Site-Specific Photolabeling of the IgG Fab Fragment Using a Small

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Site specific photo labeling of the IgG Fab fragment using a small Protein G derived domain Sara Kanje, Emma von Witting, Samuel C.C. Chiang, Yenan T. Bryceson, and Sophia Hober Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00346 • Publication Date (Web): 04 Aug 2016 Downloaded from http://pubs.acs.org on August 5, 2016

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Bioconjugate Chemistry

Site specific photo labeling of the IgG Fab fragment using a small Protein G derived domain

Sara Kanje1§, Emma von Witting1§, Samuel C.C. Chiang2, Yenan T. Bryceson2, Sophia Hober1* 1. Department of Protein Technology, KTH - Royal Institute of Technology, SE10691, Stockholm, Sweden 2. HERM, Department of Medicine Huddinge, Karolinska Institutet, SE-14157, Stockholm, Sweden §These

authors contributed equally to this paper.

*Corresponding author: [email protected]

ACS Paragon Plus Environment

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Abstract Antibodies are widely used reagents for recognition in both clinic and research laboratories all over the world. For many applications, antibodies are labeled through conjugation to different reporter molecules or therapeutic agents. Traditionally, antibodies are covalently conjugated to reporter molecules via primary amines on lysines or thiols on cysteines. While efficient, such labeling is variable and non-stoichiometric and may affect an antibody’s binding to its target. Moreover, an emerging field for therapeutics is antibody drug conjugates, where a toxin or drug is conjugated to an antibody in order to increase or incorporate a therapeutic effect. It has been shown that homogeneity and controlled conjugation is crucial in these therapeutic applications. Here we present two novel protein domains developed from an IgG-binding domain of Streptococcal Protein G. These domains show obligate Fab binding and can be used for site-specific and covalent attachment exclusively to the constant part of the Fab fragment of an antibody. The two different domains can covalently label IgG of mouse and human decent. The labeled antibodies were shown to be functional in both an ELISA and in an NK-cell antibody-dependent cellular cytotoxicity assay. These engineered protein domains provide novel tools for controlled labeling of Fab fragments and full length IgG.


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Introduction Antibodies, in particular of the immunoglobulin G (IgG) isotype, are widely used biomolecules in most biological, medical and biotechnical laboratories. The antibodies are used as means for detection and/or immobilization in standard assays such as enzyme-linked immunosorbent assay (ELISA), fluorescenceactivated cell sorting (FACS) and immunohistochemistry (IHC). Antibodies are also used in the clinic as therapeutic agents treating a variety of mainly oncological and immunological diseases1. Some of these therapies are so called antibody drug conjugates (ADC) where the antibody is conjugated to a drug or toxin, either to increase efficacy, or to use the antibody as a delivery vehicle2-4.

Beside full-length IgGs, antibody fragments such as single chain variable fragment (ScFv) and fragment antigen binding (Fab) are utilized in research. These fragments are used in particular when selecting for binders from antibody libraries with phage5 or yeast6 display for example, since full-length antibodies are too large to be easily expressed in display systems7. A few Fab fragments are approved for use in the clinic, e.g. ranibizumab for treatment of age-related macular degeneration8 and certolizumab pegol for treatment of Crohn’s disease and rheumatoid arthritis9, 10.

In order to use an antibody or antibody fragment as a detecting agent or ADC, conjugation of a functional molecule such as a fluorophore, enzyme, toxin or drug is necessary. Commonly used conjugation methods attach functional molecules to primary amines on the antibody (N-terminus or lysine side-chains), or to thiols on the side-chain of cysteine11. This can pose problems as it results in


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heterogeneous labeling where one can control neither the site of, nor the exact number of labels per antibody. If regions close to, or within, the hyper-variable loops of the antibody contain lysines, labeling may interfere with the antibody binding to its antigen, causing a less functional antibody. Cysteines on the other hand are naturally paired, forming stabilizing disulfide bridges. In order to label cysteine residues the disulfide bridges need to be reduced, decreasing antibody stability12. For ADCs, studies have shown that homogenous ADC mixtures have lower clearance and increased therapeutic index compared to heterogeneous mixtures13.

