Accepted Manuscript Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with the quantum dot conjugates Gilles Rousserie, Regina Grinevich, Kristina Brazhnik, Klervi EvenDesrumeaux, Brigitte Reveil, Thierry Tabary, Patrick Chames, Daniel Baty, Jacques H.M. Cohen, Igor Nabiev, Alyona Sukhanova PII: DOI: Reference:

S0003-2697(15)00096-2 http://dx.doi.org/10.1016/j.ab.2015.02.029 YABIO 11999

To appear in:

Analytical Biochemistry

Received Date: Revised Date: Accepted Date:

16 November 2014 23 February 2015 28 February 2015

Please cite this article as: G. Rousserie, R. Grinevich, K. Brazhnik, K. Even-Desrumeaux, B. Reveil, T. Tabary, P. Chames, D. Baty, J.H.M. Cohen, I. Nabiev, A. Sukhanova, Detection of carcinoembryonic antigen using singledomain or full-size antibodies stained with the quantum dot conjugates, Analytical Biochemistry (2015), doi: http:// dx.doi.org/10.1016/j.ab.2015.02.029

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Analytical Biochemistry / Labeling procedures Revision submitted 21 February 2015

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with the quantum dot conjugates.

Gilles Rousserie a, Regina Grinevich b, Kristina Brazhnik b, Klervi Even-Desrumeaux c, Brigitte Reveil a, Thierry Tabary a, Patrick Chames c, Daniel Baty c, Jacques H.M. Cohen a, Igor Nabiev a,b,*, Alyona Sukhanova a,b,*

a

Laboratoire de Recherche en Nanosciences, LRN-EA4682, Université de Reims

Champagne-Ardenne, 51 rue Cognacq Jay, 51100 Reims, France; b

Laboratory of Nano-Bioengineering, National Research Nuclear University MEPhI

(Moscow Engineering Physics Institute), 31 Kashirskoe shosse, 115409 Moscow, Russian Federation; c

INSERM U1068 and CNRS UMR7258, Centre de Recherche en Cancérologie de Marseille,

Institut Paoli-Calmettes and Université Aix-Marseille, 13009 Marseille, France.

*

Corresponding authors. Fax: 33-326-918-127.

E-mail addresses: [email protected] (Dr. Alyona Sukhanova, PhD, MD) or [email protected] (Prof. Igor Nabiev, PhD, DSci).

Running title: Staining of antibodies with nanocrystal conjugates

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Abstract Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability and great capacity of refolding. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labelling of different recognition molecules with QDs of different colours but excitable with the same wavelength of excitation. In this study, the abilities to specific detection of CEA expressed by tumor cells with antiCEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro were compared using flow cytometry and the conjugates of streptavidin with QDs (SAQDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs allows the sensitivity of detection of CEA on tumor cell surface to be considerably improved.

Keywords: Carcinoembryonic antigen (CEA); quantum dots; single-domain antibodies (sdAbs); biotinylation. Abbreviations used: CEA, carcinoembryonic antigen; CDRs, complementary determining regions; Fab, antigen-binding fragment; HcAbs, heavy-chain antibodies; IPTG, isopropyl-hD-thiogalactopyranoside; mAbs, monoclonal antibodies; QDs, quantum dots; SA-QD, conjugate of streptavidin with quantum dot; ScFv, single-chain variable fragment; sdAbs, single-domain antibodies. 2

