine variants, chosen so that there is at least 1 variant per immunoglobulin fold domain. Most of the variants are stable and efficiently conjugated. In several cases, we observe antibody oligomerization due to intermolecular disulfide bonding and cross-linking of the engineered cysteines. The extent of oligomerization is qualitatively and quantitatively expressed as cross-linking propensity (CLP). We also compare CLP of the variants to a recently developed spatial-aggregation propensity (SAP) parameter (18–20). SAP is a measure of the dynamic exposure of hydrophobic patches (see Experimental Procedures). SAP identifies protein aggregation prone regions, which were validated earlier by designing antibodies that were experimentally shown to be significantly more stable than the wild type (19). The analysis presented here demonstrates the utility of the SAP technology in designing antibody cysteine variants as well as the various applications of some of the new variants.

EXPERIMENTAL PROCEDURES Design of Cysteine Variants. Variants 1-13 were designed based on the X-ray structure of antibody-1. Variant 15 was selected from the structure of another IgG1, antibody-2, built by homology modeling based on the structure of antibody-1 (19). Variant 14 has the same engineered cysteine as variant 15 but in a modified antibody-2 with mutations W100K and F101K in the heavy chain (19). All engineered cysteine sites were exposed on the antibody surface. Polar residues, such as serine, threonine, and asparagine, or charged residues, such as lysine, were substituted with cysteine. Production of Cysteine Variants. DNA vectors encoding the light and heavy chains of proprietary human IgG1 antibodies

10.1021/bc900509s  2010 American Chemical Society Published on Web 01/21/2010

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1 and 2 were kindly provided by Dr. Burkhard Wilms from Novartis. The two antibodies have κ-variable light chains and H3 variable heavy chains and are indicated for the treatment of inflammatory and respiratory diseases. The light and heavy chain genes of each antibody were subcloned in vector gWIZ (Genlantis), engineered for protein expression by transient transfection of mammalian cells. Antibody variants were either de novo synthesized (GeneArt) or generated by in-house sitedirected mutagenic PCR and confirmed by sequencing. Antibody wild types and variants were expressed by transient transfection of Freestyle HEK 293 cells (Invitrogen) and purified as previously described (19) and further specified here. Cell cultures were transfected with filter-sterilized DNA, purified on DNA Maxi Prep columns (Invitrogen) and with linear polyethyleneimine MW 25 000 (PEI) (Polysciences) as the transfection reagent. For each liter of culture, 1 mg of total DNA was incubated with 20 mL of OptiPro (Invitrogen), while 2 mL of 1 mg/mL solution of PEI was incubated with 20 mL of OptiPro. After 15 min incubation at room temperature, the PEI solution was added to the DNA solution, and after another 15 min incubation, the PEI-DNA-OptiPro mix was added to the cell culture. The transfected cell cultures were incubated at 37 °C and 5% carbon dioxide atmosphere. Supernatants were collected 7-10 days post-transfection by centrifugation and filtering through 0.2 µm filters. If transfections were at higher than 1 L volume, the filtered supernatants was concentrated by diafiltration with a 30 kDa molecular weight cutoff membrane before purification. Antibodies were purified on a protein A column (GE Healthcare), eluted with 50 mM citrate buffer, pH 3.5, buffer-exchanged in 50 mM Tris pH 7.0 buffer, and concentrated to 15-50 mg/mL with 30 kDa molecular weight cutoff filters. Typically, antibody-1 wild type was expressed with yield of 25 mg/L and antibody-2 wild type at 24 mg/L. In most cases, the cysteine variants were expressed at comparable levels, with a few exceptions where the mutation affects expression levels. Thiol Reduction and Labeling. Different methods were attempted for the specific reduction of the engineered surface thiols before labeling. Tris, 2-carboxyethyl, phosphine hydrochloride (TCEP), and a stronger reducing agent, dithiothreitol (DTT), were two of the reagents used, and levels of free thiol were quantified using Ellman’s reagent (Invitrogen). We found L-cysteine to work best in our site-specific labeling experiments, so the following two-step protocol was used. First, the variants were incubated with 100-200-fold excess of L-cysteine for 4 h at 37 °C, followed by buffer exchange into TE buffer, pH 7.2. Second, the samples were incubated with 5-10-fold molar excess of Alexa488 maleimide dye (Invitrogen) for 1 h at room temperature or with 10-fold excess of pyrene maleimide dye (Invitrogen) for 12 h at room temperature. In certain cases, labeling conditions such as time of reaction and dye to protein ratio had to be optimized on an individual basis because not all engineered cysteines were equally amenable to conjugation. Free dye was removed by centrifuging the samples through Sephadex G25 (GE Healthcare) 2-3 times. Then, the labeled protein samples were buffer-exchanged in 50 mM phosphate buffer pH 7.0 and concentrated to 20-50 mg/ mL on 30 kDa molecular weight cutoff spin filters. The efficiency of protein labeling was calculated as mole of dye per mole of protein according to manufacturer’s protocols (Invitrogen). For carbohydrate labeling, the antibody variant sample was first treated with 10 mM sodium meta-periodate in 100 mM sodium acetate, pH 5.5, (Pierce) to oxidize the carbohydrates. Then, the antibody conjugation was carried out with Alexa350-hydrazide (Invitrogen) at 5-10-fold molar excess of dye at room temperature for 2 h. During labeling, exposure of the fluorophores to light was avoided as much as possible.