Lately, several techniques have been developed to circumvent the problem of unspecific antibody conjugation. These include THIOMABs that are conjugated on a particular introduced cysteine14, sortase A labeling (sortagging) that conjugates the antibody on an introduced LPXTG motif15, 16, or introduction of unnatural amino acids such as p-acetophenylalanine or p-azidophenylalanine to the antibody framework that can be labeled through oxime ligation or click chemistry17. These methods require a modification of the antibody framework that involves time-consuming optimizations. Due to this, the methods cannot be applied to conjugate already existing antibodies. Therefore systems that do not require any modification of the antibody of interest are potentially of great use. Recently Alves et al. published a method where antibodies can be sitespecifically labeled by photo-crosslinking between the nucleotide-binding site, a conserved region in the variable region of the Fab fragment site, and indole-3butyric acid with short-wave UV light of 254 nm18, department20-23 as well as others24,

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19.

Work from our

have utilized small antibody binding


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domains such as the Z domain derived from Staphylococcal Protein A26 or the C2 domain of Streptococcal Protein G27 by taking advantage of these proteins inherent affinity for the fragment crystallizable (Fc) and made variants that can covalently label the Fc through photo-crosslinking.

Protein G, via its IgG binding subdomains C1, C2 and C3, has affinity for both the Fc fragment and the Fab fragment of antibodies from many different species and subclasses28. Here, two different variants of the C2 domain were developed by a combination of directed mutagenesis and incorporation of the benzophenone containing non-canonical amino acid p-benzoylphenylalanine (BPA). These domains can site-specifically label antibodies of mouse or human origin on the Fab fragment. The introduction of BPA into the small protein domain enables covalent attachment to antibodies when exposed to UV light. Because benzophenones are activated by long-wave UV, shorter wavelengths that are damaging to proteins can be avoided29. A great potential for this molecule is that it can be used for labeling of not only full length antibodies, but also single Fab fragments.

Results To enable the design of an obligate Fab-binding protein domain, a detailed analysis of the interaction surfaces of C2 to the Fc and Fab fragments of IgG was made. When C2 binds to the Fab fragment, it interacts with the CH1 domain of the heavy chain, primarily through beta-strand contacts where backbone to backbone interactions forms an extended β-sheet between the two molecules30


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(figure 1). The Fc-interaction takes place in the region between CH2 and CH3 where three different regions of C2 on the α-helix and the third β-strand of predominantly charged and polar residues forms hydrogen bonds with the IgG31.

Figure 1. Cartoon of an IgG labeled on the Fab fragment, marked by a black circle. Also shown is the structure of the small IgG binding domain C2’s interaction with the Fab fragment (1QKZ.pdb32) with the light chain shown in pink, the heavy chain in blue and C2 in grey. The two positions used for BPA incorporation to provide covalent linkage to the antibody are marked in green. The star indicates the biotin that has been inserted to provide a mean for labeling the antibody with a molecule of interest coupled to streptavidin. The three regions involved in the C2 domain’s Fc binding are marked in dark grey (region I), dark blue (region II) and brown (region III).

In order to delete the Fc binding and obtain a protein domain that exclusively binds to the Fab fragment of IgG, double mutants were designed. These contained either the mutations at position K28 or D40 (numbering according to 1Fcc.pdb31), that had previously shown to partly delete Fc binding (data not shown), and an additional mutation. The first mutations were combined either with a mutation in the same Fc binding region of the C2 domain or a different one. The nine mutants and the corresponding Fc binding region31 of C2 to which they belong (figure 1) are presented in table 1.


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Table 1. The nine designed double mutants for deleted Fc binding of the C2 domain. C2 has three regions responsible for Fc interaction31 (figure 1). The Fc binding region of C2 to which the chosen mutation belongs is noted in the table.

Mutant

Binding region of C2

K28A K31A

Region I and II

K28A K31W

Region I and II

K28A Q32A

Region I

K28A Q32W

Region I

K28A D40A

Region I and III

K31A D40A

Region II and III

Q32A D40A

Region I and III

N35A D40A

Region III

N35W D40T

Region III

The mutants were recombinantly produced in E. coli, purified with affinity chromatography and analyzed for their binding to human IgG, Fc and Fab fragments using Surface Plasmon Resonance (SPR). Some mutants such as C2(K28A D40A) and C2(Q32A D40A) did not show eradicated affinity to the Fc fragment whereas others, such as C2( K28A Q32A) and C2(N35W D40T) lost the Fc binding while maintaining their affinity for Fab. C2(N35W D40T) hereafter denoted C2Fab, was chosen for further analyses as it demonstrated the best binding to IgG and Fab fragments while having no apparent affinity towards Fc (figure 2).