Introduction Conventional organic dyes are commonly used as fluorescent markers in biomedical studies, including diagnostics and biological imaging. These applications require specific targeting of different biomolecules, for which purpose numerous antibody-based labeling strategies have been developed. The possibility of conjugation of a fluorescent probe with an antibody (Ab) allows both specific recognition of the target and quantitative detection of the associated fluorescent signal. Detection of different fluorescent signals simultaneously would offer the possibility of multiplexed analysis and better understanding of supramolecular systems and biological processes. Routine fluorescence-based immunolabeling strategies depend on four main parameters, which impose limitations on their practical use: the affinity of Abs, the optical properties of the fluorescent probes, the labeling method, and the size of the resultant conjugate composed of the Ab, fluorescent probe, and optional linker molecules. Conjugates of single-domain Abs (sdAbs) and quantum dots (QDs) are new interesting tools which can reduce some shortcomings of the immunolabeling and detection techniques, such as the large size and poor optical properties of fluorescent probes [1–3]. Most organic dyes generally used as fluorescent probes suffer of photobleaching, a low brightness above background fluorescence, a wide overlap between the absorption and emission spectra of different dye molecules, etc. These shortcomings severely limit the use of organic dyes for detection of rare events or multiplexed imaging and analysis. In contrast, semiconductor QDs are characterized by an exceptionally bright, stable fluorescence and, hence, are particularly interesting as tools for biological imaging and diagnostics [4]. Moreover, different QD populations can be excited at the same wavelength which may be 3

very far from their respective emission bands, depending on the QD core size and composition [5, 6]. Since the first success of transfer of inorganic QDs in aqueous solutions by Bruchez [7] and Chan [8], these fluorescent nanoprobes have been among the most exciting and promising tools for fluorescent-based biological applications, such as microarray analysis [9], immunohistochemistry [10], single-particle tracking [11], flow cytometry [12], cancer research [13], and drug targeting [14, 15]. The use of QDs in different biological applications requires solubilization and chemical functionalization of the QD surface and their conjugation with recognition molecules that are responsible for specific interaction of QD conjugates with biological targets. Abs are characterized by high specificity and avidity of interaction with specific target molecules, which makes them efficient capture and detection tools for numerous biological applications. However, their complex structure, fragility and large size limit their applications. The standard conjugation procedures for attachment of a fluorescent probe to Ab may lead to conformation changes in the Ab molecular structure and decrease in antigen recognition capacity. Moreover, Abs have a high molecular weights (150 kDa) and large average size of the full-length molecule (14.5×8.5×4 nm) [16] and, hence, are not absolutely suitable for targeting. To overcome this shortcoming, small and compact antibody fragments, such as F(ab’)2 (100 kDa), Fab (50 kDa), and ScFv (25 kDa) with comparable specific recognition capacities are used (Figures 1a–1d). Despite the obvious advantages of such relatively small capture molecules, their biochemical properties, sophisticated production procedures, and a high production cost make them poorly available. In 1993, Hamers-Casterman et al. has found, in Camelidae, a new form of IgG-like antibodies, so-called heavy-chain antibodies (HcAbs), which lack the two light chains [17]. Since this discovery, a number of researchers have produced and isolated sdAbs consisting of 4

the variable domain of these HcAbs alone (Figures 1e,f) [18]. sdAbs have several important advantages over mAbs due to such characteristics as small size and weight, high stability, solubility, and expression rate [19–21 ]. These tiny antibodies (about 13 kDa) exhibit the same affinity and about the same variability as "classical" mAbs; they can also recognize and bind very small antigens, such as haptens, for example [22]. Consisting of about 110 amino acids, sdAbs include three distinct complementarity determining regions (CDR) ensuring the antigen recognition. Several recent studies have shown that sdAbs exhibit (i) a better tissue diffusion capacity than conventional Abs [23], (ii) a low immunogenicity, (iii) high thermal and chemical stabilities, and (iv) the ability to refold and recover their binding capacities after heat denaturation [24, 25]. Most importantly, the relatively simple sdAb structure allows the amplification and straightforward cloning of the corresponding genes, without requiring enzymatic cleavages of the Ab (as with the Fab fragment and F(ab')2 fragment), bicistronic constructs (as with the Fab fragment), or artificial linker peptide (as with the single-chain Fv fragment). This permits direct cloning of large sdAb repertoires from immunized camelids. The characteristics of sdAbs allow them to be produced with a high yield by prokaryotic (lactobacilli [26] and Escherichia coli [27]) and eukaryotic (Saccharomyces cerevisiae [28]) microorganisms as well as by cell lines obtained from multicellular organisms (Chinese hamster ovary cells [29]). Thus, due to their unique properties, these antibodies are promising components of specific fluorescently-labeled probes for biological and medical applications. This study was focused on the possibility to detect human carcinoembryonic antigen (CEA), a tumor-associated glycoprotein expressed in a variety of cancers, on MC38 and MC38-CEA cells with the use of biotinylated sdAbs or mAbs. Detection of CEA was carried out through interaction of biotinylated sdAbs or mAbs with streptavidin-QD conjugates (SA-QDs) followed by flow cytometry analyses of formed complexes. We have firstly optimized the 5

