Synthetic Single Domain Antibody

Abstract

The invention relates to the identification of a highly stable single domain antibody scaffold (hs2dAb) and its use in generating synthetic single domain antibody library (hs2dAb-L1). The invention also relates to antigen-binding proteins comprising said stable single domain antibody scaffold and their uses, in particular as therapeutics.

Claims

1. A method of making a synthetic single domain antibody library, said method comprising i. introducing a diversity of nucleic acids encoding CDR1, CDR2, and CDR3, between the respective framework coding regions of a synthetic single domain antibody to generate nucleic acids encoding a diversity of synthetic single domain antibodies with the same synthetic single domain antibody scaffold amino acid sequence, wherein said synthetic single domain antibody scaffold amino acid sequence contains at least the following amino acid residues: F37, E44, R45, F47, and; at least the following amino acid residues: P15, S49, S81, R93, A94, and optionally further comprising the residues Q8, Q108 and T99, wherein the positions of amino acid residues are indicated according to the Kabat nomenclature used for VH amino acid sequence, wherein said synthetic single domain antibody scaffold comprises the following framework regions consisting of FR1 of SEQ ID NO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO: 3 and FR4 of SEQ ID NO:4, with 1, 2 or 3 amino acid substitutions within one or more of the framework regions FR1-FR4.

2. The method according to claim 1, wherein the amino acid residues of the synthetic CDR1 and CDR2 of at least 70%, 80% or at least 90% of the clones of the library, are determined by the following rules: at CDR1 position 1: Y, R, S, T, F, G, A, or D; bat CDR1 position 2: Y, S, T, F, G, T, or T; at CDR1 position 3: Y, S, F, or W; at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N; at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L; at CDR1 position 6: S, T, Y, D, or E; at CDR1 position 7: S, T, G, A, D, E, N, I, or V; at CDR2 position 1: R, S, F, G, A, W, D, E, or Y; at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y; at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P; at CDR2 position 4: G, S, T, N, or D; at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M; at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, or K; at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V; and wherein CDR3 amino acid sequence comprises between 9 and 18 amino acids randomly selected among one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

3. A synthetic single domain antibody library obtainable by the method of claim 1.

4. The synthetic single domain antibody library of claim 3, comprising at least 3.10.sup.9 distinct antibody coding sequences.

5. An antigen-binding protein, comprising a synthetic single domain antibody of the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein said FR1, FR2, FR3, and FR4 have at least 90% identity to SEQ ID NOs:1-4 respectively.

6. The antigen-binding protein of claim 5, wherein said framework regions consist of FR1 of SEQ ID NO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO: 3 and FR4 of SEQ ID NO:4, with 1, 2 or 3 conservative amino acid substitutions in at least one of FR1, FR2, FR3 and FR4.

7. The antigen-binding protein of claim 5, wherein said synthetic single domain antibody can be expressed as soluble single domain antibody in E. coli periplasm to the same level as a reference single domain antibody having framework regions consisting of FR1 of SEQ ID NO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO:3, and FR4 of SEQ ID NO:4.

8. The antigen-binding protein of claim 5, wherein said synthetic single domain antibody is expressed as soluble intrabodies in E. coli cytosol, to the same level as a reference single domain antibody having framework regions consisting of FR1 of SEQ ID NO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO:3, and FR4 of SEQ ID NO:4.

9. The antigen-binding protein of claim 5, wherein said synthetic single domain antibody is as stable in a reducing environment as a reference single domain antibody having framework regions consisting of FR1 of SEQ ID NO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO:3, and FR4 of SEQ ID NO:4, as measured in a chloramphenicol acetyl transferase fusion assay.

10. The antigen-binding protein of claim 5, wherein the amino acid residues of the synthetic CDR1 and CDR2 are: at CDR1 position 1: Y, R, S, T, F, G, A, or D; at CDR1 position 2: Y, S, T, F, G, T, or T; at CDR1 position 3: Y, S, F, or W; at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N; at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L; at CDR1 position 6: S, T, Y, D, or E; at CDR1 position 7: S, T, G, A, D, E, N, I, or V; at CDR2 position 1: R, S, F, G, A, W, D, E, or Y; at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y; at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P; at CDR2 position 4: G, S, T, N, or D; at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M; at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, or K; at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V; and wherein CDR3 amino acid sequence comprises between 9 and 18 amino acids selected among one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

11. The antigen-binding protein of claim 5, which further comprises a F-box domain for targeting a protein to the proteasome.

Description

FIGURES LEGENDS

[0202] FIG. 1. (A). Chloramphenicol acetyl transferase carboxy terminal fusion is a folding reporter allowing the selection of soluble amino terminal VHH. Scheme of the construct expressed from pAO-VHH-CATHa vector (B) Relative colony growth of selected VHH on chloramphenicol selection medium (Cam). (C) Amino acid sequences alignment of the lama scaffold lsdAb with the humanized synthetic scaffold of the library hs2dAb. Positions (IMGT numbering) were highlighted according to amino acids conserved from the D10 intrabody scaffold for intrinsic solubility properties of VHH (black) and humanized residues conserved with human conventional VH3 (Grey).

[0203] FIG. 2(A) Phages presenting both scaffold were produced in E. coli and supernatant were detected in Western Blot with an anti-pIII antibody (Biolabs). Two bands are visible, one for pill and one for the fusion with single domains. (B) Production of both scaffolds in E. coli or in CHO cells analysed by Dot Blot. Serial dilutions of supernatant were revealed with an anti-HisTag antibody (Sigma). (C) Immunofluorescence of HeLa cells with recombinant Ab from both scaffolds labelling the nuclear rim structure characteristic of the nuclear lamina. (D) HeLa cells were transiently transfected with the GFP-fused anti-Lamin antibodies expression plasmids and live cells were imaged after 24 h. This showed that both synthetic antibody scaffold anable the recognition of the intracellular lamin target.