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Figure 2. SPR experiment comparing the interaction of the double mutant C2Fab and C2wt with Fc-, Fabfragments or full length IgG. The two different domains were immobilized on a chip to similar levels (85 VS. 70 Ru) and interaction with different concentrations of injected analyte, polyclonal human Fc or Fab fragments and polyclonal human IgG, was monitored. From the data it can be concluded that C2Fab has lost its Fc binding while showing a similar interaction with the Fab fragment as C2wt. The interaction with full length IgG shows lower signals for the double mutant as it has lost its affinity for two of its four binding sites on the molecule.


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To achieve a C2 domain that could covalently attach to IgG at the Fab fragment, different positions were tested for incorporation of the photo-inducible amino acid BPA. The positions were chosen by examining the interaction between C2 and Fab (figure 1, 1QKZ.pdb32), selecting positions in close proximity to the molecular interface. Protein domains based on C2Fab with BPA in eight different positions were produced using solid phase peptide synthesis. BPA was incorporated at position 11, 17, 18, 19, 29, 32, 37 or 38. Furthermore, a biotin was site specifically attached to the N-terminus of C2Fab. Most of the mutants showed no or very weak crosslinking towards IgG, the exception being the variants with BPA at position 18 or 29 that cross-linked mouse and human IgG, respectively, both exclusively on the Fab fragment (data not shown). A combination with BPA at both position 18 and 29 was produced to evaluate the possibility to achieve a protein domain with the capacity of crosslinking to both species. This new variant cross-linked Fab fragments from both species, but also human Fc, and was therefore discarded.

In order to produce larger amounts of protein, the C2 mutants were cloned and produced using a system for unnatural amino acid incorporation in Escherichia

coli33. A cysteine was added to the N- or C-terminal of the protein to be used for specific biotinylation via a maleimide coupled biotin. The proteins were purified on IgG sepharose to homogeneity, biotinylated and analyzed using mass spectrometry and SDS-PAGE (data not shown). After biotinylation of the incorporated cysteines, crosslinking experiments were performed. Having the biotin at the C-terminal provided better crosslinking efficiency compared to the


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N-terminal version (~ 50 % vs. < 30 %). Therefore, the variants with the Cterminal biotin were used for succeeding characterizations.

The proteins were tested for crosslinking to full-length antibodies as well as antibody fragments and behaved similarly to their synthesized counterparts (supporting information figure S1), showing specific labelling of mouse IgG1 and mouse IgG2b (C2Fab(T18BPA)) as well as human IgG1, human IgG2 and human IgG4 (C2Fab(V29BPA)) (figure 3). The crosslinking was confirmed by SDS-PAGE (figure 3a and c) and Western Blot using Streptavidin-HRP detection, demonstrating covalent attachment of the biotinylated C2 domain (figure 3b and d). The degree of crosslinking to each antibody subclass was quantified by image analysis (ImageJ34), where C2Fab(T18BPA) cross-linked to mouse IgG1 and IgG2b with an efficiency of 48% and 64%, respectively, whereas no crosslinking to mouse IgG2a was detected. C2Fab(V29BPA) cross-linked to human IgG1, IgG2, and IgG4 with an efficiency of 43%, 58% and 52%, respectively. A crosslinking efficiency of 50 % implies labeling of on average one heavy chain per antibody, i.e. one label per antibody.


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Figure 3. Crosslinking of the C2Fab with BPA at position 18 (A and B) or 29 (C and D) to mouse or human antibodies and antibody fragments respectively. Monoclonal IgGs and polyclonal Fc or Fab fragments were cross-linked to C2Fab and the samples were run on SDS-PAGE and Western Blot. The cross-linked proteins appear on the gel as an extra band above their unconjugated counterparts. The crosslinking was confirmed using Western Blot with streptavidin-HRP detection. Unconjugated IgG, Fab or Fc fragment are loaded in the even lanes and the cross-linked versions in the odd lanes. Polyclonal Fab fragment are loaded in lane 2 and 3, polyclonal Fc fragment in lane 4 and 5, monoclonal mouse or human IgG1 in lane 6 and 7, monoclonal mouse IgG2a or human IgG2 in lane 8 and 9 and mouse IgG2b or human IgG4 in lane 10 and 11. Pure C2 is loaded as a control in lane 12 on the SDS-PAGE in A). A) SDS-PAGE of C2Fab(T18BPA) cross-linked to polyclonal mouse Fab fragment, mouse IgG1, IgG2a and IgG2b. B) The cross-linking exclusively to the Fab fragment as well as mouse IgG1 and IgG2b is confirmed with streptavidin. C) SDS-PAGE of C2Fab(V29BPA) cross-linked to polyclonal human Fab fragment as well as human IgG1, IgG2 and IgG4. The cross-linking is confirmed by Western Blot in D).