protocol for in vitro biotinylation of both mAbs and sdAbs. In addition, the recombinant origin of sdAbs allows their in vivo site-directed enzymatic biotinylation by addition of a short peptidic tag that is recognized and modified by E. coli enzyme BirA during the sdAb biosynthesis. We have finally compared the antigen detection efficiencies by in vitro and in vivo biotinylated Abs. The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. Considering that anti-CEA mAbs and sdAbs recognize different epitopes [31], we suggested that the more sensitive cell staining with biotinylated sdAbs is a result of a higher affinity of sdAbs compared to mAbs. The results of this study allow us to conclude that sdAbs are more sensitive than mAbs, very easy to use and their combination with highly fluorescent QDs provides additional specific advantages in the immunodetection.

Materials and Methods Materials and Equipment All general chemicals were purchased from Sigma-Aldrich (St. Quentin Fallavier, France). Sulfo-NHS-biotin,

Sulfo-NHS-LC-biotin,

Sulfo-NHS-LCLC-biotin,

Pierce®

biotin

quantitation kit, Qdot®800–streptavidin conjugates were obtained from Thermo Fisher Scientific (Illkirch France). Anti-CEA monoclonal antibody, clone CLB-139 was obtained from Abcam (Cambridge, UK). PD spintrap TM G-25 and Protein G sepharose column were purchased from GE Healthcare (Velizy-Villacoublay, France). Novagen Bugbuster® Protein Extraction Reagent and Amicon ultra 5k MWCO centrifugal filter units were from MerckMillipore (Molsheim, France). TalonTM metal affinity resin was purchased from Clontech (Saint-Germain-en-Laye, France). pBir vector was from Avidity (Colorado, USA). Flow

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cytometry experiments were done with a Guava flow cytometer (Merck-Millipore, Molsheim, France). Production and Purification of Anti-CEA sdAbs E. coli strain BL21 (DE3) cells were transformed with the pET vector containing sdAbs. The transformants were inoculated in 10 mL of 2YT (16 g/L bactotryptone, 10 g/L yeast extract, 85 mM NaCl)/ampicillin (100 µg/mL) medium supplemented with 2% glucose. The bacterial culture was grown at 250 rpm at 37°C overnight. After that, the culture was diluted to an OD600 of 0.1 in 400 mL of fresh 2YT medium supplemented with ampicillin (100 µg/mL). The sdAb expression in cells was induced by the addition of 0.1 mM isopropyl-h-Dthiogalactopyranoside (IPTG) after reaching the OD600 ~ 0.5 at 30°C at 250 rpm for 20 h. Cells were harvested by centrifugation at 4000 rpm for 10 min (4°C). For periplasmic purification, the pellet was resuspended in 4 mL of TES buffer solution (0.2 M Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.5 M sucrose), and 160 µL of a 10 mg/mL stock solution of lysozyme was added to the suspension. The cells were subjected to osmotic shock by adding 16 mL of cold TES diluted in an equal volume of cold H2O. The suspension was clarified by centrifugation at 4000 rpm for 40 min at 4°C after incubation of the sample for 30 min on ice. The supernatant was incubated with 150 µL DNaseI (10 mg/mL) and MgCl2 (5 mM final) for 30 min at room temperature and dialyzed against a mixture of 50 mM sodium acetate (pH 7.0) and 0.1 M NaCl for 16 h at 4°C. For cytoplasmic purification, the pellet was frozen for 20 min at –80°C and lysed by 20 mL of Bugbuster® Protein Extraction Reagent (Novagen) for 20 min with gentle shaking. Then, sdAbs were purified from the cell extracts by means of metal affinity chromatography (Talon Metal Affinity Resin, Clontech). The protein was eluted with 250 mM imidazole and then concentrated in PBS by ultrafiltration using Amicon Ultra 5000 MWCO (Millipore) and 7