[0204] FIG. 3(A) s2dAb D5 stained microtubules by immunofluorescence in HeLa cells. Cells were fixed and stained with a VHH revealed by an anti-HisTag (Sigma) and an anti-MouseCy3 secondary antibody (Jackson). D5 detect also tubulin in Westen blot experiment with HeLa cell extracts revealed with HRP secondary antibody (Jackson). (B) Phage ELISA of clone D10 anti-Her2 on Her2 fused with a rabbit Fc versus binding on rabbit Fc at equimolar concentration. FACS analysis of HD10 anti-Her2 on SKBR3 Her2 positive cells versus MCF10A Her2 negative cells. (C) H12 is a conformational antibody binding only to the GTP bound, activated state of RhoA GTPase. A CBD tagged H12 pull down from HeLa cell extract loaded with either 100 μM GTP gamma S (GTP) or with 1 mM GDP as inputs. Western blot experiment reveals RhoA at similar level in 5% of both input but only on the GTP loaded extract in the CBD-H12 pull down. D5 anti tubulin is a negative control and the conventional GST-RBD (Rho binding domain of Rhotekin) is shown as a positive control of active Rho pull down.

[0205] FIG. 4 Intracellular expression of hs2dAb. (A) HeLa cells were cotransfected with GFP tagged Rab6 and mCherry-tagged hs2dAb anti-GFP plasmids. The hs2dAb-mCherry anti-GFP interacted with its target in vivo and the mCherry signal colocalized perfectly with the GFP-Rab6 one. (B)) HeLa cells were cotransfected with Myr-palm-mCherry and a hs2dAb -GFP anti-mCherry plasmids. The hs2dAb-GFP anti-mCherry interacted with its target in vivo and colocalized perfectly with the mCherry localized at the plasma membrane. (C) Hela cells (p53+/+) and U2OS (p53−/−) cells were transfected either with a hs2dAb-mCherry anti-p53 antibody or with a hs2dAb-mCherry anti-Lamin. While the anti-Lamin intrabody labelled both cells types, the anti-p53 intrabody labelled only the nuclei of p53-positive cells.

[0206] FIG. 5 Sensorgrams for the binding of hs2dAb anti-GFP, anti-p53 and anti-Her2 on immobilized antigens. Different concentrations of hs2dAb were loaded at 25° C. and fitted with a 1:1 langmuir interaction model.

EXAMPLES

[0207] Functional Assays

[0208] Soluble expression in E. coli periplasm

[0209] Single domain antibody fragments were subcloned in a pHEN6 derivated bacterial periplasm expression vector and expressed downstream of the pelB secretion sequence. Freshly transform colonies were grown in Terrific Broth medium supplemented with 1% glucose and 30 μg/ml kanamycin antibiotic until A600=0.6-0.8 was reached. The expression of antibody fragment tagged with 6 His was then induced with 500 μM isopropyl β-D-thiogalactopyranoside for 16 h at 28° C. then span down. After centrifugation, the cell pellets were incubated in Tris-EDTA-Sucrose osmotic shock buffer and centrifuged again. The cell lysates were cleared and loaded onto an IMAC resin affinity column for poly Histidine tag. The eluted fraction was dialyzed, and the purity of the protein was analyzed by SDS-PAGE.

[0210] Soluble Expression of Intrabodies in E. coli Cytosol

[0211] Single domain antibody fragments were subcloned in a bacterial expression vector under the control of a T7 promoter. The plasmid constructs were transformed into E. coli BL21(DE3) cells. Single colonies were grown in LB medium supplemented with 1% glucose and 30 μg/ml kanamycin antibiotic until A600=0.6-0.8 was reached. Antibody fragment expression was then induced with 500 μM isopropyl β-D-thiogalactopyranoside for 6 h to 8 h at 28° C. then span down. After centrifugation, the cell pellets were lysed and centrifuged again. The cell lysates were cleared and loaded onto an IMAC resin affinity column for poly Histidine tag. The eluted fraction was dialyzed, and the purity of the protein was analyzed by SDS-PAGE.

[0212] Chloramphenicol Acetyl Transferase Fusion Assay

[0213] Single domain antibody fragments were subcloned in the pAOCAT bacterial periplasm expression vector. Chloramphenicol resistance assay was performed using BL21(DE3) cells transformed with the pAOCAT-VHH fusion constructs. Bacteria were used for inoculating 500 μL of LB containing kanamycin (35 μg/mL) and glucose (0.2%), and were grown at 37° C. until OD600 was 0.8. The cytoplasmic expression of the VHH-CAT-fusion proteins was induced for 2 hours by the addition of 0.2 mM IPTG. At the end of the induction period, bacteria aliquots of 4 μL were plated on LB-agar plates containing IPTG (0.1 mM) and increasing chloramphenicol concentrations ranging from 0 to 500μg/ml. Bacteria were incubated at 30° C. for 20 hours before quantification of the colony formation. The resistance level was evaluated according to the colony growth rate at the different chloramphenicol concentrations. Several VHH that were giving colonies up to 500 μg/ml were compared to previously characterized intrabodies raised against GFP (nb GFP4) or Lamin (Lam) as well as to a thermostable VHH Re3 and a non intrabody C8. Liquid culture induced as above during 2 hours were diluted by serial dilution and 10 μl were spotted on agar plates containing 250 μg/ml chloramphenicol (Cam) and incubated at 30° C. for 20 hours. Colony were quantified for each dilution and normalized to the higher amount always found with the D10 clone.

[0214] Aggregation Assays in Mammalian Cell Expression System

[0215] Functional Expression as Intracellular Antibodies in Eukaryote Cells

[0216] Single domain antibody fragments were subcloned into a mammalian expression vector in order to express it as a fusion with a fluorescent protein and under the control of a CMV promoter. Mammalian cell lines were transfected and fluorescence in the cells was observed 24 h or 48 h after transfection. In comparison to non fused GFP or mCherry, the fluorescence distribution of VHH fused to one of these fluorescent proteins was homogenously spread in transfected cells, showing no obvious aggregates after 48 h of constitutive expression.