To assess the ability of the labeled antibodies to recognize their specific antigens an ELISA was performed. Site-specifically biotinylated trastuzumab was used to detect Her2 in a sandwich ELISA setup. It was tested whether combining Fab


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labeling of the antibody with a similar domain developed at our lab which can label antibodies on the Fc fragment23 with an efficiency of 90%, would increase the signal output in the assay. To ensure that the earlier developed Fc labeling domain did not interfere with C2Fab an N37Y mutation known to delete the Fab binding24 was introduced to the Fc-binder, C2Fc. Fab labeled trastuzumab detected Her2 in a concentration dependent manner, similar to the antibody labeled on the Fc fragment, but as expected with a lower signal (one vs. two biotins per antibody; figure 4). Moreover the two labeling domains could be combined to further increase the signal output in the assay. Combining C2Fab and C2Fc was shown to yield an average of three biotins per antibody (supporting information figure S2). The signal in the ELISA corresponded to the expected amount of labeling per antibody with the same limit of detection as was previously seen using this setup23.

Figure 4. Trastuzumab labeled on the Fab fragment (black circles), the Fc fragment (white squares) or a combination of both (black triangles) was used in an ELISA assay. The experiment shows that the Fab labeled antibody works well in a standard assay. It also shows that combining the Fab labeling domain with


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an Fc labeling domain yielding on average three biotins per antibody results in an increased output signal in the ELISA. The data are presented as mean values with standard deviations from three replicates.

To test whether antibodies labeled on the Fab fragment were still functional for antibody dependent cell-mediated cytotoxicity (ADCC), an exocytosis assay was performed using CD20-expressing Raji target cells35. Rituximab, an antibody that binds to CD20, was labeled either on the Fab fragment or on the Fc fragment with the C2Fab or C2Fc domains and compared in the assay to unlabeled rituximab antibody as positive control. Raji target cells were pre-incubated with the three different antibodies to allow for binding to CD20, after which they were mixed with peripheral blood mononuclear cells (PBMCs) from healthy blood donors. The antibody-coated Raji target cells triggered exocytosis of cytotoxic granules by natural killer (NK) cells, the majority of which express the low affinity Fc receptor CD16. Responses were monitored with flow cytometry by quantifying cell surface exposure of CD107a (lysosomal-associated membrane protein 1) on NK cells. CD107a is normally found within the inner membrane of cytotoxic granules but is exposed on the cell surface upon NK cell exocytosis36. Both the Fab and the Fc labeled antibodies, as well as the unlabeled control, were able to induce ADCC (figure 5). As a reference, the binding of rituximab to the Raji cells was monitored with an anti-IgG antibody, showing slightly lower binding to the cells by the labeled antibodies (supporting information figure S3).


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Figure 5. Exocytosis assay showing the CD20 binding antibody rituximab maintained high levels of ADCC

both when labeled on the Fab (black circles) and the Fc (white triangles). The labeled antibodies were compared to an unlabeled antibody (positive control, black squares). Responding NK cells were detected by staining for CD107a, which shows an induced cell surface exposure upon target cell stimulation. The plot shows ∆CD107a+(%) as a function of increasing antibody concentration incubated with the Raji target cells, where ∆CD107a+(%) is the difference in percentage of CD107a+ NK cells of the stimulated PBMCs compared to unstimulated ones. The data show a representative response from one healthy donor. Data from three additional donors are shown in Supporting information figure S4-6.

Discussion Here, we present two new molecules based on the C2 domain of Streptococcal Protein G for stoichiometric antibody labeling. Modified protein domains could covalently crosslink to the Fab fragment of mouse or human antibodies, depending on the position of incorporated BPA, the unnatural amino acid responsible for crosslinking to the Fab fragment under UV exposure. Covalent crosslinking to Fab domains have previously been reported by Alves et al.18, 19. An advantage with our method is the wavelength used for the crosslinking step.