stored at –20°C. The purity of the sample was evaluated using SDS-PAGE analysis. The protein concentration was determined spectrophotometrically using a protein assay kit (BioRad Laboratories). For additional information on anti-CEA sdAbs, see Ref. [30] for production and Ref. [31] for clone 17 (C17) characterization. Monoclonal Antibody Purification The mouse monoclonal antibody (clone CLB-139) was shipped with BSA supplement. The supplement BSA was removed by two cycles of purification on protein G column (GE healthcare). Briefly, the column was equilibrated with PBS containing 100 mM phosphate and 150 mM NaCl (pH 7.2) without potassium; then, the antibody was load on the column, and the column was washed three times with PBS. The elution was settled with 0.1 M glycine-HCl, pH 2.0. This operation was done two times to remove BSA (which contains some free primary amines) almost completely. The purity of mAbs was confirmed by SDSPAGE. In Vitro Biotinylation The anti-CEA sdAbs were biotinylated with the Sulfo-NHS-biotin, Sulfo-NHS-LC-biotin, or Sulfo-NHS-LCLC-biotin reagent according to the manufacturer's protocol. Briefly, this includes preparation of fresh 10 mM biotinylation reagent solution in ultrapure water and addition of the calculated volume of the biotinylation reagent to the sdAb solution while stirring to obtain an antibody to biotinylation reagent molar ratio of 1:20. Then, biotinylation occurs for 30 min at room temperature with gentle stirring. Finally, the unreacted biotinylation reagent excess is removed using a PD spintrap TM G-25 column. The final buffer contains 100 mM sodium phosphate and 150 mM NaCl (pH 7.2). 8

This protocol was adapted to optimize results of biotinilation for different antibody-to-SulfoNHS-LCLC-biotin molar ratios (1:10, 1:20, 1:30, 1:50, and 1:100). All other steps for procedure of mAb biotinylation were the same as those described above. In Vivo Biotinylation For in vivo biotinylation of sdAbs, the bacteria were co-transformed with the pBir vector, and the culture medium was supplemented with chloramphenicol (50 µg/mL) during sdAb production. Biotin (50 µM) was added during the induction. Briefly, E. coli were transformed to express both sdAb and the BirA enzyme, which is responsible for protein biotinylation in cells. A specific sequence recognized by BirA was included into the sdAb sequence at the Cterminal. This specific sequence became the site of in vivo biotinylation. Thus, biotinylated sdAbs had the best biotin orientation for antigen recognition and SA-QD labeling. This approach is described in full details in the previous publication [30]. Finally, the number of biotin molecules per sdAb was determined using a Pierce® biotin quantitation kit according to the manufacturer's protocol. Briefly, the HABA–avidin complex absorbance at 490 nm was determined; then, biotinylated sdAb was added to the mixture to substitute the HABA on the avidin molecule. This substitution led to a change of the absorbance peak of HABA, so, the absorption of the mixture at 490 nm was determined again to detect the quantity of HABA replaced by the biotinylated sdAb. The number of moles of biotin per mole of sdAb was calculated with the “HABA calculator” provided by Thermo Fisher Scientific (www.piercenet.com/haba/habacalc.cfm). Cell Culture The MC38 and MC38-CEA [32] cell lines were kindly provided by Dr. A. Pelegrin (INSERM, Montpellier). The cell lines were cultured in DMEM supplemented with 10% 9