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Protein Characterization. Antibody samples were analyzed by SDS-PAGE. Gels of 7.5%, 10%, and 12% were used for nonreducing analysis. Gels of 12% were used for reducing analysis of heated samples with DTT. We used a molecular weight marker “Precision Plus Protein Standards” from BioRad, containing proteins of 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kDa. Usually, samples of 5-10 µg were loaded per lane. Fluorescent images were taken under UV light before staining with Coomassie Blue. Antibody digestion was carried out by GluC (1:20 wt enzyme per wt antibody, at 25 °C for 12-24 h) and Pronase (1:20 wt enzyme per wt antibody, at 37 °C for 1 h). We also used size-exclusion high-performance liquid chromatography (SEC-HPLC) to determine monomer levels of labeled antibody variants. Samples were resolved on a TSKgel Super SW3000 column (TOSOH Bioscience), and percent monomer was calculated as the area of the monomeric peak divided by the total area of all peaks. Reporter Gene Assay. The biological activity of antibody-1 wild type and variants 1-6 was measured in a reporter gene assay, using a genetically modified cell line. This cell line was derived from human embryonic kidney cell and was stably transfected with a reporter construct of a promoter (responsive to signaling via the soluble target that antibody-1 binds) fused upstream of the luciferase gene. In this cell line, exposure to the target stimulated the expression of luciferase in a dosedependent manner. Addition of graded amounts of antibody-1 to a fixed, submaximal dose of target caused a decrease in the expression of luciferase during an incubation period of 5.5 up to 18 h. At the end of the incubation period, the amount of luciferase was quantified based on its enzymatic activity in the cell lysate. Luciferase catalyzed the conversion of the substrate luciferin to oxyluciferin, a chemiluminescent product. The resultant glow-type chemiluminescence was then determined with an appropriate luminometer. The biological potency of a test sample was determined by comparing its ability to inhibit the target-dependent induction of luciferase activity to that of antibody-1 wild type. The samples were normalized on the basis of protein content. Relative biological activity of the antibody-1 variants was then calculated using a parallel line assay according to European Pharmacopoeia. SAP Values. Spatial aggregation propensity (SAP) of amino acids in a protein is a tool that was recently developed in our lab based on amino acid surface-accessible area (SAA) and hydrophobicity (19), and we already applied to the identification of aggregation prone motifs in IgG antibodies (18). The SAP values for radius R ) 5 Å are calculated from the equation

(SAP )atomi )

∑

Simulation

(

Average

{

∑

Residues with at least one side chain atom within R from atom i

)}

SAA of side chain atoms within radius R × residue hydrophobicity SAA of side chain atoms of fully exposed residue