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When utilising BPA as the crosslinking agent, exposure to UV light around 350 nm is sufficient since this wavelength is optimal for benzophenones to form radicals. For the crosslinking of indole-3-butyric acid to the Fab-fragment as reported by Alves et al. UV light of 254 nm is necessary, a wavelength that is harmful to proteins37. Both molecules contain the double mutation N35W D40T, abolishing the inherent Fc binding of the C2 domain. Recently Unverdorben et al.38 described a Fab selective IgG binding domain based on C3, the third IgG binding domain of Protein G, differing from the C2 domain only by four amino acids38. Their mutations of positions 27, 28 and 31 to alanine showed deleted binding to the human Fc fragment. In our experiments, a domain with mutations at two of the corresponding positions, 28 and 31, rendered a protein domain with properties similar to the chosen variant C2Fab. The reason for selecting C2(N35W D40T) as our domain of choice was the higher affinity to both Fab fragments and full length IgG compared to the other designed mutants that lack Fc binding.

Incorporating BPA at position 18 of C2 rendered a molecule that covalently cross-linked to the Fab fragment of mouse IgG whereas BPA at position 29 crosslinked to that of human IgG. As C2 has affinity for IgG from multiple species and subclasses the molecules may be of use also for IgG from other animals. In fact, crosslinking experiments to polyclonal rabbit IgG demonstrated an efficiency of 50% crosslinking for C2Fab(T18BPA) (supporting information figure S7). As mouse and human antibodies are the most commonly used in research and clinic, they have been the focus of this work. The affinity of C2 for the different IgG fragments (Fab and Fc) varies for different species39, 40, which might influence


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the crosslinking efficiency of our molecules. C2Fab(V29BPA) cross-linked to all human IgG subclasses investigated (1, 2 and 4) with similar efficiency (43-58 %), corresponding to around one label per antibody. C2 Fab(T18BPA) showed diverse crosslinking patterns to different subclasses of mouse IgG, crosslinking to mouse IgG1 with a 47 % efficiency and IgG2b with a 64 % efficiency, whereas it did not crosslink to mouse IgG2a. This cannot be explained by their different affinities as the domain showed comparable affinities to both IgG2a and IgG2b (unpublished data). A possible explanation for this behavior is the differences in primary structure41 of the antibodies that might expose different side chains and thereby give different availability for crosslinking of the benzophenone group. BPA can react with various amino acids but has a preference for methionine42. The bond formation is also restricted by distance to available amino acids with BPA having a reactive volume of 3.1 Å29. Most therapeutic antibodies are human IgG143 while most monoclonal antibodies used in the lab are mouse IgG121. The C2 domains presented in this paper can crosslink to both these important antibody subclasses, yielding around on average one label per antibody.

To examine whether an antibody labeled on the Fab fragment could be used in a standard assay, an ELISA was performed23. In an attempt to increase the output signal the Fab labeller C2Fab was combined with the previously published Fc labeller23, C2Fc, that resulted in about two labels per antibody when cross-linked to human antibodies. Antibodies labeled on Fab, Fc and a combination of both, i.e. one, two or three labels per antibody, all showed binding in the ELISA in a target concentration dependent manner. The signal was increased with increasing number of labels per antibody (one, two and three respectively).


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However the signal was not increased in correspondence to the additional labeling, i.e. two labels did not yield twice as high signal as one label. This may be due to the availability of the biotin to bind the streptavidin-HRP molecule. Streptavidin is a large molecule with a hydrodynamic radius of 3.7 nm44 while the hydrodynamic radius of IgG is 5.3 nm45. Even though there are three biotins on the IgG molecule labeled on both the Fab and Fc fragment, streptavidin may not be able to bind all three of them simultaneously.

In the ADCC experiments, it was investigated whether antibodies labeled on the Fab fragment could still mediate effector functions via the Fc part of the antibody. An antibody known to induce ADCC in this assay, rituximab, was labeled on the Fab or Fc part respectively. The differently labeled antibodies were compared to each other as well as to an unlabeled antibody, as a positive control. The antibody that was labeled on the Fab fragment as well as the one labeled on the Fc both induced ADCC. The responses from the labeled antibodies were slightly lower than for the unlabeled rituximab. When monitoring the rituximab binding to the Raji cells with an anti-human IgG antibody, the labeled antibodies showed slightly lower signal than the unlabeled antibody, explaining this difference in ADCC induction (supporting information figure S3). An explanation for the lower binding to the Raji cells can be that the C2 domain interferes with the antigen binding of the antibody by constraining the antibody flexibility. Interestingly the antibody labeled on the Fc worked just as well in performing ADCC, even though it has a C2 domain covalently attached on both chains on the Fc part of the antibody. The reason for it not affecting the ADCC is probably due to its position on the Fc. C2Fc binds to the region between the CH2