(v/v) fetal calf serum and 5 µg/ml pyromicin at 37°C in a humidified atmosphere containing 5% CO2. The MC38-CEA culture medium was additionally supplemented with 0.5 mg/mL of geneticin to maintain CEA expression. Cell Immunolabeling Cell staining was performed in darkness on ice; the biotinylated antibodies were diluted with cold PBS containing 1% BSA. Typically, a total of 105 cells were incubated in a V-bottom 96-well microtiter plate for 1 h at 4°C in the dark with 50 µL of various quantities (from 100 to 0.01 ng) of biotinylated antibodies. After incubation cells were washed two times with PBS containing 1% BSA, and 1 nM of Qdot®800–streptavidine conjugates (Thermo Fisher Scientific) diluted in 50 µL of PBS-BSA (1%) were added to cells and incubated for 1 h at 4°C in the dark. After two washings flow cytometry measurements of stained cells were performed immediately. Negative controls were performed with Qdot®800–streptavidine conjugates alone and control staining of CEA negative cells (MC38). Flow Cytometry The fluorescence signal was recorded by means of a Guava easyCyte™ system (MerckMillipore) on a flat-bottom 96-well microtiter plate. The estimation was based on 5 000 events in a specified gate following the side scatter (SSC) and forward scatter (FSC) parameters of cells. The results were treated using the Cytosoft 5.2 software provided by Millipore.

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Results and discussion The CEA detection was performed on two different murine cell lines: CEA-negative MC38 cells and MC38-CEA cells stably transfected to express CEA on their surface [32]. Preliminary experiments showed that a nonspecific signal appeared on each negative control (MC38 and MC38-CEA cells stained with SA-QD800 alone or MC38 cells immunolabeled with biotinylated sdAb revealed with SA-QD800) when using SA-QD800 according to the manufacturer's recommendations. Therefore, a suitable dilution of SA-QD800 conjugates had to be established. For this purpose, a dilution range from 0.02 to 10 nM of SA-QD800, in the absence of biotinylated sdAbs, was tested for both MC38 and MC38-CEA cells. The results clearly showed that at SA-QD800 concentration of 10 nM nonspecific signal was recorded for MC38 and MC38-CEA cells with the geometric mean fluorescence signal as high as 21 a.u. However, this non-specific binding was almost completely disappeared for 1 nM conjugate concentration which was chosen for experiments (Figure 2a). We have further investigated the possibility to detect specifically membrane-bound CEA using biotinylated anti-CEA sdAbs labeled with commercial SA-QD800 conjugates. There is a panel of five sdAbs capable of binding with high affinity to CEA-expressing cells, namely C3, C17, C25, C43 and C44 [31]. One of them, C44, is also capable of binding to nonspecific cross-reacting antigen (NCA), which is expressed on several normal cell types, including granulocytes. C44 sdAb, therefore, recognizes an epitope shared by CEA and NCA, in contrast to C3, C17, C25, and C43, which are strictly specific for CEA. In our investigation, C17 sdAb was selected due to its lowest Kd value in the panel, although C43, C25, and C3 sdAbs also could be used for the study. Doing this, the anti-CEA sdAbs were biotinylated in vitro with the Sulfo-NHS-biotin reagent according to the manufacturer's protocol. Then, MC38 and MC38-CEA cells were stained 11