RESULTS

Design of a Set of 14 Antibody Variants for Site-Specific Conjugation. We designed a set of 14 IgG1 cysteine variants for site-specific conjugation. Mutations 1-13 were generated in antibody-1. Mutation 14 was generated in wild-type antibody-2 (resulting in variant 15), as well as a modified antibody-2 (resulting in variant 14), leading to a total of 15 variants

Antibody Cysteine Variants

Figure 1. A set of human IgG1 cysteine variants. (A) Relative location of the amino acids substituted with cysteines in the antibody. (B) Outline of a two-step conjugation protocol of antibody cysteine variants.

presented in this report. The idea was to have a distribution of the variants along the antibody surface, with at least one variant per immunoglobulin fold domain (Figure 1A). We substituted polar (ser, thr, asn) or charged (lys) amino acids that are exposed on the antibody surface, so that substitution with the polar amino acid cysteine does not cause a significant change in surface charge distribution and structure of the protein. Different methods for conjugating the engineered surface cysteines were applied, and the one using free cysteine in the first, reducing step worked best for us (Figure 1B). Certain Cysteine Variants Are More Amenable to Conjugation than Others. Following expression and purification of antibody variants, the engineered surface cysteines are mostly oxidized. For example, both variants 4 and 6 have less than 0.3 free thiol per antibody molecule as opposed to the anticipated 2.0 (1.0 per half antibody) for the antibodies with engineered surface cysteines. We compared the effect of a mild reducing agent, TCEP (Tris, 2-carboxyethyl, phosphine hydrochloride), and a stronger reducing agent, DTT (dithiothreitol), on two of the variants, variant 4 and variant 6. Initially, the non-oligomerizing variant 4 shows 0.13 free thiols per antibody, and the highly oligomerizing variant 6 has 0.25 free thiols per antibody. Aliquots of wild type, variant 4, and variant 6 were treated in four different conditions, 10- and 20-fold molar excess of TCEP, and 5- or 10-fold excess of DTT. After removal of the reducing agent, the samples were resolved on nonreducing PAGE (Supporting Information Figure 1A), and were quantified for free thiol (Supporting Information Figure 1B). A comparison of the results for wild type and variants indicates that the TCEP treatment is sufficient to reduce cysteines in nonoligomerized form (variant 4) with little effect on WT. However, cysteines from oligomers (variant 6) are reduced only after a harsher treatment. Treatment with DTT even at low levels leads to antibody fragmentation. Most non-oligomerizing variants like variant 4 were consequently labeled with ideal efficiency of 2.0; however, oligomerizing variants like variant 6 were underlabeled with efficiencies of 1.0-1.5. Thus, the sites where the surface cysteines are introduced have a profound effect on the ability to decap and conjugate the engineered cysteines. Distinction of Five Classes of Antibody Cysteine Variants Based on CLP. All variants were expressed by transient transfection and were labeled with Alexa-488 maleimide and Alexa-532 maleimide. Labeled and unlabeled samples were resolved on nonreducing SDS-PAGE for quality control (Figure 2 and Supporting Information Figure 2). Nonreducing gels show monomers as well as the presence of dimers, trimers, and in some cases even higher oligomers. We distinguish five classes of variants. Class I comprises variants that are monomeric and remain stable after labeling (variants 3, 4, 7, 10, 12, 14). Variants of class II contain a small percent of dimers before and after labeling (variants 1, 2, 13). Class III variants have a more pronounced propensity to oligomerize including formation of