[0217] Functional Secretion as Fc Fusion

[0218] The plasmids are based on the pFUSE-Fc2(IL2ss)™ series from Invivogen (San Diego, USA) that contains the interleukin-2 (IL2) signal sequence and allows the secretion of Fc-Fusion proteins by mammalian cells. Because the hs2dAbs were fused to the hinge domain of IgGs, the Fc domains form di-sulfide bridges and hs2dAb-Fc are expressed as dimmers. They are selectable using Zeocin™ (Zeo) both in prokaryotic and eukaryotic cells. These plasmids were modified by site directed mutagenesis and adaptor insertion (Moutel S, et al. BMC Biotechnol. 2009 Feb. 26; 9:14. doi: 10.1186/1472-6750-9-14) to allow the easy one step cassette cloning of recombinant antibodies extracted from a large collection of common recombinant antibody selection and expression plasmids (e.g pHEN, pSEX, pHAL, pCANTAB, pHOG, pOPE, pSTE). Four plasmids were constructed enabling fusion of s2dAb at their C terminus with either human IgG2 (and IgG1) (h), mouse IgG2a (m) or the rabbit IgG (r) Fc domain (Fc regions comprise the CH2 and CH3 domains of the IgG heavy chain and the hinge region).

[0219] Four days after transient transfection of CHO or HEK cells with these expression plasmids, secreted antibodies could be available using anti-IgG antibodies directed against the respective Fc species. This allows a large diversity of multiplexing.

[0220] Functional Expression in Yeast Two Hybrid System

[0221] Antigen coding sequence was cloned in yeast two hybrid bait plasmid lexA (Vojtek and Hollenberg (1995). Methods Enzymol. 255:331-42). or gal4 (Fromont-Racine, M., Rain, J. C., and Legrain, P. (1997). Nat. Genet. 16: 277-282). DNA binding domain SDAB population to be tested was transferred in yeast two hybrid prey plasmid pGADGH (Bartel, P. L., et al (1993) in Cellular interactions in development: A practical approach. ed. Hartley, D. A. (Oxford University Press, Oxford) pp. 153-179.) by PCR and Gap repair (Orr-Weaver, T. L. and Szostak, J. W. (1983). Proc. Natl. Acad. Sci. USA 80, 4417-4421): DNA prep of pHEN2-3myc plasmid pool (from 1 single clone to 3.109) was prepared. The miniprep DNA was amplify by PCR with oligonucleotide 5p 8328 CCCACCAAACCCAAAAAAAGAGATCCTAGAACTAGCTATGGCCGGACGGGCCAT GGCGGAAGTGCAGCTGCAGGCTTC (SEQ ID NO:11) and oligonucleotide 3p 8329 ACCGGGCCTCTAGACACTAGCTACTCGAGGGGCCCCAGTGGCCCTATCTATGCGG CCGCGCTACTCACAGTTAC (SEQ ID NO:12) using pFu polymerase (NEB) using 10 ng of DNA as matrix. The number of tube is depending of the need in DNA quantity and is related to the number of transformant needed. Typically to obtain 1 million yeast transformants, we carried out 8 PCR of 50 μl.

TABLE-US-00002 PCR program 45 secondes 94° C. 45 secondes 94° C. {close oversize brace} ×25 cycles 45 secondes 57° C. 3 minutes 72° C. 10 minutes 72° C. ∞  4° C.

[0222] PCRs are checked on an agarose gel and concentrated 20 times using ammonium acetate precipitation and resuspended in water.

[0223] 8 μg of plasmid prey pGADGH digested by NcoI and XhoI and 2 μl of concentrated PCR are transformed in yeast by classical LiAc/PEG transformation. The clones are spread to selective media dropt out minus Tryptophane, Leucine and Histidine. The baits specific clones, identified by this way, are intrabodies from the library that are functional in the yeast cells. Generation of synthetic single domain antibody library and characterization of binders obtained from said library

[0224] We selected a family of highly functional scaffolds, optimized for intracellular expression and high thermostability. This selection was done using fusion proteins between an antibiotic resistance gene and a collection of VHH.

[0225] Only bacteria expressing a functional VHH fusion (non aggregating, non degraded) could grow. Expression yield, solubility as GFP fusion in mammalian cells cytoplasm have been further assessed [and compared to selected chromobodies] to select a set of suitable antibodies. The sequences of these antibodies were aligned, and a consensus sequence was defined by the consensus-sdAb framework sequence of the clone D10 see SEQ ID NO:9). In addition to this llama sdAb (lsdAb) we altered the sequence so that it was more similar to human VH, an evolved consensus was thus defined as a synthetic sdAb (hereafter referred as “hs2dAb”, see SEQ ID NO:10). We kept the specific hallmarks of VHH at four FR2 positions (37, 44, 45, 47) that are conserved in conventional VH to form the hydrophobic interface with VL and that appeared crucial for intrinsic solubility properties of sdAb (Kastelic D, et al. 2009 J Immunol Methods. October 31; 350(1-2):54-62) (FIG. 1a). We then confirmed that the scaffold was robust and behaving as expected by grafting the CDRs of a known antibody into the 1sdAb and s2dAb frameworks. These experiments showed that both 1sdAb and hs2dAb allowed efficient display, efficient production in bacteria and in CHO cells, and that it allowed to keep proper reactivity of the grafted CDRs both when expressed in oxidative and in reducing condition. So we decided to select the hs2dAb as a framework to construct our library.

[0226] We then introduced a synthetic diversity in the three CDRs without affecting the functionality of the clones. Based on alignment of hundreds of llama sdAb sequences we rationally designed for each position of the CDR1 and CDR2 a set of amino acids that still mimic natural diversity. We voluntarily avoided cysteine residues because thiol groups could later interfere with proper intracellular expression and functionality. We reasoned that lowering the frequency of hydrophobic residues or arginin would avoid aggregation (De Marco A. 2011, Microb Cell Fact. June 9; 10:44 Review) and that lowering as well proline frequency would keep most flexibility in the loops. As serine, threonine and tyrosine are the most frequent residues in CDR loops involved in bonds with the epitope, aspartate and glutamate have been proposed to increase solubility (Lodish H, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman, 2000). We voluntarily enriched these five residues at some positions. In contrast we fully randomized some positions of CDR2 as well as each position of CDR3 by introducing all amino acids except cysteine. Nevertheless a diversity in length was also introduced in the CDR3 sequences to enrich binding potential to different epitope shape since nanobodies have been shown to bind both flat surface or cavity (De Genst, E., et al. Proc Natl Acad Sci, (2006) March 21; 103(12):4586-91), or even haptens (Harmsen M M, et al 2007, Appl Microbiol Biotechnol., November; 77(1):13-22. Review). In order to respect these statistics, to lower the occurrence of too hydrophobic residues or amino-acid promoting aggregation, and in contrast to enrich in polar aminoacid, and to further avoid the occurrence of in frame stop or cysteins and reduce frameshift, the synthesis of the library diversity was achieved by a unique gene synthesis technology that use double strand DNA triplet blocks corresponding to each codon (Van den Brulle J, et al. 2008. Biotechniques. September; 45(3):340-3). All codons were optimized for mammalian cell expression, a SnabI restriction site was added in FR3 for further CDR3 loop grafting or engineering whereas any other undesired restriction site were avoided in the scaffold.