with biotinylated anti-CEA sdAbs and SA-QD800 (1 nM) according to the manufacturer's recommendations for flow cytometry assays. Whereas no signal was detected for MC38 cells, the MC38-CEA cells displayed a strong shift of fluorescence (Figures 2b,c), thereby validating the proposed experimental approach employing biotinylated sdAb and SA-QD800 conjugates. On the next stage of our work, we compared the CEA detection efficiency using the antiCEA sdAb labeled with three different reagents, differing by the size of the spacer arm: Sulfo-NHS-Biotin (biotin), Sulfo-NHS-LC-Biotin (biotin-LC), and Sulfo-NHS-LCLC-Biotin (biotin-LCLC) using spacer arms of 1.35, 2.24 and 3.05 nm, respectively, which can moderate/regulate the accessibility of biotin for streptavidin-biotin interaction. After antiCEA sdAb biotinylation with each reagent according to the manufacturer's protocol under the same conditions, the ability to detect CEA on cell was assayed by flow cytometry. The comparison was carried out with the amount of biotinylated sdAbs ranging from 1 to 100 ng in order to determine whether there is some difference at very low or high sdAb amounts and compare the sensitivities of different labeling strategies (Figure 3). Note that this experiment included investigation of both the ability of the biotinylated sdAbs to specifically target the CEA and the ability of streptavidin–QDs to interact with biotinylated Abs. Biotinylation of sdAbs with biotin, biotin-LC, and biotin-LCLC reagents resulted in comparable sensitivities of CEA detection, with a slight advantage for biotin-LCLC reagent at low concentration. No nonspecific signal was detected on negative cells (MC38). Therefore, the LCLC-biotin reagent was selected for all further biotinylation procedures. One of the advantages of work with the sdAbs concerns the availability of their sequence, what permits to determine the number and the location of primary amines available for biotinylation. The anti-CEA sdAb clone used in this work is consisted of 129 amino acid residues. Five of them contain the primary amine groups (four lysines and the N-terminus) 12

which could undergo biotinylation with the biotin-LCLC reagent (Figure 4). It is worth mentioning that none of these residues belong to any of the three complementary determining regions (CDRs). Therefore, attachment of biotin to these amino acids should not directly affect the CEA detection capacity although the overbiotinylation could still have an influence on the sdAb conformation and activity. We thus investigated the effect of the number of biotin molecules per sdAb on efficiency of the CEA detection. For this purpose, anti-CEA sdAbs where biotinylated with biotin-LCLC at three sdAb to biotin-LCLC molar ratios (1:20, 1:30, and 1:50). The average number of biotin per sdAb was quantified with a Pierce® biotin quantitation kit and is presented in Table 1. The results demonstrate that the molar ratio used during the biotinylation have important effect on the average number of biotin molecules per sdAb. As expected, the biotin-LCLC molar ratio of 1:50 resulted in an almost complete biotinylation of all primary amine groups of anti-CEA sdAb. The ability to detect CEA on the cell surface with these biotinylated sdAbs was further examined. The data show that the biotinylation at the lowest sdAb-to-biotin-LCLC molar ratio 1:50 improved the quality of labeling of cells (Figure 5). This result was not unexpected, because additional biotin molecules should not prevent the detection of CEA by sdAb, whereas an increase in the number of biotins in the sdAb structure should lead to an increase in the number of nanocrystal–streptavidin conjugates bound to the sdAbs. An alternative way to biotinylation of the sdAbs relates to the recombinant origin of the sdAbs. Due to this origin, they can be directly labeled by a single biotin at their C-terminus by addition of a tag that is modified by E.coli biotin ligase during the sdAb production in E. coli. To assess the efficiency of this single but site directed biotinylation, the anti CEA-sdAb was produced in E. coli BL21DE3 strain co-transformed with a plasmid coding for BirA. In order to compare the efficiency of the detection of CEA on cells using in vitro or in vivo biotinylated sdAbs versus a conventional approach employing mAbs, chemical biotinylation 13