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some trimers (variants 5, 9). Class IV variants have an even higher propensity to oligomerize as evidenced by the presence of aggregates larger than trimer, especially after labeling (variant 6). Class V includes variants of high oligomerization propensity similar to variants of class III and IV with additional structural abnormalities such as fragmentation (variant 8), coloration of purified concentrated sample (variant 11), or extremely poor expression yield (variant 15). The oligomerization propensity of variants 1-6 was compared on nonreducing SDS-PAGE for unlabeled (Figure 2A) as well as labeled (Figure 2C) samples. The CLP of variants 7-14 was analyzed in a similar fashion (Supporting Information Figure 2A). At least three molecular weight species of unlabeled variants 8 and 11 are resolved by gel filtration and electrophoresis (Figure 2E). The observed coloration in variant 11 most likely occurs because of binding of phenol red from the tissue culture media to erroneously exposed sites of the molecule, indicative of structural abnormalities of this variant. We also note that variant 14 is expressed at a level more than 100 times higher than that of variant 15 (Supporting Information Figure 2B, and Figure 4A). The mutation S7C on the heavy chain is very poorly tolerated in an antibody with a high-SAP patch in the VH domain (variant 15) resulting in very low expression levels. In contrast, a variant with the same S7C mutation is expressed well when two amino acids in the high-SAP patch, W100 and F101, are substituted with lysines (variant 14). In addition to nonreducing SDS-PAGE, labeled variants were also analyzed by size-exclusion high-performance liquid chromatography (SEC-HPLC). Variants of class I and II are usually more than 95% but at least 80% monomeric, while variants of other classes are less than 70% monomeric, with the rest of the samples forming aggregates of various sizes (Supporting Information Figure 2C). Engineered Variants Retain Antigen-Binding Activity and Are Specifically Labeled. Labeled and unlabeled variants 1-6 were analyzed for biological activity in a reporter gene bioassay. The variants retain activity within 80% and 130% of wild type, with loss of activity in the range 1-27% of the labeled variants in comparison to their unlabeled counterparts (Figure 2B). Variants 1-14 are labeled exclusively at the light or heavy chain that carries the engineered cysteine, as shown on reducing SDS-PAGE (Figure 2D, Supporting Information Figure 2A). The specificity of labeling is also demonstrated on the differential fluorescence band patterns of variants digested with Pronase and resolved on reducing SDS-PAGE (Supporting Information Figure 2D). Proteolytic treatment of the variants with Pronase yields different fluorescence patterns for most variants but similar patterns for variants with neighboring substitutions, such as variant 3 and 12. Thus, the analyzed engineered variants retain biological activity similar to wild type and are site-specifically labeled. Correlation between CLP and SAP of the Engineered Variants Suggests a Rational Approach to the Design of Antibody Variants for Site-Specific Conjugation. To determine the correlation between the positions of the cysteinesubstituted amino acids and their SAP values, we map the substituted amino acids on the SAP plots for the Fab and Fc parts of antibody-1 (Figure 3A and B). These plots were obtained as shown before (19). All substituted residues have neutral or negative SAP. Furthermore, we map the substituted amino acids on the three-dimensional structure of the antibody with SAP (Figure 3c). The overlaid CLP and SAP threedimensional representation yields another level of informations proximity of the selected sites to hydrophobic patches (high SAP regions) indicated in red. Variants of class I CLP have SAP values between 0 and -0.07 and are not close to hydrophobic patches. Variants of class II are not close to


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Figure 2. Quality control assessment of the engineered variants. (A) Nonreducing SDS-PAGE protein gel for variants 1-6 before labeling. M is the molecular weight marker (BioRad) used in this, as well as in all other protein gels shown. The molecular weight of each individual band in the ladder is shown in kDa. (B) Biological activity of unlabeled (U) and labeled (L) antibody variants, relative to antibody-1 wild type. (C) Nonreducing SDS-PAGE protein gel for variants 1-6 after labeling: image of the Coomassie stained gel and a fluorescence image of the same gel before staining. (D) Reducing SDS-PAGE protein gel for variants 1-6 after labeling: image of the Coomassie stained gel and a fluorescence image of the same gel before staining. For each antibody variant, the protein band at about 50 kDa corresponds to the antibody heavy chain, and the protein band at about 25 kDa corresponds to the antibody light chain. (E) Gel filtration fractions of variants 9 and 11. (F) Location of each engineered variant on the antibody three-dimensional structure. Green is CLP I and II, yellow is CLP III and IV, and black is CLP V. Variants 1-13 were generated in antibody-1. Variants 14 and 15 were generated in another antibody-2.