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and CH3 domains of the Fc, while the FcγRIII protein, the receptor on the NK cell surface responsible for binding to the Fc fragment of an antibody bound to a target cell, and inducing the ADCC reaction, binds between the two CH2 domains of the Fc46, an epitope remote from where the C2 domain is covalently attached. In this paper we present a novel variant of the C2 domain of streptococcal protein G, C2Fab that exclusively binds to the Fab fragment of IgG. Furthermore the unnatural photo inducible amino acid BPA has been introduced to the protein domain that gives the domain the ability to covalently attach to either mouse IgG1 and IgG2b (C2Fab(T18BPA)) or human IgG1, IgG2, and IgG4 (C2Fab(V29BPA)). The labeled antibody can be used for detection in ELISA and by adding additional labeling molecules in a controllable manner, the signal in the ELISA assay was increased. Hence, the directed labeling of antibodies presented here enables well-defined and specific attachment of molecular tags. The method can be used either to increase a signal by multiple attachment of the same tag or by attaching different labels on the Fab an Fc fragment respectively. Furthermore, we have shown that antibodies decorated with the C2 domain, both at the Fab and Fc fragment, are able to perform effector functions through ADCC. This domain has a great potential to be used for labeling of Fab-fragments as well as full-length antibodies to facilitate their use in a number of different ways, from site directed immobilization to specific labeling.


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Experimental Procedures Protein structure images were made in the YASARA software.

Concentration determination of antibodies and crosslinking efficiencies from SDS-PAGE images was performed using the ImageJ software.

Cloning procedures, protein production with and without BPA in E. coli, protein purification on IgG sepharose and MALDI analysis were performed as described by Kanje et al. 23. SDS-PAGE analysis and Western Blot analysis were performed as described previously23 with the distinction of using the NuPAGE Novex 4-12% Bis-Tris gels with 1xMES (50 mM MES, 50 mM TRIS, 1 mM EDTA, 0.1% SDS, pH 7.3) as running buffer for SDS-PAGE and 1xTransfer buffer (25 mM Bicine, 25 mM BisTris, 1 mM EDTA, 10% Ethanol, pH 7.2) for transfer to the 0.45 µm Invitrolon PVDF Western blot membrane. PageRuler™ Plus Prestained Protein Ladder (Thermo Scientific) was used as the molecular weight marker.

Solid Phase Peptide synthesis of protein domains Peptide synthesis of the different C2 variants, removal of protecting groups and resin was performed as described by Konrad et al20. Single couplings were performed for all amino acids except the underscored ones that were double coupled (see sequence below). Amino acids marked in red were coupled as pseudo prolines (Merck). The BPA (Peptech corporation) was incorporated at position 11, 17, 18, 19, 29, 32, 37 or 38. Once synthesis was completed the Nterminal Fmoc group was removed and biotinylated as previously described20. Amino acid sequence of one of the synthesized C2 domain:


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TTYKLVINGKTLKGETTTBAVDAATAEKVFKQYAWDNGVTGEWTYDDATKTFTVTE

HPLC The synthesized peptides were purified using reverse phase high performance liquid chromatography on an Agilent 1200 series using a Reprosil Gold 300 C18 column 3 µm, 250 mm x 4.6 mm, Dr Maisch). A gradient of 20-50 % B buffer over 25 min was used. Buffer A contains 95% 100 mM triethylamine-acetate (TEAA, pH = 7) and 5% acetonitrile (ACN) and Buffer B contains 90% ACN and 10% 100 mM TEAA.

Crosslinking of antibodies and antibody fragments and separation of unconjugated probe Crosslinking of antibodies and antibody fragments were performed as described in a previous publication23 but with a 15 x molar excess of the protein domain.

The separation of unconjugated C2 from the cross-linked antibody was performed as described by Kanje et al.23 but with a 30 kDa cut-off membrane on the concentrator and with acid wash with 0.5 M HAc, pH 2.8 to separate unconjugated probe from the labeled antibodies. After the acidic wash the sample was removed from the concentrator and the pH neutralized by addition of 1/3rd volume of 1M Tris-HCl, pH 8.5. The sample was diluted to a desired final volume using 1xPBS (0.15 M NaCl, 10 mM phosphate, pH 7.4).