was also performed with a biotinylated anti-CEA mAb. The presence of an LC carbonated chain having no effect on the CEA detection capacity of this antibody (data not shown), and biotin-LCLC biotinylation reagent were selected for biotinylation of mAbs thus allowing a direct comparison. In the case of mAbs, the number of primary amine groups is unknown, but their modification is expected to be more critical for antibody activity compared to sdAb. Different mAb-tobiotin-LCLC molar ratios varying from 1:10 to 1:100 were tested in biotinylation reactions; the average number of biotins per mAb was dependent on the molar ratio used for biotinylation (Table 1). Analysis of the CEA detection on cell with these biotinylated mAbs indicated that, in this biotinylation range, the sensitivity of detection directly increased with increasing number of biotins per mAb (Figure 6). We used the same quantities of mAbs that was used in the case of sdAbs (from 0.01 to 100 ng), because we were interested in comparison of the CEA detection capacities using as little antibody material as possible. This allowed us to determine the best suitable strategy for detecting rare cells that express CEA on their surface. Finally, the efficiencies of CEA detection with biotinylated mAbs and sdAbs were compared in our cell model using flow cytometry. In this experiment in vivo biotinylated sdAbs with one biotin moiety at the C-terminus of the polypeptide chain or in vitro biotinylated sdAbs and mAb which contained an average four or eleven biotin moiety consecutively were used for comparison. Strikingly, in vitro and in vivo biotinylated sdAbs achieved up to 10-fold higher staining intensities compared to the same amount of biotinylated mAb (Figure 7). This difference can be partly explained by the difference of molecular weights of sdAbs and mAbs, providing for the same mass concentration nearly 10-fold higher molecular concentration of sdAb compared to a mAb. Taken in account that sdAb is monovalent and mAb is bivalent, one may conclude that in these experimental conditions biotinylated sdAbs 14

were at least 2-fold more efficient. Additionally, the small size of sdAb could also offer other advantages. Indeed, thanks to their small antigen binding site, sdAbs can bind sterically hindered epitopes, such as cavities. Since the CEA molecule is highly glycosylated, oligosaccharide chains may restrict the access of the bulky mAbs to the binding region of the antigen, which is not the case with the compact sdAbs. Moreover, the epitopes recognizable by anti-CEA mAbs and sdAbs are always different. Most CEA-specific mAbs can be classified into five groups according to their epitopes (so-called, five gold mAbs) [33]. As it is known that none of these five epitopes is recognized by this CEA-specific sdAb [31], the possible differences in the affinity of the anti-CEA mAb and sdAb for their respective epitopes should be also taken into consideration. In our case, the affinity of the anti-CEA sdAbs was found to be 8.3 nM [31], which is in the typical range of affinities of conventional anti-CEA mAbs. Interestingly, detection of CEA with in vivo and in vitro biotinylated sdAbs led to very similar results (Figure 7), despite the fact that in vitro biotinylated sdAbs contain four biotin moieties, whereas in vivo biotinylated sdAbs contain only one biotin moiety at the C-terminus of the polypeptide chain. The similar sensitivities of both types of biotinylated sdAbs may be attributed to the steric factor. Since an sdAb has a relatively small size, only a single biotin moiety of a biotinylated sdAb bound with the cell surface might be accessible for binding with SA-QD. Alternatively, the lower number of biotins on the in vivo biotinylated sdAb might be compensated by a better orientation of the biotin at the C-terminus of the domain, i.e. opposite to the binding site, or to an intact conformation compared to the in vitro biotinylated version of this sdAb.

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Conclusion This work demonstrates that biotinylated single-domain antibodies labeled with SA-QD800 conjugates allow specific and very sensitive flow cytometry detection of CEA on the cell surface. Moreover, the production and manipulation of these tiny antibody fragments is both precise and easy, which is important for the development of new staining strategies. The detection of CEA with the use of biotinylated sdAbs is more than 10-fold more sensitive compared to the same amount of conventional monoclonal antibody. The method developed in this study can be easily adapted to the detection of various interesting targets. For example, it would be particularly important to decrease the detection limit of cancer biomakers for diagnostic purposes. The possibility of noninvasive early detection of tumor cells could make its treatment timely, more effective, thereby decreasing the risk of recurrence, the severity of the disease, and, eventually, the cancer mortality. This work highlights the potential to explore the use of single domain antibodies and nanocrystals for the detection of rare cells, including circulating tumor cells at early stages tumors.

Acknowledgments This study was supported by the Federal Targeted Programme for Research and Development in Priority Areas of Development of the Russian Scientific and Technological Complex for 2014-2020 (grant no. 14.575.21.0065, contract no. RFMEFI57514X0065).