hydrophobic patches but have a more negative SAP value, from -0.12 to -0.23. Variants of class III have SAP like those of class I and II but are close (although not immediately adjacent) to hydrophobic sites. Variant 6 of class IV has an SAP value of -0.09 and is not adjacent to high-SAP patches. Yet, this variant is highly oligomerizing, presenting an exception to the SAP/CLP pattern. Variants of class V (variants 8 and 11) have cysteines engineered in immediate proximity to hydrophobic regions with high SAP. These variants show high CLP as well

as additional structural abnormalities, regardless of the SAP value of the substituted amino acid. Thus, the comparison of CLP, SAP of the substituted amino acid, and presence or absence of nearby high SAP site (Figure 4A) yield several straightforward and useful criteria for selecting sites for introducing surface cysteines (Figure 4B). In brief, the correlation between CLP and SAP values of the set of engineered IgG1 variants can be summarized as follows. Cysteine substitution of amino acids of partial exposure (SAP



Figure 3. SAP values for the substituted amino acids in variants 1-14 in (A) Fab and (B) Fc. (C) Mutated amino acids on the antibody-1 structure in the SAP context. For comparison, S7 of variant 14 is shown in antibody-1, although it was produced and analyzed in antibody-2. Both front and side views of the antibody molecule are shown. Red is high-SAP, and blue is low-SAP. Green is CLP I and II, yellow is CLP III and IV, and black is CLP V.

from 0.00 to -0.11), and away from high-SAP patches, show low CLP. Cysteine substitutions of amino acids of higher exposure (SAP from -0.12 to -0.23) show some CLP even in the absence of nearby high-SAP patches. The presence of adjacent high-SAP patches leads to high CLP and other structural abnormalities regardless of the SAP value of the particular substituted amino acid. Most variants were produced by transient transfection with yield between 11 and 37 mg/L with several exceptions (Figure 4A). Variants 9, 12, and 15 were expressed at lower levels. At the same time, variants even with high CLP, such as variants

6, 8, and 11, maintained high levels of expression. For comparison, antibody-1 wild type had an average yield of 25 mg/L, and antibody-2 of 24 mg/L, with our expression methods. Site-Specifically Labeled Antibody Variants Are Used to Monitor Structural Dynamics of Antibody Domains. We were also interested in applications of the antibody cysteine variants presented here. Successful labeling already indicates potentially efficient conjugation of most variants. In search of additional applications, we noticed a specific emission pattern of one of the variants conjugated with the fluorophore pyrene maleimide. When two pyrene molecules are close together there


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Figure 4. A rational approach to design antibody variants for site-specific conjugation. (A) Correlation between CLP and SAP for the 15 engineered cysteine variants. The domain that contains the mutation, the mutated amino acid, and the expression levels for each variant are shown. For comparison, antibody-1 and antibody-2 wild type were expressed at an average of 25 and 24 mg/L, respectively. Full green cell is CLP I, partial green cell is CLP II, yellow is CLP III and IV, and black is CLP V. Variants 1-13 are generated in antibody-1. Variants 14 and 15 are generated in another antibody-2, and thus, s.c. indicates separate case. Variant 14 is different from variant 15 by two surface amino acid substitutions, W100K and F101K, that is, W and F in variant 15 are replaced with lysines in variant 14. In the column “nearby high SAP”, “N” denotes absence, “Y” indicates presence, and “∼Y” denotes presence of a less pronounced high-SAP patch. (B) Application of the SAP technology to select surface amino acids for substitution to cysteines that would result in variants with low or high cross-linking propensity.