[0227] To generate a large complexity and to reduce the number of empty plasmids, we constructed a novel cloning vector bearing a suicide gene (ccdB) between non compatible cloning sites. Only plasmids were the toxic gene was lost allowed bacteria growth, hence only plasmid bearing a SDAB insert were obtained. CcdB gene is used as a positive selection marker. Most of the test done to detect binding activities, are done using a monovalent selected antibody, means of detection are then based on anti-tag immunostaining. But monovalent tags on monovalent antibodies do not allow strong detection, so we decided to add a triple tag to the novel phagemid that we constructed to increase the power of detection.

[0228] To ensure the full diversity of cloned hs2dAbs, the number of theoretical diversity according to the rational design was far above the one of synthesized molecules which was again 4 log above the number of transformed colonies. The most crucial was the very high molecular diversity of hs2dAb synthesized that reached more than 10.sup.12 sequences with a full probability of being unique since it was still above theoretical diversity imposed by the design. The fully synthetic hs2dAb-L1 library was cloned in the pHEN2-3myc vector and up to 3.10.sup.9 colonies were transformed.

[0229] The quality and functionality of the hs2dAb-L1 library has been assessed first by sequencing 10.sup.5 random clones. We then performed screening directed to various kind of Ag with several selection procedures. For the different target tested, we obtained binders with good affinities for EGFP, 13-Tubulin, actin, Rho conformational, p53 and Her2. Characterisation of the specificity, affinity and productivity of selected hs2dAb binders is described.

[0230] Materials and Methods

[0231] Plasmids and Cloning

[0232] A synthetic gene (Mister Gene) composed of a 6His-Tag and a triple c-myc Tag was inserted into the pHEN2 phagemid vector (Griffin 1. library) between NotI and BamHI sites. The ccdB gene from pENTR™4 vector (Invitrogen) was inserted into the pHEN2 vector between NcoI and NotI sites. For mammalian expression vectors, VHH or hs2dAbs were digested by NcoI and NotI and ligated into the pAOINT or the pmCherry vectors (Clontech).

[0233] Cat Assay Filter

[0234] The pAO-CAT is a cytoplasmic expression vector that enables to fuse a carboxy-terminal HA-tagged chloramphenicol acetyl transferase (CAT) to the VHH sequences. It has been constructed by cloning a VHH-CAT sequence into the pAOD-TubI-mGFP vector (Olichon A, et al. 2007. J Biol Chem. December 14; 282(50):36314-20) digested XbaI and KpnI to remove the DsbC-TubI-mGFP. The VHH-CAT sequence has been obtained by a multi-step PCR strategy. The VHH was amplified using 5′CCTTGATTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGCTG ATGTCCAGCTGCAGGCGT3′ (Fw, SEQ ID NO:13) and 5′CCACCGCTACCGCCGCTGCGG CCGCGTGAGGAGACGGTGACCTGG G3′ (Rev, SEQ ID NO:14). Two sequences of CAT were amplified independently using the pRill plasmid as a template to remove an internal NcoI site. For the N-term, the following primers were used: 5′ GCGGCCGCAGCGGCGGTAGCGGTGGCGAGAAAAAAATCACTGGATATACC 3′ (Fw, SEQ ID NO:15) and 5′ GCCCATCGTGAAAACGGGGGCG 3′ (Rev SEQ ID NO:16). The C-term was amplified using: 5′ CGCCCCCGTTTTCACGATGGGC 3′ (Fw, SEQ ID NO:17) and 5′AGAATAGGTACCAGCGTAATCTGGGACATCATAAGGGTAGCCACCCGCCCCGC CCTGCACTCATCG 3′ (Rev SEQ ID NO:18). The three sequences were assembled with a final PCR and the product was digested XbaI and KpnI before being ligated into the vector. Previously selected VHHs from naïve library were subcloned into pAOCAT using the NcoI and NotI restriction sites. Chloramphenicol resistance assay was performed using BL21(DE3) cells transformed with the pAOCAT-VHH fusion constructs. Bacteria were used for inoculating 500 μL of LB containing kanamycin (35 μg/mL) and glucose (0.2%), and were grown at 37° C. until OD600 was 0.8. The cytoplasmic expression of the VHH-CAT-fusion proteins was induced for 2 hours by the addition of 0.2 mM IPTG. At the end of the induction period, bacteria aliquots of 4 μL were plated on LB-agar plates containing IPTG (0.1 mM) and increasing chloramphenicol concentrations ranging from 0 to 500 μg/ml. Bacteria were incubated at 30° C. for 20 hours before quantification of the colony formation. The resistance level was evaluated according to the colony growth rate at the different chloramphenicol concentrations. Several VHH that were giving colonies up to 500 μg/ml were compared to previously characterized intrabodies raised against GFP (nb GFP4) or Lamin (Lam) as well as to a thermostable VHH Re3 and a non intrabody C8. Liquid culture induced as above during 2 hours were diluted by serial dilution and to 10 μl were spotted on agar plates containing 250 μg/ml chloramphenicol (Cam) and incubated at 30° C. for 20 hours. Colony were quantified for each dilution and normalized to the higher amount always found with the D10 clone.

[0235] The D10 clone was further subcloned into the pHEN6 expression vector, leading to a periplasmic expression higher than 5 mg/L of culture in E coli Xl1blue strain.

[0236] It was subcloned into the pAOint-mGFP and transfected in MRC5, HEK293 or HeLa S3 cells. Transient expression of D10-GFP under the control of a CMV promoter leads to high GFP fluorescence and no aggregation detectable compared to Lam1-GFP or Re3-GFP at 24 h and 48 h.