Surface Plasmon Resonance Surface Plasmon Resonance (SPR) experiments were run on a ProteOn™ XPR36 system (Bio-Rad). A GLC sensor chip (Bio-Rad) was activated according to the


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standard procedure. By injecting 2µg/ml protein at 30 µl/min for 300 s, the C2 variants were coupled to the chip surfaces to a response rate of 50-85 RU followed by standard deactivation with ethanolamine. Injections of analyte were performed at 100 µl/min with a 245 s association time and a 3000 s dissociation time. Polyclonal human IgG (Bethyl laboratories) and polyclonal human Fc fragments (Bethyl laboratories) were injected at 100, 33, 11, 3.7 and 1.2 nM. Polyclonal human Fab fragments (Bethyl laboratories) were injected at 900, 300, 100, 33 and 11 nM.

Maleimide biotinylation of E. coli produced proteins C2 domains containing a N- or C-terminal cysteine were biotinylated using maleimide-biotin (Sigma-Aldrich). The protein was reduced by a 10x molar excess of TCEP (Sigma-Aldrich), 30 min at room temperature. Maleimide-biotin was dissolved to a final concentration of 10 mg/ml in concentrated HAc, followed by neutralization with an equal volume of concentrated NaOH, and a 20x molar excess was added to the reduced protein sample. The reaction was incubated for 2 h at 37 °C. Excess biotin was removed by buffer exchange to water on an Illustra Nap-5 column (GE healthcare) according to the supplier’s instructions. The sample was freeze-dried before dissolving it in 1xPBS.

ELISA The ELISA was performed as previously described23 but with 200 ng/ml Herceptin® (Roche) as detecting antibody.

Antibody-dependent cellular cytotoxicity assay Rested peripheral blood mononuclear cells (PBMC) from 4 healthy donors were added to a v-bottom 96-well plate at 200 000 live cells per well. Unlabeled, Fab


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labeled and Fc labeled Rituximab were serially diluted (Ranging from 3000 ng/ml to 0.004 ng/ml) and incubated at 37°C with Raji target cells (CCL-86, ATCC) for 15 minutes. 100 000 antibody-coated Raji cells were added to wells containing PBMC and the volume of each well was adjusted to 100 µl with complete medium (RPMI supplemented with 10% heat inactivated fetal bovine serum and 2mM L-Glutamine, all Hyclone). PBMC from all four donors together with Raji target cells without any antibody stimulation served as negative controls. The plate was incubated at 37°C for 2 hours. Following incubation, the cells were stained with a viability dye and a panel of fluorescently labeled antibodies (CD3, CD56, CD107a, CD14, CD19), as previously described47 before fixing in BD Cytofix/Cytoperm. Cells were analyzed on an LSR Fortessa analyzer (BD Biosciences). Live CD3–CD56dim NK cells were gated and evaluated for surface CD107a expression. The percentage of CD107a+ expressing NK cells stimulated with varying concentrations of Rituximab was compared to the unstimulated cells, expressed as ∆CD107a(%). Viable B-cells coated with the different antibodies were also stained with a fluorescently labeled antibody towards human IgG, to detect any differences in antigen binding.

Acknowledgements The authors would like to extend their gratitude to The Scripps Research Institute for kindly providing us with the pEVOL-pBpF plasmid and to the ProNova VINN Excellence Centre for Protein Technology for funding.


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Supporting Information description •

Amino acid sequences of C2 variants

•

Figure S1 labeling cross-linking of the synthesized C2 variants

•

Figure S2 labeling of trastuzumab with C2Fc and C2Fab

•

Figure S3 amount of bound unlabeled and labeled rituximab on Raji cells

•

Figure S4-S6 additional donor results for ADCC experiment

•

Figure S7 Crosslinking of C2Fab to polyclonal rabbit IgG

Abbreviations ADCC- Antibody Dependent Cell-mediated Cytotoxicity BPA- p-benzoylphenylalanine ELISA- Enzyme Linked Immunosorbent Assay Fab- Fragment antigen binding Fc- Fragment crystallisable IgG- Immunoglobulin G NK-cells- Natural Killer cells SPR- Surface Plasmon Resonance

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Site-Specific Photolabeling of the IgG Fab Fragment Using a Small

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