Figure 5. Use of labeled cysteine variants in montoring domain structural dynamics. (A) Fluorescence emission spectra of pyrene-labeled antibody variants 4 and 7. (B) Emission spectra of variant 2 labeled at the carbohydrates, at the engineered cysteines, or at both the carbohydrates and the cysteines.

is a characteristic increase of emission at 465 nm known as excimer fluorescence. We labeled variants 4 and 7 with pyrene maleimide and monitored emission spectra. While variant 4 shows no emission at 465 nm, variant 7 shows strong excimer fluorescence (Figure 5A). Considering the position of the engineered cysteine in CH1 for variant 7, on the inner side of the Fab domains, the observed result correlates with the known scissoring effect of the Fab’s with respect to Fc. Thus, the variant can be used in the analysis of antibody domain dynamics, for example, in examining antibody pluripotence with respect to antigen targets. As another example of applications to structural dynamics, cysteine variants can be double-labeled, once at the engineered cysteines via maleimide chemistry and once at the carbohydrates via hydrazide chemistry. The engineered cysteines of variant 2 are in Fc and are proximal to the carbohydrate moiety. We observe a significant fluorescence energy transfer between donor and acceptor of double-labeled variant 2 (Figure 5B). Similar fluorescence analysis among different variants and at different conditions permits analysis of protein-carbohydrate interactions (21). An Antibody Variant with High CLP Is a Useful Molecular Weight Control. The strong oligomerization propensity of variant 6 suggested another utility of IgG1 cysteine variants and was explored in greater detail. Labeled variant 6 was subjected to gel filtration chromatography in order to separate

monomer from oligomers, and protein-containing fractions were resolved on a 7.5% nonreducing SDS-PAGE, shown after (Figure 6A) and before (Figure 6B) staining with Coomassie Blue. The gel filtration analysis on variant 6 indicates a competition between utilization of the engineered surface cysteines in labeling and in cross-linking: the higher the MW of the species (more cysteines consumed in cross-linking), the lower the labeling efficiency (fewer cysteines available for labeling) (Figure 6B). The highest MW species (five asterisks) has a labeling efficiency of 0.5, while the monomeric species (one asterisk) has a labeling efficiency of 1.0, with the original labeled sample of labeling efficiency 0.8. An antibody variant with multiple oligomers, variant 6, thus presents a suitable standard for high molecular weight proteins, with the additional functionality that it can be site-specifically labeled. Unlike commercially available markers of mixture of proteins, this standard consists of only one protein that can be expressed at very high yield and can be efficiently purified. Moreover, the currently available high molecular weight markers for denaturing gels do not exceed 250 kDa, while variant 6 covers the broad range from 150 kDa (monomer) up to 750 kDa (pentamer).

DISCUSSION Although the cysteine engineering of proteins and more specifically antibodies has been practiced for many years (10, 11, 14, 16), it is still difficult to predict whether an antibody cysteine variant



Figure 6. Variant 6 contains at least five species. (A) Gel filtration of Alexa-488 labeled variant 6 species performed on Superdex 200. Aliquots of each protein-containing fraction were analyzed on a 7.5% PAGE under nonreducing conditions. M is molecular weight markers, in kDa, and S is the original labeled sample. The gel is shown after Coomassie staining. Antibody variant 6 monomer (one asterisk) and at least four higher molecular weight species (two, three, four, and five asterisks) are resolved. (B) Fluorescence image of the same protein gel before Coomassie staining. The numbers below the fluorescent image indicate labeling efficiency for each fraction.