[0237] Library Construction

[0238] Gene collections corresponding to the FR and CDR design have been synthesized in vitro (Sloning, GeneArt). 1 μL (10 ng) of the diverse synthesis (corresponding to 1.10.sup.10 molecules, hence 10 times the target library diversity) were amplified by PCR in a total volume of 50 μL using 1 μL of Phusion DNA polymerase (New England Biolabs) with an equimolar mixture of the following primers:

TABLE-US-00003 (SEQ ID NO: 19) 5′-AACATGCCATCACTCAGATTCTCG-3′ (SEQ ID NO: 20) 5′-GTTAGTCCATATTCAGTATTATCG-3′

[0239] PCR protocol consisted of an initial denaturation step at 98° C. for 45 sec followed by 20 cycles of 98° C. for 10 sec, 55° C. for 30 sec and 72° C. for 30 sec, and a final step extension at 72° C. for 10 min. 7×150 μL of PCR were purified on 7 columns of a PCR clean-up kit (Macherey-Nagel). 55 μg of the resulting purified fragment of PCR and 80 μg of the pHEN2-ccdB-3myc phagemid were digested for 2H at 37° C. with NcoI and NotI (NEB) in a total volume of 500 μL. A dephosphorylation step was added for the phagemid with a Calf intestinal alkaline phosphatase (Sigma) 30 min at 37° C. Digestions were purified on gel with respectively 4 and 6 colums of a gel extraction kit (Macherey-Nagel) in a final volume of 80 and 120 μL. Then, purified PCR fragment was ligated into pHEN2-ccdB-3myc, between the PelB leader signal and the pIII gene. 50 μg of phagemid and 19.2 μg of insert were ligated overnight at 16° C. with 10 μL of high concentration T4 DNA ligase (NEB) in a total volume of 400 μL. Ligation was purified on 6 columns (Macherey-Nagel) with a total volume of 150 μL. The ligated DNA material was used to transform electrocompetent E. coli TG1 cells (Lucigen). 20 electroporations with 1 μl of ligation were performed according to the manufacturer's instructions (1800V; 10 μF; 600Ω). Each electroporation was resuspended with 1 mL of warm 2XYT, 1% glucose medium and incubated with a shaking agitation for 1H at 37° C. 380 mL of 2XYT, 1% glucose was added to the suspension and plated on 430 2xYT-ampicillin agar dishes (140 mm) overnight at 37° C. Library size was calculated by plating serial dilution aliquots. The colonies were scraped from the plates with liquid 2xTY and library was stored in the presence of 30% of glycerol at −80° C. with 1 mL aliquots at OD=38,4. 3.10.sup.9 individual recombinant clones were obtained.

[0240] Library Sequencing:

[0241] The heterogeneity of the individual clones from the libraries was checked by sequencing 6.10.sup.5 inserts on ion Torrent chips (Invitrogen).

[0242] IonTorrent sequencing library was prepared with the Ion Plus Fragment Library kit for AB Library Builder System (Life Technologies) following manufacturer's instructions and was controlled on the Agilent 2100 Bioanalyzer (Agilent Technologies) with the High Sensitivity DNA Kit (Agilent Technologies). Sequencing template was prepared by emulsion PCR with the Ion OneTouch 2 system and the Ion PGM Template OT2 400 Kit (Life Technologies). Sequencing was performed on a IonTorrent Personal Genome Machine using the Ion PGM Sequencing 400 Kit and a 314v2 Ion chip (Life Technologies).

[0243] Antigens

[0244] Human βActin was purchased from Sigma. RhoA GTPase fused to an amino terminal Chitin Binding Domain or a streptactin binding peptide were produced in HEK293 cells. GFP in fusion with a streptavidine binding peptide (SBP) was produced through in vitro translation system (Roche) and used directly for screening without the need for purification (Moutel S, et al. 2009. Biotechnol J. January; 4(1):38-43). Biotinylated Tubulin was purchased from Cytoskeleton. For p53, the 72 first amino acids of the NP_000537.3 isoform were produced in bacteria with a SNAP Tag and biotinylated in vitro. For Her2, the natural receptor was used as membrane protein target on SKBR3 cells.

[0245] Phage Display Selections

[0246] Screening for Bactin, H1 histone, or FITC were performed by panning in immunotubes as described in Marks J D et al, 1991 J Mol Biol. December 5; 222(3):581-97. Screening for GFP, Tubulin and p53 were performed in native condition as described in Nizak et al. 2003 Science. May 9; 300(5621):984-7. Screening on Rho was performed in native condition on a tag constitutively active mutant of RhoA expressed in HEK293 cells. Screening for Her2 was performed on surface cells as described in Even-Desrumeaux K, Chames P. 2012 Methods Mol Biol.; 907:225-35.

[0247] Enzyme-Linked Immunosorbent Assay (ELISA)

[0248] Individual clones were screened as described else-where by monoclonal phage ELISA (Lee. et al; 2007, Nat Protoc. 2(11): 3001-8.

[0249] Western-Blot

[0250] After boiling in SDS-PAGE loading buffer, the samples were separated on a 12% SDS-PAGE and transferred to nitrocellulose membranes (Whatman GmbH). Membranes were blocked in 3% non-fat milk-PBS with 0.2% Tween 20 for 1 h at room temperature or overnight at 4° C. SDAB were used at 1/100 and added to the membranes with an anti-hisTag antibody at 1/3000 (Sigma) for 90 min. Blots were then washed and incubated 1 h with secondary anti-Mouse HRP labeled antibodies (diluted at 1/10000 in PBS 0.1% Tween 20) (Jakson ImmunoResearch Laboratories). After 5 washes with PBS 0.1% Tween 20, secondary antibodies were then revealed using the SuperSignal chemoluminescent reagent (Pierce) and Hyperfilm ECL (GE HealthCare).

[0251] Immunofluorescence

[0252] Immunofluorescence screenings were performed on HeLa cells as described before (Nizak et al. 2003. Science. May 9;300(5621):984-7).

[0253] Transient Transfection

[0254] Hela Cells cultured on coverslips were transfected according to the CaPO4 procedure with 1 μg DNA per well (24 wells plate) or 10 μg DNA (10cm2 diameter dish). Cells can be observed from 12 h posttransfection on.