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Figure captions.

Figure

1:

Schematic

diagrams

of

conventional

and

camelid

antibodies.

(A) A classical IgG and its derivatives: (B) F(ab’)2, (C) Fab, and (D) ScFv. (E) Camelid heavy-chain antibody. (F) Single-domain antibody.

Figure 2: Preliminary detection of CEA with single-domain antibodies revealed by Streptavidin-QD800 (SA-QD800) conjugates. (A) Geometric mean fluorescence intensity detected for MC38 (grey bars) and MC38-CEA (black bars) stained with SA-QD800 conjugates. The fluorescence detected due to nonspecific binding of SA-QD800 conjugates on cells. (B) Overlay of fluorescence profile of MC38 cells (grey shaded area) and MC38-CEA cells (black) tagged with biotinylated sdAb and SA-QD800 conjugates. (C) Representative staining of MC38 and MC38-CEA on in flow cytometry.

Figure 3: Effects of the biotin reagent used for sdAb biotinylation on CEA detection efficiency. Anti-CEA SdAb was biotinylated with different biotinylation regents: NHS-sulfobiotin, NHS-sulfo-LC-biotin and NHS-sulfo-LCLC-biotin. The rates of CEA detection on MC38 (negative control) and MC38-CEA cells was tested by flow cytometry. Biotinylation of sdAb with different reagents results in comparable sensitivities of CEA detection. However, the biotin-LCLC reagent provides a slightly better sensitivity at low quantities of biotinylated sdAbs added to cells. The abscissa axis is in logarithmic scale.

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Figure 4: The amino acid sequence of anti-CEA sdAb (clone 17). The IMGT numbering is shown. The localization of frameworks (FR1 to FR4) and CDRs are indicated. Lysines are boldfaced and underlined. Figure 5: Effect of the number of biotins per sdAb on CEA detection efficiency. AntiCEA sdAb was biotinylated at different biotin to sdAb molar ratios: 1:20, 1:30, and 1:50. The resultant number of biotins per sdAb is represented as the sdAb to biotin ratio: 1:1, 1:3, 1:4. The capacity of biotinylated sdAbs for detecting CEA on MC38 (negative control) and MC38-CEA cells was tested by means of flow cytometry. The abscissa axis is in logarithmic scale.

Figure 6: Effect of the number of biotins per mAb on CEA detection efficiency. The number of biotins per mAb is represented as the mAb to biotin ratio: 1:4, 1:5, 1:6, 1:8, and 1:11. The capacity of biotinylated mAbs to detect CEA on MC38 (negative control) and MC38-CEA cells was tested by flow cytometry. The results show that the sensitivity of detection increases with increasing number of biotins per mAb.

Figure 7: Comparative efficiencies of different biotinylated antibodies for detecting CEA on cells. Comparison of in vitro biotinylated mAb, in vitro biotinylated sdAb and in vivo biotinylated sdAb efficiencies for the detection of CEA on MC38 (negative control) and MC38-CEA cells by flow cytometry according to the antibody quantity.

23

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Table 1. Estimated average number of biotins per antibody after in vitro biotinylation*.

Antibody to biotin-LCLC molar ratio

1:10

1:20

1:30

1:50

1:100

In vitro biotinylated single-domain antibody (number of biotins per sdAb)

-

1

3

4

-

In vitro biotinylated monoclonal antibody (number of biotins per mAb)

4

5

6

8

11

* sdAb was biotinylated with antibody to biotin-LCLC molar ratios: 1:20, 1:30, and 1:50. mAb was biotinylated with antibody to biotin-LCLC molar ratios: 1:10, 1:20, 1:30, 1:50, and 1:100. Then, the average number of biotins per antibody was estimated using a Pierce® biotin quantitation kit. The number of biotins per biotinylated antibody is the rounded mean number of moles of biotin per mole of antibody calculated by means of the HABA-calculator (www.piercenet.com/haba/habacalc.cfm) based on triplicate assay.

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