will have the desired properties and serve the intended purposes. Two of the most common problems associated with antibody cysteine variants are oligomerization and specificity of labeling. To date, antibody cysteine variants in CL, CH1, and CH3 have been designed (10, 11, 13, 14, 17). We have designed a set of human IgG1 cysteine variants that are broadly distributed on the antibody molecule with at least one variant per immunoglobulin fold domain. Most of these variants are stable and can be conjugated efficiently and specifically without significant loss of biological activity. Thus, the stable antibody variants (for example, variants 1, 3, 4, and 7) add to the repertoire of variants for site-specific conjugation of payload molecules. Engineered surface cysteines on antibodies provide functionality for conjugation of small molecules, for example, cytotoxins. The therapeutic benefit of such immunoconjugates is that, when an antibody to a cell surface antigen binds its target, the toxin or other small molecule is specifically delivered to diseased cells that express that antigen. As another application, if fluorophores are attached to the engineered cysteines, the dynamics of particular domains can be analyzed. Here, we used antibody-1 and antibody-2 as model antibodies. While the mutations in the variable regions of these two antibodies may not apply to antibodies of other allotypes, we expect that our observations on variants with mutations in the constant regions apply to most human IgG1 antibodies. The observed deviations in biological activity in variants 1-6 compared to wild type may result from variations in posttranslational modifications during expression or other minor structural changes. The consistent loss of some activity upon labeling probably reflects structural changes such as partial unfolding or oligomerization in the multiple steps of chemical and mechanical stress during labeling. Variants 6, 8, and 11 show good expression levels in comparison to wild type even though these variants have high CLP. This suggests that the observed aggregation is a result of specific cross-linking via the surface cysteines and not of a general misfolding and aggregation problem. In contrast, the lower expression levels of variants 9, 12, and 15 in comparison to the corresponding wild-type antibodies suggests that the introduced mutations lead to some folding or secretion problem. In summary, comparison of the cross-linking propensity of the variants with the SAP values of the substituted sites and the spatial regions around those sites yields a straightforward approach for selecting sites for cysteine substitution and sitespecific conjugation. First, less exposed residues (SAP from 0.00 to -0.11) are less likely to lead to oligomerization than more exposed residues (SAP from -0.12 and lower). Second, our results suggest that nearby or immediately adjacent hydrophobic sites exacerbate the cross-linking propensity even of sites with ideal SAP values. Hydrophobic patches are likely to interact

and bring adjacent molecules together, thus facilitating intermolecular cross-linking via disulfide bonding of nearby surface cysteines. The boundaries of the suggested optimal range of 0.00 to -0.11 arise from several criteria developed on the basis of our results. In this report, we considered substitution of polar or charged amino acids with cysteines as opposed to hydrophobic amino acids to avoid significant changes in surface charge; thus, the negative half of the SAP spectrum is represented. The -0.11 limit for variants with low CLP is based on the result for K248 of variant 1. That site has an SAP of -0.12. An exception to the SAP criteria and CLP observations is variant 6, possibly because of fluctuations and exposure at the tip of the CH3 domain. Our SAP results indicate a higher exposure for S440 (variant 6, SAP ) -0.09) than for S442 (variant 10, SAP ) 0.00) consistent with the observed lower CLP of variant 10.

CONCLUSION We propose the implementation of the SAP technology in designing antibody, and by extension any protein, variants for site-specific conjugation. In order to select variants, the following steps should be made: (1) an SAP map for a protein of interest is obtained as previously described (19); (2) sites with SAP values between 0.00 and -0.11 are selected; (3) the vicinity of the selected sites are inspected for the presence of high-SAP regions; (4) if such regions are absent, the selected sites are chosen for modification to cysteines. These variants are expected to have low CLP and no structural abnormalities, and thus be suitable for efficient site-specific conjugation. The results presented here demonstrate the better utility of the SAP technology to existing methods in the rational design of protein variants for site-specific conjugation, as well as possible applications of the new variants.

ACKNOWLEDGMENT We thank a number of colleagues at Novartis: Dr. Burkhard Wilms provided the DNA vectors encoding for the wild-type antibodies, Dr. Holger Heine provided training and advice on transient transfection and antibody purification, Dr. Michael Vetsch suggested using L-cysteine to reduce protein surface cysteines before labeling. We thank Prof. Wittrup from MIT and his students for discussions and for sharing lab equipment. We also thank Dr. Rajaraman Krishnan from MIT/Whitehead Institute for a discussion and suggestions on pyrene labeling, and Anna Levina for technical assistance. This work was supported by Novartis Pharma AG. Supporting Information Available: Two supplementary figures on cysteine variant reduction with TCEP and DTT,


specificity of labeling by SDS-PAGE, and determination of monomer levels by SEC-HPLC. This material is available free of charge via the Internet at http://pubs.acs.org/.

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Design and Application of Antibody Cysteine Variants - Bioconjugate

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