[0255] Flow Cytometry

[0256] Cell surface staining were performed in phosphate-buffered saline (PBS) supplemented with 1% SFV. 100 μl of supernatant (80 μl phages+20μl PBS/milk 1%) were incubated on 1.10.sup.5 cells for 1 h on ice. Phage binding was detected by a 1:300 dilution of anti-M13 Ab (GE healthcare) for 1 h on ice followed by a 1:1000 dilution of PE-conjugated anti-Mouse Ab (BD Pharmingen.) for 45 min. Samples were analyzed by flow cytometry on a FACSCalibur using CellQuest Pro software (BD Biosciences, San Jose, Calif.).

[0257] Affinity Measurement

[0258] The binding affinity of the hs2dAb antibodies selected from the library and specific for GFP and ErBB2 were performed at 25° C. using a ProteOn XPR36 (BioRad) and a Biacore T200 (GE Healthcare), respectively, and fitted with a 1:1 Langmuir interaction model. The ligand GFP (24 kDa) was diluted to 1.6 μM in sodium acetate buffer (pH 5.0) and immobilized by amine-coupling on a GLC chip (BioRad) at 730 RU. 100 μL of monovalent single-domain antibodies (14 kDa) were used as an analyte and injected at 100 μL/min at concentrations between 1000 and 3 μM (60 second injection, 600 second dissociation). The complete kinetic set was collected in a single run (one-shot) and, therefore, there was no need for surface regeneration. ErbB2 ectodomain-Fc (96 kDa) was diluted to 400 μg/mL in sodium acetate buffer (pH 5.0) and immobilized by amine-coupling on a CM5 chip (GE Healthcare) at 991 RU. Monovalent single-domain antibodies (14 kDa) were diluted in HBS-EP+ buffer and injected as analytes at 30 μL/min at concentrations between 300 and 3 μM using the single-cycle modality (120 second injection, 120 second intermediate dissociation, 600 second final dissociation). The kinetics were collected in a unique sequence of injections and surface regeneration (10 mM glycine HCl, pH 2.5, for 30 s at 30 μL/min) took place only between two successive series.

[0259] Results

[0260] Library Design

[0261] In the view of making a large single domain antibody library enriched in highly stable and functional antibody fragments, we aimed at identifying a single VHH scaffold. We previously selected several hundreds of clones from immune or naïve llama VHH libraries (Monegal A, et al. 2012 Dev Comp Immunol. January; 36(1):150-6). We screened a set of highly expressed clones using a chloramphenicol filter assay that discriminate highly stable clone from the one prone to aggregation or unfolding in bacteria cytoplasm (Olichon). We used the pAO-CAT cytoplasmic expression vector that enables to fuse a carboxy-terminal HA-tagged chloramphenicol acetyl transferase (CAT) to the VHH sequences. By comparison with published thermostable VHH (Olichon A, et al BMC Biotechnol. 2007 Jan. 26; 7:7) or intrabodies (Rothbauer U, et al Nat Methods. 2006 November; 3(11):887-9), one scaffold, clone D10, was showing higher chloramphenicol resistance (FIG. 1a). The D10 VHH was further fitting all our criteria of solubility, thermostability and no aggregation while expressed as an intrabody in mammalian cells, in the absence of any known antigen recognition. We then assessed if partial humanization (Vincke C, et al. J Biol Chem. 2009 Jan. 30; 284(5):3273-84) of the scaffold would affect its intrinsic properties by targeting seven residues that are found in human VH3. The four VHH-specific amino acids hallmarks in the framework-2 region (positions 42, 49, 50, and 52) that appeared crucial for intrinsic solubility properties (FIG. 1c) were kept untouched.

[0262] To test whether this scaffold may be suitable for antigen binding and used as a general scaffold for library construction, we grafted loops of the lam1 VHH specific of laminB protein (Rothbauer U, et al. Nat Methods. 2006 November; 3(11):887-9). FIG. 2a shows that both scaffolds (lsdAbB and hs2dAb) do not perturb the display of the recombinant antibody on phages, here labelled with an anti-pIII antidody. The yield of production from culture supernatants either from E. coli or from mammalian CHO cells (Moutel S, et al BMC Biotechnol. 2009 Feb. 26; 9:14. doi: 10.1186/1472-6750-9-14) were also high and comparable for both scaffolds, (see FIG. 2b). The grafted synthetic antibodies were then used to stain HeLa cells by indirect immunofluorescence. Both grafted single domain antibodies produced the expected staining indicating that they efficiently stained endogenous Lamin B (FIG. 2c). The two synthetic antibodies were then fused to EGFP and used as intrabodies. After transient transfection of HeLa cells, both fluorescent antibodies were soluble and labelled their intracellular target (FIG. 2d), as was observed when using the original lam1 VHH antibody (Rothbauer U, et al 2006). Both scaffold, Lama and Humanized D10, grafted with lam1 CDR loops thus retained the binding property of the parental VHH. <.

[0263] Altogether, these experiments indicated that the synthetic scaffold of humanized D10 (herein called Synthetic Single Domain Antibody or hs2dAb) is an efficient and robust framework to display CDR loops.

[0264] Library Construction

[0265] We introduced a synthetic diversity in the three CDRs by rationally designing a set of amino acids that still mimic natural diversity for each position of the CDR1 and CDR2 (based on statistical analysis of CDRs found in published VHH binders). The amino acids residues of the synthetic CDR1 and CDR2 have been determined by the following rules: [0266] at CDR1 position 1: Y, R, S, T, F, G, A, or D; [0267] at CDR1 position 2: Y, S, T, F, G, T, or T; [0268] at CDR1 position 3: Y, S, S, S F, or W; [0269] at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N; [0270] at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L; [0271] at CDR1 position 6: S, T, Y, D, or E; [0272] at CDR1 position 7: S, T, G, A, D, E, N, I, or V; [0273] at CDR2 position 1: R, S, F, G, A, W, D, E, or Y; [0274] at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y; [0275] at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P; [0276] at CDR2 position 4: G, S, T, N, or D; [0277] at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M; [0278] at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, or K; [0279] at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V;

[0280] The CDR3 amino acid sequence comprises 9, 12, 15 or and 18 amino acids selected among one or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

[0281] Synthetic DNA was amplified using a low number of PCR cycles to prevent the addition of PCR-based mutations. We started the construction with 2.10.sup.11 different molecules. We cloned the synthetic library in the pHEN2 phagemide vector modified by adding 2 supplemental myc-tags with a synthetic gene (Proteogenix) between the recombinant Ab and the pIII gene. These additional myc-tags allow better revelation of monovalent Ab (data not shown). For the cloning we added also a suicide gene (ccdB) between NcoI and NotI that allows a positive selection of clones that have inserted the library. A large amount of phagemid and insert were used to obtain a lot of material to electroporate E. coli TG1 cells. 20 electroporations were performed to produce the hs2dAb-L1 library. Transformed bacteria were plated on 430 large agar plates (140 mm). The library size was calculated by plating serial dilution aliquots and was estimated to be of 3.10.sup.9 individual clones.

[0282] Library Sequencing:

[0283] We first evaluated the functional diversity by sequencing 315 random clones with Sanger sequencing. 9 sequences were found either with stop codon or with the missing of one base, one sequence missed all the CDR1, one other missed CDR1, FR1 and CDR2, and two last sequences were found empty. Thus, only a very low number (4,1%) of defective clones were obtained. Which suggest that the vast majority of the 3 10.sup.9 clones will express a recombinant hs2dAb.

[0284] The heterogeneity of the individual clones from the libraries was further checked by sequencing 5.6.10.sup.5 inserts on ion Torrent chips (Life Technologies). The distribution of length sequences for the 4 sizes of CDR3 was homogenous. The diversity and positional amino acids frequency were in agreement with our design for all CDRs (data not shown). 3 redundant clones were found but this observation may be linked to the PCR amplification done during the Next-Generation Sequencing procedure.

[0285] Library Screening

[0286] The hs2dAb-L1 library was screened by phage display using standard methods (Hoogenboom H R, et al. 1998 June; 4(1):1-20) against a set of different antigens reported in Table1. To validate the use of hs2dAb-L1 in various screening approaches, we either carried out selection on beads (which offers a large capacity of antigen presentation and keep the antigen conformation closer to native protein), either panning on immunotubes (referenced as standard method) or on natural antigen presented naturally on the surface of mammalian cells (as often done when applying in vitro selection to therapeutic questions).

[0287] A first screening was performed in native condition (Nizak, 2005, see supra) using biotinylated tubulin (Cytoskeleton) as a target. After two rounds of selection, 40 clones were screened at random by immunofluorescence on HeLa cells fixed with methanol. 3 recombinant Ab stained the endogenous tubulin (FIG. 3A). These 3 clones were also usable in Western-Blotting of human protein samples (FIG. 3A). A second screening was performed against endogenous Her2 receptor expressed at the surface of SKBR3 cells. A pre-adsorption of the phages was done at each of the 3 rounds on Her2 negative MC10A cells to counter select non-SKBR3 specific antibodies. Using a FACS assay, 17 specific anti-SKBR3 binders were found out of 84 analysed that could be grouped in 17 non-redundant sequences. Analysis by ELISA, using Her2 ectodomain-Fc (96 kDa) as a target, showed that 12 of them were directed against HER2 (FIG. 3B). Immunofluorescence confirmed that the antibodies decorated the SKBR3 plasma membrane (data not shown). A third screening was performed directed against the tumor suppressor p53 protein. The 72 first amino acids of the NP_000537.3 isoform were produced in bacteria fused to a SNAP Tag, biotinylated in vitro and used as a target on beads. After 3 rounds of selection, 12 clones out of 80 were found positive in phage ELISA (data not shown), 6 clearly labelled endogenous p53 in immunofluorence on A431 cells (data not shown) while 2 were usable as intrabodies on HeLa cells (see FIG. 4). As expected, no staining was observed on U2OS cells which are p53−/−. A fourth screening was performed to obtain conformational binder of Rho-GTP protein. After four rounds of competitive selection (see materials and methods), 80 unique clones were tested in ELISA on using recombinant GST-RhoA proteins loaded with GDP of the non-hydrolysable analogue GTPyS. 24 antibodies gave a stronger signal against GTPγS-RhoA than against GDP-Rho, indicating conformational binding. One of the antibodies, the H12 clone, dominated the screen and was the only clone obtained upon an additional 5th round of selection. We tested these conformational binders in immunoflorescence on HeLa cells expressing SF-GFP (superfolder GFP) fusion of RhoA negative mutant N19 (GDP-bound) or the constitutively active mutant L63 (GTP-bound). None of the clones stained cells expressing the N19 negative mutant while 8 of them efficiently stained cells overexpressing the active form of RhoA. We further characterized the clone H12 and tested its ability to pull down endogenous RhoA from HeLa cell extracts incubated with GTPγS or GDP. We showed that purified H12 antibody was an effective conformational biosensor of Rho activity usable in ELISA, immonofluorescence and in vitro pull down experiments. We further observed that it was more effective than the conventional RBD activity assay were the Rho Binding Domain of an effector protein is used as a biosensor. (FIG. 3C, IF and ELISA Data not shown). A fifth screening in native conditions was performed using the GFP protein as a target. Thirty seven non redundant clones out of 80 analysed were positive in phage ELISA (data not shown). Ten of them detected GFP-myr-palm, by immunofluorescence (data not shown). Importantly, we observed that 4 of these antibodies were usable as intrabodies against recombinant GFP expressed in Hela cells (FIG. 4). A last screen was performed using immunotubes coated with native commercial bActin (Sigma). This last screening allowed the selection of 16/80 unique binders as observed by phage ELISA (data not shown), Seven of them clearly decorated endogenous actin stress fibers in HeLa cells (data not shown) and 4 were usable for Western-Blotting experiments (data not shown).

TABLE-US-00004 TABLE 1 Summary of screening Positive clones Phage Rounds of Antigen ELISA IF/FACS Intrabody panning GFP 37 10/   4/10 2 mCherry ND 6/ 2/6 3 Tubulin ND 3/ 0/3 2 Actin 11 9/ 1/7 3 p53 12 6/ 2/6 2 RhoA-GTP 24 8/ 3/8 4 Her2 15  5/10 ND 3

[0288] Affinity Measurement

[0289] Affinity of single domain antibodies against GFP, Her2 and p53 were measured using a ProteOn XPR36 (BioRad) or a Biacore T200 (GE Healthcare). Affinities were estimated to be in the nanomolar range: 3.06 10.sup.−8M for anti-GFP, 1.94 10.sup.−8M for anti-Her2 and 3.25 10.sup.−8 M for anti-p53, demonstrating that high affinity binders could be obtained from our hs2dAb-L1 library of synthetic, non immune, single domain Ab (FIG. 5).

[0290] Inhibitory Intrabodies

[0291] Identification of blocking antibodies is a challenging task. However, it is possible to functionalize non-blocking intrabodies to inhibit their target function. One approach relies on the ubiquitinylation and degradation of the recognized target as described by Caussinus et al. 2011 (Caussinus et al. 2011, Nat. Struct. Mol. Biol. 19, 117-121). This approach is based on the fusion of intrabodies to an F-box domain which allows interaction with Skip1, a member of the SCF complex, an E3 ubiquitin ligase of the complex E1/E2/E3 ubiquitinylation machinery, that target proteins to proteasome-dependent cellular degradation. This approach was initially developed to target several GFP fusion proteins in Drosophila using a single anti GFP intrabody, named GFP4, which is a robust high affinity GFP llama intrabody originally isolated from an immune library (Rothbauer, U. et al. Nat. Methods 3, 887-889). To get insight into the relative functionality of hs2dAb for such a protein knockdown approach, we fused several of our anti GFP hs2dAb at their amino terminus to the Fbox domain and compared their efficacy with the efficacy of the Fbox-GFP4 antibody. To detect cells expressing Fbox-intrabody fusion proteins (F-Ib), we constructed a bicistronic vector driving the co-expression of F-Ib together with a mitochondria-targeted mCherry (Mito-mCherry). We expressed the F-Ib antibodies in a HeLa clone stably expressing GFP fused to histone H2B (Silljé, H. H. W., Nagel, S., Körner, R. & Nigg, E. A., 2006, Curr. Biol. CB 16, 731-742) and looked for GFP-H2B depletion. As expected, F-GFP4, also known as degradFP, induced a strong reduction of H2B-GFP expression as analyzed by western blot (data not shown). Accordingly, a strong reduction in nuclear fluorescence intensity was observed in cells expressing F-GFP4. No effect was observed when expressing either GFP4 alone or a GFP4 fused to a truncated, nonfunctional, Fbox domain. When we tested anti-GFP clones selected from our novel library, we observed that several hs2dAb that were found to be efficient when used as fluorescent intrabodies failed to degrade H2B-GFP when expressed as F-Ib. This highlights the fact that not all intrabodies can efficiently be functionalized with the F-box. However, one hs2dAb anti-GFP induced a complete disappearance of nuclear H2B-GFP signal when expressed as F-Ib. FACS analysis showed a fluorescence intensity decreased as much as 70%. As expected, this effect was reversed in the presence of proteasome inhibitor treatment.

[0292] Altogether, these experiments show that the hs2dAb scaffold enables the frequent selection of antibodies that can be expressed in the mammalian cell cytoplasm to be used as fluorescent intrabodies or inhibitory intrabodies.

[0293] Useful Sequences for Practicing the Invention

TABLE-US-00005 SEQ ID NO: Description Sequence  1 single domain VQLQASGGGFVQPGGSLRLSCAASG antibody FR1  2 single domain MGWFRQAPGKEREFVSAIS antibody FR2  3 single domain YYADSVKGRFTISRDNSKNTVYLQMNSLRA antibody FR3 EDTATYYCA  4 single domain YWGQGTQVTVSS antibody FR4  5 coding nucleic ATGGCGGAAGTGCAGCTGCAGGCGAGCGGC acid sequence GGCGGCTTTGTGCAGCCGGGCGGCAGCCTG of SEQ ID NO: CGTCTGAGCTGCGCGGCGAGCGGC 1  6 coding nucleic ATGGGCTGGTTTCGTCAGGCGCCGGGCAAA acid sequence GAACGTGAATTTGTGAGCGCGATTAGC of SEQ ID NO: 2  7 coding nucleic TATTATGCGGATAGCGTGAAAGGCCGTTTT acid sequence ACCATTAGCCGTGATAACAGCAAAAACACC of SEQ ID NO: GTGTATCTGCAGATGAACAGCCTGCGTGCG 3 GAAGATACCGCTACGTATTATTGCGCG  8 coding nucleic TATTGGGGCCAGGGCACCCAGGTGACCGTG acid sequence AGCAGCGCGGCCGCA of SEQ ID NO: 4  9 Non humanized VQLQESGG GFVQAGGSLR LSCAASGFTF sequence of SSYAMGWFRQ APGKEREFVA clone D10 AISDSSGNHAYYADSVKG RFTISRDNAK NTVYLQMNSL KPEDTATYYC ARSDAAGNPS GYWGQGTQVTVSS 10 Humanized VQLQASGG GFVQPGGSLR LSCAASGFTF sequence of SSYAMGWFRQ APGKEREFVS clone D10 AISDSSGNHAYYADSVKG RFTISRDNSK NTVYLQMNSL RAEDTATYYC ARSDAAGNPS GYWGQGTQVTVSS 11 oligonucleo- CCCACCAAACCCAAAAAAAGAGATCCTAGA tide 5p ACTAGCTATGGCCGGACGGGCCATGGCGGA 8328 AGTGCAGCTGCAGGCTTC 12 oligonucleo- ACCGGGCCTCTAGACACTAGCTACTCGAGG tide 3p GGCCCCAGTGGCCCTATCTATGCGGCCGCG 8329 CTACTCACAGTTAC 13 VHH Fw primer CCTTGATTCTAGAAATAATTTTGTTTAACT TTAAGAAGGAGATATACCATGCTGATGTCC AGCTGCAGGCGT 14 VHH Rev primer CCACCGCTACCGCCGCTGCGG CCGCGTGA GGAGACGGTGACCTGG G 15 Fw N-term GCGGCCGCAGCGGCGGTAGCGGTGGCGAGA AAAAAATCACTGGATATACC 16 Rev N-term GCCCATCGTGAAAACGGGGGCG 17 Fw C-term CGCCCCCGTTTTCACGATGGGC 18 Rev C-term AGAATAGGTACCAGCGTAATCTGGGACATC ATAAGGGTAGCCACCCGCCCCGCCCTGCAC TCATCG 19 Primer library AACATGCCATCACTCAGATTCTCG 20 Primer library GTTAGTCCATATTCAGTATTATCG